Note: Descriptions are shown in the official language in which they were submitted.
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TOXIN-DERIVED DELIVERY CONSTRUCTS FOR ORAL DELIVERY
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application
Nos. 62/640,168
filed March 8, 2018; 62/640,188 filed March 8, 2018; and 62/640,194 filed
March 8, which
applications are incorporated herein by reference in their entirety for all
purposes.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing in the form of a
"paper copy"
(PDF File) and a file containing the referenced sequences (SEQ ID NO: 1 ¨ SEQ
ID NO: 221) in
computer readable form (5T25 format text file) which is submitted herein. The
Sequence Listing
is shown using standard three letter code for amino acids, as defined in 37
C.F.R. 1.822. Said
ASCII copy, created on March 8, 2019, is named 40566-711 601 SL.txt and is
350,816 bytes in
size.
BACKGROUND
[0003] The gut epithelium has thwarted efforts to orally administer large
molecule
biologics because proteins cannot diffuse across the barrier or sneak through
the tight junctions.
When they are taken up by endocytosis ¨ the only route left to them ¨ they are
typically
degraded in lysosomes rather than being transported into the body. This
inability to be readily
absorbed across the intestinal epithelium continues to be a limiting factor in
developing
commercially viable oral formulations of these agents. The most common
solution is to use
systemic administration, but that can often create considerable side effects
and reduce patient
convenience that negatively affects compliance.
INCORPORATION BY REFERENCE
[0004] All references disclosed herein are hereby incorporated by reference
in their entirety
for all purposes.
SUMMARY
[0005] The present disclosure provides methods and composition for
transport and/or
delivery of a cargo molecule to certain location(s) within a cell (e.g., a
supranuclear location) or
across a cell (e.g., epithelial cell), either in vitro or in vivo (e.g., in a
rodent or a human). Such
cargo can be directed to a set of location(s) by coupling it to a carrier
molecule. Such carrier
molecule can interact with unique receptors both on the cell surface and
intracellularly for the
targeted delivery of the cargo. Various such carrier, cargos, and uses thereof
are described
herein.
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[0006] The disclosure provides an isolated delivery construct that can
comprise: a carrier
derived from a domain I of an exotoxin and lacking a domain II, a domain lb
and a domain III of
the exotoxin; coupled to a heterologous cargo. The carrier can consist
essentially of the domain I
of the exotoxin. The delivery construct can deliver the heterologous cargo
according to one or
more of the following: across an epithelial cell via transcytosis; to the
basal side of the epithelial
cell; to a supranuclear region within the epithelial cell; or to the interior
of the epithelial cell via
endocytosis. In some aspects, the carrier is configured to deliver a
heterologous cargo to the
basal side of an epithelial cell.
[0007] The disclosure provides an isolated delivery construct that can
comprise: a chimeric
carrier comprising an intracellular epithelial targeting domain; coupled to a
heterologous cargo.
[0008] The disclosure provides an isolated delivery construct that can
comprise: a chimeric
carrier comprising a supranuclear epithelial targeting domain; coupled to the
heterologous cargo.
[0009] The disclosure provides an isolated delivery construct that can
comprise: a carrier
coupled to a heterologous cargo, wherein the carrier interacts with one or
more of ribophilin 1,
SEC24, CK-8, TMEM132, GRP75, ERGIC-53, or perlecan, and does not display
interaction
with one or more of a clathrin or GPR78, or a combination thereof. The
interaction can be a
selective interaction. The interaction can be a pH-dependent interaction. The
interaction of the
carrier with the one or more of ribophilin 1, SEC24, CK-8, TMEM132, GRP75,
ERGIC-53, or
perlecan can occur on a surface of the epithelial cell, in the interior of an
epithelial cell, or a
combination thereof. The delivery of the heterologous cargo across the
epithelial cell can occur
in vitro from the apical surface of the epithelial cell to a basolateral
compartment. The delivery
of the heterologous cargo can occur in vitro from the apical surface of the
epithelial cell to the
interior of the epithelial cell. The delivery of the heterologous cargo can
occur in vitro from the
apical surface of the epithelial cell to the supranuclear region within the
epithelial cell. The
interaction of the carrier with the one or more of ribophilin 1, SEC24, CK-
8,TMEM132, GRP75,
ERGIC-53, or perlecan, or the combination thereof can occur in vitro on the
apical surface of the
epithelial cell, in the interior of the epithelial cell, or a combination
thereof. The epithelial cell
can be a polarized epithelial cell. The polarized epithelial cell can be part
of a monolayer of
polarized epithelial cells. The polarized epithelial cell can be from a rodent
or a human. The
polarized epithelial cell can be from a human. The human polarized epithelial
cell can be a
human polarized gut epithelial cell. The human polarized gut epithelial cell
can be a Caco-2 cell.
The delivery of the heterologous cargo across the epithelial cell can occur in
vivo from a gut of a
subject to a basolateral compartment of a subject. The delivery of the
heterologous cargo can
occur in vivo from a gut of a subject to the interior of the epithelial cell
of a subject. The delivery
of the heterologous cargo can occur in vivo from a gut of a subject to the
supranuclear region
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within the epithelial cell of a subject. The interaction of the carrier with
the one or more of
ribophilin 1, SEC24, CK-8,TMEM132, GRP75, ERGIC-53, and perlecan, or the
combination
thereof, can occur in vivo on the apical surface of the epithelial cell of a
subject, in the interior of
the epithelial cell of the subject, or a combination thereof. The subject can
be a rodent or a
human. The subject can be a human and affected by one or more of the
following: inflammatory
bowel disease, psoriasis, bacterial sepsis, systemic lupus erythematosus
(SLE), pemphigus
vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenia purpura,
Grave's disease,
Sjogren's disease, dermatomyositis, Hashimoto's disease, polymyositis,
inflammatory bowel
disease, multiple sclerosis (MS), diabetes mellitus, rheumatoid arthritis,
scleroderma, non-
Hodgkin's lymphomas, Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy
cell
leukemia, acute lymphoblastic leukemia, multiple myeloma, carcinomas of the
bladder, kidney
ovary, cervix, breast, lung, nasopharynx, malignant melanoma, rituximab
resistant NHL or
leukemia, diabetes, obesity, diabetes as a consequence of obesity,
hyperglycemia, dyslipidemia,
hypertriglyceridemia, syndrome X, insulin resistance, impaired glucose
tolerance (IGT), diabetic
dyslipidemia, hyperlipidemia, growth hormone deficiency (GHD), Turner syndrome
(TS),
Noonan syndrome, Prader-Willi syndrome, short stature homeobox-containing gene
(SHOX)
deficiency, chronic renal insufficiency, or idiopathic short stature short
bowel syndrome. The
epithelial cell can be a polarized epithelial cell. The polarized epithelial
cell can be a polarized
gut epithelial cell. The carrier can be a small molecule, a polypeptide, an
aptamer, or a
combination thereof. The carrier can be a small molecule. The carrier can be a
polypeptide. The
polypeptide can be an antibody or a functional fragment thereof. The carrier
can be an aptamer.
The carrier can be derived from an exotoxin. The carrier can be derived from a
domain I of the
exotoxin and lacks a domain II, a domain lb and a domain III of the
exotoxinThe carrier that can
be derived from a domain I of an exotoxin comprises an amino acid sequence
that has at least
80% sequence identity to the amino acid sequence of the domain I of the
exotoxin, or at least
80% sequence identity to a functional fragment thereof, wherein the exotoxin
is a Cholix toxin or
a Pseudomonas exotoxin A. In some aspects, the carrier comprises at least 110
amino acid
residues of the domain I of the exotoxin. In some aspects, the carrier
comprises at least 50
contiguous amino acid residues of the domain I of the exotoxin. The carrier
that lacks the domain
II, the domain lb and the domain III of the exotoxin can comprise a portion of
the domain II, the
domain lb or the domain III of the exotoxin, or a combination thereof. The
portion can comprise
no more than 70% of the amino acid residues of the domain II, the domain lb or
the domain III
of the exotoxin. The exotoxin can be a Cholix toxin. The carrier can comprise:
an amino acid
sequence having at least 80% sequence identity to the amino acid sequence of
SEQ ID NO: 4 or
SEQ ID NO: 5 or at least 80% sequence identity to a functional fragment
thereof, and no more
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than 347 contiguous amino acid residues from SEQ ID NO: 1. The carrier can
comprise a
deletion or mutation in one or more of the amino acid residues of the amino
acid sequence set
forth in SEQ ID NO: 4 or SEQ ID NO: 5. The carrier can comprise: an amino acid
sequence
haying at least 90% sequence identity to the amino acid sequence of SEQ ID NO:
4 or SEQ ID
NO: 5 or at least 90% sequence identity to a functional fragment thereof, and
no more than 347
contiguous amino acid residues from SEQ ID NO: 1. The carrier can comprise: an
amino acid
sequence haying at least 95% sequence identity to the amino acid sequence of
SEQ ID NO: 4 or
SEQ ID NO: 5 or at least 95% sequence identity to a functional fragment
thereof, and no more
than 347 contiguous amino acid residues from SEQ ID NO: 1. The carrier can
comprise: an
amino acid sequence haying at least 99% sequence identity to the amino acid
sequence of SEQ
ID NO: 4 or SEQ ID NO: 5 or at least 99% sequence identity to a functional
fragment thereof,
and no more than 347 contiguous amino acid residues from SEQ ID NO: 1. The
carrier can
comprise: an amino acid sequence haying 100% sequence identity to the amino
acid sequence of
SEQ ID NO: 4 or SEQ ID NO: 5 or 100% sequence identity to a functional
fragment thereof, and
no more than 347 contiguous amino acid residues from SEQ ID NO: 1. The carrier
can comprise
the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a
functional fragment
thereof. The carrier can comprise the amino acid sequence set forth in SEQ ID
NO: 6 or SEQ ID
NO: 7 or a functional fragment thereof. The carrier can comprise the amino
acid sequence set
forth in SEQ ID NO: 8 or SEQ ID NO: 9 or a functional fragment thereof The
carrier can
comprise an amino acid sequence haying at least 80% sequence identity to any
one of the amino
acid sequences set forth in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150,
SEQ ID NO:
151, SEQ ID NO: 152, a functional fragment thereof, or any combination
thereof. The carrier
can comprise a spatial structure in which one or more amino acid residues of
SEQ ID NO: 148 or
SEQ ID NO: 149 are in close proximity to one or more amino acid residues of
SEQ ID NO: 151,
and one or more amino acid residues of SEQ ID NO: 148 or SEQ ID NO: 149 are in
close
proximity to one or more amino acid residues of SEQ ID NO: 152. The carrier
can comprise an
amino acid sequence haying at least 80% sequence identity to the amino acid
sequence set forth
in SEQ ID NO: 30 or SEQ ID NO: 31 or the amino acid sequence set forth in SEQ
ID NO: 10 or
SEQ ID NO: 11 or at least 80% sequence identity to a functional fragment
thereof. The carrier
can comprise a deletion or mutation in one or more of amino acid residues 1-
187 or 1-206 of
SEQ ID NO: 11 or 1-186 or 1-205 of SEQ ID NO: 10. The carrier can comprise
residues 1-187
of SEQ ID NO: 30 or 1-186 of SEQ ID NO: 31 and no more than 206 contiguous
amino acid
residues of SEQ ID NO: 1. The carrier can comprise an amino acid sequence
haying at least 80%
sequence identity to the amino acid sequence set forth in any one of SEQ ID
NO: 10 ¨ SEQ ID
NO: 31 or at least 80% sequence identity to a functional fragment thereof. The
carrier can
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comprise the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11
or a functional
fragment thereof The carrier can comprise an amino acid sequence having at
least 80% sequence
identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO:
107 or the
amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least
80% sequence
identity to a functional fragment thereof The carrier can comprise a deletion
or mutation in one
or more of amino acid residues 1-151 or 1-187 of SEQ ID NO: 4 or SEQ ID NO: 5.
The carrier
can lack any one or more of the amino acid residues 1-39 of SEQ ID NO: 5 or
amino acid
residues 1-38 of SEQ ID NO: 4. The carrier can comprise an amino acid sequence
having at least
80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 69 or
SEQ ID NO:
70 or 80% sequence identity to a functional fragment thereof. The carrier can
comprise residues
1-151 of SEQ ID NO: 5 or residues 1-150 of SEQ ID NO: 4 and no more than 187
contiguous
amino acid residues of SEQ ID NO: 1 The carrier can comprise an amino acid
sequence having
at least 80% sequence identity to the amino acid sequence set forth in any one
of SEQ ID NO: 30
¨ SEQ ID NO: 107 or at least 80% sequence identity to a functional fragment
thereof. The
carrier can comprise the amino acid sequence set forth in SEQ ID NO: 30 or SEQ
ID NO: 31 or
a functional fragment thereof The carrier can comprise an amino acid sequence
having at least
80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106
or SEQ ID NO:
107 or the amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125
or at least 80%
sequence identity to a functional fragment thereof The carrier can comprise a
deletion or
mutation in one or more of amino acid residues 1-150 of SEQ ID NO: 6 or in one
or more of
amino acid residues 1-151 of SEQ ID NO: 7. The carrier can comprise residues 1-
134 of SEQ ID
NO: 5 or residues 1-133 of SEQ ID NO: 4 and no more than 151 contiguous amino
acid residues
of SEQ ID NO: 1. The carrier can comprise an amino acid sequence having at
least 80%
sequence identity to the amino acid sequence set forth in any of SEQ ID NO:
106 ¨ SEQ ID NO:
125 or at least 80% sequence identity to a functional fragment thereof The
carrier can comprise
the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or a
functional
fragment thereof The carrier or isolated delivery construct can comprise at
least one but no more
than 20 beta strands. The exotoxin can be a Pseudomonas exotoxin A. The
carrier can comprise
an amino acid sequence having at least 80% identity to the amino acid sequence
of SEQ ID NO:
137 or at least 80% identity to a functional fragment thereof. The carrier can
comprise a deletion
or mutation in one or more of amino acid residues 1-252 of SEQ ID NO: 137. The
carrier can
comprise an amino acid sequence having at least 90% sequence identity to the
amino acid
sequence of 1-252 of SEQ ID NO: 137 or at least 90% sequence identity to a
functional fragment
thereof. The carrier can comprise an amino acid sequence having at least 95%
sequence identity
to the amino acid sequence of 1-252 of SEQ ID NO: 137 or at least 95% sequence
identity to a
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functional fragment thereof The carrier can comprise an amino acid sequence
having at least
99% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or
at least 99%
sequence identity to a functional fragment thereof The carrier can comprise an
amino acid
sequence having 100% sequence identity to the amino acid sequence of 1-252 of
SEQ ID NO:
137 or 100% sequence identity to a functional fragment thereof. The carrier
can comprise no
more than 252 contiguous amino acid residues from SEQ ID NO: 134. In some
aspects, the
carrier comprises residues 1-252 of SEQ ID NO: 134. The carrier can comprise
at least one N-
terminal methionine residue. The carrier can comprise an amino acid sequence
having at least
80% sequence identity to an amino acid sequence set forth in any one of SEQ ID
NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, SEQ ID NO:
125, or
80% sequence identity to a functional fragment thereof. The delivery construct
can form a
multimer. The multimer can be formed by multimerization of the heterologous
cargo. The
multimer can be a heteromer or a homomerThe homomer can be a homodimer. The
homodimer
can be formed by dimerization of the heterologous cargo.
[0010] The present disclosure provides an isolated delivery construct that
can comprise: a
carrier comprising a first portion and a second portion, wherein the first
portion is derived from a
first exotoxin and the second portion is derived from a second exotoxin;
coupled to a
heterologous cargo. The first exotoxin can be Cholix. The second exotoxin can
be PE. The first
portion can be derived from a domain I, a domain II, a domain lb, or a domain
III of Cholix, or
any combination thereof The first portion can comprise an amino acid sequence
having at least
80% sequence identity to any one of the amino acid sequences set forth in SEQ
ID NO: 1 ¨ SEQ
ID NO: 125 or SEQ ID NO: 133, a functional fragment thereof, or any
combination thereof. The
first portion can comprise an amino acid sequence having at least 80% sequence
identity to any
one of the amino acid sequences set forth in SEQ ID NO: 148 ¨ SEQ ID NO: 152,
a functional
fragment thereof, or any combination thereof The first portion can comprise an
amino acid
sequence having at least 80% sequence identity to any one of the amino acid
sequences set forth
in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11, a functional
fragment
thereof, or any combination thereof The second portion can be derived from a
domain I, a
domain II, a domain lb, or a domain III of PE, or any combination thereof The
second portion
can comprise an amino acid sequence having at least 80% sequence identity to
any one of the
amino acid sequences set forth in SEQ ID NO: 137 ¨ SEQ ID NO: 145, a
functional fragment
thereof, or any combination thereof The first portion can be chemically
coupled or
recombinantly coupled to the second portion. The first portion can be directly
or indirectly
coupled to the second portion. The carrier can comprise an amino acid sequence
having at least
80% sequence identity to the amino acid sequence SEQ ID NO: 146 or SEQ ID NO:
147. The
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carrier can be chemically coupled or recombinantly coupled to the heterologous
cargo. The
carrier can be covalently coupled to the heterologous cargo. The heterologous
cargo can be
coupled to the C-terminus of the carrier. The heterologous cargo can be
coupled to the N-
terminus of the carrier. The carrier can be coupled directly to the
heterologous cargo. The carrier
can be coupled indirectly to the heterologous cargo. The carrier can be
coupled to the
heterologous cargo via a spacer. The spacer can comprise an amino acid spacer.
The amino acid
spacer can be between 1 and 50 amino acid residues in length. The amino acid
spacer can
comprise one or more glycine residues and one or more serine residues. The
spacer can be a
cleavable spacer. The cleavable spacer can comprise an amino acid sequence set
forth in any one
of SEQ ID NO: 174 - SEQ ID NO: 206. The spacer can be a non-cleavable spacer.
The non-
cleavable spacer can comprise one or more of the amino acid sequences GTGGS
(SEQ ID NO:
207), GGGGS (SEQ ID NO: 208), GGGGSGGGGS (SEQ ID NO: 209),
GGGGSGGGGSGGGGS (SEQ ID NO: 210), or GGGGSGGG (SEQ ID NO: 211). The non-
cleavable spacer can comprise one or more of (GGGGS), (SEQ ID NO: 212),
wherein x = 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10. The non-cleavable spacer can comprise one or more
of (GS) x (SEQ ID
NO: 213), wherein x = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The spacer can
comprise one or more
fragments of the domain II, the domain lb or the domain III of the exotoxin,
or a combination
thereof. The spacer can comprise at most 80 amino acid residues of the domain
II, 80 amino acid
residues of the domain III, or a combination thereof The heterologous cargo
can be a
macromolecule, a small molecule, a polypeptide, a nucleic acid, a mRNA, a
miRNA, a shRNA, a
siRNA, an antisense molecule, an antibody, a DNA, a plasmid, a vaccine, a
polymer a
nanoparticle, or a catalytically-active material. The heterologous cargo can
be a biologically
active cargo. The biologically active cargo can be a cytokine, a hormone, a
therapeutic antibody,
a functional fragment thereof, or any combination thereof The cytokine can be
IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,
or IL-30. The
cytokine can have the amino acid sequence set forth in SEQ ID NO: 217 or SEQ
ID NO: 218.
The hormone can have the amino acid sequence set forth in SEQ ID NO: 215 or
SEQ ID NO:
216. The therapeutic antibody can be an anti-TNFa antibody. The anti-TNFa
antibody can be
adalimumab or infliximab. The heterologous cargo can be a detectable agent.
The detectable
agent can be a fluorophore, a contrast agent, an X-ray contrast agent, a PET
agent, a
nanoparticle, or a radioisotope. The fluorophore can be a red fluorescent
protein (RFP). The RFP
can have the amino acid sequence set forth in SEQ ID NO: 220.
[0011] A delivery construct of the present disclosure can comprise an amino
acid sequence
having at least 80% sequence identity to the amino acid sequence set forth in
any one of SEQ ID
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NO: 155, SEQ ID NO: 156, or SEQ ID NO: 158 ¨ SEQ ID NO: 165, or at least 80%
sequence
identity to a functional fragment thereof A delivery construct of the present
disclosure can
comprise an amino acid sequence having at least 90% sequence identity to the
amino acid
sequence set forth in any one of SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO:
158 ¨ SEQ
ID NO: 165, or at least 90% sequence identity to a functional fragment thereof
A delivery
construct of the present disclosure can comprise an amino acid sequence having
at least 95%
sequence identity to the amino acid sequence set forth in any one of SEQ ID
NO: 155, SEQ ID
NO: 156, or SEQ ID NO: 158 ¨ SEQ ID NO: 165, or at least 95% sequence identity
to a
functional fragment thereof A delivery construct of the present disclosure can
comprise an
amino acid sequence having at least 99% sequence identity to the amino acid
sequence set forth
in any one of SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 158¨ SEQ ID NO:
165, or at
least 99% sequence identity to a functional fragment thereof A delivery
construct of the present
disclosure can comprise an amino acid sequence having 100% sequence identity
to the amino
acid sequence set forth in any one of SEQ ID NO: 155, SEQ ID NO: 156, or SEQ
ID NO: 158 ¨
SEQ ID NO: 165, or 100% sequence identity to a functional fragment thereof
[0012] The present disclosure provides a pharmaceutical composition
comprising: an
isolated delivery construct as described herein; and a pharmaceutically
acceptable carrier. The
composition can be formulated for oral administration, topical administration,
pulmonary
administration, intra-nasal administration, buccal administration, sublingual
administration or
ocular administration. The composition can be formulated for oral
administration. The
composition can be formulated in a capsule or tablet.
[0013] The present disclosure provides a polynucleotide that can encode an
isolated delivery
construct as described herein.
[0014] In various aspects, the present disclosure provides a vector
comprising a
polynucleotide encoding an isolated delivery construct as described herein.
[0015] The present disclosure provides a host cell that can comprise a
vector that expresses
a delivery construct, wherein the host cell comprises a vector comprising a
polynucleotide
encoding an isolated delivery construct as described herein.
[0016] The present disclosure provides a method of delivering a
heterologous cargo across
an epithelial cell, the method can comprise: applying a delivery construct to
the apical surface of
the epithelial cell; and delivering the delivery construct to the basal side
of the epithelial cell at a
rate greater than 10-6 cm/sec, wherein the delivery construct comprises: a
carrier; coupled to the
heterologous cargo. In some aspects, the method further comprises releasing
the delivery
construct from the basal side of the epithelial cell following delivery across
the epithelial cell. In
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some aspects, the carrier is configured to deliver a heterologous cargo to the
basal side of an
epithelial cell.
[0017] The present disclosure provides a method of delivering a
heterologous cargo to the
interior of an epithelial cell via endocytosis, the method can comprise:
applying a delivery
construct to the apical surface of the epithelial cell; and delivering the
delivery construct to the
interior of the epithelial cell via endocytosis, wherein the delivery
construct comprises: a carrier;
coupled to the heterologous cargo.
[0018] The present disclosure provides a method of delivering a
heterologous cargo to a
supranuclear region within an epithelial cell via endocytosis, the method can
comprise: applying
a delivery construct to the apical surface of an epithelial cell; and
delivering the delivery
construct to the supranuclear region within the epithelial cell via
endocytosis, wherein the
delivery construct comprises: a carrier; coupled to the heterologous cargo.
[0019] The present disclosure provides a method of interacting with
ribophilin 1, SEC24,
CK-8, TMEM132, GRP75, ERGIC-53, or perlecan, or a combination thereof, the
method can
comprise: applying a delivery construct to the apical surface of the
epithelial cell; and interacting
the delivery construct with the ribophilin 1, SEC24, CK-8, TMEM132, GRP75,
ERGIC-53, or
perlecan, or the combination thereof, wherein the delivery construct
comprises: a carrier;
coupled to a heterologous cargo. The carrier can interact with one or more of
ribophilin 1,
SEC24, CK-8, TMEM132, GRP75, ERGIC-53, or perlecan, or the combination
thereof, and does
not display interaction with one or more of a clathrin or GPR78, or a
combination thereof The
interaction can be a selective interaction or a pH-dependent interaction, or a
combination thereof.
The interaction of the carrier with the one or more of ribophilin 1, SEC24, CK-
8, TMEM132,
GRP75, ERGIC-53, or perlecan can occur on a surface of an epithelial cell, in
the interior of an
epithelial cell, or a combination thereof.
[0020] The present disclosure provides a method of treating a disease in a
subject in need
thereof, the method can comprise administering to the subject a delivery
construct comprising: a
carrier; coupled to a heterologous cargo; wherein the delivery construct is
capable of delivering
the heterologous cargo to the interior of an epithelial cell.
[0021] The present disclosure provides a method of treating a disease in a
subject in need
thereof, the method can comprise administering to the subject a delivery
construct comprising: a
carrier; coupled to a heterologous cargo; wherein the delivery construct is
capable of delivering
the heterologous cargo to a supranuclear region within an epithelial cell.
[0022] The present disclosure provides a method of diagnosing a disease in
a subject in need
thereof, the method can comprise administering to the subject a delivery
construct comprising: a
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carrier; coupled to a heterologous cargo; wherein the delivery construct is
capable of delivering
the heterologous cargo to the interior of an epithelial cell.
[0023] The present disclosure provides a method of diagnosing a disease in
a subject in need
thereof, the method can comprise administering to the subject a delivery
construct comprising: a
carrier; coupled to a heterologous cargo; wherein the delivery construct is
capable of delivering
the heterologous cargo to a supranuclear region within an epithelial cell.
[0024] The present disclosure provides a method of treating a disease in a
subject in need
thereof, the method can comprise administering to the subject a delivery
construct comprising: a
carrier derived from a domain I of an exotoxin and lacking a domain II, a
domain lb and a
domain III of the exotoxin; coupled to a heterologous cargo; wherein the
delivery construct is
capable of delivering the heterologous cargo via transcytosis across an
epithelial cell.
[0025] The present disclosure provides a method of diagnosing a disease in
a subject in need
thereof, the method can comprise administering to the subject a delivery
construct comprising: a
carrier derived from a domain I of an exotoxin and lacking a domain II, a
domain lb and a
domain III of the exotoxin; coupled to a heterologous cargo; wherein the
delivery construct is
capable of delivering the heterologous cargo via transcytosis across an
epithelial cell. The
delivery of the heterologous cargo across the epithelial cell can occur in
vitro from the apical
surface of the epithelial cell to a basolateral compartment. The delivery of
the heterologous cargo
can occur in vitro from the apical surface of the epithelial cell to the
interior of the epithelial cell.
The delivery of the heterologous cargo can occur in vitro from the apical
surface of the epithelial
cell to the supranuclear region within the epithelial cell. The interaction of
the carrier with the
one or more of ribophilin 1, SEC24, CK-8,TMEM132, GRP75, ERGIC-53, or
perlecan, or the
combination thereof, can occur in vitro on the apical surface of the
epithelial cell, in the interior
of the epithelial cell, or a combination thereof The epithelial cell can be a
polarized epithelial
cell. The polarized epithelial cell can be part of a monolayer of polarized
epithelial cells. The
polarized epithelial cell can be from a rodent. The polarized epithelial cell
can be from a human.
The human polarized epithelial cell can be a human polarized gut epithelial
cell. The human
polarized gut epithelial cell can be a Caco-2 cell. The delivery of the
heterologous cargo across
the epithelial cell can occur in vivo from a gut of a subject to a basolateral
compartment of the
subject. The delivery of the heterologous cargo can occur in vivo from a gut
of a subject to the
interior of the epithelial cell of the subject. The delivery of the
heterologous cargo can occur in
vivo from a gut of a subject to the supranuclear region within the epithelial
cell of the subject.
The interaction of the carrier with the one or more of ribophilin 1, SEC24, CK-
8,TMEM132,
GRP75, ERGIC-53, and perlecan, or the combination thereof, can occur in vivo
on the apical
surface of the epithelial cell of a subject, in the interior of the epithelial
cell of the subject, or a
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combination thereof. The subject can be a rodent or a human. The epithelial
cell can be a
polarized epithelial cell. The polarized epithelial cell can be a polarized
gut epithelial cell. The
method further can comprise formulating the delivery construct for
administration to the subject.
The formulation can comprise one or more pharmaceutically acceptable carriers.
