Note: Descriptions are shown in the official language in which they were submitted.
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AMINO ACID COPOLYMER COMPOSITIONS AND USES THEREOF
CROSS REFERENCE TO PRIOR APPLICATIONS
This application claims priority to and the benefit of U.S. provisional patent
.. application serial numbers 62/357,800 filed on July 1, 2016 and 62/478,410
filed on March
29, 2017, the disclosures of which are incorporated herein by reference in
their entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted
via EFS-Web
and is hereby incorporated by reference in its entirety. Said ASCII copy,
created on June 30,
2017, is named 109044-0048-W01 SL.txt, and is 102,602 bytes in size.
BACKGROUND OF THE DISCLOSURE
Many challenges exist in designing and manufacturing polypeptide
vaccines/therapeutics for immunotherapy. Although the specific challenges may
vary across
applications, some of the challenges include (i) inducing antibodies against
relevant target(s);
(ii) preserving the normal functional protein where applicable; (iii)
eliciting immunogenicity
across a broad patient population; (iv) achieving efficacious antibody titer;
(v) reaching
target(s) in the brain; (vi) avoiding or minimizing a Thl/pro-inflammatory
immunity and
.. meningoencephalitis, where applicable; (vii) avoiding microhemorrhages and
vasogenic
edema; and (viii) significant costs associated with passive immunity. In light
of the modest
success of existing approaches for some applications, there remains a vast
need for a better
approach for generating effective polypeptide/peptide compositions suitable
for use as
vaccines or immunogens. Such an approach would provide compositions and
methods for
eliciting beneficial immune responses toward pathological proteins or
polypeptides related to
a disease or condition, or against infectious pathogens such as pathogens,
which tend to evade
the host immune system.
SUMMARY OF THE DISCLOSURE
The present disclosure provides compositions and methods that aim to address
the
limitations of existing technologies. In certain embodiments, the present
disclosure provides
compositions comprising high complexity mixtures (e.g., mixtures of greater
than at least 500
polypeptides) of polypeptides based on a template arrangement comprising one
or more
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antigenic regions (ARs) and one or more random copolymer regions (RCRs) (e.g.,
compositions of the disclosure; amino acid polymer compositions of the
disclosure). In
certain embodiments, such compositions are capable of inducing CCL22 release
by
monocytes and/or of inducing CD4+ T cell proliferation and/or of inducing
specific
antibodies, such as antibodies to an epitope within an antigenic region. In
certain
embodiments, such compositions are capable of inducing antibodies against a
relevant target
(e.g., a target from which one or more ARs of the composition are derived; an
antigen from
which one or more ARs of the composition are derived). In certain embodiments,
the CCL22
release, CD4+ T-cell proliferation and/or antibody induction properties of the
composition
are better, as compared to (i) a single polypeptide based on one or more of
the same AR
and/or (ii) the same or similar polypeptide or polypeptide composition in the
absence of
RCRs and/or (iii) the same or similar polypeptide composition having a net
charge or
estimated net charge of less than 1 or less than 2. In certain embodiments of
any of the
aspects or embodiments described herein, the CCL22 release, CD4+ T-cell
proliferation
and/or antibody induction properties of the composition are better, as
compared to (i) a single
polypeptide based on one or more of the same AR and (ii) the same or similar
polypeptide or
polypeptide composition in the absence of RCRs.
In one aspect, the disclosure provides a composition comprising a mixture of
at least
500 different polypeptides each having a length of between about 25 to 100
amino acids,
wherein each polypeptide, such as at least 500 polypeptides, comprises one or
more
antigenic regions (each an AR) linked to one or more random copolymer regions
of 3-15
amino acids in length (each an RCR), wherein the one or more AR and the one or
more RCR
are arranged according to a linear template arrangement (together a "complex
polypeptide
mixture component"). In certain embodiments, at least one AR comprises a
sequence of
amino acid positions corresponding to a first base peptide sequence derived
from an antigen
associated with a disease and for each amino acid position of said base
peptide sequence,
each polypeptide has an amino acid independently selected from one or more of:
an original
amino acid found at the corresponding amino acid position of the first base
peptide sequence,
alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved
substitution for
the original amino acid, and wherein the distribution of the amino acids at a
given position
among the polypeptides in the mixture is determined by a pre-determined molar
input ratio
(and/or by a relative molar input percentage) of the amino acids available for
that position
and is independently selected. In certain embodiments, for each amino acid
position of an
RCR, each polypeptide has an amino acid selected from (i) A and (ii) at least
one of K,
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arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic
acid (D), glutamic
acid (E), or phenylalanine (F), and wherein, for each amino acid position of
each RCR, the
relative molar input percentage of A for each position is less than or equal
to 65%, the
relative molar input percentage of positively charged amino acids for each
position is at least
35% and the relative molar input percentage of negatively charged amino acids
for each
position is less than or equal to 20%, and wherein the distribution of the
amino acids at a
given position of the one or more RCRs among the polypeptides is determined by
a pre-
determined molar input ratio of the amino acids available for that position
and is
independently selected for each position, and wherein, if the polypeptides of
the mixture
comprise more than one RCR, amino acid content and molar input percentage of
each RCR is
independently selected.
In another aspect, the disclosure provides a composition comprising a mixture
of at
least 500 different polypeptides each having a length of between about 25 to
100 amino acids,
wherein each polypeptide, such as at least 500 polypeptides, comprises one or
more
antigenic regions (each an AR) linked to two or more random copolymer regions
of 3-7
amino acids in length (each an RCR), wherein the one or more AR and the two or
more
RCRs are arranged according to a linear template arrangement (together a
"complex
polypeptide mixture component"). In certain embodiments, at least one AR
comprises a
sequence of amino acid positions corresponding to a first base peptide
sequence derived from
an antigen associated with a disease and for each amino acid position of said
base peptide
sequence, each polypeptide has an amino acid independently selected from one
or more of:
an original amino acid found at the corresponding amino acid position of the
first base
peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid
serving as a
conserved substitution for the original amino acid, and wherein the
distribution of the amino
.. acids at a given position among the polypeptides is determined by a pre-
determined molar
input ratio (or by a relative molar input percentage) of the amino acids
available for that
position and is independently selected. In certain embodiments, for each amino
acid position
of the two or more RCRs, each polypeptide has an amino acid selected from (i)
A and (ii) at
least one of K, arginine (R) or histidine (H), and, optionally, (iii) at least
one of aspartic acid
(D) or glutamic acid (E), and wherein, for each amino acid position of the two
or more RCRs,
the relative molar input percentage of A for each position is less than or
equal to 65%, the
relative molar input percentage of positively charged amino acids for each
position is at least
35% and the relative molar input percentage of negatively charged amino acids
for each
position is less than or equal to 20%, and wherein the distribution of the
amino acids at a
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given position of the one or more RCRs among the polypeptides is determined by
a pre-
determined molar input ratio of the amino acids available for that position
and is
independently selected for each position, and wherein amino acid content and
molar input
percentage of each RCR is independently selected.
In another aspect, the disclosure provides a composition comprising a mixture
of at
least 500 different polypeptides each having a length of between about 25 to
100 amino acids,
wherein each polypeptide, such as at least 500 polypeptides, comprises one or
more
antigenic regions (each an AR) linked to one or more random copolymer regions
of 3-15
amino acids in length (each an RCR), wherein the one or more AR and the one or
more RCR
are arranged according to a linear template arrangement (together a "complex
polypeptide
mixture component"). In certain embodiments, at least one AR comprises a
sequence of
amino acid positions corresponding to a first base peptide sequence derived
from an antigen
associated with a disease and for each amino acid position of said base
peptide sequence,
each polypeptide has an amino acid independently selected from one or more of:
an original
amino acid found at the corresponding amino acid position of the first base
peptide sequence,
alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved
substitution for
the original amino acid, and wherein the distribution of the amino acids at a
given position
among the polypeptides in the mixture is determined by a pre-determined molar
input ratio
(and/or by a relative molar input percentage) of the amino acids available for
that position
and is independently selected. In certain embodiments, for each amino acid
position of an
RCR, each polypeptide has an amino acid selected from (i) A and (ii) at least
one of K,
arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic
acid (D), glutamic
acid, or phenylalanine (F), and wherein the distribution of the amino acids at
a given position
of the one or more RCRs among the polypeptides is determined by a pre-
determined molar
input ratio of the amino acids available for that position and is
independently selected for
each position, and wherein, if the polypeptides of the mixture comprise more
than one RCR,
amino acid content and molar input percentage of each RCR is independently
selected. In
certain embodiments, the ratio of the percentage of alanine to the percentage
of lysine, on a
molar basis, in the polypeptides of the composition having a length of between
about 25 to
100 amino acids (the "output" ratio) is greater than or equal to 1.5 and less
than or equal to
5.0 (as expressed as a quotient, which ratio may also be expressed as a
relative ratio of 1.5:1
to 5.0:1).
In another aspect, the disclosure provides a composition comprising a mixture
of at
least 500 different polypeptides each having a length of between about 25 to
100 amino acids,
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wherein each polypeptide, such as at least 500 polypeptides, comprises one or
more antigenic
regions (each an AR) linked to two or more random copolymer regions of 3-7
amino acids in
length (each an RCR), wherein the one or more AR and the two or more RCRs are
arranged
according to a linear template arrangement (together a "complex polypeptide
mixture
component"). In certain embodiments, at least one AR comprises a sequence of
amino acid
positions corresponding to a first base peptide sequence derived from an
antigen associated
with a disease and for each amino acid position said base peptide sequence,
each polypeptide
has an amino acid independently selected from one or more of: an original
amino acid found
at the corresponding amino acid position of the first base peptide sequence,
alanine (A),
lysine (K), arginine (R), or an amino acid serving as a conserved substitution
for the original
amino acid, and wherein the distribution of the amino acids at a given
position among the
peptides is determined by a pre-determined molar input ratio (and/or by a
relative molar input
percentage) of the amino acids available for that position and is
independently selected. In
certain embodiments, for each amino acid position of the two or more RCRs,
each
polypeptide has an amino acid selected from (i) A and (ii) at least one of K,
arginine (R) or
histidine (H), and, optionally, (iii) at least one of aspartic acid (D) or
glutamic acid (E), and
wherein the distribution of the amino acids at a given position of the one or
more RCRs
among the polypeptides is determined by a pre-determined molar input ratio of
the amino
acids available for that position and is independently selected for each
position, and wherein
amino acid content and molar input percentage of each RCR is independently
selected. In
certain embodiments, the ratio of the percentage of alanine to the percentage
of lysine, on a
molar basis, in the polypeptides of the composition having a length of between
about 25 to
100 amino acids (the output ratio) is greater than or equal to 1.5 and less
than or equal to 5Ø
In another aspect, the disclosure provides a composition comprising a mixture
of at
least 500 different polypeptides each having a length of between about 25 to
100 amino acids,
wherein each polypeptide, such as at least 500 polypeptides, comprises one or
more
antigenic regions (each an AR) linked to one or more random copolymer regions
of 3-15
amino acids in length (each an RCR), wherein the one or more AR and the one or
more RCR
are arranged according to a linear template arrangement (together a "complex
polypeptide
mixture component"), which composition has an estimated net charge of greater
than 2.0 and
less than 4.0 at pH7. In certain embodiments, at least one AR comprises a
sequence of amino
acid positions corresponding to a first base peptide sequence derived from an
antigen
associated with a disease and for each amino acid position of said base
peptide sequence,
each polypeptide has an amino acid independently selected from one or more of:
an original
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amino acid found at the corresponding amino acid position of the first base
peptide sequence,
alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved
substitution for
the original amino acid, and wherein the distribution of the amino acids at a
given position
among the polypeptides in the mixture is determined by a pre-determined molar
input ratio
(and/or by a relative molar input percentage) of the amino acids available for
that position
and is independently selected. In certain embodiments, for each amino acid
position of an
RCR, each polypeptide has an amino acid selected from (i) A and (ii) at least
one of K,
arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic
acid (D), glutamic
acid (E), or phenylalanine (F), and wherein, for each amino acid position of
each RCR, the
distribution of the amino acids at a given position among the polypeptides in
the mixture is
determined by a pre-determined molar input ratio of the amino acids available
for that
position and is independently selected for each position of each RCR.
In another aspect, the disclosure provides a composition comprising a mixture
of at
least 500 different polypeptides each having a length of between about 25 to
100 amino acids,
wherein each polypeptide comprises one or more antigenic regions (each an AR)
linked to
two or more random copolymer regions of 3-7 amino acids in length (each an
RCR), wherein
the one or more AR and the two or more RCRs are arranged according to a linear
template
arrangement (together a "complex polypeptide mixture component"), which
composition has
an estimated net charge of greater than 2.0 and less than 4.0 at pH7. In
certain embodiments,
at least one AR comprises a sequence of amino acid positions corresponding to
a first base
peptide sequence derived from an antigen associated with a disease and for
each amino acid
position of said base peptide sequence, each polypeptide has an amino acid
independently
selected from one or more of: an original amino acid found at the
corresponding amino acid
position of the first base peptide sequence, alanine (A), lysine (K), arginine
(R), or an amino
acid serving as a conserved substitution for the original amino acid, and
wherein the
distribution of the amino acids at a given position among the polypeptides is
determined by a
pre-determined molar input ratio (and/or by a relative molar input percentage)
of the amino
acids available for that position and is independently selected. In certain
embodiments, for
each amino acid position of the two or more RCRs, each polypeptide has an
amino acid
selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H),
and, optionally,
(iii) at least one of aspartic acid (D) or glutamic acid (E), and wherein, for
each amino acid
position of the two or more RCRs, the distribution of the amino acids at a
given position
among the polypeptides in the mixture is determined by a pre-determined molar
input ratio of
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the amino acids available for that position and is independently selected for
each position of
each RCR.
In certain embodiments of any of the foregoing or following, one or more of
the
positively charged amino acids of an RCR may instead be a non-naturally
occurring amino
acid and/or one or more of the negatively charged amino acids of an RCR may
instead be a
non-naturally occurring amino acid.
In certain embodiments of any of the foregoing or following, the relative
molar input
percentage of A is between about 5% and 40%, between about 10% and 40%,
between about
15% and 40%, between about 10% and 35%, or between about 15% and 35% of the
total
input amino acid composition of the complex polypeptide mixture component. In
certain
embodiments, the relative molar input percentage of A is less than 30%, such
as between 10
and 30% or 15 and 30% of the total input amino acid composition of the complex
polypeptide
mixture. In certain embodiments, the relative molar input percentage of A is
between 15 and
25% of the total input amino acid composition. In certain embodiments, the
relative molar
input percentage of A is between 15 and 20% of the total input amino acid
composition. In
certain embodiments, the relative molar input percentage of A is between 20
and 25% of the
total input amino acid composition.
In certain embodiments of any of the foregoing or following, the estimated net
charge
of the polypeptides of the composition having a length of between about 25 to
100 amino
acids is greater than or equal to 2 at pH7. In certain embodiments of any of
the foregoing or
following, the estimated net charge is greater than or equal to 2 and less
than or equal to 4 at
pH7. In certain embodiments of any of the foregoing or following, the
estimated net charge
is greater than 2.1 and less than or equal to 4 at pH7.
In certain embodiments of any of the foregoing or following, at least 55%, at
least
60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or
greater than at
least 90% of the polypeptides in the complex polypeptide mixture based on the
linear
template arrangement are substantially full-length polypeptides or full-length
polypeptides.
In certain embodiments, at least 95% (or even greater) of the polypeptides in
the complex
mixture are full-length or substantially full-length polypeptides (based on
the linear template
arrangement).
In certain embodiments of any of the foregoing or following, amino acid
sequence of
at least one AR does not vary among the polypeptides in the composition. In
certain
embodiments of any of the foregoing or following, the amino acid sequence of
at least one
AR does not vary among the polypeptides in the composition, and the amino acid
sequence of
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said AR comprises a sequence of amino acid positions corresponding to the base
peptide
sequence and the polypeptides in the composition have an amino acid at each
position of the
AR selected from an original amino acid found at the corresponding amino acid
position of
first base peptide sequence.
In certain embodiments of any of the foregoing or following, the amino acid
sequence
of at least one AR varies with respect to the base peptide sequence among the
polypeptides of
the composition, various polypeptides having an amino acid at each position
independently
selected from: an original amino acid found at the corresponding amino acid
position of the
base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid
serving as a
conserved substitution for the original amino acid.
In certain embodiments of any of the foregoing or following, the polypeptides
in the
composition, such as the substantially full-length polypeptides in the
composition, comprise
two ARs (ARa and ARb), and each AR may be derived from the same or a different
base
peptide sequence, said different base peptide sequence a second base peptide
sequence, and
wherein ARa may be either N-terminal or C-terminal to ARb. In some
embodiments, the full-
length or substantially full-length polypeptides in the composition comprise
two ARs (ARa
and ARb), and each AR may be derived from the same or a different base peptide
sequence,
said different base peptide sequence a second base peptide sequence, and
wherein ARa may
be either N-terminal or C-terminal to ARb.
In certain embodiments of any of the foregoing or following, the polypeptides
in the
composition, such as the substantially full-length polypeptides in the
composition, comprise
two or more ARs (ARa, ARb, and ARm), and each of these ARs may be derived from
the
same or a different base peptide sequence, and wherein m is an integer from 0-
3. In some
embodiments, the full-length or substantially full-length polypeptide in the
composition
comprise two or more ARs (ARa, ARb, and ARm), and each of these ARs may be
derived
from the same or a different base peptide sequence, and wherein m is an
integer from 0-3.
In certain embodiments of any of the foregoing or following, ARa and ARb are
derived from a different base peptide sequence. In certain embodiments, all of
the ARs are
from the same antigen, but from a different base peptide sequence.
In certain embodiments of any of the foregoing or following, m is 1, such that
the
polypeptides in the composition, such as the substantially full-length
polypeptides in the
composition, each comprises three ARs.
In certain embodiments of any of the foregoing or following, the amino acid
sequence
of at least one AR does not vary among the polypeptides in the composition,
and the amino
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acid sequence of said AR comprises a sequence of amino acid positions
corresponding to the
base peptide sequence and the polypeptides in the composition have an amino
acid at each
position of the at least one AR selected from an original amino acid found at
the
corresponding amino acid position of the base peptide sequence. In certain
embodiments, the
amino acid sequence of at least one AR varies with respect to its
corresponding base peptide
sequence among the polypeptides of the composition, various polypeptides
having an amino
acid at each position independently selected from: an original amino acid
found at the
corresponding amino acid position of the base peptide sequence, alanine (A),
lysine (K),
arginine (R), or an amino acid serving as a conserved substitution for the
original amino acid.
In certain embodiments of any of the foregoing or following, the amino acid
sequence
of ARa and ARb varies independently with respect to the first and second base
peptide
sequence, respectively, among the polypeptides of the composition, various
polypeptides
having an amino acid at each position independently selected from: an original
amino acid
found at the corresponding amino acid position of the base peptide sequence,
alanine (A),
lysine (K), arginine (R), or an amino acid serving as a conserved substitution
for the original
amino acid.
In certain embodiments of any of the foregoing or following, at least one base
peptide
sequence and/or at least one AR is 9-30, 13-26, 15-20, or 13-20 amino acids in
length. In
certain embodiments, the foregoing applies to at least two, at least three, or
three ARs and/or
base peptide sequences, wherein the length of each is independently selected.
In certain embodiments of any of the foregoing or following, the polypeptides
in the
composition comprise two or more ARs (ARa, ARb, and ARm), and each of these
ARs may be
derived from the same or a different base peptide sequence (a first, a second,
and an mth base
peptide sequence, respectively), and wherein m is an integer from 0-3, and
wherein the length
of each of the base peptides sequences and/or ARs is independently selected
from 9-30, 13-
26, 15-20, or 13-20 amino acids.
In certain embodiments of any of the foregoing or following, the polypeptides
in the
composition, such as the full-length polypeptides in the composition, comprise
two RCRs
(RCRa and RCRb), and each RCR may have the same or differing length and/or
amino acid
distribution, and wherein the distribution of amino acids at each position is
independently
selected. In certain embodiments, the full-length polypeptides in the
composition, comprise
two or more RCRs (RCRa, RCRb, and RCRO, and each of these RCRs may have the
same or
differing length and/or amino acid distribution, wherein the distribution of
amino acids at
each position is independently selected, and wherein n is an integer from 0-3.
In certain
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embodiments, RCRa and RCRb have the same amino acid distribution. In other
embodiments, the polypeptides in the composition comprise three RCRs (RCRa,
RCRb, and
RCRc), and each RCR may have the same or differing length and/or amino acid
distribution,
and wherein the distribution of amino acids at each position is independently
selected. In
certain embodiments, RCRa, RCRb, and RCRc have the same amino acid
distribution.
In certain embodiments of any of the foregoing or following, the template
arrangement comprises RCRa-ARa-RCRb-ARb or ARa-RCRa-ARb-RCRb or ARa-RCRa-ARb-
RCRb-ARc , RCRa-ARa-RCRb-ARb-RCRc-ARc , or ARa-RCRa-ARb-RCRb-ARc-RCRc. In
certain embodiments, one or more of (a) an RCRa and ARa, (b) an ARa and RCRb,
(c) an
RCRa and ARb, (d) an RCRb and ARb, or (e) an RCRb and ARC, or (f) an RCR c and
AR are
interconnected via a linker of at least one amino acid residues.
In certain embodiments of any of the foregoing or following, the polypeptides,
such
as the substantially full-length polypeptides in the composition based on the
template
arrangement, comprise, at one or both ends, 1-8 positively charged residues
selected
independently, for each position, from Lysine (K), Arginine (R), or Histidine
(H). In certain
embodiments, the polypeptides, such as the substantially full-length
polypeptides in the
composition based on the template arrangement, comprise, at one terminus, 2-5
positively
charged residues independently, for each position, selected from Lysine (K),
Arginine (R), or
Histidine (H). In other embodiments, one or more of these positively charged
amino acids
may be a non-naturally occurring positively charged amino acid. In certain
embodiments, the
positively charged residue is the same at each position.
In certain embodiments of any of the foregoing or following, at least 80%, at
least
90% or at least 95% of the polypeptides in the composition having a length of
between 25 to
100 amino acids are of substantially the same length, such as full-length. In
certain
embodiments, at least 80% of said polypeptides in the composition having a
length of
between 25 to 100 amino acids are about 58-65 amino acids in length. In
certain
embodiments, polypeptides having a full-length of between 25 to 100 amino
acids represents
at least 90%, at least 95% or close to 100% of the composition.
In another aspect, the disclosure provides a method of manufacturing a
composition
.. by solid phase peptide synthesis. The method comprises, in certain
embodiments, selecting
one or more antigens associated with a disease or condition; selecting one or
more base
peptide sequences based on said antigens; selecting, for each position of each
AR and for
each position of each RCR, one or more amino acid residues permissible at that
position
among the polypeptides in the composition and, for each possible permissible
amino acid at
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each position, select a pre-determined molar input ratio of the amino acids
available for that
position; synthesizing the complex mixture of polypeptides, on a polypeptide-
by-polypeptide
basis, by solid phase peptide synthesis based on the available molar input
ratio of permissible
amino acids available for each position, wherein for each polypeptide in the
complex
mixture, synthesis is from C-terminus to N-terminus.
In certain embodiments of any of the foregoing or following, the C-terminal
most
amino acid residue for each polypeptide in the complex polypeptide mixture is
Norleucine
(Nle) or another non-naturally occurring amino acid.
In certain embodiments of any of the foregoing or following, the method or
composition is such that at least 55% or at least 60% of the polypeptides in
the complex
polypeptide mixture that are based on the template arrangement are
substantially full-length
polypeptides. In certain embodiments, at least 80%, at least 90%, or at least
95% of the
polypeptides in the complex polypeptide mixture that are based on the template
arrangement
are full-length or substantially full-length polypeptides
In certain embodiments of any of the foregoing or following, for each position
of the
one or more RCRs, the relative molar input percentages of amino acids are,
independently for
each position of each of the RCRs, selected from 15% to 55% A, 35% to 75% K,
R, or H, or
combinations thereof, and 5% to 15% E or D, or combinations thereof. In other
such
embodiments, for each position of the one or more RCRs, the relative molar
input
percentages of amino acids are, independently for each position of each of the
RCRs, selected
from 20% to 50% A, 40% to 70% K, R, or H, or combinations thereof, and 5% to
10% E or
D, or combinations thereof. In certain embodiments, the relative molar input
percentage, for
each position of and/or across each RCR, of A is less than or equal to 50%,
such as 15%-
50%. In certain embodiments, the relative molar input percentage, for each
position of and/or
.. across each RCR, of K (or R or H) is 50-70%. In other such embodiments, the
relative molar
input percentages of amino acids are the same for each position of at least
one RCR or for
each RCR. In other such embodiments, the relative molar input percentages are
independently selected and may vary for one or more positions of at least one
RCR. In some
embodiments, the molar input percentage at one position of an RCR varies
versus the other
positions. In some embodiments, the same pattern is repeated across the
different RCRs.
In certain embodiments of any of the foregoing or following, the amino acid
available
at each position of at least one RCR or of each RCR is selected from A, K and
E. In other
such embodiments, the amino acid available at each position of at least one
RCR or of each
RCR is selected from A and K.
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In certain embodiments of any of the foregoing or following, the amino acid
available
at each position of each RCR is independently selected from (i) A, K and E;
(ii) A and K; (iii)
A, K and D; (iv) A and R; (v) A, R, and E; (vi) A, R and D; (vii) A and H;
(viii) A, H and E;
(ix) A, H and D.
In certain embodiments of any of the foregoing or following, the template
arrangement comprises two or more RCRs each of 3-7 amino acids in length with
at least one
AR interposed between two of the RCRs, and wherein the length of each RCR is
independently selected. In certain embodiments, the two RCRs are 5, 6, or 7
amino acids in
length.
In certain embodiments of any of the foregoing or following, the template
arrangement comprises three RCRs each of 3-7 amino acids in length with at
least two ARs
interposed, and wherein the length of each RCR is independently selected, and
wherein the
length of each AR is independently selected. In certain embodiments, the three
RCRs are 3,
4, or 5 amino acids in length.
In certain embodiments of any of the foregoing or following, the one or more
ARs are
or comprise base peptide sequences of 9-25 amino acids in length, and wherein,
if two or
more ARs are present, the length of each is independently selected.
In certain embodiments of any of the foregoing or following, the template
sequence
comprises two or more ARs, wherein: (i) the ARs are each based on the first
base peptide
sequence, or (ii) one of the ARs is based on the first base peptide sequence
and another AR is
based on a second base peptide derived from a second sequence of the antigen.
In certain embodiments of any of the foregoing or following, the antigen is
associated
with a protein conformational disorder affecting the central and/or peripheral
nervous system.
In certain embodiments of any of the foregoing or following, the antigen is
associated
with a disease selected from Alzheimer's disease (AD); frontotemporal dementia
with
parkinsonism linked to chromosome 17 (FTDP-17); Dutch hereditary cerebral
hemorrhage
with amyloidosis (a.k.a cerebrovascular amyloidosis); congophilic angiopathy;
Pick's disease;
progressive supranuclear palsy; familial British dementia; Parkinson's disease
(PD); Lewy
body dementias; multiple system atrophy; Hallervorden-Spatz disease;
amyotrophic lateral
sclerosis (ALS); Huntington's disease (HD); spinocerebellar ataxia; neuronal
intranuclear
inclusion disease; hereditary dentatorubral-pallidoluysian atrophy; prion-
related diseases such
as scrapie, bovine spongiform encephalopathy, variant Creutzfeldt Jakob
disease, Gerstmann-
Straussler-Scheinker syndrome, kuru, fatal familial insomnia, and related
disorders;
hereditary cystatin c amyloid angiopathy; dementia pugilistica;
neurodegenerative diseases
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characterized by cerebral and nerve atrophy; spinal and bulbar muscular
atrophy; hereditary
systemic and cerebral amyloidosis; Finnish-type familial amyloidosis; senile
systemic
amyloidosis (a.k.a. senile cardiac amyloidosis); familial amyloid
polyneuropathy; Type-2
diabetes, in particular pancreatic islet amyloidosis; dialysis-related
amyloidosis (DRA);
inflammation-associated reactive systemic amyloidosis (a.k.a. AA amyloidosis);
aortic
medial amyloidosis; medullary carcinoma of the thyroid; hereditary renal
amyloidosis; light
chain associated amyloidosis, light chain deposition disease, light chain cast
nephropathy,
light chain cardiomyopathy; atrial amyloidosis; injection-localized
amyloidosis; cystic
fibrosis (CF); and sickle cell anemia.
In certain embodiments of any of the foregoing or following, the first base
peptide
sequence and/or any base peptide sequence comprises a sequence from an antigen
selected
from prion protein, amyloid beta precursor protein, ABri peptide, tau protein,
alpha-
synuclein, alpha-synuclein central fragment, SOD1, TDP-43, repeat-associated
non-ATG
(RAN)-translated peptides of the C90RF72 locus, islet amyloid polypeptide
(a.k.a. amylin),
prothymosin alpha, amino-terminal domain of androgen receptor protein, ataxin-
1, DRPLA
protein (a.k.a. atrophin-1), calcitonin, cystatin c, transthyretin, beta 2
microglobulin, serum
amyloid A protein, huntingtin, exon I of huntingtin, immunoglobulin light
chain variable
domains, insulin, lysozyme, alpha lactalbumin, monellin, ligand- and DNA-
binding domains
of androgen receptor protein, lactadherein, lactadherein fragment (a.a.
residue 245-294, a.k.a.
medin), gelsolin, apolipoprotein Al, fibrinogen, atrial natriuretic factor,
and fragments
thereof.
In certain embodiments of any of the foregoing or following, the first base
peptide
sequence, and optionally and if present, the second and third base peptide
sequence, is a
sequence selected from SEQ ID NOs: 1 through 20 or a portion thereof of at
least 9 amino
acid residues, or any of the other sequences provided in tables or the
sequence listing herein.
In certain embodiments of any of the foregoing or following, one or more base
peptide sequences comprise or permit variability based on one or more
phosphorylated,
nitrated, or acetylated amino acids. In certain embodiments of any of the
foregoing or
following, at least one base peptide sequence comprises or permits variability
based on at
least one phosphorylated residue corresponding to a phosphorylation site in
the native antigen
that is associated with a pathological state.
In certain embodiments of any of the foregoing or following, the antigen is
associated
with a pathogenic infection. In other such embodiments, the pathogen is an
intracellular
pathogen. In certain embodiments of any of the foregoing or following, the
pathogen is
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selected from viruses or bacteria. In certain such embodiments, the pathogen
is of the genus
selected from Chlamydia, Rickettsia, Coxiella, Mycobacterium, Francisella,
Listeria,
Salmonella, Brucella, Legionella, Nocardia, Rhodococcus, Yersinia, and
Neisseria. In
certain embodiments, the pathogen is human papilloma virus (HPV).
In certain embodiments of any of the foregoing or following, the one or more
base
peptide sequences comprise or permit variability based on one or more non-
natural amino
acids.
In certain embodiments of any of the foregoing or following, the ratio of the
molar
input percentage of Alanine to that of Lysine, in the total amino acid input
concentration of
the composition, is between 1.25 to 2.5 (as expressed as a quotient, which can
also be
expressed as 1.25:1 to 2.5:1).
In certain embodiments of any of the foregoing or following, the ratio of the
percentage of alanine to the percentage of lysine, on a molar basis, across
the polypeptides of
the composition having a length of between about 25 to 100 amino acids is
greater than or
equal to 2.5 and less than or equal to 7. Such a ratio refers to the output.
In certain embodiments of any of the foregoing or following, the percentage of
alanine, on a molar basis, across the polypeptides of the composition having a
length of
between about 25 to 100 amino acids is greater than or equal to 12 and less
than or equal to
30. In certain embodiments, this refers to the output.
In certain embodiments of any of the foregoing or following, the percentage of
lysine,
on a molar basis, across the polypeptides of the composition having a length
of between
about 25 to 100 amino acids is greater than or equal to 4 and less than or
equal to 9. In
certain embodiments, this refers to the output.
In certain embodiments of any of the foregoing or following, the length of
each or of
at least 60% of the polypeptides in the composition based on the template
arrangement (and
template sequence) is about 40-80 residues. In certain embodiments, the length
of each or of
at least 60% of the polypeptides in the composition based on the template
arrangement is
about 45-65 residues.
In certain embodiments of any of the foregoing or following, the conserved
substitution comprises replacing an original amino with a similar or
interchangeable amino
acid defined according to amino acid similarity. In certain embodiments, at
least one
conserved substitution is selected from an amino acid present in a naturally
occurring variant
of the antigen. In certain embodiments, the conserved substitution comprises
replacing an
original amino acid with a phosphorylated or nitrated form of that amino acid.
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In certain embodiments, one or more RCRs have an amphipathic alpha-helical
structure.
In one aspect, the disclosure provides a pharmaceutical composition comprising
an
amino acid copolymer composition of the disclosure and one or more
pharmaceutically
acceptable carriers and/or excipients. In some embodiments, the pharmaceutical
composition
comprises an adjuvent.
In one aspect, the disclosure provides a method for prophylactic or
therapeutic
treatment of a protein conformational disorder or a pathogenic infection,
comprising
administering to a subject in need thereof an effective amount of a
composition according to
the disclosure, wherein the antigen is an antigen associated with the protein
conformational
disorder or the pathogenic infection.
In one aspect, the disclosure provides a kit comprising a composition
according to the
disclosure and instructions for use in the treatment of a protein
conformational disorder.
In one aspect, the disclosure provides a method of eliciting or otherwise
promoting a
release of a Th2-associated cytokine or chemokine from monocytes, comprising
contacting
the monocytes with a composition according to the disclosure, wherein the Th2-
associated
cytokine or chemokine is selected from: IL-4, IL-5, IL-6, IL-10, IL-13, CCL17,
and CCL22.
In one aspect, the disclosure provides a method of eliciting or otherwise
promoting a
CCL22 release from monocytes, comprising contacting the monocytes with a
composition
according to the disclosure.
In one aspect, the disclosure provides a method of eliciting or otherwise
promoting
CD4+ T cell proliferation, comprising contacting peripheral blood mononuclear
cells with a
composition according to the disclosure.
In one aspect, the disclosure provides a method of eliciting or otherwise
promoting
CD8+ T cell proliferation, comprising contacting peripheral blood mononuclear
cells with a
composition according to the disclosure.
In another aspect, the disclosure provides a kit comprising a composition of
the
disclosure and instructions for use in the treatment of a protein
conformational disorder or
pathogenic infection.
In another aspect, the disclosure provides a method of targeting a
phosphorylated or
nitrated site on a target antigen, comprising administering to a subject or
contacting a sample
with a composition of the disclosure or a pharmaceutical composition according
to the
disclosure.
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In another aspect, the disclosure provides a method for generating antibodies
comprising the steps of (i) administering a composition of the disclosure to
an animal; and
(ii)(a) isolating antibodies immunoreactive with said composition from said
animal, or (ii)(b)
isolating cells that produce antibodies immunoreactive with said composition
from said
animal, and then isolating antibodies immunoreactive with said composition
from said
isolated cells. In certain embodiments, the method further comprises
administering the
antibodies so isolated or contacting cells or tissues with said antibodies.
The disclosure contemplates that any of the compositions of the disclosure,
described
using any one or more structural and/or functional features, can be used in
any of the in vitro
or in vivo methods described herein. Moreover, compositions may be screened
using
methods described herein to identify compositions having suitable
characteristics.
The disclosure contemplates all combinations of any of the foregoing aspects
and
embodiments, as well as combinations with any of the embodiments set forth in
the detailed
description, examples and/or claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schematic representation of a high-complexity amino acid
copolymer composition of the disclosure. In this exemplary representation,
polypeptides in
the composition are based on the following template arrangement: 2 RCRs (RCRa
and RCRb
) associated with 2 ARs (ARa and ARb), in an arrangement of RCRa-ARa-RCRb-ARb.
Though not shown, for any given polypeptide in the composition, the RCRs and
ARs are
associated and may be associated directly (e.g., they are contiguous) or via a
linker (e.g., via
an amino acid residue that is neither part of the RCR nor part of the AR).
Also, though not
shown, the polypeptides may include additional sequence at the N- or C-
terminus, such as a
tag to facilitate detection or manufacture. The template arrangement, template
sequence and
input amino acid percentages permitted at each position influence the possible
sequences of
the particular individual polypeptides within the composition. This is
depicted in Figure 1 by
the output polypeptides. Each output peptide will comprise a specific amino
acid sequence
within each of the RCRs and ARs, and thus, will have a specific amino acid
sequence
influenced by the linear template arrangement, input amino acids, and input
percentages (e.g.
molar input percentage). The RCRs of individual peptides of the mixture are
depicted as:
RCRai(SEQ ID NO: 46), RCRa2(SEQ ID NO: 47), RCRa3 (SEQ ID NO: 48) (e.g., the N-
terminal RCR (RCRa) of peptides 1, 2 and 3, respectively, of the mixture) and
RCRbi (SEQ
ID NO: 49), RCRb2(SEQ ID NO: 50), RCRb3 (SEQ ID NO: 51) (e.g., the second RCR
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(RCRO of peptides 1, 2 and 3, respectively, of the mixture). The ARs of
individual peptides
of the mixture are depicted as: ARai(SEQ ID NO: 40), ARa2 (SEQ ID NO: 41),
ARa3 (SEQ
ID NO: 42) (e.g., the N-terminal AR (ARa) of peptides 1, 2 and 3,
respectively, of the
mixture) and ARbi(SEQ ID NO: 39), ARb2(SEQ ID NO: 39), ARb3 (SEQ ID NO: 39)
(e.g.,
the second AR (ARb) of peptides 1, 2 and 3, respectively, of the mixture).
Figure 2 shows an example of a schematic corresponding to the design of an
amino
acid copolymer composition with antigenic specificity to C-terminal portions
of Tau (e.g., an
example of a composition of the disclosure, compositions based on this
template arrangement
and amino acid input referred to as DP-0016.B). Figure 2, as modified by the
description
provided in the example, discloses SEQ ID NO: 52. An example of compositions
of the
disclosure is a composition comprising a plurality of polypeptides (such as a
plurality of at
least 500 different polypeptides) comprising on amino acid sequence set forth
in SEQ ID NO:
52 (in the presence or absence of a terminal Norleucine (Nle) or similar non-
naturally
occurring amino acid). As described herein, a plurality of the individual
polypeptides within
the composition are complex and differ in sequence based on the position-
specific variability
depicted in Figure 2 and SEQ ID NO: 52. For example, for the positions in the
RCRs, the
amino acid at each position can be K/A/E, and for particular positions within
the ARs, the
amino acid at each position varies, as depicted and described.
Figure 3 shows another example of a schematic corresponding to the design of
another amino acid copolymer composition with antigenic specificity to C-
terminal portions
of Tau (e.g., an example of a composition of the disclosure, compositions
based on this
template arrangement and amino acid input referred to as DP-0016.C). Figure 3,
as modified
by the description provided in the example, discloses SEQ ID NO: 60. An
example of
compositions of the disclosure is a composition comprising a plurality of
polypeptides (such
as a plurality of at least 500 different polypeptides) comprising on amino
acid sequence set
forth in SEQ ID NO: 60 (in the presence or absence of a terminal Norleucine
(Nle) or similar
non-naturally occurring amino acid). As described herein, a plurality of the
individual
polypeptides within the composition are complex and differ in sequence based
on the
position-specific sequence variability depicted in Figure 3 and SEQ ID NO: 60.
For example,
for the positions in the RCRs, the amino acid at each position can be K/A/E,
and for
particular positions within the ARs, the amino acid at each position varies,
as depicted and
described.
Figure 4 shows the effect of DP-0016.B and DP-0016.0 on a monocyte assay using
RAW cells. DP-0016.A (which is an amino acid copolymer composition lacking any
RCRs
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but with an AR spanning the same region of Tau as the two ARs present in DP-
0016.B and
DP-0016.C), COPAXONE , and single phosphorylated (Phospho Peptide Cnt) and non-
phosphorylated (Non-Phospho Peptide Cnt) control peptides spanning the same
region of Tau
as the two ARs present in DP-0016.B and DP-0016.0 were also assayed for
comparison.
This assay can be readily used to confirm that activity of other compositions.
Figures 5A and 5B show the effect of DP-0016.A, DP-0016.B, DP-0016.C,
COPAXONE , and single phosphorylated (Phospho Peptide Cnt) and non-
phosphorylated
(Non-Phospho Peptide Cnt) control peptides spanning the same region of Tau as
the two ARs
present in DP-0016.B and DP-0016.0 on CD4+ T-cell proliferation in a PBMC CFSE
assay.
Figure 5A shows flow cytometry data after staining with anti-CD3 and anti-CD4.
Figure 5B
is a graphical representation of the proliferation index of CD4+ T-cells when
stimulated with
DP-0016.A, DP-0016.B, DP-0016.C, COPAXONE or single phosphorylated (Phospho
Peptide Cnt) and non-phosphorylated (Non-Phospho Peptide Cnt) control peptides
spanning
the same region of Tau as the two ARs present in DP-0016.B and DP-0016.C. A
PBS-only
negative control and an anti-CD3/CD28 positive control were also included for
comparison.
Figures 6A and 6B show the immunogenic effects of DP-0016.A, DP-0016.B and
DP-0016.C. Figure 6A shows the splenocyte recall response when splenocytes
from mice
injected with DP-0016.A, DP-0016.B or DP-0016.0 are restimulated with the
respective
immunogen. Figure 6B shows the results of antibody ELISA to measure the IgG1
response
against respective immunogen.
Figure 7 shows an example of a schematic corresponding to the design of an
amino
acid copolymer composition with antigenic specificity to human a-synuclein
(e.g., an
example of a composition of the disclosure, compositions based on this
template arrangement
and amino acid input referred to as DP-0003.A). Figure 7 discloses SEQ ID NO:
53. An
example of compositions of the disclosure is a composition comprising a
plurality of
polypeptides (such as at least 500 different polypeptides) comprising on amino
acid sequence
set forth in SEQ ID NO: 53 (in the presence or absence of a terminal
Norleucine (Nle) or
similar non-naturally occurring amino acid). As described herein, a plurality
of the
individual polypeptides within the composition are complex and differ in
sequence based on
the position-specific sequence variability depicted in Figure 7 and SEQ ID NO:
53.
Figure 8 shows an example of a schematic corresponding to the design of an
amino
acid copolymer composition with antigenic specificity to human a-synuclein
(e.g., an
example of a composition of the disclosure, compositions based on this
template arrangement
and amino acid input referred to as DP-0003.B). Figure 8 discloses SEQ ID NO:
54. An
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example of compositions of the disclosure is a composition comprising a
plurality of
polypeptides (such as at least 500 different polypeptides) comprising on amino
acid sequence
set forth in SEQ ID NO: 54 (in the presence or absence of a terminal
Norleucine (Nle) or
similar non-naturally occurring amino acid). As described herein, a plurality
of the
individual polypeptides within the composition are complex and differ in
sequence based on
the position-specific sequence variability depicted in Figure 8 and SEQ ID NO:
54
Figure 9 shows an example of a schematic corresponding to the design of an
amino
acid copolymer composition with antigenic specificity to human a-synuclein
(e.g., an
example of a composition of the disclosure, compositions based on this
template arrangement
and amino acid input referred to as DP-0003.C). Figure 9 discloses SEQ ID NO:
55. An
example of compositions of the disclosure is a composition comprising a
plurality of
polypeptides (such as at least 500 different polypeptides) comprising on amino
acid sequence
set forth in SEQ ID NO: 55 (in the presence or absence of a terminal
Norleucine (Nle) or
similar non-naturally occurring amino acid). As described herein, a plurality
of the
individual polypeptides within the composition are complex and differ in
sequence based on
the position-specific sequence variability depicted in Figure 9 and SEQ ID NO:
55
Figure 10 shows an example of a schematic corresponding to the design of an
amino
acid copolymer composition with antigenic specificity to human a-synuclein
(e.g., an
example of a composition of the disclosure, compositions based on this
template arrangement
and amino acid input referred to as DP-0003.D). Figure 10 discloses SEQ ID NO:
56. An
example of compositions of the disclosure is a composition comprising a
plurality of
polypeptides (such as at least 500 different polypeptides) comprising on amino
acid sequence
set forth in SEQ ID NO: 56 (in the presence or absence of a terminal
Norleucine (Nle) or
similar non-naturally occurring amino acid). As described herein, a plurality
of the
individual polypeptides within the composition are complex and differ in
sequence based on
the position-specific sequence variability depicted in Figure 10 and SEQ ID
NO: 56
Figure 11 shows an example of a schematic corresponding to the design of an
amino
acid copolymer composition with antigenic specificity to C-terminal portions
of Tau (e.g., an
example of a composition of the disclosure, compositions based on this
template arrangement
and amino acid input referred to as DP-0016.E). Figure 11 discloses SEQ ID NO:
57. An
example of compositions of the disclosure is a composition comprising a
plurality of
polypeptides (such as at least 500 different polypeptides) comprising on amino
acid sequence
set forth in SEQ ID NO: 57 (in the presence or absence of a terminal
Norleucine (Nle) or
similar non-naturally occurring amino acid). As described herein, a plurality
of the
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individual polypeptides within the composition are complex and differ in
sequence based on
the position-specific sequence variability depicted in Figure 11 and SEQ ID
NO: 57
Figure 12 shows an example of a schematic corresponding to the design of an
amino
acid copolymer composition with antigenic specificity to C-terminal portions
of Tau (e.g., an
example of a composition of the disclosure, compositions based on this
template arrangement
and amino acid input referred to as DP-0016.F). Figure 12 discloses SEQ ID NO:
58. An
example of compositions of the disclosure is a composition comprising a
plurality of
polypeptides (such as at least 500 different polypeptides) comprising on amino
acid sequence
set forth in SEQ ID NO: 58 (in the presence or absence of a terminal
Norleucine (Nle) or
similar non-naturally occurring amino acid). As described herein, a plurality
of the
individual polypeptides within the composition are complex and differ in
sequence based on
the position-specific sequence variability depicted in Figure 12 and SEQ ID
NO: 58
Figure 13 shows tau isoforms, domains, and potential phosphorylation sites for
targeting when designing an amino copolymer composition.
Figure 14 shows the effect of DP-0016.B, DP-0016.D, DP-0016.E, DP-0016.F, and
COPAXONE , on CD4+ T-cell proliferation in a PBMC CFSE assay.
Figure 15A, 15B, and 15C show the immunogenic effects of DP-0016.C, DP-0016.F,
or IFA alone as measured by antibody-ELISA. Figure 15A shows the anti-DP-0016
and
anti-PHF1 total IgG response on Day 29. Figure 15B shows the anti-p5422 and
anti-
recombinant Tau antibody titers. Figure 15C shows the anti-PHF-tau antibody
titers and the
anti-PHF-Tau total IgG response on Day 29.
Figure 15D shows the binding of antibodies induced by DP-0016.0 or DP-0016.F
to
human Alzheimer's disease brain tissue. Antibodies are shown to specifically
bind to
neurofibrillary tangles (NFT), Neuropil threads, and beta-amyloid plaques.
Figure 16 shows an example of a schematic corresponding to the design of an
amino
acid copolymer composition with antigenic specificity to HPV L2 (e.g., an
example of a
composition of the disclosure referred to as DP-0O24.1). Figure 16 discloses
SEQ ID NO:
59. An example of compositions of the disclosure is a composition comprising a
plurality of
polypeptides (such as at least 500 different polypeptides) comprising on amino
acid sequence
set forth in SEQ ID NO: 59 (in the presence or absence of a terminal
Norleucine (Nle) or
similar non-naturally occurring amino acid). As described herein, a plurality
of the
individual polypeptides within the composition are complex and differ in
sequence based on
the position-specific sequence variability depicted in Figure 16 and SEQ ID
NO: 59.