The delivery
construct can be formulated for oral administration, topical administration,
pulmonary
administration, intra-nasal administration, buccal administration, sublingual
administration or
ocular administration. The composition can be formulated for oral
administration. The disease
can be an inflammatory disease, an autoimmune disease, a cancer, a metabolic
disease, a fatty
liver disease, or a growth hormone deficient growth disorder. The inflammatory
disease can be
an inflammatory bowel disease, psoriasis or bacterial sepsis. The inflammatory
bowel disease
can be Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic
colitis, ischemic
colitis, diversion colitis, Behcet's syndrome or indeterminate colitis. The
autoimmune disease
can be systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia
gravis, hemolytic
anemia, thrombocytopenia purpura, Grave's disease, Sjogren's disease,
dermatomyositis,
Hashimoto's disease, polymyositis, inflammatory bowel disease, multiple
sclerosis (MS),
diabetes mellitus, rheumatoid arthritis, or scleroderma. The cancer can be a
non-Hodgkin's
lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy cell
leukemia, acute
lymphoblastic leukemia, multiple myeloma, carcinomas of the bladder, kidney
ovary, cervix,
breast, lung, nasopharynx, malignant melanoma, rituximab resistant NHL, or
leukemia. The
metabolic disease can be diabetes, obesity, diabetes as a consequence of
obesity, hyperglycemia,
dyslipidemia, hypertriglyceridemia, syndrome X, insulin resistance, impaired
glucose tolerance
(IGT), diabetic dyslipidemia, or hyperlipidemia. The carrier can be a small
molecule. The carrier
can be a polypeptide. The polypeptide can be an antibody or a functional
fragment thereof. The
carrier can be an aptamer. The carrier can be derived from an exotoxin. The
carrier can be
derived from a domain I of the exotoxin and lacks a domain II, a domain lb and
a domain III of
the exotoxin. The carrier can be derived from a domain I of an exotoxin
comprises an amino acid
sequence that has at least 80% sequence identity to the amino acid sequence of
the domain I of
the exotoxin, or at least 80% sequence identity to a functional fragment
thereof, wherein the
exotoxin is a Cholix toxin or a Pseudomonas exotoxin A. The carrier can
comprise at least 130
amino acid residues of the domain I of the exotoxin. The carrier can comprise
at least 150
contiguous amino acid residues of the domain I of the exotoxin. The carrier
that lacks the domain
II and domain III of the exotoxin can comprise a portion of the domain II or
the domain III of the
exotoxin, or a combination thereof. The portion comprises no more than 82 of
the amino acid
residues of the domain II or the domain III of the exotoxin. The exotoxin can
be a Cholix toxin.
The carrier can comprise: an amino acid sequence having at least 80% sequence
identity to the
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amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 80% sequence
identity to a
functional fragment thereof, and no more than 347 contiguous amino acid
residues from SEQ ID
NO: 1. The carrier can comprise a deletion or mutation in one or more of amino
acid residues of
the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5. The carrier
can comprise:
an amino acid sequence haying at least 90% sequence identity to the amino acid
sequence of
SEQ ID NO: 4 or SEQ ID NO: 5 or at least 90% sequence identity to a functional
fragment
thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO:
1. The carrier
can comprise: an amino acid sequence haying at least 95% sequence identity to
the amino acid
sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 95% sequence identity to
a functional
fragment thereof, and no more than 347 contiguous amino acid residues from SEQ
ID NO: 1.
The carrier can comprise: an amino acid sequence haying at least 99% sequence
identity to the
amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 99% sequence
identity to a
functional fragment thereof, and no more than 347 contiguous amino acid
residues from SEQ ID
NO: 1. The carrier can comprise: an amino acid sequence haying 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or 100% sequence identity
to a
functional fragment thereof, and no more than 347 contiguous amino acid
residues from SEQ ID
NO: 1. The carrier can comprise the amino acid sequence set forth in SEQ ID
NO: 4 or SEQ ID
NO: 5 or a functional fragment thereof. In some aspects, the carrier comprises
the amino acid
sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 or a functional fragment
thereof The
carrier can comprise the amino acid sequence set forth in SEQ ID NO: 8 or SEQ
ID NO: 9 or a
functional fragment thereof The carrier can comprise an amino acid sequence
haying at least
80% sequence identity to any one of the amino acid sequences set forth in SEQ
ID NO: 148,
SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, a functional
fragment
thereof, or any combination thereof The carrier can comprise an amino acid
sequence haying at
least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:
30 or SEQ ID
NO: 31 or the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11
or at least
80% sequence identity to a functional fragment thereof. The carrier can
comprise a deletion or
mutation in one or more of amino acid residues 1-187 or 1-206 of SEQ ID NO: 5
or one or more
of amino acid residues 1-186 or 1-205 of SEQ ID NO: 4. The carrier can
comprise residues 1-
187 of SEQ ID NO: 5 or residues 1-186 of SEQ ID NO: 4 and no more than 206
contiguous
amino acid residues of SEQ ID NO: 1. The carrier can comprise an amino acid
sequence haying
at least 80% sequence identity to the amino acid sequence set forth in any one
of SEQ ID NO: 1
¨ SEQ ID NO: 31 or at least 80% sequence identity to a functional fragment
thereof. The carrier
can comprise the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO:
11 or a
functional fragment thereof The carrier can comprise an amino acid sequence
haying at least
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80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106
or SEQ ID NO:
107 or the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or
at least 80%
sequence identity to a functional fragment thereof The carrier can comprise a
deletion or
mutation in one or more of amino acid residues 1-151 or 1-187 of SEQ ID NO: 5
or in one or
more of amino acid residues 1-150 or 1-186 of SEQ ID NO: 4. The carrier can
lack any one or
more of the amino acid residues 1-39 of SEQ ID NO: 5 or residues 1-38 of SEQ
ID NO: 4. The
carrier can comprise an amino acid sequence haying at least 80% sequence
identity to the amino
acid sequence set forth in SEQ ID NO: 69 or SEQ ID NO: 70 or 80% sequence
identity to a
functional fragment thereof The carrier can comprise residues 1-151 of SEQ ID
NO: 5 or
residues 1-150 of SEQ ID NO: 4 and no more than 187 contiguous amino acid
residues of SEQ
ID NO: 1. The carrier can comprise an amino acid sequence haying at least 80%
sequence
identity to the amino acid sequence set forth in any of SEQ ID NO: 30 ¨ SEQ ID
NO: 107 or at
least 80% sequence identity to a functional fragment thereof The carrier can
comprise the amino
acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or a functional
fragment thereof.
The carrier can comprise an amino acid sequence haying at least 80% sequence
identity to the
amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125 or the amino
acid
sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or at least 80%
sequence identity to a
functional fragment thereof The carrier can comprise a deletion or mutation in
one or more of
amino acid residues 1-151 of SEQ ID NO: 5 or in one or more of amino acid
residues 1-150 of
SEQ ID NO: 4. The carrier can comprise residues 1-134 of SEQ ID NO: 5 or
residues 1-133 of
SEQ ID NO: 4 and no more than 151 contiguous amino acid residues of SEQ ID NO:
1. The
carrier can comprise an amino acid sequence haying at least 80% sequence
identity to the amino
acid sequence set forth in any one of SEQ ID NO: 106 ¨ SEQ ID NO: 125 or at
least 80%
sequence identity to a functional fragment thereof The carrier can comprise
the amino acid
sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or a functional
fragment thereof. The
carrier can comprise at least one but no more than 20 beta strands. The
exotoxin can be a
Pseudomonas exotoxin A. The carrier can comprise an amino acid sequence haying
at least 80%
identity to the amino acid sequence of SEQ ID NO: 137 or at least 80% identity
to a functional
fragment thereof The carrier can comprise a deletion or mutation in one or
more of amino acid
residues 1-252 of SEQ ID NO: 137. The carrier can comprise an amino acid
sequence haying at
least 90% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO:
137 or at least
90% sequence identity to a functional fragment thereof. The carrier can
comprise an amino acid
sequence haying at least 95% sequence identity to the amino acid sequence of 1-
252 of SEQ ID
NO: 137 or at least 95% sequence identity to a functional fragment thereof.
The carrier can
comprise an amino acid sequence haying at least 99% sequence identity to the
amino acid
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sequence of 1-252 of SEQ ID NO: 137 or at least 99% sequence identity to a
functional fragment
thereof. The carrier can comprise an amino acid sequence having 100% sequence
identity to the
amino acid sequence of 1-252 of SEQ ID NO: 137 or 100% sequence identity to a
functional
fragment thereof The carrier can comprise residues 1-252 of SEQ ID NO: 135.
The carrier can
comprise a first portion and a second portion, wherein the first portion is
derived from a first
exotoxin and the second portion is derived from a second exotoxin. The first
exotoxin can be
Cholix and the second exotoxin can be PE. The first portion can be derived
from a domain I, a
domain II, a domain lb, or a domain III of Cholix, or any combination thereof.
The first portion
can comprise an amino acid sequence having at least 80% sequence identity to
any one of the
amino acid sequences set forth in SEQ ID NO: 1 ¨ SEQ ID NO: 125, a functional
fragment
thereof, or any combination thereof The first portion can comprise an amino
acid sequence
having at least 80% sequence identity to any one of the amino acid sequences
set forth in SEQ
ID NO: 148 ¨ SEQ ID NO: 152, a functional fragment thereof, or any combination
thereof. The
first portion can comprise an amino acid sequence having at least 80% sequence
identity to any
one of the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ
ID NO: 10, or
SEQ ID NO: 11, a functional fragment thereof, or any combination thereof. The
second portion
can be derived from a domain I, a domain II, a domain lb, or a domain III of
PE, or any
combination thereof. The second portion can comprise an amino acid sequence
having at least
80% sequence identity to any one of the amino acid sequences set forth in SEQ
ID NO: 137 ¨
SEQ ID NO: 145, a functional fragment thereof, or any combination thereof. The
first portion
can be chemically coupled or recombinantly coupled to the second portion. The
first portion can
be directly or indirectly coupled to the second portion. The carrier can
comprise an amino acid
sequence having at least 80% sequence identity to the amino acid sequence SEQ
ID NO: 146 or
SEQ ID NO: 147. The carrier can further comprise at least one N-terminal
methionine residue.
The carrier can comprise an amino acid sequence having at least 80% sequence
identity to an
amino acid sequence set forth in any one of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 9, SEQ
ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, SEQ ID NO: 125, or 80% sequence
identity to a
functional fragment thereof The delivery construct can form a multimer. The
multimer can be
formed by multimerization of the heterologous cargo. The multimer can be a
heteromer or a
homomer. The homomer can be a homodimer. The homodimer can be formed by
dimerization of
the heterologous cargo. The carrier can be chemically coupled or recombinantly
coupled to the
heterologous cargo. The carrier can be covalently coupled to the heterologous
cargo. The
heterologous cargo can be coupled to the C-terminus of the carrier. The
heterologous cargo can
be coupled to the N-terminus of the carrier. The carrier can be coupled
directly to the
heterologous cargo. The carrier can be coupled indirectly to the heterologous
cargo. The carrier
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can be coupled to the heterologous cargo via a spacer. The spacer can comprise
an amino acid
spacer. The amino acid spacer can comprise one or more glycine residues and
one or more serine
residues. The amino acid spacer can be between 1 and 50 amino acid residues in
length. The
spacer can be a cleavable spacer. The cleavable spacer can comprise an amino
acid sequence
selected from the group consisting of the amino acid sequences set forth in
SEQ ID NO: 174 -
SEQ ID NO: 206. The spacer can be a non-cleavable spacer. The non-cleavable
spacer can
comprise one or more of the amino acid sequences GTGGS (SEQ ID NO: 207), GGGGS
(SEQ
ID NO: 208), GGGGSGGGGS (SEQ ID NO: 209), GGGGSGGGGSGGGGS (SEQ ID NO:
210), or GGGGSGGG (SEQ ID NO: 211). The non-cleavable spacer can comprises one
or more
of (GGGGS)x(SEQ ID NO: 212), wherein x = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The
non-cleavable
spacer can comprise one or more of (GS)x(SEQ ID NO: 213), wherein x = 1, 2, 3,
4, 5, 6, 7, 8, 9,
or 10. The spacer can comprise one or more fragments of the domain II, a
domain lb or the
domain III of the exotoxin, or a combination thereof. The spacer can comprise
at most 82 amino
acid residues of the domain II, 82 amino acid residues of the domain III, or a
combination
thereof. The heterologous cargo can be a macromolecule, a small molecule, a
polypeptide, a
nucleic acid, a mRNA, a miRNA, a shRNA, a siRNA, an antisense molecule, an
antibody, a
DNA, a plasmid, a vaccine, a polymer a nanoparticle, or a catalytically-active
material. The
heterologous cargo can be a biologically active cargo. The biologically active
cargo can be a
cytokine, a hormone, a therapeutic antibody, a functional fragment thereof, or
any combination
thereof. The cytokine can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,
IL-23, IL-24, IL-25,
IL-26, IL-27, IL-28, IL-29, or IL-30. The cytokine can have the amino acid
sequence set forth in
SEQ ID NO: 217 or SEQ ID NO: 218. The hormone can have the amino acid sequence
set forth
in SEQ ID NO: 215 or SEQ ID NO: 216. The therapeutic antibody can be an anti-
TNFa
antibody. The anti-TNFa antibody can be adalimumab or infliximab. The
heterologous cargo can
be a detectable agent. The detectable agent can be a fluorophore, a contrast
agent, an X-ray
contrast agent, a PET agent, a nanoparticle, or a radioisotope. The
fluorophore can be a red
fluorescent protein (RFP). The RFP can have the amino acid sequence set forth
in SEQ ID NO:
220.
[0026] The present disclosure relates to novel non-naturally occurring
delivery constructs
that can comprise a bacterial toxin-derived chimeric carrier coupled to a
biologically active
cargo; wherein the chimeric carrier is derived from a domain I but does not
comprise a domain
II, a domain lb, or a domain III of the bacterial toxin (e.g., an exotoxin);
and wherein the
delivery construct is capable of delivering a heterologous (e.g., a
biologically active) cargo via
transcytosis transport across an epithelial cell (e.g., an intestinal
epithelial cell).
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[0027] The carrier can be derived from a domain I of an exotoxin and is
capable of
recognizing and interacting with one or more receptors on the luminal (e.g.,
apical) surface of
intestinal epithelial cells. The receptor can be selective or non-selective.
In some aspects, the
receptor that a carrier interacts with is a non-selective scavenger receptor
or a transmembrane
receptor 132 (TMEM132) receptor. Interaction of the delivery constructs with a
cell surface
receptor that is present on the apical membrane of a polarized epithelial cell
can occur with
sufficient affinity to allow endocytosis of the delivery construct. The
carrier that a delivery
construct of the present disclosure is comprised of can bind to receptor(s)
known to be present on
the apical membrane of an epithelial cell by one of skill in the art without
limitation. In various
embodiments, the receptor binding domain of the delivery construct can bind to
low density
lipoprotein receptor-related protein 1 (LRP1) or TMEM132 receptor.
[0028] A delivery construct as described herein can be capable of
delivering a heterologous
(e.g., a biologically active) cargo across an epithelial cell from the apical
side to a basolateral
compartment and/or the lamina propria. A delivery construct as described
herein can be capable
of delivering a heterologous (e.g., a biologically active) cargo into an
epithelial cell (e.g., a
polarized gut epithelial cell), such as an intracellular vesicle or
compartment or the cytosol of the
epithelial cell, thereby allowing for accumulation of the heterologous (e.g.,
biologically active)
cargo in the epithelial cell. The carrier can be derived from the domain I of
an exotoxin selected
from the group consisting of cholix carrier (Cholix) and Pseudomonas exotoxin
A (PE).
[0029] The carrier can be a polypeptide derived from Cholix and/or PE and
having: at most
amino acid residues; at most 10 amino acid residues; at most 15 amino acid
residues; at most
20 amino acid residues; at most 30 amino acid residues; at most 40 amino acid
residues; at most
50 amino acid residues; at most 60 amino acid residues; at most 70 amino acid
residues; at most
80 amino acid residues; at most 90 amino acid residues; at most 100 amino acid
residues; at most
110 amino acid residues; at most 120 amino acid residues; at most 130 amino
acid residues; at
most 140 amino acid residues; at most 150 amino acid residues; at most 160
amino acid residues;
at most 170 amino acid residues; at most 180 amino acid residues; at most 190
amino acid
residues; at most 200 amino acid residues; at most 210 amino acid residues; at
most 220 amino
acid residues; at most 230 amino acid residues; at most 240 amino acid
residues; at most 250
amino acid residues; at most 260 amino acid residues; and at most 265 amino
acid residues.
[0030] The carrier can be derived from a domain I of a Cholix exotoxin and
can comprise an
amino acid sequence selected from the group consisting of an amino acid
sequence having
greater than 50% homology to SEQ ID NO: 4, having greater than 60% homology to
SEQ ID
NO: 4, having greater than 70% homology to SEQ ID NO: 4, having greater than
80% homology
to SEQ ID NO: 4, having greater than 85% homology to SEQ ID NO: 4, having
greater than
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90% homology to SEQ ID NO: 4, and having greater than 95% homology to SEQ ID
NO: 4. In
some cases, the delivery construct is derived from cholix exotoxin (Cholix)
and comprises the
receptor binding domain polypeptide having the amino acid sequence set forth
in SEQ ID NO: 4.
The carrier can comprise an amino acid sequence with greater than 90% homology
to SEQ ID
NO: 4. The carrier can comprise an amino acid sequence with greater than 95%
homology to
SEQ ID NO: 4. The carrier can comprise a receptor binding domain polypeptide
wherein one or
more amino residues of SEQ ID NO: 4 is substituted with another amino acid.
The carrier can
comprise a receptor binding domain polypeptide that is a truncated portion of
the amino acid
sequence set forth in SEQ ID NO: 4.
[0031] The carrier can be derived from a domain I of a Pseudomonas exotoxin
A (PE) and
can comprise a polypeptide having the amino acid sequence set forth in SEQ ID
NO: 137. The
delivery construct can comprise an amino acid sequence with greater than 90%
homology to
SEQ ID NO: 137. The carrier can comprise an amino acid sequence with greater
than 95%
homology to SEQ ID NO: 137. The carrier can comprise a receptor binding domain
polypeptide
wherein one or more amino residues of SEQ ID NO: 137 is substituted with
another amino acid.
The carrier can comprise a receptor binding domain polypeptide that is a
truncated portion of the
amino acid sequence set forth in SEQ ID NO: 137.
[0032] A delivery construct can comprise a carrier, wherein the carrier
comprises one or
more amino acid residues of one exotoxin domain I (e.g., a Cholix or PE domain
I) is replaced
by one or more amino acid residues of a second exotoxin domain I (e.g., a
Cholix or PE domain
I), (also referred to hereinafter as a hybrid or chimeric carrier). The
carrier can comprise an
amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 4 is
replaced by
one or more amino acid residues of SEQ ID NO: 137. The carrier can comprise an
amino acid
sequence wherein one or more amino acid residues of SEQ ID NO: 137 is replaced
by one or
more amino acid residues of SEQ ID NO: 4. The carrier can comprise an amino
acid sequence
wherein amino acid residues 77-87 of SEQ ID NO: 4 are replaced by amino acid
residues of a
second bacterial carrier receptor binding domain polypeptide. The carrier can
comprise an amino
acid sequence wherein amino acid residues 188-236 of SEQ ID NO: 4 are replaced
by amino
acid residues of a second bacterial carrier receptor binding domain
polypeptide. The carrier can
comprise an amino acid sequence wherein amino acid residues 69-71 of SEQ ID
NO: 137 are
replaced by amino acid residues of a second bacterial carrier receptor binding
domain
polypeptide. The carrier can comprise an amino acid sequence wherein amino
acid residues 177-
228 of SEQ ID NO: 137 are replaced by amino acid residues of a second
bacterial carrier
receptor binding domain polypeptide.
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[0033] A carrier of the present disclosure that can be derived from a
domain I of an exotoxin
and can further comprise a portion of a domain II, a portion of a domain lb,
and/or a portion of a
domain III of the same or another exotoxin. Thus, a carrier can comprise a
domain I of an
exotoxin, or a truncated and/or modified version thereof, and one or more
portions derived from
a domain II, domain lb, and/or domain III of the same or a different exotoxin.
The domain II, or
modified domain II, and domain III, or modified domain III, can be derived
from the same
bacterial toxin. The domain II, or modified domain II, and domain III, or
modified domain III,
can be derived from a bacterial carrier selected from the group consisting of
cholix carrier
(Cholix) and Pseudomonas exotoxin A (PE), botulinum toxin, diptheria toxin,
pertussis toxin,
cholera toxin, heat-labile E. coli entero-toxin, shiga toxin, and shiga-like
toxin. Toxicity of the
bacterial carrier (e.g., Cholix or PE) may not be required for transport
across epithelial layers
such as the gut epithelium. For example, a delivery construct as described
herein can comprise a
carrier coupled to a heterologous cargo, and wherein the carrier is derived
from a Cholix domain
I (e.g., having an amino acid sequence set forth in any one of SEQ ID NO: 4 ¨
SEQ ID NO: 125)
and further comprising portions of a domain II (e.g., SEQ ID NO: 126 or SEQ ID
NO: 138), a
domain lb (e.g., SEQ ID NO: 127 or SEQ ID NO: 139), and/or a domain III (e.g.,
SEQ ID NO:
128 or SEQ ID NO: 140) of an exotoxin (e.g., Cholix and/or PE).
[0034] A delivery construct can comprise a carrier having the amino acid
sequence derived
from the sequence set forth in SEQ ID NO: 4, a translocation domain having the
amino acid
sequence derived from the sequence set forth in SEQ ID NO: 126, and a non-
toxic catalytic
domain having the amino acid sequence derived from the sequence set forth in
SEQ ID NO: 128.
A delivery constructs can comprise a receptor binding domain polypeptide
having the amino acid
sequence derived from the sequence set forth in SEQ ID NO: 136, a
translocation domain having
the amino acid sequence derived from the sequence set forth in SEQ ID NO: 137,
and a non-
toxic catalytic domain having the amino acid sequence derived from the
sequence set forth in
SEQ ID NO: 139. The delivery construct can comprises the amino acid sequence
set forth in
SEQ ID NO: 146. In various embodiments, the delivery construct comprises the
amino acid
sequence set forth in SEQ ID NO: 147.
[0035] The delivery constructs of the present disclosure can comprise a
carrier coupled to a
heterologous cargo. The heterologous cargo can be a biologically active cargo.
The heterologous
cargo can be a detectable agent. The carrier can be coupled to a biologically
active cargo to
produce a delivery construct that is capable of delivering the biologically
active cargo via
transcytosis transport across an intestinal epithelium. The biologically
active cargo can be
selected from e.g., a macromolecule, small molecule, peptide, polypeptide,
nucleic acid, mRNA,
miRNA, shRNA, siRNA, antisense molecule, antibody, DNA, plasmid, vaccine,
polymer
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nanoparticle, or catalytically-active material. The biologically active cargo
can be an enzyme
selected from hyaluronidase, streptokinase, tissue plasminogen activator,
urokinase, or PGE-
adenosine deaminase. The biologically active cargo can comprises an amino acid
sequence
selected from the group consisting of the amino acid sequences set forth in
SEQ ID NO: 214,
SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO:
219,
or any combination thereof
[0036] The delivery constructs can comprise a carrier directly coupled to a
heterologous
(e.g., a biologically active) cargo. The heterologous (e.g., a biologically
active) cargo can be
directly coupled to the C-terminus of the delivery construct. The heterologous
(e.g., a
biologically active) cargo can be directly coupled to the N-terminus of the
delivery construct.
[0037] The delivery constructs can comprise a carrier chemically coupled to
a heterologous
(e.g., a biologically active) cargo. The delivery constructs can comprise a
carrier recombinantly
coupled to a heterologous (e.g., a biologically active) cargo. A delivery
construct of the present
disclosure can be produced partly synthetically (e.g., via solid-phase
synthesis) or recombinantly
(e.g., bacterially expressed (e.g., E. coli) or in a mammalian cell (e.g., CHO
cell)). A delivery
construct of the present disclosure can be produced partly synthetic and
partly recombinant.
[0038] The delivery constructs can comprise a delivery construct coupled to
a biologically
active cargo by a cleavable spacer. The spacer can be cleavable by an enzyme
that is present at a
basolateral membrane of a polarized epithelial cell. The spacer can be
cleavable by an enzyme
that is present in the plasma. The cleavable spacer can comprise the amino
acid sequence set
forth in any one of SEQ ID NO: 174 ¨ SEQ ID NO: 206. The cleavable spacer can
be a spacer
that comprises an amino acid sequence that can be a known substrate for the
tobacco etch virus
(TEV) protease. The cleavable spacer comprises the amino acid sequence set in
forth in SEQ ID
NO: 193. The spacer can be cleavable by an enzyme that is present at a basal-
lateral membrane
of a polarized epithelial cell. The spacer can be cleavable by an enzyme that
is present in the
plasma of a subject.
[0039] The cleavable spacers can comprise a peptide sequence (or like
domain), which
serves to inhibit, interfere with, or block the ability of the biologically
active cargo to bind to
receptors at the surface of epithelial cells, but wherein the delivery
construct retains the ability of
the cargo to activate it's receptor after the delivery construct is
transported across the epithelial
barrier and the cargo is released from the delivery construct and spacer
components of the
construct. The cleavable spacer can comprise the amino acid sequence set forth
in, e.g., SEQ ID
NO: 194 ¨ SEQ ID NO: 206.
[0040] The present disclosure also relates to pharmaceutical compositions
that can comprise
a novel non-naturally occurring delivery construct of the present disclosure
and one or more
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pharmaceutically acceptable carriers, formulated for oral administration,
topical administration,
pulmonary administration, intra-nasal administration, buccal administration,
sublingual
administration or ocular administration.
[0041] The present disclosure provides a method of treating an inflammatory
disease in a
subject that can comprise administering a pharmaceutical composition of the
present disclosure
to the subject. In various embodiments, the inflammatory disease is selected
from an
inflammatory bowel disease, psoriasis or bacterial sepsis. In various
embodiments, the
inflammatory bowel disease is Crohn's disease, ulcerative colitis, collagenous
colitis,
lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome or
indeterminate
colitis.
[0042] The present disclosure provides a method of treating an autoimmune
disease in a
subject that can comprise administering a pharmaceutical composition of the
present disclosure
to the subject. In various embodiments, the autoimmune disease is systemic
lupus erythematosus
(SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia,
thrombocytopenia purpura,
Grave's disease, Sjogren's disease, dermatomyositis, Hashimoto's disease,
polymyositis,
inflammatory bowel disease, multiple sclerosis (MS), diabetes mellitus,
rheumatoid arthritis, or
scleroderma.
[0043] The present disclosure provides a method of treating a cancer in a
subject that can
comprise administering a pharmaceutical composition of the present disclosure
to the subject. In
various embodiments, the cancer to be treated includes, but is not limited to,
non-Hodgkin's
lymphomas, Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy cell
leukemia, acute
lymphoblastic leukemia, multiple myeloma, carcinomas of the bladder, kidney
ovary, cervix,
breast, lung, nasopharynx, malignant melanoma and rituximab resistant NHL and
leukemia.
[0044] The present disclosure provides a method of treating a subject
having a metabolic
disorder, said method can comprise administering a pharmaceutical composition
of the present
disclosure in an amount sufficient to treat said disorder, wherein said
metabolic disorder is
diabetes, obesity, diabetes as a consequence of obesity, hyperglycemia,
dyslipidemia,
hypertriglyceridemia, syndrome X, insulin resistance, impaired glucose
tolerance (IGT), diabetic
dyslipidemia, or hyperlipidemia.
[0045] The present disclosure provides a method of treating a subject
having a fatty liver
disease (e.g., nonalcoholic fatty liver disease (NAFLD); nonalcoholic
steatohepatitis (NASH)), a
gastrointestinal disease, or a neurodegenerative disease, said method
comprising orally
administering a pharmaceutical composition of the present disclosure in an
amount sufficient to
treat said disease.
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[0046] The present disclosure provides a method of treating a subject
having a GH deficient
growth disorder, said method can comprise administering a pharmaceutical
composition of the
present disclosure in an amount sufficient to treat said disorder, wherein
said disorder is growth
hormone deficiency (GHD), Turner syndrome (TS), Noonan syndrome, Prader-Willi
syndrome,
short stature homeobox-containing gene (SHOX) deficiency, chronic renal
insufficiency, and
idiopathic short stature short bowel syndrome, GH deficiency due to rare
pituitary tumors or
their treatment, and muscle-wasting disease associated with HIV/AIDS.
[0047] A delivery construct can comprise a carrier comprising an amino acid
sequence
having at least 80% sequence identity to any one or more of the amino acid
sequences set forth
in SEQ ID NO: 1 ¨ SEQ ID NO: 133 or SEQ ID NO: 137¨ SEQ ID NO: 147. A delivery
construct can comprise a carrier comprising an amino acid sequence having at
least 90%
sequence identity to any one or more of the amino acid sequences set forth in
SEQ ID NO: 1 ¨
SEQ ID NO: 133 or SEQ ID NO: 137 ¨ SEQ ID NO: 147. A delivery construct can
comprise a
carrier comprising an amino acid sequence having at least 95% sequence
identity to any one or
more of the amino acid sequences set forth in SEQ ID NO: 1 ¨ SEQ ID NO: 133 or
SEQ ID NO:
137 ¨ SEQ ID NO: 147. A delivery construct can comprise a carrier comprising
an amino acid
sequence having at least 99% sequence identity to any one or more of the amino
acid sequences
set forth in SEQ ID NO: 1 ¨ SEQ ID NO: 133 or SEQ ID NO: 137 ¨ SEQ ID NO: 147.
The
carrier can be derived from a Cholix domain I and can comprise an amino acid
sequence having
at least 80% sequence identity to any one or more of the amino acid sequences
set forth in SEQ
ID NO: 4 ¨ SEQ ID NO: 125 and/or SEQ ID NO: 148 ¨ SEQ ID NO: 152. The carrier
can be
derived from a Cholix domain I and can comprise an amino acid sequence having
at least 90%
sequence identity to any one or more of the amino acid sequences set forth in
SEQ ID NO: 4 ¨
SEQ ID NO: 125 and/or SEQ ID NO: 148 ¨ SEQ ID NO: 152. The carrier can be
derived from a
Cholix domain I and can comprise an amino acid sequence having at least 95%
sequence identity
to any one or more of the amino acid sequences set forth in SEQ ID NO: 4 ¨ SEQ
ID NO: 125
and/or SEQ ID NO: 148 ¨ SEQ ID NO: 152. The carrier can be derived from a
Cholix domain I
and can comprise an amino acid sequence having at least 99% sequence identity
to any one or
more of the amino acid sequences set forth in SEQ ID NO: 4 ¨ SEQ ID NO: 125
and/or SEQ ID
NO: 148 ¨ SEQ ID NO: 152. Any one of these carriers can be combined with any
heterologous
cargo described and disclosed herein, e.g., those having at least 80% sequence
identity to an
amino acid sequence set forth in any one of SEQ ID NO: 214 ¨ SEQ ID NO: 220.