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Figure 17 shows an example of MALDI-TOF-MS results of two different
preparations of an amino acid copolymer composition with antigenic specificity
to Tau (e.g.,
an example of a composition of the disclosure referred to as DP-0016.F). The
relative purity
of the amino acid copolymer composition is calculated on the right.
Figure 18 shows the full MALDI-TOF spectrum of DP-0016.F recovered from PLA
particles. The DP-0016.F was previously loaded onto Polylactic Acid (PLA)
particles,
particles which protect foreign antigens (Ags) from degradation and dilution,
for 4 months.
Figure 19A shows CD4+ T-cell proliferation assays wherein healthy donor PBMC
were cultured in the presence of 7.5 tM DP-0016.F.
Figure 19B shows CD8+ T-cell proliferation assays wherein healthy donor PBMC
were cultured in the presence of 7.5 tM DP-0016.F.
Figure 19C shows that DP-0016.F loaded onto PLA particles significantly
induced a
CD4+ T-cell response. PLA particles (90 and 260 ug/mL final concentration in
the culture
medium) significantly boosted the CD4+ T-cell proliferation against DP-0016.F
at 0.25 uM
and 0.75 uM. The PLA particles alone were cytotoxic at higher concentrations
(860 ug/mL).
The final concentrations of PLA particles were 860, 260, and 90 pg/mL in the
cell culture
wells containing 2.5, 0.75, and 0.25 i.tM of DP-0016.F, respectively.
Figure 19D shows 0.25 i.tM DP-0016.F loaded onto PLA particles shows a
significant
increase in CD4+ and CD8+ T-cell response as compared to DP-0016.F only
diluted in PBS.
Figure 20A shows the results of an antibody ELISA against PHF-tau, which are
paired helical filaments otherwise defined as hyper-phosphorylated tau
oligomers isolated
from AD patients' brain autopsies, and full-length non-phosphorylated
recombinant Tau
(recTau) in mice immunized with DP-0016.F at 0.3 or 1.2mg/kg either loaded
onto PLA
particles or emulsified in Incomplete Freund's Adjuvant (IFA). Figure 20A also
shows that
high titers of anti-PHF-tau antibodies are induced by DP-0016.F at 1.2mg/kg
emulsified in
IFA.
Figure 20B shows the results of an antibody ELISA against a short linear
peptide
covering PHF1 (p5396/pS404) and p5422 epitopes in mice immunized with DP-
0016.F at
0.3 mg/kg or 1.2 mg/kg either loaded onto PLA particles or emulsified with
IFA. Figure 20B
also shows that the highest titers of anti-PHF1 antibodies are induced by DP-
0016.F, 1.2
mg/kg emulsified in IFA.
Figure 20C shows the antibody response on day 168 as compared to day 28 in
mice
treated with DP-0016.F 0.3 mg/kg loaded onto PLA particles. The graph shows
the results of
the antibody ELISA against a short linear peptide covering PHF1, a short
peptide covering
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pS422, PHF-tau isolated from AD patients' autopsies, and recombinant Tau
(recTau),
respectively.
Figure 20D shows the effect of DP-0016.F loaded onto PLA particles on hind
limb
clasping as a measure of neurodegeneration and motor impairment in JNPL3 mice.
The
graph depicts the correlation between anti-PHF1 antibody titer and hind limb
clasping in
JNPL3 mice treated with DP-0016.F (boxes) or hind limb clasping in all the
mice in the
study, treated with DP-0016.F and non-treated controls (black circles).
Figure 21A shows the effect of DP-0016.F, 0.8 mg/kg emulsified in IFA or DP-
0016.F, 0.8 mg/kg loaded onto PLA particles on the production of anti-
immunogen
antibodies which were detected using both anti-IgG1 and anti-IgG2a isotype
secondary
antibodies, in separate reactions.
Figure 21B shows the effect of DP-0016.F, 0.8 mg/kg emulsified in IFA or DP-
0016.F, 0.8 mg/kg loaded onto PLA particles on the production of anti-Tau
antibodies which
were detected using both PHF-tau and recombinant Tau (recTau) as antigens, in
separate
reactions.
Figure 21C shows the effect of DP-0016.F, 0.8 mg/kg emulsified in IFA, DP-
0016.F,
0.8 mg/kg loaded onto PLA particles or PLA particles alone (vehicle) on total
soluble tau
content in ug/mg of total proteins in the cortex, hindbrain and hippocampus of
JNPL3 mice.
Figure 21D shows the effect of DP-0016.F, 0.8 mg/kg emulsified in IFA, DP-
0016.F,
.. 0.8 mg/kg loaded onto PLA particles or PLA particles alone (vehicle) on the
relative
percentages of hyperphosphorylated soluble tau, using 3 monoclonal antibodies
PHF1, CP13
and RZ3 specific for pS396/pS404, pS202 and pT231 respectively, in the
hindbrain of JNPL3
mice.
Figure 21E shows the effect of DP-0016.F, 0.8 mg/kg emulsified in IFA, DP-
0016.F,
0.8 mg/kg loaded onto PLA particles or PLA particles alone (vehicle) on the
relative
percentages of hyperphosphorylated soluble tau, using 3 monoclonal antibodies
PHF1, CP13
and RZ3 specific for pS396/pS404, pS202 and pT231, respectively, in the
hippocampus of
JNPL3 mice.
Figure 21F shows the effect of DP-0016.F, 0.8 mg/kg emulsified in IFA, DP-
0016.F,
0.8 mg/kg loaded onto PLA particles or PLA particles alone (vehicle) on the
relative
percentage of insoluble total tau within the whole total DA31-positive tau
content in the
hindbrain and cortex of JNPL3 mice.
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Figure 21G shows the effect of DP-0016.F, 0.8 mg/kg emulsified in IFA, DP-
0016.F,
0.8 mg/kg loaded onto PLA particles or PLA particles alone (vehicle) on
aggregated tau
content in the hippocampus, cortex, and hindbrain of JNPL3 mice.
Figure 21H shows the effect of DP-0016.F, 0.8 mg/kg emulsified in IFA, DP-
0016.F,
0.8 mg/kg loaded onto PLA particles or PLA particles alone (vehicle) on hind
limb clasping,
and its correlation with total soluble tau in the cortex of JNPL3 mice.
Figure 22 shows an example of a schematic corresponding to the design of an
amino
acid copolymer composition with antigenic specificity to human a-synuclein
(e.g., an
example of a composition of the disclosure, compositions based on this
template arrangement
and amino acid input referred to as DP-0003.E). Figure 22 discloses SEQ ID NO:
61. An
example of compositions of the disclosure is a composition comprising a
plurality of
polypeptides (such as at least 500 different polypeptides) comprising on amino
acid sequence
set forth in SEQ ID NO: 61 (in the presence or absence of a terminal
Norleucine (Nle) or
similar non-naturally occurring amino acid). As described herein, a plurality
of the
individual polypeptides within the composition are complex and differ in
sequence based on
the position-specific sequence variability depicted in Figure 22 and SEQ ID
NO: 61.
DETAILED DESCRIPTION
I. Overview
The present disclosure provides a novel approach to polypeptide vaccines. The
disclosure provides compositions that are, in certain embodiments, capable of
inducing or
otherwise stimulating a Th2 chemokine (e.g., CCL22 and/or CCL17) release by
monocytes
and/or of inducing CD4+ T cell proliferation and/or of inducing antibodies
against a relevant
target (e.g., a target from which one or more ARs of the composition are
derived). In certain
embodiments, compositions of the disclosure are capable of inducing CD8+ T
cell
proliferation. In certain embodiments, the Th2 chemokine (e.g., CCL22 and/or
CCL17)
release, CD4+ T-cell proliferation and/or antibody induction properties of the
composition
are better, as compared to (i) a single polypeptide based on one or more of
the same AR
and/or (ii) the same or similar polypeptide or polypeptide composition in the
absence of
RCRs2. In certain embodiments, such compositions are capable of stimulating
CCL22
release by monocytes. In certain embodiments, such compositions, additionally
or
alternatively, are capable of stimulating CCL17 release by monocytes. In
certain
embodiments, the compositions of the disclosure do not require in vivo priming
to induce
strong T-cell proliferation in vitro. As provided herein and without being
bound by theory,
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the presence of the RCRs imparts and/or contributes to numerous functional
benefits to the
compositions (e.g. CCL22 release from monocytes, broad T cell proliferation,
activation of
monocytes, increased induction of antibody titers, etc.). These and other
benefits of RCRs
can be readily seen in the examples.
Compositions of the disclosure are useful in numerous methods in vitro and in
vivo.
In certain embodiments, compositions of the disclosure provide an improved
approach to
immunotherapy and may be suitable, for example, for studying disease
mechanism, for
raising antibodies, for promoting an anti-inflammatory Th2 immunity, for
promoting or
otherwise stimulating Th2 chemokine (e.g., CCL22 and/or CCL17) release by
monocytes, for
promoting CD4+ T-cell proliferation, for promoting CD8+ T-cell proliferation,
and/or in
methods of treating various diseases or conditions related to targets,
portions of which are the
basis for the ARs of the composition of the disclosure (e.g. protein
conformational disorders,
pathogenic infections, conditions amenable to treatment via an immunotherapy,
etc.).
Briefly, the amino acid copolymer compositions of the disclosure comprise
novel
mixtures of polypeptides having high complexity (e.g., compositions of greater
than 500,
greater than 1 x 103, greater than 1 x 104, greater than 1 x 105, greater than
1 x 106, or greater
than 1 x 108 polypeptides based on the same linear template arrangement). The
compositions
may be manufactured using solid-phase peptide synthesis, such as in a single
solid-phase
peptide synthesis step, wherein the polypeptides of the mixture have one or
more antigenic
regions from a target (e.g., referred to as a protein or antigen) related to
the disease or
condition of interest that are presented in association with one or more
(e.g., 1, 2, 3, or more
than 3) short random copolymer regions (RCRs). The disclosure is based on the
surprising
finding that the use of one or more short RCRs provides significant and
surprising
improvements in properties consistent with the use of the compositions as
immunotherapeutics (e.g., significant improvements versus those obtained in
the absence of
RCRs; surprising in view of the state of the art). Without being bound by
theory, the
presence of the RCRs imparts and/or contributes to numerous functional
benefits to the
compositions (e.g. CCL22 release from monocytes, broad T cell proliferation,
activation of
monocytes, increased induction of antibody titers, etc.). These and other
benefits of RCRs
can be readily seen in the examples.). Particularly surprising, were the
improvements seen
when using very short RCRs, such as those of 3-6 (and as short as 3) residues.
Given the
improvements seen with 8-9 residue RCRs, it was unexpected that the same or
even
improved effects could be obtained using RCRs of only a third the length. In
certain
embodiments, the disclosure contemplates that, in certain embodiments, when an
RCR of 3 or
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even 4 residues is used, the template arrangement includes 3 such RCRs (e.g.,
for a total RCR
length of 9-12 residues). As elsewhere, the amino acid percentage make-up can
vary across
the RCR as described throughout. On the other hand, in certain embodiments,
the disclosure
contemplates that when larger RCRs are used, such as RCRs of 8 or 9 residues,
two RCRs are
used ¨ although 1 or 3 RCRs can also be used. For RCRs of 5 or 6 or 7 residues
in length, in
certain embodiments, the disclosure contemplates that two or three RCRs are
used.
As described in detail herein, the compositions of the disclosure include one
or more
RCRs, each of which is short (e.g., 3-15 amino acid residues, such as 3-14
residues or 3-11
residues or 3-9 residues or even just 3-7 residues or three to six residues)
and corresponds to
a mixture of alanine and positively charged amino acids at every position of
the RCR. The
length of composition of each RCR is independently selected. Optionally, the
RCRs also
include, in a lesser proportion, an amount of negatively charged amino acid
to, for example,
increase solubility and/or to promote an alpha-helical configuration and/or a
lesser proportion
of certain other amino acids like phenylalanine. Without being bound by
theory, the short
random repeats increase the immunogenicity of the compositions and/or induce
an immune
response and, as provided herein, can be provided in association with one or
more ARs of
interest (e.g., ARs relevant to any of a range of diseases or conditions).
Moreover, without
being bound by theory and in certain embodiments, the RCRs and other portions
of the
composition may be designed for the whole composition to have an estimated net
charge at
pH7, such as to have an estimated net charge above 1 or even above 2, such as
from 2 to 4.
Although, in other embodiments, particularly where solubility of the compound
is not an
issue or the ARs provide solubility and alpha-helix structures through
negatively charged
amino acids such as glutamic acid (Glu) residues in particular, estimated net
charge of the
whole composition may be less positive or even negative. Without being bound
by theory, in
certain embodiments, even when net charge is not above 2, the RCR compositions
are
selected to increase the net charge of the composition versus the same ARs in
the absence of
the RCRs. In some embodiments, the net positive charge at pH7 of the
composition may be
negative, but less negative than an equivalent composition containing
identical ARs without
the RCRs.
The compositions of the disclosure are, in certain embodiments, capable of
inducing a
safe, anti-inflammatory Th2 immunity (e.g., safe in comparison to other
compositions which
may induce a significant pro-inflammatory Thl immunity). In other embodiments,
compositions of the disclosure are capable of inducing Thl immunity. In
certain
embodiments, compositions of the disclosure are capable, such as with or
without also
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inducing T cell proliferation, of producing antibodies against an antigen of
interest (e.g., an
antigen associated with a disease or condition suitable for intervention via
an
immunotherapeutic approach). In certain embodiments, such compositions of the
disclosure
may be referred to as immunotherapeutics or polypeptide vaccines. In certain
embodiments,
an advantage of the compositions of the disclosure is the ability to induce an
immune
response against an antigen and certain variants of an antigen, or across a
range of particular
variants or strains, and/or against more than one epitope of a target. This
may be useful for
providing compositions that both promote a more robust immune response and are
effective
across a broader population. For example, a composition of the disclosure can
be designed to
target hyper-phosphorylated Tau (e.g., a design in which one or more ARs, such
as two ARs,
are based on a base peptide sequence corresponding to a portion of hyper-
phosphorylated
Tau, optionally, where a position in the AR is selected from an amino acid or
the
phosphorylated form of that amino acid). Here the target is hyper-
phosphorylated Tau and
base peptide sequences correspond to one or more antigenic portions of the
target. Such a
composition is capable of inducing production of specific antibodies against
many variants of
phosphorylated Tau proteins found in oligomers and paired helical filaments
(PHFs) and can
be used to treat various tauopathies such as but not limited to Alzheimer's
disease (AD),
frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17),
and
progressive supranuclear palsy. The compositions of the disclosure may be
capable of
inducing specific antibodies that inhibit early phosphorylation and
accumulation of toxic
precursor tau oligomers. In some embodiments, the compositions of the
disclosure target
glial tau pathology in astrocytes. In some embodiments, the compositions of
the disclosure
target glial tau pathology in microglia.
The compositions and methods of the present disclosure overcome many of the
challenges currently faced in designing polypeptide vaccines/therapeutics and
outlined above
in the Background section. The compositions of the disclosure comprise
mixtures of
polypeptides based on a linear template arrangement and comprising one or more
RCRs and
one or more ARs. The composition has a complexity (e.g., is composed of at
least a certain
number of different polypeptides, such as more than 500) where the
polypeptides in the
composition are related by sequence and the complexity and variability is
based on the
permitted level of sequence differences at each position, as well as the pre-
determined molar
input percentages of the amino acids permitted at each position. The
predominance of a
particular residue at a position is described, at least in part, by the molar
input percentage of
one or more amino acids available at each position. Compositions of the
disclosure may also
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be described based on amino acid output (e.g., percentage of one or more
particular amino
acids, on a molar basis, in the amino acid composition).
Compositions of the disclosure are mixtures of polypeptides based on a linear
template arrangement. The polypeptides comprise one or more random copolymer
regions
(RCRs) linked (directly or via an amino acid linker) to one or more antigenic
regions (ARs).
Optionally, additional amino acid sequences may be present at the N- or C-
terminus, such as
additional positively charge residues. In addition, in certain embodiments,
compositions of
the disclosure may be described based on a linear template arrangement and
template
sequence, and optionally, on amino acid input ratios/percentages at each
position or in total
and/or on output ratios and/or on estimated net charge at pH7. Without being
bound by
theory, in certain embodiments, the antigenic regions (ARs) of the composition
promote the
induction of antibodies against the relevant disease targets (e.g., against
antigens/protein
typically associated with a disease or condition), optionally, while
preserving the normal
functional protein (if present). In certain embodiments, compositions of the
disclosure are
.. capable of promoting or otherwise stimulating Th2 chemokine (e.g., CCL22
and/or CCL17)
release by monocytes (e.g., CCL22 release by monocytes) and/or promoting CD4+
T-cell
proliferation (e.g., CD4+ T-cell proliferation among human peripheral blood
mononuclear
cells), such as via the RCRs and/or the combined activity of the ARs and RCRs.
In certain
embodiments, compositions of the disclosure do not require in vivo priming to
induce strong
.. T-cell proliferation in vitro. In certain embodiments, compositions of the
disclosure exhibit
epitope promiscuity, and are thus suitable for eliciting immunogenicity in
patients with
different HLA phenotypes.
The amino acid copolymer compositions of the present disclosure improve upon
existing designs and comprise mixtures of polypeptides made of one or more
short random
copolymer regions (RCRs) linked to one or more antigenic regions (ARs). In
certain
embodiments, the compositions of the disclosure contain a mixture of
polypeptides
containing 3 RCRs of 3, 4, or 5 amino acids each, linked to two or more ARs.
In certain
embodiments, the compositions of the disclosure contain a mixture of
polypeptides
containing 2 RCRs of 3, 4, 5, 6, or 7 amino acids each, linked to one or more
ARs. In certain
embodiments, the disclosure contemplates the use of slightly longer RCRs, such
as RCRs of
8 or 9 residues. As described herein, longer RCRs are also contemplated (e.g.,
10-15
residues) and they may be particularly suitable to embodiments where only a
single RCR is
used in the template arrangement. In certain embodiments, compositions of the
disclosure can
be synthesized by solid phase peptide synthesis, such as in a single
manufacturing step. In
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certain embodiments, compositions of the disclosure contain a mixture of
polypeptides
wherein at least 55% or at least 60% of the polypeptides in the complex
mixture based on the
linear template arrangement are substantially full-length polypeptides. In
other embodiments,
the percentage of such full-length or substantially full-length polypeptides
is at least 75% or
at least 80%. In certain embodiments, the first amino acid at the C-terminal
position of each
polypeptide in the mixture that is based on the template arrangement is
Norleucine (Nle). In
certain embodiments, the first amino acid at the C-terminal position is Nle or
another non-
naturally occurring amino acid. Thus, even when not shown in the examples,
presence of a
Nle or other non-naturally occurring amino acid at the C-terminus is
contemplated in certain
embodiments for all compositions. Compositions of the disclosure have numerous
beneficial
properties, including having combined B- and T-cell determinants. Further, in
certain
embodiments, the antigenic regions (ARs) of the compositions of the present
disclosure
incorporate alanine to confer epitope promiscuity and, in certain embodiments,
to improve
solubility. The random copolymer regions (RCRs) incorporate alanine,
positively charged
residues, and optionally, negatively charged residues for monocyte activation
and broad T-
cell proliferation (and optionally a negatively charged and/or certain other
residues). The
improvement in one or more properties of compositions of the disclosure
containing one or
more short RCRs versus single polypeptides or mixtures of polypeptides that
are similar to
compositions of the disclosure but lack RCRs was surprising. In other words,
it was
unexpected that the introduction of such short, random sequences would instill
the desirable
properties observed, such as adjuvant-like properties.
In certain embodiments, particularly where solubility of the composition is an
issue,
such as due to characteristics of the ARs, increasing net charge at pH7 via
the use and
composition of the RCRs improves solubility and thus provides improved
compositions. In
certain embodiments, the compositions of the disclosure comprising one or more
RCRs have
an estimated net charge (e.g., based on predicted net charge of full-length
polypeptides and
given input percentages (e.g. molar input percentages) and amino acid
distributions) of
greater than 2 (e.g., 2.1 or even about 3) at pH7. In some embodiments the
estimated net
charge is greater than 2.0 and less than 4.0 at pH7. However, positive charge
is also a proxy
for alpha helical confirmation and/or solubility, and thus, the nature and
composition of
certain ARs permit good solubility or promote alpha helical conformation even
at a different
charge. Accordingly, in certain embodiments, although the use of RCRs in
compositions of
the disclosure increases the estimated net charge of the composition, versus a
composition of
the same complex ARs without RCRs, the estimated net charge at pH7 can be less
than 2.
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Accordingly, the disclosure contemplates the use of the RCRs to increase the
estimated net
charge of the composition at pH7 and this can be modulated to improve
solubility of the
composition. In some embodiments, the ARs may have a negative charge at pH7,
and the
RCR compositions increase the net positive charge at pH7 of the composition as
a whole
(although not necessarily to a positive charge). It will be understood that
the impact of the
RCRs is not merely on charge, as described herein, as compositions of the
disclosure have
numerous functional features indicative of their use as a vaccine and improved
functional
properties owing to the addition of the RCRs.
The estimated net charge is a proxy for the predicted charge (e.g., estimated
charge) at
pH7 and is based on the net charge of the ARs and RCRs within the linear
template
arrangement. The estimated net charge for a composition of the disclosure is
based on the
template sequence where: (i) the net charge of the ARs, is calculated based on
the base
peptide sequence without the inclusion of potential variation at each amino
acid position of
the AR, (ii) the net charge of the RCRs, is calculated for a hypothetical
polypeptide in which
for every 1 position in which a positively charged amino acid is assumed to
occur, alanine is
assumed to occur in two positions (e.g., if template arrangement and template
sequence for a
polypeptide mixture includes 1 RCR of 3 amino acid residues in length,
estimated net charge
is calculated based on the assumption that the RCR contains 1 positively
charged residue and
two alanines); and (iii) and additional amino acids outside of the ARs and
RCRs and present
in the design are included (e.g., a 4 residue N- or C- terminal tail in the
design is accounted
for based on its sequence and charge). For the purpose of this calculation,
negatively charged
amino acids are not considered for the RCR.
In certain embodiments, compositions of the disclosure have one or more of the
following advantages: (i) ability to target multiple antigenic targets and
adaptability across
targets and disease indications; (ii) sustained immunogenicity; (iii) anti-
inflammatory
properties; (iv) antigen-independent T-cell help; (v) IgG4 antibody response;
(vi) HLA
promiscuity; (vii) intrinsic adjuvant properties that eliminate/decrease the
need for the
addition of a strong adjuvant or carrier protein for therapeutic efficacy;
(viii) no/decreased
risk of neutralizing antibodies; (ix) no IV administration; (x) cost-
effectiveness; (xi)
satisfactory safety profile; (xii) PK/PD correlation; (xiii) improved
solubility; (ix) net positive
charge at pH7 (e.g., preferably net charge or estimated net charge of greater
than 2 or of
greater than 2 and less than 4). As noted above, (ix) may vary depending on
the nature of the
ARs. However, the compositions do have an increase in net charge versus that
of a similar
composition based on the complex ARs alone. Improved properties of the
compositions of
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the disclosure may be measured versus, for example, a similar composition
having the same
or similar ARs but in the absence of RCRs. As provided herein and without
being bound by
theory, the presence of the RCRs imparts and/or contributes to numerous
functional benefits
to the compositions (e.g. CCL22 release from monocytes, broad T cell
proliferation,
activation of monocytes, increased induction of antibody titers, etc.). These
and other
benefits of RCRs can be readily seen in the examples.
As described herein, polypeptide mixture compositions of the disclosure,
designed as
described herein, have numerous advantageous immunological properties, while
also, in
certain embodiments, inducing antigen-specific antibodies (e.g., antibodies
against targets
consistent with the ARs). The compositions of the disclosure are, in certain
embodiments,
able to stimulate Th2 chemokine (e.g., CCL22 and/or CCL17) release from
monocytic cells
and promote broad CD4+ T-cell proliferation. Monocyte activation and T-cell
proliferation
activities of the compounds demonstrate their intrinsic adjuvant properties
and their Th2
immunity profile. As an example, DP-0016.B and DP-0016.C, two compositions
according
to the disclosure that target the C-terminal region of Tau (e.g., ARs
correspond to base
peptides derived from human Tau-F), induced anti-hyper-phosphorylated tau
antibodies,
while promoting CCL22 release from monocytes and promoting CD4+ T-cell
proliferation.
Other examples are also provided. This approach and design structure, based on
incorporation of RCRs of 3-15 amino acids in length is applicable to any
target of interest
(e.g., a target for which the generation of antibodies, such as to use for
study or as an
immunotherapy, would be useful), and not just targets limited to protein
conformational
disorders. Exemplary suitable targets are described herein. Moreover, further
description of
the linear template arrangements, RCRs, and ARs is described herein. The
disclosure
contemplates compositions based on any combination of structural and/or
functional features
described herein. Any such compositions may be made, for example, based on the
methods
described herein. Any such compositions may be used in methods, in vitro
and/or in vivo.
The disclosure provides methods of making (e.g., manufacturing) compositions
of the
disclosure, and any such compositions may be so described based on any one or
combination
of features or embodiments described herein. For example, the disclosure
provides a
composition made by a method of manufacture, comprising synthesizing a high
complexity
polypeptide composition based on a template arrangement, as described herein.
For example, the disclosure provides a composition comprising:
a mixture of at least 500 different polypeptides each having a length of
between about 25 to
100 amino acids, wherein each polypeptide is manufactured based on a template
arrangement
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comprising one or more antigenic regions (each an AR) linked to two or more
random
copolymer regions of 3-7 amino acids in length (each an RCR), wherein the one
or more ARs
and the two or more RCRs are arranged according to a linear template
arrangement (together
a "complex polypeptide mixture component"),
wherein at least one AR comprises a sequence of amino acid positions
corresponding to a
first base peptide sequence derived from an antigen associated with a disease
and for each
amino acid position of said base peptide sequence, each polypeptide has an
amino acid
independently selected from one or more of: an original amino acid found at
the
corresponding amino acid position of the first base peptide sequence, alanine
(A), lysine (K),
arginine (R), or an amino acid serving as a conserved substitution for the
original amino acid,
and wherein the distribution of the amino acids at a given position among the
polypeptides is
determined by a pre-determined molar input ratio of the amino acids available
for that
position and is independently selected,
wherein for each amino acid position of the two or more RCRs, each polypeptide
has an
amino acid selected from (i) A and (ii) at least one of lysine (K), arginine
(R) or histidine (H),
and, optionally, (iii) at least one of aspartic acid (D) or glutamic acid (E),
and wherein, for
each amino acid position of the two or more RCRs, the relative molar input
percentage of A
for each position is less than or equal to 65%, the relative molar input
percentage of
positively charged amino acids for each position is at least 35% and the
relative molar input
percentage of negatively charged amino acids for each position is less than or
equal to 20%,
and wherein the distribution of the amino acids at a given position of the one
or more RCRs
among the polypeptides is determined by a pre-determined molar input ratio of
the amino
acids available for that position and is independently selected for each
position, and wherein
amino acid content and molar input percentage of each RCR is independently
selected.
In certain embodiments, the foregoing composition may include or be described
based
on any of the features of the compositions of the disclosure provided herein.
Similarly, the disclosure provides a composition comprising:
a mixture of at least 500 different polypeptides each having a length of
between about 25 to
100 amino acids, wherein each polypeptide is manufactured based on a template
arrangement
comprising one or more antigenic regions (each an AR) linked to two or more
random
copolymer regions of 3-7 amino acids in length (each an RCR), wherein the one
or more AR
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and the two or more RCRs are arranged according to a linear template
arrangement (together
a "complex polypeptide mixture component"),
wherein at least one AR comprises a sequence of amino acid positions
corresponding to a
first base peptide sequence derived from an antigen associated with a disease
and for each
amino acid position said base peptide sequence, each polypeptide has an amino
acid
independently selected from one or more of: an original amino acid found at
the
corresponding amino acid position of the first base peptide sequence, alanine
(A), lysine (K),
arginine (R), or an amino acid serving as a conserved substitution for the
original amino acid,
and wherein the distribution of the amino acids at a given position among the
peptides is
determined by a pre-determined molar input ratio of the amino acids available
for that
position and is independently selected,
wherein for each amino acid position of the two or more RCRs, each polypeptide
has an
amino acid selected from (i) A and (ii) at least one of K, arginine (R) or
histidine (H), and,
optionally, (iii) at least one of aspartic acid (D) or glutamic acid (E), and
wherein the
distribution of the amino acids at a given position of the one or more RCRs
among the
polypeptides is determined by a pre-determined molar input ratio of the amino
acids available
for that position and is independently selected for each position, and wherein
amino acid
content and molar input percentage of each RCR is independently selected,
wherein the ratio of the percentage of alanine to the percentage of lysine, on
a molar basis, in
the polypeptides of the composition having a length of between about 25 to 100
amino acids
(the output ratio) is greater than or equal to 1.5 and less than or equal to
5Ø
In certain embodiments, the foregoing composition may include or be described
based
on any of the features of the compositions of the disclosure provided herein.
Similarly, the disclosure provides a composition comprising:
a mixture of at least 500 different polypeptides each having a length of
between about 25 to
100 amino acids, wherein each polypeptide is manufactured based on a template
arrangement
comprising one or more antigenic regions (each an AR) linked to one or more
random
copolymer regions of 3-15 amino acids in length (each an RCR), wherein the one
or more AR
and the one or more RCR are arranged according to a linear template
arrangement (together a
"complex polypeptide mixture component"),
wherein at least one AR comprises a sequence of amino acid positions
corresponding to a
first base peptide sequence derived from an antigen associated with a disease
and for each
amino acid position of said base peptide sequence, each polypeptide has an
amino acid
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independently selected from one or more of: an original amino acid found at
the
corresponding amino acid position of the first base peptide sequence, alanine
(A), lysine (K),
arginine (R), or an amino acid serving as a conserved substitution for the
original amino acid,
and wherein the distribution of the amino acids at a given position among the
polypeptides in
the mixture is determined by a pre-determined molar input ratio of the amino
acids available
for that position and is independently selected,
wherein for each amino acid position of an RCR, each polypeptide has an amino
acid selected
from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and,
(iii) optionally, at least
one of aspartic acid (D), glutamic acid (E), or phenylalanine (F), and
wherein, for each amino
acid position of each RCR, the relative molar input percentage of A for each
position is less
than or equal to 65%, the relative molar input percentage of positively
charged amino acids
for each position is at least 35% and the relative molar input percentage of
negatively charged
amino acids for each position is less than or equal to 20%, and wherein the
distribution of the
amino acids at a given position of the one or more RCRs among the polypeptides
is
determined by a pre-determined molar input ratio of the amino acids available
for that
position and is independently selected for each position, and wherein, if the
polypeptides of
the mixture comprise more than one RCR, amino acid content and molar input
percentage of
each RCR is independently selected.
In certain embodiments, the foregoing composition may include or be described
based
on any of the features of the compositions of the disclosure provided herein.
The disclosure further provides a composition comprising:
a mixture of at least 500 different polypeptides each having a length of
between about 25 to
100 amino acids, wherein each polypeptide is manufactured based on a template
arrangement
compriseing one or more antigenic regions (each an AR) linked to one or more
random
copolymer regions of 3-15 amino acids in length (each an RCR), wherein the one
or more AR
and the one or more RCR are arranged according to a linear template
arrangement (together a
"complex polypeptide mixture component"),
wherein at least one AR comprises a sequence of amino acid positions
corresponding to a
first base peptide sequence derived from an antigen associated with a disease
and for each
amino acid position of said base peptide sequence, each polypeptide has an
amino acid
independently selected from one or more of: an original amino acid found at
the
corresponding amino acid position of the first base peptide sequence, alanine
(A), lysine (K),
arginine (R), or an amino acid serving as a conserved substitution for the
original amino acid,
and wherein the distribution of the amino acids at a given position among the
polypeptides in
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the mixture is determined by a pre-determined molar input ratio of the amino
acids available
for that position and is independently selected,
wherein for each amino acid position of an RCR, each polypeptide has an amino
acid selected
from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and,
(iii) optionally, at least
one of aspartic acid (D), glutamic acid, or phenylalanine (F), and wherein the
distribution of
the amino acids at a given position of the one or more RCRs among the
polypeptides is
determined by a pre-determined molar input ratio of the amino acids available
for that
position and is independently selected for each position, and wherein, if the
polypeptides of
the mixture comprise more than one RCR, amino acid content and molar input
percentage of
each RCR is independently selected, and
wherein the ratio of the percentage of alanine to the percentage of lysine, on
a molar basis, in
the polypeptides of the composition having a length of between about 25 to 100
amino acids
(the "output" ratio) is greater than or equal to 1.5 and less than or equal to
5.0 (as expressed
as a quotient, which ratio may also be expressed as a relative ratio of 1.5:1
to 5.0:1).
In certain embodiments, the foregoing composition may include or be described
based
on any of the features of the compositions of the disclosure provided herein.
The disclosure further provides a composition comprising:
a mixture of at least 500 different polypeptides each having a length of
between about 25 to
100 amino acids, wherein each polypeptide is manufactured based on a template
arrangement
comprising one or more antigenic regions (each an AR) linked to one or more
random
copolymer regions of 3-15 amino acids in length (each an RCR), wherein the one
or more AR
and the one or more RCR are arranged according to a linear template
arrangement (together a
"complex polypeptide mixture component"), which composition has an estimated
net charge
of greater than 2.0 and less than 4.0 at pH7,
wherein at least one AR comprises a sequence of amino acid positions
corresponding to a
first base peptide sequence derived from an antigen associated with a disease
and for each
amino acid position of said base peptide sequence, each polypeptide has an
amino acid
independently selected from one or more of: an original amino acid found at
the
corresponding amino acid position of the first base peptide sequence, alanine
(A), lysine (K),
arginine (R), or an amino acid serving as a conserved substitution for the
original amino acid,
and wherein the distribution of the amino acids at a given position among the
polypeptides in
the mixture is determined by a pre-determined molar input ratio of the amino
acids available
for that position and is independently selected,
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wherein for each amino acid position of an RCR, each polypeptide has an amino
acid selected
from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and,
(iii) optionally, at least
one of aspartic acid (D), glutamic acid (E), or phenylalanine (F), and
wherein, for each amino
acid position of each RCR, the distribution of the amino acids at a given
position among the
polypeptides in the mixture is determined by a pre-determined molar input
ratio of the amino
acids available for that position and is independently selected for each
position of each RCR.
In certain embodiments, the foregoing composition may include or be described
based
on any of the features of the compositions of the disclosure provided herein.
The disclosure further provides a composition comprising:
a mixture of at least 500 different polypeptides each having a length of
between about 25 to
100 amino acids, wherein each polypeptide is manufactured based on a template
arrangement
comprising one or more antigenic regions (each an AR) linked to two or more
random
copolymer regions of 3-7 amino acids in length (each an RCR), wherein the one
or more AR
and the two or more RCRs are arranged according to a linear template
arrangement (together
a "complex polypeptide mixture component"), which composition has an estimated
net
charge of greater than 2.0 and less than 4.0 at pH7,
wherein at least one AR comprises a sequence of amino acid positions
corresponding to a
first base peptide sequence derived from an antigen associated with a disease
and for each
amino acid position of said base peptide sequence, each polypeptide has an
amino acid
independently selected from one or more of: an original amino acid found at
the
corresponding amino acid position of the first base peptide sequence, alanine
(A), lysine (K),
arginine (R), or an amino acid serving as a conserved substitution for the
original amino acid,
and wherein the distribution of the amino acids at a given position among the
polypeptides is
determined by a pre-determined molar input ratio of the amino acids available
for that
position and is independently selected,
wherein for each amino acid position of the two or more RCRs, each polypeptide
has an
amino acid selected from (i) A and (ii) at least one of K, arginine (R) or
histidine (H), and,
optionally, (iii) at least one of aspartic acid (D) or glutamic acid (E), and
wherein, for each
amino acid position of the two or more RCRs, the distribution of the amino
acids at a given
position among the polypeptides in the mixture is determined by a pre-
determined molar
input ratio of the amino acids available for that position and is
independently selected for
each position of each RCR.
In certain embodiments, the foregoing composition may include or be described
based
on any of the features of the compositions of the disclosure provided herein.
For example, in
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certain embodiments, the RCRs are 8 or 9 residues in length, and the template
arrangement
comprises at least two RCRs or two RCRs. In other embodiments, the RCRs are 3
or 4
residues in length, and the template arrangement comprises three RCRs. In
other
embodiments, the RCRs are 5, 6, or 7 amino acids in length, and the template
arrangement
comprises two or three RCRs. Of course, as set forth in the examples and
description, these
are exemplary arrangements.
Similarly, the disclosure provides compositions comprising a plurality of
polypeptides
based on any of the template arrangements and designs disclosed herein, such
as in the
figures and sequence listing. These are examples of compositions of the
disclosure. Such
compositions comprise a plurality of related polypeptides (such as at least
500 or more than
500 polypeptides) each based on the sequences provided. For example, the
linear sequence
of the template arrangements provided in the examples describe the variety of
polypeptides in
the complex compositions, and thus, can be used to describe that composition,
as described
herein. Such polypeptides may be used and formulated, just as any other
compositions of the
disclosure, as described herein, including with an adjuvant and/or for the
various routes of
delivery provided herein.
Any of the compositions of the disclosure, whether described by template
arrangement or sequence, may be formulated, as described herein, and may be
used in any of
the in vitro or in vivo methods described herein. Compositions of the
disclosure may be
described based on any combination of one or more features disclosed herein
(e.g., sequence,
template arrangement, molar input percentage, amino acid selection, ARs, base
peptide
sequence(s), antigen, number of RCRs, etc.).
II. Definitions
The term "associated with" means "coexistent with" or "in correlation with."
The
term does not necessarily indicate causal relationship, though such
relationship may exist.
The term "binding" refers to a direct association between two molecules, due
to, for
example, covalent, electrostatic, hydrophobic, ionic and/or hydrogen-bond
interactions under
physiological conditions, and including interactions such as salt bridges and
water bridges.
The term "HLA" molecule means any class II major histocompatibility complex
glycoproteins.
The term "patient" refers to an animal, preferably a mammal, including humans
as
well as livestock and other veterinary subjects. In certain embodiments, the
patient is a
human.
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The terms "peptide", "polypeptide" and "protein" are used interchangeably
herein.
These terms refer to unmodified amino acid chains, and also include minor
modifications,
such as phosphorylations, glycosylations and lipid modifications. The terms
"peptide" and
"peptidomimetic" are not mutually exclusive and include substantial overlap.
A "peptidomimetic" includes any modified form of an amino acid chain, such as
a
phosphorylation, capping, fatty acid modification and including unnatural
backbone and/or
side chain structures. As described below, a peptidomimetic comprises the
structural
continuum between an amino acid chain and a non-peptide small molecule.
Peptidomimetics
generally retain a recognizable peptide-like polymer unit structure. Thus, a
peptidomimetic
may retain the function of binding to a HLA protein forming a complex, which
activates
autoreactive T cells in a patient suffering from an autoimmune disease.
The term "substantially full-length" , with respect to a polypeptide, refers
to a
polypeptide that varies in length by less than 5% versus a comparator. For
example, with
respect to polypeptides within a complex mixture that are based on a template
arrangement
and design having a length of 60 amino acids, a substantially full-length
polypeptide
produced following solid phase synthesis would be a polypeptide that is
anywhere from 57
amino acids to 63 amino acids. However, polypeptides in the composition are,
in certain
embodiments, full-length (based on a particular template arrangement, as
described herein).
The method is based on synthesis using particular input molar percentages of
particular amino acid residue(s) at each position, including each position of
each RCR and
each position of each AR. This can be readily described for any of the
compositions of the
disclosure, and thus, the disclosure contemplates that products by process and
methods of
manufacture are similarly described based on the characteristics of the
compositions
described herein. By way of example, the disclosure provides complex
compositions of
greater than 500 polypeptides manufactured based on any of the template
arrangements of
RCRs and ARs, as provided herein, and based on any of the molar input
percentages and
amino acid distributions and rules described herein.
The term "amino acid residue" is known in the art. In general the
abbreviations used
herein for designating the amino acids and the protective groups are based on
recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see
Biochemistry (1972) 11:1726-1732). In certain embodiments, the amino acids
used in the
application of this disclosure are those naturally occurring amino acids found
in proteins, or
the naturally occurring anabolic or catabolic products of such amino acids,
which contain
amino and carboxyl groups. Particularly suitable amino acid side chains
include side chains
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selected from those of the following amino acids: glycine, alanine, valine,
cysteine, leucine,
isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid,
glutamine, asparagine,
lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.
The term "amino acid residue" further includes analogs, derivatives and
congeners of
any specific amino acid referred to herein, as well as C-terminal or N-
terminal protected
amino acid derivatives (e.g. modified with an N-terminal or C-terminal
protecting group).
For example, the present disclosure contemplates the use of amino acid analogs
wherein a
side chain is lengthened or shortened while still providing a carboxyl, amino
or other reactive
precursor functional group for cyclization, as well as amino acid analogs
having variant side
chains with appropriate functional groups). For instance, the subject compound
can include
an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic
acid,
norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-
hydroxytryptophan, 1-
methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or
diaminobutyric acid.
Other naturally occurring amino acid metabolites or precursors having side
chains, which are
suitable herein will be recognized by those skilled in the art and are
included in the scope of
the present disclosure.
Most of the amino acids used in the polypeptide compositions of the present
disclosure may exist in particular geometric or stereoisomeric forms. In
preferred
embodiments, the amino acids used to form the subject polypeptide mixtures are
(0-isomers,
although (D)-isomers may be included in the polypeptides such as at non-anchor
positions or
in the case of peptidomimetic versions of the polypeptides.
"Naturally occurring variations", as used herein in reference to amino acids
are allelic
variations, isomeric and species differences of functionally equivalent
proteins, naturally
occuring amino acid modifications, whether or not incorporated while synthesis
or post-
synthesis (i.e. post-translation modification in vivo and post-synthesis
modification in vitro)
such as preformed phosphorylations, preformed nitrations, preformed
glycosylations,
methylation, modification by deamination, modification by deimination,
modification by
fatty acids (such as myristoylation), modified amino acid side chains
including modification
to produce amino acid analogs as described in paragraph defining "amino acid
residue",
cross-linking such as disulfide bonds, and other known modifications.
"Prevent", as used herein, means to delay or preclude the onset of, for
example, one
or more symptoms, of a disorder or condition.
"Treat", as used herein, means at least lessening the severity or ameliorating
the
effects of, for example, one or more symptoms, of a disorder or condition.
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"Treatment regimen" as used herein, encompasses therapeutic, palliative and
prophylactic modalities of administration of one or more compositions of the
disclosure. A
particular treatment regimen may last for a period of time at a particular
dosing pattern,
which will vary depending upon the nature of the particular disease or
disorder, its severity
and the overall condition of the patient, and may extend from once daily, or
more preferably
once every 36 hours or 48 hours or longer, to once every month or several
months.
The terms "structure-activity relationship" or "SAR" refer to the way in which
altering
the molecular structure of drugs alters their interaction with a receptor,
enzyme, etc.
The practice of the present disclosure will employ, where appropriate and
unless
otherwise indicated, conventional techniques of cell biology, cell culture,
molecular biology,
transgenic biology, microbiology, virology, recombinant DNA, and immunology,
which are
within the skill of the art. Such techniques are described in the literature.
See, for example,
Molecular Cloning: A Laboratory Manual, 3rd Ed., ed. by Sambrook and Russell
(Cold
Spring Harbor Laboratory Press: 2001); the treatise, Methods In Enzymology
(Academic
Press, Inc., N.Y.); Using Antibodies, Second Edition by Harlow and Lane, Cold
Spring
Harbor Press, New York, 1999; Current Protocols in Cell Biology, ed. by
Bonifacino, Dasso,
Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons, Inc., New York,
1999; and
PCR Protocols, ed. by Bartlett et al. , Humana Press, 2003; PHARMACOLOGY A
Pathophysiologic Approach Edited by Josehp T. DiPiro, Robert Talbert, Gary,
Yee, Gary
Matzke, Barbara Wells, and L. Michael Posey. 5th edition 2002 McGraw Hill;
Pathologic
Basis of Disease. Ramzi Cotran, Vinay Kumar, Tucker Collins. 6th Edition 1999.
Saunders.
III. Amino acid Copolymers with Antigenic Specificity
In part, the present disclosure is based on the discovery that antigenic
sequences can
be modified by linking them to short cationic polypeptides in high-complexity
mixtures to
elicit a robust immune response. In certain embodiments, the compositions of
the disclosure
have the further advantage of being able to stimulate the generation of
specific antibodies
against an antigen and, in certain embodiments, against variants of the
antigen (including
against all significant variants of the antigen).
Briefly, the amino acid copolymer compositions of the disclosure comprise
novel
mixtures of polypeptides having high complexity (e.g., compositions of greater
than 500
polypeptides based on the same linear template arrangement). The compositions
may be
manufactured using solid-phase peptide synthesis, such as in a single solid-
phase peptide
synthesis step, wherein the polypeptides of the mixture have a portion based
on one or more
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antigenic sequences from a target (e.g., referred to as a protein or antigen)
related to the
disease or condition of interest that are presented in association with one or
more short
random copolymer repeats (RCRs). The disclosure is based on the surprising
finding that the
use of one or more short RCRs, such as use of 2, 3 or more than 3 RCRs,
provides significant
and surprising improvements in properties consistent with the use of the
compositions as
immunotherapeutics (e.g., significant improvements versus those obtained in
the absence of
RCRs; surprising in view of the state of the art). In certain embodiments, the
compositions of
the disclosure contain a mixture of polypeptides made of 3 RCRs of 3, 4, or 5
amino acids
each, linked to one or more ARs. The length of each RCR may be the same or
independent
from that of the other RCRs. In certain embodiments, the compositions of the
disclosure
contain a mixture of polypeptides made of 2 RCRs of 3, 4, 5, 6, 7, 8, or 9,
such as 5, 6, 7, 8,
or 9 amino acids each, linked to one or more ARs. The length of each RCR may
be the same
or independent from that of the other RCRs. As described herein, the
compositions of the
disclosure contain a mixture of polypeptides wherein at least 55% or at least
60% of the
polypeptides in the complex mixture based on the linear template arrangement
are full-length
or substantially full-length polypeptides. Furthermore, the compositions of
the disclosure
include one or more RCRs, each of which is short (e.g., 3-15 amino acid
residues, such as 3-
12 residues or 3-9 residues or 3-7 residues or 3-6 residues or 3-4 residues)
and corresponds to
a mixture of alanine and positively charged amino acids at every position of
the RCR.
Optionally, the RCRs also include, in a lesser proportion, an amount of
negatively charged
amino acid to, for example, increase solubility and/or to promote an alpha-
helical
configuration and/or a lesser proportion of certain other amino acids like
phenylalanine. The
short random copolymer repeats may increase the immunogenicity of the
compositions
and/or induce an immune response, such as a T cell response and, as provided
herein, can be
provided in association with one or more ARs of interest (e.g., ARs relevant
to any of a range
of diseases or conditions).