Any one of
these carriers can be combined with any heterologous cargo described and
disclosed herein, e.g.,
those having at least 90% sequence identity to an amino acid sequence set
forth in any one of
SEQ ID NO: 214 ¨ SEQ ID NO: 220. Any one of these carriers can be combined
with any
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heterologous cargo described and disclosed herein, e.g., those having at least
95% sequence
identity to an amino acid sequence set forth in any one of SEQ ID NO: 214 ¨
SEQ ID NO: 220.
Any one of these carriers can be combined with any heterologous cargo
described and disclosed
herein, e.g., those having at least 99% sequence identity to an amino acid
sequence set forth in
any one of SEQ ID NO: 214 ¨ SEQ ID NO: 220. A delivery construct described
herein can
comprise an amino acid sequence having at least 80% sequence indentity to an
amino acid
sequence set forth in any one of SEQ ID NO: 153 ¨ SEQ ID NO: 165. A delivery
construct
described herein can comprise an amino acid sequence having at least 90%
sequence indentity to
an amino acid sequence set forth in any one of SEQ ID NO: 153 ¨ SEQ ID NO:
165. A delivery
construct described herein can comprise an amino acid sequence having at least
95% sequence
indentity to an amino acid sequence set forth in any one of SEQ ID NO: 153 ¨
SEQ ID NO: 165.
A delivery construct described herein can comprise an amino acid sequence
having at least 99%
sequence indentity to an amino acid sequence set forth in any one of SEQ ID
NO: 153 ¨ SEQ ID
NO: 165. A construct can be capable of endocytosis (e.g., apical endocytosis).
A construct can
be capable of apical-to-basal transcytosis.
[0048] The present disclosure provides isolated delivery constructs that
can be capable of
binding a receptor on the luminal surface of intestinal epithelial cells with
sufficient affinity to
allow endocytosis; wherein the domain is a polypeptide comprising an amino
acid sequence
wherein one or more amino acid residues of one bacterial toxin domain I
polypeptide is replaced
by one or more amino acid residues of a second bacterial toxin (e.g., an
exotoxin) domain I
polypeptide. The domain I of a first exotoxin can comprise a polypeptide which
comprises an
amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 4
can be replaced
by one or more amino acid residues of a second bacterial toxin (e.g., an
exotoxin) receptor
binding domain polypeptide. The receptor binding domain can be a polypeptide
which comprises
an amino acid sequence wherein one or more amino acid residues of SEQ ID NO: 4
is replaced
by one or more amino acid residues of SEQ ID NO: 137. The receptor binding
domain can be a
polypeptide which comprises an amino acid sequence wherein one or more amino
acid residues
of SEQ ID NO: 137 is replaced by one or more amino acid residues a second
bacterial toxin
receptor binding domain polypeptide. The receptor binding domain can be a
polypeptide which
comprises an amino acid sequence wherein one or more amino acid residues of
SEQ ID NO: 136
is replaced by one or more amino acid residues of SEQ ID NO: 4. A chimeric
carrier can
comprise a biologically active cargo coupled to the polypeptide to produce a
chimeric construct;
wherein the chimeric construct is capable of delivering the biologically
active cargo.
[0049] The present disclosure provides a chimeric construct comprising a
bacterial toxin-
derived delivery construct; and a biologically active cargo; wherein the
delivery construct is
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capable of delivering the biologically active cargo into an epithelial cell;
and wherein the
delivery construct does not comprise a bacterial toxin-derived translocation
domain or a bacterial
toxin-derived catalytic (cytotoxic) domain. The present disclosure provides a
chimeric construct
consisting of a receptor binding domain of a bacterial toxin; and a
biologically active cargo;
wherein the delivery construct is capable of delivering the biologically
active cargo into an
epithelial cell, and wherein the chimeric construct is capable of binding a
receptor on the luminal
surface of intestinal epithelial cells.
[0050] The present disclosure further provides polynucleotides that encode
the non-
naturally occurring delivery constructs and/or delivery constructs of the
present
disclosure; vectors comprising polynucleotides encoding the non-naturally
occurring delivery
constructs and/or delivery constructs of the present disclosure; optionally,
operably-linked to
control sequences recognized by a host cell transformed with the vector; host
cells comprising
vectors comprising polynucleotides encoding the non-naturally occurring
delivery constructs
and/or delivery constructs of the present disclosure; a process for producing
the non-naturally
occurring delivery constructs and/or delivery constructs of the present
disclosure comprising
culturing host cells comprising vectors comprising polynucleotides encoding
the non-naturally
occurring delivery constructs and/or delivery constructs of the present
disclosure such that the
polynucleotide is expressed; and, optionally, recovering the non-naturally
occurring delivery
constructs and/or delivery constructs from host cell culture medium.
[0051] Disclosed herein is a use of a non-naturally occurring delivery
construct of the
present disclosure for the preparation of a medicament for treatment,
prophylaxis and/or
prevention of an inflammatory disease in a subject in need thereof
[0052] Disclosed herein is a use of a non-naturally occurring delivery
construct of the
present disclosure for the preparation of a medicament for treatment,
prophylaxis and/or
prevention of an autoimmune disease in a subject in need thereof.
[0053] Disclosed herein is a use of a non-naturally occurring delivery
construct of the
present disclosure for the preparation of a medicament for treatment,
prophylaxis and/or
prevention of a cancer in a subject in need thereof
[0054] Disclosed herein is a use of a non-naturally occurring delivery
construct of the
present disclosure for the preparation of a medicament for treatment,
prophylaxis and/or
prevention of a metabolic disorder in a subject in need thereof
[0055] Disclosed herein is a use of a non-naturally occurring delivery
construct of the
present disclosure for the preparation of a medicament for treatment,
prophylaxis and/or
prevention of a fatty liver disease in a subject in need thereof.
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[0056] Disclosed herein is a use of a non-naturally occurring delivery
construct of the
present disclosure for the preparation of a medicament for treatment,
prophylaxis and/or
prevention of GH deficient growth disorder in a subject in need thereof
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The novel features of the present disclosure are set forth with
particularity in the
appended claims. A better understanding of the features and advantages of the
present disclosure
will be obtained by reference to the following detailed description that sets
forth illustrative
aspects, in which the principles of the disclosure are utilized, and the
accompanying drawings
(also "Figure" and "FIG." herein), of which:
[0058] FIG. 1 and FIG. 2 depict fluorescence microscopic detection of
Constructs 7-12
(prepared as described in EXAMPLE 1 herein) observed 20 min after intra-
luminal injection
using a rat intra-luminal injection model. FIG. 1 depicts (top to bottom)
localization of
Constructs 12, 11 and 10. FIG. 2 depicts (top to bottom) Constructs 9, 8 and
7. Left to right:
fluorescence image, dark field illumination, composite of fluorescence image
and dark field
illumination (white arrow #1 highlights the apical surface, and white arrow #2
highlights the
basal surface).
[0059] FIG. 1A shows localization (fluorescence image) of construct 12
observed 20 min
after intra-luminal injection using a rat intra-luminal injection model.
[0060] FIG. 1B shows localization (white light image) of construct 12
observed 20 min
after intra-luminal injection using a rat intra-luminal injection model.
[0061] FIG. 1C shows localization (merge image, with DAPI) of construct 12
observed 20
min after intra-luminal injection using a rat intra-luminal injection model.
[0062] FIG. 1D shows localization (fluorescence image) of construct 11
observed 20 min
after intra-luminal injection using a rat intra-luminal injection model.
[0063] FIG. 1E shows localization (white light image) of construct 11
observed 20 min
after intra-luminal injection using a rat intra-luminal injection model.
[0064] FIG. 1F shows localization (merge image, with DAPI) of construct 11
observed 20
min after intra-luminal injection using a rat intra-luminal injection model.
[0065] FIG. 1G shows localization (fluorescence image) of construct 10
observed 20 min
after intra-luminal injection using a rat intra-luminal injection model.
[0066] FIG. 1H shows localization (white light image) of construct 10
observed 20 min
after intra-luminal injection using a rat intra-luminal injection model.
[0067] FIG. 11 shows localization (merge image, with DAPI) of construct 10
observed 20
min after intra-luminal injection using a rat intra-luminal injection model.
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[0068] FIG. 2A shows localization (fluorescence image) of construct 9
observed 20 min
after intra-luminal injection using a rat intra-luminal injection model.
[0069] FIG. 2B shows localization (white light image) of construct 9
observed 20 min after
intra-luminal injection using a rat intra-luminal injection model.
[0070] FIG. 2C shows localization (merge image, with DAPI) of construct 9
observed 20
min after intra-luminal injection using a rat intra-luminal injection model.
[0071] FIG. 2D shows localization (fluorescence image) of construct 8
observed 20 min
after intra-luminal injection using a rat intra-luminal injection model.
[0072] FIG. 2E shows localization (white light image) of construct 8
observed 20 min after
intra-luminal injection using a rat intra-luminal injection model.
[0073] FIG. 2F shows localization (merge image, with DAPI) of construct 8
observed 20
min after intra-luminal injection using a rat intra-luminal injection model.
[0074] FIG. 2G shows localization (fluorescence image) of construct 7
observed 20 min
after intra-luminal injection using a rat intra-luminal injection model.
[0075] FIG. 211 shows localization (white light image) of construct 7
observed 20 min after
intra-luminal injection using a rat intra-luminal injection model.
[0076] FIG. 21 shows localization (merge image, with DAPI) of construct 7
observed 20
min after intra-luminal injection using a rat intra-luminal injection model.
[0077] FIG. 3 depicts fluorescence microscopic detection of Construct 6
(prepared as
described in EXAMPLE 1 herein) observed 20 min after intra-luminal injection
using a rat
intra-luminal injection model. Left to right: fluorescence image, dark field
illumination,
composite of fluorescence image and dark field illumination (white arrow #1
highlights the
apical surface, and white arrow #2 highlights the basal surface).
[0078] FIG. 3A depicts fluorescence microscopic detection of Construct 6
(anti-Cho,
1/500).
[0079] FIG. 3B depicts dark field illumination detection of Construct 6
(anti-Cho, 1/500).
[0080] FIG. 3B depicts a composite of fluorescence image and dark field
illumination
detection of Construct 6 (anti-Cho, 1/500).
[0081] FIG. 3D depicts fluorescence microscopic detection of Construct 6
(anti-RPF, 1/50).
[0082] FIG. 3E depicts dark field illumination detection of Construct 6
(anti-RPF, 1/50).
[0083] FIG. 3F depicts a composite of fluorescence image and dark field
illumination
detection of Construct 6 (anti-RPF, 1/50).
[0084] FIG. 4 and FIG. 5 depict fluorescence microscopic detection of
Construct 13 (SEQ
ID NO: 146, prepared as described in EXAMPLE 5 herein) observed after 1 min
(FIG. 4) and
20 min (FIG. 5) after intra-luminal injection using a rat intra-luminal
injection model.
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[0085] FIG. 4A shows fluorescence microscopic detection of Construct 13
after 1 min.
[0086] FIG. 4B shows fluorescence microscopic detection of Construct 13
after 1 min.
[0087] FIG. 4C shows fluorescence microscopic detection of Construct 13
after 1 min.
[0088] FIG. 4D shows fluorescence microscopic detection of Construct 13
after 1 min.
[0089] FIG. 4E shows fluorescence microscopic detection of Construct 13
after 1 min
(white arrow #1 highlights the apical surface, and white arrow #2 highlights
the basal surface).
[0090] FIG. 5A shows fluorescence microscopic detection of Construct 13
after 20 min.
[0091] FIG. 5B shows fluorescence microscopic detection of Construct 13
after 20 min.
[0092] FIG. 5C shows fluorescence microscopic detection of Construct 13
after 20 min.
[0093] FIG. 5D shows fluorescence microscopic detection of Construct 13
after 20 min.
[0094] FIG. 5E shows fluorescence microscopic detection of Construct 13
after 20 min
(white arrow #1 highlights the apical surface, and white arrow #2 highlights
the basal surface).
[0095] FIG. 6 and FIG. 7 depict fluorescence microscopic detection of
Construct 14 (SEQ
ID NO: 147, prepared as described in EXAMPLE 5 herein) observed after 1 min
(FIG. 6) and
20 min (FIG. 7) after intra-luminal injection using a rat intra-luminal
injection model (white
arrow #1 highlights the apical surface, and white arrow #2 highlights the
basal surface).
[0096] FIG. 6A shows fluorescence microscopic detection of Construct 14
after 1 min.
[0097] FIG. 6B shows fluorescence microscopic detection of Construct 14
after 1 min.
[0098] FIG. 6C shows fluorescence microscopic detection of Construct 14
after 1 min.
[0099] FIG. 6D shows fluorescence microscopic detection of Construct 14
after 1 min.
[0100] FIG. 6E shows fluorescence microscopic detection of Construct 14
after 1 min
(white arrow #1 highlights the apical surface, and white arrow #2 highlights
the basal surface).
[0101] FIG. 7A shows fluorescence microscopic detection of Construct 14
after 20 min.
[0102] FIG. 7B shows fluorescence microscopic detection of Construct 14
after 20 min.
[0103] FIG. 7C shows fluorescence microscopic detection of Construct 14
after 20 min.
[0104] FIG. 7D shows fluorescence microscopic detection of Construct 14
after 20 min.
[0105] FIG. 7E shows fluorescence microscopic detection of Construct 14
after 20 min
(white arrow #1 highlights the apical surface, and white arrow #2 highlights
the basal surface).
[0106] FIG. 8A depicts fluorescence microscopic detection of the construct
comprising an
amino acid sequence set forth in SEQ ID NO: 165 (M+Cholix39-186-(spacer with
SEQ ID NO:
210)-HGH) observed 5 min after intra-luminal injection using a rat intra-
luminal injection model
(white arrow #1 highlights the apical surface, and white arrow #2 highlights
the basal surface).
[0107] FIG. 8B depicts fluorescence microscopic detection of the construct
comprising an
amino acid sequence set forth in SEQ ID NO: 165 (M+Cholix39-186-(spacer with
SEQ ID NO:
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210)-HGH) observed 10 min after intra-luminal injection using a rat intra-
luminal injection
model.
[0108] FIG. 8C depicts fluorescence microscopic detection of the construct
comprising an
amino acid sequence set forth in SEQ ID NO: 165 (M+Cholix39-186-(spacer with
SEQ ID NO:
210)-HGH) observed 15 min after intra-luminal injection using a rat intra-
luminal injection
model.
[0109] FIG. 9A depicts fluorescence microscopic detection of the construct
comprising an
amino acid sequence set forth in SEQ ID NO: 160 (Cholix1187-(spacer with SEQ
ID NO: 210)-
HGH) observed 5 min after intra-luminal injection using a rat intra-luminal
injection model
(white arrow #1 highlights the apical surface, and white arrow #2 highlights
the basal surface).
[0110] FIG. 9B depicts fluorescence microscopic detection of the construct
comprising an
amino acid sequence set forth in SEQ ID NO: 160 (Cholix1187-(spacer with SEQ
ID NO: 210)-
HGH) observed 10 min after intra-luminal injection using a rat intra-luminal
injection model.
[0111] FIG. 9C depicts fluorescence microscopic detection of the construct
comprising an
amino acid sequence set forth in SEQ ID NO: 160 (Cholix1187-(spacer with SEQ
ID NO: 210)-
HGH) observed 15 min after intra-luminal injection using a rat intra-luminal
injection model
(white arrow #2 highlights the basal surface).
[0112] FIG. 10A shows Non-toxic Cholix (ntChx) transcytosis across human
polarized
intestinal epithelium in vitro. FIG. 10A depicts the amount of non-toxic
Cholix (ntChx, SEQ ID
NO: 3) detected in the basal compartment by ELISA at 2 h after an apical
application of 2.5 -200
mg/mL ntChx (N=2; mean SE., white arrow #1 highlights the apical surface,
and white arrow
#2 highlights the basal surface).
[0113] FIG. 10B depicts a Western blot analysis of basal compartment
contents 2 h after an
apical application of 2.5 -200 mg/mL ntChx that were concentrated
approximately 10-fold prior
to analysis showing that the ntChx that transported was not significantly
altered (e.g., chemically
altered) during transport.
[0114] FIG. 10C depicts basal quantities of ntChx detected over a time
course of 2 h by
ELISA. The graph shows a delay of ¨20-25 min in detectable quantities and
comparable rates of
transport for apical applications of 5-20 mg/mL at 37 C, and a significant
reduction in transport
rate at 4 C.
[0115] FIG. 11 shows that apical to basal transport of 5-20 mg/mL ntChx
(SEQ ID NO: 3),
as measured by ELISA, was more efficient with apical compartment at pH 7
compared to pH 5
(N=2; mean SE).
[0116] FIG. 12A depicts transcytosis of non-toxic Cholix (ntChx, SEQ ID NO:
3) in vivo
after 1 minutes following intraluminal injection (ILI) into rat jejunum
visualized by
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immunofluorescence microscopy. Open arrow = apical enterocyte domain; solid
arrow = pen-
nuclear region of cell; dashed line = epithelial cell-basement membrane
demarcation; GC =
goblet cell.
[0117] FIG. 12B depicts transcytosis of non-toxic Cholix (ntChx, SEQ ID NO:
3) in vivo
after 5 minutes following ILI into rat jejunum visualized by
immunofluorescence microscopy.
Open arrow = apical enterocyte domain; solid arrow = pen-nuclear region of
cell; dashed line =
epithelial cell-basement membrane
[0118] demarcation; GC = goblet cell.
[0119] FIG. 12C depicts transcytosis of non-toxic Cholix (ntChx, SEQ ID NO:
3)/n vivo
after 15 minutes following ILI into rat jejunum visualized by
immunofluorescence microscopy.
Co-localization with clathrin shows the villus tip area. Open arrow = apical
enterocyte domain;
solid arrow = pen-nuclear region of cell; dashed line = epithelial cell-
basement membrane
[0120] demarcation; GC = goblet cell.
[0121] FIG. 13A shows in vivo ntChx transcytosis at 15 min after ILI of
ntChx (SEQ ID
NO: 3) into rat jejunum with simultaneous staining of early endosomal antigen
1 (EEA1) (white
arrow #1 highlights the apical surface, and white arrow #2 highlights the
basal surface). GC =
goblet cell; l-p = lamina propria.
[0122] FIG. 13B shows in vivo ntChx transcytosis at 15 min after ILI of
ntChx (SEQ ID
NO: 3) into rat jejunum with simultaneous staining of Ras-related protein Rab
1 1 a (white arrow
#1 highlights the apical surface, and white arrow #2 highlights the basal
surface). GC = goblet
cell; l-p = lamina propria.
[0123] FIG. 13C shows in vivo ntChx transcytosis at 15 min after ILI of
ntChx (SEQ ID
NO: 3) into rat jejunum with simultaneous staining of trans-Golgi network
(TGN)-38 protein.
[0124] FIG. 13D shows in vivo ntChx transcytosis at 15 min after ILI of
ntChx (SEQ ID
NO: 3) into rat jejunum with simultaneous staining of calnexin (white arrow #1
highlights the
apical surface, and white arrow #2 highlights the basal surface). GC = goblet
cell; l-p = lamina
propria.
[0125] FIG. 13E shows in vivo ntChx transcytosis at 15 min after ILI of
ntChx (SEQ ID
NO: 3) into rat jejunum with simultaneous staining of Ras-related protein Rab
7 (white arrow #1
highlights the apical surface, and white arrow #2 highlights the basal
surface). GC = goblet cell;
l-p = lamina propria.
[0126] FIG. 13F shows in vivo ntChx transcytosis at 15 min after ILI of
ntChx (SEQ ID
NO: 3) into rat jejunum with simultaneous staining of Golgi-associated 58kDa
formiminotransferase cyclodeaminase protein (FTCD). GC = goblet cell; l-p =
lamina propria.
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[0127] FIG. 14A shows small amounts of RFP (negative control) reaching
cells within the
lamina propria (1-p) with no detectable RFP in the villus epithelium (solid
arrows point to apical
surface of epithelium) at 30 min post ILI. A polyclonal antibody to RFP
demonstrates small
amounts of RFP can reach cells within the lamina propria (1-p) with no
detectable RFP in the
villus epithelium ; (solid arrows point to apical surface of epithelium) at 30
min post ILI. Solid
arrow (#1) = luminal (apical) membrane; white arrow #2 = basal membrane; GC =
goblet cell; 1-
p = lamina propria.
[0128] FIG. 14B shows that a construct comprising an amino acid sequence
set forth in
SEQ ID NO: 157 comprising full-length of non-toxic Cholix (ntChx, SEQ ID NO:
3) genetically
fused to or conjugated to red fluorescent protein (RFP, SEQ ID NO: 220) is
capable of efficient
apical-to-basal transcytosis. Solid arrow (#1) = luminal (apical) membrane;
white arrow #2 =
basal membrane; GC = goblet cell; 1-p = lamina propria. Full-length of non-
toxic Cholix (ntChx,
SEQ ID NO: 3) was genetically conjoined to the red fluorescent protein (RFP,
SEQ ID NO:
220).
[0129] FIG. 14C shows that Cholix domain I is sufficient for apical to
basal transcytosis
after intraluminal injection (ILI) into rat jejunum in vivo. FIG. 14C shows
that a Cholix
truncated at the termination of domain I (amino acid residue 265 of SEQ ID NO:
1, plus an N-
terminal methionine residue resulting in SEQ ID NO: 5) and that is genetically
fused to RFP
(e.g., thus having an amino acid sequence set forth in SEQ ID NO: 156) is
capable of efficient
apical-to-basal transcytosis, suggesting that Cholix domain I may be
sufficient for apical to basal
transcytosis after intraluminal injection (ILI) into rat jejunum in vivo.
Solid white arrows #1
indicate the apical epithelial surface, and white arrow #2 highlights the
basal surface. Cholix
domain I (SEQ ID NO: 5) was genetically conjoined to the red fluorescent
protein (RFP, SEQ ID
NO: 220).
[0130] FIG. 15 depicts apical-to-basal transport of human growth hormone
(HGH, SEQ ID
NO: 214) compared to chimeras of Cholix domain I and truncated elements of
this domain
(designated by amino acids) that were genetically conjoined to HGH. Western
blotting for HGH
qualitatively assessed the capacity of these proteins to undergo apical-to-
basal transport across
polarized monolayers of primary human small intestinal epithelial cells in
vitro after 2 h. The
amounts of apically-applied materials were equivalent on a molar basis for HGH
content and
basal collections were concentrated ¨10-fold prior to analysis.
[0131] FIG. 15A shows that background apical-to-basal transport of HGH
alone in this
model was minimal compared to that observed for the delivery construct
comprising the amino
acid sequence set forth in SEQ ID NO: 164, comprising a Cholix domain I (SEQ
ID NO: 5), a
spacer (SEQ ID NO: 210), and HGH (SEQ ID NO: 214). Cholix domain I (SEQ ID NO:
5)
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truncations at positions 134, 151, 187 or at 40-187 of SEQ ID NO: 5 were
incapable of
facilitating apical-to-basal transport of conjoined HGH.
[0132] FIG. 15B shows that truncations of Cholix domain I (SEQ ID NO: 5) at
positions
206, 245, or 251 demonstrated apical-to-basal transport of conjoined HGH.
While truncations as
positions 245 and 251 resulted in apical-to-basal transport comparable to that
of the construct
comprising the carrier with SEQ ID NO: 5, the chimera where Cholix domain I is
truncated at
position 206 showed a significant enhancement of apical-to-basal transport.
[0133] FIG. 16A shows the assessment of Cholix domain I truncation-human
growth
hormone (HGH) chimera transport across rat jejunum epithelia monolayers in
vivo 15 min after
intraluminal injection as demonstrated by immunofluorescence microscopy. FIG.
16A shows
that the Cholix-HGH construct with SEQ ID NO: 211 (comprises Cholix1-133+ N-
term.
methionine) did not enter epithelial cells, suggesting that the functional
peptide fragment having
an amino acid sequence set forth in SEQ ID NO: 148 may be required for
endocytosis.
[0134] FIG. 16B shows the assessment of Cholix domain I truncation-human
growth
hormone (HGH) chimera transport across rat jejunum epithelia monolayers in
vivo 15 min after
intraluminal injection as demonstrated by immunofluorescence microscopy. FIG.
16B shows
that the Cholix-HGH construct with SEQ ID NO: 212 (comprises Cholix1-15 + N-
term.
methionine) did enter epithelial cells (as opposed to protein with SEQ ID NO:
211) but remained
in apical and basal vesicular pools and did not enter the lamina propria, thus
enabling delivery
the interior of an epithelial cell (e.g., a compartment at the basal side of
the epithelial cell).
[0135] FIG. 16C shows the assessment of Cholix domain I truncation-human
growth
hormone (HGH) chimera transport across rat jejunum epithelia monolayers in
vivo 15 min after
intraluminal injection as demonstrated by immunofluorescence microscopy. FIG.
16C shows
that the Cholix-HGH construct with SEQ ID NO: 213 (comprises Cholix1-186 + N-
term.
methionine) entered epithelial cells, reached apical and basal compartments
and, significantly,
also a supra-nuclear region of the cell, yet still remained inside the
epithelial cell, suggesting that
the functional peptide fragment having an amino acid sequence set forth in SEQ
ID NO: 151
may allow access and delivery to supranuclear regions, yet does not allow
release of the
construct into a basolateral compartment (e.g., lamina propria).
[0136] FIG. 16D shows the assessment of Cholix domain I truncation-human
growth
hormone (HGH) chimera transport across rat jejunum epithelia monolayers in
vivo 15 min after
intraluminal injection as demonstrated by immunofluorescence microscopy. FIG.
16D shows
that the Cholix-HGH construct with SEQ ID NO: 218 (comprises Cholix39-186 + N-
term.
Methionine) entered epithelial cells but remained in the apical compartment
and did not appear
to reach the basal or supra-nuclear compartments.
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[0137] FIG. 16E shows the assessment of Cholix domain I truncation-human
growth
hormone (HGH) chimera transport across rat jejunum epithelia monolayers in
vivo 15 min after
intraluminal injection as demonstrated by immunofluorescence microscopy. FIG.
16E shows
that the Cholix-HGH construct with SEQ ID NO: 161 (comprises Cholix1-205 + N-
term.
methionine) completed the transcytosis process as indicated by delivery of the
chimera to cells
within the lamina propria, suggesting that the functional peptide fragment
having an amino acid
sequence set forth in SEQ ID NO: 152 (e.g., amino acid residues 187-206 of SEQ
ID NO: 5)
may allow release of the construct from the epithelium into a basolateral
compartment (e.g.,
lamina propria).
[0138] FIG. 16F shows the assessment of Cholix domain I truncation-human
growth
hormone (HGH) chimera transport across rat jejunum epithelia monolayers in
vivo 15 min after
intraluminal injection as demonstrated by immunofluorescence microscopy. FIG.
16F shows
that the Cholix-HGH construct with SEQ ID NO: 164 (comprises Cholix1-265 + N-
term.
Methionine, SEQ ID NO: 5) completed the transcytosis process as indicated by
delivery of the
chimera to cells within the lamina propria similar to the Cholix-HGH construct
with SEQ ID
NO: 164.
[0139] FIG. 17 shows that selected amino acid fragments of Cholix domain I
achieve apical
to basal transcytosis in vitro and in vivo. A polymer framework containing
peptide sequences of
amino acids from positions 1-39, 134-151, 151-178, and 178-206 of Cholix
domain I with SEQ
ID NO: 5 in various combinations were labeled with different forms of quantum
dots (e.g.,
cadmium sulfide, lead sulfide, etc.).
[0140] FIG. 17A shows transcytosis of various truncated Cholix (Chx)
constructs across
polarized intestinal epithelium in vitro after 2 h. The amount of transported
material is reported
as the florescence-fold increase relative to polymer-quantum dot preparation
lacking any Chx
peptides. (N=2; mean SE.).
[0141] FIG. 17B shows in vivo transcytosis at 15 min of the Cholix39-186 -
HGH construct
(SEQ ID NO: 218) (white arrow #1 highlights the apical surface, and white
arrow #2 highlights
the basal surface).
[0142] FIG. 17C shows in vivo transcytosis at 15 min of the (SEQ ID NO: 5)-
(4N)-RFP
construct labeled with quantum dots (white arrow #1 highlights the apical
surface, and white
arrow #2 highlights the basal surface).
[0143] FIG. 18 shows the amino acid sequence set forth in SEQ ID NO: 221 of
a Cholix
domain 1 (incl. and N-terminal methionine) having a spacer with an amino acid
sequence set
forth in SEQ ID NO: 210 attached to its C-terminus. This spacer can be used to
attach cargo
moieties (e.g., therapeutic agents) to the Cholix carrier for transport across
epithelial layers (e.g.,
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the gut epithelium). The highlighted amino acid fragments can provide certain
functionalities in
relation to transcytosis across epithelial layers. For example, the fragment
with the amino acid
residues of positions 134-151 of the sequence set forth in SEQ ID NO: 5 can
promote apical
entry of Cholix constructs into epithelial cells. The highlighted fragment
with amino acid
residues 151-187 (e.g., of SEQ ID NO: 5) can promote early endosomal sorting.
The highlighted
fragment with amino acid residues 187-206 (e.g., of SEQ ID NO: 5) can promote
complete
transcytosis of a Chx construct as described herein.
[0144] FIG. 19 shows potential glycosylation sites of the asparagine
residues located at
positions N98, N154, N165, and N224 of Cholix domain I having an amino acid
sequence set
forth in SEQ ID NO: 221.
[0145] FIG. 20 shows a general 3D structure of Cholix domain I (SEQ ID NO:
5) with
highlighted functional fragments.
[0146] FIG. 20A shows a general 3D structure of Cholix domain I (SEQ ID NO:
5) with the
highlighted functional fragment having an amino acid sequence of SEQ ID NO:
148 (residues
134-151 of SEQ ID NO: 5).
[0147] FIG. 20B shows a general 3D structure of Cholix domain I (SEQ ID NO:
5) with the
highlighted functional fragment having an amino acid sequence of SEQ ID NO:
149 (e.g.,
residues 151-187 of SEQ ID NO: 5).
[0148] FIG. 20C shows a general 3D structure of Cholix domain I (SEQ ID NO:
5) with the
highlighted functional fragment having an amino acid sequence of SEQ ID NO:
152 (e.g.,
residues 187-206 of SEQ ID NO: 5).
[0149] FIG. 21 illustrates a trafficking pathway analysis for the Cholix
derived delivery
construct having the amino acid sequence set forth in SEQ ID NO: 154 (the
delivery construct is
M+Cholix386-GGGGSGGGGSGGGGS (SEQ ID NO: 210)-IL-10, from N- to C-terminus).
The delivery construct comprising Cholix domain (SEQ ID NO: 5) and human
growth hormone
(HGH) as cargo could also be used to show similar results as shown for the
construct comprising
M+Cholix386.
[0150] FIG. 21A shows that M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery
construct
strongly co-localized with the EEA1 antigen in cellular locations consistent
with trafficking at
both the apical and basal domains of enterocytes, suggesting the presence of
the Cholix derived
delivery constructs in early endosome compartments (white arrow #1 highlights
the apical
surface, and white arrow #2 highlights the basal surface).
[0151] FIG. 21B show that the M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery
construct
(top right) strongly co-localizes with the Rab7 (top left) predominantly in
the apical
compartment of enterocytes, but with only limited co-localization in cells
within the lamina
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propria, suggesting the presence of the Cholix derived delivery constructs in
late endosome
compartments (bottom left shows white light image, and bottom right shows
merged staining
with DAPI).
[0152] FIG. 21C shows that LANIP1 was identified in large, specific
vesicles consistent
mature lysosomes that were devoid of M+Cholix386 -IL-10 (SEQ ID NO: 154)
delivery
constructs (white arrows). M+Cholix386 -IL-10 (SEQ ID NO: 154), however, also
co-localizes
with the LAMP1 antigen in cellular locations other than lysosome-like
structures, consistent with
vesicle trafficking at both the apical and basal domains of enterocytes,
suggesting the presence of
the Cholix derived delivery constructs in late endosomal compartments.
[0153] FIG. 21D shows that M+Cholix386 -IL-10 (SEQ ID NO: 154) chimera also
strongly
co-localized with clathrin-coated vesicles, particularly in areas adjacent to
the nucleus and in the
Rabl 1 predominantly in the basal compartment of enterocytes as well as in
selected cells within
the lamina propria.
[0154] FIG. 21E shows that M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery
construct co-
localizes with the endoplasmic reticulum as demonstrated by calnexin in a
pattern adjacent to the
nucleus in enterocytes and in a large fraction of cells with in the lamina
propria. The
M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery construct strongly co-localizes
with the
endoplasmatic reticulum Golgi intermediate compartment (ERGIC) and the LAMN1
antigen
appeared to re-distribute in response to carrier endocytosis and transcytosis,
as shown for 1
(FIG. 21F), 5 (FIG. 21G), 10 (FIG. 2111), and 15 minutes after injection (FIG.
211).
[0155] FIG. 21F shows that the M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery
construct
strongly co-localizes with the endoplasmatic reticulum Golgi intermediate
compartment
(ERGIC) and the LAMN1 antigen appeared to re-distribute in response to carrier
endocytosis
and transcytosis, as shown for 1 minute after injection (white arrow #1
highlights the apical
surface, and white arrow #2 highlights the basal surface).
[0156] FIG. 21G shows M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery
construct co-
localization with LAMN1 antigen 5 minutes after injection (white arrow #1
highlights the apical
surface, and white arrow #2 highlights the basal surface).
[0157] FIG. 2111 shows M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery
construct co-
localization with LAMN1 antigen 10 minutes after injection.
[0158] FIG. 211 shows M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery
construct co-
localization with LAMN1 antigen 15 minutes after injection.
[0159] FIG. 21J shows that M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery
construct does
not co-localize with the low levels of giantin present in enterocytes. Some
giantin co-localized
with the chimera in a subset of cells present in the lamina propria,
suggesting that the Cholix
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derived carrier does not locate with the Golgi compartment (white arrow #1
highlights the apical
surface, and white arrow #2 highlights the basal surface).
[0160] FIG. 21K shows that the 58K antigen localizes in enterocytes at a
site apical to the
nucleus and the M+Cholix386-IL-10 (SEQ ID NO: 154) delivery construct shows
some co-
localization with this antigen in a manner that suggests a brief movement
through this
compartment. No 58K antigen was observed in cells within the lamina propria
(white arrow #1
highlights the apical surface, and white arrow #2 highlights the basal
surface).
[0161] FIG. 21L shows that the M+Cholix386-IL-10 (SEQ ID NO: 154) delivery
construct
showed some level of co-localization with the TGN38 antigen (top right), which
showed a
cellular distribution that was restricted to the apical side of nuclei in
enterocytes and adjacent to
the nucleus in a few cells within the lamina propria (white light and merge
images shown bottom
left and bottom right, respectively).
[0162] FIG. 21M shows that the M+Cholix386-IL-10 (SEQ ID NO: 154) delivery
construct
(staining shown in green, top right) strongly co-localizes with Rabl 1 (top
left) predominantly in
the basal compartment of enterocytes and in selected cells within the lamina
propria (white light
and merge images shown bottom left and bottom right, respectively).
[0163] FIG. 22 illustrates a 1D SDS-PAGE showing that an efficient protocol
using nano-
sized magnetic beads (25 nm or 100 nm diameter) decorated with non-toxic
Cholix derived
carrier elements can be used for specific protein capture to analyze proteins
that interact with
Cholix or carriers derived therefrom.
[0164] FIG. 23 illustrates that, after multiple washings, the magnetic bead-
enriched vesicles
can be solubilized in lysis buffer and the protein components present can be
separated by 2-D
SDS-PAGE for analysis.
[0165] FIG. 24 shows that patterns of these proteins can be compared to the
total protein
content of the cells and that mass spectrometry can be used to identify
specific elements
associated with vesicular structures accessed by the Cholix derived delivery
constructs.
[0166] FIG. 25 shows a comparison of outcomes from repeats of the above
described
protocol used to identify a set of interaction candidates. The interacting
proteins can then
examined for their content in Caco-2 cells and in rat small intestine.
Interaction of Cholix with
the identified candidate proteins can be confirmed using Cholix carrier-coated
magnetic beads
and purified candidate protein.
[0167] FIG. 26 shows that incubation of the Cholix carrier (having the
amino acid set forth
in SEQ ID NO: 154)-coated beads with the pure proteins and subsequent Western
Blots or
ELISA can enable detection of Cholix-protein interaction. For example, this
figure shows
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interaction of Cholix carrier with heparan sulfate proteoglycan (HSPG),
Dickkopf-related protein
1 (DKK1), the chaperone glucose-regulated protein 75 (GRP75), and cytokeratin-
8 (K8 or CK8).
[0168] FIG. 27 shows microscopic co-localization of candidate proteins and
Cholix derived
delivery construct in rat jejunum. Co-localization of a delivery construct
comprising a Cholix
carrier protein coupled to IL-10 (SEQ ID NO: 154, M+Cholix386-GGGGSGGGGSGGGGS
(SEQ
ID NO: 210)-IL-10) with CK8 was shown in vivo.
[0169] FIG. 27A shows co-localization after rat jejunum was treated with a
luminal
application of M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery construct for 1
minute (white
arrow #1 highlights the apical surface, and white arrow #2 highlights the
basal surface).
[0170] FIG. 27B shows co-localization after rat jejunum was treated with a
luminal
application of M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery construct for 5
minute.
[0171] FIG. 27C shows co-localization after rat jejunum was treated with a
luminal
application of M+Cholix386 -IL-10 (SEQ ID NO: 154) delivery construct for 10
minutes. Thus,
co-localization in the supra-nuclear region was shown to increase over time.
[0172] FIG. 28 shows a comparison of results obtained in an IHC study with
the human
atlas to ensure that the receptor distribution is consistent between rat in
vivo studies and human
intestine. Here, two of the receptors identified by mass spectrometry and
verified in rat jejunum
are examined.
[0173] FIG. 28A shows that the intestinal localization of GRP75 is
consistent between rat
and human intestine.
[0174] FIG. 28B shows that the intestinal localization of HSPC is
consistent between rat
and human intestine.
[0175] FIG. 29 shows effects of HSPG knockout by CRISPR on transport
function of the
delivery construct (SEQ ID NO: 164) comprising Cholix domain I (SEQ ID NO: 5)
coupled to
HGH (SEQ ID NO: 214) via a polyglycine-serine spacer (SEQ ID NO: 210) and HGH
alone as
internal control of non-selective transport. Cells were seeded at 1.5x105
cells/mL in transwelIs.
On day 18, transepithelial/transendothelial electrical resistance (TEER) was
measured and PBS
containing 20 ug/mL of the delivery construct was added to the apical
chambers. After 3 h,
basolateral samples were collected and concentrated. The extent of protein
transport was
analyzed by Western blotting using anti-HGH antibody. The results shown in
FIG. 29
demonstrate that transcytosis and active, selective transport of Cholix
derived carrier proteins is
HSPG-dependent, as the Cholix carrier showed significantly less transcytosis
function in HSPC-
knock-down cells compared to normal, HSPG-positive Caco-2 cells.
[0176] FIG. 30 shows knockout effects of K8, HSPC, and GRP75 on the
transcytosis
function of Cholix domain I derived delivery constructs. Stable cell lines of
Caco-2 cells lacking
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the expression of specific candidate proteins were used as monolayers in vitro
to verify their
requirement for carrier transcytosis using active and selective endogenous
transport mechanisms.
The specific transport of the HGH-containing delivery construct vs non-
selective transport of
HGH alone was reduced in HSPG and GRP75 knockouts, but not the K8 knockout.
[0177] FIG. 30A shows knockout effects of K8.
[0178] FIG. 30B shows knockout effects of HSPC.
[0179] FIG. 30C shows knockout effects of GRP75.
[0180] FIG. 30D shows the control experiment.
[0181] FIG. 31 shows Biacore binding interactions used to examine the pH-
dependency of
Cholix carrier-GRP75 interactions. Cholix carrier proteins were attached to
magnetic beads
using the biotin-streptavidin bioconjugation and incubated with purified GRP75
protein in buffer
solutions with pH 5.5, 6.5, and 7.5, respectively. Highest binding affinity
was shown at pH 6.5.
[0182] FIG. 32 shows an exemplary surface model of Cholix domain I (SEQ ID
NO: 5) was
used to highlight selected areas of potential interest in this transcytosis
process due to their
projection from the protein surface. It is interesting to note that two amino
acids regions between
M1 and G4 are adjacent to surface exposed amino acids D151-A187 and A187-
1_,206.
Specifically,
128-126 (domain X1) and T171-1176 (domain X2) coordinate to form a pocket
surrounded by
several negative charges. Similarly, 1(187-E1203 (domain X3) coordinates with
132-E4 (domain X4)
to form a continuous ridge structure
[0183] FIG. 32A shows the proximity of domains X3 and X4.
[0184] FIG. 32B shows the proximity of domains X1 and X2, as well as X3 and
X4.
[0185] FIG. 32C shows the proximity of domains X1 and X2.
[0186] FIG. 32D shows the proximity of domains X1 and X2, as well as X3 and
X4.
DETAILED DESCRIPTION
Introduction
[0187] While various embodiments of the invention have been shown and
described herein,
it will be obvious to those skilled in the art that such embodiments are
provided by way of
example only. Numerous variations, changes, and substitutions may occur to
those skilled in the
art without departing from the invention. It should be understood that various
alternatives to the
embodiments of the invention described herein may be employed.
[0188] The present disclosure provides methods and compositions for
transport and/or
delivery of a cargo molecule to certain location(s) within a cell (e.g., a
supranuclear location) or
across a cell (e.g., epithelial cell), either in vitro or in vivo (e.g., in a
rodent or a human). Such
cargo can be directed to a set of location(s) by coupling it to a carrier
molecule. Such carrier
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molecule can interact with unique receptors both on the cell surface and
intracellularly for the
targeted delivery of the cargo. Various such carrier, cargos, and uses thereof
are described
herein.
[0189] Unless otherwise defined herein, scientific and technical terms used
in connection
with the present disclosure shall have the meanings that are commonly
understood by those of
ordinary skill in the art. Further, unless otherwise required by context,
singular terms shall
include pluralities and plural terms shall include the singular. Generally,
nomenclatures used in
connection with, and techniques of, cell and tissue culture, molecular
biology, immunology,
microbiology, genetics, protein and nucleic acid chemistry, and hybridization
described herein
are those commonly used and well known in the art. The methods and techniques
of the present
disclosure are generally performed according to conventional methods well
known in the art and
as described in various general and more specific references that are cited
and discussed
throughout the present specification unless otherwise indicated. See, e.g.,
Green and Sambrook,
Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory
Press, Cold
Spring Harbor, N.Y. (2012) and Ausubel et al., Current Protocols in Molecular
Biology, Greene
Publishing Associates (1992), each incorporated herein by reference. Enzymatic
reactions and
purification techniques are performed according to manufacturer's
specifications, as commonly
accomplished in the art or as described herein. The nomenclature used in
connection with, and
the laboratory procedures and techniques of, analytical chemistry, synthetic
organic chemistry,
and medicinal and pharmaceutical chemistry described herein are those commonly
used and well
known in the art. Standard techniques are used for chemical syntheses,
chemical analyses,
pharmaceutical preparation, formulation, and delivery, and treatment of
patients.
[0190] As described herein, an amino acid sequence can comprise one or more
modification
to the amino acid sequences at the N-terminus. An amino acid sequence as
disclosed herein can
comprise an "N-cap." Generally, an N-cap as disclosed herein can refer to a
modification of an
N-terminus of a peptide or polypeptide in a variety of ways, and particularly
can refer to (i) the
addition of one or more amino acid sequences or other moieties (e.g., affinity
handles, cell-
penetrating peptide sequences, etc.), and (ii) a modification of one or more
amino acid residues
within the first 1-10 N-terminal amino acids of a peptide or polypeptide,
wherein the amino acid
modification is relative to a reference sequence or a consensus sequence (see
e.g., comparison of
the first 4 N-terminal amino acid residues of polypeptide sequences set forth
in SEQ ID NO: 4
and SEQ ID NO: 5, or SEQ ID NO: 1 and SEQ ID NO: 2 as described herein). An N-
cap can
comprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 or 100 additional
amino acid residues that
are attached to (e.g., chemically coupled to) to the N-terminus of an amino
acid sequence, such
as a Cholix derived carrier molecule. An N-cap can further comprise one or
more variations in
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the amino acid sequence at the N-terminus. For example, a Cholix domain I
derived carrier can
comprise an N-cap. The N-cap can comprise substituting one or more N-terminal
amino acid
residues with other amino acid residues. An N-cap can further comprise an N-
terminal
methionine residue. One or more of these modifications can be a result of
producing the Cholix
domain I amino acid sequence in a bacterial production system (e.g., E. coli).
As an example,
Cholix domain I can comprise amino acid residues 1-265 of SEQ ID NO: 1 which
is set forth in
SEQ ID NO: 4. A bacterially expressed Cholix domain can comprise an amino acid
sequence set
forth in SEQ ID NO: 5, which as SEQ ID NO: 4 plus an N-terminal methionine
residues, which
can also be referred to herein as M+Cholix1-265 or M+Cholix265.
[0191] As described herein, the term "lacks a domain" or "lacking a domain"
generally
refers to not comprising a complete domain, but optionally comprising a
portion or fragment
thereof. For example, a carrier that is derived from a domain I of an exotoxin
but lacks a domain
II, a domain lb, and a domain III of said exotoxin generally refers to a
carrier that does not
comprise the full amino acid sequences of (e.g., 100% sequence identity to)
any one of the
domains II, lb, and III, but which can optionally comprise portions or
fragments thereof Thus, a
carrier derived from a Cholix domain and lacking a Cholix domain II as
described herein can
comprise Cholix domain I having an amino acid sequence set forth in SEQ ID NO:
4 or SEQ ID
NO: 5 and an additional 50-80 amino acid residues of Cholix domain II (e.g.,
amino acids 1-50
or 1-80 of SEQ ID NO: 126), and or an additional 50-80 amino acid residues of
Cholix domain
III (e.g., amino acids 1-50 or 1-80 of SEQ ID NO: 128).
[0192] As described herein, the terms "attached to", "coupled to", "linked
to", "conjugated
to" and "fused to" can be used interchangeably and generally mean that a first
molecule (e.g., a
polypeptide) is associated with a second molecule (e.g., a polypeptide, small
molecule, etc.). The
association can be via a chemical linkage, wherein the chemical linkage can be
covalently or
non-covalently. A covalent chemical linkage between a first polypeptide and a
second
polypeptide can be produced by synthetically coupling the first polypeptide to
the second
polypeptide, or it can be produced by recombinant fusion of the first
polypeptide to the second
polypeptide. Thus, a first (e.g., a first polypeptide) molecule can be
chemically (e.g.,
synthetically) or recombinantly coupled to a second molecule (e.g., a second
polypeptide).
[0193] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to
refer to a polymer of amino acid residues. In addition, the terms "toxin",
"carrier", "delivery
construct", "chimeric construct", "protein", and "polypeptide" can be used
interchangeably and
generally refer to a molecule that can be coupled to a heterologous cargo.
Generally, "delivery
constructs" and "chimeric constructs" are "peptides", "polypeptides", or
"proteins", are
described herein as chains of amino acids whose alpha carbons are linked
through peptide bonds.
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The terminal amino acid at one end of the chain (amino terminal) therefore has
a free amino
group, while the terminal amino acid at the other end of the chain (carboxy
terminal) has a free
carboxyl group. As used herein, the term "amino terminus" (abbreviated N-
terminus) refers to
the free a-amino group on an amino acid at the amino terminal of a peptide or
to the a-amino
group (imino group when participating in a peptide bond) of an amino acid at
any other location
within the peptide. Similarly, the term "carboxy terminus" refers to the free
carboxyl group on
the carboxy terminus of a peptide or the carboxyl group of an amino acid at
any other location
within the peptide. Peptides also include essentially any polyamino acid
including, but not
limited to, peptide mimetics such as amino acids joined by an ether bond as
opposed to an amide
bond. Generally, peptides, polypeptide, and proteins as described herein can
be recombinantly
produced or chemically synthesized (e.g., using solid-phase synthesis), or a
combination thereof
[0194] As disclosed herein, the term "delivery" generally refers to the
presence of a
molecule (e.g., a heterologous cargo) at a location (e.g., an intracellular
compartment or a
supranuclear region) for a certain period of time. The term "delivery" can
refer to the presence of
a molecule (e.g., a heterologous cargo) at a location (e.g., an intracellular
compartment or a
supranuclear region) for a time that is sufficient to elicit a certain
biological effect, such as an
interaction (e.g., binding) with a protein (e.g., an enzyme or a receptor) at
that location. The
delivery of a molecule (e.g., a heterologous cargo) to a location (e.g., an
intracellular
compartment or a supranuclear region) can refer to the retention of the
molecule at that location.
Retention of a molecule at a certain intracellular or extracellular region or
compartment can be
for a certain amount of time, e.g., at least 2 minutes, at least 5 minutes, at
least 10 minutes, at
least 15 minutes, at 30 minutes, or at least 60 minutes. Retention of a
molecule can depend on
various factors such as the location where the molecule is retained and/or the
types of molecular
interactions that occur between the molecule (e.g., a carrier, a delivery
construct, and/or a
heterologous cargo). For example, delivery of a heterologous cargo to a
basolateral compartment
via transcytosis across a polarized epithelial cell can comprise retaining the
heterologous cargo at
the basolateral location for a time sufficient to elicit a certain effect,
such as a therapeutic effect
in case of a therapeutic and/or biologically active cargo.
[0195] Polypeptides of the disclosure include polypeptides that have been
modified in any
way and for any reason, for example, to: (1) reduce susceptibility to
proteolysis, (2) reduce
susceptibility to oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter
binding affinities, and (5) confer or modify other physicochemical or
functional properties. For
example, single or multiple amino acid substitutions (e.g., conservative amino
acid substitutions)
can be made in the naturally occurring sequence (e.g., in the portion of the
polypeptide outside
the domain(s) forming intermolecular contacts). A "conservative amino acid
substitution" refers
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to the substitution in a polypeptide of an amino acid with a functionally
similar amino acid. The
following six groups each contain amino acids that are conservative
substitutions for one
another:
1) Alanine (A), Serine (S), and Threonine (T)
2) Aspartic acid (D) and Glutamic acid (E)
3) Asparagine (N) and Glutamine (Q)
4) Arginine (R) and Lysine (K)
5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)
6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)
[0196] A "non-conservative amino acid substitution" refers to the
substitution of a member
of one of these classes for a member from another class. In making such
changes, according to
various embodiments, the hydropathic index of amino acids can be considered.
Each amino acid
has been assigned a hydropathic index on the basis of its hydrophobicity and
charge
characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine
(-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and
arginine (-4.5). In other
embodiments, the carrier of a delivery constructs is a chimeric carrier
comprising a peptide,
polypeptide, small molecule, aptamer, fragments thereof, or any combination
thereof.
[0197] The importance of the hydropathic amino acid index in conferring
interactive
biological function on a protein is understood in the art (see, for example,
Kyte et al., 1982, J.
Mol. Biol. 157:105-131). It is known that certain amino acids can be
substituted for other amino
acids having a similar hydropathic index or score and still retain a similar
biological activity. In
making changes based upon the hydropathic index, in various embodiments, the
substitution of
amino acids whose hydropathic indices are within + 2 is included. In various
embodiments, those
that are within + 1 are included, and in various embodiments, those within +
0.5 are included.
[0198] It is also understood in the art that the substitution of like amino
acids can be made
effectively on the basis of hydrophilicity, particularly where the
biologically functional protein
or peptide thereby created is intended for use in immunological applications,
as disclosed herein.
In various embodiments, the greatest local average hydrophilicity of a
protein, as governed by
the hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and
antigenicity, i.e., with a biological property of the protein.
[0199] The following hydrophilicity values have been assigned to these
amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +/- .1); glutamate
(+3.0 +/- .1); serine
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(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5 +/- .1);
alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-
1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-
3.4). In making changes
based upon similar hydrophilicity values, in various embodiments, the
substitution of amino
acids whose hydrophilicity values are within + 2 is included, in various
embodiments, those that
are within + 1 are included, and in various embodiments, those within + 0.5
are included.
[0200] Exemplary amino acid substitutions are set forth in TABLE 1.
TABLE 1 ¨ Exemplary Amino Acid Substitutions
Original Residues Exemplary Substitutions Preferred Substitutions
Ala Val, Leu, Ile Val
Arg Lys, Gln, Asn Lys
Asn Gln Gln
Asp Glu Glu
Cys Ser, Ala Ser
Gln Asn Asn
Glu Asp Asp
Gly Pro, Ala Ala
His Asn, Gln, Lys, Arg Arg
Leu, Val, Met Ala, Phe,Ile Leu
Norleucine
Norleucine, Ile, Val, Met,Leu Ile
Ala, Phe
Arg, 1,4-diamino-butyric
Lys Arg
acid, Gln, Asn
Met Leu, Phe, Ile Leu
Phe Leu, Val, Ile, Ala, Tyr Leu
Pro Ala Gly
Ser Thr, Ala, Cys Thr
Thr Ser Ser
Trp Tyr, Phe Tyr
Tyr Trp, Phe, Thr, Ser Phe
Ile, Met Leu, Phe, Ala,Val Leu
Norleucine
[0201] A skilled artisan will be able to determine suitable variants of
polypeptides as set
forth herein using well-known techniques. One skilled in the art can identify
suitable areas of the
molecule that can be changed without destroying activity by targeting regions
not believed to be
important for activity. The skilled artisan can identify residues and portions
of the molecules that
are conserved among similar polypeptides. Areas of these materials that can be
important for
biological activity or for structure could be subjected to conservative amino
acid substitutions
without destroying the biological activity or without adversely affecting the
polypeptide
structure.
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[0202] Additionally, one skilled in the art can review structure-function
studies identifying
residues in similar polypeptides that are important for activity or structure.
In view of such a
comparison, the skilled artisan can predict the importance of amino acid
residues in a
polypeptide that correspond to amino acid residues important for activity or
structure in similar
polypeptides. One skilled in the art can opt for chemically similar amino acid
substitutions for
such predicted important amino acid residues.
[0203] One skilled in the art can also analyze the three-dimensional
structure and amino
acid sequence in relation to that structure in similar polypeptides. In view
of such information,
one skilled in the art can predict the alignment of amino acid residues of a
polypeptide with
respect to its three-dimensional structure. One skilled in the art can choose
to not make radical
changes to amino acid residues predicted to be on the surface of the
polypeptide, since such
residues can be involved in important interactions with other molecules.
Moreover, one skilled in
the art can generate test variants containing a single amino acid substitution
at each desired
amino acid residue. The variants can then be screened using activity assays
known to those
skilled in the art. Such variants could be used to gather information about
suitable variants. For
example, if one discovered that a change to a particular amino acid residue
resulted in destroyed,
undesirably reduced, or unsuitable activity, variants with such a change in
the amino acid
sequence can be avoided. In other words, based on information gathered from
such routine
experiments, one skilled in the art can readily determine the amino acids
where further
substitutions should be avoided either alone or in combination with other
mutations.
[0204] The term "polypeptide fragment" and "truncated polypeptide" as used
herein refers
to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion
as compared to a
corresponding full-length protein. In various embodiments, fragments can be,
e.g., at least 5, at
least 10, at least 25, at least 50, at least 100, at least 150, at least 200,
at least 250, at least 300, at
least 350, at least 400, at least 450, at least 500, at least 600, at least
700, at least 800, at least 900
or at least 1000 amino acids in length. In various embodiments, fragments can
also be, e.g., at
most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at
most 450, at most
400, at most 350, at most 300, at most 250, at most 200, at most 150, at most
100, at most 50, at
most 25, at most 10, or at most 5 amino acids in length. A fragment can
further comprise, at
either or both of its ends, one or more additional amino acids, for example, a
sequence of amino
acids from a different naturally-occurring protein (e.g., an Fc or leucine
zipper domain) or an
artificial amino acid sequence (e.g., an artificial spacer sequence).
[0205] The terms "polypeptide variant" and "polypeptide mutant" as used
herein refer to a
polypeptide that comprises an amino acid sequence wherein one or more amino
acid residues are
inserted into, deleted from and/or substituted into the amino acid sequence
relative to another
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polypeptide sequence. The number of amino acid residues to be inserted,
deleted, or substituted
can be, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at
least 10, at least 25, at least 50,
at least 75, at least 100, at least 125, at least 150, at least 175, at least
200, at least 225, at least
250, at least 275, at least 300, at least 350, at least 400, at least 450 or
at least 500 amino acids in
length. Variants of the present disclosure include fusion proteins.
[0206] A "derivative" of a polypeptide is a polypeptide that has been
chemically modified,
e.g., conjugation to another chemical moiety such as, for example,
polyethylene glycol, albumin
(e.g., human serum albumin), phosphorylation, and glycosylation.
[0207] The term "% sequence identity" is used interchangeably herein with
the term "%
identity" and refers to the level of amino acid sequence identity between two
or more peptide
sequences or the level of nucleotide sequence identity between two or more
nucleotide
sequences, when aligned using a sequence alignment program. For example, as
used herein, 80%
identity means the same thing as 80% sequence identity determined by a defined
algorithm, and
means that a given sequence is at least 80% identical to another length of
another sequence. In
various embodiments, the % identity is selected from, e.g., at least 60%, at
least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or
at least 99% or more
sequence identity to a given sequence. In various embodiments, the % identity
is in the range of,
e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%,
about 85% to
about 90%, about 90% to about 95%, or about 95% to about 99%.
[0208] The term "% sequence homology" is used interchangeably herein with
the term "%
homology" and refers to the level of amino acid sequence homology between two
or more
peptide sequences or the level of nucleotide sequence homology between two or
more nucleotide
sequences, when aligned using a sequence alignment program. For example, as
used herein, 80%
homology means the same thing as 80% sequence homology determined by a defined
algorithm,
and accordingly a homologue of a given sequence has greater than 80% sequence
homology over
a length of the given sequence. In various embodiments, the % homology is
selected from, e.g.,
at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at
least 95%, or at least 99% or more sequence homology to a given sequence. In
various
embodiments, the % homology is in the range of, e.g., about 60% to about 70%,
about 70% to
about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about
95%, or
about 95% to about 99%.