Each composition of the disclosure is a high-complexity mixture (e.g. >500
different
polypeptides) of related polypeptides based on a linear template arrangement
of one or more
antigenic regions (each an AR, such as ARa, ARb...and so on) linked, directly
or indirectly, to
one or more random copolymer regions (each an RCR, such as RCRa, RCRb... and
so on). A
linear template arrangement refers to the arrangement of ARs and RCRs in a
linear sequence
that is common to all of the full-length polypeptides in a composition and
upon each of the
complex mixture of polypeptides of the composition is based. An "antigenic
region" or
"AR" as used herein refers to a portion having an amino acid sequence based on
a sequence
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derived from an antigen of interest (a base peptide derived from a target of
interest). We note
that, unless context indicates otherwise, the terms "antigenic region" and
"AR" are used
interchangeably herein. In general, an antigenic region is anywhere from about
9-60 amino
acids long and could be based on any relevant portion of a protein of interest
that is
associated with a disease or condition. For example, an antigenic region may
be based on a
portion of a protein that is associated with a disease or condition, such as a
portion containing
an immunodominant determinant. The amino acid sequence of an AR may remain
constant
amongst the polypeptides of the composition, i.e. each polypeptide of the
composition may
have the same amino acid sequence as the base peptide in that region.
Alternatively, the
amino acid sequence of an AR may be a source of complexity, such that the
specific
sequence of the AR may vary across the polypeptides of the composition, i.e.
the
polypeptides may differ from the base peptide and from one another at one or
more amino
acid positions in that region. For example, an amino acid at each position may
be selected
from an original amino acid found at the corresponding amino acid position of
the first base
peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid
serving as a
conserved substitution for the original amino acid. The template AR refers to
the sequence
and permissible amino acid substitutions present across all of the
polypeptides in the mixture,
and are referred to generically as AR (or antigenic region) or using letters
to specifically refer
to a particular AR present in a linear template arrangement (ARa, ARb, ARE,
where n is an
integer from 0-3). When referring to a specific polypeptide within the amino
acid copolymer
composition, the ARs of that specific polypeptide are depicted using numbers
(ARai, ARa2,
ARa3
ARa500 ... etc., where the first AR of each of the specific polypeptides 1-500
of the
composition are thus represented).
A "random copolymer region" or "RCR" as used herein refers to short random
repeats
(e.g. anywhere between 3-15 amino acids long, such as 3-14 amino acids long or
3-12 or 3-9
or 3-7 amino acids long or three to six amino acids) generated from mixtures
(at each
position) of alanine, at least one positively charged amino acid (e.g. lysine,
arginine,
histidine, non-naturally occurring positively charged residue), and
optionally, at least one
negatively charged amino acid (e.g. aspartic acid or glutamic acid). In some
embodiments,
the RCR may optionally further comprise phenylalanine. In some embodiments,
the RCR
may optionally further comprise one or more amino acids that promote an alpha-
helical
secondary structure (e.g. methionine or leucine). Unless context indicates
otherwise, the
terms "random copolymer region" and "RCR" are used interchangeably herein.
Exemplary
RCRs are each 3, 4, 5, 6, 7, 8, or 9 amino acids in length.
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The template RCR refers to the sequence and permissible amino acid
substitutions
present across the polypeptides in the mixture, and are referred to
generically as RCR (or
random copolymer region) or using letters to specifically refer to a
particular RCR present in
a linear template arrangement (e.g., RCRa, RCRb, RCR, where m is an integer
from 0-3;
e.g., there may be 1, 2, 3, 4, or 5 RCRs). When referring to a specific
polypeptide within the
amino acid copolymer composition, the RCRs of that specific polypeptide are
depicted using
numbers (RCRai, RCRa2, RCRa3 RCRa500 ... etc., where the first RCR of
each of the
specific polypeptides 1-500 of the composition are thus represented). As
described herein,
exemplary numbers of RCRs are two or three, although more and fewer are
contemplated.
Thus, each polypeptide, such as the full-length or substantially full-length
polypeptides in the composition, will comprise one or more antigenic regions
based on the
one or more template antigenic regions (each an AR,, wherein x corresponds to
the position
of the AR in the linear template arrangement and describes that template, and
wherein y
identifies the individual polypeptide, e.g. a composition with 500 different
polypeptides will
have a first AR that is identified as ARai, ARa2, ARa3...ARa500 for each
polypeptide of the
composition) and one or more random copolymer regions (each an RCR,, wherein x
corresponds to the position of the RCR in the linear template arrangement and
wherein y
identifies the individual polypeptide, e.g. a composition with 500 different
polypeptides could
have a first RCR that is identified as RCRai, RCRa2, RCRa3...RCRa500 in each
polypeptide of
the composition).
A hypothetical amino acid copolymer composition is shown schematically in Fig.
1
for illustrative purposes. In this example, the linear template arrangement of
polypeptides in
the composition, such as the substantially full-length polypeptides in the
composition,
comprises: two RCRs and 2 ARs configured as RCRa-ARa-RCRb-ARb.
The complexity of the composition derives from the diversity of input amino
acids
provided for certain positions along the length of the polypeptides, the
diversity of output
amino acids, and the diversity in polypeptide length. In the example shown in
Fig. 1, the
random copolymer regions (RCRs) of each polypeptide in the composition are
made of A, K,
or E, at each position. The distribution of A, K, and E at each position of a
random
copolymer region will depend on the relative molar input percentages of A, K,
and E (not
shown in Figure 1). The first antigenic region (ARa) in the example is based
on base peptide
sequence "STLYA" and the second antigenic region (ARb) is based on base
peptide sequence
"HIQRW". In the example, multiple input amino acids are provided for certain
positions
along the first antigenic region to permit variability at those positions
across the specific
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polypeptides in the composition, while only the base peptide residue is
provided for each
position of the second antigenic region. Distribution at each position will
depend on the
relative molar input percentage (or relative molar input ratio) of each
possible amino acid at
that position of the AR.
Each output polypeptide comprises a specific amino acid sequence within each
of the
RCRs and ARs, and thus, will have a specific amino acid sequence influenced by
the linear
template arrangement, input amino acids, input and output percentages. The
RCRs of
individual polypeptides of the composition are depicted as: RCRai, RCRa2,
RCRa3 (e.g., the
first RCR (RCRa) of polypeptides 1, 2 and 3, respectively, of the composition)
and RCRbi,
RCRb2, RCRb3 (e.g., the second RCR (RCRb) of polypeptides 1, 2 and 3,
respectively, of the
mixture). The ARs of individual polypeptides of the composition are depicted
as: ARai,
ARa2, ARa3 (e.g., the first AR (ARa) of polypeptides 1, 2 and 3, respectively,
of the
composition) and ARbi, ARb2, ARb3 (e.g., the first AR (ARb) of polypeptides 1,
2 and 3,
respectively, of the composition). In the example, the amino acids provided in
the
composition for position 1 of ARa include (i) S, the original amino acid at
the corresponding
position of the base peptide, (ii) A, and (iii) T, a conserved substitution
for S. For positions 2
and 5 of ARa, only T or only A, the original amino acids at the corresponding
positions of the
base peptide are provided. For position 3 of ARa, L, the original amino acid
at the
corresponding position of the base peptide, and M, a conserved substitution
for L, are
provided. For position 4 of ARa, Y, the original amino acid at the
corresponding position of
the base peptide, and A are provided. Thus, the polypeptides of the
composition may differ
from each other in their first antigenic sequence (e.g., the first antigenic
region of polypeptide
1 (ARai) has the sequence ATLAA, the first antigenic region of polypeptide 2
(ARa2) has the
sequence TTMYA, the first antigenic region of polypeptide 3 (ARa) has the
sequence
STMYA. Since only the base peptide residue is provided for each position of
the second
antigenic region, the second antigenic region remains constant amongst the
polypeptides of
the composition (e.g., the second antigenic regions of polypeptides 1-3,
designated by ARbi,
ARb2, and ARb3, respectively, all have the sequence HIQRW). The distribution
of amino
acids at each position of the antigenic regions depends on the relative molar
input percentages
of amino acids provided for that position and on the molar output percentages
for that
position (not shown on Figure 1). The schematic in Fig. 1 is for illustrative
purposes only
and is not to be construed as limiting the full scope of the disclosure.
In some embodiments, the relative molar input percentage of A is between about
5%
and 65%, between about 10% and 65%, between about 15% and 65%, between about
10%
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and 35%, or between about 15% and 35% of the total input amino acid
composition of the
polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments,
the relative
molar input percentage of alanine is between about 15-65% of the total input
amino acid
composition of the polypeptide mixture, i.e., across all the RCRs and ARs. In
some
.. embodiments, the relative molar input percentage of alanine is between
about 20-35% of the
total input amino acid composition of the polypeptide mixture, i.e., across
all the RCRs and
ARs. In some embodiments, the relative molar input percentage of alanine is
between about
20-30% of the total input amino acid composition of the polypeptide mixture,
i.e., across all
the RCRs and ARs. In some embodiments, the relative molar input percentage of
alanine is
between about 10-25% of the total input amino acid composition of the
polypeptide mixture,
i.e., across all the RCRs and ARs. In some embodiments, the relative molar
input percentage
of alanine is between about 15-30% of the total input amino acid composition
of the
polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments,
the relative
molar input percentage of alanine is less than 50% and greater than 10%, less
than 40% and
greater than 10%, or less than 30% and greater than 15% of the total input
amino acid
composition of the polypeptide mixture, i.e., across all the RCRs and ARs. In
some
embodiments, the relative molar input percentage of alanine is 10-25% or 15-
25% of the
total input amino acid composition of the polypeptide mixture, i.e., across
all the RCRs and
ARs.
In some embodiments, the relative molar output percentage of A is between
about 5%
and 75%, between about 10% and 75%, between about 15% and 75%, between about
10%
and 35%, or between about 15% and 35% of the total output amino acid
composition of the
polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments,
the relative
molar output percentage of alanine is between about 15-75% of the total output
amino acid
.. composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
In some
embodiments, the relative molar output percentage of alanine is between about
20-35% of the
total output amino acid composition of the polypeptide mixture, i.e., across
all the RCRs and
ARs. In some embodiments, the relative molar output percentage of alanine is
between about
20-30% of the total output amino acid composition of the polypeptide mixture,
i.e., across all
the RCRs and ARs. In some embodiments, the relative molar output percentage of
alanine is
between about 10-25% of the total output amino acid composition of the
polypeptide mixture,
i.e., across all the RCRs and ARs. In some embodiments, the relative molar
output
percentage of alanine is between about 15-30% of the total output amino acid
composition of
the polypeptide mixture, i.e., across all the RCRs and ARs.
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In some embodiments, the relative molar output percentage of lysine (K) is
between
about 100 and 40%, between about 300 and 40%, between about 5% and 40%,
between about
5% and 20%, between about 5% and 15%, between about 5% and 10%, between about
'7 A
and 150o, between about 6% and 8% or between about 150o and 350 of the total
output
amino acid composition of the polypeptide mixture, i.e., across all the RCRs
and ARs, and
inclusive of the end points of these ranges. In some embodiments, the relative
molar output
percentage of alanine is about 1-40% of the total output amino acid
composition of the
polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments,
the relative
molar output percentage of alanine is about 10%-40%, or about 10%-35%, or
about 12%-
30%, or about 7-15% of the total output amino acid composition of the
polypeptide mixture,
i.e., across all the RCRs and ARs. In some embodiments, the relative molar
output
percentage of alanine is between about 5-40% of the total output amino acid
composition of
the polypeptide mixture, i.e., across all the RCRs and ARs. In certain
embodiments, any of
the foregoing molar output percentages of lysine may referred to the molar
output percentage
of any other single positively charged amino acid, such as arginine,
histidine, or a non-
naturally occurring positively charged amino acid, or may refer to the
percentage of all
positively charged amino acid residues present (e.g., R+K+H).
In some embodiments, the relative molar output ratio of the percentage of A to
the
percentage of K, on a molar basis is greater than or equal to 1.5 and less
than or equal to 5.0,
between about 1 and 5, between about 1 and 4, between about 1.5 and 5.5,
between about 1.5
and 6, or between about 1.5 and 3. In some embodiments, the relative output
ratio of the
percentage of A to the percentage of K, on a molar basis is between about 1
and 4.5. In some
embodiments, the relative output ratio of the percentage of A to the
percentage of K, on a
molar basis is between about 1.5 and 3.5. In some embodiments, the relative
output ratio of
the percentage of A to the percentage of K, on a molar basis is between about
1 and 7. In
some embodiments, the relative output ratio of the percentage of A to the
percentage of K, on
a molar basis is between about 1 and 6. In some embodiments, the relative
output ratio of the
percentage of A to the percentage of K, on a molar basis is between about 1.5
and 6.5. Note
that the foregoing relative molar output ratio is expressed as a quotient. An
output ratio of
percentage of A to the percentage of K expressed as a quotient of 1.5 can
interchangeably be
expressed as 1.5:1.
Similarly, any of the foregoing relative molar output ratio of the percentage
of A to
the percentage of K, may have the K be replaced with other positively charged
amino acids
(i.e. arginine (R) or histidine (H)). The relative ratio can also be expressed
as a quotient. For
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example, a relative ratio of of A:K of 4:1 is equivalent to a quotient of 4.
In certain
embodiments, any of the foregoing output ratios may refer to the molar output
percentage of
any other single positively charged amino acid, such as arginine, histidine,
or a non-naturally
occurring positively charged amino acid, or may refer to the percentage of all
positively
charged amino acid residues present (e.g., R+K+H).
The input ratios of amino acids may be adjusted in view of the ratio of
alanine to
positively charged amino acids across the entire composition (RCRs and ARs).
In some
embodiments, the molar input ratio of A:(K+R+H) in the total amino acid
concentration of
the composition is about 1.25:1 to 2.5:1. In some embodiments, the molar input
ratio of
A:(K+R+H) in the total amino acid concentration of the composition is about
1:1,
1.25:1,1.35:1, 1.5:1, 1.75:1, 1.8:1, 2:1, 2.25:1, 2.3:1, 2.4:1, or 2.5:1. In
some embodiments,
the molar input ratio of A:(K+R+H) in the total amino acid concentration of
the composition
is about 1:1 to 1.25:1, 1.25:1 to 1.5:1, 1.5:1 to 1.75:1, 1.75:1 to 2:1, 2:1
to 2.25:1, or 2.25:1 to
2.5:1. The relative ratio can also be expressed as a quotient. For example, a
relative ratio of
A:(K+R+H) of 2.5:1 is equivalent to a quotient of 2.5, and this quotient may
also be referred
to as the relative ratio.
In some embodiments, the polypeptides comprise two or more RCRs and two or
more
ARs. The two or more RCRs may be of the same or differing lengths and/or amino
acid
distributions (e.g., content and/or input percentages (e.g. molar input
percentages)). In some
embodiments, the combined length of the two or more RCRs is less than 20 amino
acids (e.g.,
the combined length of RCRa + RCRb is less than 20 amino acid residues). The
two or more
ARs may be based on the same base peptide or different base peptides. In some
embodiments, the different base peptides are from the same antigen. In some
embodiments,
the different base peptides are from different antigens.
The one or more random copolymer regions and the one or more antigenic regions
can be arranged in any order, with respect to each other, in the linear
template arrangement.
The regions are associated (e.g., directly or indirectly interconnected), thus
giving rise to
contiguous polypeptides. In some embodiments, where two or more random
copolymer
regions are present, at least one antigenic region is interposed between them.
In some
embodiments, where two or more random copolymer regions are present, no
antigenic region
is interposed between them. In some embodiments, the linear template
arrangement is an
alternating string of random copolymer regions and antigenic regions, wherein
there are two
or more random copolymer regions and two or more antigenic regions. In some
embodiments, the linear template arrangement comprises RCRa-ARa-RCRb-ARb or
ARa-
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RCRa-ARb-RCRb. In some embodiments, the linear template arrangement comprises
RCRa-
ARa-RCRb-ARb-RCItc-ARc or ARa-RCRa-ARb-RCRb-ARc-RCRc or ARa-RCRa-ARb-RCRb-
ARc In some embodiments, the linear template arrangement comprises RCRa-ARa-
RCRb or
ARa-RCRa-ARb.
In some embodiments, the length of each of the polypeptides in the
composition, i.e.,
the length across all of the RCRs and ARs for a single polypeptide, is about
40-80 residues.
In some embodiments, length of each of the polypeptides in the composition,
i.e., the length
across all of the RCRs and ARs, is about 40-100 or 50-100 residues. In some
embodiments,
length of each of the polypeptides in the composition, i.e., the length across
all of the RCRs
and ARs, is about 30-80, 30-60, 40-60, 40-75, 45-65, or 45-60 residues. In
some
embodiments, the length of each polypeptide in the composition, i.e., the
length across each
polypeptide, varies between 1-100 residues. In some embodiments, the length of
each
polypeptide in the composition, i.e., the length across each polypeptide,
varies between 40-80
residues. In some embodiments, the length of each polypeptide in the
composition, i.e., the
length across each polypeptide, varies between 40-65 residues.
The complexity of a composition of the disclosure is greater than 5 x 102
different
polypeptides (e.g., polypeptides that differ in sequence at at least one
position but that are
related based on linear template arrangement and the characteristics of the
RCRs and ARs).
In certain embodiments, the complexity of the composition is greater than 1 x
104 different
polypeptides. In certain embodiments, the complexity of the composition is
greater than 1 x
106 different polypeptides. In some embodiments, the complexity of a
composition of the
disclosure is greater than 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x
108, 1 x 109, or 1 x
101 .
Designing an amino acid copolymer composition of the disclosure comprises
selecting a target (e.g., an antigen), and selecting one or more base peptide
sequences from
that target for use as the ARs. A linear template arrangement of the one or
more ARs and one
or more RCRs is also selected. The length, sequence composition and input
percentage (e.g.
molar input percentage) for the RCRs are also selected.
Diversity in the high-complexity polypeptide mixtures of the compositions
arises
from defined substitution rules for one or more positions of the antigenic
regions and from
the variation and input percentage (e.g. molar input percentage) amongst the
random
copolymer regions of the polypeptides. The composition is synthesized by
applying a set of
synthesis rules that define the amino acid variations along the length of the
linear template
arrangement and the input ratio of occurrence of introduction of such amino
acid residues at
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any given position. Thus, the compositions of the disclosure are not
synthesized as single
polypeptides, but are synthesized as mixtures of multiple related
polypeptides, related to one
another and based on the linear template arrangement, the overall composition
of which is
reproducible and consistent with the rules of synthesis that were applied.
The overall composition of amino acids that make up the antigenic regions may
be
modified via the introduction of different, related amino acids at one or more
positions, such
introduction made in accordance with a defined set of rules. In some
embodiments, the
antigenic regions are not modified and the diversity comes from the random
copolymer
regions alone. The result is a mixture of related polypeptides useful in and
of itself as a
therapeutic, and which is useful to induce production of antibodies that react
with specificity
with the known sequence, but are not easily elicited by a simple immunization
using the
known sequence.
For the solid phase synthesis procedure of the instant disclosure, the mixture
of amino
acids for a given position in the polypeptide is defined by a ratio one to
another. Prior to
starting the synthesis, such ratio is determined for each position along the
linear template
arrangement. A limitation of solid phase peptide synthesis is that large
polypeptides may be
more difficult to synthesize due to incomplete coupling reactions. As such,
each resulting
complex polypeptide mixture may contain some percentage of polypeptides that
are less than
full-length (e.g., polypeptides which comprise, for example, only about 75% or
85% of the
full-length polypeptide based on the original template arrangement). Because
the solid phase
peptide synthesis proceeds from C-terminus to N-terminus, these shorter
polypeptides in a
composition are generally truncated from the N-terminus (e.g., missing a more
N-terminal
portion). That said, such truncated polypeptides, when present, can be
minimized or
eliminated by either post synthesis purification steps or by increasing
synthesis times or other
methods described briefly herein. Accordingly, compositions of the disclosure
comprising
very high percentages of full length polypeptides (based on the template
arrangement) are
provided, such as compositions in which greater than 75%, greater than 80%,
greater than
85%, greater than 90%, or even 95% or greater than 95% of the polypeptides in
the complex
mixture are full length. Furthermore, synthesis of polypeptides by solid phase
peptide
synthesis may result in a preferential incorporation of alanine (A) over
lysine (K) in the
polypeptide. Thus, the resulting polypeptide mixture comprises a multiplicity
of related
polypeptide sequences.
In some embodiments, the polypeptide compositions of the present disclosure
have
a net positive charge. In some embodiments, the polypeptide compositions of
the present
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disclosure have a net charge that is greater than or equal to 2 at pH7. In
some embodiments,
the polypeptide compositions of the present disclosure have a net charge that
is between 2
and 4 at pH7. In some embodiments, the polypeptide compositions of the present
disclosure
have a net charge that is greater than 2.1 at pH7. In some embodiments, the
polypeptide
compositions of the present disclosure have an increased estimated net charge
versus a
similar composition based on ARs of the same complexity but in the absent of
the RCRs.
Positively charged proteins may have such attributes as being more soluble and
more
immunogenic. The net charge of the protein affects protein solubility, and in
turn, the
immunogenicity of the polypeptide composition. At pH7, proteins, which carry a
positive
.. charge, have an improved solubility profile. Numerous factors in the base
peptide sequence
may influence the net charge of a polypeptide composition at pH7. These
factors in the base
peptide sequence may include the length and distribution along the copolymers
of the RCR
motifs, the alanine content, the lysine content, and the arginine content.
Additionally, the
output ratio of alanine, lysine, and arginine may influence the net charge of
the polypeptide
composition at pH7. However, for polypeptides in which solubility is not an
issue, net charge
at pH7 may be negative and glutamic acid (Glu) residues might provide alpha-
helix
configurations and the use of such compositions is also provided for, as
described herein.
In certain embodiments, the compositions described herein have sustained
immunogenicity. In certain embodiments, the compositions described herein also
act as
intrinsic T-cell adjuvants and exhibit HLA promiscuity. In certain
embodiments, the
compositions promote the release of Th2 chemokines and also promoted CD4+ T
cell
proliferation. In some embodiments, the compositions promote the release of a
Th2-
associated cytokine or chemokine from monocytes, wherein the Th2-associated
cytokine or
chemokine is selected from at least one of: IL-4, IL-5, IL-6, IL-10, IL-13,
CCL17, and
CCL22. In some embodiments, the compositions promote the release of CCL22. In
some
embodiments, the compositions promote the release of CCL17.
Various additional embodiments of the RCRs and ARs are described below. Any of
the embodiments of RCR, linear template arrangement and AR described herein
may be
combined.
a) Random Copolymer Regions (RCRs)
The compositions of the disclosure comprise mixtures (e.g., greater than 500)
of
polypeptides with one or more random copolymer regions (e.g., amino acid
copolymer
compositions of the disclosure). As described above, a "random copolymer
region" or
"RCR" refers to short random repeats (e.g. anywhere between 3-15 amino acids
long, such as
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3-14 amino acids long or 3-7 or 3-6 or 3-4 amino acids long) generated from
mixtures of
alanine, at least one positively charged amino acid (e.g. lysine, arginine, or
histidine), and
optionally, at least one negatively charged amino acid (e.g. aspartic acid or
glutamic acid). In
some embodiments, the RCR may optionally further comprise phenylalanine. In
some
embodiments, the RCR may optionally further comprise one or more amino acids
that
promote an alpha-helical secondary structure in the repeats (e.g. methionine
or leucine). The
contribution of the various residues to polypeptides in the composition is
determined based
on, for example, the molar input percentage of each amino acid residues at
each position of
the RCR.
Each polypeptide in a composition of the disclosure has one or more random
copolymer regions corresponding to the random copolymer regions of the linear
template
arrangement (each an RCRxy, wherein x corresponds to the position of the RCR
in the linear
template arrangement and wherein y identifies the individual polypeptide, e.g.
a composition
with 500 different polypeptides could have a first RCR that is identified as
RCRal, RCRa2,
RCRa3...RCRa500 in each polypeptide of the mixture). The distribution of the
amino acids
at a given position of the one or more random copolymer regions among the
polypeptides is
determined by a pre-determined molar input ratio of the amino acids available
for that
position.
In some embodiments, an RCR (such as each RCR) is 3-15 or 3-14 amino acids
long.
In some embodiments, the RCRs are 5-15 amino acids long. In some embodiments,
the
RCRs are 3-12 or 3-10 amino acids long. In some embodiments, the RCRs are 3-8
or 3-9
amino acids long. In some embodiments, the RCRs are 3-7, 7-9, 9-11, 11-13, or
13-15 amino
acids long. In some embodiments, the RCRs are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, or 15
amino acids long. In certain embodiments, where the linear template
arrangement comprises
.. more than one RCR, the length of each is independently selected and may be
the same or
different. In certain embodiments, the total length of the RCRs (e.g., the
length of template
RCRa and template RCRb) is less than 15 amino acids in length. By way of
example, if the
linear template arrangement comprises two RCRs, one of which is 8 amino acids
long and
one of which is 9 amino acids long, the total length of the RCRs across the
linear template
arrangement is less than 20 amino acids. As a result, the length of the RCRs
in each
individual polypeptide of the composition is less than 15 amino acids.
In some embodiments, the linear template arrangement is based on one RCR, and
thus, polypeptides in the composition comprise one RCR. In some embodiments,
the linear
template arrangement is based on two or more RCRs, and thus polypeptides in
the
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composition comprise two or more random copolymer regions. In some
embodiments, the
polypeptides comprise three or more random copolymer regions. In some
embodiments, the
polypeptides comprise four, or five random copolymer regions. In some
embodiments, the
two or more RCRs each have the same length and amino acid distribution (e.g.,
amino acids
and molar input percentage). In some embodiments, the random copolymers
regions have the
same or differing length and/or amino acid distribution. In some embodiments,
at least one
antigenic region is interposed between two of the RCRs. In some embodiments,
no antigenic
region is interposed between the RCRs. In some embodiments, the template
arrangement
comprises two RCRs (or optionally three RCRs) and the RCRs are 8 or 9 (or more
than 9)
residues in length, as illustrated in the examples. In other embodiments, the
2 RCRs are 5, 6,
7, 8 or 9 residues in length (as illustrated in the examples). In some
embodiments, the
template arrangement comprises three RCRs (or optionally more) and the RCRs
are 3 or 4 or
5 or 6 residues in length, as illustrated in the examples.
In some embodiments, the amino acids available for each position of an RCR
include
(i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii)
optionally, at least one
of aspartic acid (D), glutamic acid (E), or phenylalanine (F). Thus, at a
particular amino acid
position, the polypeptides of the compositions will have a mixture of (i) A
and (ii) at least one
of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of
aspartic acid (D),
glutamic acid (E), or phenylalanine (F). Moreover, across the length of a
random copolymer
region of a given polypeptide, one can expect a random polypeptide composed of
(i) A and
(ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally,
at least one of aspartic
acid (D), glutamic acid (E), or phenylalanine (F). Note that in certain
embodiments, a
positively charged residue in an RCR may be a non-naturally occurring,
positively charged
residue.
It is preferable that one or more random copolymer regions of the composition
are
cationic in nature and/or have an amphipathic alpha-helical structure. Thus,
in some
embodiments, the relative molar input percentage of A for each position of at
least one
random copolymer region is less than or equal to 65% (but greater than or
equal to 30%), the
relative molar input percentage of positively charged amino acids for each
position is at least
35% (but less than or equal to 70%) and the relative molar input percentage of
negatively
charged amino acids for each position is less than or equal to 20%. In some
embodiments,
the relative molar input percentage of alanine for each position of a random
copolymer region
is 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, or 60-65%. In some embodiments, the
relative molar input percentage of alanine for each position of a random
copolymer region is
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35-45%, 45-55%, or 55-65%. In some embodiments, the relative molar input
percentage of
positively charged amino acids (K, R or H) for each position is 30-35%, 40-
45%, 45-50%,
50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75-80%. In some embodiments, the
relative
molar input percentage of positively charged amino acids (K, R or H) for each
position is 35-
75%. In some embodiments, the relative molar input percentage of positively
charged amino
acids (K, R or H) for each position is 30-80% or 40-70% or 50-70%. In some
embodiments,
only K is provided as the positively charged amino acid. In some embodiments,
only R is
provided as the positively charged amino acid. In some embodiments, a
combination of K
and R, K and H, or R and H are provided as the positively charged amino acids.
In some
embodiments, the relative molar input percentage of negatively charged amino
acids for each
position is 0-5%, 5-10%, 10-15%, or 15-20%. In some embodiments, the relative
molar input
percentage of negatively charged amino acids for each position is 0-10%, 5-
15%, 10-20%, or
5-20%. In some embodiments, only E is provided as the negatively charged amino
acid. In
some embodiments, only D is provided as the negatively charged amino acid. In
some
embodiments, a combination of D and E is provided as the negatively charged
amino acids.
The input percentages of the various amino acids for each position add up to a
100%. It will
be understood that where the input percentages of A, K/R/H, and optionally DIE
specified in
a composition do not add up to a 100%, the remaining percentage is made up of
one or more
optional amino acids such as F, M, or L or any other amino acid that assists
in the
maintenance of an overall amphipathic alpha-helical configuration. In some
embodiments,
the relative molar input percentage of F at one or more positions of a random
copolymer
region is 1-3%, 3-5%, 1-5%, 5-7%, or 7-10%. In some embodiments, such as
embodiments
of any of the foregoing, the relative molar input percentages of amino acids
are the same for
each position of a random copolymer region. In some embodiments, the relative
molar input
percentages of amino acids may be different for each position or for one or
more positions of
a random copolymer region. In some embodiments, the relative molar input
percentages of
amino acids are the same for each position across a given RCR, but may vary
between two
RCRs. In some embodiments, the composition comprises more than two RCRs, and
each
RCR may have the same or differing length and/or amino acid distribution.
In some embodiments, one or more random copolymer regions are composed of A
and K. In other words, the amino acids available at each position of the RCR
are A and K,
each provided at a given input percentage. In some embodiments, the random
copolymer
regions are composed of A, K, and E. In some embodiments, the random copolymer
regions
are composed of (i) A, K, E and F; (ii) A, K and D; (iii) A, K, D and F; (iv)
A and R; (v) A,
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R, and E; (v) A, R, E and F; (vi) A, R and D; and (vii) A, R, D and F. In some
embodiments,
the composition, such as the substantially full-length polypeptide, comprises
more than two
RCRs, and each RCR may have the same amino acid composition and/or amino acid
distribution (e.g., the linear template arrangement comprises more than two
RCRs). In some
.. embodiments, the composition, such as the substantially full-length
polypeptide, comprises
more than two RCRs, and each RCR may have the same or differing length and/or
amino
acid composition. In some embodiments, the composition, such as the
substantially full-
length polypeptide, comprises three RCRs (e.g., the linear template
arrangement comprises
three RCRs.)
In some embodiments, the relative molar input percentages of amino acids for
each
position of a random copolymer region are 45% to 55% A, 35% to 50% K or R, and
5% to
20% E or D. In some embodiments, relative molar input percentages of amino
acids for each
position of a random copolymer region are 45-50% A, 40-45% K or R, and 5-15% E
or D. In
some embodiments, relative molar input percentages of amino acids for each
position of a
.. random copolymer region are 45-50% A, 40-45% K or R, and 10% E or D. In
some
embodiments, the relative molar input percentages of amino acids for each
position of a
random copolymer region are 50% A, 40% K, and 10% E. In some embodiments, the
relative
molar input percentages of amino acids for each position of a random copolymer
region are
30-80% K, 15-55% A, and 5-15% E. In some embodiments, the relative molar input
percentages of amino acids for each position of a random copolymer region are
35-75% K,
15-50% A, and 5-15% E. In some embodiments, the relative molar input
percentages of
amino acids for each position of a random copolymer region are 35-75% K, 15-
55% A, and
5-15% E. In some embodiments, such as embodiments of any of the foregoing, the
relative
molar input percentage of amino acids for each position of a random copolymer
region are
.. 35%-75% K (e.g., 45%, 50%, 55%, 60%, 65%, 70%), 15-50% A (e.g., 20%, 25%,
30%, 35%,
40%), and 0-15% E (e.g., 0, 5%, 10%). The input percentages of the various
amino acids for
each position add up to a 100%. It will be understood that where the input
percentage of A,
K/R/H, and optionally D/E specified in a composition do not add up to 100%,
the remaining
percentage is made up of one or more optional amino acids such as F, M, or L
or any other
.. amino acid that assists in the maintenance of an overall amphipathic alpha-
helical
configuration. In some embodiments, the relative molar input percentages of
amino acids are
the same for each position of a random copolymer region. In some embodiments,
the relative
molar input percentages of amino acids are the different for each position of
a random
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copolymer region. In some embodiments, the composition comprises more than two
RCRs,
and each RCR may have the same or differing length and/or amino acid
distribution.
In some embodiments, the relative molar input percentages of amino acids for
each
position of a random copolymer region are 45-55% A, and 45-55% K, with the
total adding
up to 100%. In some embodiments, the relative molar input percentages of amino
acids for
each position of a random copolymer region are 40-65% A, and 35-60% K, with
the total
adding up to 100%. In other embodiments, the total of A and K adds up to less
than 100%,
and the RCR comprises relative molar input percent of D or E of 0-20% and/or
of F of 0-
10%. In certain embodiments of any of the foregoing, the RCR may comprise R
instead of
K.
In some embodiments, the relative molar input percentages of amino acids for
each
position of a random copolymer region are 45-55% A, 35-45% K, and 5-15% E,
with the
total adding up to 100%. In some embodiments, the relative molar input
percentages of amino
acids for each position of a random copolymer region are 40-65% A, 35-60% K,
and 5-10%
E, with the total adding up to 100%. In other embodiments, the total of A and
K and E adds
up to less than 100%, and the RCR comprises relative molar input percent of F
of 0-10%. In
certain embodiments of any of the foregoing, the RCR may comprise R instead of
K and/or D
instead of E. In some embodiments, the relative molar input percentage of A at
each position
of the RCR and/or across the RCR is less than or equal to 55%, such as less
than or equal to
50%. In some embodiments, the relative molar input percentage of K at each
position of the
RCR and/or across the RCR is greater than or equal to 35%, such as greater
than or equal to
40% or 50%.
In some embodiments, the molar input ratio of A:(K+R+H) in the total amino
acid
concentration of an RCR is about 0.8:1 to 2:1. In some embodiments, the molar
input ratio of
A:(K+R+H) in the total amino acid concentration of an RCR is about 0.75:1,
0.8:1, 0.9:1, 1:1,
1.25:1,1.35:1, 1.5:1, 1.75:1, or 1.8:1 or 2:1. In some embodiments, the molar
input ratio of
A:(K+R+H) in the total amino acid concentration of an RCR is about 0.75:1 to
1:1, 1.1:1 to
1.25:1, 1.25:1 to 1.5:1, 1.5:1 to 1.75:1, or 1.75:1 to 2:1. In some
embodiments, the molar
input ratio of A:K in the total amino acid concentration of an RCR is about
0.8:1 to 2:1, with
no R or H being present. In some embodiments, the molar input ratio of A:R in
the total
amino acid concentration of an RCR is about 0.8:1 to 2:1, with no K or H being
present. In
some embodiments, the molar input ratio of A:(K+R) in the total amino acid
concentration of
an RCR is about 0.75:1, 0.8:1, 0.9:1, 1:1, 1.25:1,1.35:1, 1.5:1, 1.75:1, or
1.8:1 or 2:1. In
some embodiments, the molar input ratio of A:(K+R) in the total amino acid
concentration of
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an RCR is about 0.75:1 to 1:1, 1.1:1 to 1.25:1, 1.25:1 to 1.5:1, 1.5:1 to
1.75:1, or 1.75:1 to
2:1. The ratio can also be expressed as a quotient. For example, a ratio of
A:(K+R+H) of 2:1
is equivalent to a ratio of 2, when expressed as a quotient.
In some embodiments, the molar output ratio of A:(K+R+H) in the total amino
acid
concentration of the composition is about 1.5:1 to 7:1. In some embodiments,
the molar
output ratio of A:(K+R+H) in the total amino acid concentration of the
composition is about
7:1, 6.5:1, 6:1, 5.5:1, 5:1, 4:1, 3:1, 2.5:1, 2:1, or 1.5:1. In some
embodiments, the molar
output ratio of A:(K+R+H) in the total amino acid concentration of the
composition is about
6.75:1, 6.25:1, 5.75:1, 5.25:1, 4.75:1, 4.5:1, 3.75:1, 2.25:1, 1.75:1, and
1.25:1. In some
.. embodiments, the molar output ratio of A:K in the total amino acid
concentration of the
composition is about 7:1, 6.5:1, 6:1, 5.5:1, 5:1, 4:1, 3:1, 2.5:1, 2:1, or
1.5:1. In some
embodiments, the molar output ratio of A:K in the total amino acid
concentration of the
composition is about 6.75:1, 6.25:1, 5.75:1, 5.25:1, 4.75:1, 4.5:1, 3.75:1,
2.25:1, 1.75:1, and
1.25:1. These ratios may interchangeably be expressed as a quotient. For
example, a ratio of
A:(K+R+H) of 1.5:1 is equivalent to a ratio of 1.5, expressed as a quotient.
In specific embodiments, the compositions of the disclosure comprise the
polypeptide
sequences disclosed in SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO:
55,
SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or
SEQ
ID NO: 61. For example, the disclosure provides compositions comprising a
plurality of
polypeptides (such as at least or more than 500 different polypeptides)
comprising the amino
acid sequence set forth in any of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,
SEQ ID
NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:
60, or SEQ ID NO: 61.
b) Antigenic regions
The compositions of the disclosure comprise high complexity mixtures (e.g.,
greater
than 500, greater than 1 x 103, greater than 1 x 104, greater than 1 x 105,
greater than 1 x 106,
or greater than 1 x 108) of polypeptides with one or more antigenic regions
(ARs) in addition
to one or more random copolymer regions (RCRs). To create amino acid copolymer
compositions that can meaningfully serve as disease-specific therapeutics and
provoke a
specific antibody response against one or more protein targets associated with
a disease or
condition, one first needs to define the base peptide sequences of the
antigenic regions of the
composition. The base peptide sequences can be derived in many ways. A
polypeptide
sequence useful for this purpose is a polypeptide sequence that is associated
with a disease or
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condition of interest. The base peptide sequence can be derived from viral,
bacterial or
parasitic proteins known to be associated with a pathogenic infection of a
mammal.
Alternatively, the base peptide sequences can be derived from proteins known
to be
associated with protein conformational disorders. A polypeptide sequence
useful for this
.. purpose may be related to an immune response in a mammal. These polypeptide
sequences
are derived from, for example, partial sequences of any protein or antigen
associated with the
disease or condition of interest, or empirically derived polypeptide
sequences, such as
through screening of a library created by combinatory chemistry.
As described above, an "antigenic region" or "AR" (used interchangeably
herein) as
.. used herein refers to a sequence based on a sequence derived from an
antigen of interest (a
base peptide). Each polypeptide in the mixture will comprise one or more ARs
based on the
one or more template ARs (each an ARxy, wherein x corresponds to the position
of the AR in
the linear template arrangement and wherein y identifies the individual
polypeptide, e.g. a
composition with 500 different polypeptides will have a first AR that is
identified as ARal,
.. ARa2, ARa3...ARa500 in each polypeptide of the mixture). In general, an AR
is anywhere
between 8-60 amino acids long and could be based on any relevant portion of a
protein of
interest that is associated with a disease or condition. In some embodiments,
an AR is at least
8 amino acids, at least 10 amino acids, at least 12 amino acids, at least 15
amino acids, at
least 20 amino acids, at least 25 amino acids, or at least 30 amino acids (but
in each case less
.. than 80 amino acids long). In some embodiments, an AR is between 8-30, 8-
25, 10-25, 10-
30, 10-40, 10-50, 10-60, 10-80, 15-25, 15-30, 20-30, 20-40, 20-50, 20-60, or
20-80 amino
acids long. An AR may remain constant amongst the polypeptides of the
composition, i.e.
each polypeptide of the composition has the same amino acid sequence as the
base peptide in
that region or an AR may vary across the polypeptides of the composition, i.e.
the
polypeptides may differ from the base peptide and from one another at one or
more amino
acid positions. The distribution of the amino acids at a given position among
the
polypeptides is determined by a pre-determined molar input ratio and molar
output ratio of
the amino acids available for that position (Fig. 1).
It has previously been shown that mixtures of related polypeptides may be
.. therapeutically more effective than a single polypeptide. Lustgarten et
at., I Immunol. 2006,
176: 1796-1805; Quandt et at., Molec. Immunol. 2003, 40: 1075-1087. The
effectiveness of a
polypeptide mixture as opposed to a single polypeptide is the likelihood of
interaction with
the relevant epitopes that are not yet fully defined, particularly in terms of
the
conformationally specific interactions. Further, in some cases, although
immunization with
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an immunogen having a single epitope may induce multiple antibodies having
complementarity determining regions (CDR) different from each other, it may be
difficult to
strongly elicit (and thus detect and identify) all varieties of antibodies. In
addition, even if
antibodies are induced, the most easily inducible and detectable antibodies
against such
epitope may not include those antibodies with a high specificity towards the
particular
pathological conformation as described in the preceding paragraphs. In an
attempt to
overcome these challenges, investigators have designed polypeptides with
sequences similar
to the target polypeptides. These variations of the target polypeptides may
induce generation
of antibodies that are different from those induced by the target
polypeptides, but may cross-
react sufficiently with the target polypeptides. Thus, these related
polypeptides may be
desirable and/or required to identify an antibody that may not be induced by
an epitope of the
original sequence.
Moreover, single polypeptides may not be very immunogenic and strong adjuvant
and/or carrier proteins are frequently used to increase the antibody response.
Single
.. polypeptides are also limited in terms of MEW binding and only bind to
specific HLA alleles.
The compositions of the instant disclosure overcome these hurdles.
In one aspect, one or more ARs of the composition are synthesized with one of
several amino acids at one or more amino acid positions. In one aspect, one or
more ARs are
not varied, i.e., the compositions are synthesized with just one amino acid at
each amino acid
position.
The amino acid to be incorporated into a particular position of a polypeptide
is
selected from one or more of: an original amino acid found at the
corresponding amino acid
position of the corresponding base peptide sequence, alanine (A), lysine (K),
arginine (R), or
an amino acid serving as a conserved substitution for the original amino acid,
and the
distribution of the amino acids at a given position among the polypeptides is
determined by a
pre-determined molar input ratio of the amino acids available for that
position. In some
embodiments, the amino acid sequence of at least one AR does not vary among
the
polypeptides in the composition, and the amino acid sequence of said AR
comprises a
sequence of amino acid positions corresponding to a first base peptide
sequence and the
polypeptides in the composition have an amino acid at each position of the AR
selected from
an original amino acid found at the corresponding amino acid position of the
first base
peptide sequence. In some embodiments, the amino acid sequence of at least one
AR varies
with respect to the first base peptide sequence among the polypeptides of the
composition,
various polypeptides having an amino acid at each position selected from one
or more of: an
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original amino acid found at the corresponding amino acid position of the base
peptide
sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a
conserved
substitution for the original amino acid. In some embodiments, the first amino
acid at the C-
terminal position is a Norleucine (Nle). In some embodiments, the first amino
acid at the N-
terminal position is Nle. In some embodiments, the first amino acid at the C-
terminal
position or the N-terminal position is a non-naturally occurring amino acid.
In some embodiments, the polypeptides comprise two or more ARs. In some
embodiments, the polypeptides comprise three or more ARs. In some embodiments,
the
polypeptides comprise four, five, six, seven, or eight ARs. In some
embodiments, the two or
more ARs are each derived from the same base peptide sequences. In some
embodiments,
the ARs are each derived from different base peptide sequences, with the
different base
peptide sequences being derived from the same or different antigens. In some
embodiments,
two or more of the ARs share a base peptide sequence that differs from the
base peptide
sequence(s) of the other antigenic regions. In some embodiments, the lengths
of the different
ARs are independently selected. In some embodiments, at least one RCR is
interposed
between two of the ARs. In some embodiments, two or more ARs based on the same
or
different base peptide sequences are arranged consecutively.
In some embodiments, one or more AR includes little or no complexity (e.g.,
the input
amino acids correspond to the base peptide sequence). In some embodiments, Ala
is present
at one or more positions of one or more ARs, but the input percentage of Ala
is kept
relatively low, such as less than or equal to 20%, less than or equal to 15%
or less than or
equal to 10% at each position (independently selected at each position), other
than
(optionally) positions where Ala is the native amino acid at that position
(where the input
percentage is independently selected at each position). This helps keep the
overall molar
input percentage of Ala across ARs and RCRs less than, for example in certain
embodiments,
50%, less than 45%, less than 40%, less than 35%, less than 30%, less than
25%, or less than
or equal to 20% (e.g., 10-25% or 15-25%, etc.).
A base peptide sequence that is derived from an antigen of interest can be
modified in
one or more ways with respect to the corresponding sequence of the original
antigen. One or
more amino acid residues present in the original antigen may be absent in a
base peptide
sequence. One or more amino acids of the base peptide can be modified in
accordance with
an inflammatory or oxidative environment, for example, with nitration,
nitrosylation, or
phosphorylation. One or more amino acids of the base peptide can be modified
by
acetylation. One or more additional amino residues, not present in the
corresponding
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sequence of the original antigen, e.g. one or more cysteine residues, may be
introduced into
the base peptide sequence. In some embodiments, the base peptide sequence
comprises a
Norleucine (Nle) residue. The base peptide sequence may comprise one or more
non-
naturally occurring amino acids or amino acid derivatives. In some
embodiments, the amino
acid derivative is pyroglutamate.
In some embodiments, specific disulfide bridges between cysteine residues of
individual polypeptides are generated. In some embodiments, the post-
translation formation
of the cysteine bridge is performed using the reduction of a disulfide bridge
between two
cysteine residues. In some embodiments, reducing due to the presence of
disulfide bond
reductases (e.g. thioredoxins and glutaredoxins) are generated.
Peptide sequences with some significance to a disease state or an adverse
reaction
may be identified through experimental investigation of a relevant epitope.
These sequences
may include non-naturally occurring peptide sequences that proved to be useful
in treating a
disease or a condition, an example found in the international patent
application publication
WO 2006/031727, US Pat. No. 6,930,168 and the related scientific publication
Stern et al.,
Proc. Nat. Acad. Sci. USA, 2005, 102:1620-25.
Further, epitopes or antigenic determinants are empirically determined by
identifying
candidate sequences by positional scanning of synthetic combinatorial peptide
libraries (see,
for example, D. Wilson et al., above; R. Houghten et al., above; Hernandez et
al., Eur
Immunol., 2004, 34:2331-41), or by making overlapping polypeptide sequences of
the entire
protein of interest, and testing those polypeptides for immune reactivity
(using, for example,
any readout assay useful for such purposes, described in Current Protocols in
Immunology
Edited by John E Coligan, Ada M Kruisbeek, David H Margulies, Ethan M Shevach,
Warren
Strober NIH, John Wiley & Sons) in an in vitro or in vivo assay system
appropriate for the
disease and species the epitope is sought for.
Each amino acid position is kept constant or subjected to change based on a
defined
set of rules. As described above, in one aspect, one or more ARs of the
composition are
synthesized with one of several amino acids at one or more amino acid
positions. The amino
acid to be incorporated to a particular position of a polypeptide is selected
from one or more
of: an original amino acid found at the corresponding amino acid position of
the
corresponding base peptide sequence, alanine (A), lysine (K), arginine (R), or
an amino acid
serving as a conserved substitution for the original amino acid, and the
distribution of the
amino acids at a given position among the polypeptides is determined by a pre-
determined
molar input ratio and molar output ratio of the amino acids available for that
position. In
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some embodiments, the amino acid to be incorporated into a particular position
of a
polypeptide is the original amino acid found at the corresponding amino acid
position of the
corresponding base peptide sequence. In some embodiments, the amino acid to be
incorporated into a particular position of a polypeptide is selected from the
original amino
acid found at the corresponding amino acid position of the corresponding base
peptide
sequence and alanine (A). In some embodiments, the amino acid to be
incorporated to a
particular position of a polypeptide is selected from the original amino acid
found at the
corresponding amino acid position, alanine (A), and lysine (K). In some
embodiments, the
amino acid to be incorporated to a particular position of a polypeptide is
selected from the
.. original amino acid found at the corresponding amino acid position, and
lysine (K). In some
embodiments, the amino acid to be incorporated to a particular position of a
polypeptide is
selected from the original amino acid found at the corresponding amino acid
position of the
corresponding base peptide sequence, alanine (A), and a conserved
substitution. In some
embodiments, the amino acid to be incorporated to a particular position of a
polypeptide is
selected from the original amino acid found at the corresponding amino acid
position of the
corresponding base peptide sequence, and a conserved substitution. In some
embodiments,
only the original amino acid found at the corresponding amino acid position of
the
corresponding base peptide sequence is provided for one or more, or all of the
amino acid
positions of a polypeptide. In certain embodiments, an amino acid to be
incorporated at a
particular position some proportion of the time is phosphorylated (e.g.,
either the original
amino acid found at the corresponding position is or can be phosphorylated, or
such
phosphorylated amino acid is deemed a conservative substitution). In certain
embodiments,
an amino acid to be incorporated at a particular position some proportion of
the time is
nitrated (e.g., either the original amino acid found at the corresponding
position is or can be
nitrated, or such nitrated amino acid is deemed a conservative substitution).