[0209] Exemplary computer programs which can be used to determine identity
between two
sequences include, but are not limited to, the suite of BLAST programs, e.g.,
BLASTN,
BLASTX, TBLASTX, BLASTP, and TBLASTN, publicly available on the Internet at
the NCBI
website. See also Altschul et al., 1990, J. Mol. Biol. 215:403-10 (with
special reference to the
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published default setting, i.e., parameters w=4, t=17) and Altschul et al.,
1997, Nucleic Acids
Res., 25:3389-3402. Sequence searches are typically carried out using the
BLASTP program
when evaluating a given amino acid sequence relative to amino acid sequences
in the GenBank
Protein Sequences and other public databases. The BLASTX program is preferred
for searching
nucleic acid sequences that have been translated in all reading frames against
amino acid
sequences in the GenBank Protein Sequences and other public databases. Both
BLASTP and
BLASTX are run using default parameters of an open gap penalty of 11.0, and an
extended gap
penalty of 1.0, and utilize the BLOSUM-62 matrix. See id.
[0210] In addition to calculating percent sequence identity, the BLAST
algorithm also
performs a statistical analysis of the similarity between two sequences (see,
e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of
similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)), which
provides an
indication of the probability by which a match between two nucleotide or amino
acid sequences
would occur by chance. For example, a nucleic acid is considered similar to a
reference sequence
if the smallest sum probability in a comparison of the test nucleic acid to
the reference nucleic
acid is, e.g., at most 0.1, at most 0.01, or at most 0.001.
[0211] "Polynucleotide" refers to a polymer composed of nucleotide units.
Polynucleotides
include naturally occurring nucleic acids, such as deoxyribonucleic acid
("DNA") and
ribonucleic acid ("RNA") as well as nucleic acid analogs. Nucleic acid analogs
include those
which include non-naturally occurring bases, nucleotides that engage in
linkages with other
nucleotides other than the naturally occurring phosphodiester bond or which
include bases
attached through linkages other than phosphodiester bonds. Thus, nucleotide
analogs include, for
example and without limitation, phosphorothioates, phosphorodithioates,
phosphorotriesters,
phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl
phosphonates, 2-0-
methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such
polynucleotides can be
synthesized, for example, using an automated DNA synthesizer. The term
"nucleic acid"
typically refers to large polynucleotides. The term "oligonucleotide"
typically refers to short
polynucleotides, generally no greater than about 50 nucleotides. It will be
understood that when
a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C),
this also includes an
RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."
[0212] Conventional notation is used herein to describe polynucleotide
sequences: the left-
hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-
hand direction of a
double-stranded polynucleotide sequence is referred to as the 5'-direction.
The direction of 5' to
3' addition of nucleotides to nascent RNA transcripts is referred to as the
transcription direction.
The DNA strand having the same sequence as an mRNA is referred to as the
"coding strand";
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sequences on the DNA strand having the same sequence as an mRNA transcribed
from that
DNA and which are located 5' to the 5'-end of the RNA transcript are referred
to as "upstream
sequences"; sequences on the DNA strand having the same sequence as the RNA
and which are
3' to the 3' end of the coding RNA transcript are referred to as "downstream
sequences."
[0213] "Complementary" refers to the topological compatibility or matching
together of
interacting surfaces of two polynucleotides. Thus, the two molecules can be
described as
complementary, and furthermore, the contact surface characteristics are
complementary to each
other. A first polynucleotide is complementary to a second polynucleotide if
the nucleotide
sequence of the first polynucleotide is substantially identical to the
nucleotide sequence of the
polynucleotide binding partner of the second polynucleotide, or if the first
polynucleotide can
hybridize to the second polynucleotide under stringent hybridization
conditions.
[0214] "Hybridizing specifically to" or "specific hybridization" or
"selectively hybridize
to", refers to the binding, duplexing, or hybridizing of a nucleic acid
molecule preferentially to a
particular nucleotide sequence under stringent conditions when that sequence
is present in a
complex mixture (e.g., total cellular) DNA or RNA. The term "stringent
conditions" refers to
conditions under which a probe will hybridize preferentially to its target
subsequence, and to a
lesser extent to, or not at all to, other sequences. "Stringent hybridization"
and "stringent
hybridization wash conditions" in the context of nucleic acid hybridization
experiments such as
Southern and northern hybridizations are sequence-dependent, and are different
under different
environmental parameters. An extensive guide to the hybridization of nucleic
acids can be found
in Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology--
Hybridization
with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of
hybridization and the
strategy of nucleic acid probe assays", Elsevier, N.Y.; Sambrook et al., 2001,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3<sup>rd</sup> ed., NY;
and Ausubel
et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene
Publishing
Associates and Wiley Interscience, NY.
[0215] Generally, highly stringent hybridization and wash conditions are
selected to be
about 5 C lower than the thermal melting point (Tm) for the specific sequence
at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50%
of the target sequence hybridizes to a perfectly matched probe. Very stringent
conditions are
selected to be equal to the Tm for a particular probe. An example of stringent
hybridization
conditions for hybridization of complementary nucleic acids which have more
than about 100
complementary residues on a filter in a Southern or northern blot is 50%
formalin with 1 mg of
heparin at 42 C, with the hybridization being carried out overnight. An
example of highly
stringent wash conditions is 0.15 M NaCl at 72 C for about 15 minutes. An
example of stringent
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wash conditions is a 0.2 x SSC wash at 65 C for 15 minutes. See Sambrook et
al. for a
description of SSC buffer. A high stringency wash can be preceded by a low
stringency wash to
remove background probe signal. An exemplary medium stringency wash for a
duplex of, e.g.,
more than about 100 nucleotides, is 1 x SSC at 45 C for 15 minutes. An
exemplary low
stringency wash for a duplex of, e.g., more than about 100 nucleotides, is 4-6
x SSC at 40 C for
15 minutes. In general, a signal to noise ratio of 2 x (or higher) than that
observed for an
unrelated probe in the particular hybridization assay indicates detection of a
specific
hybridization.
[0216] "Primer" refers to a polynucleotide that is capable of specifically
hybridizing to a
designated polynucleotide template and providing a point of initiation for
synthesis of a
complementary polynucleotide. Such synthesis occurs when the polynucleotide
primer is placed
under conditions in which synthesis is induced, i.e., in the presence of
nucleotides, a
complementary polynucleotide template, and an agent for polymerization such as
DNA
polymerase. A primer is typically single-stranded, but can be double-stranded.
Primers are
typically deoxyribonucleic acids, but a wide variety of synthetic and
naturally occurring primers
are useful for many applications. A primer is complementary to the template to
which it is
designed to hybridize to serve as a site for the initiation of synthesis, but
need not reflect the
exact sequence of the template. In such a case, specific hybridization of the
primer to the
template depends on the stringency of the hybridization conditions. Primers
can be labeled with,
e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable
moieties.
[0217] "Probe," when used in reference to a polynucleotide, refers to a
polynucleotide that
is capable of specifically hybridizing to a designated sequence of another
polynucleotide. A
probe specifically hybridizes to a target complementary polynucleotide, but
need not reflect the
exact complementary sequence of the template. In such a case, specific
hybridization of the
probe to the target depends on the stringency of the hybridization conditions.
Probes can be
labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used
as detectable
moieties. In instances where a probe provides a point of initiation for
synthesis of a
complementary polynucleotide, a probe can also be a primer.
[0218] A "vector" is a polynucleotide that can be used to introduce other
nucleic acids
linked to it into a cell. One type of vector is a "plasmid," which refers to a
linear or circular
double stranded DNA molecule into which additional nucleic acid segments can
be ligated.
Another type of vector is a viral vector (e.g., replication defective
retroviruses, adenoviruses and
adeno-associated viruses), wherein additional DNA segments can be introduced
into the viral
genome. Certain vectors are capable of autonomous replication in a host cell
into which they are
introduced (e.g., bacterial vectors comprising a bacterial origin of
replication and episomal
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mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into
the genome of a host cell upon introduction into the host cell, and thereby
are replicated along
with the host genome. An "expression vector" is a type of vector that can
direct the expression of
a chosen polynucleotide.
[0219] A "regulatory sequence" is a nucleic acid that affects the
expression (e.g., the level,
timing, or location of expression) of a nucleic acid to which it is operably
linked. The regulatory
sequence can, for example, exert its effects directly on the regulated nucleic
acid, or through the
action of one or more other molecules (e.g., polypeptides that bind to the
regulatory sequence
and/or the nucleic acid). Examples of regulatory sequences include promoters,
enhancers and
other expression control elements (e.g., polyadenylation signals). Further
examples of regulatory
sequences are described in, for example, Goeddel, 1990, Gene Expression
Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif and Baron et al., 1995,
Nucleic Acids
Res. 23:3605-06. A nucleotide sequence is "operably linked" to a regulatory
sequence if the
regulatory sequence affects the expression (e.g., the level, timing, or
location of expression) of
the nucleotide sequence.
[0220] Generally, a cell of the present disclosure can be a eukaryotic cell
or a prokaryotic
cell. A cell can be an epithelial cell. An epithelial cell can be a polarized
epithelial cell (e.g., a
Caco-2 cell or a Chinese Hamster Ovary (CHO) cell). A cell can be an animal
cell or a plant cell.
An animal cell can include a cell from a marine invertebrate, fish, insects,
amphibian, reptile, or
mammal. A mammalian cell can be obtained from a primate, ape, equine, bovine,
porcine,
canine, feline, or rodent. A mammal can be a primate, ape, dog, cat, rabbit,
ferret, or the like. A
rodent can be a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea
pig. A bird cell can be
from a canary, parakeet or parrots. A reptile cell can be from a turtles,
lizard or snake. A fish cell
can be from a tropical fish. For example, the fish cell can be from a
zebrafish (e.g., Danino
rerio). A worm cell can be from a nematode (e.g., C. elegans). An amphibian
cell can be from a
frog. An arthropod cell can be from a tarantula or hermit crab.
[0221] A mammalian cell can also include cells obtained from a primate
(e.g., a human or a
non-human primate). A mammalian cell can include a blood cell, a stem cell, an
epithelial cell,
connective tissue cell, hormone secreting cell, a nerve cell, a skeletal
muscle cell, or an immune
system cell. In preferred embodiments, the methods and compositions of the
present disclosure
are used in combination with one or more mammalian blood cells.
[0222] A "host cell" is a cell that can be used to express a polynucleotide
of the disclosure.
A host cell can be a prokaryote, for example, E. coli, or it can be a
eukaryote, for example, a
single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a
tobacco or tomato plant
cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a
rat cell, a mouse cell, or
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an insect cell) or a hybridoma. Typically, a host cell is a cultured cell that
can be transformed or
transfected with a polypeptide-encoding series of nucleic acids, which can
then be expressed in
the host cell. The phrase "recombinant host cell" can be used to denote a host
cell that has been
transformed or transfected with a polypeptide-encoding series of nucleic acids
to be expressed. A
host cell also can be a cell that comprises series of nucleic acids but does
not express these at a
desired level unless a regulatory sequence is introduced into the host cell
such that it becomes
operably linked with the nucleic acid. It is understood that the term host
cell refers not only to
the particular subject cell but to the progeny or potential progeny of such a
cell. Because certain
modifications can occur in succeeding generations due to, e.g., mutation or
environmental
influence, such progeny may not, in fact, be identical to the parent cell, but
are still included
within the scope of the term as used herein.
[0223] The term "isolated molecule" (where the molecule is, for example, a
polypeptide
such as a carrier or a delivery construct, or a polynucleotide) is a molecule
that by virtue of its
origin or source of derivation (1) is not associated with naturally associated
components that
accompany it in its native state, (2) is substantially free of other molecules
from the same species
(3) is expressed by a cell from a different species, or (4) does not occur in
nature. Thus, a
molecule that is chemically synthesized, or expressed in a cellular system
different from the cell
from which it naturally originates, will be "isolated" from its naturally
associated components. A
molecule also can be rendered substantially free of naturally associated
components by isolation,
using purification techniques well known in the art. Molecule purity or
homogeneity can be
assayed by a number of means well known in the art. For example, the purity of
a polypeptide
sample can be assayed using polyacrylamide gel electrophoresis and staining of
the gel to
visualize the polypeptide using techniques well known in the art. For certain
purposes, higher
resolution separation techniques can be provided by using HPLC or other means
well known in
the art for purification.
[0224] As disclosed herein, the terms "complete transcytosis", "efficient
transcytosis", or
"transcytosis", or "transport" can be used interchangeably and can refer to
the transport of toxin-
derived delivery constructs across epithelial layers such as the gut
epithelium. These terms can
refer to a complete transport of these construct as determined in the
respective experiment using
various techniques to assess transcytosis efficiency, such as fluorescence
microscopy.
[0225] As used herein, the terms "comprising" and "having" can be used
interchangeably.
For example, the terms "a polypeptide comprising an amino acid sequence of SEQ
ID NO: 1"
and "a polypeptide having an amino acid sequence of SEQ ID NO: 1" can be used
interchangeably.
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[0226] A protein or polypeptide is "substantially pure," "substantially
homogeneous," or
"substantially purified" when at least about 60% to 75% of a sample exhibits a
single species of
polypeptide. The polypeptide or protein can be monomeric or multimeric. A
substantially pure
polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90%
W/W of a
protein sample, more usually about 95%, and e.g., will be over 99% pure.
Protein purity or
homogeneity can be indicated by a number of means well known in the art, such
as
polyacrylamide gel electrophoresis of a protein sample, followed by
visualizing a single
polypeptide band upon staining the gel with a stain well known in the art. For
certain purposes,
higher resolution separation techniques can be provided by using HPLC or other
means well
known in the art for purification.
[0227] As disclosed herein, a "spacer" refers to a molecule that joins two
other molecules,
either covalently, or through ionic, van der Waal s or hydrogen bonds, e.g., a
nucleic acid
molecule that hybridizes to one complementary sequence at the 5' end and to
another
complementary sequence at the 3' end, thus joining two non-complementary
sequences. A
"cleavable spacer" refers to a spacer that can be degraded or otherwise
severed to separate the
two components connected by the cleavable spacer. Cleavable spacers are
generally cleaved by
enzymes, typically peptidases, proteases, nucleases, lipases, and the like.
Cleavable spacers can
also be cleaved by environmental cues, such as, for example, specific
enzymatic activities,
changes in temperature, pH, salt concentration, etc. when there is such a
change in environment
following transcytosis of the delivery constructs across a polarized
epithelial membrane. Thus, a
heterologous cargo (e.g., a biologically active cargo) can be released from
the carrier in a pH-
dependent and/or enzyme-dependent manner.
[0228] "Pharmaceutical composition" refers to a composition suitable for
pharmaceutical
use in an animal. A pharmaceutical composition comprises a pharmacologically
effective
amount of an active agent and a pharmaceutically acceptable carrier.
"Pharmacologically
effective amount" refers to that amount of an agent effective to produce the
intended
pharmacological result.
[0229] "Pharmaceutically acceptable carrier" refers to any of the standard
pharmaceutical
carriers, vehicles, buffers, and excipients, such as a phosphate buffered
saline solution, 5%
aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil
emulsion, and
various types of wetting agents and/or adjuvants. Suitable pharmaceutical
carriers and
formulations are described in Remington's Pharmaceutical Sciences, 21st Ed.
2005, Mack
Publishing Co, Easton. A "pharmaceutically acceptable salt" is a salt that can
be formulated into
a compound for pharmaceutical use including, e.g., metal salts (sodium,
potassium, magnesium,
calcium, etc.) and salts of ammonia or organic amines.
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[0230] The terms "treat", "treating" and "treatment" refer to a method of
alleviating or
abrogating a biological disorder and/or at least one of its attendant
symptoms. As used herein, to
"alleviate" a disease, disorder or condition means reducing the severity
and/or occurrence
frequency of the symptoms of the disease, disorder, or condition. Further,
references herein to
"treatment" include references to curative, palliative and prophylactic
treatment.
[0231] As used herein, the term "subject," generally refers to a human or
to another animal.
A subject can be of any age, for example, a subject can be an infant, a
toddler, a child, a pre-
adolescent, an adolescent, an adult, or an elderly individual.
[0232] Ranges can be expressed herein as from "about" one particular value,
and/or to
"about" another particular value. When such a range is expressed, another
embodiment includes
from the one particular value and/or to the other particular value. Similarly,
when values are
expressed as approximations, by use of the antecedent "about," it will be
understood that the
particular value forms another embodiment. It will be further understood that
the endpoints of
each of the ranges are in relation to the other endpoint, and independently of
the other endpoint.
The term "about" as used herein refers to a range that is 15% plus or minus
from a stated
numerical value within the context of the particular usage. For example, about
10 can include a
range from 8.5 to 11.5.
Carriers
[0233] Contemplated herein are various carriers that can be used to deliver
a cargo to a
location within a cell (e.g., epithelial cell) or across a cell (e.g.,
epithelial cell). Such carriers can
be a small molecule, a polypeptide, an aptamer, an antibody, a nucleic acid a
fragment of any of
the above, or a combination of any of the above.
[0234] Examples of a polypeptide contemplated herein include any
polypeptide that is
derived from a domain I of an exotoxin and lacking a domain II, a domain lb
and a domain III of
the exotoxin. Such domain I's include but are not limited to amino acid
sequences set forth in
SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 137. Polypeptides that are derived
from any of
the above sequences include those that have a high sequence homology to the
above sequences
(e.g., greater than 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity as
defined in more detail
herein). Polypeptides that are derived from any of the above sequences include
those that are
fragments of the above which function to deliver a cargo to a defined location
within a cell or
across a cell (e.g., epithelial cell).
[0235] Examples of small molecules contemplated herein include those that
are rationally
designed to interact with one or more of the following receptors ribophilin 1,
5EC24, CK-8,
TMEM132, GRP75, ERGIC-53, and/or perlecan and/or to have a similar or the same
3D
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structure of a domain I of an exotoxin (e.g., Cholix or PE), or a functional
fragment of a domain
I of an exotoxin.
[0236] Examples of antibodies, or functional binding fragments thereof,
that are
contemplated herein include those that are rationally designed to interact
with one or more of the
following receptors ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53,
and/or perlecan
and/or to have a similar or the same 3D structure of a domain I of an exotoxin
(e.g., Cholix or
PE), or a functional fragment of a domain I of an exotoxin.
[0237] Examples of nucleic acids that are contemplated herein include those
that are
rationally designed to interact with one or more of the following receptors
ribophilin 1, SEC24,
CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan and/or to have a similar or
the same 3D
structure of a domain I of an exotoxin (e.g., Cholix or PE), or a functional
fragment of a domain
I of an exotoxin. The nucleic acid can be a mRNA, a siRNA, shRNA, or a cDNA.
[0238] The methods and compositions of the present disclosure are based on
the inventors'
surprising finding that a carrier capable of interacting with one or more
endogenous receptors
(e.g., ribophilin 1, SEC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan)
can provide
rapid and efficient delivery of cargo into and/or across a cell such an
epithelial cell.
[0239] The methods and compositions described herein allow rapid and
efficient transport
and/or delivery of cargo molecules across epithelial cells and/or to the
interior (e.g., to the
intracellular vesicle or compartment or the cytosol) of epithelial cells
(e.g., polarized gut
epithelial cells). The present disclosure provides constructs (e.g., isolated
delivery constructs)
that can comprise a carrier coupled to a heterologous cargo. A carrier as
disclosed herein can
vary in molecular size and composition as well as other physicochemical
parameters such as
isoelectric point, overall molecular net charge, etc. Generally, and as
further described herein, a
carrier can be a small molecule, a polypeptide, an aptamer, a nucleic acid, a
fragment and/or any
combination thereof.
[0240] A carrier can be derived from an exotoxin, e.g., any exotoxin
described herein. For
example, a carrier can be a non-naturally occurring form of Cholix exotoxin
(Cholix) or
Pseudomonas exotoxin A (PE) comprising only a domain I (i.e., lacking a domain
II (sometimes
referred to as translocation domain), a domain lb and a domain III (sometimes
referred to as
cytotoxic domain)) and can be capable of transporting and/or delivering a
cargo (e.g., a
heterologous cargo such as biological, therapeutic, or diagnostic molecules)
across intact
epithelial cells (e.g., polarized gut epithelial cells) and epithelial cell
barriers (e.g., Caco-2 cell
monolayers or the gut epithelium of a subject) via transcytosis and/or to the
interior of an
epithelial cell (e.g., via apical endocytosis and subsequent endosomal sorting
and trafficking).
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[0241] The present disclosure provides methods and compositions comprising
a carrier,
wherein the carrier can be coupled to a cargo, and as such, can deliver the
cargo into or across
epithelial cells. The carrier can be a polypeptide, wherein the polypeptide
can be derived from an
exotoxin. The exotoxin can be Cholix or PE, or any combination thereof (e.g.,
a carrier
comprising one or more domains Cholix and PE, or truncated versions thereof).
Thus, a carrier
as described herein can comprise elements or portions derived from both Cholix
and PE, which
can be referred to a chimeric carrier. As further described herein, it was
surprisingly found that a
Cholix domain I or a PE domain I (e.g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
137), or a
combination thereof, can be sufficient for rapid and efficient transport and
delivery of cargo
across an epithelial cell. Such transport and delivery may even be superior to
the transcytosis
function of the respective full-length Cholix or PE exotoxins (e.g., SEQ ID
NO: 1, SEQ ID NO:
2 or SEQ ID NO: 135, respectively). The exotoxin-derived carrier polypeptides
described herein
can utilize endogenous trafficking pathways, including endogenous receptors
and receptor
complexes, to achieve apical-to-basal transcytosis and/or uptake into the
interior of a cell, such
as an epithelial cell (e.g., enterocytes). The delivery carriers of the
present disclosure can access
a basolateral compartment (e.g., lamina propria) and/or the interior of an
epithelial cell without
damaging the cell or cell layer and without being altered, degraded or
modified (e.g., chemically
or enzymatically altered or modified). The carrier constructs (e.g., isolated
delivery constructs)
of the present disclosure can further utilize specific intracellular
compartments during
transcytosis and/or intracellular delivery to achieve the described transport
efficiency.
[0242] The present disclosure provides methods and compositions that can
comprise carriers
that use (or interaction with) a set of endogenous proteins and receptors
involved in the apical-to-
basal transcytosis process across epithelial cells, such as polarized
intestinal epithelial cells (e.g.,
enterocytes), to mediate transcytosis of a carrier coupled to a cargo from a
lumen bordering the
apical surface of a mucous membrane to the basolateral side of a mucous
membrane. The
delivery constructs disclosed herein can engage in interactions with such
proteins and receptors
to provide efficient transport and delivery of various cargo molecules to
locations within an
epithelial cell and/or across an epithelial cell to the basal side of an
epithelium (e.g., a gut
epithelium of a subject). The constructs described herein, such as delivery
constructs, can
comprise a carrier coupled to a heterologous cargo, wherein the carrier and/or
the heterologous
cargo can interact with proteins and/or receptors during intracellular
delivery (e.g., to a
supranuclear region or to a compartment located at the basal side within the
epithelial cell) or
during transcytosis (e.g., vesicular transcytosis). The carrier can interact
with one or more
proteins (e.g., receptors or enzymes). These interactions can be dynamical
and/or pH-dependent.
It is pointed out that the herein described interactions are examples only and
are not limiting the
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methods and compositions of this disclosure to other interactions (e.g., with
other proteins or
receptors).
[0243] The compositions and methods disclosed herein provide efficient
delivery and
transport of various cargo molecules (e.g., small molecules as well as
macromolecules) across
epithelial cells and/or into epithelial cells. The carriers described herein
achieve such efficient
delivery of cargo in a manner that does not impair the epithelial cell barrier
nor the delivery
construct itself Thus, the functional properties of the delivery constructs
(e.g., those of the
carrier as well as the functions of the cargo) can be retained during
transport, allowing a fast and
efficient delivery of such cargo. The presently described carriers utilize
endogenous trafficking
pathways to deliver exogenous or endogenous cargo molecules to specific
locations. Those
locations can be inside an epithelial cell and/or in basolateral compartments
outside epithelial
cells on the basal side, e.g., the lamina propria.
[0244] The carriers of the present disclosure comprise can be derived from
an exotoxin.
Bacterial protein toxins are well known in the art, and are discussed in such
sources as Burns, D.,
et al., eds., BACTERIAL PROTEIN TOXINS, ASM Press, Herndon Va. (2003),
Aktories, K.
and Just, I., eds., BACTERIAL PROTEIN TOXINS (HANDBOOK OF EXPERIMENTAL
PHARMACOLOGY), Springer-Verlag, Berlin, Germany (2000), and Alouf, J. and
Popoff, M.,
eds., THE COMPREHENSIVE SOURCEBOOK OF BACTERIAL PROTEIN TOXINS,
Academic Press, Inc., San Diego, Calif (3rd Ed., 2006).
[0245] As further described herein, an exotoxin can comprise one or more
domains. As
disclosed herein, an exotoxin can be Cholix or PE. For Cholix, the following
nomenclature is
used herein to describe its various domains (N- to C-terminus) and using the
functional Cholix
variant having the amino acid sequence set forth in SEQ ID NO: 1 as a
reference sequence: (i)
domain I (amino acid residues 1-265, SEQ ID NO: 4), (ii) domain II (amino acid
residues 266-
386, SEQ ID NO: 126), (iii) domain lb (amino acid residues 387-425, SEQ ID NO:
127), and
(iv) domain III (amino acid residues 426-634, SEQ ID NO: 128). For PE, the
following
nomenclature is used herein to describe its various domains (N- to C-terminus)
and using the
functional PE variant having the amino acid sequence set forth in SEQ ID NO:
135 as a
reference sequence: (i) domain I (amino acid residues 1-252, SEQ ID NO: 137),
(ii) domain II
(amino acid residues 253-364, SEQ ID NO: 138), (iii) domain lb (amino acid
residues 365-404,
SEQ ID NO: 139), and (iv) domain III (amino acid residues 405-613, SEQ ID NO:
140).
Moreover, the ranges of amino acid residues defining these domains can be
flexible and
variations of about 5-10 amino acid residues may still fall within the scope
of this disclosure,
e.g., describing an amino acid sequence comprising the amino acid residues 5-
265 or 5-270, or 1-
260, or 5-260 of full-length Cholix may still be understood as a Cholix domain
I and so forth. As
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disclosed herein, the terms "domain I" and "receptor binding domain" of an
exotoxin can be
used interchangeably. As disclosed herein, the terms "domain II" and
"translocation domain" of
an exotoxin can be used interchangeably. As disclosed herein, the terms
"domain III", "catalytic
domain" and "cytotoxic domain" of an exotoxin can be used interchangeably.
[0246] Pseudomonas aeruginosa exotoxin A (PE), Corynebacterium diphtheria
Diphtheria
carrier (DT), and Vibrio cholera Cholix make up a family of bacterial protein
toxins that act as
ADP-ribosyltransferases. Thus, the carrier can be derived from a Cholix toxin.
The carrier can be
derived from a PE.
[0247] A Cholix polypeptide as described herein may be rendered non-toxic
by one or more
amino acid substitutions. A Cholix derived polypeptide or carrier as described
herein may be
rendered non-toxic by substituting a glutamic acid residue at position 581 of
the amino acid
sequence set forth in SEQ ID NO: 2 with alanine, resulting in a Cholix
construct comprising an
amino acid sequence set forth in SEQ ID NO: 3.
[0248] As further described herein, Cholix and PE are organized into
distinct domains (I, II,
lb, and III) that are denoted based upon their structural relationships.
Domain I appears to
facilitate exotoxin internalization and transcytosis, whereas domains II, lb,
and III provide other
functions as, for example, enzymatic activity in case of domain III that can
ADP-ribosylate
elongation factor 2 to induce cell apoptosis via blockade of protein
synthesis. It has previously
been unknown what components of PE and Cholix proteins are involved in the
trans-epithelial
transcytosis process.
[0249] Cholix is secreted by Vibrio cholera as a 70.7 kDa protein composed
of three
prominent globular domains (Ia, II, and III) and one small subdomain (lb)
connecting domains II
and III similar to the structure of PE (Jorgensen, R. et al., J Biol Chem
283(16):10671-10678,
2008). Mature Cholix comprises a genus of functional variants, wherein each
variant can differ
in one or more amino acid residues compared to another variant. However, all
Cholix variants
disclosed herein and encompassed in this disclosure are functional Cholix
variants. As used
herein, Cholix is a 634-residue protein, and two functional variants are
specifically included
herein, which are those having the amino acid sequences set forth in SEQ ID
NO: 1 and SEQ ID
NO: 1. A nucleic acid encoding the mature Cholix as used herein is set forth
in SEQ ID NO: 134.
[0250] Pseudomonas exotoxin A or "PE" is secreted by Pseudomonas aeruginosa
as a 67
kDa protein composed of three prominent globular domains (Ia, II, and III) and
one small
subdomain (lb) connecting domains II and III (see Allured et. al., Proc. Natl.
Acad. Sci. 83:1320
1324, 1986). Mature PE as used herein is a 613-residue protein, whose sequence
is set forth in
SEQ ID NO: 134. A nucleic acid encoding mature PE as used herein is set forth
in SEQ ID NO:
135.
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[0251] The amino acid sequence of the mature Cholix toxin is set forth in
SEQ ID NO: 1
and is used as the reference sequence, unless specified otherwise. For
example, the amino acid
sequence set forth in SEQ ID NO: 4 contains the amino acid residues 1-265 of
the amino acid
sequence of mature Cholix toxin set forth in SEQ ID NO: 1 and is defined as
Cholix domain I.
Thus, the polypeptide having the amino acid sequence set forth in SEQ ID NO: 4
can also be
described as "Chx1-265" (or "Cholix1-265" or "Cholix265" or "Cholix domain
I"). In addition to the
Cholix reference sequence set forth in SEQ ID NO: 1, any other, functionally
active, Cholix
exotoxin variants are encompassed in the present disclosure, e.g., those that
comprise a
consensus sequence defining the functional activity of the Cholix exotoxins.
(See e.g., Awasthi
et al. Novel Cholix toxin variants, ADP-ribosylating toxins in Vibrio Cholerae
Non-01/Non-
0139 strains, and their pathogenicity, Infection and Immunity, 81(2), p. 531-
541 (2013)). As an
example, the polypeptide having the amino acid sequence set forth in SEQ ID
NO: 2 is a
functional variant of SEQ ID NO: 1. As such, a domain I derived from that
Cholix exotoxin
sequence, or a truncated version thereof, can be used as a carrier for the
rapid and efficient
delivery of cargo. Using the nomenclature described herein with the reference
sequence being
SEQ ID NO: 1, a domain I polypeptide of the Cholix exotoxin with SEQ ID NO: 2
can also be
described as amino acid residues 1-4 of SEQ ID NO: 2 + Cholix5-265.
[0252] In other cases, and as described herein, a first carrier and a
second carrier are
produced in a different expression system (e.g., a bacterial or a mammalian
expression system).
Bacterial expression systems include E. coli, and mammalian expression systems
include CHO
cells, for example. A bacterially produced polypeptide can comprise an N-cap,
wherein the N-
cap can comprise one more modifications at the N-terminal of the polypeptide.
An N-cap can
comprise an N-terminal methionine residue. Examples of Cholix domain I derived
carrier
polypeptides that can be bacterially produced and that comprise such N-
terminal methionine
include those comprising the amino acid sequences set forth in SEQ ID NO: 5,
SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, and SEQ ID NO:
125.
[0253] The present disclosure contemplates isolated non-naturally occurring
and bacterial
toxin derived carriers (e.g., an exotoxin derived) that can be coupled to a
cargo (e.g., a
biologically active); wherein the carrier is capable of delivering the cargo
(e.g., a biologically
active) via transcytosis transport across the intestinal epithelium. The
carrier can be derived from
a domain I of an exotoxin (e.g., Cholix or PE). A carrier that is derived form
a domain I of an
exotoxin can lack a domain II (e.g., SEQ ID NO: 126 or SEQ ID NO: 138), a
domain lb (e.g.,
SEQ ID NO: 127 or SEQ ID NO: 139), or a domain III (e.g., SEQ ID NO: 128 or
SEQ ID NO:
140) of an exotoxin (e.g., Cholix or PE).
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[0254] As described herein, a carrier that "lacks" a domain II, domain lb,
and a domain III
of an exotoxin (e.g., Cholix and/or PE) can still comprise a portion of the
domain II, a domain lb,
or the domain III of the exotoxin, or a combination thereof Thus, the term
"lacking" as referred
to herein means that a carrier does not comprise a complete domain II, a
complete domain lb, or
a complete domain III. A carrier can comprise no more than 70% of the amino
acid residues of a
domain II, a domain lb, or a domain III of an exotoxin. For example, a carrier
can comprise a
Cholix domain I (e.g., SEQ ID NO: 4 or SEQ ID NO: 5) or a truncated version
thereof, and
further comprise the amino acid residues 1-82 of Cholix domain II (SEQ ID NO:
126). A carrier
can comprise no more than 60% of the amino acid residues of a domain II, a
domain lb, or a
domain III of an exotoxin. A carrier can comprise no more than 50% of the
amino acid residues
of a domain II, a domain lb, or a domain III of an exotoxin. A carrier can
comprise no more than
25% of the amino acid residues of a domain II, a domain lb, or a domain III of
an exotoxin. A
carrier can comprise no more than 10% of the amino acid residues of a domain
II, a domain lb,
or a domain III of an exotoxin.
[0255] The present disclosure contemplates isolated non-naturally occurring
and bacterial
toxin derived carriers (e.g., an exotoxin derived) that can be coupled to a
cargo (e.g., a
biologically active); wherein the carrier is capable of delivering the cargo
(e.g., a biologically
active) to the interior of an epithelial cell, such as an intracellular
vesicle or compartment or the
cytosol. Regions and/or compartments in the interior of an epithelial cell can
include regions
and/or compartments on the apical side of the interior of an epithelial cell,
regions and/or
compartments on the basal side of the interior of an epithelial cell,
supranuclear regions of an
epithelial cell, or any combination thereof. The epithelial cell can be a
polarized gut epithelial
cell. The polarized gut epithelial cell can be part of a polarized epithelial
cell monolayer (e.g.,
comprising Caco-2 cells) or it can be part of a gut epithelium of a subject
(e.g., a rodent or a
human).
[0256] A carrier can be derived from a bacterial carrier such as an
exotoxin (e.g., Cholix
and/or PE) and can be derived from a domain I of said exotoxin and can lack a
domain II (e.g.,
SEQ ID NO: 126 or SEQ ID NO: 138), a domain Ib (e.g., SEQ ID NO: 127 or SEQ ID
NO:
139), or a domain III (e.g., SEQ ID NO: 128 or SEQ ID NO: 140) of an exotoxin
(e.g., Cholix or
PE). The carrier can comprise a receptor binding domain or binding fragment,
which can be a
domain, region, or fragment within the exotoxin derived domain I, and which
allows binding of
the delivery construct to one or more selective or non-selective receptors on
the luminal surface
of an epithelial cell. A receptor can be a selective receptor or a non-
selective receptor, such as a
non-selective scavenger receptor on the luminal surface of intestinal
epithelial cells. The one or
more receptors that a carrier can interact with on the surface of an
epithelial cell and/or during
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endocytosis can include a low density lipoprotein receptor-related protein 1
(LRP1) or a
transmembrane protein 132 (TMEM132). Thus, the delivery construct can bind to
one or more
cell surface receptor that can be present on the apical membrane of an
epithelial cell with
sufficient affinity to allow endocytosis. The delivery construct can bind to
any receptor known to
be present on the apical membrane of an epithelial cell by one of skill in the
art without
limitation. The carrier can bind to LRP1. The carrier can bind to TMEM132.
Alternatively, the
carrier can bind to LRP1 and TMEM132.
[0257] A carrier can be derived from a domain I of an exotoxin. The
exotoxin is selected
from the group consisting of Cholix and PE. A carrier as described herein is
derived from a
domain I of an exotoxin, wherein the exotoxin is Cholix. Thus, a carrier as
described herein can
comprise an amino acid sequence that is derived from that of Cholix domain I
(e.g., SEQ ID NO:
4 or SEQ ID NO: 5). A Cholix Domain I (e.g., SEQ ID NO: 4) can comprise amino
acids 1-265
of SEQ ID NO: 1 or it can comprise amino acid sequence set forth in SEQ ID NO:
5 (e.g., when
bacterially produced comprising an N-terminal methionine residue) and can be
described as a
"receptor binding domain" that functions as a ligand for a cell surface
receptor and mediates
Cholix binding and endocytosis. A carrier can comprise an amino acid sequence
with greater
than 50% homology to any one of SEQ ID NO: 4 ¨ SEQ ID NO: 125. A carrier can
comprise an
amino acid sequence with greater than 60% homology to any one of SEQ ID NO: 4
¨ SEQ ID
NO: 125. A carrier can comprise an amino acid sequence with greater than 70%
homology to
any one of SEQ ID NO: 4 ¨ SEQ ID NO: 125. A carrier can comprise an amino acid
sequence
with greater than 80% homology to any one of SEQ ID NO: 4 ¨ SEQ ID NO: 125. A
carrier can
comprise an amino acid sequence with greater than 90% homology to any one of
SEQ ID NO: 4
¨ SEQ ID NO: 125. A carrier can comprise an amino acid sequence with greater
than 95%
homology to any one of SEQ ID NO: 4 ¨ SEQ ID NO: 125. Conservative or non-
conservative
substitutions can be made to an amino acid sequence of any one of SEQ ID NO: 4
¨ SEQ ID
NO: 125. As described herein, an amino acid residue substitution will be
identified by reference
to the particular amino acid substitution at a specific amino acid residue.
Thus, e.g., the term
"530A" indicates that the "S" (serine, in standard single letter code) residue
at position 30 in
SEQ ID NO: 4 has been substituted with an "A" (alanine, in standard single
letter code), and the
modified carrier will be identified as "Cholixs3 A". A carrier can be a
truncated version of a
Cholix domain I sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5. Thus, a
carrier
comprising a truncated version of a Cholix domain I can comprise an amino acid
sequence set
forth in any one of SEQ ID NO: 6 ¨ SEQ ID NO: 125. A carrier can comprise an
amino acid
sequence of any one of SEQ ID NO: 4 ¨ SEQ ID NO: 125, wherein one or more
amino residues
of such sequence is deleted. A carrier can comprise an amino acid sequence of
any one of SEQ
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ID NO: 4 ¨ SEQ ID NO: 125, wherein one or more amino acid residues can be
substituted with
another amino acid. As described herein, a truncated carrier can be identified
by reference to the
amino acid residues comprising the truncated toxin, e.g., a truncated Cholix
carrier consisting of
amino acid residues 1-260 of SEQ ID NO: 4 will be identified as Cholix26 and
so forth,
according to nomenclature described herein.
[0258] Exemplary nucleotide and amino acid sequences of carriers as
described herein are
shown below in TABLE 2. In various embodiments, a carrier comprises any of the
amino acid
sequences shown in TABLE 2, or fragment, or a combination thereof.
TABLE 2¨ Exemplary Nucleotide and Amino Acid Sequences of Carriers
SEQ ID NO Sequence
SEQ ID NO: 1 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
SWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYN
YITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRI
HFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQ
AQNIVSLEVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINE
NNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADIL
SLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGV
TNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAP
VPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLP
TRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIG
HSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAY
EELAIDEEAVAKEQSISTKPPYKERKDELK
SEQ ID NO: 2 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
SWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYN
YITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRI
HFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQ
AQNIVSLEVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINE
NNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADIL
SLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGV
TNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAP
VPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLP
TRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIG
HSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAY
EELAIDEEAVAKEQSISTKPPYKERKDELK
SEQ ID NO: 3 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
SWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYN
YITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRI
HFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQ
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AQNIVSLFVATRILF SHLD SVF TLNLDEQEPEVAERL SDLRRINE
NNPGMVTQVLTVARQIYNDYVTHHPGLTPEQT S AGAQAAD IL
SLFCPDADK S C VA SNND Q ANINIE SRS GRSYLPENRAVITPQGV
TNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAP
VPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLP
TRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIG
HSLPLRNEAF TGPESAGGEDATVIGWDMAIHAVAIP S T IP GNA
YEELAIDEEAVAKEQ SISTKPPYKERKDELK
SEQ ID NO: 4
VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT GLAL CWLVPMDAIYN
YITQQNCTLGDNWF GGSYETVAGTPKVITVKQGIEQKPVEQRI
HF SK
SEQ ID NO: 5 MVEEALNIFDECR SP C SLTPEPGKPIQ SKL S IP SDVVLDEGVLYY
S MT INDE QND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GV
IHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDE
LDQQRNIIEVPKLYSIDLDNQTLEQWKTQ GNV SF SVTRPEHNIA
I SWP S V S YKAAQKEGSRHKRWAHWHT GLALCWLVPMDAIYN
YITQQNCTLGDNWF GGSYETVAGTPKVITVKQGIEQKPVEQRI
HF SK
SEQ ID NO: 6 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT GLAL CWLVPMDAIYN
YITQQNCTLGDNWF GGSYETVAGTPKVITVKQ
SEQ ID NO: 7 MVEEALNIFDECR SP C SLTPEPGKPIQ SKL S IP SDVVLDEGVLYY
S MT INDE QND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GV
IHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDE
LDQQRNIIEVPKLYSIDLDNQTLEQWKTQ GNV SF SVTRPEHNIA
I SWP S V S YKAAQKEGSRHKRWAHWHT GLALCWLVPMDAIYN
YITQQNCTLGDNWF GGSYETVAGTPKVITVKQ
SEQ ID NO: 8 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT GLAL CWLVPMDAIYN
YITQQNCTLGDNWF GGSYETVAGTPK
SEQ ID NO: 9 MVEEALNIFDECR SP C SLTPEPGKPIQ SKL S IP SDVVLDEGVLYY
S MT INDE QND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GV
IHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDE
LDQQRNIIEVPKLYSIDLDNQTLEQWKTQ GNV SF SVTRPEHNIA
I SWP S V S YKAAQKEGSRHKRWAHWHT GLALCWLVPMDAIYN
YITQQNCTLGDNWF GGSYETVAGTPK
SEQ ID NO: 10 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
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SWP S V S YKAAQKEGSRHKRWAHWHT GL
SEQ ID NO: 11 MVEEALNIFDECR SP C SLTPEPGKPIQ SKL S IP SDVVLDEGVLYY
SMT INDEQND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GV
IHLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKIS VDE
LDQQRNIIEVPKLYSIDLDNQTLEQWKTQ GNV SF SVTRPEHNIA
I SWP S V S YKAAQKEGSRHKRWAHWHT GL
SEQ ID NO: 12 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT G
SEQ ID NO: 13 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT
SEQ ID NO: 14 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWH
SEQ ID NO: 15 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHW
SEQ ID NO: 16 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAH
SEQ ID NO: 17 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWA
SEQ ID NO: 18 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRW
SEQ ID NO: 19 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKR
SEQ ID NO: 20 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHK
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SEQ ID NO: 21 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRH
SEQ ID NO: 22 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSR
SEQ ID NO: 23 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGS
SEQ ID NO: 24 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEG
SEQ ID NO: 25 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKE
SEQ ID NO: 26 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQK
SEQ ID NO: 27 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQ
SEQ ID NO: 28 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAA
SEQ ID NO: 29 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKA
SEQ ID NO: 30 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWPSVSYK
SEQ ID NO: 31 MVEEALNIFDECR SP C SLTPEPGKPIQ SKL S IP SDVVLDEGVLYY
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SMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGV
IHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDE
LD Q QRNIIEVPKLY S IDLDNQ TLEQWK T Q GNV SF SVTRPEHNIA
ISWPSVSYK
SEQ ID NO: 32 EEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSM
TINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIH
LD IT TENGTKTY S YNRKEGEFAINWLVPIGED SPA S IKI S VDELD
Q QRNIIEVPKLY S ID LDNQ TLE QWK T Q GNV SF SVTRPEHNIAIS
WPSVSYK
SEQ ID NO: 33 EALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMT
INDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHL
DITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDELD
Q QRNIIEVPKLY S ID LDNQ TLE QWK T Q GNV SF SVTRPEHNIAIS
WPSVSYK
SEQ ID NO: 34 ALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTI
NDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLD
IT TENGTKTYS YNRKE GEF AINWLVP IGED SPA S IKIS VDELD Q
QRNIIEVPKLY S IDLDNQ TLEQWK T Q GNV SF SVTRPEHNIAISW
PSVSYK
SEQ ID NO: 35 LNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTIN
DEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDI
TTENGTKTYSYNRKEGEFAINWLVPIGED SPA SIKIS VDELDQ Q
RNIIEVPKLY S IDLDNQ TLEQWK T Q GNV SF SVTRPEHNIAISWP
SVSYK
SEQ ID NO: 36 NIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTIND
EQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITT
ENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRN
IIEVPKLY S IDLDNQ TLEQWKTQ GNV SF SVTRPEHNIAISWP S V S
YK
SEQ ID NO: 37 IFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDE
QNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTE
NGTKTYSYNRKEGEFAINWLVPIGED SPA S IKIS VDELDQ QRNII
EVPKLY S IDLDNQ TLE QWK T Q GNV SF SVTRPEHNIAISWP S V S
YK
SEQ ID NO: 38 FDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQ
NDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTEN
GTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIE
VPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSY
K
SEQ ID NO: 39 DECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQ
NDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTEN
GTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIE
VPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSY
K
SEQ ID NO: 40 ECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQN
DIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENG
TKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDELD Q QRNIIEV
PKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 41 CRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDI
KDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGT
KTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDELD Q QRNIIEVP
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KLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 42 RSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIK
DEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTK
TYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPK
LYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 43 SPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKD
EDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKT
YSYNRKEGEFAINWLVPIGED SPA S IKIS VDELD Q QRNIIEVPKL
YSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 44 PCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDE
DKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTY
SYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLY
SIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 45 CSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDE
DKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTY
SYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLY
SIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 46 SLTPEP GKPIQ SKL S IP SDVVLDEGVLYY SMTINDEQNDIKDED
KGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYS
YNRKEGEFAINWLVPIGED SPASIKISVDELDQQRNIIEVPKLYS
IDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 47 LTPEP GKPIQ SKL SIP SDVVLDEGVLYY SMTINDEQNDIKDEDK
GESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSY
NRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSI
DLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 48 TPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKG
ESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYN
RKEGEFAINWLVPIGED SPAS IKIS VDELDQQRNIIEVPKLYS IDL
DNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 49 PEP GKP IQ SKL S IP SDVVLDEGVLYY SMTINDEQNDIKDEDKGE
SIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNR
KEGEFAINWLVPIGED SPASIKISVDELDQQRNIIEVPKLYSIDL
DNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 50 EP GKP IQ SKL S IP SDVVLDEGVLYYSMTINDEQNDIKDEDKGES
IITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRK
EGEFAINWLVPIGED S PA S IKI S VDELD Q QRNIIEVPKLY S IDLD
NQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 51 PGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESII
TIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKE
GEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDN
QTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 52 GKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIIT
IGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEG
EFAINWLVPIGED SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ
TLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 53 KPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGE
FAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQT
LEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 54 PIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIG
EFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEF
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AINWLVPIGED SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ TL
EQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 55 IQ SKL SIP SDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGE
FAT VRATRHYVNQDAPF GVIELD ITTENGTKTY S YNRKEGEF A
INWLVPIGED SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ TLE
QWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 56 Q SKL SIP SDVVLDEGVLYY SMTINDEQND IKDEDKGE S ITT IGEF
ATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAI
NWLVPIGED SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ TLEQ
WKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 57 SKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFA
TVRATRHYVNQDAPF GVIHLD IT TENGTKTY S YNRKEGEF AIN
WLVPIGED SPA S IKI S VDELDQQRNIIEVPKLYSIDLDNQTLEQW
KT QGNVSF SVTRPEHNIAISWP S VS YK
SEQ ID NO: 58 KLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFAT
VRATRHYVNQDAPF GVIELDITTENGTKTYSYNRKEGEFAINW
LVPIGED SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWK
TQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 59 LSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATV
RATRHYVNQDAPFGVIELDITTENGTKTYSYNRKEGEFAINWL
VPIGED SPA S IKIS VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKT
QGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 60 SIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVR
ATRHYVNQDAPF GVIELDITTENGTKTYSYNRKEGEFAINWLV
PIGED SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKTQ
GNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 61 IPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVR
ATRHYVNQDAPF GVIELDITTENGTKTYSYNRKEGEFAINWLV
PIGED SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKTQ
GNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 62 PSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRA
TRHYVNQDAPF GVIELD IT TENGTKTY S YNRKEGEF AINWLVP
IGED SPA S IKIS VDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQG
NV SF SVTRPEHNIAISWP S VS YK
SEQ ID NO: 63 SDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRAT
RHYVNQDAPFGVIELDITTENGTKTYSYNRKEGEFAINWLVPI
GED SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q G
NV SF SVTRPEHNIAISWP S VS YK
SEQ ID NO: 64 DVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATR
HYVNQDAPF GVIHLD IT TENGTKTY S YNRKEGEF AINWLVP IG
ED SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GN
VSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 65 VVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRH
YVNQDAPF GVIELD IT TENGTKTY S YNRKEGEF AINWLVP IGE
D SPA S IKI S VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKTQ GNV
SF SVTRPEHNIAISWP S VS YK
SEQ ID NO: 66 VLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHY
VNQDAPF GVIELDITTENGTKTYSYNRKEGEFAINWLVPIGED S
PASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSF
SVTRPEHNIAISWPSVSYK
SEQ ID NO: 67 LDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYV
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NQDAPFGVIELDITTENGTKTYSYNRKEGEFAINWLVPIGEDSP
ASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFS
VTRPEHNIAISWPSVSYK
SEQ ID NO: 68 DEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVN
QDAPFGVIELDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPA
SIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSV
TRPEHNIAISWPSVSYK
SEQ ID NO: 69 EGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQ
DAPFGVIELDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASI
KI S VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWK T Q GNV SF S VTR
PEHNIAISWPSVSYK
SEQ ID NO: 70 GVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQ
DAPFGVIELDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASI
KI S VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWK T Q GNV SF S VTR
PEHNIAISWPSVSYK
SEQ ID NO: 71 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
SWPSVSY
SEQ ID NO: 72 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
SWPSVS
SEQ ID NO: 73 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
SWPSV
SEQ ID NO: 74 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
SWPS
SEQ ID NO: 75 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
SWP
SEQ ID NO: 76 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
SW
SEQ ID NO: 77 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAI
S
SEQ ID NO: 78 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
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MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SEQ ID NO: 79 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIA
SEQ ID NO: 80 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNI
SEQ ID NO: 81 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHN
SEQ ID NO: 82 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEH
SEQ ID NO: 83 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPE
SEQ ID NO: 84 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRP
SEQ ID NO: 85 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF S VTR
SEQ ID NO: 86 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF S VT
SEQ ID NO: 87 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF S V
SEQ ID NO: 88 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF S
SEQ ID NO: 89 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSF
SEQ ID NO: 90 VEEALNIFDECRSPC SL TPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SP A S IKI S VDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVS
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SEQ ID NO: 91 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGNV
SEQ ID NO: 92 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQGN
SEQ ID NO: 93 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQG
SEQ ID NO: 94 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKTQ
SEQ ID NO: 95 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWKT
SEQ ID NO: 96 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQWK
SEQ ID NO: 97 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQW
SEQ ID NO: 98 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLEQ
SEQ ID NO: 99 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTLE
SEQ ID NO: 100 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQTL
SEQ ID NO: 101 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQT
SEQ ID NO: 102 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
DQQRNIIEVPKLYSIDLDNQ
SEQ ID NO: 103 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDEL
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DQQRNIIEVPKLYSIDLDN
SEQ ID NO: 104 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEVPKLYSIDLD
SEQ ID NO: 105 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEVPKLYSIDL
SEQ ID NO: 106 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEVPKLYSID
SEQ ID NO: 107 MVEEALNIFDECR SP C SLTPEPGKPIQ SKL S IP SDVVLDEGVLYY
S MT INDE QND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GV
IHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDE
LD Q QRNIIEVPKLY S ID
SEQ ID NO: 108 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEVPKLYSI
SEQ ID NO: 109 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEVPKLYS
SEQ ID NO: 110 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEVPKLY
SEQ ID NO: 111 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEVPKL
SEQ ID NO: 112 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEVPK
SEQ ID NO: 113 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEVP
SEQ ID NO: 114 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIEV
SEQ ID NO: 115 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNIIE
SEQ ID NO: 116 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
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HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNII
SEQ ID NO: 117 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRNI
SEQ ID NO: 118 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQRN
SEQ ID NO: 119 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQR
SEQ ID NO: 120 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQQ
SEQ ID NO: 121 VEEALNIFDECRSPC SLTPEP GKP IQ SKL SIP SDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
DQ
SEQ ID NO: 122 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D
SEQ ID NO: 123 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
SEQ ID NO: 124 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDE
SEQ ID NO: 125 MVEEALNIFDECR SP C SLTPEPGKPIQ SKL S IP SDVVLDEGVLYY
S MT INDE QND IKDEDK GE S ITT IGEF AT VRATRHYVNQD APF GV
IHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDE
SEQ ID NO: 126 GNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNI
V S LF VATRILF SHLD S VFTLNLDEQEPEVAERLSDLRRINENNP
GMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQA
SEQ ID NO: 127 ADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPEN
SEQ ID NO: 128 RAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVA
AQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIK
EGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENA
EEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAI
PSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK
SEQ ID NO: 129 VEEALNIFDECRSPC SLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDK GE S IIT IGEF AT VRATRHYVNQD APF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT GLAL CWLVPMDAIYN
YITQQNCTLGDNWF GGSYETVAGTPKVITVKQGIEQKPVEQRI
HF SK GNAM S ALAAHRVC GVP LE TLAR S RKPRDL TDD L SCAYQ
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AQNIVSLFVATRILF SHLD SVF TLNLDEQEPEVAERL SDLRRINE
NNPGMVTQVLTVARQIYNDYVTHHPGLTPEQT S AGAQAAD IL