After identifying a candidate base peptide, a probable set of additional
related
polypeptides may be generated using modeling and prediction algorithms
described in readily
available references, for example WO 2000/042559, align and analyze the
predicted binding
of these probable epitopes using available prediction methods described in,
for example, WO
2005/103679, WO 2002/073193 and WO 99/45954. In some embodiments, amino acid
substitutions at one or more positions of the base peptide are determined as
follows: select
from the polypeptides having the highest predicted activity/binding, take 40%
of the
predicted sequences and acquire the percentage of any given amino acid at each
position.
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Use those percentages to create the rules for amino acid incorporation into
the synthesis
scheme.
In one aspect, incorporation of alanine and/or other substitutions into the
polypeptides
of the mixture may increase immunogenicity by creating artificial T- and B-
cell epitopes and
.. HLA/MEIC promiscuity.
In some embodiments, a "conserved substitution" in the context of the
antigenic
region may refer to a conservative substitution as understood by the skilled
artisan. For
example, an amino acid may be substituted with an alternative amino acid
having similar
properties, for example, another basic amino acid, another acidic amino acid,
another neutral
amino acid, another charged amino acid, another hydrophilic amino acid,
another
hydrophobic amino acid, another polar amino acid, another aromatic amino acid
or another
aliphatic amino acid. In some embodiments, a conserved substitution in the
context of an
antigenic region is selected according to the methods of rational comparison
and findings of
similarity described in Kosiol et al., I Theoretical Biol., 2004, 228:97-106.
Amino acids are
.. grouped together in a matrix, referred therein as PAM replacement matrix.
In some
embodiments, a conserved substitution, as used herein, is based on amino acid
similarity and
means the relationship of those amino acids grouped together according to
Kosiol, based on
the characteristics of the residues such as size, charge, hydrophobicity, etc.
A conserved
substitution can be selected in accordance with the methods set forth in US
Patent
Application Publication No. 2008/0146504 or can refer to the exemplary
substitutions
described in PCT/U52004/032598, page 10-11. A conserved substitution may also
be
selected in accordance with the differences in amino acid composition at a
given position
between species. A conserved substitution may be an orthologous substitution,
such as a
substitution present in an ortholog of the gene encoding the protein of
interest. Alternatively,
amino acids can be changed in accordance with the differences at a given
position between
individual examples within the same species. In some embodiments, a conserved
substitution
refers to a modified form of the amino acid. In some embodiments, a conserved
substitution
refers to a phosphorylated form of the amino acid. In some embodiments, a
conserved
substitution refers to a nitrated form of the amino acid. In some embodiments,
the amino
acids can be modified in accordance with an inflammatory or oxidative
environment, for
example, with nitration, or phosphorylation. Alternatively, the amino acids
can be modified
by acetylation. In some embodiments, a conserved substitution is an isomer of
the amino
acid. In some embodiments, the amino acids can be changed or modified in order
to
promote, delay, accelerate or inhibit amlyoidogenesis. In some embodiments, a
conserved
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substitution includes a small, polar, or charged amino acid. In some
embodiments, a
"conserved substitution" includes the absence of an amino acid at a given
position.
In some embodiments, a conserved substitution provided for a particular
position of
an antigenic region is are defined according either: (1) to a rational
comparison and finding of
similarities of relevant characteristics of the original amino acid found at
the corresponding
amino acid position of the base peptide sequence with those of the substitute
residue, (2) in
accordance with the differences at a given position between species, (3) in
accordance with
the differences at a given position within individuals of the same species, or
(4) to a
comparison of reported experimental results on the relative activities of
actual polypeptides
having slight variations from the base sequence, or (5) is a conservative
substitution. The
substitute residues defined in any of these approaches are termed "conserved
substitution"
herein.
A comparison of experimental results showing the relative activities of
polypeptides
having slight variations from the base sequence can also be used as a basis
for the rule for
substitution. The sequences of the polypeptides responsible for observed
changes are aligned
and the type and percent presence of the new amino acid are noted. If there is
more than one
amino acid substitution at any given position of the polypeptide, the
frequency of occurrence
of an amino acid and the magnitude of activity change compared to the original
sequence are
taken into account to determine the order of prevalent substitution. Examples
of the overall
process leading up to the rule generation for amino acid copolymer synthesis
can be found
using libraries (Molec. Immunol. 40:1047-1055; Molec. Immunol. 40:1063-74; J
Autoimmunity 20:199-201; and I Immunol 163:6424-34), by making altered
polypeptide
ligands of overlapping polypeptides representing the entire protein of
interest (Atkinson et at.,
Cl/n. Invest. 94:2125-29; Meini et at., I Cl/n. Invest. 92:2633-43) or de novo
(US Patents
7,058,515; 6,376,246; 6,368,861; 7,024,312; 6,376,246; 7,024,312; 6,961,664;
6,917,882).
Briefly, a cellular material of interest is chosen as the assay system to rank
the
immunoreactivity of the polypeptides to be interrogated. Such an assay system
can be either
an in vitro or in vivo system, and can comprise adaptive or innate immune
reactivity.
Readouts for the assay system can be the up- or down¨regulation of the status
of the
activation state of a protein, a change in the localization of a protein, the
expression of the
mRNA encoding for the protein, the relative concentration of a protein,
changes in the
generation of specific cell types, changes in cellular phenotype, changes in
cellular activation,
changes in cell number, changes in organ size or function, changes in animal
behavior or
phenotype. Once the assay or assays are performed the results are analyzed to
determine the
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prevalence of any particular amino acid as a conserved substitution. If more
than three
residues in a given position within the polypeptide sequence are identified as
generating a
change in immunologic function, the top three residues first by frequency of
representation in
the interrogated polypeptides, and second by the magnitude of changes
elicited. Once chosen,
the relative amounts of the residues are defined.
It will be understood by the skilled artisan that the base peptides can be
derived from
any protein or antigen of interest that is associated with a disease or
condition of interest (e.g.,
a target). The compositions of the disclosure are broadly applicable to
numerous diseases
and indications, for example, by selecting ARs corresponding to portions of a
target of
interest. In some embodiments, the antigen (e.g., target of interest) is
associated with a
protein conformational disorder. In some embodiments, the antigen is prion
protein, amyloid
beta precursor protein, ABri peptide, Tau protein, alpha-synuclein, alpha-
synuclein central
fragment, SOD1, TDP-43, repeat-associated non-ATG (RAN)-translated peptides of
the
C90RF72 locus, islet amyloid polypeptide (a.k.a. amylin), prothymosin alpha,
amino-
terminal domain of androgen receptor protein, ataxin-1, DRPLA protein (a.k.a.
atrophin-1),
calcitonin, cystatin c, transthyretin, beta 2 microglobulin, serum amyloid A
protein,
huntingtin, exon I of huntingtin, immunoglobulin light chain variable domains,
insulin,
lysozyme, alpha lactalbumin, monellin, ligand- and DNA-binding domains of
androgen
receptor protein, lactadherein, lactadherein fragment (a.a. residue 245-294,
a.k.a. medin),
gelsolin, apolipoprotein Al, fibrinogen, atrial natriuretic factor, or
fragments thereof. In some
embodiments, the antigen is Tau. In some embodiments, the antigen is alpha-
synuclein. In
some embodiments, the antigen is Abeta. In some embodiments, the antigen is
SOD1. In
some embodiments, the antigen is TDP-43. In some embodiments, the antigen is
RAN
peptides of the C90RF72 locus. In some embodiments, the antigen is associated
with a
pathogenic infection. In some embodiments, the antigen is a bacterial protein.
In some
embodiments, the antigen is a viral protein. In some embodiments, the antigen
is a protein
from a parasitic agent. In some embodiments, the antigen is a protein from
human
cytomegalovirus (HCMV). In some embodiments, the antigen is a protein from
human
immunodeficiency virus (HIV). In some embodiments, the antigen is a protein
from human
papillomavirus (HPV). The amino acid sequences of exemplary targets are
provided in the
sequence listing. Base peptide sequences for use in deriving an AR can be
selected from, for
example, these targets. Exemplary base peptide sequences suitable as ARs are
also provided
in the sequence listing.
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In some embodiments, the antigen is associated with one of the diseases or
conditions
described in this disclosure and the compositions of the disclosure are useful
in treating or
preventing one of the diseases or conditions described below. Other suitable
uses include, for
example, to study the disease or condition with which the antigen is
associated, to raise
antibodies reactive with a target (e.g., antigen), to identify other binders
of the target, to
induce CD4+ T-cell proliferation in vitro or in vivo, to induce a Th2 immune
response in
vitro or in vivo, and the like.
IV. Exemplary Diseases or Conditions Associated with Targets
a) Protein Conformational Disorders
It has been recognized in the recent years that there is a class of diseases
and disorders
that correlates with the presence of aggregates, whether intra- or extra-
cellular, of misfolded
or conformationally altered proteins. These proteins exist in a non-diseased
environment. In
a disease state, however, through certain alterations in the conformation,
they adopt a
secondary/tertiary structure different from those in the non-diseased state.
The amino acid
sequence is often unaltered. The misfolded proteins tend to self-associate,
aggregating in an
ordered fashion, form toxic precipitates, and deposit into tissues. The
aggregated protein
often takes a fibrillar appearance.
Examples of these disorders, known as "protein conformational disorders"
(PCDs),
include but are not limited to Alzheimer's disease (AD), Parkinson disease
(PD),
amyotrophic lateral sclerosis (ALS), frontotemporal dementia and parkinsonism
linked to
chromosome 17 (FTDP-17), progressive supranuclear palsy, dialysis-related
amyloidosis
(DRA), reactive amylosis, Type-2 diabetes, cystic fibrosis (CF), sickle cell
anemia,
Huntington's disease (HD), Creutzfeldt-Jakob disease (CJD) and related
disorders, and
systemic and cerebral hereditary amyloidosis. Examples of globular proteins
that undergo
fibrillogenesis include transthyretin, beta 2 microglobulin, serum amyloid A
protein, Ig light
chains, insulin, human lysozyme, alpha lactalbumin, and monellin. Examples of
natively
unfolded proteins that undergo fibrillogenesis include amyloid beta protein,
tau protein,
alpha-synuclein, amylin, and prothymosin alpha.
Investigators have correlated protein aggregate deposition with the
degeneration of
tissue. Although there remains controversy with regard to the "cause or
effect" of the
presence of aggregate and the manifestation of the disease pathology, evidence
is
accumulating that the pathology is caused by aggregates, perhaps by direct
toxicity due to the
aggregation or by a loss of biological function of the misfolded protein.
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The formation of aggregates is referred to as "fibrillogenesis." Before the
start of
fibrillogenesis, the protein relevant to PCD pathology is in a naturally
folded conformation
and in monomeric or defined oligomeric forms, each polypeptide comprising a
mixture of
alpha-helices, some beta-sheets, and random coils. By the end of
fibrillogenesis, the protein
is aggregated, and the polypeptide has adopted an altered conformation, i.e.
mostly a beta-
pleated sheet conformation. The conformational changes of the polypeptides and
aggregation
appear to coincide, but the cause and effect of conformational change and
aggregation, and
the sequence of events, remain to be elucidated.
When considering the pathogenesis of a PCD, it has been proposed that the
fibrillogenesis is a crystallization-like process: after a "seed" of oligomers
forms, an
aggregate grows over time through self-association. The protein may take an
altered
conformation because the aggregate exists and serves as a template, or it may
take the altered
conformation because of other factors, but once in that conformation, easily
participates in
fibrillogenesis. In contrast, another proposal hypothesizes that the
conformational alterations
alone may not cause or promote aggregation, and there is a factor that induces
the
aggregation. Such underlying factors that promote or induce structural changes
in the protein
include inflammatory or oxidative environments, nitration, phosphorylation,
pH, or metal ion
exposure (high concentrations of copper ions can induce the oligomerization of
J32
microglobulin monomers, which in turn leads to fibril formation (Eakin et at.,
Biochemistry
2004, 43, 7808-7815)).
Various treatment modes and possible therapeutic agents for PCDs are currently
being
investigated. Whether conformational change precedes the start of the
fibrillogenesis or vice
versa will influence the effectiveness of a treatment strategy. For example, a
treatment mode
with an assumption that fibrillogenesis is caused by the beta-sheet
conformation will attempt
to inhibit the beta-sheet formation. In contrast, if the assumption was that
aggregation
promotes further formation of proteins with a degenerative conformation, a
treatment mode
may aim to inhibit aggregation by various means. An illustration of the former
approach
includes an attempt to inhibit the formation of, or to break, beta-sheets,
using polypeptides.
Such polypeptides are designed from the sequences of areas of proteins most
likely involved
in the process of nucleation and aggregation, such as the hydrophic core of
amyloid-beta, a
polypeptide intimately involved in the pathology of Alzheimer's disease. An
illustration of
the latter approach is an attempt to manipulate protein conformation and
prohibit nucleation
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and subsequent formation of amyloids, or, "amyloidogenesis," by creating mini-
chaperone
polypeptides from outside of the beta-sheet regions.
Another promising approach, regardless of the mechanism of aggregate
formation, is
to focus on the aggregates themselves. There have been attempts to reduce the
level of
aggregated protein of interest by antibodies: given sufficient specificity and
ability to
promote clearance, an antibody has a potential to be an effective therapeutic.
To overcome
delivery challenges, attempts have been made to express such antibodies
intracellularly from
a delivered gene. However, despite its potential, currently, the existing
antibody therapeutics,
if any, do not sufficiently prevent, improve, or even slow progression of the
pathology, and
there remain largely unmet needs for an effective treatment for a PCD.
In PCD, for therapeutic, prophylactic, and diagnostic purposes, the antibodies
that are
desirable recognize and specifically bind to proteins of certain altered
conformation. The
difficulty lies in the fact that these proteins exist as normal parts of the
patient's system, were
it not for the altered conformation that they are in. Thus, even though these
proteins are
pathological, they may not elicit strong natural immune responses in the
afflicted individuals,
and it may be difficult to elicit an immune response (thus to raise
antibodies) using the native
sequence of the target protein in other subjects of the same species, or in an
individual with
similar immunological profile, which is often desirable due to the lower
probability of
adverse immunological reaction.
Another challenge is that the antibody should differentiate between the same
protein
in a non-pathological conformation and in a pathological conformation. A
protein relevant to
a PCD may have the same primary structure, whether in a non-pathological
condition or in
pathological condition. Without the ability to distinguish, the antibody
intended for
therapeutic purposes may adversely affect the patient by eliminating or
interfering with the
normal, functioning protein. Thus, a high specificity towards the particular
conformation, or
series of alterations, is required. For example, abnormal hyperphosphorylation
of Tau is a
hallmark of AD and several other related neurodegenerative disorders, called
tauopathies.
Hyperphosphorylation of Tau causes conformational changes and leads to the
formation of
pathogenic species such as paired helical filaments (PHF) and neurofibrillary
tangles (NFT).
Effects of Tau hyperphosphorylation include dissociation of Tau from
microtubules, toxicity,
and synaptic dysfunction. Thus, a method of targeting variants of
hyperphosphorylated Tau
in a specific manner will lead to efficient treatment. The present disclosure
provides
compositions uniquely suitable to treating and studying PCDs.
Peptide sequences related to protein conformational diseases
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In some embodiments, one or more base peptide sequences that form the basis
for the
antigenic regions of the amino acid copolymer compositions of the disclosure
are derived
from an antigen relevant to the pathology of protein conformational disorders
affecting the
central and/or peripheral nervous system, selected from the group consisting
of: Alzheimer's
disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17
(FTDP-
17), Dutch hereditary cerebral hemorrhage with amyloidosis (a.k.a
cerebrovascular
amyloidosis), congophilic angiopathy; Pick's disease, progressive supranuclear
palsy; familial
British dementia; Parkinson's disease (PD), Lewy-body related diseases,
multiple system
atrophy, Hallervorden- Spatz disease; amyotrophic lateral sclerosis (ALS);
Huntington's
disease (HD); spinocerebellar ataxia; neuronal intranuclear inclusion disease;
hereditary
dentatorubral-pallidoluysian atrophy; prion-related diseases such as scrapie,
bovine
spongiform encephalopathy, chronic traumatic encephalopathy (CTE), variant
Creutzfeldt¨
Jakob disease, Gerstmann-Straussler-Scheinker syndrome, kuru, fatal familial
insomnia, and
related disorders; hereditary cystatin c amyloid angiopathy; dementia
pugilistica; and other
neurodegenerative diseases characterized by cerebral and nerve atrophy and
detection of
intracellular and/or extracellular fibrillar aggregates as the disorder
progresses.
In other embodiments, a base peptide sequence is derived from an antigen
relevant to
the pathology of protein conformational disorders affecting multiple organs or
organs other
than the central nervous system, selected from the group consisting of: spinal
and bulbar
muscular atrophy; hereditary systemic and cerebral amyloidosis, Finnish-type
familial
amyloidosis; senile systemic amyloidosis (a.k.a. senile cardiac amyloidosis),
familial amyloid
polyneuropathy; Type-2 diabetes, in particular pancreatic islet amyloidosis;
dialysis-related
amyloidosis (DRA); inflammation-associated reactive systemic amyloidosis
(a.k.a. AA
amyloidosis); aortic medial amyloidosis; medulary carcinoma of the thyroid;
hereditary renal
amyloidosis; light chain associated amyloidosis, light chain deposition
disease, light chain
cast nephropathy, light chain cardiomyopathy; atrial amyloidosis; injection-
localized
amyloidosis; cystic fibrosis (CF); sickle cell anemia, and other disorders
wherein
fibrillogenesis is observed in the affected organs or tissues.
Examples of natively unfolded proteins and polypeptides, and those suspected
to be
natively unfolded, that undergo fibrillogenesis, and therefore associated with
protein
conformational disorders and that may be use as the source sequences of the
base peptides for
the preparation of a composition of the disclosure, include: prion protein and
its fragments,
amyloid beta protein and its fragments, ABri peptide, tau protein, alpha-
synuclein and its
central fragment, islet amyloid polypeptide (a.k.a. amylin), exon I of
huntingtin, prothymosin
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alpha, amino-terminal domain of androgen receptor protein, ataxin-1, DRPLA
protein (a.k.a.
atrophin-1), and calcitonin.
Examples of globular proteins that undergo fibrillogenesis and therefore
associated
with protein conformational disorders and that may be use as the source
sequences of the
base peptides for the preparation of a composition of the disclosure, include:
cystatin c,
transthyretin, beta 2 microglobulin, serum amyloid A protein and its
fragments, huntingtin
and its fragments (including exon I of huntingtin), imunoglobulin light chain
variable
domains, insulin, lysozyme (in particular human lysozyme), alpha lactalbumin,
and monellin,
ligand- and DNA-binding domains of androgen receptor protein, lactadherein and
more
specifically its fragments (for example, a.a. residue 245-294, a.k.a. medin),
gelsolin,
apolipoprotein Al, fibrinogen and its fragments, and atrial natriuretic
factor. Fragments of all
the proteins in this paragraph may also be used as source sequences.
As a specific example, in Alzheimer's disease, pathology correlates strongly
with the
presence of a 4 kDa amyloid beta (AP) peptide that is enzymatically cleaved
from the
Amyloid precursor protein (APP) by 13-secretase and y-secretase. The majority
of AP
peptides are 40 amino acids long, and designated A(340, A(340, A(31-40, or,
having varied amino
terminal, A(3x-40. Studies have indicated that the fibrillar form of A131-40
stimulates the
microglia, which cell type is currently thought to play an important role in
the pathogenesis
of Alzheimer's disease. (Jekabsone, A. et al., I Neuroinflammation 3:24
(2006)). The
peptide sequence of A131-40 is shown as SEQ ID NO: 1 in Table I. On the other
hand, A(31-42 ,
which is a minor fraction of plaque-forming AP, is thought to contribute to
the initiation of
the formation of fibrillar AP. This "long form" of the peptide is described as
SEQ ID NO: 2
in Table 1. Therefore, a base peptide sequence of an antigenic region is
derived from such an
AP peptide, exemplified by SEQ ID NO: 2. The base peptide sequence may also be
derived
from a shorter peptide, i.e. A(3x-40, A(31-11, which has been reported in some
cases to have
clinical significance, A(314-23, or A(316-20. Tjernberg, L.O. et at., Biochem.
1 366:343-351
(2002). In some embodiments, a base peptide sequence of an antigenic region is
a portion of
SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the base peptide sequence
is
modified with respect to the above sequences to include one or more nitrated,
acetylated or
phosphorylated residues, one or more insertions or deletions, or non-natural
amino acids.
A further specific example is Parkinson's Disease (PD). PD is a degenerative
neurological disorder currently without a cure affecting 1-2% of the
individuals over 50 years
of age. The neuropathological hallmarks are characterized by progressive loss
of
neuromelanin containing dopaminergic neurons in the substantia nigra pars
compacta (SNpc)
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with the presence of eosinophillic, intracytoplamic, proteinaceous inclusions
termed Lewy
Bodies (LB). a¨Synuclein is the most abundant protein in Lewy Bodies, and
appears to be an
important mediator, perhaps even a causal factor, of toxicity in PD. Thus,
reduction of toxic
a¨Synuclein is thought to be beneficial to PD patients. The sequence of one
such mouse a-
Synuclein polypeptide, derived from the C-terminal region of the full-length
protein, is
shown as SEQ ID NO: 3 in Table 1. (Benner, E.J. et al., PLoS ONE 3(1): e1376
(2008)).
Further, elimination or sequestration of nitrated a¨Synuclein and fragments
thereof, appear to
have favorable effects on the patients suffering from PD. Therapeutically
effective
antibodies are said to be directed at the nitrated a¨Synuclein but not native.
Therefore, the
base peptide sequence of an antigenic region is derived from, for example, SEQ
ID NO: 3, or
a portion thereof In another embodiment of the instant disclosure, the base
peptide sequence
is derived from a fragment comprising amino acids 121-137 of human a¨Synuclein
(DNEAYEMPSEEGYQDYE)(SEQ ID NO: 4). In yet other embodiments, the a¨Synuclein
fragment (121-137) sequence is substituted at positions 121 and 122 in
different species, tri-
nitrated at each Y (tyrosine) position, and/or phosphorylated at S129. In
another embodiment,
the base peptide sequence is derived from a fragment most closely
corresponding to amino
acids 123-137 of human a¨Synuclein, the fragment of which is set forth in SEQ
ID NO: 36
(GSEAYEMPSEEGYQDYE; SEQ ID NO: 36). In another embodiment of the instant
disclose, the base peptide sequence is derived from a fragment comprising
amino acids 121-
137 of human a¨Synuclein, and this fragment is set forth in SEQ ID NO: 37
(DNEAYEMPSEEGYQDYE; SEQ ID NO: 37). In some embodiments, the base peptide
sequence is modified with respect to the above sequences to include one or
more nitrated,
acetylated or phosphorylated residues, one or more insertions or deletions, or
non-natural
amino acids.
As defined here, "a portion thereof' corresponds to a contiguous portion or
fragment
of the antigen of interest. In some embodiments, "a portion thereof' refers to
a portion of an
antigen that is at least 6 amino acid residues, at least 8, at least 9, at
least 10 amino acid
residues, at least 15 amino acids, at least 20 amino acids, at least 25 amino
acids, or at least
amino acids. In some embodiments, "a portion thereof' refers to a portion or
fragment of
30 an antigen between 8-20, 9-30, 10-30, 10-40, 10-60, 10-80, 20-30, 20-40,
20-50, 20-60, 20-
80, or 20-100 amino acids long. In certain embodiments, a portion of an
antigen or target of
interest is suitable for use as a base peptide sequence, and thus, forms the
basis of an AR.
Another specific example is the group of neurodegenerative diseases known as
tauopathies (e.g. Alzheimer's disease (AD), FTDP-17, progressive supranuclear
palsy (PSP),
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PSP-like tauopathy, etc.) that are characterized by pathological aggregation
of Tau proteins.
Hyperphosphorylation of Tau, particularly in the C-terminal region, leads to
the formation of
pathological PHFs and NFTs and it is desirable to design and manufacture
compositions that
target all significant variants of phosphorylated Tau protein found in early
oligomers, PHFs
and NFTs. An immune response capable of eliminating toxic species of Tau will
slow down
disease progression in patients suffering from a tauopathy. Phosphorylation at
AT8 and
PHF1 sites induces conformational changes and normally phosphorylation at the
AT8 and
PHF determinants occurs early in the disease process. PHF-Tau is made of
phosphorylated
Tau peptides interacting through MTBD R2 and R3 repeats. Normally, the N- and
C-
terminal regions of Tau inhibit aggregation. However, phosphorylation of the N-
and C-
terminal regions (at the AT8 and PHF1 sites) neutralizes the inhibitory
effect. The PHF-1
site includes S396 and S404 of Tau-F. Other known phosphorylation sites in the
same region
include Y394, S400, T403, S409, S412, S413, T414, S416, S422, T427, S433, or
S435. In
some embodiments, a base peptide of an antigenic region is derived from the
hyperphosphorylated region of Tau and spans the PHF1 site. A base peptide
sequence can be
derived from a partial sequence of residues 388-441 of Tau-F (SEQ ID NO: 6).
In some
embodiments a base peptide sequence is derived from residues 388-408 of Tau-F
(SEQ ID
NO: 7) or a portion thereof. In some embodiments a base peptide sequence is
derived from
residues 417-441 of Tau-F (SEQ ID NO: 8) or a portion thereof In some
embodiments, a
base peptide sequence is derived from residues 390-408 of Tau-F (SEQ ID NO: 9)
or a
portion thereof. In some embodiments a base peptide sequence is derived from
residues 418-
441 of Tau-F (SEQ ID NO: 10) or a portion thereof In some embodiments, a base
peptide
sequence is derived from residues 414-438 of Tau-F (SEQ ID NO: 31). In some
embodiments,
a base peptide sequence is derived from residues 414-429 of Tau-F (SEQ ID NO:
32). In
some embodiments, a base peptide sequence is derived from residues 424-438 of
Tau-F (SEQ
ID NO: 33). In some embodiments, one or more of the phosphorylation sites are
phosphorylated in all the polypeptides of a mixture. In some embodiments,
there is a mixture
of phosphorylated and non-phosphorylated residues at a given phosphorylation
site amongst
the polypeptides of the composition. In some embodiments, the antibodies
produced are
capable of decreasing intracellular Tau. In some embodiments, the antibodies
produced are
capable of decreasing extracellular Tau. In certain embodiments, the
disclosure provides a
method of decreasing the presence of intracellular Tau, such as Tau present in
the hindbrain,
cortex, or hippocampus. In certain embodiments, the disclosure provides a
method of
decreasing the presence of extracellular Tau, such as Tau present in the
hindbrain, cortex, or
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hippocampus. In certain embodiments, the disclosure provides a method of
decreasing the
presence of soluble Tau. In certain embodiments, the disclosure provides a
method of
decreasing the presence of insoluble Tau. In certain embodiments, the
disclosure provides a
method of decreasing the presence of aggregated Tau. In certain embodiments on
any of the
foregoing, the decrease in Tau is in the hindbrain or hippocampus. In certain
embodiments,
the compositions of the disclosure are capable of inducing specific antibodies
that inhibit
early phosphorylation and accumulation of toxic precursor tau oligomers. In
some
embodiments, the compositions of the disclosure target glial tau pathology in
astrocytes. In
some embodiments, the compositions of the disclosure target glial tau
pathology in microglia.
Exemplary amino acid copolymer compositions designed according to the methods
described herein (e.g. DP-0016.B and DP-0016.C) have been shown to induce a
specific Th2
immune response capable of eliminating toxic species of Tau. Examples of
tauopathies may
include, but are not limited to, AD, FTDP-17, PSP, PSP-like tauopathy,
corticobasal
degeneration (CBD), pseudobulbar palsy, traumatic brain injury, chronic
traumatic
encephalopathy (CTE), Pick's disease, Niemann-Pick disease type C,
postencephalitic
parkinsonism, dementia pugilistica, chronic traumatic encephalopathy, lytico-
Boding disease,
ganglioglioma, gangliocytoma, subacute sclerosing panencephalitis,
meningioangiomatosis,
lead encephalopathy, tuberous sclerosis, Hallervorden- Spatz disease, and
lipofuscinosis.
In another embodiment of the disclosure, a base peptide sequence of an
antigenic
region is derived from a protein sequence relevant to prion-diseases. SEQ ID
NO: 13 is
human prion protein sequence. A relevant base peptide may be derived from
partial
sequences of SEQ ID NO: 13. Various species' prion sequences are disclosed by
Harmeyer,
S. et al., J Gen Virol. 79(Pt 4):937-45 (1998), the entirety of which is
incorporated herein by
reference. The amino acid variations by species can be used to design the
substituting amino
acids. In some embodiments, the base peptide sequence is modified with respect
to the above
sequences to include one or more nitrated, acetylated or phosphorylated
residues, one or more
insertions or deletions, or non-natural amino acids.
In yet another embodiment of the disclosure, a base peptide sequence of an
antigenic
region is derived from superoxide dismutase I (SOD1). SOD1 mutation is known
to have
causal relationship with the pathology of some forms of familial ALS. It has
been reported
that the antisera raised against a mutant form of SOD1, human G93A SOD1
recombinant
protein, had protective effect on a mouse model of ALS carrying G37R mutant
SOD1 (line
29), which overexpress human SOD1 protein by 4-fold higher than endogenous
mouse SOD1.
Urushitani, M. et al., Proc. Nat. Acad. Sci. USA, 104(7): 2495-2500 (2007). An
example of
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SOD1 protein sequence is SEQ ID NO: 14. Therefore, in one embodiment, a base
peptide
sequence is derived from SEQ ID NO: 14 or a portion thereof
In another embodiment of the disclosure, a base peptide sequence of an
antigenic
region is derived from TAR DNA-binding protein 43 (TDP-43). TDP-43
proteinopathies,
consisting of several neurodegenerative diseases including frontotemporal
lobar dementia
(FTLD) and amyotrophic lateral sclerosis (ALS), are characterized by the
presence of
inclusion bodies made of hyperphosphorylated and poly-ubiquitinated full-
length and
truncated TDP-43. TDP-43 abnormalities also occur in a subset of Alzheimer's
disease
patients. Therefore, in one embodiment, a base peptide sequence of an
antigenic region is
derived from SEQ ID NO: 18 or a portion thereof.
In another embodiment of the disclosure, a base peptide sequence of an
antigenic
region is derived from dipeptide repeat proteins (DPR) translated from the
GGGGCC repeats
upstream of the chromosome 9 orf 72 (C9orf7 2) coding region. Recently, it has
been
discovered that expansion of a GGGGCC hexanucleotide repeat expansion located
upstream
of the C9orf72 coding region is the most common cause of both ALS and familial
frontotemporal lobar degeneration (FTLD). Pathological aggregating DPRs,
formed by
unconventional repeat-associated non-ATG (RAN) translation of the GGGGCC
repeats in all
three frames are the characteristic pathological findings in many patients
(Mori et al. Science
2013, 339:1335-1338; Zu et al. PNAS 2013, E4968-477). Most of the
characteristic
intracellular inclusions of misfolded proteins contain poly-(Gly-Ala)
dipeptide repeats.
Inclusions containing poly-(Gly-Pro) and poly-(Gly-Arg) dipeptide repeats are
also found,
but to a lesser extent. Therefore, in one embodiment, a base peptide sequence
of an antigenic
region is derived from RAN-translated peptides of the C9orf7 2 locus and
comprises a poly-
(Gly-Ala) dipeptide repeat sequence. In one embodiment, a base peptide
sequence of an
antigenic region comprises a poly-(Gly-Pro) dipeptide repeat sequence. In one
embodiment,
a base peptide sequence of an antigenic region comprises a poly-(Gly-Arg)
dipeptide repeat
sequence. In some embodiments, the base peptide sequences derived from the RAN-
translated dipeptide repeats of the C9orf72 locus are 10-20 amino acid
residues in length. In
some embodiments, the base peptide sequences derived from the RAN-translated
dipeptide
repeats of the C9orf72 locus are 10-15, 10-25, or 10-30 amino acid residues in
length.
Misfolded protein also plays a role in Huntington's disease, a genetic
disorder caused
by the pathological expansion of a polyglutamine (polyQ) tract in the
huntingtin (htt) protein
(SEQ ID NO: 15), resulting in neurodegeneration and premature death of the
afflicted
individual. A single-chain antibody that binds to an epitope formed by the N-
terminal 17
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amino acids of htt (Lecerf, J.-M. et al., Proc Natl Acad Sci USA. 98(8): 4764-
4769 (2001)
SEQ ID NO: 5) has been shown to reduce symptoms in a Drosophila model of
Huntington's
disease. (Wolfgang, W.J. et al., Proc Natl Acad Sci USA. 102(32): 11563-11568
(2005))
Therefore, a base peptide sequence is derived from SEQ ID NO: 5. In some
embodiments, a
base peptide sequence of an antigenic region is derived from a portion of SEQ
ID NO: 15.
A further specific example is Dialysis-related Amyloidosis (DRA). DRA may be
caused by different forms of blood filtration, such as haemodialysis,
hemofiltration, or
Continuous Ambulatory Peritoneal Dialysis (CAPD). DRA has an incidence of
greater than
95% of patients on dialysis for more than 15 years with beta-2-microglobulin
(B2M, SEQ ID
NO: 12) amyloidosis being prevalent and predictably increasing over time.
Conformational
isomers of B2M have been observed in a clinical setting (Uji et at. Nephron
Clin Pract
2009;111:c173¨c181). B2M is part of the human leukocyte antigen (HLA) class I
molecule,
and has a prominent beta-pleated structure characteristic of amyloid fibrils.
B2M is known to
circulate as an unbound monomer distributed in the extracellular space. B2M
undergoes
fibrillogenesis to form amyloid deposits in a variety of tissues. This
deposition causes renal
failure, which causes an increase in synthesis and release of B2M,
exacerbating the condition.
Thus, in an embodiment of the disclosure, a protein the base sequence of which
is used for
preparation of a composition of the disclosure is beta 2 microglobulin (SEQ ID
NO: 12) and
fragments thereof. An exemplary fragment of B2M is that spanning amino acid
residues 21-
40, SEQ ID NO: 11 in Table 1, useful as a base peptide for DRA.
In some embodiments, the composition (for example, a composition used to treat
or
diagnose DRA) is modified with advanced glycation end (AGE) products, useful
to elicit
immune responses and to generate antibodies against certain AGE products.
AGE products are a heterogeneous group of carbohydrate molecules formed by non-
enzymatic glycation and oxidative reactions between reducing sugars and
protein amino
groups. As described in Niwa (Seminars in Dialysis, 14(2) (March¨April) 2001
pp. 123-126),
AGE-modification of B2M is often observed in DRA patients, and appears to
contribute to
the pathology of DRA. In particular, the author observed imidazolone, NE-
(carboxymethyl)lysine (CIVIL), and pentosidine modifications. As AGE-modified
B2M
accumulates, chemotaxis is enhanced, stimulating macrophages to release pro-
inflammatory
cytokines and interfering with collagen synthesis. Furthermore, AGE-B2M
interacts with
mononuclear phagocytes (MPs), cells important in the pathogenesis of
inflammatory
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arthropathy. This interaction prompts the MPs to secrete elevated levels of
TNFcc and
interleukin-1, worsening inflammation (Rashid et at., IMAJ 2006;8:36-39).
To date, both haemodialysis and peritoneal dialysis have been found
unsatisfactory in
removing AGE products from the bloodstream; thus, new methods are needed to
lower the
levels of AGE products in DRA pateints. Advanced glycation end products may be
formed
primarily on B2M aggregates rather than monomers, and thus may be useful in
producing
antibodies with specificity to the pathogenic aggregate form of B2M.
Alternatively,
oxidation of B2M may enhance amyloid deposition.
Examples of epitopes identified as part of a naturally occurring, full-length
protein or
synthetic peptides that were identified to have similar activities as such
epitopes are shown in
the table below. In certain embodiments, an AR or base peptide sequence
comprises or
consists of all or a portion of a sequence set forth in the tables below or as
described above.
Table 1: Examples of peptides associated with protein conformational disorders
Exemplary base
Application peptide sequences
derived from an Source/ Target Residue
antigen (antigen) Number
Protein
conformational DAEFRHDSGYEVHH
disorders; QKLVFFAEDVGSNK
neuro- GAIIGLMVGGVV
degeneration; (SEQ ID NO: 1) Abeta 1-40 N/A
Protein DAEFRHDSGYEVHH
conformational QKLVFFAEDVGSNK
disorders; GAIIGLMVGGVVIA
neuro- (SEQ ID NO: 2)
degeneration; Abeta 1-42 N/A
Protein MGKGEEGYPQEGIL
conformational EDMPVDPGSEAYEM
disorders; PSEEGYQDYEEA
neuro- (SEQ ID NO: 3) Mouse alpha 100-140 of SEQ
degeneration; synuclein ID No. 16
Protein
conformational
disorders; DNEAYEMPSEEGYQ
neuro- DYE (SEQ ID NO: 4) Human alpha 121-137 of SEQ
degeneration; synuclein ID No. 20
Protein
conformational
disorders; MATLEKLMKAFESL
neuro- KSF (SEQ ID NO: 5) 1-17 of SEQ ID
degeneration; Huntingtin No. 15
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HGAEIVYKSPVVSG
Protein DT SPRHLSNVS STGS
conformational IDMVDSPQLATLAD
disorders; EVSASLAKQGL
neuro- (SEQ ID NO: 6) 388-441 of SEQ
degeneration; Human Tau-F ID No. 17
Protein
conformational HGAEIVYKSPVVSG
disorders; DTSPRHL (SEQ ID
neuro- NO: 7) 388-408 of SEQ
degeneration; Human Tau-F ID No. 17
Protein
conformational IDMVDSPQLATLAD
disorders; EVSASLAKQGL
neuro- (SEQ ID NO: 8) 417-441 of SEQ
degeneration; Human Tau-F ID No. 17
Protein
conformational
disorders; AEIVYKSPVVSGDTS
neuro- PRHL (SEQ ID NO: 9) 390-408 of SEQ
degeneration; Human Tau-F ID No. 17
Protein
conformational DMVDSPQLATLADE
disorders; VSASLAKQGL (SEQ
neuro- ID NO: 10) 418-441 of SEQ
degeneration; Human Tau-F ID No. 17
Protein
conformational IQRTPKIQVYSRHPA
disorders; ENGKS (SEQ ID NO:
dialysis-related 11) Beta-2 21-40 of SEQ ID
amyloidosis microglobulin No. 12
Protein TGSIDMVDSPQLAT Human Tau-F 414-438 of
conformational LADE VSASLAK SEQ ID No. 17
disorders; (SEQ ID NO: 31)
neuro-
degeneration;
Protein TGSIDMVDSPQLAT Human Tau-F 414-429 of
conformational LA (SEQ ID NO: 32) SEQ ID No. 17
disorders;
neuro-
degeneration;
Protein QLATLADEVSASL Human Tau-F 424-438 of
conformational AKRRR (SEQ ID SEQ ID No. 17
disorders; NO: 33)
neuro-
degeneration;
Protein GSEAYEMPSEEGY Human alpha 123-137 of
conformational
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disorders; QDYE synuclein SEQ ID No. 35
neuro-
degeneration; (SEQ ID NO: 36)
Protein DNEAYEMPSEEGY Human alpha 121-137 of
conformational QDYE (SEQ ID NO: synuclein SEQ ID No. 35
disorders; 37)
neuro-
degeneration;
Pathogenic infections
The infection of a host by an infectious pathogen is a complex event
comprising a
series of coordinated events in which the pathogen attempts to evade both the
host's innate
and adaptive immune systems. In their attempts to replicate and survive, the
invading
pathogens cause damage to the host. The destruction generally takes the form
of cell death,
either from pathogen entry into the cell or from endo/exo-toxins produced by
the pathogen, as
well as the induction of host cellular responses that have the ability to
cause further tissue
damage, scarring or hypersensitivity. Although between 1938 and 1952, the
decline in
infectious disease-related mortalities decreased 8.2% per year (Armstrong, GL
et at., AMA
1999, 281:61-6,), the death rates due to infectious disease have been
increasing since the
1980s. A study performed at the National Center for Infectious Diseases
demonstrated that
the death rate due to infectious diseases as the underlying cause of death
increased 58% from
1980 to 1992. In 1992, 64 of every 1,000 deaths were attributable to
infectious disease
(Pinner, RW et al., AMA 1996, 275:3).
The mechanisms of infectious diseases comprise two distinct but interconnected
aspects: (1) the various organisms attempting to invade a host, each different
class of
infectious agents having distinct means by which they attempt to evade the
host's immune
system, and (2) the host's immune response to the invading pathogen. Humans
have warded
off infectious diseases in both of these aspects, in the first aspect, by
preventing the access of
infectious agents (e.g. by sanitary habits) and in the second aspect, by
assisting and boosting
the immune system by, for example, vaccination.
To make vaccination more effective, advances in eliciting stronger immune
responses
have been made with the development of antigen/epitope non-specific treatments
that boost
immune activity, such as the adjuvant alum (see Vaccine Adjuvants and Delivery
Systems,
edited by Manmohan Singh 2007 Wiley & Sons ISBN: 978-0-471-73907-4,
incorporated by
reference herein, for an extensive review of vaccine adjuvants), as well as
development of
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immunogens based on the understanding of the genetic basis of these pathogens
(GenBank, a
database managed by the National Center for Biotechnology Information, now has
over 85
billion base pairs in its database. Searching based on pathogen is widely used
(http://www.ncbi.nlm.nih.gov/Genbank/). The latter has opened up the
possibility of utilizing
discrete sequences of proteins derived from the pathogen as immunizing agents
in the context
of cell-mediated (T cell via MHC class I and/or II), but not humoral
(Bcell/antibody-mediated
immunity). These polypeptide-based vaccines are specific to epitopes (also
called antigenic
determinants) that are the precise moieties within antigenic materials to
interact with immune
system components, intended to boost immune reactivity, and are administered
using
methods designed to excite immune function. One major limitation of the
epitope/polypeptide-based approach is the variability with which the human MHC
class I and
II receptors bind the polypeptides.
While improvements to the techniques have been made in the form of differing
types
of inactivation of pathogen or in the use of adjuvants to enhance
immunogenicity, there
remain infectious diseases that are generally refractory to traditional
vaccine therapies.
A need remains in the development of vaccines that can handle the infectious
agents
such as human immunodeficiency virus (HIV), cytomegalovirus, and severe acute
respiratory
syndrome coronavirus, as well as bacteria such as Pseudomonas aeruginosa,
Neisseria
gonorrhea, or Mycobacterium tuberculosis or parasitic diseases such as malaria
or hookworm
disease. In the context of influenza, the current licensed vaccines are
produced in eggs, and
make use of half-century old technology (Ben-Yedidia 2007, above).
These infectious agents, bacteria, and parasitic diseases are harder to treat
using the
inactive pathogen vaccine approach because of the organism's ability to evade
host detection.
The HIV or the flu virus has the ability to alter its immune profile multiple
times in the
amount of time less than a calendar year, progressively marginalizing the
effectiveness of
immunity gained by previous infection and/or even the most recently created
vaccine.
In the context of developing vaccines in the prevention of infectious disease,
the
invading pathogens have acquired by natural selection the ability to quickly
create variability
in the relevant immunogenic epitope. Examples of viral mutants generating
escape variants
that avoid immune detection have been reported for Hepatitis B, influenza, and
HIV (Tabor,
E, Journal of Med. Virol., 2006, 78:S43-S47; Daniels, RS et al., EMBO Journal
1987,
6:1459-65; D'Costa, S et al., AIDS Res Hum Retroviruses 2001, 17:1205-9). This
variation
progressively dampens the utility of single-epitope vaccines.
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However, a simple mixture of several soluble polypeptide epitopes faced
certain
manufacturing issue of soluble polypeptide mixtures, such as difficulties in
delivering a
consistent ratio and quantity of each of the polypeptides in the mixture.
However, not all known and significant infectious agents are readily treated
with
vaccines created by conventional methods. Epitopes that are therapeutically
and
prophylactically effective may be cryptic, or they may change so frequently as
to render a
developed vaccine ineffective and irrelevant. Rapidly evolving viruses would
mean the
number of people carrying the virus with same immunological profile becomes
small,
fragmenting the patient population against which a vaccine may be effective.
Moreover,
chronic infections caused by agents such as HIV, hepatitis C virus (HCV), and
human
papillomavirus (HPV) may require a different approach to immune elicitation
due to the fact
that they evolved to evade immune systems and are poor antigens.
Current therapeutic regimens for HIV treatment comprise immunization using
clade B
gag, protease, reverse transcriptase, gp120 and nef peptides and lipopeptides,
both by
expression of these proteins using expression vectors and by direct
immunization. Other
immunogenic and therapeutically promising HIV proteins are gp41 and env
proteins. In
some embodiments, a base peptide sequence of an antigenic region may be
derived from an
HIV protein such as, but not limited to, gag, protease, reverse transcriptase,
gp120, nef
peptides and lipopeptide, gp41, or env proteins or portions thereof
Unlike Hepatitis A and Hepatitis B, for which there are effective vaccines and
antibody treatments available, Hepatitis C has evaded immune responses.
Vaccines based on
envelope glycoprotein (E1/E2) and virus-like particles have met limited
success. In some
embodiments, a base peptide sequence of an antigenic region may be derived
from a
Hepatitis C protein such as, but not limited to, glycoprotein E1/E2 or a
portion thereof
The peptide sequences of these proteins and epitopes have been extensively
investigated and are readily available to one skilled in the art. See, for
example, Berzofsky et
at., I Cl/n. Invest. 114:450-462 (2004) for a review with citation of these
proteins. See also,
Immune Epitope Database and Analysis Resources (http://www.immuneepitope.org).
In another embodiment of the disclosure, a base peptide sequence of an
antigenic
region is derived from epitopes associated with human papilloma virus (HPV).
In other
words, the target antigen is based on an HPV protein and the base peptide
and/or AR
corresponds to a portion of that HPV protein. Exemplary such proteins that may
be the target
from which the base peptide is derived include, for example, Li and L2.
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HPV is a non-enveloped double stranded DNA virus, the genome of which encodes
two protein types: 'Early proteins' (El, E2, E4, E5, E6, and E7) which
regulate the
replication of viral DNA and 'Late proteins' (L1 and L2) which are the
structural components
of the viral capsid. Targets for compositions of the disclosure may be based
on structural
components or other components of the virus. More than 170 different types of
HPV have
been identified.
Currently, there are two vaccines that are intended for use in HPV naïve
individuals:
(1) CervarixTM, a bivalent vaccine which is effective in preventing HPV types
16 & 18 and
(2) GardasilTM, a quadrivalent vaccine effective in preventing HPV 6, 11, 16,
and 18. Both
the Gardasil and Cervarix vaccine are made from purified virus-like particles
(VLPs) of the
Ll capsid protein. The Ll protein is highly HPV type specific, and thus does
not provide
broad protection against the full range of HPV type infections. Another
limitation of targeting
Ll is that Ll proteins are only expressed during the initial infection with
the virus. Thus,
vaccines targeting Ll are ineffective once the infection becomes established
and systemic.
Other HPV associated targets may be the basis of compositions of the
disclosure. For
example, base peptides based on or derived from, for example, L2 may provide
protection
against more HPV types. Furthermore, the use of L2, although more similar
mechanistically
to using Ll, based vaccination approaches may provide protection against
additional HPV
types.
In some embodiments, a base peptide sequence is derived from residues most
closely
corresponding to 13-40 of HPV L2 protein (SEQ ID NO: 34). The foregoing is
exemplary,
and base peptides may be derived from any portion of a HPV L2 protein.
Examples of epitopes identified as part of a naturally occurring, full-length
protein or
synthetic peptides that were identified to have similar activities as such
epitopes are shown in
the table below. In certain embodiments, an AR or base peptide sequence
comprises or
consists of all or a portion of a sequence set forth in the tables below or as
described above
Table 2: Examples of peptides associated with infectious diseases
Application Exemplary base Source/Target Residue
peptide sequences (antigen) Number
derived from a target
antigen
Pathogenic ILARNLVPMV (SEQ human 481-490 of
infection ID NO: 21) cytomegalovirus SEQ ID No. 29
HCMVpp65
Pathogenic ELEGVWQPA (SEQ HCMVpp65 516-524 of
infection
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ID NO: 22) SEQ ID No. 29
Pathogenic RIFAELEGV (SEQ ID HCMVpp65 512-520 of
infection NO: 23) SEQ ID No. 29
Pathogenic NLVPMVATV (SEQ HCMVpp65 485-493 of
infection ID NO: 24) SEQ ID No. 29
Pathogenic RIQRGPGRAFVTIGK HIV-gp120 V3 loop
infection (SEQ ID NO: 25)
Pathogenic SVTQLYKTCKQSGTC HPV L2 Most closely
infection PPDVIPKVEGTTL corresponding
(SEQ ID NO: 34) to 13-40 of
SEQ ID No. 30
In some embodiments, one or more base peptide sequences that form the basis
for the
antigenic regions of the amino acid copolymer compositions of the disclosure
are derived
from an antigen associated with a pathogenic infection. In some embodiments,
the pathogen
is a virus, bacteria, or a parasite. In some embodiments, the pathogen is
virus selected from
HIV, porcine reproductive and respiratory syndrome (PRRS), foot-and-mouth
diseasese, or
HSV. In some embodiments, the pathogen is a bacterium selected from
Salmonella,
Mycobacterium (e.g., M tuberculosis), Legionella, Coxiella, Chlamydia,
Pseudomonas,
Staphylococcus, Typhimurium, Yersinia, or Listeria. In some embodiments, the
pathogen is a
parasite such as a protozoan selected from a trypanosome (e.g., Trypanosoma
brucei,
Trypanosoma cruzi, Leishmania), or Plasmodium (e.g., P. falciparum, P. vivax,
P. knowlesi,
P. malariae, P. ovale, P. brasilianum, P. cynomolgi, P. inui, P. rhodiani, P.
schweitzi, P.
semiovale, P. simium) or Toxoplasma (e.g., T. gondii). In some embodiments,
the pathogen
is a parasite such as a helminth (e.g. monogeneans, cestodes, trematodes,
nematodes). In
some embodiments, the antigens are derived from an intracellular pathogen. In
some
embodiments, the pathogen is of the genus selected from Chlamydia, Rickettsia,
Coxiella,
Mycobacterium, Francisella, Listeria, Salmonella, Brucella, Legionella,
Nocardia,
Rhodococcus, Yersinia, and Neisseria. In some embodiments, the peptide
sequences are
derived from SEQ ID Nos. 21-29 or portions thereof. In some embodiments, the
one or more
base peptide sequences that form the basis for the antigenic regions of the
amino acid
copolymer compositions of the disclosure are derived from an antigen
associated with any of
the pathogen disclosed herein. The peptide sequences of these proteins and
epitopes have
been extensively investigated and are readily available to one skilled in the
art.
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V. Polyp eptide Synthesis Methods
Any known solid phase synthesis appropriate for polypeptide synthesis may be
used
to synthesize a composition of the disclosure, for example, as originally
described by
Merrifield (J. Am. Chem. Soc., 1963, 85:2149) and any variation thereof More
specifically,
the synthesis is done in multiple steps from C-terminus to N-terminus by the
Solid Phase
Peptide Synthesis (SPPS) approach using Fmoc protected amino acids. The first
amino acid
at the C-terminal position may be a Norleucine (Nle) covalently coupled to the
resin support.
SPPS is based on sequential addition of protected amino acid derivatives, with
side chain
protection where appropriate, to a polymeric support (bead). The base-labile
Fmoc group is
used for N-protection. After removing the protecting group (via piperidine
hydrolysis) the
next amino acid mixture is added using a coupling reagent (TBTU). At each
position and for
each polypeptide generated, Fmoc may be cleaved and an amino acid is randomly
selected
from the amino acid solution described in the linear template arrangement for
each position.
After the final amino acid is coupled, the N-terminus is acetylated.
The resulting polypeptides (attached to the polymeric support through the C-
terminus)
are cleaved with TFA to yield the crude polypeptide. During this cleavage
step, all of the
side chains protecting groups are also cleaved. After precipitation with
diisopropyl ether, the
solid is filtered and dried. The resulting polypeptides are analyzed and
stored at 2-8 C.
We note that SPSS of large polypeptides may be more difficult to synthesize
due to
incomplete coupling reactions. As such, each resulting complex polypeptide
mixture may
contain some percentage of polypeptides that are less than full-length or less
than
substantially full-length (e.g., polypeptides which comprise, for example,
only about 75% or
85% of the full-length polypeptide based on the original template
arrangement). Because the
SPPS proceeds from C-terminus to N-terminus, these shorter polypeptides, if
any, in a
composition are generally truncated from the N-terminus (e.g., missing a more
N-terminal
portion). That said,such truncated polypeptides, when present, can be
minimized or
eliminated by either post synthesis purification steps or by increasing
synthesis times or other
methods described briefly herein. Accordingly, compositions of the disclosure
comprising
very high percentages of full length polypeptides (based on the template
arrangement) are
provided, such as compositions in which greater than 75%, greater than 80%,
greater than
85%, greater than 90%, or even 95% or greater than 95% of the polypeptides in
the complex
mixture are full length. Moreover, although not done here, full-length or
substantially full-
length polypeptides may be separated from the mixture. Accordingly, high
complexity
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mixtures of polypeptides may comprise truncated polypeptides based on a
portion of the
complete template arrangement, or may be enriched for a higher percentage of
full-length or
substantially full-length polypeptides based on the template arrangement. In
addition, to
improve the coupling efficiency and generate a higher yield in each step,
longer coupling
times or the addition of redundant coupling reactions may be used.
Accordingly,
compositions comprising greater than 90% or even greater than 95% full-length
polypeptide
can be obtained.
Additionally, any polypeptide synthesis method that allows synthesis
incorporating
more than one amino acid species at a controlled ratio in any given position
of the
polypeptide sequence is suitable for use with this disclosure. Further, as
described below, the
polypeptides of a composition according to the disclosure may be
peptidomimetics or include
unnatural or modified amino acids, necessitating adaptation to allow addition
of such
chemical species to the polymers synthesized up to that point.
The synthesis may include unnatural amino acids, or amino acid analogs. In
some
embodiments, the polypeptides of the compositions of the disclosure are
comprised of
naturally occurring and synthetic derivatives, for example, selenocysteine.
Amino acids
further include amino acid analogs. An amino acid "analog" is a chemically
related form of
the amino acid having a different configuration, for example, an isomer, or a
D-configuration
rather than an L-configuration, or an organic molecule with the approximate
size and shape
of the amino acid, or an amino acid with modification to the atoms that are
involved in the
peptide bond, so as to be protease resistant when polymerized in a
polypeptide. The
polypeptides of the compositions can be composed of stereoisomers (e.g., D-
amino acids,
Nle, Nva, Cha, Orn, Hle, Chg, Hch, or Har) of the twenty conventional amino
acids,
unnatural amino acids such as cc-, cc-disubstituted amino acids, N-alkyl amino
acids, lactic
acid, and other unconventional amino acids can also be suitable components for
the
polypeptides of the present disclosure. Examples of unconventional amino acids
include (i.e.,
are not limited to): 4-hydroxyproline, y-carboxyglutamate, c-N,N,N-
trimethyllysine, c -N-
acetyllysine, 0-phosphoserine, N-acetylserine, N-formylmethionine, 3-
methylhistidine, 5-
hydroxylysine, a-N-methylarginine, and other similar amino acids and imino
acids (e.g., 4-
hydroxyproline).
In some embodiments, the polypeptides include Norleucine (Nle) at the C-
terminus.
In some embodiments, the polypeptides include analogs of phosphorylated amino
acids. In some embodiments, the analog is (a,a-difluoroalkyl)phosphonate
analog of
phosphoserine, phosphothreonine or allo-threonine. In some embodiments, the
analog is
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(a,a-difluoromethylene) phosphonate mimic of phosphoserine (pCF2Ser). In some
embodiments, the analog is p-aminobenzylphosphonate or p-
aminobenzylphosphonate or
derivates thereof.
The polypeptides of the compositions according to the present disclosure can
be
composed of L- or D-amino acids or mixtures thereof. As is known by those of
skill in the
art, L-amino acids occur in most natural proteins. However, D-amino acids are
commercially
available and can be substituted for some or all of the amino acids used to
make polypeptides
of the present disclosure. The present disclosure contemplates polypeptides
containing both
D- and L-amino acids, as well as polypeptides consisting essentially of either
L- or D-amino
acids.
In some embodiments, the polypeptides of the present disclosure include such
linear
polypeptides that are further modified by substituting or appending different
chemical
moieties. In one embodiment, such modification is at a residue location and in
an amount
sufficient to inhibit proteolytic degradation of the polypeptides in a
subject. For example, the
amino acid modification may be the presence of at least one proline residue in
the sequence;
the residue is present in at least one of carboxy- and amino termini; further,
the proline can be
present within four residues of at least one of the carboxy- and amino-
termini. Further, the
amino acid modification may be the presence of a D-amino acid.
In some embodiments, the subject polypeptides are peptidomimetics.
Peptidomimetics are compounds based on, or derived from, polypeptides and
proteins. The
peptidomimetics of the present disclosure typically can be obtained by
structural modification
of one or more native amino acid residues, e.g., using one or more unnatural
amino acids,
conformational restraints, isosteric replacement, and the like. The subject
peptidomimetics
constitute the continuum of structural space between peptides and non-peptide
synthetic
structures.
Such peptidomimetics can have such attributes as being non-hydrolyzable (e.g.,
increased stability against proteases or other physiological conditions which
degrade the
corresponding polypeptides), increased specificity and/or potency. For
illustrative purposes,
peptide analogs of the present disclosure can be generated using, for example,
benzodiazepines (e.g., see Freidinger et at. in "Peptides: Chemistry and
Biology," G.R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma
lactam
rings (Garvey et at. in "Peptides: Chemistry and Biology," G.R. Marshall ed.,
ESCOM
Publisher: Leiden, Netherlands, 1988, p123), C-7 mimics (Huffman et at. in
"Peptides:
Chemistry and Biology," G.R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988, p.
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105), keto-methylene pseudopeptides (Ewenson et al. I Med. Chem., 1986,
29:295; and
Ewenson et at. in "Peptides: Structure and Function (Proceedings of the 9th
American
Peptide Symposium)," Pierce Chemical Co. Rockland, IL, 1985), 13-turn
dipeptide cores
(Nagai et al., Tetrahedron Lett., 1985 26:647; and Sato et al. I Chem. Soc.
Perkin Trans.,
1986,1:1231), (3-aminoalcohols (Gordon et al. Biochem. Biophys. Res. Commun.,
1985,
126:419; and Dann et al. Biochem. Biophys. Res. Commun., 1986, 134:71),
diaminoketones
(Nataraj an et at. Biochem. Biophys. Res. Commun., 1984, 124:141), and
methyleneamino-
modified (Roark et at. in "Peptides: Chemistry and Biology," G.R. Marshall
ed., ESCOM
Publisher: Leiden, Netherlands, 1988, p134). Also, see generally, Session III:
Analytic and
synthetic methods, in "Peptides: Chemistry and Biology," G.R. Marshall ed.,
ESCOM
Publisher: Leiden, Netherlands, 1988.
In some embodiments, the polypeptides of the composition are synthesized in a
single
manufacturing step. In some embodiments, different template regions of the
polypeptide
composition are synthesized separately and coupled by peptide ligation (e.g.,
an RCR and an
AR can be synthesized separately and coupled together by peptide ligation). In
some
embodiments, one or more RCRs are synthesized separately and couple to a
recombinant
protein or antigen of interest or portion thereof to generate an amino acid
copolymer of the
composition. In some embodiments, the peptide ligation is Native Chemical
Ligation or
bis(2-sulfanylethyl)amino (SEA) Native Peptide Ligation.
The molecular weight of a polypeptide composition can be adjusted during
polypeptide synthesis or after the polypeptides have been synthesized. To
adjust the
molecular weight during polypeptide synthesis, the synthetic conditions or the
amounts of
amino acids are adjusted so that synthesis stops when the polypeptide reaches
the
approximate length, which is desired. After synthesis, polypeptides with the
desired
molecular weight can be obtained by any available size selection procedure,
such as
chromatography of the polypeptides on a molecular weight sizing column or gel,
and
collection of the molecular weight ranges desired. The present polypeptides
can also be
partially hydrolyzed to remove high molecular weight species, for example, by
acid or
enzymatic hydrolysis, and then purified to remove the acid or enzymes.
In some embodiments, polypeptides with a desired molecular weight may be
prepared
by a process which includes reacting a protected polypeptide with hydrobromic
acid to form a
trifluoroacetyl-polypeptide having the desired molecular weight profile. The
reaction is
performed for a time and at a temperature, which is predetermined by one or
more test
reactions. During the test reaction, the time and temperature are varied and
the molecular
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weight range of a given batch of test polypeptides is determined. The test
conditions, which
provide the optimal molecular weight range for that batch of polypeptides, are
used for the
batch. Thus, a trifluoroacetyl-polypeptide having the desired molecular weight
profile can be
produced by a process which includes reacting the protected polypeptide with
hydrobromic
acid for a time and at a temperature predetermined by test reaction. The
trifluoroacetyl-
polypeptide with the desired molecular weight profile is then further treated
with an aqueous
piperidine solution to form a low toxicity polypeptide having the desired
molecular weight.
In some embodiments, a test sample of protected polypeptide from a given batch
is
reacted with hydrobromic acid for about 10-50 hours at a temperature of about
20-28 C. The
best conditions for that batch are determined by running several test
reactions. For example,
in one embodiment, the protected polypeptide is reacted with hydrobromic acid
for about 17
hours at a temperature of about 26 C.
In some embodiments, specific glycogenated forms of a polypeptide composition
are
generated. In some embodiments, the post-translational modification of a
polypeptide
composition is performed using glycogen synthase, or alternatively using
chemical
complexation techniques well known in the art.
In some embodiments, specific glycosylated forms of a polypeptide composition
are
generated. In some embodiments, the glycosylation is selected from one or more
of N-linked
glycosylation, 0-linked glycosylation, phospho-serine glycosylation, C-
mannosylation, and
glypiation. In some embodiments, the post-translational modification of a
polypeptide is
performed using glycosyl transferases, or alternatively using chemical
complexation
techniques well known in the art.
In some embodiments, specific nitrated forms of a polypeptide composition are
generated.
In some embodiments, specific nitrosylated forms of a polypeptide composition
are
generated. In some embodiments, S-nitrosylation of a polypeptide composition
are generated.
In some embodiments, specific disulfide bridges between cysteine (disulfide
bridge)
residues of individual polypeptides are generated. In some embodiments, the
post-translation
formation of the cysteine bridge is performed using the reduction of a
disulfide bridge
between two cysteine residues. In some embodiments, reducing due to the
presence of
disulfide bond reductases (e.g. thioredoxins and glutaredoxins) are generated.
The net charge on a protein can be either positively (cation) or negatively
(anion)
charged depending on both the amino acid composition of the protein and the pH
conditions.
The net charge of the protein may affect protein solubility, and in turn, the
immunogenicity
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of the polypeptide composition. Accordingly, depending on the nature of the
ARs, the RCRs
can, in addition to their other specific immunogenic properties, be used to
increase the
estimated net charge of the composition as a whole.
While the input method comprising base peptide design and synthesis have been
described herein, the output of the polypeptide composition may vary from
input due to
several factors. One of these factors is the synthesis of the polypeptide
composition by solid
phase peptide synthesis (SPSS). SPSS can lead to two types of variation in the
output of the
polypeptide composition: (1) polypeptides with varied lengths due to
incomplete polypeptide
synthesis (which, as noted here, can be controlled) and (2) variation in the
relative ratio of
amino acid composition at each position due to a preferential incorporation of
a particular
amino acid (which can be evaluated based on output ratios, as described
herein).
As described above, Solid Phase Peptide Synthesis (SPSS) is a polypeptide
synthesis
process done in multiple steps using Fmoc protected amino acids. A limitation
of SPSS is
that large polypeptides may be more difficult to synthesize due to incomplete
coupling
reactions. As such, each resulting complex polypeptide mixture may contain
some
percentage of polypeptides that are less than full-length or substantially
full-length (e.g.,
polypeptides which comprise, for example, only about 75% or 85% of the full-
length
polypeptide based on the original template arrangement). Because the SPPS
proceeds from
C-terminus to N-terminus, these shorter polypeptides, when present in a
composition, are
generally truncated from the N-terminus (e.g., missing a more N-terminal
portion). That said,
such truncated polypeptides, can be minimized or eliminated by either post
synthesis
purification steps or by increasing synthesis times or other methods described
briefly herein.
Accordingly, compositions of the disclosure comprising very high percentages
of full length
polypeptides (based on the template arrangement) are provided, such as
compositions in
which greater than 75%, greater than 80%, greater than 85%, greater than 90%,
or even 95%
or greater than 95% of the polypeptides in the complex mixture are full
length.
The variations in polypeptide length may be evaluated using a myriad of
techniques.
MALDI-TOF-MS analysis may be used to evaluate the percentage of full-length
proteins
found within the copolymer composition. As described in Example 12 and Figure
17,
MALDI-TOF-MS may be used to determine the mass/charge (m/z) value of various
copolymers within the copolymer composition. As can be seen in Figure 17, the
full-length
polypeptides based on the DP-0016.F template are about 63 amino acids in
length and have a
mass of about 6900 Daltons. The peaks which occur prior to the 6900 Dalton
peak are due to
synthesis of polypeptides based on this template arrangement but which are
shorter due to
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incomplete protein synthesis. By calculating the relative ratio between the
masses of
polypeptides in the composition having a substantially full-length polypeptide
sequence and
the masses of the polypeptides having masses indicative of a less than full-
length sequence, it
may be determined at between which positions peptide synthesis was disrupted.
As can be
seen in Figure 17, in one batch of DP-0016.F composition (a composition based
on the DP-
0016.F template arrangement, sequence, and input amino acid input
distribution), the
resulting composition synthesized had a purity of about 53% (e.g., calculated
at about
52.61%). Purity, in this case, refers to the percentage of polypeptides in the
composition
based on the template arrangement that are full-length or substantially full-
length
polypeptides. Thus, "about 53% purity" refers to a composition in which about
53% of the
polypeptides in the composition that are based on the template arrangement are
full-length or
substantially full-length polypeptides.
While purity may be determined by MALDI-TOF-MS analysis, the purity may be
increased by separating the substantially full-length polypeptides from the
copolymer
mixture. This may be achieved by a multitude of protein purification methods
based on size,
which may include but is not limited to techniques such as SDS-polyacrylamide
gel
electrophoresis, liquid chromatography, or gel filtration chromatography.
Furthermore, the
substantially full-length polypeptide purity may be increased by modifying the
synthesis
protocol based on the copolymer template and incomplete polypeptide synthesis
information
gathered from MALDI-TOF-MS analysis. For example, in Example 12 and Figure 17,
MALDI-TOF analysis was used to determine that incomplete synthesis was
occurring
between positions corresponding to positions 16-17 and positions 24-25 of the
DP-0016.F
polypeptide. In order to increase the percentage of full-length proteins in
the composition,
modifications to the synthesis protocol such as the strategic addition of
coupling reactions at
particular positions to improve the coupling efficiency and generate a higher
yield in each
step, longer coupling times or the addition of redundant coupling reactions
may be used.
Furthermore, synthesis of polypeptides by SPSS may result in a preferential
incorporation of alanine (A) over lysine (K) in the polypeptide. For example,
when
comparing the A/K relative input ratio to the A/K relative output ratio in DP-
0016.0 and an
embodiment of DP-0016.F, the A/K relative output ratio is at least double the
A/K relative
input ratio in both polypeptide compositions are shown in the Table below.
Table 3: Alanine to Lysine input and out ratio in copolymer compositions
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A/K A/K A/K
Peptide Length (aa)
Repeat input ratio output ratio
DP-0016.13 61 2 x 9 KAE 1.98 4.92
DP-0016.F 63 3 x 3 KAE 1.82 3.82
The A/K input ratio and A/K output ratio may be determined by using a
combination
of the information gained from the MALID-TOF-MS analysis and amino acid
analysis
(AAA). Amino acid analysis is a technique, which can be used to determine the
amino acid
composition of each individual amino acid in a polypeptide. Amino acid
analysis can take
advantage of an internal standard to which the individual amino acid may be
compared to
determine the molar concentration of each amino acid. Norleucine may provide
an internal
standard for amino acid analaysis. As discussed above, solid phase peptide
synthesis
synthesizes each polypeptide in a C-terminus to N-terminus direction.
Norleucine (Nle) may
be covalently coupled to the resin support and thus incorporated into the C-
terminal most
position of each polypeptide. Considering that SPSS proceeds in a C-terminus
to N-terminus
direction, and Norleucine will be in the C-terminal most position, it can be
assumed that one
Norleucine will be incorporated into each polypeptide. Thus, Norleucine may be
used as an
internal standard to determine the composition of the individual polypeptides.
The output
analysis of amino acid that may be obtained from a copolymer composition is
shown in the
Table below.
Table 4: Amino acid percentage analysis in copolymer compositions
Nle% Ala% Lys% Asx% Glx%
Pro%
(input) (input) (Input) (input)
(input) (input)
vs vs vs vs vs vs
(output) (output) (output) (output)
(output) (output)
DP C016.13 1.64 27.87 14.10 5.90 8.69
4.43
-
1.93 27.29 5.55 5.38 5.83
ND
DP C016 .F 1.59 20.79 11.43 5.71 7.14
4.29
-
1.66 24.89 7.16 5.38 6.88
3.77
The Table above describes the percent input and percent output of amino acids
in
three different copolymer compositions (DP-0016.B and an embodiment of DP-
0016.F). DP-
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C016.B represents analysis of one preparation of the DP-0016.B template
described in Figure
2. DP-0016.F represents analysis of one preparation of the DP-0016.F template
described in
Figure 12. Due to the hydrolysis solution used during amino acid analysis, the
concentrations
of glutamine and glutamic acid may not be differentiated and is indicated as
Glx.
Furthermore, the concentrations of asparagine and aspartic acid may not be
differentiated and
is indicated as Asx. As can be seen across these examples, lysine may be
counter-selected
against and alanine may be preferentially selected during some SPSS coupling
reactions.
This results in a variation of the A/K input ratio and the A/K output ratio
across the
copolymer compositions.
V/. Antibody Production
The method is drawn to increasing the diversity of antibodies generated to
react with a
ligand. Further, the method is drawn to overcoming the problem of creating
antibodies
against ligands with low immunogenicity. Still further, the method is drawn to
overcoming
problems relating to generating antibodies having reactivities to only a
single species. The
instant disclosure comprises a method of creating antibody reagents for use in
research
studies. The instant disclosure comprises a method of creating antibody
reagents for use as
diagnostic tools. The instant disclosure further comprises a method for the
generation of
antibodies useful as therapeutic agents for the treatment of disease. Using
the same principle,
antibodies may be produced in vivo, i.e., the compositions for stimulating
antibody
production may be used as vaccines. Immunization steps of all the
representative methods
described below can be modified for in vivo use of the immunogens of the
present disclosure
as vaccines.
A method of preparing antibodies using a known antigen or a mixture of
antigens is
well known in the art. Antibodies are produced by designing and synthesizing
the amino acid
copolymer compositions as described above, creating antibodies by introducing
the
compositions into an in vivo setting, or alternatively introducing the
compositions into an in
vitro setting, or still alternatively contacting the composition with a system
of maintaining the
connection between antibody phenotype and genotype such as phage display,
determining the
activity of the generated antibodies by contacting the antibodies with the
native molecule of
interest, selecting antibodies having desired activity, such activity being
either of a higher
affinity antibody, or alternatively a lower affinity antibody, a single
species reactivity, or
alternatively a multi-species reactivity, a single-molecule of interest
reactivity or alternatively
a multi-molecule reactivity.
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The instant disclosure also comprises a process for producing antibodies that
are
therapeutically or prophylactically useful in the treatment of various
diseases or conditions
(e.g. protein conformational disorders or pathogenic infections), or useful
for use as research
reagents, and as diagnostic tools for such diseases or conditions, by
eliciting immune
responses using a composition comprising directed epitope polypeptide
mixtures. The
disclosure also encompasses composition comprising antibodies thus produced.
The method of the instant disclosure also encompasses an augmentation of the
paratopes associated with an antibody response to an antigen of interest. The
method of the
instant disclosure further encompasses the generation of novel functioning
antibodies having
antigen binding properties that elicit a varied amount of downstream
consequences to the
binding event.
Briefly, the method comprises the steps of selecting a protein relevant to the
disease
or condition of interest (e.g. a protein conformational disorder, a pathogenic
infection, etc.),
determining relevant epitopes within the protein known or suspected to be
closely associated
with the disease or condition, selecting the relevant antigenic region(s),
selection the number
and/or arrangement of one or more random copolymer regions, designing directed
permutations of the antigenic regions and random copolymer regions so as to
create an
expanded yet related series of polypeptides, performing solid phase synthesis
thus creating a
high-complexity polypeptide mixture, using the polypeptide mixture
collectively as a set of
antigens by placing the mixture in contact with a means of antibody
generation, determining
the activity of the generated antibodies, selecting antibodies having the
desired activity, and
utilizing the antibody as a single species reagent, multi-species reagent,
single species
diagnostic, multi-species diagnostic, or alternatively as a therapeutic. The
means of antibody
generation is, for example, an animal to be immunized by the polypeptide
mixture and cells
from such an animal (e.g. spleen cells from a mouse for monoclonal antibody
production), a
phage display library, or a B cell library.
Alternatively, the instant disclosure encompasses methods of producing
antibodies,
the methods comprising: selecting the relevant antigenic region(s), selection
the number
and/or arrangement of one or more random copolymer regions, designing directed
permutations of the antigenic regions and random copolymer regions so as to
create an
expanded yet related series of polypeptides, performing solid phase synthesis
thus creating a
high-complexity polypeptide mixture, preparing the polypeptide mixture as a
pharmaceutically acceptable salt, introducing the polypeptide mixture into a
host, harvesting
primary tissue containing antibody from the host after one week, alternatively
harvesting
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primary tissue containing antibody from the host after a time greater one
week, determining
the activity of the generated antibodies, selecting, and utilizing the
antibody as a reagent,
diagnostic, or alternatively as a therapeutic.
Thus produced, another aspect of the present disclosure is a composition
comprising
antibodies generated against an amino acid copolymer composition as described
above,
wherein the base peptide sequence of at least one antigenic region is derived
from a sequence
of a protein known to be associated with a disease or condition (e.g. protein
conformation
disorder, pathogenic infection, etc.). In some embodiments, the base peptide
sequence is
derived from a protein known to be associated with a protein conformational
disorder. More
particularly, such protein is known to form an aggregate or fibril. In
particular, antibodies
thus generated are specific to the pathological conformation of such protein.
In some
embodiments, the base peptide sequence is derived from a protein known to be
associated
with a pathogenic infection.
In an embodiment of this aspect of the disclosure, the antibodies are modified
antibodies having an engineered Fc region, wherein the engineered Fc region
confers
favorable pharmacodynamic profiles. In one embodiment, the Fc region enhances
clearance
of antibody-antigen complex. In another embodiment, the Fc region is not
immunogenic to
the subject. Such modified antibodies may be created after antibodies with
certain desired
(complementarity determining regions) are identified, by replacing chemically
or by
molecular biological means the Fc region with an IgA, IgG, IgE, IgM, or IgD
region.
In another embodiment, the antibodies are humanized antibodies having desired
CDRs (complementarity determining regions), such CDRs having been identified
using the
compositions of the disclosure or antibodies having such CDRs having been
generated using
the compositions of the disclosure. Humanized antibodies may be made according
to any
means known in the art, including CDR grafting and the introduction of point
mutations to
reduce immunogenicity. In yet another embodiment, the antibodies are single
chain variable
fragment (scFv), either engineered from an identified antibody, or generated
using a phage
display library and other means and screened for desired antibodies using the
compositions of
the disclosure. Methods of scFv production and phage display are known in the
art.
The antibodies may also have a detectable label, such as a radiolabel, an
enzymatic
label, or a fluorescent label. In some embodiments, the fluorescent label is
selected from the
group consisting of Texas Red, phycoerythrin (PE), cytochrome c, and
fluorescent
isothiocyanate (FITC). In addition, labels such as biotin followed by
streptavidin-alkaline
phosphatase (AP), horseradish peroxidase (HRP) are contemplated.
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This disclosure also provides antibodies with at least 70%, 80%, 90%, 95%, or
99%
amino acid sequence identity to the antibodies described above. Antibodies in
general have
well characterized structure-activity relationships, and one of skill in the
art would be well
aware that certain mutations would be unlikely to disrupt the antigen-binding
function of an
antibody. For example, conservative substitutions in the constant region would
be unlikely to
disrupt antigen binding, while substitutions in the CDRs would be more likely
to disrupt
antigen binding.
An aspect of the disclosure is a composition comprising a scaffold or support
material
to which antibodies are attached, which antibodies are generated against an
amino acid
copolymer composition as described above, wherein the base peptide sequence is
derived
from a sequence of a protein known to be associated with a protein
conformational disorder.
In one embodiment, the scaffold is a membrane compatible with haemodialysis.
Membranes
for haemodialysis are typically semi-permeable, allowing for water and some
dissolved
solutes to pass through. The membranes can have different pore sizes and are
thus
categorized as low-flux or high-flux. Membranes can be made from a variety of
materials,
including cellulose acetate, polyarylethersulfone, polyamide,
polyvinylpyrrolidone,
polycarbonate, and polyacrylonitrile. In a particular embodiment, the
antibodies are
conjugated to such membrane. This will allow for removal of specified proteins
at while
haemodialysis is carried out. This process is useful, inter al/a, for treating
removing amyloid
forms of B2M and treating DRA. In another embodiment, the antibodies are
conjugated to a
resin, such as CN-Br agarose resin (for example CN-Br Sepharose (Pharmacia),
to create an
immunoaffinity resin.
VII. Exemplary Uses
a) Administration of amino acid copolymer compositions
The instant disclosure provides compositions of the disclosure (also referred
to as
amino acid copolymer compositions of the disclosure). Such compositions
comprise a
plurality of polypeptides having the same template arrangement and comprising
one or more
RCRs and one or more ARs. Sequence complexity is contributed by the RCRs
and/or ARs,
such that the composition comprises a mixture of polypeptides that are closely
related by
sequence.
Compositions of the disclosure may be administered in vitro or in vivo. Any of
the
compositions of the disclosure, described based on any combination of
structural and/or
functional features provided herein, may be used in any of the methods
described herein.
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Compositions of the disclosure may be administered in vitro or in vivo alone,
or in
combination with one or more other agents. In certain embodiments, the one or
more other
agent is an adjuvant. Although in other embodiments, an additional adjuvant is
not required
(e.g., the method does not include use of an adjuvant other than a composition
of the
disclosure).
In certain embodiments, compositions of the disclosure are suitable for use as
an
immunotherapy. The compositions of the disclosure are useful in the treatment
of a disease
or condition that would benefit from induction of an antigen-specific immune
response (e.g.
protein conformational disorders, pathogenic infections, etc.) and/or from
stimulation of the
immune system. The amino acid copolymer compositions of the disclosure can be
dynamically administered based on the ability of the composition to achieve an
increased
immune activation, while generating a Thl immune posture, or a Th2 immune
posture, and
while producing anti-composition antibodies at either a low or a high level.
In some
embodiments, the amino acid copolymer composition generates a Th2 immune
posture.
Dynamic administration of amino acid copolymer compositions of the disclosure
is
comprised of any combination of dose, regimen, route of administration, and/or
formulation.
This dynamic immunomodulation provides for increased effectiveness at any of
the multiple
stages of a disease within a particular patient, as well as the ability to
treat multiple,
pathogenic antigenic-determinant unrelated diseases more effectively.
The disclosure provides methods for the treatment or prevention of a disease
in a
subject, preferably in a human, which subject is afflicted with or is
suspected to be afflicted
with the disease. One embodiment of the present disclosure is a method for
prophylactically
treating a subject at risk of developing a pathogenic infection by
administering an amino acid
copolymer composition of the disclosure. One embodiment of the present
disclosure is a
method for prophylactically treating a subject at risk of developing a protein
conformational
disorder by administering an amino acid copolymer composition of the
disclosure. One
embodiment of the present disclosure is a method for therapeutically treating
a subject at risk
of developing a protein conformational disorder by administering an amino acid
copolymer
composition. A subject at risk is identified by, for example, determining the
genetic
susceptibility to a protein conformational disorder by testing for alleles of
HLA that are
associated with such disorder, and/or based on familial history, or other
genetic markers that
correlate with such disorder. In addition, many patients receiving dialysis or
other form of
blood filtration are at risk for developing dialysis related amyloidosis
(DRA), especially if the
blood filtration is performed over a long period of time, such as more than 3,
5, 7, or 10 years.
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Further, subjects that are asymptomatic but show biochemical markers of a
protein
conformational disorder are at risk of developing such disorder.
One aspect of the disclosure provides methods of treating or preventing a
disease, the
method comprising administering to said subject a dosing regimen of an
effective amount of
an amino acid copolymer composition of the disclosure for the amelioration of
a disease
treatable with the amino acid copolymer composition, said effective amount
delivered to said
subject at time intervals greater than 24 hours, 36 hours, or more preferably
greater than 48
hours. A related aspect of the disclosure provides a method for the treatment
of a subject in
need thereof, comprising administering to said subject a dosing regimen of an
effective
amount of an amino acid copolymer composition of the disclosure for the
amelioration of a
disease treatable with the amino acid copolymer composition, said effective
amount delivered
to the subject using a sustained-release formulation which administers the
amino acid
copolymer composition over a period of at least 2 days, at least 4 days, or at
least 6 days,
wherein the effective amount is an amount that is effective if delivered
daily. In some
embodiments, an amino acid copolymer composition of the disclosure is
administered
encapsulated in poly(lactide) microparticles, poly(lactide-co-glycolide)
microparticles,
liposomes, archaeosome adjuvants, mucosal adjuvants, polyphosphazenes. In some
embodiments, an amino acid copolymer composition of the disclosure is
encapsulated in
poly(lactide) microparticles. Compositions of the disclosure may be
administered with an
adjuvant ¨ either co-formulated or administered in separate formulations at
the same or
different time. In some embodiments, an amino acid copolymer composition of
the
disclosure is formulated and/or administered with alum as an adjuvant. In some
embodiments, an amino acid copolymer composition of the disclosure is
formulated and/or
administered with aluminum hydroxide or aluminum phosphate.
One aspect of the disclosure is the administration of an amino acid copolymer
composition of the disclosure to a subject in need there of, as described
above, in
combination with other therapeutic agents that are effective in treating the
conditions that are
treated by administration of amino acid copolymer composition, or conditions
that
accompany or occur concurrently with the conditions that are treated by
administration of the
amino acid copolymer composition. The additional therapeutically active agents
may treat
the same or related disease as the amino acid copolymer composition, or may be
intended to
treat an undesirable side effect of administration of the amino acid copolymer
composition,
such as to reduce swelling at a site of intradermal injection. Alternatively,
the other
therapeutic agents enhance the activity of the amino acid copolymer
compositions. Such
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additional therapeutic agents are, by way of example, antibodies, cytokines,
growth factors,
enzyme inhibitors, antibiotics, antiviral agents, anti-inflammatory including
steroids, immune
boosters, antimetabolites, soluble cytokine receptors, and vitamin D or agents
that increase
the level of circulating vitamin D, toll-like receptor agonists, CpG
oligodeoxynucleotides,
surface charged poly(lactide-co-glycolide) microparticles, poly(lactide)
microparticles, any of
the above encapsulated into liposomes, archaeosome adjuvants, mucosal
adjuvants,
polyphosphazenes. Additional therapeutically active agents also include
copolymers, which
bind to a HLA molecule associated with the disease such as another amino acid
copolymer
composition of the disclosure. The HLA molecule may be an HLA-DQ molecule, an
HLA-
DP molecule or an HLA-DR molecule. The enzyme inhibitor may be a protease
inhibitor or
a cyclooxygenase inhibitor. Examples of the therapeutically active agents to
be administered
in conjunction with the amino acid copolymer composition are recited in
Section IV,
"Pharmaceutical Composition" section, though the administration of these
agents are not
limited to co-administration as a single composition. The additional
therapeutic agents may
be administered before, concomitantly with, or after the administration of the
amino acid
copolymer composition, at such time that the effect of the additional
therapeutic agents and
the effect of the amino acid copolymer composition overlap at some time point.
Alternatively, antigen/epitope non-specific treatments and therapies directly
targeted
at controlling T lymphocytes or their functions may be administered in
conjunction with an
amino acid copolymer composition of the disclosure. The therapeutic agents
useful for such
treatment include Muromonab-CD3 (OKT3), antilymphocyte globulin (ALG),
antithymocyte
globulin (ATG), or interleukin-2 receptor monoclonal antibody ("mAb")
daclizumab or
basiliximab. Other agents include soluble CTLA-4, an anti-CD154 mAb; anti-CD11
a; a
humanized mAb which inhibits VLA-4; anti-CD2, 3, or 4 antibodies; and anti-
CD152
antibodies (J.B. Matthews et al., Amer. I Transplantation, 2003, 3: 794-80).
When treating protein conformation diseases (e.g. Alzheimer's disease (AD),
frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17),
progressive
supranuclear palsy, ALS, PD, etc.) it may be advantageous to administer the
amino acid
copolymer therapeutic or amino acid copolymer-specific antibody therapeutic in
combination
with one or more additional therapy.
In one embodiment of the methods described herein, the route of administration
can
be oral, intraperitoneal, intradermal, transdermal, subcutaneous, by
intravenous or
intramuscular injection, by inhalation, topical, intralesional, or by
infusion; liposome-
mediated delivery; intrathecal, gingival pocket, rectal, intravaginal,
intrabronchial, nasal,
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transmucosal, intestinal, ocular or otic delivery, or any other methods known
in the art as one
skilled in the art may easily perceive. Administration can be systemic or
local. In the event
more than one amino acid copolymer composition of the disclosure is being
administered to a
subject during the same or overlapping time period, such additional
therapeutic agent may be
administered by the same or a route different rote of administration. In
certain embodiments,
the composition is formulated for any of the foregoing routes of
administration, such as
intradermal, transdermal, intramuscular or subcutaneous.
In general, an embodiment of the disclosure is to administer a suitable dose
of a
therapeutic amino acid copolymer composition of the disclosure that will be
the lowest
effective dose to produce a therapeutic effect, for example, mitigating
symptoms. The
therapeutic amino acid copolymer compositions are preferably administered at a
dose per
subject, which corresponds to a dose per month of at least about 0.5 mg, at
least about 1 mg,
at least about 2 mg, at least about 5 mg, at least about 10 mg, or at least
about 20 mg as
appropriate minimal starting dosages, or about x mg, wherein x is an integer
between 0.5 and
20. In some embodiments, the compositions are administered once a month, twice
a month,
three times a month, once a week, or once daily. In some embodiments, the
compositions are
administered once a month. In one embodiment of the methods described herein,
a dose of
about 0.01 to about 10 mg/kg can be administered. In some embodiments, the
effective
dosage of the amino acid copolymer composition of the present disclosure is
about 50 to
about 400 micrograms of the composition per kilogram of the subject per day.
In some
embodiments, each individual dosage in the treatment regimen is from about 0.5
to 20, or
more preferably from about 1 to 10mg/dose. In some embodiments, the preferred
dosing
regimen is 1-10 mg once a month. In some embodiments, the preferred dosing
regimen is 1-5
mg, 2-6 mg, 3-7 mg, 4-8 mg, or 5-10 mg once a month. In some embodiments, the
preferred
dosing regiment is 0.5-5 mg twice a month. In some embodiments, the preferred
dosing
regimen is 0.25-2.5 mg once a week. In some embodiments, the preferred dosing
regimen is
0.5-5 mg twice a month. In some embodiments, the preferred dosing regimen is
0.03-0.3 mg
daily.
However, it is understood by one skilled in the art that the dose of the amino
acid
copolymer composition of the disclosure will vary depending on the subject,
the condition
being treated and upon the particular route of administration used. It is
routine in the art to
adjust the dosage to suit the individual subjects. Additionally, the effective
amount may be
based upon, among other things, the size of the polypeptides or amino acid
copolymers, the
biodegradability of the polypeptides or amino acid copolymers, the bioactivity
of the
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polypeptides or amino acid copolymers and the bioavailability of the
polypeptides or amino
acid copolymers. If the amino acid copolymer compositions do not degrade
quickly, such as
is expected when the compositions comprise unnatural amino acids or comprise
peptidomimetics, is bioavailable and highly active, a smaller amount will be
required to be
effective. The actual dosage suitable for a subject can easily be determined
as a routine
practice by one skilled in the art, for example a physician or a veterinarian
given a general
starting point. For example, the physician or veterinarian could start doses
of an amino acid
copolymer composition of the disclosure employed in the pharmaceutical
composition at a
level lower than that required in order to achieve the desired therapeutic
effect, and increase
the dosage with time until the desired effect is achieved. The dosage of the
amino acid
copolymer composition may either be increased in the event the patient does
not respond
significantly to current dosage levels, or the dose may be decreased if an
alleviation of the
symptoms of the disorder or disease state is observed, or if the disorder or
disease state has
been ablated, or if an unacceptable side effects are seen with the starting
dosage.
In one embodiment, a therapeutically effective amount of an amino acid
copolymer
composition of the disclosure is administered to the subject in a treatment
regimen
comprising intervals administering more than one dose at intervals over some
period of time.
Treatment regimens with longer dosing intervals (time between
administrations),
consequently often with lower total exposure of polypeptides or amino acid
copolymers, are
expected to induce lower titers of antibodies against the polypeptides or
amino acid
copolymers themselves, while still inducing desired protective effects. Such
reduction of
neutralizing antibodies are desirable because it is considered likely to help
the amino acid
copolymer compositions of the disclosure to retain their effectiveness without
being
neutralized, and it is associated with reduced risk of anaphylactic shocks,
providing safer
treatments of diseases.
In one embodiment, an amino acid copolymer composition of the disclosure is
administered to be subject at least three times during a treatment regimen,
such that there are
at least two time intervals between administrations.
In other embodiments of the disclosure, any of the methods of the disclosure
may be
practiced using a sustained release formulation comprising an amino acid
copolymer
composition of the disclosure. For such sustained release administration, such
method
comprises applying a sustained-release transdermal patch or implanting a
sustained-release
capsule or a coated implantable medical device so that a therapeutically
effective dose of the
copolymer of the present disclosure is delivered at defined time intervals to
a subject of such
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a method. The amino acid copolymer composition of the subject disclosure may
be delivered
via a capsule, which allows regulated-release of the compositions over a
period of time.
Controlled or sustained-release compositions include formulation in lipophilic
depots (e.g.,
fatty acids, waxes, oils). Also comprehended by the disclosure are particulate
compositions
coated with polymers (e.g., poloxamers or poloxamines). In certain
embodiments, a source
of an amino acid copolymer composition is stereotactically provided within or
proximate to
the area where pathology is observed or suspected.
An improvement in the symptoms of a subject afflicted with a disease as a
result of
administration of the amino acid copolymer composition may be noted by a
decrease in
frequency of recurrences of episodes of the disease symptoms, by decrease in
severity of
symptoms, and by elimination of recurrent episodes for a period of time after
the start of
administration. A therapeutically effective dosage preferably reduces symptoms
and
frequency of recurrences by at least about 20%, for example, by at least about
40%, by at
least about 60%, and by at least about 80%, or by about 100% elimination of
one or more
symptoms, or elimination of recurrences of the autoimmune disease, relative to
untreated
subjects. The period of time can be at least about one month, at least about
six months, or at
least about one year.
b) Administration of antibodies generated using the amino acid copolymer
compositions of
the disclosure
An aspect of the present disclosure is a method of treating a subject
afflicted with a
disease or condition that would benefit for an antigen-specific immune
response (e.g. protein
conformational disorders, pathogenic infections, etc.), comprising the steps
of administering
an antibody prepared using an amino acid copolymer composition of the
disclosure.
One aspect of the present disclosure is a method of treatment using antibodies
against
a composition of the disclosure related to a disease, in particular, a protein
conformational
disease. The antibodies useful for such method of treatment are antibodies
generated against
an amino acid copolymer composition of the disclosure, wherein the base
peptide sequence is
a sequence of a protein known to be associated with a protein conformational
disorder. More
particularly, such protein is known to form an aggregate or fibril. In
particular, antibodies
thus generated are specific to the pathological conformation of such protein.
The list of
relevant diseases is recited above in this specification.
One aspect of the present disclosure is a method of treatment using antibodies
against
a composition of the disclosure related to a disease, in particular, a
pathogenic infection. The
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antibodies useful for such method of treatment are antibodies generated
against an amino acid
copolymer composition of the disclosure, wherein the base peptide sequence is
a sequence of
a protein known to be associated with a pathogenic infection. In some
embodiments, the
pathogen is an intracellular pathogen. In particular, antibodies thus
generated are specific to
the pathogenic protein. The list of relevant diseases is recited above in this
specification.
In an embodiment of this aspect of the disclosure, the antibodies for the use
in the
method of treatment are modified antibodies having an engineered Fc region,
wherein the
engineered Fc region confers favorable pharmacodynamic profiles. In one
embodiment, the
Fc region enhances clearance of antibody-antigen complex. In another
embodiment, the Fc
region is not immunogenic to the subject.
An aspect of the present disclosure is a method of treating a subject
afflicted with a
protein conformational disorder, comprising the steps of administering an
antibody prepared
using an amino acid copolymer composition of the disclosure. In a particular
embodiment,
the protein conformational disorder is Parkinson's disease. In another
embodiment, the
protein conformational disorder is ALS. In another embodiment, the protein
conformational
disorder is Alzheimer's disease.
An aspect of the present disclosure is a method of treating a subject
afflicted with a
pathogenic infection, comprising the steps of administering an antibody
prepared using an
amino acid copolymer composition as described above. In some embodiments, the
pathogen
is a bacterium or a virus. In some embodiments, the pathogen is of the genus
selected from
Chlamydia, Rickettsia, Coxiella, Mycobacterium, Francisella, Listeria,
Salmonella, Brucella,
Legionella, Nocardia, Rhodococcus, Yer sinia, and Neisseria.