SLF CPDADK S C VA SNND QANINIE SRS GR S YLPEN
SEQ ID NO: 130 VEEALNIFDECRSPC SLTPEP GKP IQ SKL S IP SDVVLDEGVLYYS
MT INDEQND IKDEDKGE S IITIGEFATVRATRHYVNQDAPF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT GLAL CWLVPMDAIYN
YITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRI
HF SKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDL SCAYQ
AQNIVSLFVATRILF SHLD SVF TLNLDEQEPEVAERL SDLRRINE
NNPGMVTQVLTVARQIYNDYVTHHPGLTPEQT S AGAQAAD IL
SLF CPDADK S C VA SNND QANINIE S
SEQ ID NO: 131 VEEALNIFDECRSPC SLTPEPGKPIQ SKL S IP SDVVLDEGVLYYS
MT INDEQND IKDEDKGE S IITIGEFATVRATRHYVNQDAPF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT GLAL CWLVPMDAIYN
YITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRI
HF SKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDL SCAYQ
AQNIVSLFVATRILF SHLD SVF TLNLDEQEPEVAERL SDLRRINE
NNPGMVTQVLTVARQIYNDYVTHHPGLTPEQT S AGAQAAD IL
SLFCPDA
SEQ ID NO: 132 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYS
MT INDEQND IKDEDKGE S IITIGEFATVRATRHYVNQDAPF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT GLAL CWLVPMDAIYN
YITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRI
HF SKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDL SCAYQ
AQNIVSLFVATRILF SHLD SVF TLNLDEQEPEVAERL SDLRRINE
NNPGMVTQVLTVARQIYNDYVTHHPGLTPEQT SAGAQA
SEQ ID NO: 133 VEEALNIFDECRSPC SLTPEP GKP IQ SKL S IP SDVVLDEGVLYYS
MT INDEQND IKDEDKGE S IITIGEFATVRATRHYVNQDAPF GVI
HLDITTENGTKTYSYNRKEGEFAINWLVPIGED SPA S IKI S VDEL
D Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRPEHNIAI
SWP S V S YKAAQKEGSRHKRWAHWHT GLAL CWLVPMDAIYN
YITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRI
HF SKGNAMSALAAHRVCGVPLETLARSRKP
SEQ ID NO: 134 GTCGAAGAAGCTTTAAACATCTTTGATGAATGCCGTTCGCC
AT GT TC GTT GACC CC GGAACC GGGTAAGCC GATT CAAT CAA
AAC T GTC TAT CC C TAGTGAT GTT GT TC TGGATGAAGGTGT TC
TGTATTACTCGATGACGATTAATGATGAGCAGAATGATATT
AAGGATGAGGACAAAGGC GAGT C C ATTAT CAC TAT TGGT GA
AT TTGC CAC AGTAC GC GC GAC TAGAC ATTAT GTTAATC AAG
AT GC GCC TT TT GGTGTC ATC CAT TTAGATATTAC GACAGAA
AATGGTACAAAAACGTACTCTTATAACCGCAAAGAGGGTG
AATT TGC AATC AATT GGT TAGT GC C TAT TGGTGAAGATT C T C
CTGCAAGCATCAAAATCTCCGTTGATGAGCTCGATCAGCAA
CGCAATAT CAT CGAGGTGC C TAAAC T GTATAGTATT GAT C T
C GATAAC C AAAC GT TAGAGCAGT GGAAAAC C CAAGGTAAT
GT TTC TT TT TC GGTAACGC GTC C T GAACATAATAT CGC TAT C
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TCTTGGCCAAGCGTGAGTTACAAAGCAGCGCAGAAAGAGG
GTTCACGCCATAAGCGTTGGGCTCATTGGCATACAGGCTTA
GCACTGTGTTGGCTTGTGCCAATGGATGCTATCTATAACTAT
ATCACCCAGCAAAATTGTACTTTAGGGGATAATTGGTTTGG
TGGCTCTTATGAGACTGTTGCAGGCACTCCGAAGGTGATTA
CGGTTAAGCAAGGGATTGAACAAAAGCCAGTTGAGCAGCG
CATCCATTTCTCCAAGGGGAATGCGATGAGCGCACTTGCTG
CTCATCGCGTCTGTGGTGTGCCATTAGAAACTTTGGCGCGC
AGTCGCAAACCTCGTGATCTGACGGATGATTTATCATGTGC
CTATCAAGCGCAGAATATCGTGAGTTTATTTGTCGCGACGC
GTATCCTGTTCTCTCATCTGGATAGCGTATTTACTCTGAATC
TTGACGAACAAGAACCAGAGGTGGCTGAACGTCTAAGTGA
TCTTCGCCGTATCAATGAAAATAACCCGGGCATGGTTACAC
AGGTTTTAACCGTTGCTCGTCAGATCTATAACGATTATGTCA
CTCACCATCCGGGCTTAACTCCTGAGCAAACCAGTGCGGGT
GCACAAGCTGCCGATATCCTCTCTTTATTTTGCCCAGATGCT
GATAAGTCTTGTGTGGCTTCAAACAACGATCAAGCCAATAT
CAACATCGAGTCTCGTTCTGGCCGTTCATATTTGCCTGAAAA
CCGTGCGGTAATCACCCCTCAAGGCGTCACAAATTGGACTT
ACCAGGAACTCGAAGCAACACATCAAGCTCTGACTCGTGAG
GGTTATGTGTTCGTGGGTTACCATGGTACGAATCATGTCGCT
GCGCAAACCATCGTGAATCGCATTGCCCCTGTTCCGCGCGG
CAACAACACTGAAAACGAGGAAAAGTGGGGCGGGTTATAT
GTTGCAACTCACGCTGAAGTTGCCCATGGTTATGCTCGCAT
CAAAGAAGGGACAGGGGAGTATGGCCTTCCGACCCGTGCT
GAGCGCGACGCTCGTGGGGTAATGCTGCGCGTGTATATCCC
TCGTGCTTCATTAGAACGTTTTTATCGCACGAATACACCTTT
GGAAAATGCTGAGGAGCATATCACGCAAGTGATTGGTCATT
CTTTGCCATTACGCAATGAAGCATTTACTGGTCCAGAAAGT
GCGGGCGGGGAAGACGAAACTGTCATTGGCTGGGATATGG
CGATTCATGCAGTTGCGATCCCTTCGACTATCCCAGGGAAC
GCTTACGAAGAATTGGCGATTGATGAGGAGGCTGTTGCAAA
AGAGCAATCGATTAGCACAAAACCACCTTATAAAGAGCGC
AAAGATGAACTTAAG
SEQ ID NO: 135 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG
VLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKP
VRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLS
HMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHA
GVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLA
QQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEG
GSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLV
ALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQAR
LALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGEC
AGPAD S GDALLERNYP T GAEFLGD GGDV SF STRGTQNWTVER
LLQAHRQLEERGYVEVGYHGTFLEAAQSIVEGGVRARSQDLD
AIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPR
SSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEG
GRLETILGWPLAERTVVIP SAIPTDPRNVGGDLDP S SIPDKEQAI
SALPDYASQPGKPPREDLK
SEQ ID NO: 136 GCCGAGGAAGCCTTCGACCTCTGGAACGAATGCGCCAAGG
CCTGCGTGCTCGACCTCAAGGACGGCGTGCGTTCCAGCCGC
ATGAGCGTCGACCCGGCCATCGCCGACACCAACGGCCAGG
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GCGTGCTGCACTACTCCATGGTCCTGGAGGGCGGCAACGAC
GCGCTCAAGCTGGCCATCGACAACGCCCTCAGCATCACCAG
CGACGGCCTGACCATCCGCCTCGAAGGTGGCGTCGAGCCGA
ACAAGCCGGTGCGCTACAGCTACACGCGCCAGGCGCGCGG
CAGTTGGTCGCTGAACTGGCTGGTGCCGATCGGCCACGAGA
AGCCTTCGAACATCAAGGTGTTCATCCACGAACTGAACGCC
GGTAACCAGCTCAGCCACATGTCGCCGATCTACACCATCGA
GATGGGCGACGAGTTGCTGGCGAAGCTGGCGCGCGATGCC
ACCTTCTTCGTCAGGGCGCACGAGAGCAACGAGATGCAGCC
GACGCTCGCCATCAGCCATGCCGGGGTCAGCGTGGTCATGG
CCCAGGCCCAGCCGCGCCGGGAAAAGCGCTGGAGCGAATG
GGCCAGCGGCAAGGTGTTGTGCCTGCTCGACCCGCTGGACG
GGGTCTACAACTACCTCGCCCAGCAGCGCTGCAACCTCGAC
GATACCTGGGAAGGCAAGATCTACCGGGTGCTCGCCGGCA
ACCCGGCGAAGCATGACCTGGACATCAAGCCCACGGTCATC
AGTCATCGCCTGCATTTCCCCGAGGGCGGCAGCCTGGCCGC
GCTGACCGCGCACCAGGCCTGCCACCTGCCGCTGGAGACCT
TCACCCGTCATCGCCAGCCGCGCGGCTGGGAACAACTGGAG
CAGTGCGGCTATCCGGTGCAGCGGCTGGTCGCCCTCTACCT
GGCGGCGCGGCTGTCGTGGAACCAGGTCGACCAGGTGATCC
GCAACGCCCTGGCCAGCCCCGGCAGCGGCGGCGACCTGGG
CGAAGCGATCCGCGAGCAGCCGGAGCAGGCCCGTCTGGCC
CTGACCCTGGCCGCCGCCGAGAGCGAGCGCTTCGTCCGGCA
GGGCACAGGCAACGACGAGGCCGGCGCGGCCAGCGCCGAC
GTGGTGAGCCTGACCTGCCCGGTCGCCGCCGGTGAATGCGC
GGGCCCGGCGGACAGCGGCGACGCCCTGCTGGAGCGCAAC
TATCCCACTGGCGCGGAGTTCCTCGGCGACGGCGGCGACAT
CAGCTTCAGCACCCGCGGCACGCAGAACTGGACGGTGGAG
CGGCTGCTCCAGGCGCACCGCCAACTGGAGGAGCGCGGCT
ATGTGTTCGTCGGCTACCACGGCACCTTCCTCGAAGCGGCG
CAAAGCATCGTCTTCGGCGGGGTGCGCGCGCGCAGCCAGG
ACCTCGACGCGATCTGGCGCGGTTTCTATATCGCCGGCGAT
CCGGCGCTGGCCTACGGCTACGCCCAGGACCAGGAACCCG
ACGCGCGCGGCCGGATCCGCAACGGTGCCCTGCTGCGGGTC
TATGTGCCGCGCTCGAGTCTGCCGGGCTTCTACCGCACCGG
CCTGACCCTGGCCGCGCCGGAGGCGGCGGGCGAGGTCGAA
CGGCTGATCGGCCATCCGCTGCCGCTGCGCCTGGACGCCAT
CACCGGCCCCGAGGAGGAAGGCGGGCGCCTGGAAACCATT
CTCGGCTGGCCGCTGGCCGAGCGCACCGTGGTGATTCCCTC
GGCGATCCCCACCGACCCGCGCAACGTCGGCGGCGACCTCG
ACCCGTCCAGCATCCCCGACAAGGAACAGGCGATCAGCGC
CCTGCCGGACTACGCCAGCCAGCCCGGCAAACCGCCGCGCG
AGGACCTGAAG
SEQ ID NO: 137 AEEAFDLWNEC AK AC VLDLKD GVRS SRMSVDPAIADTNGQG
VLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKP
VRYSYTRQARGSW SLNWLVPIGHEKP SNIKVFIHELNAGNQLS
HMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHA
GVSVVMAQAQPRREKRW SEWA SGKVLCLLDPLDGVYNYLA
QQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPE
SEQ ID NO: 138 GGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRL
VALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQA
RLALTLAAAESERFVRQGTGNDEAGAAS
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SEQ ID NO: 139 ADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDG
SEQ ID NO: 140 GDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEA
AQ SIVF GGVRARS QDLDAIWRGFYIAGDPALAYGYAQDQEPD
ARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLI
GHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIP SAIPTDPR
NVGGDLDP S SIPDKEQAISALPDYAS QPGKPPREDLK
SEQ ID NO: 141 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG
VLHYSMVLEGGNDALKLAIDNAL SIT SD GL TIRLEGGVEPNKP
VRYSYTRQARGSW SLNWLVPIGHEKP SNIKVFIHELNAGNQLS
HM S PIYT IEMGDELLAKLARDATFF VRAHE SNEMQP TLAIS HA
GVSVVMAQAQPRREKRW SEWA SGKVLCLLDPLDGVYNYLA
QQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEG
GSLAALTAHQACHLPLETF TRHRQPRGWEQLEQC GYP VQRLV
ALYLAARLSWNQVD QVIRNALA SP GS GGDLGEAIREQPEQAR
LAL TLAAAE SERF VRQ GTGNDEAGAA S AD VV SL TCP VAAGEC
AGPAD SGDALLERNYPTGAEFLGDG
SEQ ID NO: 142 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG
VLHYSMVLEGGNDALKLAIDNAL SIT SD GL TIRLEGGVEPNKP
VRYSYTRQARGSW SLNWLVPIGHEKP SNIKVFIHELNAGNQLS
HM S PIYT IEMGDELLAKLARDATFF VRAHE SNEMQP TLAIS HA
GVSVVMAQAQPRREKRW SEWA SGKVLCLLDPLDGVYNYLA
QQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEG
GSLAALTAHQACHLPLETF TRHRQPRGWEQLEQC GYP VQRLV
ALYLAARLSWNQVD QVIRNALA SP GS GGDLGEAIREQPEQAR
LAL TLAAAE SERF VRQ GTGNDEAGAA S AD VV SL TCP VAAGEC
AGPAD SGDALLERNYP
SEQ ID NO: 143 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG
VLHYSMVLEGGNDALKLAIDNAL SIT SD GL TIRLEGGVEPNKP
VRYSYTRQARGSW SLNWLVPIGHEKP SNIKVFIHELNAGNQLS
HM S PIYT IEMGDELLAKLARDATFF VRAHE SNEMQP TLAIS HA
GVSVVMAQAQPRREKRW SEWA SGKVLCLLDPLDGVYNYLA
QQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEG
GSLAALTAHQACHLPLETF TRHRQPRGWEQLEQC GYP VQRLV
ALYLAARLSWNQVD QVIRNALA SP GS GGDLGEAIREQPEQAR
LAL TLAAAE SERF VRQ GTGNDEAGAA S AD VV SL TCP VA
SEQ ID NO: 144 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG
VLHYSMVLEGGNDALKLAIDNAL SIT SD GL TIRLEGGVEPNKP
VRYSYTRQARGSW SLNWLVPIGHEKP SNIKVFIHELNAGNQLS
HM S PIYT IEMGDELLAKLARDATFF VRAHE SNEMQP TLAIS HA
GVSVVMAQAQPRREKRW SEWA SGKVLCLLDPLDGVYNYLA
QQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEG
GSLAALTAHQACHLPLETF TRHRQPRGWEQLEQC GYP VQRLV
ALYLAARLSWNQVD QVIRNALA SP GS GGDLGEAIREQPEQAR
LAL TLAAAE SERF VRQ GTGNDEAGAA S
SEQ ID NO: 145 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG
VLHYSMVLEGGNDALKLAIDNAL SIT SD GL TIRLEGGVEPNKP
VRYSYTRQARGSW SLNWLVPIGHEKP SNIKVFIHELNAGNQLS
HM S PIYT IEMGDELLAKLARDATFF VRAHE SNEMQP TLAIS HA
GVSVVMAQAQPRREKRW SEWA SGKVLCLLDPLDGVYNYLA
QQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEG
GSLAALTAHQACHLPLETF TRHRQ
SEQ ID NO: 146 MVEEALNIFDECR SP C SLTPEPGKPIQ SKL S IP SDVVLDEGVLYY
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SMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGV
IHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDE
LDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIA
ISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYN
YITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRI
HFSKGGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGY
PVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIRE
QPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCP
VAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQ
NWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRA
RSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALL
RVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAIT
GPEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPD
KEQAISALPDYASQPGKPPREDLK
SEQ ID NO: 147 MAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQ
GVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNK
PVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQL
SHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISH
AGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLA
QQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEG
GNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNI
VSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNP
GMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAAEILSLFC
PDADKSCVATNNDQANINIESRSGRSYLPENRAVITPQGVTNW
TYQELEATHQALTREGYVFVGYTNHVAAQTIVNRIAPVPRGN
NTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAER
DARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLR
NEAFTGPESAGGEDTVIGWDMAIHAVAIPSTIPGNAYEELAIDE
EAVAKEQSISTKPPYKERKDELK
[0259] A carrier can be a polypeptide that is derived from a Cholix
exotoxin and having: at
most 5 amino acid residues; at most 10 amino acid residues; at most 15 amino
acid residues; at
most 20 amino acid residues; at most 30 amino acid residues; at most 40 amino
acid residues; at
most 50 amino acid residues; at most 60 amino acid residues; at most 70 amino
acid residues; at
most 80 amino acid residues; at most 90 amino acid residues; at most 100 amino
acid residues; at
most 110 amino acid residues; at most 120 amino acid residues; at most 130
amino acid residues;
at most 140 amino acid residues; at most 150 amino acid residues; at most 160
amino acid
residues; at most 170 amino acid residues; at most 180 amino acid residues; at
most 190 amino
acid residues; at most 200 amino acid residues; at most 210 amino acid
residues; at most 220
amino acid residues; at most 230 amino acid residues; at most 240 amino acid
residues; at most
250 amino acid residues; at most 260 amino acid residues; and at most 265
amino acid residues
of SEQ ID NO: 4 or SEQ ID NO: 5. The bacterial carrier receptor binding domain
can be a
polypeptide derived from Cholix and having at least 10%, 20%, 30%, 40%, 50%,
60%, 70%,
80%, 90%, 95%, 99%, or more sequence homology with SEQ ID NO: 4 or SEQ ID NO:
5. The
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carrier can be a polypeptide derived from Cholix and having at most 10%, 20%,
30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence homology with any one of SEQ ID
NO: 1 -
SEQ ID NO: 133. The amino acid residues can be consecutive. The amino acid
residues are also
be non-consecutive. A carrier can be derived from a domain I of a Cholix
exotoxin. A carrier that
is derived from a domain I of an exotoxin can comprise an amino acid having at
least 80%
sequence identity to any one of the amino acid sequences set forth in SEQ ID
NO: 4 - SEQ ID
NO: 125, or at least 80% sequence identity to a functional fragment thereof. A
carrier that is
derived from a domain I of an exotoxin can comprise an amino acid having at
least 90%
sequence identity to any one of the amino acid sequences set forth in SEQ ID
NO: 4 - SEQ ID
NO: 125, or at least 90% sequence identity to a functional fragment thereof. A
carrier that is
derived from a domain I of an exotoxin can comprise an amino acid having at
least 95%
sequence identity to any one of the amino acid sequences set forth in SEQ ID
NO: 4 - SEQ ID
NO: 125, or at least 95% sequence identity to a functional fragment thereof. A
carrier that is
derived from a domain I of an exotoxin can comprise an amino acid having at
least 99%
sequence identity to any one of the amino acid sequences set forth in SEQ ID
NO: 4 - SEQ ID
NO: 125, or at least 99% sequence identity to a functional fragment thereof. A
carrier that is
derived from a domain I of an exotoxin can comprise an amino acid having 100%
sequence
identity to any one of the amino acid sequences set forth in SEQ ID NO: 4 -
SEQ ID NO: 125,
or 100% sequence identity to a functional fragment thereof.
[0260] A carrier can be artificially synthesized. A carrier can be an
artificially synthesized
polypeptide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
99%, or
more amino acid sequence homology to a Cholix domain I (e.g., any one of SEQ
ID NO: 4 -
SEQ ID NO: 125). A carrier can be a synthetic polypeptide having at most 10%,
20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% amino acid sequence homology to a
Cholix
domain I (e.g., any one of SEQ ID NO: 4 - SEQ ID NO: 125). The polypeptide
that a carrier can
be comprises of can be synthesized using solid-phase synthesis.
[0261] A carrier can be a polypeptide derived from Cholix and having at
most 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence homology with any one
of SEQ
ID NO: 4 - SEQ ID NO: 125. Certain fragments within the amino acid sequence of
the carrier
can have specific functions that can be related to one or more aspects of the
transcytosis process.
These functions can comprise crossing a polarized monolayer of primary human
small intestinal
epithelial cells or an intact gut epithelium, enabling or promoting
endocytosis into an epithelial
cell, apical-to-basal transport, release of the delivery construct from the
basal membrane into a
basolateral compartment, delivery into an intracellular vesicle or compartment
or the cytosol of
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an epithelial cell, and/or delivery to a supranuclear region of an epithelial
cell (e.g., a polarized
gut epithelial cell).
[0262] Thus, the present disclosure provides carriers that can have various
functions (e.g.,
one or more of endocytosis, transcytosis, intracellular delivery, etc.). Such
a carrier can comprise
an amino acid sequence having at least 80% sequence identity to the amino acid
sequence of
SEQ ID NO: 4 or SEQ ID NO: 5 or at least 80% sequence identity to a functional
fragment
thereof, and no more than 347 contiguous amino acid residues from SEQ ID NO:
1. Such a
carrier can comprise a deletion or mutation in one or more of amino acid
residues of the amino
acid sequence set forth in SEQ ID NO: 4 (e.g., a Cholix domain I expressed in
a mammalian
cell) or SEQ ID NO: 5 (e.g., a Cholix domain I expressed in a bacterial cell).
Such a carrier can
comprise an amino acid sequence having at least 90% sequence identity to the
amino acid
sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 90% sequence identity to
a functional
fragment thereof, and no more than 347 contiguous amino acid residues from SEQ
ID NO: 1. A
carrier can comprise an amino acid sequence having at least 95% sequence
identity to the amino
acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 95% sequence
identity to a
functional fragment thereof, and no more than 347 contiguous amino acid
residues from SEQ ID
NO: 1. A carrier can comprise an amino acid sequence having at least 99%
sequence identity to
the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or at least 99%
sequence identity to
a functional fragment thereof, and no more than 347 contiguous amino acid
residues from SEQ
ID NO: 1. A carrier can comprise an amino acid sequence having 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or 100% sequence identity
to a
functional fragment thereof, and no more than 347 contiguous amino acid
residues from SEQ ID
NO: 1. A carrier disclosed herein can comprise the amino acid sequence set
forth in SEQ ID NO:
4 or SEQ ID NO: 5 or a functional fragment thereof. The carrier can comprises
the amino acid
sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 or a functional fragment
thereof The
carrier can comprises the amino acid sequence set forth in SEQ ID NO: 8 or SEQ
ID NO: 9 or a
functional fragment thereof
[0263] A functional carrier of the present disclosure can comprise an amino
acid sequence
having at least 80% sequence identity to any one of the amino acid sequences
set forth in SEQ
ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152,a
functional fragment thereof, or any combination thereof. A carrier can
comprise an amino acid
sequence having at least 90% sequence identity to any one of the amino acid
sequences set forth
in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:
152, a
functional fragment thereof, or any combination thereof. A carrier can
comprise an amino acid
sequence having at least 95% sequence identity to any one of the amino acid
sequences set forth
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in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:
152, a
functional fragment thereof, or any combination thereof. A carrier can
comprise comprises an
amino acid sequence having at least 99% sequence identity to any one of the
amino acid
sequences set forth in SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID
NO: 151,
SEQ ID NO: 152, a functional fragment thereof, or any combination thereof. A
carrier can
comprise a spatial structure in which one or more amino acid residues of SEQ
ID NO: 148 or
SEQ ID NO: 149 are in close proximity to one or more amino acid residues of
SEQ ID NO: 151,
and one or more amino acid residues of SEQ ID NO: 148 or SEQ ID NO: 149 are in
close
proximity to one or more amino acid residues of SEQ ID NO: 152.
[0264] A carrier of the present disclosure can be capable of delivering a
cargo across an
epithelial cell (e.g., a polarized epithelial cell). Such a carrier can
comprise an amino acid
sequence having at least 80% sequence identity to the amino acid sequence set
forth in SEQ ID
NO: 30 or SEQ ID NO: 31 or the amino acid sequence set forth in SEQ ID NO: 10
or SEQ ID
NO: 11 or at least 80% sequence identity to a functional fragment thereof. A
carrier can
comprise an amino acid sequence having at least 90% sequence identity to the
amino acid
sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or the amino acid
sequence set forth in
SEQ ID NO: 10 or SEQ ID NO: 11 or at least 90% sequence identity to a
functional fragment
thereof. A carrier can comprise an amino acid sequence having at least 95%
sequence identity to
the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or the
amino acid
sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or at least 95% sequence
identity to a
functional fragment thereof A carrier can comprise an amino acid sequence
having at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ
ID NO: 31 or
the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11 or at
least 99% sequence
identity to a functional fragment thereof A carrier can further comprise a
deletion or mutation in
one or more of amino acid residues 1-187 or 1-205 of SEQ ID NO: 10 or 1-186 or
1-206 of SEQ
ID NO: 11. A carrier can comprise residues 1-186 of SEQ ID NO: 30 or 1-187 of
SEQ ID NO:
31 and no more than 206 contiguous amino acid residues of SEQ ID NO: 1.
[0265] The methods and compositions of the present disclosure can comprise
a carrier
comprising an amino acid sequence having at least 80% sequence identity to the
amino acid
sequence set forth in any one of SEQ ID NO: 10 ¨ SEQ ID NO: 31 or at least 80%
sequence
identity to a functional fragment thereof A carrier can comprise an amino acid
sequence having
at least 90% sequence identity to the amino acid sequence set forth in any one
of SEQ ID NO: 10
¨ SEQ ID NO: 31 or at least 90% sequence identity to a functional fragment
thereof. In some
instances, the carrier comprises an amino acid sequence having at least 95%
sequence identity to
the amino acid sequence set forth in any one of SEQ ID NO: 10 ¨ SEQ ID NO: 31
or at least
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95% sequence identity to a functional fragment thereof. A carrier can comprise
an amino acid
sequence having at least 99% sequence identity to the amino acid sequence set
forth in any one
of SEQ ID NO: 10 ¨ SEQ ID NO: 31 or at least 99% sequence identity to a
functional fragment
thereof. The carrier can comprises the amino acid sequence set forth in SEQ ID
NO: 10 or SEQ
ID NO: 11 or a functional fragment thereof.
[0266] A carrier can comprise an amino acid sequence having at least 80%
sequence
identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO:
107 or to the
amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least
80% sequence
identity to a functional fragment thereof Such a carrier can be capable of
delivering cargo to an
intracellular location. Such an intracellular location may be a supranuclear
region. Such a carrier
can comprise an amino acid sequence having at least 90% sequence identity to
the amino acid
sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or to the amino acid
sequence set
forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least 90% sequence identity to a
functional
fragment thereof A carrier can comprise an amino acid sequence having at least
95% sequence
identity to the amino acid sequence set forth in SEQ ID NO: 106 or SEQ ID NO:
107 or to the
amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 or at least
95% sequence
identity to a functional fragment thereof A carrier can comprise an amino acid
sequence having
at least 99% sequence identity to the amino acid sequence set forth in SEQ ID
NO: 106 or SEQ
ID NO: 107 or to the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID
NO: 31 or at
least 99% sequence identity to a functional fragment thereof A carrier can
comprise a deletion
or mutation in one or more of amino acid residues 1-151 or 1-187 of SEQ ID NO:
4 or SEQ ID
NO: 5.
[0267] The methods and compositions of the present disclosure provide a
carrier that can
lack any one or more of the amino acid residues 1-39 of SEQ ID NO: 5 or amino
acid residues 1-
38 of SEQ ID NO: 4. Such a carrier can be capable of delivering cargo to an
intracellular
location via endocytosis. Such a location can be an apical region or
compartment. A carrier can
lack all of the amino acid residues 1-39 of SEQ ID NO: 5 or amino acid
residues 1-38 of SEQ ID
NO: 4. A carrier can comprises an amino acid sequence having at least 80%
sequence identity to
the amino acid sequence set forth in SEQ ID NO: 70 or 80% sequence identity to
a functional
fragment thereof A carrier can comprises an amino acid sequence having at
least 90% sequence
identity to the amino acid sequence set forth in SEQ ID NO: 70 or 90% sequence
identity to a
functional fragment thereof A carrier can comprises an amino acid sequence
having at least 95%
sequence identity to the amino acid sequence set forth in SEQ ID NO: 70 or 95%
sequence
identity to a functional fragment thereof A carrier can comprises an amino
acid sequence having
at least 99% sequence identity to the amino acid sequence set forth in SEQ ID
NO: 70 or 99%
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sequence identity to a functional fragment thereof A carrier can comprises
residues 1-151 of
SEQ ID NO: 5 or residues 1-150 of SEQ ID NO: 4 and no more than 187 contiguous
amino acid
residues of SEQ ID NO: 1.
[0268] A carrier of the present disclosure can comprise a truncated version
of a Cholix
domain I. Thus, a carrier can comprise an amino acid sequence having at least
80% sequence
identity to the amino acid sequence set forth in any one of SEQ ID NO: 30 ¨
SEQ ID NO: 107 or
at least 80% sequence identity to a functional fragment thereof. Such a
carrier can be capable of
delivering cargo to an intracellular location via endocytosis. Such a location
can be an apical
and/or a basal region or compartment. A carrier can comprise an amino acid
sequence having at
least 90% sequence identity to the amino acid sequence set forth in any one of
SEQ ID NO: 30 ¨
SEQ ID NO: 107 or at least 90% sequence identity to a functional fragment
thereof A carrier
can comprise an amino acid sequence having at least 95% sequence identity to
the amino acid
sequence set forth in any one of SEQ ID NO: 30 ¨ SEQ ID NO: 107 or at least
95% sequence
identity to a functional fragment thereof A carrier can comprise an amino acid
sequence having
at least 99% sequence identity to the amino acid sequence set forth in any one
of SEQ ID NO: 30
¨ SEQ ID NO: 107 or at least 99% sequence identity to a functional fragment
thereof. A carrier
can comprise the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO:
31 or a
functional fragment thereof
[0269] A carrier of the present disclosure can comprises an amino acid
sequence having at
least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:
106 or SEQ ID
NO: 107 or the amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO:
125 or at least
80% sequence identity to a functional fragment thereof. Such a carrier can be
capable of
delivering cargo to an intracellular location via endocytosis. Such a location
can be an apical
region or compartment. A carrier can comprise an amino acid sequence having at
least 90%
sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 or
SEQ ID NO: 107
or the amino acid sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125 or at
least 90%
sequence identity to a functional fragment thereof A carrier can comprise an
amino acid
sequence having at least 95% sequence identity to the amino acid sequence set
forth in SEQ ID
NO: 106 or SEQ ID NO: 107 or the amino acid sequence set forth in SEQ ID NO:
124 or SEQ
ID NO: 125 or at least 95% sequence identity to a functional fragment thereof.
A carrier can
comprise an amino acid sequence having at least 99% sequence identity to the
amino acid
sequence set forth in SEQ ID NO: 106 or SEQ ID NO: 107 or the amino acid
sequence set forth
in SEQ ID NO: 124 or SEQ ID NO: 125 or at least 99% sequence identity to a
functional
fragment thereof A carrier can further comprise a deletion or mutation in one
or more of amino
acid residues 1-151 of SEQ ID NO: 6 or in one or more of amino acid residues 1-
150 of SEQ ID
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NO: 7. A carrier as described herein can comprise residues 1-134 of SEQ ID NO:
5 or residues
1-133 of SEQ ID NO: 4 and no more than 151 contiguous amino acid residues of
SEQ ID NO: 1.
A carrier can comprise an amino acid sequence having at least 80% sequence
identity to the
amino acid sequence set forth in any of SEQ ID NO: 106 ¨ SEQ ID NO: 125 or at
least 80%
sequence identity to a functional fragment thereof A carrier can comprise an
amino acid
sequence having at least 90% sequence identity to the amino acid sequence set
forth in any of
SEQ ID NO: 106 ¨ SEQ ID NO: 125 or at least 90% sequence identity to a
functional fragment
thereof. A carrier can comprise an amino acid sequence having at least 95%
sequence identity to
the amino acid sequence set forth in any of SEQ ID NO: 106 ¨ SEQ ID NO: 125 or
at least 95%
sequence identity to a functional fragment thereof A carrier can comprise an
amino acid
sequence having at least 99% sequence identity to the amino acid sequence set
forth in any of
SEQ ID NO: 106 ¨ SEQ ID NO: 125 or at least 99% sequence identity to a
functional fragment
thereof. A carrier can comprise the amino acid sequence set forth in SEQ ID
NO: 106 or SEQ ID
NO: 107 or a functional fragment thereof
[0270] A carrier of the present disclosure can be derived from a domain I
of an exotoxin.
The exotoxin can be Cholix. A carrier that is derived from a Cholix domain I
can comprise at
least one but no more than 20 beta strands. A carrier that is derived from a
Cholix domain I can
comprise at least one but no more than 15 beta strands. A carrier that is
derived from a Cholix
domain I can comprise between 10 and 15 beta strands. A carrier that is
derived from a Cholix
domain I can comprise at least one but less than 10 a-helices. A carrier that
is derived from a
Cholix domain I can comprise between 1 and 5 a-helices.
[0271] A carrier of the present disclosure can comprise an amino acid
fragment of Cholix
domain I that can enable, promote, and/or enhance apical entry of the Cholix
derived carrier into
epithelial cells such as polarized gut epithelial cells. Such a fragment can
comprise the amino
acid sequence set forth in SEQ ID NO: 148 and can promote and/or enhance
apical entry of the
Cholix derived carrier into epithelial cells on the apical epithelial/luminal
surface. This may
enhance the delivery and/or transport function of the carrier and increase the
amount cargo
molecules delivered and/or transported into and/or across an epithelial cell.