In an aspect of the disclosure, an antibody or antibodies identified by the
method to
generate antibodies against antigens associated with a disease or condition is
cloned. The
nucleic acids encoding such antibodies are cloned into an appropriate
expression vector and
delivered to a cellular site where such antibodies are desirable, at which
site the antibodies
are expressed.
Another aspect of the present disclosure is a method of treating a subject
afflicted
with a disease or condition, comprising the steps of contacting under sterile
conditions the
blood of the subject to a membrane or a resin having conjugated with
antibodies specific to a
protein associated with the disease or condition and prepared using an amino
acid copolymer
composition of the disclosure, such antibodies described above, wherein the
protein
associated with the disease or condition binds to such antibodies and is
removed from the
blood, and returning the blood to the subject. In some embodiments, the
disease or condition
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is a protein conformational disorder. In some embodiments, the disease or
condition is a
pathogenic infection.
An embodiment of the disclosure is a method of prophylactic treatment of a
subject at
risk for developing a disease or condition by contacting under sterile
conditions the blood of
the subject to a membrane or a resin having conjugated with antibodies
specific to a protein
associated with the disease or condition and prepared using an amino acid
copolymer
composition of the disclosure, such antibodies described above, wherein the
protein
associated with the disease or condition binds to such antibodies and is
removed from the
blood, and returning the blood to the subject, whereby preventing the onset of
such disease or
condition. In some embodiments, the disease or condition is a protein
conformational
disorder. In some embodiments, the disease or condition is a pathogenic
infection.
An aspect of the disclosure is a composition comprising a scaffold to which
antibodies are attached, which antibodies are generated against an amino acid
copolymer
composition of the disclosure, wherein the base peptide sequence is derived
from a sequence
of a protein known to be associated with a disease or condition. In some
embodiments, the
base peptide sequence is derived from a sequence of a protein known to be
associated with a
protein conformational disorder. In some embodiments, the base peptide
sequence is derived
from a sequence of a protein known to be associated with a pathogenic
infection. In one
embodiment, the scaffold is a membrane compatible with haemodialysis. In a
particular
embodiment, the antibodies are conjugated to such membrane. In another
embodiment, the
antibodies are conjugated to a resin, such as CN-Br agarose resin (for example
CN-Br
Sepharose (Pharmacia), to create an immunoaffinity resin.
An aspect of the disclosure is a composition comprising antibodies against a
composition of the disclosure related to a disease, in particular, a protein
conformational
disease, or a target (e.g., an antigen). In a particular embodiment, an
antibody of the
disclosure is an antibody, or combination of antibodies, that binds to at
least one protein
associated with a protein conformational disorder. In certain embodiments, an
antibody of
the disclosure is an antibody, or combination of antibodies, that binds to at
least one protein
associated with Parkinson's disease. In certain embodiments, an antibody of
the disclosure is
an antibody, or combination of antibodies, that binds to at least one protein
associated with
ALS. In certain embodiments, an antibody of the disclosure is an antibody, or
combination
of antibodies, that binds to at least one protein associated with Alzheimer's
disease. Also
provided are methods in which a composition of the disclosure is used to
raise, induce or
generate antibodies, such as by administering to a human or non human subject.
In certain
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embodiments, a composition of the disclosure can be used to isolate
antibodies. For example,
a composition of the disclosure may be affixed to a column or used in another
screening
system to isolate antibodies, such as antibodies generated following
administration of that
same composition to a subject.
c) Other methods and uses
Compositions of the disclosure have numerous other uses. For example,
compositions of the disclosure are useful for generating antibodies, such as
for use as
reagents, and the instant disclosure also comprises a method of generating
antibody reagents
for use in, for example, research studies in vitro or in vivo. For example,
compositions of the
disclosure can be used to immunize a host, such as a mouse or rabbit, and
serum from the
animal is harvested. Similarly, monoclonal antibodies may be generated using
known
techniques following challenge with a composition of the disclosure.
Certain antibodies generated or selected by their specific binding to an amino
acid
copolymer composition of the disclosure are useful to identify specific
conformation of a
protein in its pathological and non-pathological state. Such antibodies, when
conjugated to
scaffolds, are further useful for isolating and purifying the target proteins
and polypeptides.
Such antibodies are also useful in preclinical investigations of candidate
pharmaceutical
agents, wherein such agent may disrupt or disturb the binding of such
antibodies to the target
proteins. The antibodies can also be used to detect certain pathological
antibodies and to
measure the effects of such candidate pharmaceutical agents. Compositions of
the disclosure
may also be used to isolate antibodies of interest.
The instant disclosure also comprises a method of creating antibody reagents
for use
as diagnostic tools.
An embodiment of the disclosure provides, a method of diagnosing a disease or
condition, comprising: (i) contacting a biological sample from a subject with
an antibody of
the disclosure (e.g., a monoclonal or polyclonal antibody raised by immunizing
a host with a
composition of the disclosure); (ii) contacting a control sample with the
antibody; and (iii)
measuring specific binding of the antibody to an antigen in the sample;
wherein specific
binding of the antibodies to the antigen is indicative of the subject being
afflicted with the
disorder. A number of methods for measuring antibody-protein binding are known
in the art,
including ELISA, Western blotting, and spot-blot. The control sample may be a
standard
sample, a sample from a second subject known to be free of the pathology that
is being
investigated, or a sample from the same subject at a different time point to
determine the
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chronological changes of the disease conditions. In some embodiments, tests
are performed
simultaneously using the antibody of the disclosure and a positive control
antibody that
confirms that the biological sample contains sufficient material. The positive
control
antibody may recognize any protein that is reasonably expected to be present
in all samples
(i.e. from both healthy and diseased patients), and may recognize a
housekeeping enzyme (for
example). In some embodiments, the binding of the antibody of the disclosure
is quantified;
in other embodiments, the binding is evaluated qualitatively. In some
embodiments, the
disease or condition is a protein conformational disorder. In some
embodiments, the disease
or condition is a pathogenic infection.
More particularly, such disorder to be detected is one of the disorders
enumerated
elsewhere in this application.
In some embodiments, the diagnostic test may be performed in vivo, identifying
the
affected locations within the body. The antibody may be labeled in such a
manner that it can
be detected within a patient's body, e.g. with MRI. This label may be an iron-
containing
compound, such as ferrous and ferric-containing compounds, e.g. ferric-oxides.
Specific
examples include Fe2O3 and Fe304. Antibodies labeled with iron-containing
compounds may
also be used for in vitro diagnosis, e.g. when MRI is performed on a
biological sample. In a
variation of the foregoing, antibodies generated using compositions of the
disclosure can be
used for imaging analysis, such as to examine biodistribution of a target in
vitro or in vivo.
In one aspect, the compositions of the disclosure are useful for eliciting the
release of
a Th2-associated cytokine or chemokine from cells of the immune system in
vitro or in vivo.
In some embodiments, the immune cells are monocytes. In some embodiments, the
Th2
associated cytokine is IL-4, IL-5, IL-6, IL-10, or IL-13. In some embodiments,
the Th2
associated chemokine is CCL17 or CCL22. In some embodiments, the compositions
of the
disclosure are useful for eliciting the release of CCL22 from immune cells.
Indeed,
exemplary compositions described herein (e.g. DP-0016.B, DP-0016.C, etc.) have
been
shown to induce CCL22 release from monocytic cells. Accordingly, the
disclosure provides
such methods.
In one aspect, the compositions of the disclosure are useful for eliciting
preferential
proliferation of CD4+ T-cells T in vitro or in vivo. In some embodiments, the
compositions
of the disclosure elicit preferential CD4+ T-cell proliferation amongst
peripheral blood
monocytes contacted with the compositions. In some embodiments, the
compositions of the
disclosure elicit preferential CD4+ T-cell proliferation amongst splenocytes
contacted with
the compositions. Indeed, exemplary compositions described herein (e.g. DP-
0016.B, DP-
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C016.C, etc.) have been shown to induce preferential CD4+ T-cell
proliferation. Accordingly,
the disclosure provides such methods.
Compositions of the disclosure may be used to study a disease or condition.
For
example, compositions of the disclosure may be used to identify other proteins
involved in a
disease or condition. Compositions of the disclosure may be used to evaluate
disease
pathology and progression in cells or animal models of the condition.
Compositions of the
disclosure may be used to generate reagents suitable for further studying a
disease or
condition in vitro or in vivo. Compositions of the disclosure may be used to
induce/promote
CD4+ T-cell proliferation in vitro or in vivo, such as in animal models of
disease.
Ha Methods of Administration
Various delivery systems are known and can be used to administer compositions
of
the disclosure (whether provided alone or with an adjuvant or other agent ¨
and including
when provided as a pharmaceutical composition), e.g., various formulations,
encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable of
expressing the
compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.
Chem.
262:4429-4432). Methods of introduction can be enteral or parenteral,
including but not
limited to, intradermal, transdermal, intramuscular, intraperitoneal,
intravenous,
subcutaneous, pulmonary, intranasal, intraocular, epidural, and oral routes.
In particular
embodiments, parenteral introduction includes intramuscular, subcutaneous,
intravenous,
intravascular, and intrapericardial administration. In certain embodiments,
introduction is
intracranially or intraventricularly or into the cerebrospinal fluid.
The present disclosure provides systemic delivery of one or more doses of
composition of the disclosure. Systemic delivery includes, for example,
subcutaneous,
intravenous, or intramuscular. In certain embodiments, delivery comprises
subcutaneous
delivery.
In the case of in vitro methods, administration may comprise contacting a
culture of
cells with a composition of the disclosure, e.g., by adding composition to the
culture media.
The composition and route of administration is chosen depending on the
particular
use of the technology. For example, a different composition and/or route of
administration
may be appropriate when using the compositions of the disclosure for research
purposes, such
as in vitro or in an animal model, versus when using for diagnostic or
therapeutic purposes in
human patients or animal subjects. Similarly, the route of administration may
vary
depending on the target (e.g., the antigen upon which the ARs are based). One
of skill in the
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art can select the appropriate route of administration depending on the
particular application
of the technology.
IX Pharmaceutical Compositions
One aspect of the present disclosure is a pharmaceutical composition
comprising an
amino acid copolymer composition of the disclosure (also referred to as a
"composition of the
disclosure"). In certain embodiments, the compositions of the disclosure are
useful in the
treatment of a disease or condition amenable to treatment with an
immunotherapy (e.g.,
where it would be beneficial to both increase the immune response, preferably
T-cell
proliferation and/or to generate an antigen-specific immune response. Suitable
diseases or
conditions include, for example, protein conformational disorders, pathogenic
infections,
other conditions amenable to treatment via immunotherapy, etc.). In other
embodiments,
compositions of the disclosure are suitable for use in animal models of
disease and/or to cells
in culture. In certain embodiments, compositions of the disclosure are
provided as
.. pharmaceutical compositions.
Compositions of the disclosure may be formulated as a pharmaceutical
composition
comprising one or more pharmaceutically acceptable carriers and/or excipients.
The compositions and pharmaceutical compositions of the disclosure may be
formulated for administration in any convenient way for, for example, use in
human or
veterinary medicine. Wetting agents, emulsifiers and lubricants, such as
sodium lauryl
sulfate and magnesium stearate, as well as coloring agents, release agents,
coating agents,
sweetening, flavoring and perfuming agents, preservatives and antioxidants can
also be
present in the compositions.
Formulations of the compositions of the disclosure include those suitable for
oral,
nasal, topical, parenteral, rectal, and/or intravaginal administration. The
formulations may
conveniently be presented in unit dosage form and may be prepared by any
methods well
known in the art of pharmacy. The amount of active ingredient, which can be
combined with
a carrier material to produce a single dosage form, will vary depending upon
the host being
treated and the particular mode of administration. The amount of active
ingredient, which
can be combined with a carrier material to produce a single dosage form, will
generally be
that amount of the compound which produces a therapeutic effect.
In certain embodiments, methods of preparing these formulations or
compositions
include combining the therapeutic agent and a carrier and, optionally, one or
more accessory
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ingredients. In general, the formulations can be prepared with a liquid
carrier, or a finely
divided solid carrier, or both, and then, if necessary, shaping the product.
Pharmaceutical compositions suitable for parenteral administration may
comprise one
or more compositions of the disclosure in combination with one or more
pharmaceutically
acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions,
suspensions or
emulsions, or sterile powders which may be reconstituted into sterile
injectable solutions or
dispersions just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes
which render the formulation isotonic with the blood of the intended recipient
or suspending
or thickening agents. Examples of suitable aqueous and non-aqueous carriers,
which may be
employed in the pharmaceutical compositions of the disclosure include water,
ethanol,
polyols (such as glycerol, propylene glycol, polyethylene glycol, and the
like), and suitable
mixtures thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl
oleate. Proper fluidity can be maintained, for example, by the use of coating
materials, such
as lecithin, by the maintenance of the required particle size in the case of
dispersions, and by
the use of surfactants.
These compositions may also contain adjuvants, such as preservatives, wetting
agents,
emulsifying agents and dispersing agents. Prevention of the action of
microorganisms may
be ensured by the inclusion of various antibacterial and antifungal agents,
for example,
paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into the
compositions. In
addition, prolonged absorption of the injectable pharmaceutical form may be
brought about
by the inclusion of agents, which delay absorption, such as aluminum
monostearate and
gelatin.
In certain embodiments of the present disclosure, the compositions of the
disclosure
are formulated in accordance with routine procedures as a pharmaceutical
composition
adapted for intravenous or subcutaneous administration to human beings. Where
necessary,
the composition may also include a solubilizing agent and a local anesthetic
such as lidocaine
to ease pain at the site of the injection.
The amount of the compositions of the disclosure for use in the methods of the
present disclosure can be determined by standard clinical techniques and may
vary depending
on the particular indication or use. Effective doses may be extrapolated from
dose-response
curves derived from in vitro or animal model test systems.
In certain embodiments, compositions of the disclosure, including
pharmaceutical
preparations, are sterile and/or non-pyrogenic. In other words, in certain
embodiments, the
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compositions are sterile and/or substantially pyrogen free. In one embodiment
the
formulations of the disclosure are pyrogen-free formulations, which are
substantially free of
endotoxins and/or related pyrogenic substances. Endotoxins include toxins that
are confined
inside a microorganism and are released only when the microorganisms are
broken down or
die. Pyrogenic substances also include fever-inducing, thermostable substances
(glycoproteins) from the outer membrane of bacteria and other microorganisms.
Both of
these substances can cause fever, hypotension and shock if administered to
humans. Due to
the potential harmful effects, even low amounts of endotoxins must be removed
from
intravenously administered pharmaceutical drug solutions. The Food & Drug
Administration
("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram
body weight
in a single one hour period for intravenous drug applications (The United
States
Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When
therapeutic
proteins are administered in relatively large dosages and/or over an extended
period of time
(e.g., such as for the patient's entire life), even small amounts of harmful
and dangerous
.. endotoxin could be dangerous. In certain specific embodiments, the
endotoxin and pyrogen
levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or
less then 1
EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001
EU/mg.
The foregoing applies to any of the compositions and methods described herein.
The
disclosure specifically contemplates any combination of the features of
compositions of the
.. present disclosure (alone or in combination) with the features described
for the various
pharmaceutical compositions and route of administration described in this
section.
Exemplary pharmaceutically acceptable carrier includes any solvents,
dispersion
media, or coatings that are physiologically compatible. Preferably, the
carrier is suitable for
oral, rectal, transmucosal (including by inhalation), parenteral, intravenous,
intramuscular,
intraperitoneal, intradermal, transdermal, topical, or subcutaneous
administration. One
exemplary pharmaceutically acceptable carrier is physiological saline. Other
pharmaceutically acceptable carriers and their formulations are well-known and
generally
described in, for example, Remington 's Pharmaceutical Science (18th Ed., ed.
Gennaro, Mack
Publishing Co., Easton, PA, 1990). Various pharmaceutically acceptable
excipients are well-
known in the art and can be found in, for example, Handbook of Pharmaceutical
Excipients
(4th ed., Ed. Rowe et at. Pharmaceutical Press, Washington, D.C.). The
composition can be
formulated as a solution, microemulsion, liposome, capsule, tablet, or other
suitable forms.
The active component, which comprises the copolymer, may be coated in a
material to
protect it from inactivation by the environment prior to reaching the target
site of action. The
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pharmaceutical compositions of the present disclosure are preferably sterile
and non-
pyrogenic at the time of delivery, and are preferably stable under the
conditions of
manufacture and storage. When desirable, the composition further comprises
components to
enhance stability, permeability, and/or bioavailability, such as particulate
forms protective
coatings, protease inhibitors or permeation enhancers for various routes of
administration,
including parenteral, pulmonary, nasal and oral.
In some embodiments, the pharmaceutical compositions also include additional
therapeutically active agents. Such additional ingredient can be one or more
of: an additional
amino acid copolymer composition that binds to a different target, an
additional amino acid
copolymer composition where the ARs are derived from a different portion of
the same target,
an antibody which activates inflammatory molecules, or cytokines. Further
additional
ingredient can be activating cytokines and chemokines (as described in Shaw,
Jennifer,
Infection and Immunity, 69:4667-4672, 2001) taken from the group consisting of
Mipl 0,
Mip 1 a, Mip-2, Mip3a, IP-10, MCP-1, TCA-3, IL-1, IL-18, IL-6, IFNy, MIF, IL-
12, CCL19,
and CCL21. Alternatively, the pharmaceutical composition does not include
these additional
agents, but any one or more of them can be administered or provided in a
separate
composition as part of a method of the disclosure (e.g., co-administration at
the same time or
at differing times).Further, a form of vitamin D that is or becomes
biologically active within
the body of the subject receiving such form of vitamin D may also be used as
an additional
ingredient. The two main forms of vitamin D are: vitamin D3 or
cholecalciferol, which is
formed in the skin after exposure to sunlight or ultraviolet light, and
ergocalciferol or vitamin
D2 which is obtained by irradiation of plants or plant materials or foods. The
differences are
situated in the side chain. Vitamin D3 may be obtained from natural sources
such as fatty
fish such as herring and mackerel. In the body, two other forms of vitamin D3
can be found.
Vitamin D3 is hydroxylated in the liver into 25-hydroxyvitamin D3 (25(OH)D),
and
subsequently in the kidney into 1,25-dihydroxyvitamin D3 (1,25(OH)2D), which
is the active
metabolite that stimulates the calcium absorption from the gut (Feldman et al.
, 2005). When
1,25(OH)2D is sufficiently available, 24,25-dihydroxyvitamin D (24,25(OH)2D)
is formed
in the kidney, which is further catabolized. Alternatively, the pharmaceutical
composition
does not include these additional agents, but any one or more of them can be
administered or
provided in a separate composition as part of a method of the disclosure
(e.g., co-
administration at the same time or at differing times).
In certain embodiments, the composition has intrinsic adjuvant properties and
is
capable of raising an immune response without an adjuvant. In some
embodiments, only a
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minimal or greatly reduced amount of adjuvant is required. In either case, in
certain
embodiments, pharmaceutical compositions of the disclosure optionally contain
an additional
adjuvant. In some embodiments, the adjuvant minimizes a broad inflammatory
response (e.g.,
does not promote a broad inflammatory response when administered). In some
embodiments,
the adjuvant promotes a broad inflammatory response. Alternatively, in certain
embodiments,
any of the methods described herein may optionally comprise administering an
additional
adjuvant before, concurrently with, or after administration of a compositions
or
pharmaceutical composition of the disclosure. In some embodiments, the
adjuvant minimizes
a broad inflammatory response. In some embodiments, the adjuvant promotes a
broad
inflammatory response. In some embodiments, an amino acid copolymer
composition of the
disclosure is formulated and/or administered with alum as an adjuvant. In some
embodiments, an amino acid copolymer composition of the disclosure is
formulated and/or
administered with aluminum hydroxide or aluminum phosphate. In some
embodiments, the
composition, whether formulated and/or administered with or without an
adjuvant, is
formulated for intradermal, transdermal, intramuscular or subcutaneous
injection. In some
embodiments, the composition, whether formulated and/or administered with or
without an
adjuvant, is formulated for intramuscular delivery. In some embodiments, the
composition,
whether formulated and/or administered with or without adjuvant, is formulated
for
transdermal delivery.
Another aspect of the disclosure is a pharmaceutical composition comprising
one or
more antibodies generated and produced using the process described herein
elsewhere. An
antibody or antibodies that react to a protein conformational disease can be
used to neutralize
pathological proteins that such antibodies specifically bind, or to facilitate
clearing from the
body of a patient afflicted with such disease.
Pharmaceutically acceptable carriers and their formulations are well-known and
generally described in, for example, Remington's Pharmaceutical Science (18th
Ed., ed.
Gennaro, Mack Publishing Co., Easton, PA, 1990). Various pharmaceutically
acceptable
excipients are well-known in the art and can be found in, for example,
Handbook of
Pharmaceutical Excipients (4th ed., Ed. Rowe et at. Pharmaceutical Press,
Washington, D.C.).
Further, formulations suitable for antibodies are generally known in the art,
including buffers
and excipients, and preservative agents such as protease inhibitors that are
suitable for
pharmaceutical use. The pharmaceutical compositions of the present disclosure
are
preferably sterile and non-pyrogenic at the time of delivery.
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The compositions may be formulated for administration by injection, e.g., by
bolus
injection or continuous infusion in a parenteral, intravenous,
intraperitoneal, intramuscular, or
subcutaneous manner. Formulations for injection may be presented in unit
dosage form, e.g.,
in ampoules or in multi-dose containers, with an added preservative. The
compositions may
take such forms as suspensions, solutions or emulsions in oily or aqueous
vehicles, and may
contain formulatory agents such as suspending, stabilizing and/or dispersing
agents.
Alternatively, the active ingredient may be in powder form for reconstitution
with a suitable
vehicle, e.g., sterile pyrogen free water, before use.
In certain embodiments, antibodies are single chain variable fragments, to
facilitate
transport into the tissues due to its smaller size compared to naturally
occurring antibodies.
Such antibodies may further be associated with a carrier or agent to cross the
blood brain
barrier, for example, an anti-transferring antibody. See, for example, Friden
et al., Anti-
transferrin receptor antibody and antibody-drug conjugates cross the blood-
brain barrier,
PNAS June 1, 1991 vol. 88 no. 11 4771-4775.
X Methods of Screening for Amino acid Copolymers with Antigenic Specificity
In one aspect, the disclosure provides methods of identifying suitable amino
acid
copolymer compositions for use in one or more methods of the disclosure. In
some
embodiments, suitable amino acid copolymer compositions are identified based
on their
ability to induce proliferation of CD4+ T-cells, such as in assays using
peripheral blood
mononuclear cells (PBMCs). In some embodiments, the PBMCs are human PBMCs. In
some embodiments, suitable amino acid copolymer compositions are identified
based on their
ability to stimulate Th2 chemokine (e.g., CCL22, CCL17, etc.) secretion from
monocytes. In
some embodiments, the Th2 chemokine is CCL22. However, it is contemplated that
although
one chemokine may be used to identify suitable characteristics of a
composition, the
composition may be capable of stimulate secretion/release of additional
chemokines. In some
embodiments, the monocytes are mouse monocytes. In some embodiments, the
monocytes
are human monocytes.
In one aspect, a method of screening for suitable amino acid copolymer
compositions
comprises testing candidate compositions in one or both of the assays
described above (i.e., a
CD4+ T cell proliferation assay and a Th2 chemokine (e.g., CCL22, CCL17, etc.)
release
assay).
In one aspect, the ability of an amino acid copolymer composition to stimulate
Th2
chemokine (e.g., CCL22, CCL17, etc.) release is predictive of its CD4+ T-cell
proliferative
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ability. Thus, in some embodiments, a Th2 chemokine (e.g., CCL22, CCL17, etc.)
release
assay alone is sufficient to identify suitable compositions.
More generally, for any of the foregoing, in certain embodiments, the method
comprises providing a candidate amino acid copolymer composition and
evaluating the
composition in one or both of an assay to evaluate CD4+ T-cell proliferation
or Th2
chemokine (e.g., CCL22, CCL17, etc.) release from monocytes. The effect of the
candidate
composition may be evaluated versus one or more relevant controls, such as a
single
polypeptide or a mixture of polypeptides lacking RCR region(s). A candidate
amino acid
composition that tests positive, relative to suitable controls, in one or both
assays is identified
as a suitable amino acid copolymer composition (e.g., suitable for use in
vitro or in vivo to
promote CD4+ T-cell proliferation or Th2 chemokine (e.g., CCL22, CCL17, etc.)
release
from monocytes; suitable for use as an immunotherapeutic; suitable to study an
immune
stimulatory response in vitro and/or in vivo; etc.).
EXEMPLIFICATION
The disclosure now being generally described, it will be more readily
understood by
reference to the following examples, which are included merely for purposes of
illustration of
certain aspects and embodiments of the present disclosure, and are not
intended to limit the
disclosure.
Example 1. Preparation of an amino acid copolymer composition with antigenic
specificity to Tau protein (DP-0016.B)
Figure 2 shows a schematic for the design of an amino acid copolymer
composition (a
composition based on the DP-0016.B template arrangement, sequence, and input
amino acid
input distribution), comprising a high complexity (500 or greater peptides)
mixture of
polypeptides based on antigenic sequences derived from Tau. DP-0016.B is
designed to
target multiple phosphorylation patterns among 9 potential phosphorylation
sites in the C-
terminus of Tau and can be used to generate specific antibodies against many
variants of
early phosphorylated Tau protein in oligomers and paired helical filaments
(PHFs). The
substantially full-length polypeptides of DP-0016.B in this schematic are 60
amino acids
long and the amino acid positions, relative to full-length polypeptides based
on the depicted
template arrangement, of the polypeptides of the composition are indicated in
Row 1 of the
top and bottom panels (the row labeled peptide #). The DP-0016.B polypeptides
(e.g., the
peptides in the mixture of polypeptides comprising the DP-0016.B composition)
are based on
a template arrangement having 2 random copolymer regions (RCRs) wherein the
amino acid
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at each position is selected from Alanine, Lysine or Glutamic Acid, i.e., the
RCRs comprise
A/K/E motifs (N-terminal RCRa (corresponding to positions 1-8 in Figure 2) and
RCRb
(corresponding to positions 28-36 in Figure 2)) and two antigenic regions
(ARs) based on
sequences derived from human Tau-F (ARa (corresponding to positions 9-27 in
Figure 2) and
ARb (corresponding to positions 37-60 in Figure 2)). In this example, the base
peptide
sequence upon which ARa is based is set forth in SEQ ID NO: 9, and the base
peptide
sequence upon which ARb is based is set forth in SEQ ID NO: 10. The template
arrangement
of DP-0016.B comprises: RCRa-ARa-RCRb-ARb, although other template
arrangements are
described herein. In this example, RCRa and RCRb differ in length, and ARa and
ARb
correspond to or comprise different base peptide sequences, although each
derived from
human Tau-F. In this example, complexity across the polypeptides in the
composition comes
from both the RCRs and the ARs although, in other examples, complexity may
come only
from the RCRs or from the RCRs and one, but not both, ARs. In this example,
the RCRs and
ARs are contiguous. However, they may be interconnected by one or more
linkers, such as
by one or more intervening amino acid residues. Moreover, the polypeptides in
the
composition may include one or more additional amino acid residues at the N-
or C-terminus,
including natural or non-natural amino acids.
Row 2 of the top and bottom panels (the row labeled Tau-F #) indicates the
positions
in human Tau-F (SEQ ID No. 17) that correspond to residues in the AR, which
are or may be
phosphorylated in this composition. Row 3 of the top and bottom panels
indicates the two
random copolymer regions (RCRs) corresponding to positions 1-8 (RCRa) and
positions 28-
36 (RCRb) (designated by the residue A ¨ although the actual sequence in an
individual
peptide will vary according to the input % of A/K/E; where for this example,
the input % is
provided in Rows 5-7), and the base peptide sequences for the two antigenic
regions derived
from human Tau-F (ARa (corresponding to positions 9-27 of Figure 2) and ARb
(corresponding to positions 37-60 of Figure 2)). The sequence of the AR given
in Row 3
corresponds to the base peptide sequence and reflects original amino acid
residues derived
from the corresponding portion of the target, human Tau-F.
Row 4 of the top and bottom panels (the row labeled Phospho %) indicates the
input
percentage of phosphorylation at the residues in the first AR at positions
corresponding to
positions 13, 15, 19, 22 and 23 (corresponding to Y394, S396, S400, T403, and
S404 of
human Tau-F), and in the second AR at positions corresponding to positions 41,
46, 52 and
54 (corresponding to S422, T427, S433 and S435 of human Tau-F). For instance,
phosphorylated tyrosine is provided at an input percentage of 45% at the
position
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corresponding to Y394 and phosphorylated serine is provided at an input
percentage of 100%
at the position corresponding to S396.
Rows 5-7 of the top and bottom panels indicate the different proportions of
alanine,
lysine, and glutamic acid that are provided at each position for generating
the individual
polypeptides of the mixture. Additional amino acids such as conserved
substitutions may be
provided at any of the positions in the antigenic region. In this example, the
input percentage
for each of the two RCRs is 50% A, 40% K and 10% E, for each position of each
RCR. As
described herein, other ratios, distributions, and input percentages for the
RCRs are also
contemplated. Similarly, RCRs of differing sizes (e.g., shorter or longer than
depicted) are
contemplated, as described herein.
Rows 8 and 9 of the top and bottom panels indicate the percentage of positive
and
negatively charged residues available at each position. The estimated
percentages of alanine,
positively charged residues and negatively charged residues across the entire
length of the
polypeptides across the composition are indicated in the rightmost column of
the bottom
panel. In this example, for each position of the two random copolymer regions,
the relative
molar input percentages of alanine, lysine, and glutamic acid, were 50%, 40%,
and 10%,
respectively. Therefore, each polypeptide has an A, K, or E at each position
of its random
copolymer regions, and the distribution in the polypeptides in the composition
is influenced
by these input percentages.
The AR at positions corresponding to positions 9-27 is based on a base peptide
sequence which corresponds to positions 390-408 of human Tau-F (SEQ ID No. 17)
and the
AR at positions corresponding to positions 37-60 is based on a base peptide
sequence which
corresponds to positions 418-441 of human Tau-F (SEQ ID No. 17). For this
particular
example, most positions of the ARs (e.g. positions 10-12, 14, 16-18, 20, 21,
24-27, 37-40, 42-
44, 47, 49-51, 55, 57-60) specify a relative molar input percentage of 10%
alanine for
generating complexity at those positions across the mixture of polypeptides,
with the original
amino acid (relative to the native human Tau-F sequence) making up the
remaining 90% of
the input molar percentage of amino acid at each position and for generating
complexity at
those positions across the mixture. Therefore, in this example, at these
positions, each
polypeptide has either the original amino acid indicated in Row 3 or alanine
in the ARs.
However, additional complexity could be provided by, for example, also
permitting variation
at a position based on a conserved substitution, such as an orthologous
substitution, a residue
that is present in a different species, a phosphorylated or nitrated residue,
a residue that is
observed based on polymorphism or natural variation across humans, or a
substitution with a
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similar amino acid residue that is considered interchangeable or a
conservative substitution.
For certain other positions in this example, such as positions 9, 45, 48, 53,
and 56, no amino
acids other than the residue present at the corresponding position of the base
peptide are
provided. For the positions corresponding to Y394, S400, T403, T427, S433, and
S435,
relative molar input percentages of 10% alanine, 45% non-phosphorylated amino
acid, and
45% phosphorylated amino acid are provided for the generation of the mixture.
Therefore, at
these positions, each polypeptide has the phosphorylated amino acid, the non-
phosphorylated
amino acid, or alanine. For the positions corresponding to S396, S400, S404
and S422, the
relative molar input percentage of phospho-serine is 100%. Additional amino
acids such as
conserved substitutions may be provided at any of the positions. This provides
a description
of this particular composition. However, other compositions based on, for
example, differing
template arrangements, input percentages in the RCRs or ARs, and/or base
peptide sequence
or antigen are contemplated and provided.
A polypeptide mixture according to the schematic shown in Figure 2 was
successfully
synthesized using solid-phase peptide synthesis (SPPS). Briefly, polypeptides
based on this
template arrangement and input percentages were synthesized from C-terminus to
N-terminus
using Fmoc-based SPPS. Automated peptide synthesis is performed on a
continuous flow
peptide synthesizer. The first amino acid at the C-terminal position is a
Norleucine (Nle)
covalently coupled to the resin support (not shown in Figure 2). At each
position and for each
polypeptide generated, Fmoc is cleaved and an amino acid is randomly selected
from the
amino acid solution described in Figure 2 for each position. For example, at
position 60, the
amino acid solution for coupling contains 10% alanine (A) and 90% leucine (L),
and thus, for
each polypeptide, alanine or leucine is incorporated at a position
corresponding to position
60. The final step involves cleavage of the linear polypeptide from the solid
resin support.
Further, DP-0016.B was recognized by an anti-Tau paired helical filament pS396
monoclonal antibody (data not shown). This demonstrates that the polypeptides
of the
mixture are in the correct conformation to induce antibodies against paired
helical filaments.
Example 2. Preparation of an amino acid copolymer composition with antigenic
specificity to Tau protein (DP-0016.C)
Figure 3 shows a schematic for the design of an amino acid copolymer
composition (a
composition based on the DP-0016.0 template arrangement, sequence, and input
amino acid
input distribution) comprising a high complexity mixture of polypeptides based
on antigenic
sequences derived from Tau. DP-0016.C, like DP-0016.B, is designed to target
multiple
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phosphorylation patterns among 14 potential phosphorylation sites in the C-
terminus of Tau
and can be used to generate specific antibodies against many variants of early
phosphorylated
Tau protein in oligomers and paired helical filaments (PHFs). Full-length
polypeptides of
DP-0016.0 in this schematic are about 60 amino acids long and the amino acid
positions,
relative to full-length polypeptides based on the depicted template
arrangement, of the
polypeptides of the composition are indicated in Row 1 of the top and bottom
panels (the row
labeled peptide #). DP-0016.0 polypeptides (e.g., the polypeptides in the
mixture of
polypeptides comprising the DP-0016.0 composition) are each based on a
template
arrangement having 2 random copolymer regions (RCRs) wherein the amino acid at
each
position is selected from Alanine, Lysine, Glutamic Acid or Phenylalanine,
i.e., the RCRs
comprise A/K/E/F motifs (N-terminal RCRa (corresponding to positions 1-8 in
Figure 3) and
RCRb (corresponding to positions 28-36 in Figure 3)) and two antigenic regions
(ARs) based
on sequences derived from human Tau-F (ARa (corresponding to positions 9-27 in
Figure 3)
and ARb (corresponding to positions position 37-60 in Figure 3)). In this
example, the base
peptide sequence upon which ARa is based is set forth in SEQ ID NO: 9, and the
base
peptide sequence upon which ARb is based is set forth in SEQ ID NO: 10. The
template
arrangement of DP-0016.0 comprises: RCRa-ARa-RCRb-ARb, although other template
arrangements are described herein. In this example, RCRa and RCRb differ in
length, and
ARa and ARb correspond to or comprise different base peptide sequences,
although each
derived from human Tau-F. In this example, complexity across the polypeptides
in the
composition comes from both the RCRs and the ARs although, in other examples,
complexity may come only from the RCRs or from the RCRs and one, but not both,
ARs. In
this example, the RCRs and ARs are contiguous. However, they may be
interconnected by a
linker, such as by one or more intervening amino acid residues. Moreover, the
polypeptides
in the composition may include one or more additional amino acid residues at
the N- or C-
terminus, including natural or non-natural amino acids.
Row 2 of the top and bottom panels (the row labeled Tau-F #) indicates the
positions
in human Tau-F (SEQ ID No. 17) that correspond to residues in the AR which are
or may be
phosphorylated in this composition. Row 3 of the top and bottom panels
indicates the two
random copolymer regions (RCRs) corresponding to positions 1-8 (RCRa) and
positions 28-
36 (RCRb) (designated by the residue A ¨ although the actual sequence in an
individual
polypeptide will vary according to the input % of A/K/E/F; where for this
example, the
input % is provided in Rows 5-8), and the base peptide sequences for the two
antigenic
regions derived from human Tau-F (ARa (positions 9-27) and ARb (positions 37-
60)).
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Row 4 of the top and bottom panels (the row labeled Phospho %) indicates the
input
percentage of phosphorylation at the residues in the first AR at positions
corresponding to
positions 13, 15, 19, 22 and 23 (corresponding to Y394, S396, S400, T403, and
S404 of
human Tau-F), and in the second AR at positions corresponding to positions 41,
46, 52 and
.. 54 (corresponding to S422, T427, S433 and S435 of human Tau-F). For
instance,
phosphorylated tyrosine is provided at an input percentage of 45% at the
position
corresponding to Y394 and phosphorylated serine is provided at an input
percentage of 100%
at the position corresponding to S396.
Rows 5-8 of the top and bottom panels indicate the different proportions of
alanine,
lysine, glutamic acid, and phenylalanine that are provided at each position
for generating the
individual polypeptides of the mixture. Additional amino acids such as
conserved
substitutions may be provided at any of the positions in the antigenic region.
In this example,
the input percentage for each of the two RCRs is 50% A, 40% K, 5% E, and 5% F,
for each
position of each RCR. As described herein, other ratios, distributions, and
input percentages
for the RCRs are also contemplated. Similarly, RCRs of differing sizes (e.g.,
shorter or
longer than depicted) are contemplated, as described herein.
Rows 9 and 10 of the top and bottom panels indicate the percentage of positive
and
negatively charged residues available at each position. The estimated
percentages of alanine,
positively charged residues and negatively charged residues across the entire
length of the
.. polypeptides across the composition are indicated in the rightmost column
of the bottom
panel. In this example, for each position of the two random copolymer regions,
the relative
molar input percentages of alanine, lysine, glutamic acid, and phenylalanine,
were 50%, 40%,
5%, and 5% respectively. Therefore, each polypeptide has an A, K, E, or F at
each position
of its random copolymer regions, and the distribution in the polypeptides in
the composition
is influenced by these input percentages.
The ARa at positions corresponding to positions 9-27 is based on a base
peptide
sequence which corresponds to positions 390-408 of human Tau-F (SEQ ID No. 17)
and the
ARb at positions corresponding to positions 37-60 is based on a base peptide
sequence which
corresponds to positions 418-441 of human Tau-F (SEQ ID No. 17). For this
particular
example, most positions of the ARs (e.g. positions 10-12, 14, 16-18, 20, 21,
24-27, 37-40, 42-
44, 47, 49-51, 55, 57-60) specify a relative molar input percentage of 10%
alanine for
generating complexity at those positions across the mixture of polypeptide,
with the original
amino acid (relative to the native human Tau-F sequence) making up the
remaining 90% of
the input molar percentage of amino acid at each position and for generating
complexity at
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those positions across the mixture. Therefore, in this example, at these
positions, each
polypeptide has either the original amino acid indicated in Row 3 or alanine
in the ARs.
However, additional complexity could be provided by, for example, also
permitting variation
at a position based on a conserved substitution, such as an orthologous
substitution, a residue
.. that is present in a different species, a residue that is observed based on
polymorphism or
natural variation across humans, or substitution with a similar residue. For
certain other
positions in this example, such as positions 9, 45, 48, 53, and 56, no amino
acids other than
the residue present at the corresponding position of the base peptide are
provided. For the
positions corresponding to Y394, S400, T403, T427, S433, and S435, relative
molar input
percentages of 10% alanine, 45% non-phosphorylated amino acid, and 45%
phosphorylated
amino acid are provided for the generation of the mixture. Therefore, at these
positions, each
polypeptide has the phosphorylated amino acid, the non-phosphorylated amino
acid, or
alanine. For the positions corresponding to S396, S400, S404 and S422, the
relative molar
input percentage of phosphor-serine is 100%. Additional amino acids such as
conserved
substitutions may be provided at any of the positions. The first amino acid at
the C-terminal
position is a Norleucine (Nle) covalently coupled to the resin support (not
shown in Figure 3).
This provides a description of this particular composition. However, other
compositions
based on, for example, differing template arrangements, input percentages in
the RCRs or
ARs, and/or base peptide sequence or antigen are contemplated and provided.
DP-0016.0 differs from DP-0016.B based on the RCRs (e.g., the RCRs in DP-
0016.0 include an input percentage of phenylalanine). However, DP-0016.B and
DP-
0016.0 span the same antigenic regions. A polypeptide mixture according to the
schematic
shown in Figure 3 was successfully synthesized using solid-phase peptide
synthesis. Briefly,
polypeptides based on this template arrangement and input percentages were
synthesized
from C-terminus to N-terminus using Fmoc-based SPPS, as described above. To
improve the
coupling efficiency and generate a higher yield in each step, longer coupling
times or the
addition of redundant coupling reactions may be used at one or more positions.
Further, DP-
0016.0 was recognized by an anti-Tau paired helical filament pS396 monoclonal
antibody
(data not shown). This demonstrates that the polypeptides of the mixture are
in the correct
conformation to induce antibodies against paired helical filaments.
Example 3. Effect of DP-0016.B and DP-0016.0 on monocytes
The biological activity of exemplary amino acid copolymer compositions (DP-
0016.B and DP-0016.C) was evaluated by measuring their ability to induce CCL22
secretion
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in macrophage-derived RAW264.7 cells. Biological activity of these amino acid
copolymer
compositions was compared to that of (i) DP-0016.A, which is a complex mixture
of
peptides with one antigenic region corresponding to residues 388-441 of SEQ ID
No. 17 but
which lacks any random copolymer regions (e.g., the AR spans the ARa and ARb
of DP-
C016-.B), (ii) two single control peptides (Non-Phospho Peptide cnt or Phospho
Peptide Cnt)
spanning residues 388-441 of SEQ ID No. 17, one lacking any phosphorylated
residues and
one that is phosphorylated at both S396 and S404, and (iii) COPAXONE .
Cell culture. Briefly, RAW264.7 cells were incubated in 96-well round-bottom
plates
(3E+05 cells/well) in the presence of increasing concentrations (0; 0.0128;
0.064; 0.32; 1.6; 8
and 40[tM) of a polypeptide mixture (DP-0016.A, DP-0016.B, or DP-0016.C),
single
control peptides (Non-Phospho Peptide cnt or Phospho Peptide Cnt), or COPAXONE
(0;
1.25 ; 2.5 ; 5 ; 10 ; 20 ; and 40[tM).
As a positive control, LPS at 1 g/mL was used for the CCL22 assay. Each
condition
was tested in triplicate per run. Plates were duplicated for testing of
cytotoxicity and of
CCL22 production.
Determination of CCL22 concentration by ELISA. After incubation, the plate was
centrifuged and 150 of supernatants was transferred and aliquoted into two
96-well V-
bottom plates (75 L per plate). Supernatants were frozen at -80 C for > 16h
prior to CCL22
testing.
504, of supernatant per well were tested undiluted for CCL22 by "Quantikineg
Mouse CCL22/MDC Immunoassay" kit. Briefly, after a 2 hour incubation in CCL22
coated
plates, wells were washed then sequentially incubated for 2 hours with HRP-
coupled anti-
MDC polyclonal antibody and for 30 minutes in the dark with substrate
solution.
Absorbance at 450 nm was measured using the Infinite 200 ELISA reader. Each
cell culture
replicate was tested independently in one replicate and average of
triplicates.
An analytical batch included 8 calibration samples run in duplicate to
generate the
standard curve and study samples tested once. Negative control (supernatants
of
unstimulated RAW cells), positive control (supernatants of RAW cells
stimulated with
1 g/mL LPS) were run in parallel with supernatants of RAW cells stimulated
with increasing
concentrations of control peptides/copolymer compositions or COPAXONE . A
quality
control provided with the kit was tested in duplicate.
An experimental run was validated if 75% of back-calculated calibration points
were
within 75-125% of the nominal value, except for lower and upper standard
points for which
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limits were between 70 and 130% and if quality control of the kit is within
the range given by
manufacturer.
Remaining supernatant was stored at -80 C for potential re-testing if needed
(out of
range values for example).
Results. Figure 4 shows CCL22 production by RAW monocytic cells stimulated
with
DP-0016.A, DP-0016.B, DP-0016.C, single control peptides covering the same
region of
Tau, or COPAXONE . It can be seen from Figure 4 that compositions of the
disclosure
represented by DP-0016.B and DP-0016.0 are comparable to or better than
COPAXONE
in terms of CCL22 release by the tested monocytic cell line. DP-0016.B and DP-
0016.0
induced CCL22 production by RAW cells at 40, 8, and 1.6 M. Control
compositions
comprising single phosphorylated or non-phosphorylated peptides spanning the
same region
of Tau covered by DP-0016 did not stimulate CCL22 production in the RAW
monocytic
cells. Further, DP-0016.A, which has an antigenic region spanning the same
region of Tau
covered by DP-0016.B and DP-0016.0 but lacks any random copolymer regions
(i.e. DP-
C016.A has no K/A or K/A/E or K/A/E/F RCRs sequences), did not stimulate CCL22
production in the RAW monocytic cells. Thus, DP-0016.B and DP-0016.C, which
have
relatively short 8-9 amino acid RCRs linked to ARs, promote CCL22 release.
Moreover, DP-
0016.B and DP-0016.0 also induce anti-hyper-phosphorylated Tau specific
antibodies (data
not shown).
Example 4. DP-0016.B and DP-0016.0 induce broad CD4+ T-cell proliferation
The effect of DP-0016.B and DP-0016.0 on the proliferation of human peripheral
blood mononuclear cells (PBMCs) was evaluated using a CFSE proliferation
assay. Their
proliferative activity was compared to that of (i) DP-0016.A, which is a
peptide mixture with
an antigenic region corresponding to residues 388-441 of SEQ ID No. 17 but
which lacks any
RCRs, (ii) two single control peptides (Non-Phospho Peptide cnt or Phospho
Peptide Cnt)
spanning residues 388-441 of SEQ ID No. 17, one lacking any phosphorylated
residues and
one that is phosphorylated at both S396 and S404, and (iii) COPAXONE .
The impact of tested single control peptides and copolymer mixtures on PBMCs
proliferation was assessed on PBMCs purified from citrate-phosphate-dextrose
(CPD) blood
bags obtained from 12 healthy volunteers (EFS / French blood center). After
purification,
PBMCs were frozen in DMSO and stored in liquid nitrogen until proliferation
testing.
After thawing and cell counting, PBMCs were stained with 5 M of
Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE). After washing and
counting,
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CF SE-labeled PBMCs were seeded at 2E+05 PBMC per well in 96-well culture
plates in the
presence or absence 2.5 tM of single control peptide (Non-Phospho Peptide cnt
or Phospho
Peptide Cnt), or peptide composition (DP-0016.A, DP-0016.B, or DP-0016.C), or
COPAXONE , or immobilized anti-CD3 (10 pg/mL) plus soluble anti-CD28 (5 pg/mL)
antibodies as a positive control and incubated for 6 days at 37 C, 5% CO2 in
the dark.
After 6 days of culture, proliferating cells were characterized by flow
cytometry after
staining with anti-CD3, anti-CD4 and anti-CD8.
Figures 5A and 5B show the results of the proliferation assays. The
proliferation
index combines % of proliferating cells with mean fluorescence intensity (MFI)
((% CD4+ T-
cells/MFI) x 100). DP-0016.B has proliferative properties similar to COPAXONE
based on
% proliferative CD4+ T-cells and shift in mean fluorescence intensity (MFI)
and induces
potent CD4+ T-cell proliferation. In fact, DP-0016.B showed higher median
levels of CD4+
T-cell proliferation than COPAXONE . 5.8% CD4+ T-cells divide up to once a day
when
cultured with DP-0016.B at 2.5 tM over 6 days. DP-0016.0 also induced
significant CD4+
T-cell proliferation. Very limited proliferation of CD8+ T-cells is observed
with either DP-
0016.B or DP-0016.C. In contrast, DP-0016.A, which has an antigenic region
spanning the
same region of Tau covered by DP-0016.B and DP-0016.0 but lacks any RCRs (i.e.
DP-
0016.A has no K/A or K/A/E or K/A/E/F RCR sequences), does not induce
significant CD4+
T-cell proliferation. The single control peptides also do not significantly
induce CD4+ T-cell
proliferation.
Thus, DP-0016.B and DP-0016.C, which have short 8-9 amino acid long random
copolymer regions linked to antigenic regions, induce CD4+ T-cell
proliferation, while also
inducing anti-hyper-phosphorylated Tau specific antibodies (data not shown).
Example 5. DP-0016.B and DP-0016.0 induce sustained immunogenicity in mice
The immunogenic profile of murine spleen cells in the presence of DP-0016.A,
DP-
0016.B, or DP-0016.0 was evaluated in vitro. Further, the ability of DP-
0016.A, DP-
0016.B, or DP-0016.0 to raise immunogen-specific antibodies in mice injected
with the
compositions was evaluated.
Mouse treatment and injection. Thirty-five (35) female SJL/J mice, 8-12-weeks-
old,
were divided into seven groups of five mice (n=5 per group): DP-0016.A (0.25
mg/kg and
2.5mg/kg); DP-0016.B (0.25 mg/kg and 2.5mg/kg); DP-0016.0 (0.25 mg/kg and
2.5mg/kg); and incomplete Freund's adjuvant (IFA) (200 uL in 0.01 M of
ammonium
bicarbonate). Each ml of IFA contained 0.85 ml of paraffin oil and 0.15 ml of
mannide
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monooleate. The mice were injected once a week for a total of 4 subcutaneous
injections
without the use of anesthetics. The injection was carried out in the form of
an emulsion in
IFA, using glass syringes with metal connectors. The compounds were injected
at 2
concentrations, one concentration per group. A control group injected with IFA
alone was
also tested. In addition, there were three un-injected mice. One week after
the fourth
injection, all the mice were sacrificed. The naive mice were also sacrificed
and their sera
were used as negative control in the anti-DP-0016.A Ab assay.
Splenocyte proliferation assay. On the day of sacrifice, splenocyte
suspensions were
prepared and cultured in 96-well plates for three days with medium alone or 6
concentrations
of each of the corresponding copolymer composition (100, 30, 10, 3, 1 and
0.3m/mL), or
ammonium bicarbonate 0.2 mM, which corresponded to the bicarbonate
concentration in the
highest compound concentration tested, or phytohaemagglutinin (PHA) (at 5 or
201.tg/mL).
DP-0016.B was used for the recall response with splenocytes from IFA-treated
mice. Each
culture condition was performed in quadruplicate (4 wells on a 96-well plate)
except for the
PHA tested in duplicates. On day 3 of the proliferation assay, 3H-thymidine
was added and
cells were cultured for an additional ¨18 hours. Prior to thymidine
incorporation, 50 [IL of
the cell culture supernatant was collected for possible future analysis.
Anti -immunogen antibody determination. Sera from the 35 injected mice and
from the
3 un-injected mice were collected. The 30 sera from mice injected with the
compounds were
assayed for the determination of anti-immunogen antibodies using a titer assay
approach.
The anti-immunogen antibody assay plate consisted of test sample(s), and
negative
control (sera collected from non-injected mice or from mice injected with
IFA), titered down
(8 dilutions) with dilution buffer. All the plates were coated with the
respective composition
used for the injection. The presence of anti-immunogen antibodies was detected
using both
anti-IgG1 and anti-IgG2a antibodies, in separate reactions.
Results. Figure 6A shows the results of the splenocyte proliferation assay in
mice
immunized with 0.25 mg/kg. When splenocytes from mice injected with DP-0016.A,
DP-
0016.B, or DP-0016.0 were collected and re-stimulated in vitro with various
concentrations
of the corresponding composition, the T-cell response induced by DP-0016.B and
DP-
C016. C was greater than that of DP-0016.A. Therefore, DP-0016.B and DP-0016.0
were
able to induce specific T-cells in the spleen of mice immunized with the
compositions while
DP-0016.A was not, indicating that the relatively short 8-9 amino acid long
random
copolymer regions (RCRs) of DP-0016.B and DP-0016.0 lead to the increased
splenocyte
recall response. Further, splenocytes from the control group injected with IFA
alone were re-
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stimulated with DP-0016.B and, as can be seen from Fig. 6A, DP-0016.B was able
to induce
strong proliferation of naive T-cells. This supports the conclusion that DP-
0016.B (an amino
acid copolymer composition of the disclosure) does not require in vivo priming
to induce
strong T-cell proliferation.
Figure 6B shows the results of antibody ELISA against the respective
immunogen.
Serum samples from mice injected with the compositions were assayed for the
presence of
anti-immunogen antibodies, and significantly higher titers of the anti-
immunogen IgG1
antibodies were present in the sera of mice injected with DP-0016.B and DP-
0016.0
compared to the sera of mice injected with DP-0016.A. The presence of IgG1
antibodies is
indicative of a Th2 response.
Thus, DP-0016.B and DP-0016.C, which comprise relatively short 8-9 amino acid
long random copolymer regions (RCRs), were significantly more immunogenic than
DP-
0016.A, a composition that lacks random copolymer regions. The greater
immunogenicity of
DP-0016.B and DP-0016.0 indicates that the compositions have potential value
in
generating a robust immune response in elderly patients whose immune systems
generally
tend to be weaker.
Example 6. Preparation of a mixture of amino acid copolymers with antigenic
specificity to a-synuclein protein
DP-0003.E, an amino acid copolymer composition (a composition based on the DP-
0003 .E template arrangement, sequence, and input amino acid input
distribution) comprising
a high complexity mixture of polypeptides based on antigenic sequences derived
from a-
synuclein, was designed and synthesized according to the methods of the
disclosure. Figure
22 shows a schematic for the design of DP-0003.E. DP-0003.E is designed to
target
multiple phosphorylation and nitration patterns covering Asp121, Asn122,
Tyr125, Tyr133,
and Tyr 136 of human a-synuclein and can be used to generate specific
antibodies. The full-
length polypeptides of DP-0003 .E are about 61 amino acids long. The template
arrangement
of DP-0003.E comprises: ARa-RCRa-ARa-RCRb-ARc. The DP-0003.E polypeptides
(e.g.,
the polypeptides in the mixture of peptides comprising the DP-0003 .E
composition) are each
based on a template arrangement having two 5 amino acid-long random copolymer
regions
(RCRs) wherein the amino acid at each position is selected from Alanine or
Lysine i.e., the
RCRs comprises K/A motifs and three antigenic regions that are all based on
the same base
peptide sequence corresponding to residues 121-137 of SEQ ID No. 20. For each
position of
the two random copolymer regions, the relative molar input percentages of
alanine and lysine
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were 50% and 50%, respectively. Therefore, each peptide has an A or K at each
position of
its RCRs with the distribution according to these percentages. The AR at
positions
corresponding to 1-17, the AR at position corresponding to 23-39, and the AR
at positions
corresponding to 41-57, of Figure 22 are based on a base peptide sequence
which
corresponds to positions 121-137 of human a-synuclein (human a-synuclein set
forth in SEQ
ID No. 20.
Two other amino acid copolymer compositions, DP-0003.F and DP-0003.G,
comprising high complexity mixtures of polypeptides based on antigenic
sequences derived
from a-synuclein, were designed and synthesized according to the methods of
the disclosure.
The full-length polypeptides of DP-0003.F and DP-0003.G, like those of DP-
0003.E, are
about 61 amino acids long and their template arrangements also comprise: ARa-
RCRa-ARa-
RCRb-ARc. The DP-0003.F and DP-0003.G polypeptides (e.g., the polypeptides in
the
mixture of peptides comprising the DP-0003.F or DP-0003.G compositions) are
each based
on a template arrangement having two 5 amino acid-long random copolymer
regions (RCRs)
wherein the amino acid at each position is selected from Alanine or Lysine
i.e., the RCRs
comprises K/A motifs and three antigenic regions that are all based on the
same base peptide
sequence corresponding to residues 121-137 of SEQ ID No. 20. For each position
of the two
random copolymer regions, the relative molar input percentages of alanine and
lysine were
50% and 50%, respectively, similar to DP-00003.E. Therefore, each peptide has
an A or K
at each position of its RCRs with the distribution according to these
percentages. However,
whereas DP-0003 .E had a relative molar input percentage of alanine of 10%
along each of
the positions in the ARs, with the exception of any native alanine residues or
serine residues
(positions 1-3, 5-8, 10-17, 23-25, 27-30, 32-39, 45-47, 49-52, 54-61), DP-
0003.7 and DP-
0003.8, respectively, had relative molar input percentages of alanine of 30%
and 50%, at
each of these positions of the AR. Thus, DP-0003.F and DP-0003.G differed from
DP-
0003.E in the composition of the ARs, with DP-0003.F and CP-0003.G having a
substantially higher input percentage of alanine across the ARs, and thus, a
substantially
higher total input percent of alanine across the ARs and RCRs.
The ability of DP-0003.E, DP-0003.F and DP-0003.G to induce CCL22 release by
monocytes was tested. DPC003.E stimulated CCL22 release from RAW monocytic
cells
more effectively than either DPC003.F or DPC003.G. Thus, an increase in
alanine content
across the antigenic regions, leading to overall increase in alanine content
in the
compositions, was correlated with a loss of efficacy in a CCL22 production
assay. Data not
shown.
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Figure 7 shows a schematic for the design of another amino acid copolymer
composition (a composition based on the DP-0003 .A template arrangement,
sequence, and
input amino acid input distribution) comprising a high complexity of
polypeptides based on
antigenic sequences derived from a-synuclein. DP-0003 .A is designed to target
multiple
phosphorylation and nitration patterns covering Tyr125, Tyr133, Tyr136 and
Ser129 of
human a-synuclein and can be used to generate specific antibodies.
The full-length polypeptides of DP-0003.A based on this template arrangement
are
about 53 amino acids long and the amino acid positions, relative to full-
length polypeptides
based on the depicted template arrangement, of the composition are indicated
in Row 1 (the
row labeled peptide #). The DP-0003.A polypeptides (e.g. the polypeptides in
the mixture of
polypeptides comprising the DP-0003 .A composition) are based on a template
arrangement
having a maximum of 2 random copolymer regions (RCRs) wherein the amino acid
at each
position is selected from lysine, alanine or glutamic acid, i.e., the RCRs
comprise K/A/E
motifs (N-terminal RCRa (corresponding to positions 1-9 in Figure 7) and RCRb
(corresponding to positions 27-35 in Figure 7)), and two antigenic regions
(ARs) based on
sequences derived from human a-synuclein (ARa (corresponding to positions 10-
26 in Figure
7) and ARb (corresponding to positions 36-52 in Figure 7)). The template
arrangement of
DP-0003 .A comprises: RCRa-ARa- RCRb-ARb, although other template arrangements
are
described herein. In this example, complexity across the polypeptides in the
composition
comes from both the RCRs and ARs although, in other examples, complexity may
come only
from the RCRs or from the RCRs and one, but not both, ARs. In this example,
the RCRs and
ARs are contiguous. However, they may be interconnected by a linker, such as
by one or
more intervening amino acid residues. Moreover, the polypeptides in the
composition may
include on or more additional amino acid residues at the N- or C-terminus,
including
naturally occurring or non-natural amino acids.
Row 2 of the top and bottom panels (the row labeled aSyn #) indicates the
positions in
human a-synuclein (SEQ ID No. 35) that correspond to residues that may be
phosphorylated
or nitrated, and for which phosphorylation or nitration is among the diversity
available in the
polypeptides in the mixture. Rows 3, 4, and 5 indicate the possible amino
acids, which are
or may be incorporated at each position in the peptide. The two copolymer
regions (RCRs)
correspond to positions 1-9 (RCRa) and positions 27-35 (RCRb). The two
antigenic regions
(ARs) correspond to positions 10-26 (ARa) and to positions 36-52 (ARb). The
sequence of the
AR given in Row 3 corresponds to the base peptide sequence and reflects
original amino acid
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residues derived from the corresponding portion of the target, human a-
synuclein (SEQ ID
No. 35).
Rows 6, 7, and 8 (the rows labeled 1" Row %, 2nd Row%, and 3rd Row %,
respectively) indicate the molar input percentages of the corresponding, color-
coded, amino
acid above. For example, at position 10, the relative molar amino acid input
percentages of
glycine (G) and alanine (A) are 90% and 10%, respectively. Additional amino
acids such as
conserved substitutions may be provided at any of the positions in the
antigenic region.
The two RCRs in this example are 9 amino acids in length and contain either
lysine
(K), alanine (A), or glutamic acid (E) at each position. RCRs of differing
sizes (e.g. shorter or
longer than depicted) are contemplated, as described herein. The relative
molar input
percentages of lysine, alanine, or glutamic acid were 40%, 50%, or 10%
relatively. The
relative molar input percentage of lysine, alanine, or glutamic acid was
selected from within
the foregoing ranges, independently for each position.
The AR at positions corresponding to 10-26 of Figure 7 is based on a base
peptide
sequence which corresponds to positions 123-137 of human a-synuclein (human a-
synuclein
set forth in SEQ ID No. 35; corresponding base peptide sequence used in this
example set
forth in SEQ ID NO: 36). The AR at positions corresponding to 36-52 of Figure
7 is based
on a base peptide sequence which corresponds to positions 121-137 of human a-
synuclein
(SEQ ID No. 35; corresponding base peptide sequence used in this example set
forth in SEQ
ID NO: 37).
For this particular example, most positions of the ARs (e.g. positions 10-12,
15-17,
19-21, 23-24, 26, 36-38, 41-43, 45-47, 49-50, and 52) specify a relative molar
input
percentage of 10% alanine for generating complexity at those positions across
the mixture of
polypeptides, with the original amino acid (relative to the human a-synuclein
sequence)
making up the remaining 90% of the input molar percentage of amino acid at
each position
and for generating complexity at those positions across the mixture.
Therefore, in this
example, at these positions, each polypeptide has either the original amino
acid indicated in
Row 3 or alanine in the ARs. However, additional complexity could be provided
by, for
example, also permitting variation at a position based on a conserved
substitution, such as an
orthologous substitution, a residue that is present in a different species, a
residue that is
observed based on polymorphism or natural variation across humans or
substitution with a
similar residue. For certain other positions in this example, such as
positions 13, 18, 39, and
44, no amino acids other than the residue present at the corresponding
position of the base
peptide are provided. For the positions corresponding to Y125, Y133, and Y136,
relative
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molar input percentages of 10% alanine, 45% non-phosphorylated amino acid, and
45%
nitrated amino acid are provided for the generation of the mixture. Therefore,
at these
positions, each polypeptide has the nitrated amino acid, the non-nitrated
amino acid, or
alanine. For the position corresponding to S129, the relative molar input
percentage of
phosphor-serine is 100%. Norleucine is found at the C-terminal most amino acid
at position
53 of Figure 7. Additional amino acids such as conserved substitutions may be
provided at
any of the positions. This provides a description of this particular
composition. However,
other compositions based on, for example, differing template arrangements,
input percentages
in the RCRs or ARs, and/or base peptide sequence or antigen are contemplated
and provided.
A polypeptide mixture according to the schematic shown in Figure 7 was
successfully
synthesized using solid-phase peptide synthesis. Briefly, the polypeptides
based on this
template arrangement and input percentages were synthesized from C-terminus to
N-terminus
using Fmoc-based SPPS, as described above. To improve the coupling efficiency
and
generate a higher yield in each step, longer coupling times or the addition of
redundant
coupling reactions may be used at one or more positions. The disclosure
provides
compositions manufactured based on this template arrangement, sequence and
input amino
acid distribution.
Other exemplary copolymer compositions based on antigenic sequences derived
from
a-synuclein include DP-0003.B (Figure 8), DP-0003.0 (Figure 9), and DP-0003.D
(Figure
10). Each are briefly described herein.
DP-0003.B, an amino acid copolymer composition (a composition based on the DP-
0003 .B template arrangement, sequence, and input amino acid input
distribution) comprising
a high complexity mixture of peptides based on antigenic sequences derived
from a-
synuclein, is designed and synthesized according to the methods of the
disclosure. The full-
length peptides of DP-0003.B are about 57 amino acids long. DP-0003.B is
designed to
target multiple phosphorylation and nitration patterns covering Asp121,
Asn122, Tyr125,
Tyr133, and Tyr 136 of human a-synuclein and can be used to generate specific
antibodies.
The template arrangement of DP-0003.B comprises: ARa-RCRa-ARb-RCRb-ARc. The DP-
0003.B peptides (e.g., the peptides in the mixture of peptides comprising the
DP-0003.B
composition) each have two 3 amino acid-long random copolymer regions (RCRs)
wherein
the amino acid at each position is selected from lysine, alanine, or glutamic
acid i.e., the
RCRs comprises K/A/E motifs and three antigenic regions that are all based on
the same base
peptide sequence corresponding to residues 121-137 of SEQ ID No. 35. For each
position of
the two random copolymer regions, the relative molar input percentages of
lysine, alanine,
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and glutamic acid were 35-75%, 15-55%, and 10%, respectively. Therefore, each
peptide has
a K, A, or E at each position of its RCRs with the distribution according to
these percentages,
and wherein the actual input percentage is independently selected for each
position.
The AR at positions corresponding to 1-17 and the AR at positions
corresponding to
41-57 of Figure 8 is based on a base peptide sequence which corresponds to
positions 121-
137 of human a-synuclein (human a-synuclein set forth in SEQ ID No. 35;
corresponding
base peptide sequence used in this example set forth in SEQ ID NO: 37). The AR
at
positions corresponding to 21-37 of Figure 8 is based on a base peptide
sequence which most
closely corresponds to positions 123-137 of human a-synuclein (SEQ ID No. 35;
corresponding base peptide sequence used in this example set forth in SEQ ID
NO: 36).
DP-0003.C, an amino acid copolymer composition (a composition based on the DP-
0003.0 template arrangement, sequence, and input amino acid input
distribution) comprising
a high complexity mixture of peptides based on antigenic sequences derived
from a-
synuclein, is designed and synthesized according to the methods of the
disclosure. The full-
length polypeptides of DP-0003.0 are about 63 amino acids long. DP-0003.0 is
designed to
target multiple phosphorylation and nitration patterns covering Tyr125,
5er129, Tyr133, and
Tyr 136 of human a-synuclein and can be used to generate specific antibodies.
The template
arrangement of DP-0003.0 comprises: RCRa-ARa- RCRb-ARb-RCRc-ARc-RCRd. The
DP-0003.0 polypeptides (e.g., the polypeptides in the mixture of polypeptides
comprising
the DP-0003 .0 composition) each have four 3 amino acid-long random copolymer
regions
(RCRs) wherein the amino acid at each position is selected from lysine,
alanine, or glutamic
acid i.e., the RCRs comprises K/A/E motifs and three antigenic regions that
are all based on
the same base peptide sequence corresponding to residues 121-137 of SEQ ID No.
35. For
each position of the four random copolymer regions, the relative molar input
percentages of
lysine, alanine, and glutamic acid were 35-75%, 15-55%, and 10%, respectively.
Therefore,
each peptide has a K, A, or E at each position of its RCRs with the
distribution according to
these percentages and wherein the actual input percentage is independently
selected for each
position.
The AR at positions corresponding to 4-20 and the AR at positions
corresponding to
44-60 of Figure 9 is based on a base peptide sequence which corresponds to
positions 121-
137 of human a-synuclein (human a-synuclein set forth in SEQ ID No. 35;
corresponding
base peptide sequence used in this example set forth in SEQ ID NO: 37). The AR
at
positions corresponding to 24-40 of Figure 9 is based on a base peptide
sequence which most
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closely corresponds to positions 123-137 of human a-synuclein (SEQ ID No. 35;
corresponding base peptide sequence used in this example set forth in SEQ ID
NO: 36).
DP-0003.D, an amino acid copolymer composition (a composition based on the DP-
0003 .D template arrangement, sequence, and input amino acid input
distribution) comprising
a high complexity mixture of peptides based on antigenic sequences derived
from a-
synuclein, is designed and synthesized according to the methods of the
disclosure. The full-
length polypeptides of DP-0003.D are about 63 amino acids long. DP-0003.D is
designed to
target multiple phosphorylation and nitration patterns covering Tyr125,
5er129, Tyr133, and
Tyr 136 of human a-synuclein and can be used to generate specific antibodies.
The template
arrangement of DP-0003.D comprises: RCRa-ARa- RCRb-ARb-RCRc-ARc-RCRd. The
DP-0003.D polypeptides (e.g., the polypeptides in the mixture of peptides
comprising the
DP-0003 .D composition) each have four 3 amino acid-long random copolymer
regions
(RCRs) wherein the amino acid at each position is selected from arginine,
alanine, or
glutamic acid i.e., the RCRs comprises R/A/E motifs and three antigenic
regions that are all
based on the same base peptide sequence corresponding to residues 121-137 of
SEQ ID No.
35. For each position of the four random copolymer regions, the relative molar
input
percentages of arginine, alanine, and glutamic acid were 35-75%, 15-55%, and
10%,
respectively. Therefore, each peptide has a R, A, or E at each position of its
RCRs with the
distribution according to these percentages and wherein the actual input
percentage is
independently selected for each position.
The AR at positions corresponding to 4-20 and the AR at positions
corresponding to
44-60 of Figure 10 is based on a base peptide sequence which corresponds to
positions 121-
137 of human a-synuclein (human a-synuclein set forth in SEQ ID No. 35;
corresponding
base peptide sequence used in this example set forth in SEQ ID NO: 37). The AR
at
positions corresponding to 24-40 of Figure 9 is based on a base peptide
sequence which most
closely corresponds to positions 123-137 of human a-synuclein (SEQ ID No. 35;
corresponding base peptide sequence used in this example set forth in SEQ ID
NO: 36). The
disclosure provides compositions manufactured based on any of the foregoing
template
arrangement, sequence and input amino acid distribution.
Any of the foregoing compositions with antigenic specificity to a-synuclein
could
comprise RCRs (such as two or three RCRs) that are 3 amino acids, or 4 amino
acids, or 5
amino acids, or 6 amino acids, or 7 amino acids in length (or even 8 or 9 as
described herein).
The length of each RCR may be selected independently from length of the other
RCRs. All
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of the foregoing compositions may have a Norleucine or another non-naturally
occurring
amino acid at either the C-terminus or the N-terminus.
Example 7. Preparation of an amino acid copolymer composition with antigenic
specificity to Tau protein (DP-0016.E)
Figure 11 shows a schematic for the design of an amino acid copolymer
composition
(a composition based on the DP-0016.E template arrangement, sequence, and
input amino
acid input distribution) comprising a high complexity of polypeptides based on
antigenic
sequences derived from Tau. DP-0016.E is designed to target multiple
phosphorylation
patterns among 11 potential phosphorylation residues found in the C-terminus
of human Tau-
F and can be used to generate specific antibodies against many variants of
hyperphosphorylated Tau protein found in neuropathological hallmarks such as
neurofibrillary tangles (NFTs), Neuropil threads, plaques and paired helical
filaments (PHFs).
The full-length polypeptides of DP-0016.E in this schematic are about 51 amino
acids long and the amino acid positions, relative to full-length polypeptides
based on the
depicted template arrangement, of the composition are indicated in Row 1 (the
row labeled
peptide #). The DP-0016.E polypeptides (e.g. the polypeptides in the mixture
of
polypeptides comprising the DP-0016.E composition) are based on a template
arrangement
having a maximum of 2 random copolymer regions (RCRs) wherein the amino acid
at each
position is selected from lysine, alanine or glutamic acid, i.e., the RCRs
comprise K/A/E
motifs (N-terminal RCRa (corresponding to positions 1-3 in Figure 11) and RCRb
(corresponding to positions 23-25 in Figure 11), and two antigenic regions
(ARs) based on
sequences derived from human Tau-F (ARa (corresponding to positions 4-22 in
Figure 11)
and ARb (corresponding to positions 26-50 in Figure 11). The template
arrangement of DP-
C016.F comprises: RCRa-ARa- RCRb-ARb, although other template arrangements are
described herein. In this example, ARa corresponds to or comprises a region of
PHF1
epitope, and ARb corresponds to or comprises a region comprising the pS422 and
the pS433
epitopes of Tau-F. In this example, complexity across the polypeptides in the
composition
comes from both the RCRs and ARs although, in other examples, complexity may
come only
from the RCRs or from the RCRs and one, but not both, ARs. In this example,
the RCRs and
ARs are contiguous. However, they may be interconnected by a linker, such as
by one or
more intervening amino acid residues. Moreover, the polypeptides in the
composition may
include on or more additional amino acid residues at the N- or C-terminus,
including
naturally occurring or non-natural amino acids.
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Row 2 of the top and bottom panels (the row labeled Tau-F #) indicates the
positions
in human Tau-F (SEQ ID No. 17) that correspond to residues in the AR which are
or may be
phosphorylated in this composition. Rows 3, 4, and 5 indicate the possible
amino acids
which are or may be incorporated at each position in the peptide. The two
copolymer regions
(RCRs) correspond to positions 1-3 (RCRa) and positions 23-25 (RCRb). The two
antigenic
regions (ARs) correspond to positions 4-22 (ARa) and to positions 26-50 (ARb).
The
sequence of the AR given in Row 3 corresponds to the base peptide sequence and
reflects
original amino acid residues derived from the corresponding portion of the
target, human
Tau-F.
Rows 6, 7, and 8 (the rows labeled 1" Row %, 2nd Row%, and 3rd Row %,
respectively) indicate the molar input percentages of the corresponding, color-
coded, amino
acid above. For example, at position 5, the relative molar amino acid input
percentages of
glutamic acid (E) and alanine (A) are 90% and 10%, respectively. Additional
amino acids
such as conserved substitutions may be provided at any of the positions in the
antigenic
region.
The two RCRs in this example are 3 amino acids in length and contain either
lysine
(K), alanine (A), or glutamic acid (E) at each position. RCRs of differing
sizes (e.g. shorter or
longer than depicted) are contemplated, as described herein. The relative
molar input
percentages of lysine, alanine, or glutamic acid were 30%-80%, 15%-55%, or 5%-
15%
relatively. The relative molar input percentage of lysine, alanine, or
glutamic acid was
selected from within the foregoing ranges, independently for each position.
The AR at positions corresponding to 4-22 of Figure 11 is based on a base
peptide
sequence which corresponds to positions 390-408 of human Tau-F (human Tau-F
sequence
set forth in SEQ ID No. 17; corresponding base peptide sequence used in this
example set
forth in SEQ ID NO: 9). The AR at positions corresponding to 26-50 of Figure
11 is based
on a base peptide sequence which corresponds to positions 414-438 of human Tau-
F (human
Tau-F sequence set forth in SEQ ID No. 17; corresponding base peptide sequence
used in this
example set forth in SEQ ID NO: 31). For this particular example, most
positions of the ARs
(e.g. positions 5-7,9, 11-13, 15-16, 19-22, 27, 29-33, 35-37, 40, 42-44, 48,
and 50) specify a
relative molar input percentage of 10% alanine for generating complexity at
those positions
across the mixture of polypeptides, with the original amino acid (relative to
the human Tau-F
sequence) making up the remaining 90% of the input molar percentage of amino
acid at each
position and for generating complexity at those positions across the mixture.
Therefore, in
this example, at these positions, each polypeptide has either the original
amino acid indicated
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in Row 3 or alanine in the ARs. However, additional complexity could be
provided by, for
example, also permitting variation at a position based on a conserved
substitution, such as an
orthologous substitution, a residue that is present in a different species, a
residue that is
observed based on polymorphism or natural variation across humans or
substitution with a
.. similar residue. For certain other positions in this example, such as
positions 4, 10, 18, 34,
38, 41, 45-46, 49, and 51, no amino acids other than the residue present at
the corresponding
position of the base peptide are provided. For the positions corresponding to
Y394, S400,
T403, T414, S416, T427, and S435, relative molar input percentages of 10%
alanine, 45%
non-phosphorylated amino acid, and 45% phosphorylated amino acid are provided
for the
generation of the mixture. Therefore, at these positions, each polypeptide has
the
phosphorylated amino acid, the non-phosphorylated amino acid, or alanine. For
the positions
corresponding to S396, S404, S422, and S433, the relative molar input
percentage of
phosphor-serine is 100%. Norleucine is found at the C-terminal most amino acid
at position
51of Figure 11. Additional amino acids such as conserved substitutions may be
provided at
any of the positions. This provides a description of this particular
composition. However,
other compositions based on, for example, differing template arrangements,
input percentages
in the RCRs or ARs, and/or base peptide sequence or antigen are contemplated
and provided.
A polypeptide mixture according to the schematic shown in Figure 11 was
successfully synthesized using solid-phase peptide synthesis. Briefly, the
polypeptides based
on this template arrangement and input percentages were synthesized from C-
terminus to N-
terminus using Fmoc-based SPPS, as described above. To improve the coupling
efficiency
and generate a higher yield in each step, longer coupling times or the
addition of redundant
coupling reactions may be used at one or more positions. The disclosure
provides
compositions manufactured based on this template arrangement, sequence and
input amino
acid distribution.
Example 8 Preparation of an amino acid copolymer composition with antigenic
specificity to Tau protein (DP-0016.F)
Figure 12 shows a schematic for the design of an amino acid copolymer
composition
(a composition based on the DP-0016.F template arrangement, sequence, and
input amino
acid input distribution) comprising a high complexity of polypeptides based on
antigenic
sequences derived from Tau. The disclosure provides compositions manufactured
based on
this template arrangement, sequence and input amino acid distribution.
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DP-0016.F is designed to target multiple phosphorylation patterns among 14
potential
phosphorylation residues found in the C-terminus of human Tau-F and can be
used to
generate specific antibodies against many variants of hyperphosphorylated Tau
protein found
in neuropathological hallmarks such as neurofibrillary tangles (NFTs),
Neuropil threads,
plaques and paired helical filaments (PHFs).
The full-length polypeptides of DP-0016.F in this schematic are about 63 amino
acids
long and the amino acid positions, relative to full-length polypeptides based
on the depicted
template arrangement, of the composition are indicated in Row 1 (the row
labeled peptide #).
The DP-0016.F polypeptides (e.g. the polypeptides in the mixture of
polypeptides
comprising the DP-0016.F composition) are based on a template arrangement
having a
maximum of three random copolymer regions (RCRs) wherein the amino acid at
each
position is selected from lysine, alanine or glutamic acid, i.e., the RCRs
comprise K/A/E
motifs (N-terminal RCRa (corresponding to positions 1-3 in Figure 12), RCRb
(corresponding
to positions 23-25 in Figure 12), and RCRc (corresponding to positions 42-44
in Figure 12))
and three antigenic regions (ARs) based on sequences derived from human Tau-F
(ARa
(corresponding to positions 4-22 in Figure 12) ARb (corresponding to positions
26-41 in
Figure 12), and AR c (corresponding to positions 45-59 in Figure 12)). The
template
arrangement of DP-0016.F comprises: RCRa-ARa- RCRb-ARb- RCItc-AR, although
other
template arrangements are described herein. In this example, ARa corresponds
to or
.. comprises a region of a PHF1 epitope, ARb correspond to or comprises a
region comprising
the pS422 epitope of Tau-F, and AR c corresponds to or comprises a region
comprising the
pS433 epitope of Tau-F. In this example, complexity across the polypeptides in
the
composition comes from both the RCRs and ARs although, in other examples,
complexity
may come only from the RCRs or from the RCRs and one, but not both, ARs. In
this
example, the RCRs and ARs are contiguous. However, they may be interconnected
by a
linker, such as by one or more intervening amino acid residues. Moreover, the
polypeptides
in the composition may include one or more additional amino acid residues at
the N- or C-
terminus, including naturally occurring or non-natural amino acids. In this
example,
additional C-terminal amino acids are included.
Row 2 of the top and bottom panels (the row labeled Tau-F #) indicates the
positions
in human Tau-F (SEQ ID No. 17) that correspond to residues in the AR, which
are or may be
phosphorylated in this composition. Rows 3, 4, and 5 indicate the possible
amino acids
which are or may be incorporated at each position in the peptide. The three
copolymer
regions (RCRs) correspond to positions 1-3 (RCRa), positions 23-25 (RCRb), and
regions 42-
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44 (RCItc). The three antigenic regions (ARs) correspond to positions 4-22
(ARa), positions
26-41 (ARb), and positions 45-59 (AR:), in each case as depicted for full-
length sequences in
Figure 12. The sequence of the AR given in Row 3 corresponds to the base
peptide sequence
and reflects original amino acid residues derived from the corresponding
portion of the target,
human Tau-F.
Rows 6, 7, and 8 (the rows labeled 1" Row %, 2nd Row%, and 3rd Row %,
respectively) indicate the molar input percentages of the corresponding, color-
coded, amino
acid above. For example, at position 5, the relative molar amino acid input
percentages of
glutamic acid (E) and alanine (A) were 90% and 10%, respectively. Additional
amino acids
such as conserved substitutions may be provided at any of the positions in the
antigenic
region.
The three RCRs in this example are 3 amino acids in length and contain either
lysine
(K), alanine (A), or glutamic acid (E) at each position. RCRs of differing
sizes (e.g. shorter or
longer than depicted) are contemplated, as described herein. The relative
molar input
percentages of lysine, alanine, or glutamic acid were 30%-80%, 15%-55%, or 5%-
15%,
relatively. In some embodiments, the relative molar input percentage of lysine
is 35%-75%,
40-75%, or 50-70%. The relative molar input percentage of lysine, alanine or
glutamic acid
was selected from within the foregoing ranges, independently for each
position.
The AR at positions corresponding to 4-22 of Figure 12 is based on a base
peptide
sequence which corresponds to positions 390-408 of human Tau-F (human Tau-F
sequence
set forth in SEQ ID No. 17; corresponding base peptide sequence used in this
example set
forth in SEQ ID NO: 9). The AR at positions corresponding to 26-41 of Figure
12 is based
on a base peptide sequence which corresponds to positions 414-429 of human Tau-
F (human
Tau-F sequence set forth in SEQ ID No. 17; corresponding base peptide sequence
used in this
example set forth in SEQ ID NO: 32). The AR at positions corresponding to
positions 45-59
of Figure 12 is based on the base peptide sequence which corresponds to
positions 424-438 of
human Tau-F (human Tau-F sequence set forth in SEQ ID No. 17; corresponding
base
peptide sequence used in this example set forth in SEQ ID NO: 33). For this
particular
example, most positions of the ARs (e.g. positions 5-7, 9, 11-13, 15-16, 19-
22, 27, 35-37, 40,
45-46, 49, 51-53, 57, and 59) specify a relative molar input percentage of 10%
alanine for
generating complexity at those positions across the mixture of polypeptides,
with the original
amino acid (relative to the human Tau-F sequence) making up the remaining 90%
of the
input molar percentage of amino acid at each position and for generating
complexity at those
positions across the mixture. Therefore, in this example, at these positions,
each polypeptide
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has either the original amino acid indicated in Row 3 or alanine in the ARs.
However,
additional complexity could be provided by, for example, also permitting
variation at a
position based on a conserved substitution, such as an orthologous
substitution, a residue that
is present in a different species, a residue that is observed based on
polymorphism or natural
variation across humans or substitution with a similar residue. For certain
other positions in
this example, such as positions 4, 10, 18, 29-34, 38, 41, 47, 50, 54-55, 58,
and 60-63, no
amino acids other than the residue present at the corresponding position of
the base peptide
are provided. For the positions corresponding to Y394, S400, T403, T414, S416,
T427, and
S435, relative molar input percentages of 10% alanine, 45% non-phosphorylated
amino acid,
and 45% phosphorylated amino acid are provided for the generation of the
mixture.
Therefore, at these positions, each polypeptide has the phosphorylated amino
acid, the non-
phosphorylated amino acid, or alanine. For the positions corresponding to
S396, S404, S422,
and S433, the relative molar input percentage of phosphor-serine is 100%.
Norleucine is
found at the C-terminal most amino acid at position 63 of Figure 12. We note
the relatively
low input percentages of alanine used in the ARs, which is, in certain
embodiments, an
important feature. Additional amino acids such as conserved substitutions may
be provided
at any of the positions. This provides a description of this particular
composition. However,
other compositions based on, for example, differing template arrangements,
input percentages
in the RCRs or ARs, and/or base peptide sequence or antigen are contemplated
and provided.
For instance, alternate phosphorylation sites and phosphorylation patterns in
the C-terminal
region of tau may be targeted (Figure 13). The examples depict experiments
with
embodiments of DP-0016.F.
DP-0016.E (Figure 11) differs from DP-0016.F (Figure 12) based on the both the
RCRs and ARs. Full-length polypeptides based on DP-0016.E template arrangement
contains
two RCRs as compared to the three RCRs in DP-0016.F. The one antigenic region
(ARa)
(which corresponds to positions 390-408 of human Tau-F (SEQ ID No. 17)) is
identical
between DP-0016.E and DP-0016.F. However, where ARb and AR c of DP-0016.F
correspond to positions 414-429 and 424-438 of human Tau-F (SEQ ID No. 17), DP-
0016.E
combines these regions into one antigenic region (ARb) which corresponds to
positions 414-
438 of human Tau-F (SEQ ID No. 17). These differences contribute to DP-0016.E
having an
estimated net charge of 1.1 at pH7 and DP-0016.F having an estimated net
charge of 3.1 at
pH7 (data not shown). The net charge and nature of the ARs contributes to
overall solubility
and lack of solubility prevents evaluation of immunogenicity of the complex
polypeptide
mixture. Accordingly, where the ARs are of a character that contributes to
poor solubility
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and/or alpha-helical structure, increasing charge helps improve solubility so
that the
immunogenic properties may be fully evaluated. The estimated net charge is a
proxy for the
predicted charge (e.g., estimated charge) at pH7 and is based on the net
charge of the ARs
and RCRs within the linear template arrangement. The estimated net charge for
a
composition of the disclosure is based on the template sequence where: (i) the
net charge of
the ARs, is calculated based on the base peptide sequence without the
inclusion of potential
variation at each amino acid position of the AR, (ii) the net charge of the
RCRs, is calculated
for a hypothetical polypeptide in which for every 1 position in which a
positively charged
amino acid is assumed to occur, alanine is assumed to occur in two positions
(e.g., if template
arrangement and template sequence for a polypeptide mixture includes 1 RCR of
3 amino
acid residues in length, estimated net charge is calculated based on the
assumption that the
RCR contains 1 positively charged residue and two alanines); and (iii) and
additional amino
acids outside of the ARs and RCRs and present in the design are included
(e.g., a 4 residue
N- or C- terminal tail in the design is accounted for based on its sequence
and charge). For
.. the purpose of this calculation, negatively charged amino acids are not
considered for the
RCR.
Polypeptide mixtures according to the schematics shown in Figures 11 and 12
were
successfully synthesized using solid-phase peptide synthesis. Briefly, the
polypeptides based
on this template arrangement and input percentages were synthesized from C-
terminus to N-
terminus using Fmoc-based SPPS, as described above. To improve the coupling
efficiency
and generate a higher yield in each step, longer coupling times or the
addition of redundant
coupling reactions may be used at one or more positions. The disclosure
provides
compositions manufactured based on these template arrangements, sequences and
input
amino acid distributions.
Example 9. Effect of DP-0016.E and DP-0016.F on CD4+ T-cell proliferation
The effect of DP-0016.E and DP-0016.F on the proliferation of human peripheral
blood mononuclear cells (PBMCs) was evaluated using a CFSE proliferation
assay. Their
proliferative activity was compared to that of (i) DP-0016.B, which is a
polypeptide mixture
based on the template arrangement, amino acid sequence, and amino acid input
percentages
described in Figure 2, (ii) DP-0016.D, which is a polypeptide mixture with a
template
arrangement comprising first antigenic region corresponding to residues 387-
407 of SEQ ID
No. 17 and second antigenic region corresponding to 417-432 of SEQ ID No. 17,
and two 5
amino acid long RCRs, and (iii) COPAXONE .
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The impact of tested copolymer mixtures on PBMCs proliferation was assessed on
PBMCs purified from citrate-phosphate-dextrose (CPD) blood bags obtained from
12 healthy
volunteers (EFS / French blood center). After purification, PBMCs were frozen
in DMSO
and stored in liquid nitrogen until proliferation testing.
After thawing and cell counting, PBMCs were stained with 5 [NI of
Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE). After washing and
counting,
CF SE-labeled PBMCs were seeded at 2E+05 PBMC per well in 96-well culture
plates in the
presence or absence 2.511M of polypeptide composition (DP-0016.B, DP-0016.D,
DP-
0016.E, or DP-0016.F), or COPAXONE as a positive control and incubated for 6
days at
37 C, 5% CO2 in the dark.
After 6 days of culture, proliferating cells were characterized by flow
cytometry after
staining with anti-CD3, anti-CD4 and anti-CD8.
Figure 14 shows the results of the proliferation assays. The stimulation index
combines % of proliferating cells with mean fluorescence intensity (MFT) ((%
CD4+ T-
cells/MFI) x 100). DP-0016.F has proliferative properties similar to DP-0016.B
based on %
proliferative CD4+ T-cells and shift in mean fluorescence intensity (MFT) and
induces potent
CD4+ T-cell proliferation. 2.2% CD4+ T-cells divide up to once a day when
cultured with
DP-0016.F at 2.511M over 6 days. DP-0016.D did not significantly induce CD4+ T-
cell
proliferation in this experiment, although we note that it was insoluble, and
thus, no
conclusions can be drawn regarding DP-0016.D based on this experiment. In
addition, DP-
0016.D has an estimated net negative charge of -3.8 at pH7. Similarly, DP-
0016.E also had
poor solubility.
DP-0016.F and DP-0016.B induce CD4+ T-cell proliferation, while also inducing
anti-hyper-phosphorylated Tau specific antibodies (data not shown).
Example 10. DP-0016.F induces a strong and specific immune response in mice
The ability of DP-0016.F to raise immunogen-specific antibodies in mice
injected
with the compositions was evaluated.
Mouse treatment and injection. Fifteen (15) female CS 7B1/6 mice, 8-12-weeks-
old,
were divided into three groups of five mice (n=5 per group): DP-0016.0 (1.2
mg/kg); DP-
0016.F (1.2 mg/kg); and incomplete Freund's adjuvant (IFA) (200 uL in 0.01 M
of
ammonium bicarbonate). Each ml of IFA contained 0.85 ml of paraffin oil and
0.15 ml of
mannide monooleate. The mice were injected once a week for a total of 4
subcutaneous
injections without the use of anesthetics. The injection was carried out in
the form of an
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emulsion in IFA, using glass syringes with metal connectors. The compounds
were injected
at one concentration per group. A control group injected with IFA alone was
also tested. In
addition, there were three un-injected mice. DP-0016.0 and DP-0016.F are
examples of
compounds of the disclosure. One week after the fourth injection (Day 29), all
the mice were
.. sacrificed. The naive mice were also sacrificed and their sera were used as
negative control in
the anti-DP-0016 and Anti-PHF1 Ab assay.
Anti-immunogen antibody determination. Sera from the 15 injected mice and from
the
3 un-injected mice were collected. The sera from mice injected with the
compounds were
assayed for the determination of anti-immunogen antibodies using a titer assay
approach.
The anti-immunogen antibody assay plate included test sample(s), and negative
control (sera collected from non-injected mice or from mice injected with
IFA), titered down
(8 dilutions) with dilution buffer. All the plates were coated with the
respective composition
used for the injection. The presence of anti-immunogen antibodies was detected
using both
anti-IgG1 and anti-IgG2a antibodies, in separate reactions.
Results. Figure 15A shows the results of antibody ELISA against DP-0016 or
PHF1.
Serum samples from mice injected with the compositions were assayed for the
presence of
anti-DP-0016 or anti-PHF1 antibodies, and significantly higher titers of the
anti- DP-0016 or
anti-PHF1 IgG1 antibodies were present in the sera of mice injected with DP-
0016.0 or DP-
0016.F compared to the sera of mice injected with IFA alone. The presence of
IgG1
antibodies is indicative of a Th2 response.
DP-0016.F and DP-0016.0 induced a similar immunogenic profile. The significant
immunogenicity indicates that such compositions have potential value in
generating a robust
immune response, including in elderly patients whose immune systems generally
tend to be
weaker.
Figure 15B shows the results of the antibody ELISA against a short linear
peptide
covering pS422 and recombinant Tau. One of the key biomarkers for
hyperphosphorylation
of Tau is the phosphorylation of position pS422 (corresponding to position 422
of SEQ ID
No. 17). In contrast, recombinant Tau represents the major microtubule
associated protein,
which is part of a normal mature neuron. Figure 15B shows that high titers of
anti-p5422
.. antibodies are present, while very low titers of anti-recombinant Tau
antibodies are present.
This indicates that the high complexity polypeptides of DP-0016.F and, to a
lesser extent,
DP-0016.C, induce a robust, but specific antibody response against pathologic
Tau.
Figure 15C shows the results of the antibody ELISA against PHF-Tau. In
patients,
hyperphosphorylated Tau leads to conformational changes and, ultimately, to
the formation
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of pathogenic species such as paired helical filaments (PHFs). Figure 15C
shows that high
titers of anti-PHF Tau antibodies are induced by DP-0016.F. The significant
immunogenicity of DP-0016.F and DP-0016.0 is consistent with generation of a
robust
immune response against the pathological species of Tau (PHF-Tau).
Figure 15D shows the specificity of the antibodies induced by DP-0016.0 and DP-
0016.F against human Alzheimer's disease tissue. As described above, C57B1/6
mice were
injected once a week for four weeks with the DP-0016.0 and DP-0016.F
compositions
emulsified in IFA. On day 29, terminal bleeding was conducted and sera were
collected. The
antibodies induced by DP-0016.0 and DP-0016.F were isolated from sera samples.
These
antibodies were applied to post-mortem brain tissue from Alzheimer's disease
patients.
Antibodies bound to antigens were visualized using immunofluorescence antibody
assay
(IFA). Antibodies induced by the administration of each of the two
compositions showed
high affinity and specific binding to hyperphosphorylated tau found in amyloid
plaques and
neurofibrillary tangles. This indicates that these antibodies have the
specificity to bind to the
pathologic lesions which occur in the brains of Alzheimer's disease patients.
Example 11. Preparation of an amino acid copolymer composition with antigenic
specificity to HPV L2 protein (DP-0O24.1)
Figure 16 shows a schematic for the design of an amino acid copolymer
composition
(a composition based on the DP-0O24.1 template arrangement, sequence, and
input amino
acid input distribution) comprising a high complexity of polypeptides based on
antigenic
sequences derived from HPV L2. DP-0O24.1 is designed to target the minor
capsid protein
L2, which localizes along the inner surface of the virion, and plays a role in
capsid
stabilization through interaction with the major capsid protein Ll. The DP-
0O24.1
polypeptides can be used to generate specific antibodies against many variants
of human
papilloma virus, which provide protection against a broad range of HPV
variants. Without
being bound by theory, antibodies to L2 may be particularly useful in a
prophylactic vaccine,
such as in subject who has not already been infected with HPV.
The full-length polypeptides of DP-0O24.1 based on the template arrangement,
sequence, and amino acid input distribution shown in this schematic are about
69 amino acids
long and the amino acid positions, relative to full-length polypeptides based
on the depicted
template arrangement, of the composition are indicated in Row 1 (the row
labeled peptide #).
The DP-0O24.1 polypeptides (e.g. the polypeptides in the mixture of
polypeptides comprising
the DP-0O24.1 composition) are based on a template arrangement having a
maximum of 3
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random copolymer regions (RCRs) wherein the amino acid at each position is
selected from
lysine, alanine or glutamic acid, i.e., the RCRs comprise K/A/E motifs (N-
terminal RCRa
(corresponding to positions 1-4 in Figure 16), RCRb (corresponding to
positions 33-36 in
Figure 16), and RCRc (corresponding to positions 65-68 in Figure 16), and two
antigenic
regions (ARs) based on sequences derived from HPV L2 (ARa (corresponding to
positions 5-
32 in Figure 16) and ARb (corresponding to positions 37-64 in Figure 16). The
template
arrangement of DP-0O24.1 comprises: RCRa-ARa- RCRb-ARb-RCItc, although other
template
arrangements are described herein. In this example, both ARa and ARb
correspond to or
comprise a region of an L2 protein of HPV (e.g., a base peptide sequence based
on a portion
a L2 protein). In this example, complexity across the polypeptides in the
composition comes
from both the RCRs and ARs although, in other examples, complexity may come
only from
the RCRs or from the RCRs and one, but not both, ARs. In this example, the
RCRs and ARs
are contiguous. However, they may be interconnected by a linker, such as by
one or more
intervening amino acid residues. Moreover, the polypeptides in the composition
may include
on or more additional amino acid residues at the N- or C-terminus, including
naturally
occurring or non-natural amino acids.
Row 2 of the top and bottom panels (the row labeled HPV L2 #) indicates the
positions that correspond to residues, which may undergo post-translational
modifications to
form cysteine bridges (positions 21 and 27), or residues, which represent
boundaries of the
L2 protein epitope in this example (positions 13 and 40). Rows 3, 4, 5, and 6
indicate the
possible amino acids, which are or may be incorporated at each position in the
peptide. The
three copolymer regions (RCRs) correspond to positions 1-4 (RCRa), positions
33-36 (RCRb),
and positions 65-68 (RCItc). The two antigenic regions (ARs) correspond to
positions 5-32
(ARa) and to positions 37-64 (ARb). The sequence of the AR given in Row 3
corresponds to
the base peptide sequence and reflects original amino acid residues derived
from the
corresponding portion of the target, an HPV L2.
Rows 7, 8, 9, and 10 (the rows labeled 1" Row %, 2nd Row%, 3rd Row %, and 4th
Row
% respectively) indicate the molar input percentages of the corresponding,
color-coded,
amino acid above. For example, at position 5, the relative molar amino acid
input percentages
of serine (S) and alanine (A) are 90% and 10%, respectively. Additional amino
acids such as
conserved substitutions may be provided at any of the positions in the
antigenic region.
The three RCRs in this example are 4 amino acids in length and contain either
lysine
(K), alanine (A), or glutamic acid (E) at each position. RCRs of differing
sizes (e.g. shorter or
longer than depicted) are contemplated, as described herein. The relative
molar input
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percentages of lysine, alanine, or glutamic acid were 40%, 50%, or 10%,
relatively. The
relative molar input percentage of lysine, alanine, or glutamic acid was
selected from within
the foregoing ranges, independently for each position
The AR at positions corresponding to 5-32 of Figure 16 and the AR
corresponding to
positions 37-64 of Figure 16 are based on a base peptide sequence which
substantially
corresponds to positions 13-40 of an HPV L2 (exemplary longer L2 protein set
forth in SEQ
ID No. 30; corresponding base peptide sequence used in this example set forth
in SEQ ID
NO: 34). For this particular example, most positions of the ARs (e.g.
positions 5, 7-10, 12,
14, 17-18, 20, 22-28, 31, 37, 39-42, 44, 46, 49-50, 52, 54-60, and 63) specify
a relative molar
input percentage of 10% alanine for generating complexity at those positions
across the
mixture of polypeptides, with the original amino acid (relative to the HPV L2
sequence)
making up the remaining 90% of the input molar percentage of amino acid at
each position
and for generating complexity at those positions across the mixture.
Therefore, in this
example, at these positions, each polypeptide has either the original amino
acid indicated in
Row 3 or alanine in the ARs. For some positions in the AR (6, 8, 11, 15-16,
21, 23-25, 27,
29-30, 32, 38, 40, 43, 47-48, 53, 57, 59, 61-62, and 64), additional
complexity is provided by,
for example, also permitting variation at a position based on a conserved
substitution, such as
an orthologous substitution, a residue that is present in a different strain,
a residue that is
observed based on polymorphism or natural variation, or substitution with a
similar residue.
For certain other positions in this example, such as positions 13, 19, 45, and
51 no amino
acids other than the residue present at the corresponding position of the base
peptide are
provided. Norleucine is found at the C-terminal most amino acid at position 69
of Figure 16.
Additional amino acids such as conserved substitutions may be provided at any
of the
positions. This provides a description of this particular composition.
However, other
compositions based on, for example, differing template arrangements, input
percentages in
the RCRs or ARs, and/or base peptide sequence or antigen are contemplated and
provided.
A polypeptide mixture according to the schematic shown in Figure 16 can be
successfully synthesized using solid-phase peptide synthesis. Briefly, the
polypeptides based
on this template arrangement and input percentages may be synthesized from C-
terminus to
N-terminus using Fmoc-based SPPS, as described above. To improve the coupling
efficiency
and generate a higher yield in each step, longer coupling times or the
addition of redundant
coupling reactions at one or more positions may be used. The disclosure
provides
compositions manufactured based on this template arrangement, sequence and
input amino
acid distribution.
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Example 12. MALDI-TOF-MS analysis of an amino acid copolymer composition with
antigenic specificity to Tau protein (DP-0016.F)
Figure 17 shows a representative example of a MALDI-TOF-MS spectra comparing 2
different preparations of an amino acid copolymer composition, DP-0016.F,
comprising a
high complexity of polypeptides based on antigenic sequences derived from Tau
and the
template arrangement and amino acid inputs for DP-0016.F set forth in Example
8.
MALDI-TOF-MS Sample Preparation. An amino acid copolymer composition was
synthesized using solid phase peptide synthesis according to the method
previously described
in Example 8. Matrix solutions for MALDI-TOF-MS were prepared using the amino
acid
copolymer composition. Each sample contained about 10mg/m1 of 3,5-dimethoxy-4-
hydroxy
cinnamic acid (sinapinic acid) in an aqueous solvent solution comprising 0.1%
acqueous
trifluoroacetic acid (TFA) and acetronitrile. The amino acid copolymer
composition was
mixed with the matrix solution to create cocrystallized samples for analysis.
MALDI-TOF-MS Analysis. The cocrystallized samples were deposited onto the
stainless
steel autosampler pins of a MALDI-TOF-MS instrument and allowed to air dry.
The sample
was irradiated at 337 nm with a nitrogen laser resulting in the analytes being
protonated and
desorbed into the gas phase. The mass/charge (m/z) value was determined in a
TOF mass
analyzer.
MALDI-TOF-MS Results. The resulting full MALDI-TOF spectrum of two different
batches
of DP-0016.F compositions is shown in Figure 17. Full-length polypeptides
based on this
template arrangement are about 63 amino acids and have a mass of about 6900
Daltons. The
peaks which occur prior to the 6900 Dalton peak are due to synthesis of
polypeptides based
on this template arrangement but which are shorter due to incomplete protein
synthesis. By
calculating the ratio between the masses of polypeptides in the composition
having a full-
length polypeptide sequence and the masses of the polypeptides having masses
indicative of a
less than full-length sequence, it was determined that peptide synthesis may
be disrupted
between the positions corresponding to positions 16-17 and positions 24-25 of
Tau-F in
Figure 12. As can be seen in Figure 17, in one batch of DP-0016.F composition
(a
composition based on the DP-0016.F template arrangement, sequence, and input
amino acid
input distribution), the resulting composition synthesized had a purity of
about 53% (e.g.,
calculated at about 52.61%). Purity, in this case, refers to the percentage of
polypeptides in
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the composition based on the template arrangement that are substantially full-
length
polypeptides. Thus, "about 53% purity" refers to a composition in which about
53% of the
polypeptides in the composition that are based on the template arrangement are
substantially
full-length polypeptides.
In the second batch of DP-0016.F polypeptides, redundant coupling reactions
were
added at the positions corresponding to the incomplete truncations (between
positions
corresponding to positions 16-17 and positions 24-25) of Tau-F. As can be seen
in the
bottom panel of Figure 17, the addition of these additional coupling reactions
resulted in an
improvement in the percentage of full-length polypeptides in the composition
based on the
same template arrangement, sequence and amino acid inputs to about 83% purity
(e.g.,
83.47%).
Example 13. Preparation of an amino acid copolymer composition with antigenic
specificity to Tau protein (DP-0016.F) loaded onto Polylactic Acid (PLA)
particles
DP-0016.F was prepared as described in Example 8. DPC016.F was then loaded
onto
Polylactic Acid (PLA) particles, particles which protect foreign antigens
(Ags) from
degradation and dilution. PLA particles are efficiently captured by phagocytes
and improve
Ag processing and presentation to T-cells. Figure 18 shows a representative
example of a
MALDI-TOF-MS spectra of DP-0016.F loaded onto PLA particles.
MALDI-TOF-MS Sample Preparation. An amino acid copolymer composition was
synthesized using solid phase peptide synthesis according to the method
previously described
in Example 8. The DP-0016.F was then loaded onto PLA particles. Matrix
solutions for
MALDI-TOF-MS were prepared using the amino acid copolymer composition loaded
onto
PLA particles. Each sample contained about 10mg/m1 of 3,5-dimethoxy-4-hydroxy
cinnamic
acid (sinapinic acid) in an aqueous solvent solution comprising 0.1% acqueous
trifluoroacetic
acid (TFA) and acetronitrile. The amino acid copolymer composition was mixed
with the
matrix solution to create cocrystallized samples for analysis.
MALDI-TOF-MS Analysis. The cocrystallized samples were deposited onto the
stainless
steel autosampler pins of a MALDI-TOF-MS instrument and allowed to air dry.
The sample
was irradiated at 337 nm with a nitrogen laser resulting in the analytes being
protonated and
desorbed into the gas phase. The mass/charge (m/z) value was determined in a
TOF mass
analyzer.
MALDI-TOF-MS Results. The resulting full MALDI-TOF spectrum of DP-0016.F
recovered
from 4 months of loading onto PLA particles is shown in Figure 18. Full-length
polypeptides
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based on this template arrangement are about 63 amino acids and have a mass of
about 6900
Daltons, and represents 83% purity.
Example 14. DP-0016.F is immunogenic in CD4+ and CD8+ T-cell proliferation
assays
DP-0016.F was tested for its ability to induce human T-cell proliferation in
vitro. CD4+ and
CD8+ T-cell proliferation assays were conducted using healthy donor PBMC as
described
above in Example 9. Figure 19A and 19B shows the relative fraction of the
proliferating
CD4+ and CD8+ T-cells, respectively.
DP-0016.F, 7.5 tM induced CD4+ T-cell proliferation in the majority of healthy
donors'
PBMCs, significantly better than the tetanus toxoid peptide TET 830 (TT) and
as well as the
flu HA peptide 317-319 (HA) (Fig. 19A). DP-0016.F did not significantly induce
CD8+ T-
cell proliferation as compared to TT, indicating the potential for a CD4+
specific response.
DP-0016.F was loaded onto PLA particles and was tested for its ability to
induce a
CD4+ T-cell response. The final concentrations of PLA particles were 860, 260,
and 90
pg/mL in the cell culture wells containing 2.5, 0.75, and 0.25 tM of DP-
0016.F,
respectively. In cultures containing 0.25 uM and 0.75 uM of DP-0016.F, the PLA
particles
significantly boosted the CD4+ T-cell proliferation. However, at the 860 pg/mL
concentration, the PLA particles were cytotoxic. (Fig. 19C). Loading of DP-
0016.F, 0.25 tM
onto PLA particles restored CD4+ T-cell proliferation above background in 18
subjects
(range 0.03 to +5.34% versus -1.47 to +0.10% for DP-0016 alone). Furthermore,
a
comparison of DP-0016.F in either PBS or loaded onto PLA particles showed a
significant
additional effect of PLA particles on both CD4+ and CD8+ T-cell response to DP-
0016.F.
See Figure 19D.
Example 15. DP-0016.F administered with IFA or loaded onto PLA particles
induces
antibody production in JNPL3 mice
The ability of DP-0016.F to raise immunogen-specific antibodies in mice
injected
with the compositions was evaluated.
Mouse treatment and injection. JNPL3 mice were used which express hTau ON4R
(383 amino acids) with the P301L mutation under mouse prion promoter. Twenty-
five (25)
female JNPL3 mice, 12 weeks-old, were divided into five groups of five mice
(n=5 per
group): DP-0016.F with Incomplete Freund's Adjuvant (IFA) (0.3mg/kg); DP-
0016.F with
IFA (1.2 mg/kg); DP-0016.F with PLA particles (0.3mg/kg); DP-0016.F with PLA
particles
(1.2 mg/kg); PLA particles alone. Each ml of IFA contained 0.85 ml of paraffin
oil and 0.15
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ml of mannide monooleate. The mice were injected once a week for a total of 4
subcutaneous
injections (day 0, 7, 14, and 21) without the use of anesthetics. The
injection was carried out
in the form of an emulsion in IFA or loaded onto PLA particles, using glass
syringes with
metal connectors. The compounds were injected at one concentration per group.
One week
after the fourth injection (Day 28), the mice were bled. The groups treated
with DP-0016.F
with PLA particles (0.3mg/kg) and with DP-0016.F with PLA particles (1.2
mg/kg) were
further treated on day 49, 77, 105, and 133. On day 168, the DP-0016.F with
PLA particles
(0.3mg/kg) and DP-0016.F with PLA particles (1.2 mg/kg) treated groups were
bled again.
Anti -immunogen antibody determination. Sera from the 25 injected mice were
collected. The sera from mice injected with the compounds were assayed for the
determination of anti-immunogen antibodies using a titer assay approach.
The anti-immunogen antibody assay plate included test samples titered down (8
dilutions) with dilution buffer. All the plates were coated with the one of
the following
antigens: PHF1, pS422, recTau and PHF-tau. The presence of anti-immunogen
antibodies
was detected using an antibody ELISA.
Hind Limb Clasping Score. Hind limb clasping is an assay which allows for the
physical assessment of disease severity in a mouse model. The JNPL3 mouse
model is
known to develop hind limb dysfunction with age and increased disease
severity. To
evaluate the hind limb clasping, the mice were suspended by the base of the
tail on Day 168.
Each mouse was rated for hind limb clasping on a scale of 0 to 2; a score of 0
being no
clasping, 1 moderate clasping and 2 severe clasping.
Results. Figure 20A shows the results of the antibody ELISA against PHF-tau,
which
are paired helical filaments isolated from AD patients' autopsies, and
recombinant Tau
(recTau). Paired helical filaments (PHF) are one of the key pathological
hallmarks in
Alzheimer's disease and other tauopathies. In contrast, recombinant Tau
represents the major
microtubule associated protein, which is part of a normal mature neuron.
Figure 20A shows
that high titers of anti-PHF-tau antibodies are present. This indicates that
the high
complexity polypeptides of DP-0016.F emulsified in IFA 1.2 mg/kg induces a
robust
antibody response against pathologic Tau. The response to recTau is much lower
in non-
transgenic mice (data not shown).
Figure 20B shows the results of the antibody ELISA against short linear
peptides
covering either PHF1 or pS422. The PHF-1 site includes pS396 and pS404 of Tau-
F, and are
commonly phosphorylated in Alzheimer's disease and other tauopathies. In
contrast, position
pS422 (corresponding to position 422 of SEQ ID No. 17) represents a key
biomarkers for
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hyperphosphorylated Tau and this position is targeted by DP-0016.F. Figure 20B
shows that
high titers of anti-PHF1 antibodies are present. There is also an increase in
pS422 antibody
titer when DP-0016.F is emulsified in IFA (1.2 mg/kg).
Figure 20C shows the antibody response on day 168 as compared to day 28. The
graph shows the results of the antibody ELISA against short linear peptide
covering PHF1,
short peptide covering pS422, PHF-tau, and recombinant Tau (recTau),
respectively. Mice
treated with DP-0016.F loaded onto PLA particles (0.3 mg/kg) showed a
sustained anti-tau
response over 168 days, with the last treatment occurring on day 133.
Figure 20D shows the effect of DP-0016.F loaded onto PLA particles on hind
limb
clasping. JNPL3 mice were treated as described above. The results show a
significant inverse
correlation between anti-PHF1 antibody titer and hind limb clasping as
compared to controls.
Example 16. DP-0016.F emulsified in IFA or loaded onto PLA particles increases
soluble tau, decreases hyperphosphorylated tau, and decreases tau content in
various
brain regions of JNPL3 mice
Mice were administered DP-0016.F with either Incomplete Freund's Adjuvant
(IFA)
or polylactic acid (PLA) particles, and the effect of on tau phosphorylation,
total tau contents,
and disease state was evaluated.
PLA formulation. DP-0016.F was loaded onto PLA particles. Formulation was
optimized to achieve a compromise between yield of adsorption (% of loaded
peptides) and loading capacity (mg of peptides per g of particles or m2 of
particle surface).
Hydrodynamic diameter, polydispersity index (PdI) and Zeta potential were
measured.
Mouse treatment and injection. Female JNPL3 mice were used. Forty-eight (48)
female JNPL3 mice, 8-12 weeks-old, were divided into three groups of sixteen
mice (n=16
per group): DP-0016.F emulsified in IFA (0.8 mg/kg); DP-0016.F with PLA
particles (0.8
mg/kg); and PLA particles alone. Each ml of IFA contained 0.85 ml of paraffin
oil and 0.15
ml of mannide monooleate. The mice treated with DP-0016.F with IFA (0.8 mg/kg)
received
a total of 5 subcutaneous injections (Day 0, 14, 42, 70, and 98). The mice
treated with either
DP-0016.F with PLA particles (0.8 mg/kg) or PLA particles alone received a
total of 7
subcutaneous injections (Day 0, 7, 14, 28, 42, 70, and 98). The injections
were carried out in
the form of an emulsion in IFA or loaded onto PLA particles. On Day 119, the
mice were
measured for hind limb clasping, antibody response, and were euthanized for
histological
analysis of tau in the cortex, hindbrain and hippocampus. Soluble, insoluble,
and aggregated
tau contents were measured.
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Anti -immunogen antibody determination. Sera from the 48 injected mice were
collected. The sera from mice injected with the compounds were assayed for the
determination of anti-immunogen antibodies using a titer assay approach.
The anti-immunogen antibody assay plate included test sample(s) titered down
(8
dilutions) with dilution buffer. All the plates were coated with the one of
the following
antigens: DP-0016.F, recTau and PHF-tau. The presence of anti-immunogen
antibodies was
detected using both anti-IgG1 and anti-IgG2a antibodies, in separate
reactions.
Extraction and measurement of soluble and total tau. The cortex, hindbrain and
hippocampus of the 48 injected mice were homogenized in a solution of TBS,
pH7.4
.. containing 10 mM NaF, 1 mM NaV03, and 2 mM EGTA, including a complete Mini
protease inhibitor. Soluble tau, insoluble tau, and aggregated tau were
isolated, and further
analyzed by quantitative ELISA using the following antibodies: DA31 (total
tau), CP13
(p5202), RZ3 (pT231), PHF1 (p5396 +1)5404), and DA9 (aggregated tau).
Hind Limb Clasping Score. Hind limb clasping is an assay which allows for the
physical assessment of disease severity in a mouse model. The JNPL3 mouse
model is
known to develop hind limb dysfunction with age and increased disease
severity. To
evaluate the hind limb clasping, the mice were suspended by the base of the
tail on Day 119.
Each mouse was rated for hind limb clasping on a scale of 0 to 2; a score of 0
being no
clasping, 1 moderate clasping and 2 severe clasping.
Results. Mice were injected with DP-0016.F emulsified in IFA (0.8 mg/kg), DP-
0016.F with PLA particles (0.8 mg/kg) or PLA particles alone. On day 119, sera
were
collected. The sera were assayed for antibody response against DP-0016.F. The
anti-
immunogen antibodies were detected using both anti-IgG1 and anti-IgG2a isotype
secondary
antibodies, in separate reactions (Figure 21A). The sera were further assayed
for antibody
response to PHF-tau and recTau (Figure 21B).
The cortex, hindbrain and hippocampus of the 47 injected mice were homogenized
and the total soluble tau was measured in each region. Figure 21C shows the
effect of DP-
0016.F emulsified in IFA (0.8 mg/kg); DP-0016.F with PLA particles (0.8
mg/kg); or PLA
particles alone on total soluble tau in the cortex, hindbrain and hippocampus
of JNPL3 mice.
Abnormal accumulation of pathological tau species in the brain of JNPL3
transgenic mice
has been associated with tauopathic disease. Soluble tau (prefibrillar forms
of tau) are
potentially more toxic than insoluble tau (tangles), which may serve as a
protective
mechanism.
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The effect of DP-0016.F emulsified in IFA (0.8 mg/kg); DP-0016.F with PLA
particles (0.8 mg/kg); or PLA particles alone (vehicle) on hyperphosphorylated
tau oligomer
levels was measured in the hindbrain (Figure 21D), cortex (data not shown), or
hippocampus
(Figure 21E) of JNPL3 mice. Various markers of tau phosphorylation were used:
PHF1
(pS396/pS404), CP13 (pS202), RZ3 (pT231), and DA31 (total tau). The group
treated with
DP-0016.F emulsified in IFA (0.8 mg/kg) showed the largest decrease in soluble
phosphoTau in the hindbrain. The DP-0016.F emulsified in IFA (0.8 mg/kg)
treatment group
also showed the largest decrease in soluble phosphoTau marked by PHF1 and DA31
antibodies in the hindbrain.
Insoluble tau in the hindbrain and cortex were measured in mice treated with
DP-
0016.F emulsified in IFA (0.8 mg/kg); DP-0016.F with PLA particles (0.8
mg/kg); or PLA
particles alone (vehicle). There was a 48% decrease in insoluble tau in the
hindbrain of mice
treated with DP-0016.F emulsified in IFA (0.8 mg/kg) (Figure 21F).
Furthermore, there was
a 24% decrease in insoluble tau in the cortex of mice treated with DP-0016.F
with PLA
.. particles (0.8 mg/kg). Insoluble total tau represents 1.5% and 3.0% of
total tau in the
hindbrain and cortex, respectively.
Tau aggregate contents in the hippocampus, cortex, and hindbrain were measured
using a quantitative ELISA with anti-total tau DA9 antibody. A respective
decrease of 34%
(50% based on mean value) and 21% (62% based on mean value) in tau aggregates
were
measured in the hippocampus and hindbrain of mice treated with DP-0016.F
emulsified with
IFA (0.8 mg/kg) (Figure 21G). These results show that administration of DP-
0016.F results
in a reduction in hyperphosphorylated soluble, insoluble and aggregated tau.
While it
remains to be determined which form of tau is particularly toxic, treatment
with DP-0016.F
clears each type of tau, distinguishing itself from prior antibody therapies
which only
decrease insoluble and aggregated tau. Furthermore, the antibodies produced by
this
approach may clear tau by entering neurons and binding to intracellular tau.
Alternatively,
antibodies may bind to extracellular tau and promote its clearance.
Figure 21H shows the effect of DP-0016.F emulsified in IFA (0.8 mg/kg); DP-
0016.F with PLA particles (0.8 mg/kg); or PLA particles alone on hind limb
clasping, and its
.. correlation with total soluble tau in the cortex of JNPL3 mice. The results
show that total tau
content drops in DP-0016.F treated mice with severe clasping, and that total
tau content
increases with clasping severity in vehicle treated mice.
INCORPORATION BY REFERENCE
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All publications and patents mentioned herein are hereby incorporated by
reference in
their entirety as if each individual publication or patent was specifically
and individually
indicated to be incorporated by reference.
While specific embodiments of the subject disclosure have been discussed, the
above
specification is illustrative and not restrictive. Many variations of the
disclosure will become
apparent to those skilled in the art upon review of this specification and the
claims below.
The full scope of the disclosure should be determined by reference to the
claims, along with
their full scope of equivalents, and the specification, along with such
variations.
EXEMPLARY SEQUENCES
Sequences from Tables 1 and 2
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO: 1);
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 2);
MGKGEEGYPQEGILEDMPVDPGSEAYEMPSEEGYQDYEEA (SEQ ID NO: 3);
DNEAYEMPSEEGYQDYE (SEQ ID NO: 4);
MATLEKLMKAFESLKSF (SEQ ID NO: 5);
HGAEIVYKSPVVSGDISPRHLSNVSSIGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO: 6);
HGAEIVYKSPVVSGDTSPRHL (SEQ ID NO: 7);
IDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO: 8);
AEIVYKSPVVSGDTSPRHL (SEQ ID NO: 9);
DMVDSPQLATLADEVSASLAKQGL (SEQ ID NO: 10);
IQRTPKIQVYSRHPAENGKS (SEQ ID NO: 11);
ILARNLVPMV (SEQ ID NO: 21);
ELEGVWQPA (SEQ ID NO: 22);
.. RIFAELEGV (SEQ ID NO: 23);
NLVPMVATV (SEQ ID NO: 24);
RIQRGPGRAFVTIGK (SEQ ID NO: 25);
TGSIDMVDSPQLATLADEVSASLAK (SEQ ID NO: 31);
TGSIDMVDSPQLATLA (SEQ ID NO: 32);
QLATLADEVSASLAKRRR (SEQ ID NO: 33);
SVTQLYKICKQSGTCPPDVIPKVEGTIL (SEQ ID NO: 34);
GSEAYEMPSEEGYQDYE (SEQ ID NO: 36);
DNEAYEMPSEEGYQDYE (SEQ ID NO: 37);
Further Exemplary targets
SEQ ID NO: 12
Beta-2-microglobulin - human (P61769-1)
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MSRSVALAVL ALLSLSGLEA IQRTPKIQVY SRHPAENGKS NFLNCYVSGF HPSDIEVDLL
KNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM
SEQ ID NO: 13
HUMAN PRION PROTEIN (AAH22532)
MANLGCWMLV LFVATWSDLG LCKKRPKPGG WNTGGSRYPG QGSPGGNRYP PQGGGGWGQP
HGGGWGQPHG GGWGQPHGGG WGQPHGGGWG QGGGTHSQWN KPSKPKTNMK HMAGAAAAGA
VVGGLGGYVL GSAMSRPIIH FGSDYEDRYY RENMHRYPNQ VYYRPMDEYS NQNNFVHDCV
NITIKQHTVT TTTKGENFTE TDVKMMERVV EQMCITQYER ESQAYYKRGS SMVLFSSPPV
ILLISFLIFL IVG
SEQ ID NO: 14
HUMAN SOD1 (CAG46542)
MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE FGDNTAGCTS
AGPHFNPLSR KHGGPKDEER HVGDLGNVTA DKDGVADVSI EDSVISLSGD HCIIGRTLVV
HEKADDLGKG GNEESTKTGN AGSRLACGVI GIAQ
SEQ ID NO: 15
HUMAN HUNTINGTIN (NP 002102.4)
1 matleklmka feslksfqqq qqqqqqqqqq qqqqqqqqqq pppppppppp pqlpqpppqa
61 ullpqpqpp ppppppppgp avaeeplhrp kkelsatkkd rvnhcltice nivaqsvrns
121 pefqkllgia melfllcsdd aesdvrmvad eclnkvikal mdsnlprlql elykeikkng
181 aprslraalw rfaelahlvr pqkcrpylvn 11pcltrtsk rpeesvget1 aaavpkimas
241 fgnfandnei kvllkafian lksssptirr taagsaysic ghsrrtgyfy swllnvllgl
301 lvpvedehst llilgv11t1 rylvpllqqq vkdtslkgsf gvtrkemevs psaeqlvqvy
361 eltlhhtqhq dhnvvtgale llqqlfrtpp pellqtltav ggigqltaak eesggrsrsg
421 siveliaggg sscspvlsrk qkgkvllgee ealeddsesr sdvsssalta svkdeisgel
481 aassgvstpg saghdiiteq prsqhtlqad svdlascdlt ssatdgdeed ilshsssqvs
541 avpsdpamdl ndgtqasspi sdssqttteg pdsavtpsds seivldgtdn gylglgiggp
601 qdedeeatgi 1pdeaseafr nssmalqqah llknmshcrq psdssvdkfv lrdeatepgd
661 genkperikg digqstddds aplvhcvrll sasflltggk nvlvpdrdvr vsvkalalsc
721 vgaavalhpe sffsklykvp ldtteypeeq yvsdilnyid hgdpqvrgat ailcgtlics
781 ilsrsrfhvg dwmgtirtlt gntfsladci pllrktlkde ssvtcklact avrncvmslc
841 sssyselglq liidvltlrn ssywlvrtel letlaeidfr lvsfleakae nlhrgahhyt
901 gllklgervl nnvvihllgd edprvrhvaa aslirlvpkl fykcdqgqad pvvavardqs
961 svylkllmhe tqppshfsys titriyrgyn llpsitdvtm ennlsrviaa vshelitstt
1021 raltfgccea lcllstafpv ciwslgwhcg vpplsasdes rksctvgmat miltllssaw
1081 fpldlsahqd alilagnlla asapkslrss waseeeanpa atkqeevwpa lgdralvpmv
1141 eqlfshllkv inicahvldd vapgpaikaa 1psltnppsl spirrkgkek epgegasvp1
1201 spkkgseasa asrqsdtsgp vttskssslg sfyhlpsylk lhdvlkatha nykvtldlqn
1261 stekfggflr saldvlsqil elatlqdigk cveeilgylk scfsrepmma tvcvqqllkt
1321 lfgtnlasqf dglssnpsks ggragrlgss svrpglyhyc fmapythftq aladaslrnm
1381 vgaegendts gwfdvlqkvs tqlktnitsv tknradknai hnhirlfepl vikalkqytt
1441 ttcvqlqkqv ldllaglvq1 rvnyclldsd qvfigfv1kg feyievgqfr eseaiipnif
1501 fflvllsyer yhskqiigip kiiqlcdgim asgrkavtha ipalqpivhd lfvlrgtnka
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1561 dagkeletqk evvvsmllrl igyhqvlemf ilvlqqchke nedkwkrlsr giadiilpml
1621 akqqmhidsh ealgvintlf eilapsslrp vdmllrsmfv tpntmasyst vqlwisgila
1681 ilrvlisqst edivlsriqe lsfspylisc tvinr1rdgd ststleehse gkqiknlpee
1741 tfsrfllqlv gilledivtk qlkvemseqg htfycqelgt llmclihifk sgmfrritaa
1801 atrlfrsdgc ggsfytldsl nlrarsmitt hpalvllwcq illlvnhtdy rwwaevqqtp
1861 krhslsstkl lspqmsgeee dsdlaaklgm cnreivrrga lilfcdyvcq nlhdsehltw
1921 livnhiqdli slsheppvqd fisavhrnsa asglfigaig srcenlstpt mlkktlqcle
1981 gihlsgsgav ltlyvdr1lc tpfrvlarmv dilacrrvem llaanlqssm aqlpmeelnr
2041 igeylcissgl agrhgrlysl ldrfrlstmq dslspsppvs shpldgdghv sletvspdkd
2101 wyvhlvksqc wtrsdsalle gaelvnripa edmnafmmns efnlsllapc 1s1gmseisg
2161 gqksalfeaa revtlarvsg tvqqlpavhh vfqpelpaep aaywsklndl fgdaalyqs1
2221 ptlaralaqy lvvvsklpsh lhlppekekd ivkfvvatle alswhliheq iplsldlqag
2281 ldccclalql pglwsvvsst efvthacsli ycvhfileav avqpgeqlls perrtntpka
2341 iseeeeevdp ntqnpkyita acemvaemve slgsvlalgh krnsgvpafl tpllrniiis
2401 larlplvnsy trvpplvwkl gwspkpggdf gtafpeipve flqekevfke fiyrintlgw
2461 tsrtqfeetw atllgvlvtq plvmegeesp peedtertqi nvlavqaits lvlsamtvpv
2521 agnpayscle qqprnkplka ldtrfgrkls iirgivegei qamvskreni athhlyclawd
2581 pvpslspatt galishekll lqinperelg smsyklgqvs ihsvwlgnsi tplreeewde
2641 eeeeeadapa psspptspvn srkhragvdi hscsqfllel ysrwilpsss arrtpailis
2701 evvrsllvvs dlfternqfe lmyvtltelr rvhpsedeil aqylvpatck aaavlgmdka
2761 vaepvsrlle stlrsshlps rvgalhgvly vlecdllddt akqlipvisd yllsnlkgia
2821 hcvnihsqqh vlvmcatafy lienypldvg pefsasiiqm cgvmlsgsee stpsiiyhca
2881 lrglerllls eqlsrldaes lvklsvdrvn vhsphramaa lglmltcmyt gkekvspgrt
2941 sdpnpaapds esvivamery svlfdrirkg fpcearvvar ilpqflddff ppgclimnkvi
3001 geflsnqqpy pqfmatvvyk vfqtlhstgq ssmvrdwvml slsnftqrap vamatwslsc
3061 ffvsastspw vaailphvis rmgkleqvdv nlfclvatdf yrhqieeeld rrafqsvlev
3121 vaapgspyhr lltclrnvhk vttc
SEQ ID NO: 16
MOUSE ALPHA SYNUCLEIN (NP 001035916)
MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVTTVAEKTK
EQVTNVGGAV VTGVTAVAQK TVEGAGNIAA ATGFVKKDQM GKGEEGYPQE GILEDMPVDP
GSEAYEMPSE EGYQDYEPEA
SEQ ID NO: 17
Microtubule-associated protein tau isoform 2 (Tau-F) [Homo sapiens]
NP 005901.2
1 maeprgefev medhagtygl gdrkdqggyt mhqdgegdtd aglkesplqt ptedgseepg
61 setsdakstp taedvtaplv degapgkqaa aqphteipeg ttaeeagigd tpsledeaag
121 hvtqarmvsk skdgtgsddk kakgadgktk iatprgaapp gqkgqanatr ipaktppapk
181 tppssgeppk sgdrsgyssp gspgtpgsrs rtpslptppt repkkvavvr tppkspssak
241 srlqtapvpm pdlknvkski gstenlkhqp gggkvqiink kldlsnvqsk cgskdnikhv
301 pgggsvgivy kpvdlskvts kcgslgnihh kpggggvevk sekldfkdry gskigsldni
361 thvpgggnkk iethkltfre nakaktdhga eivykspvvs gdtsprhlsn vsstgsidmv
421 dspqlatlad evsaslakqg 1
SEQ ID NO: 18
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HUMAN TAR DNA-BINDING PROTEIN 43 (TDP-43) (NP 031401)
MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGN
LVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYESTFGEVLMVQVKKDLK
TGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREF
.. FSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNP
GGEGNQGGEGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPS
GNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGENGGFGSSMDSKSSGWGM
SEQ ID NO: 19
TAU HUMAN (UNIPROTKB/SWISS-PROT P10636.5)
MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTP
TAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQEPESGKVVQEGFLREP
GPPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPEDTEGGRHAPELLKHQLLGDLHQEGPPLKGAGGKE
RPGSKEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPVDFLSKVSTEIP
ASEPDGPSVGRAKGQDAPLEFTFHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLGEDTKEAD
LPEPSEKQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTRSSAKTLKNRPCLSPKHPTPGSS
DPLIQPSSPAVCPEPPSSPKYVSSVTSRTGSSGAKEMKLKGADGKTKIATPRGAAPPGQKGQANATRIPA
KTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRL
QTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPV
DLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAK
AKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL
SEQ ID NO: 20
alpha-synuclein isoform NACP140 [Homo sapiens] (NP 000336.1)
1 mdvfmkglsk akegvvaaae ktkqgvaeaa gktkegvlyv gsktkegvvh gvatvaektk
61 eqvtnvggav vtgvtavaqk tvegagsiaa atgfvkkdql gkneegapqe giledmpvdp
121 dneayempse egyqdyepea
SEQ ID NO: 26
TRUNCATED ENVELOPE GLYCOPROTEIN HUMAN IMMUNODEFICIENCY VIRUS 2
ACCESSION ABC39624
MAHEGTQLLI AFLLTSACLI YCKQYVTVFY GIPAWKNASI PLFCATRNRD TWGTIQCLPD
NDDYQEIVLN VAEAFDAWDN TVTEQAIEDV WNLFETSTKP CVKLTPLCVT MRCNTSTTTT
TTAPTSTSAG STATPKPIMV NENTSCMHAN NCSGLGEEEV VNCEFNMTGL VRDKPKTYNE
TWYSRDVDCE PDSTTNSRKC YMNHCNTSVI TESCDKHYWD DIRFRYCAPP GYALLRCNDT
NYSGFAPNCS KVVASTCTRM METQTSTWFG FNGTRAENRT YLYWHSKSDR TIISLNKYYN
LSIHCKRPGN KTVTPITLMS GYKFHSRPVI NTRPKQAWCW FKGRWKDAMQ EVKKTVAEHP
RAVTKDPKNI TFAAPGKGSD PEVEYMWTNC RGEFLYCDMT WFLDWIENRP TRKAWRNYVP
CHIRQIINTW HKVGKHVYLP PREGELTCNS TVTSIIANID VNEIDKRTNI TFSAEVAELY
RVELGDYKLV EVTPIGFAPT SEKRYSSGHR ETYKRCARAR ILGFSRNSRF CNGRSVLDAV
RSAPDFTGRD SAATATAVGR GQETTRNVAT DRLGNEKSPG KSHCYREIPK GSGAAKFMGM
CV
SEQ ID NO: 27
GP120 HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 ACCESSION AAF69492
VPVWRDADTT LFCASDAKSH VTEAHNVWAT HACVPTDPNP QEIHLENVTE NFNMWKNNMV
EQMQEDVISL WEQSLKPCVK LTPLCVTLNC TNANLTNANL TNANNITNVE NITDEVRNCS
FNVTTDLRDK QQKVHALFYR LDIVQINSKN SSDYRLINCN TSVIKQACPK ISFDPIPIHY
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CTPAGYAILK CNDKNFNGTG PCKNVSSVQC THGIKPVVST QLLLNGSLAE EEIIIRSENL
INNVKIIIVH LNKSVEINCT RPSNNTRTSI TIGPGQVFYR TGDIIGDIRK VSCELNGTKW
NEVLKQVKEK LKEHFNKNIS FQPPSGGDLE ITMHHFSCRG EFFYCNTTQL FNNTYSNGTI
TLPCKIKQII NMWQGVGQAM YAPPISGRIN CLSNITGLLL TRDGNNGTNE TFRPGGGNIK
DNWRSELYKC KVVQIEPLGI APTRAKRRVV EREKK
SEQ ID NO: 28
ENVELOPE GLYCOPROTEIN I HUMAN HERPESVIRUS 3 ACCESSION NP 040189
MFLIQCLISA VIFYIQVTNA LIFKGDHVSL QVNSSLTSIL IPMQNDNYTE IKGQLVFIGE
QLPTGTNYSG TLELLYADTV AFCFRSVQVI RYDGCPRIRT SAFISCRYKH SWHYGNSTDR
ISTEPDAGVM LKITKPGIND AGVYVLLVRL DHSRSTDGFI LGVNVYTAGS HHNIHGVIYT
SPSLQNGYST RALFQQARLC DLPATPKGSG TSLFQHMLDL RAGKSLEDNP WLHEDVVTTE
TKSVVKEGIE NHVYPTDMST LPEKSLNDPP ENLLIIIPIV ASVMILTAMV IVIVISVKRR
RIKKHPIYRP NTKIRRGIQN ATPESDVMLE AAIAQLATIR EESPPHSVVN PFVK
SEQ ID NO: 29
human cytomegalovirus HCMVpp65 (GenBank: AAA45994.1)
1 masvlgpisg hvlkavfsrg dtpvlphetr 11qtgihvry sqpslilvsq ytpdstpchr
61 gdnqlqvght yftgsevenv svnvhnptgr sicpsqepms iyvyalplkm lnipsinvhh
121 ypsaaerkhr hlpvadavih asgkqmwqar ltvsglawtr qqnqwkepdv yytsafvfpt
181 kdvalrhvvc ahelvcsmen tratkmqvig dqyvkvyles fcedvpsgkl fmhvtlgsdv
241 eedltmtrnp qpfmrphern gftvlcpknm iikpgkishi mldvaftshe hfgllcpksi
301 pglsisgnll mngqqiflev qairetvelr qydpvaalff fdidlllqrg pqysehptft
361 sqyriqgkle yrhtwdrhde gaaqgdddvw tsgsdsdeel vtterktpry tgggamagas
421 tsagrkrksa ssatactagv mtrgrlkaes tvapeedtde dsdneihnpa vftwppwqag
481 ilarnlvpmv atvqgqnlky qeffwdandi yrifaelegv wqpaaqpkrr rhrqdalpgp
541 ciastpkkhr g
SEQ ID NO: 30
L2 HUMAN PAPILLOMAVIRUS TYPE 18 ACCESSION ADC35722.1 (exemplary L2
sequence)
1 mvshraarrk rasvtdlykt ckgsgtcppd vvpkvegttl adkilqwssl giflgglgig
61 tgsgtggrtg yiplggrsnt vvdvgptrpp vviepvgptd psivtlieds svvtsgaprp
121 tftgtsgfdi tsagtttpav lditpsstsv sisttnftnp afsdpsiiev pqtgevsgnv
181 fvgtptsgth gyeeiplqtf assgtgeepi sstplptvrr vagprlysra yqqvsvanpe
241 fltrpsslit ydnpafepvd ttltfdprsd vpdsdfmdii rlhrpaltsr rgtvrfsrlg
301 qratmftrsg tqigarvhfy hdispiapsp eyielulvs atedndlfdi yaddmdpavp
361 vpsrsttsfa lskysptiss assysnvtvp ltsswdvpvy tgpditlpst tsvwpivspt
421 apastqyigi hgthyylwpl yyfipkkrkr vpyffadgfv aa
SEQ ID NO: 35
NACP/ALPHA-SYNUCLEIN [HOMO SAPIENS] (GenBank: AACO2114.1)
MDVFMXGLSKAKEGVVAAAEKTKQGVAEAAGKIKEGVLYVGSKTKEGVVHGVATVAEKTKEQVINVGGAV
VTGVTAVAQKTVEGAGSIAAXTGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA
SEQ ID NO: 52
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
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20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa Ala
35 40 45
Xaa Xaa Xaa Xaa Ala Xaa Xaa Ala Xaa Xaa Xaa Xaa
50 55 60
Where residues 1-8 are Lys, Ala or Glu,
residue 10 is Glu or Ala,
residue 11 is Ile or Ala,
residue 12 is Val or Ala,
residue 13 is Tyr, phosphoTyr or Ala,
residue 14 is Lys or Ala,
residue 15 is phosphoSer,
residue 16 is Pro or Ala,
residues 17-18 are Val or Ala,
residue 19 is Ser, phosphoSer or Ala,
residue 20 is Gly or Ala,
residue 21 is Asp or Ala,
residue 22 is Tyr, phosphoTyr or Ala,
residue 23 is phosphoSer,
residue 24 is Pro or Ala,
residue 25 is Arg or Ala,
residue 26 is His or Ala,
residue 27 is Leu or Ala,
residues 28-36 are Lys, Ala or Glu,
residue 37 is Asp or Ala,
residue 38 is Met or Ala,
residue 39 is Val or Ala,
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residue 40 is Asp or Ala,
residue 41 is phosphoSer,
residue 42 is Pro or Ala,
residue 43 is Gln or Ala,
residue 44 is Leu or Ala,
residue 46 is Tyr, phosphoTry or Ala,
residue 47 is Leu or Ala,
residue 49 is Asp or Ala,
residue 50 is Glu or Ala,
residue 51 is Val or Ala,
residue 52 is Ser, phosphoSer or Ala,
residue 54 is Ser, phosphoSer or Ala,
residue 55 is Leu or Ala,
residue 57 is Lys or Ala,
residue 58 is Gln or Ala,
residue 59 is Gly or Ala,
residue 60 is Leu or Ala.
SEQ ID NO: 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa Ala
35 40 45
Xaa Xaa Xaa Xaa Ala Xaa Xaa Ala Xaa Xaa Xaa Xaa
50 55 60
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Where residues 1-8 are Lys, Ala, Glu or Phe
residue 10 is Glu or Ala,
residue 11 is Ile or Ala,
residue 12 is Val or Ala,
residue 13 is Tyr, phosphoTyr or Ala,
residue 14 is Lys or Ala,
residue 15 is phosphoSer,
residue 16 is Pro or Ala,
residues 17-18 are Val or Ala,
residue 19 is Ser, phosphoSer or Ala,
residue 20 is Gly or Ala,
residue 21 is Asp or Ala,
residue 22 is Tyr, phosphoTyr or Ala,
residue 23 is phosphoSer,
residue 24 is Pro or Ala,
residue 25 is Arg or Ala,
residue 26 is His or Ala,
residue 27 is Leu or Ala,
residues 28-36 are Lys, Ala, Glu or Phe,
residue 37 is Asp or Ala,
residue 38 is Met or Ala,
residue 39 is Val or Ala,
residue 40 is Asp or Ala,
residue 41 is phosphoSer,
residue 42 is Pro or Ala,
residue 43 is Gln or Ala,
residue 44 is Leu or Ala,
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residue 46 is Tyr, phosphoTry or Ala,
residue 47 is Leu or Ala,
residue 49 is Asp or Ala,
residue 50 is Glu or Ala,
.. residue 51 is Val or Ala,
residue 52 is Ser, phosphoSer or Ala,
residue 54 is Ser, phosphoSer or Ala,
residue 55 is Leu or Ala,
residue 57 is Lys or Ala,
.. residue 58 is Gln or Ala,
residue 59 is Gly or Ala,
residue 60 is Leu or Ala.
SEQ ID NO: 61
.. Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa
25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala
20 35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Where residue 1 is Asp, Gly or Ala,
Residue 2 is Asn, Ser or Ala,
.. Residue 3 is Glu or Ala,
Residue 5 is Tyr, nitroTyr or Ala,
Residue 6 is Glu or Ala,
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Residue 7 is Met or Ala,
Residue 8 is Pro or Ala,
Residue 9 is phosphoSer,
Residues 10-11 are Glu or Ala,
Residue 12 is Gly or Ala,
Residue 13 is Tyr, nitroTyr or Ala,
Residue 14 is Gln or Ala,
Residue 15 is Asp or Ala,
Residue 16 is Tyr, nitroTyr or Ala,
Residue 17 is Glu or Ala,
Residues 18-22 are Lys or Ala,
Residue 23 is Asp, Gly or Ala,
Residue 24 is Asn, Ser or Ala,
Residue 25 is Glu or Ala,
Residue 27 is Tyr, nitroTyr or Ala,
Residue 28 is Glu or Ala,
Residue 29 is Met or Ala,
Residue 30 is Pro or Ala,
Residue 31 is phosphoSer,
Residues 32-33 are Glu or Ala,
Residue 34 is Gly or Ala
Residue 35 is Tyr, nitroTyr or Ala
Residue 36 is Gln or Ala,
Residue 37 is Asp or Ala
Residue 38 is Tyr, nitroTyr or Ala
Residue 39 is Glu or Ala
Residues 40-44 are Lys or Ala
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Residue 45 is Asp, Gly or Ala,
Residue 46 is Asn, Ser or Ala,
Residue 47 is Glu or Ala,
Residue 49 is Tyr, nitroTyr or Ala,
Residue 50 is Glu or Ala,
Residue 51 is Met or Ala,
Residue 52 is Pro or Ala,
Residue 53 is phosphoSer,
Residues 54-55 are Glu or Ala,
Residue 56 is Gly or Ala,
Residue 57 is Tyr, nitroTyr or Ala,
Residue 58 is Gln or Ala,
Residue 59 is Asp or Ala,
Residue 60 is Tyr, nitroTyr or Ala,
Residue 61 is Glu or Ala.
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