[0272] A carrier of the present disclosure can comprise an amino acid
fragment of Cholix
domain I that can enable, promote, and/or enhance apical-to-basal transcytosis
of a Cholix-
derived carrier as described herein. Such an amino acid fragment that enables,
promotes, and/or
enhances apical-to-basal transcytosis of the delivery construct can comprise
an amino acid
sequence set forth in SEQ ID NO: 149 or SEQ ID NO: 150. This may enhance the
delivery
and/or transport function of the carrier and increase the amount cargo
molecules delivered and/or
transported into and/or across an epithelial cell. For example, a carrier
comprising such fragment
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with SEQ ID NO: 149 or SEQ ID NO: 150 can increase the amount cargo molecules
delivered
and/or transported to a basal compartment. This may further enhance basal
release of the carrier.
[0273] A carrier of the present disclosure can comprise an amino acid
fragment of Cholix
domain I that can enable, promote, and/or enhance early and/or late endosomal
sorting, thereby
enabling, promoting, and/or enhancing transport of the Cholix-derived carrier
to a supranuclear
region within an epithelial cell. Such a peptide fragment of Cholix domain I
comprising the
amino acid sequence set forth in SEQ ID NO: 151 and can enable, promote,
and/or enhance early
endosomal sorting of a Cholix-derived delivery construct as described herein.
Supranuclear
regions that may be targeted using such a carrier can include the
endoplasmatic reticulum, the
Golgi apparatus, and/or endosomes. Thus, a carrier capable of accessing such
region can provide
efficient delivery of cargo to such regions.
[0274] A carrier of the present disclosure can comprise an amino acid
fragment of Cholix
domain I that can enable, promote, and/or enhance complete transcytosis of the
Cholix-derived
delivery construct across an intact epithelial layer such as the gut
epithelium. Such a fragment
comprising the amino acid sequence set forth in SEQ ID NO: 152 can enable,
promote, and/or
enhance complete transcytosis of the Cholix-derived delivery construct by
enabling basal release
of the carrier and/or the delivery construct from the epithelial cell.
Complete transcytosis of the
Cholix-derived delivery construct can be determined, for example, by measuring
the presence of
the delivery construct in a basolateral compartment or the lamina prop/a. The
ability of such
carriers to delivery cargo across intact epithelial cell layers can be of high
significance as it
allows the oral administration of drugs that would not be able to cross such
epithelial layers by
themselves.
[0275] As described herein, the present disclosure contemplates the
surprising finding that a
carrier that is derived from a Cholix domain I and that lacks a Cholix domain
II, domain lb, and
domain III, is sufficient for rapid and efficient apical-to-basal transcytosis
(e.g., and sufficient for
rapid and efficient apical-to-basal transport of cargo via transcytosis).
Furthermore, it is shown
that certain portions of the amino acid sequence of Cholix domain I can have
specific functions
related to apical-to-basal transcytosis across an epithelial cell, and/or the
delivery into the cytosol
or interior of an epithelial cell. A carrier of the present disclosure can
comprise any one of the
amino acid sequences set forth in SEQ ID NO: 4 ¨ SEQ ID NO: 125. A carrier of
the present
disclosure can comprise one or more of the functional amino acid peptide
fragments of a Cholix
domain I set forth in SEQ ID NO: 148 ¨ SEQ ID NO: 152. A carrier of the
present disclosure can
comprise a Cholix domain I with an amino acid sequence set forth in SEQ ID NO:
4 and/or SEQ
ID NO: 5.
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[0276] The present disclosure further contemplates carriers that comprise
one or more
functional fragments of a Cholix domain I. The functional fragments can be in
the same order as
in the mature Cholix amino acid sequence, or the functional fragments can be
in a different order
without impairing the functions(s) of such functional fragments. Thus, a
carrier can comprise one
or more of the functional amino acid sequences derived a Cholix domain I and
set forth in SEQ
ID NO: 148 ¨ SEQ ID NO: 152. Such amino acid sequences can be linked together
to form a
polymeric polypeptide comprising a plurality of Cholix domain I derived
peptide fragments.
Such a carrier of the present disclosure can comprise one or more amino acid
sequences set forth
in SEQ ID NO: 148 ¨ SEQ ID NO: 152, or any combination thereof, that form a
polymeric
polypeptide capable of efficient transcytosis across epithelial layers such as
the gut epithelium.
Such a polymeric peptide can comprise a plurality of amino acid fragment
derived from Cholix
domain I and such polymeric polypeptide can be used instead of and/or in
addition to a Cholix
domain I polypeptide or a truncated version thereof Such a non-naturally
occurring synthetic
polymeric peptide can possess superior or inferior transcytosis capabilities
when used as a
delivery construct compared to Cholix domain I or a truncated version thereof.
The functionality
of such synthetic polypeptides can depend on several factors such spatial
structure and geometry,
stability, and/or the cargo that may be coupled to such polypeptide.
[0277] A carrier of the present disclosure may not be significantly altered
in a chemical,
structural, and/or conformational manner during the transcytosis process
across an epithelial cell.
Thus, the Cholix toxin-derived carrier as disclosed herein (including
polymeric peptides
comprising a plurality of Cholix-derived amino acid fragments) can be used as
an efficient
delivery vehicle for various cargo molecules (e.g., therapeutic cargo
molecules) as described
herein. A Cholix-derived carrier as described herein does not contain the
domains II and III, but
instead is attached to one or more cargo moieties (e.g., therapeutic cargo
molecules) without
having reduced transport and/or transcytosis capabilities compared to mature
ntChx.
[0278] Transport of a carrier as described herein across an epithelial
layer (e.g., a gut
epithelium) can comprise multiple steps. Transport of a delivery construct
(e.g., a Cholix-derived
delivery construct) can comprise elements of Cholix domain I functioning in a
multistep process.
For example, transport or transcytosis can include apical endocytosis,
vesicular trafficking
involving apical, basal, and/or supra-nuclear regions of enterocytes, and
release from the basal
membrane to reach the lamina propria. Furthermore, a Cholix-derived delivery
construct as
described herein can utilize a receptor-mediated-type endocytosis process.
Receptor-mediated
endocytosis can involve an amino acid sequence having at least 80% sequence
identity to the
amino acid set forth in SEQ ID NO: 148 or a fragment or derivative thereof,
which, can provide
access to an early endosomal vesicular compartment in the apical portion of
enterocytes, e.g., via
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endocytosis. An amino acid sequence having at least 80% sequence identity to
the amino acid set
forth in SEQ ID NO: 151 or a fragment or derivative thereof, can allow,
promote, or enhance the
movement of a Cholix-derived delivery construct to a supranuclear region
consistent with a
sorting site in the cell for secretory events. Movement of a delivery
construct comprising a
Cholix-derived carrier to the basal compartment of the cells can be more
efficient when the
carrier comprises an amino acid sequence having at least 80% sequence identity
to the amino
acid set forth in SEQ ID NO: 149 or SEQ ID NO: 150, or a fragment or
derivative thereof An
amino acid sequence having at least 80% sequence identity to the amino acid
set forth in SEQ ID
NO: 149, a fragment or derivative thereof, can provide a mechanism for
secretion from the basal
membrane that releases an intact and functional delivery construct (e.g.,
including the cargo
moiety) into a basolateral compartment or the lamina propria from where it can
reach various
other locations (e.g., cells, tissues or organs) within an organism (e.g., in
a human or in a rodent).
[0279] In addition to leaving the carrier unaltered or unmodified during
transcytosis,
transport of a delivery construct as disclosed herein across an epithelial
barrier (e.g., an intact
intestinal epithelium) generally does not involve enterocyte intoxication or
disruption. Thus, a
delivery construct as disclosed herein can comprise a Cholix domain I, a
fragment or truncated
version thereof (e.g., any one of SEQ ID NO: 6 ¨ SEQ ID NO: 125), or a
polymeric peptide
comprising a plurality of amino acid fragments derived from a Cholix domain I
(e.g., SEQ ID
NO: 148 ¨ SEQ ID NO: 152), and wherein the other domains (e.g., domains II,
domain lb, and
III) can be replaced by various other moieties, such as spacers, heterologous
cargos (e.g.,
therapeutic and/or biologically active cargo), small molecules, nucleic acids
(e.g., aptamers or
interfering RNAs), or any combination thereof, as further described herein.
[0280] A carrier of the present disclosure can be used to deliver various
cargo molecules
into and/or across epithelial cells in an efficient manner, e.g., when
comprising an amino acid
sequence having at least 80% sequence identity to an amino acid sequence set
forth in any one of
SEQ ID NO: 4 ¨ SEQ ID NO: 125). Thus, a carrier of the present disclosure can
enable efficient
endocytosis on the apical site and transport into the interior of an
epithelial cell (e.g., an
enterocyte and/or a polarized gut epithelial cell) such as an intracellular
vesicle or compartment
or the cytosol and/or a supranuclear region. Such a carrier can comprise an
amino acid sequence
set forth in any one of SEQ ID NO: 30¨ SEQ ID NO: 125. Thus, constructs for
delivery of cargo
molecules into epithelial cells may comprise a truncated Cholix domain I or
fragment of a Cholix
domain I that does not comprise the amino acid sequence set forth in SEQ ID
NO: 151, and/or
the amino acid amino acid sequence set forth in SEQ ID NO: 152, or any
combination thereof
[0281] A carrier of the present disclosure can comprise one or more
potential glycosylation
sites. The one or more glycosylation sites can be located within a Cholix
domain I (e.g., SEQ ID
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NO: 4 or SEQ ID NO: 5). A carrier as described herein can comprise the amino
acid sequence
set forth in SEQ ID NO: 5, wherein the asparagine residues N98, N154, N165,
N224, or any
combination thereof, can be potential glycosylation sites. Variation or
mutation of one or more
of these amino acid residues that can act as glycosylation sites can affect or
reduce a function
related to transcytosis of a delivery construct. TABLE 3 shows exemplary
functional peptide
fragments of Cholix domain I that were identified to provide one or more
functions related to
apical-to-basal transcytosis.
TABLE 3¨ Exemplary Functional Peptide Fragments Derived from Cholix Domain I
SEQ ID NO Amino acid sequence
SEQ ID NO: 148 ELDQQRNIIEVPKLYSID
SEQ ID NO: 149 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLD
SEQ ID NO: 150 VEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLD
SEQ ID NO: 151 DLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK
SEQ ID NO: 152 KAAQKEGSRHKRWAHWHTGLAL
As further described herein, a carrier of the present disclosure can comprise
one or more
functional fragments. Such functional fragments can include those listed in
TABLE 3. Thus, a
carrier can comprise an amino acid sequence having at least 80% sequence
identity to one or
more of the amino acid sequences set forth in SEQ ID NO: 148 ¨ SEQ ID NO: 152.
A carrier can
comprise an amino acid sequence having at least 90% sequence identity to one
or more of the
amino acid sequences set forth in SEQ ID NO: 148 ¨ SEQ ID NO: 152. A carrier
can comprise
an amino acid sequence having at least 95% sequence identity to one or more of
the amino acid
sequences set forth in SEQ ID NO: 148 ¨ SEQ ID NO: 152. A carrier can comprise
an amino
acid sequence having at least 99% sequence identity to one or more of the
amino acid sequences
set forth in SEQ ID NO: 148 ¨ SEQ ID NO: 152. A carrier as described herein
can comprise at
least one of the amino acid sequences set forth in SEQ ID NO: 148 ¨ SEQ ID NO:
152. A carrier
as described herein can comprise at least two of the amino acid sequences set
forth in SEQ ID
NO: 148 ¨ SEQ ID NO: 152. A carrier as described herein can comprise at least
three of the
amino acid sequences set forth in SEQ ID NO: 148 ¨ SEQ ID NO: 152. A carrier
as described
herein can comprise at least four of the amino acid sequences set forth in SEQ
ID NO: 148 ¨
SEQ ID NO: 152. A carrier as described herein can comprise all of the amino
acid sequences set
forth in SEQ ID NO: 148 ¨ SEQ ID NO: 152. Thus, a carrier as described herein
can comprise
the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5, or a
functional fragment
thereof.
[0282] As disclosed herein, the PE exotoxin domain I (SEQ ID NO: 137)
comprises amino
acids 1-252 of SEQ ID NO: 135 and has been described as a "receptor binding
domain" that
functions as a ligand for a cell surface receptor and mediates binding of PE
to a cell. Thus, a
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carrier of the present disclosure can be derived from PE and can comprise the
receptor binding
domain polypeptide having the amino acid sequence set forth in SEQ ID NO: 137.
A carrier can
comprise an amino acid sequence with greater than 50% homology to SEQ ID NO:
137. A
carrier can comprise an amino acid sequence with greater than 60% homology to
SEQ ID NO:
137. A carrier can comprise an amino acid sequence with greater than 70%
homology to SEQ ID
NO: 137. A carrier can comprise an amino acid sequence with greater than 80%
homology to
SEQ ID NO: 137. A carrier can comprise an amino acid sequence with greater
than 90%
homology to SEQ ID NO: 137. A carrier can comprise an amino acid sequence with
greater than
95% homology to SEQ ID NO: 137. Moreover, conservative or non-conservative
substitutions
can be made to the amino acid sequence of SEQ ID NO: 7, so long as the ability
to mediate
binding of the delivery construct to a cell is not substantially eliminated. A
carrier can comprise
a receptor binding domain that is a truncated version of SEQ ID NO: 137. A
carrier can comprise
a receptor binding domain polypeptide wherein one or more amino residues of
SEQ ID NO: 137
are deleted. A carrier can comprise a receptor binding domain polypeptide
wherein one or more
amino residues of SEQ ID NO: 137 are substituted with another amino acid.
[0283] A carrier of the present disclosure that is derived from a PE domain
I can comprise
an amino acid sequence having at least 80% identity to the amino acid sequence
of SEQ ID NO:
137 or at least 80% identity to a functional fragment thereof. A carrier can
comprise a deletion or
mutation in one or more of amino acid residues 1-252 of SEQ ID NO: 137. A
carrier can
comprise an amino acid sequence having at least 90% sequence identity to the
amino acid
sequence of 1-252 of SEQ ID NO: 137 or at least 90% sequence identity to a
functional fragment
thereof. A carrier can comprise an amino acid sequence having at least 95%
sequence identity to
the amino acid sequence of 1-252 of SEQ ID NO: 137 or at least 95% sequence
identity to a
functional fragment thereof A carrier can comprise an amino acid sequence
having at least 99%
sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 137 or at
least 99%
sequence identity to a functional fragment thereof A carrier can comprise an
amino acid
sequence having 100% sequence identity to the amino acid sequence of 1-252 of
SEQ ID NO:
137 or 100% sequence identity to a functional fragment thereof.
[0284] A carrier (e.g., a bacterial carrier receptor binding domain) of the
present disclosure
can be a polypeptide derived from PE and having: at most 5 amino acid
residues; at most 10
amino acid residues; at most 15 amino acid residues; at most 20 amino acid
residues; at most 30
amino acid residues; at most 40 amino acid residues; at most 50 amino acid
residues; at most 60
amino acid residues; at most 70 amino acid residues; at most 80 amino acid
residues; at most 90
amino acid residues; at most 100 amino acid residues; at most 110 amino acid
residues; at most
120 amino acid residues; at most 130 amino acid residues; at most 140 amino
acid residues; at
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most 150 amino acid residues; at most 160 amino acid residues; at most 170
amino acid residues;
at most 180 amino acid residues; at most 190 amino acid residues; at most 200
amino acid
residues; at most 210 amino acid residues; at most 220 amino acid residues; at
most 230 amino
acid residues; at most 240 amino acid residues; at most 250 amino acid
residues; at most 260
amino acid residues; and at most 265 amino acid residues of SEQ ID NO: 137.
The bacterial
carrier receptor binding domain can be a polypeptide derived from PE and
having at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more sequence homology
with SEQ
ID NO: 137. The bacterial carrier receptor binding domain can be a polypeptide
derived from PE
and having at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%
sequence
homology with SEQ ID NO: 137. The amino acid residues can be consecutive. The
amino acid
residues can be non-consecutive.
[0285] A carrier of the present disclosure can comprise a binding domain
that is an
artificially synthesized polypeptide having at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%,
90%, 95%, 99%, or more amino acid sequence homology to PE domain I. The
carrier comprising
a receptor binding domain can be a synthetic polypeptide having at most 10%,
20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, or 99% amino acid sequence homology to PE domain
I set
forth in SEQ ID NO: 137. The polypeptide can be synthesized using solid-phase
synthesis or
recombinant expression.
[0286] A carrier of the present disclosure capable of delivering cargo
across epithelial cells
(e.g., polarized epithelial cells) can comprise a receptor binding domain
polypeptide (e.g., a
domain I or a derivative thereof) wherein one or more amino acid residues of
one bacterial
carrier receptor binding domain polypeptide is replaced by one or more amino
acid residues of a
second bacterial carrier receptor binding domain polypeptide (also referred to
hereinafter as "a
hybrid receptor binding domain polypeptide"). For example, a carrier can
comprise an amino
acid sequence wherein one or more amino acid residues of SEQ ID NO: 4 are
replaced by one or
more amino acid residues of SEQ ID NO: 137. Alternatively, a carrier can
comprise an amino
acid sequence wherein one or more amino acid residues of SEQ ID NO: 137 are
replaced by one
or more amino acid residues of SEQ ID NO: 4. Furthermore, such a carrier can
comprise an
amino acid sequence wherein amino acid residues 77-87 of SEQ ID NO: 4 (Cholix)
are replaced
by amino acid residues of a second bacterial carrier receptor binding domain
polypeptide (e.g., a
PE domain I). A carrier can comprise an amino acid sequence wherein amino acid
residues 188-
236 of SEQ ID NO: 4 are replaced by amino acid residues of a second bacterial
carrier receptor
binding domain polypeptide. A carrier can comprise an amino acid sequence
wherein amino acid
residues 69-71 of SEQ ID NO: 137 are replaced by amino acid residues of a
second bacterial
carrier receptor binding domain polypeptide. A carrier can also comprise an
amino acid sequence
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wherein amino acid residues 177-228 of SEQ ID NO: 137 are replaced by amino
acid residues of
a second bacterial carrier receptor binding domain polypeptide. Thus, a
carrier of the present
disclosure can comprise an amino acid sequence having at least 10%, 20%, 30%,
40%, 50%,
60%, 70%, 80%, 90%, 95%, 99%, or more sequence homology with SEQ ID NO: 4 and
at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more sequence
homology
with SEQ ID NO: 137.
[0287] A carrier of the present disclosure that is derived from a domain I
of an exotoxin can
further comprise a portion of an exotoxin translocation domain, or modified
translocation
domain elements. A translocation domain can be a domain II of an exotoxin. A
carrier of the
present disclosure that is derived from a domain I of an exotoxin can further
comprise a portion
of a non-toxic catalytic domain or modified non-toxic catalytic domain
elements. A non-toxic
catalytic domain can be a modified domain III of an exotoxin, e.g., those that
comprise one or
more amino acid variations and/or a deletion of one or more amino acid
residues rendering the
domain III non-toxic (e.g., an E581A substitution (e.g., SEQ ID NO: 3) or a
AE581deletion). A
translocation domain, or a modified translocation domain, and a non-toxic
catalytic domain, or a
modified non-toxic catalytic domain, can be derived from the same bacterial
toxin. Alternatively,
a translocation domain, or a modified translocation domain, and a non-toxic
catalytic domain, or
a modified non-toxic catalytic domain can be derived from a bacterial carrier
selected from the
group consisting of Cholix carrier (Cholix) and Pseudomonas exotoxin (PE),
botulinum toxin,
diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli entero-
toxin, shiga toxin, and
shiga-like toxin.
[0288] As described herein, Cholix domain II (SEQ ID NO: 126) comprises
amino acids
266-386 of SEQ ID NO: 1). A carrier of the present disclosure can comprise a
Cholix derived
carrier comprising the entire amino acid sequence of SEQ ID NO: 126, or can
comprise a
portion(s) of SEQ ID NO: 126. Further, conservative or non-conservative
substitutions can be
made to SEQ ID NO: 126. A representative assay that can routinely be used by
one of skill in the
art to determine whether a transcytosis domain has transcytosis activity is
described herein. A
carrier can comprise at least 1%, at least 5%, at least 10%, at least 20%, at
least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, or at least 99% amino acid residues of the entire amino
acid sequence of SEQ
ID NO: 126. A carrier of the present disclosure can comprise a truncated
Cholix domain II, e.g.,
those identified as Cholix425(SEQ ID NO: 129), Cholix415(SEQ ID NO: 130),
Cholix397(SEQ ID
NO: 131), Cholix386 (SEQ ID NO: 132), Cholix291(SEQ ID NO: 133), and
Cholix265(SEQ ID
NO: 4).
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[0289] As described herein, a PE domain II (SEQ ID NO: 138) comprises amino
acids 253-
364 of SEQ ID NO: 135). A carrier of the present disclosure can comprise a PE
carrier
comprising the entire amino acid sequence of SEQ ID NO: 137, or can comprise a
portion(s) of
SEQ ID NO: 137. For example, it is demonstrated herein that, similar to the
Cholix exotoxin
domain I, PE domain I can be sufficient for rapid and efficient apical-to-
basal transcytosis. Thus,
as described above for Cholix derived carriers, portion(s) of PE domain II can
be used as a
spacer to attach further payload, such as a heterologous cargo. Further,
conservative or non-
conservative substitutions can be made to SEQ ID NO: 137. A representative
assay that can
routinely be used by one of skill in the art to determine whether a
transcytosis domain has
transcytosis activity is described herein. As used herein, the transcytosis
activity is not
substantially eliminated so long as the activity is, e.g., at least 1%, at
least 5%, at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as
compared to a PE carrier
comprising the entire amino acid sequence of SEQ ID NO: 137. Thus, a carrier
of the present
disclosure can comprise a truncated PE domain II, e.g., those identified as
PE404(SEQ ID NO:
141), PE395(SEQ ID NO: 142), PE376(SEQ ID NO: 143), PE364(SEQ ID NO: 144),
PE277(SEQ
ID NO: 145), and PE252(SEQ ID NO: 146).
[0290] A carrier of the present disclosure can comprise a receptor binding
domain, and a
translocation domain (e.g., a domain II), or a modified translocation domain
(e.g., a modified
domain II), and can further comprise a non-toxic catalytic domain (e.g., a
domain III), or
modified non-toxic catalytic domain (e.g., a modified domain III). The non-
toxic catalytic
domain, or modified non-toxic catalytic domain can be derived from a bacterial
carrier selected
from the group consisting of Cholix carrier (Cholix) and Pseudomonas exotoxin
(PE), botulinum
toxin, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli
entero-toxin, shiga toxin,
and shiga-like toxin. In various embodiments, the translocation domain, or
modified
translocation domain, and the non-toxic catalytic domain, or modified non-
toxic catalytic
domain, are derived from the same bacterial toxin.
[0291] As described herein, Cholix domain III (SEQ ID NO: 128) comprises
amino acids
426-634 of SEQ ID NO: 1 and has been described as a catalytic domain
responsible for
cytotoxicity and includes an endoplasmic reticulum retention sequence. Domain
III mediates
ADP ribosylation of elongation factor 2 ("EF2"), which inactivates protein
synthesis. A carrier
that "lacks endogenous ADP ribosylation activity" or a "detoxified Cholix"
refers to any Cholix
derived carrier described herein (including modified variants) that does not
comprise the entire
amino acid sequence set forth in SEQ ID NO: 128 (e.g., a portion of a domain
III). Such a carrier
can comprise one or more modifications within SEQ ID NO: 128 in a manner which
detoxifies
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the molecule. For example, deletion of the glutamic acid (Glu) residue at
amino acid position
156 of SEQ ID NO: 128 detoxifies the molecule. In various embodiments, the
portion of SEQ ID
NO: 128 other than the ER retention signal can be replaced by another amino
acid sequence.
This amino acid sequence can itself be non-immunogenic, slightly immunogenic,
or highly
immunogenic. A highly immunogenic ER retention domain is preferable for use in
eliciting a
humoral immune response. For example, Cholix domain III is itself highly
immunogenic and can
be used in delivery constructs where a robust humoral immune response is
desired.
[0292] As described herein, PE Domain III (SEQ ID NO: 140) comprises amino
acids 405-
613 of SEQ ID NO: 3) and has been described as a catalytic domain responsible
for cytotoxicity
and includes an endoplasmic reticulum retention sequence. Domain III mediates
ADP
ribosylation of elongation factor 2 ("EF2"), which inactivates protein
synthesis. A PE derived
carrier that "lacks endogenous ADP ribosylation activity" or a "detoxified PE"
refers to any PE
described herein (including modified variants or derivatives) that does not
comprise SEQ ID NO:
140 and/or which has been modified within SEQ ID NO: 140 in a manner which
detoxifies the
molecule. For example, deletion of the glutamic acid (Glu) residue at amino
acid position 149 of
SEQ ID NO: 140 detoxifies the molecule. In various embodiments, the portion of
PE domain III
other than the ER retention signal can be replaced by another amino acid
sequence. This amino
acid sequence can itself be non-immunogenic, slightly immunogenic, or highly
immunogenic. A
highly immunogenic ER retention domain is preferable for use in eliciting a
humoral immune
response. For example, PE domain III is itself highly immunogenic and can be
used in delivery
constructs where a robust humoral immune response is desired.
[0293] The present disclosure contemplates carriers that can comprise a
receptor binding
domain polypeptide having the amino acid sequence derived from the sequence
set forth in SEQ
ID NO: 137, a translocation domain having the amino acid sequence derived from
the sequence
set forth in SEQ ID NO: 138, and a non-toxic catalytic domain having the amino
acid sequence
derived from the sequence set forth in SEQ ID NO: 140. The present disclosure
contemplates
carriers that can comprise a receptor binding domain polypeptide having the
amino acid
sequence derived from the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5,
a translocation
domain having the amino acid sequence derived from the sequence set forth in
SEQ ID NO: 126,
and a non-toxic catalytic domain having the amino acid sequence derived from
the sequence set
forth in SEQ ID NO: 128.
[0294] In addition to carriers comprising a domain I and/or portions of a
domain II and a
domain III of an exotoxin, the present disclosure provides carriers that can
comprise a Cholix
domain lb (SEQ ID NO: 127), or a portion thereof. Cholix domain lb (SEQ ID NO:
127) consists
of amino acids 387-425 of SEQ ID NO: 1. Thus, a carrier that is derived from a
domain I of an
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exotoxin, can further comprise the amino acid sequence set forth in SEQ ID NO:
127, or a
modified sequence truncated at an amino acid residue within SEQ ID NO: 127.
The herein
described PE domain lb (SEQ ID NO: 139) consists of amino acids 365-404 of SEQ
ID NO:
135. Thus, a PE derived carrier that comprises a receptor binding domain, and
a translocation
domain, or a modified translocation domain, and a non-toxic catalytic domain,
or modified non-
toxic catalytic domain, can further comprise the amino acid sequence set forth
in SEQ ID NO:
139, or a modified sequence truncated at an amino acid residue within SEQ ID
NO: 139.
[0295] A carrier of the present disclosure can comprise portion(s) of one
or more of a
domain II, a domain lb, or a domain III, wherein those portions (e.g., certain
amino acid
sequences thereof) can be part of a spacer as further described herein.
[0296] The methods and compositions of the present disclosure contemplate
carriers that
can comprise a first portion and a second portion, wherein the first portion
is derived from a first
exotoxin and the second portion is derived from a second exotoxin; and wherein
the carrier can
be coupled to a cargo (e.g., a heterologous cargo such as a biologically
active cargo). The first
exotoxin can be Cholix, and the second exotoxin can be PE. The first portion
can be derived
from a domain I, a domain II, a domain lb, or a domain III of Cholix, or any
combination
thereof. The first portion can comprise an amino acid sequence having at least
80% sequence
identity to any one of the amino acid sequences set forth in SEQ ID NO: 1 ¨
SEQ ID NO: 133, a
functional fragment thereof, or any combination thereof. The first portion can
comprise an amino
acid sequence having at least 80% sequence identity to any one of the amino
acid sequences set
forth in SEQ ID NO: 148 ¨ SEQ ID NO: 152, a functional fragment thereof, or
any combination
thereof. The first portion can comprise an amino acid sequence having at least
80% sequence
identity to any one of the amino acid sequences set forth in SEQ ID NO: 4, SEQ
ID NO: 5, SEQ
ID NO: 10, or SEQ ID NO: 11, a functional fragment thereof, or any combination
thereof The
second portion can be derived from a domain I, a domain II, a domain lb, or a
domain III of PE,
or any combination thereof The second portion can comprise an amino acid
sequence having at
least 80% sequence identity to any one of the amino acid sequences set forth
in SEQ ID NO: 137
¨ SEQ ID NO: 145, a functional fragment thereof, or any combination thereof.
The first portion
can be chemically coupled or recombinantly coupled to the second portion. The
first portion can
further be directly or indirectly coupled to the second portion. Such a
carrier can comprise an
amino acid sequence having at least 80% sequence identity to the amino acid
sequence SEQ ID
NO: 146 or SEQ ID NO: 147.
[0297] Generally, a carrier of the present disclosure can comprise a
polypeptide, wherein the
polypeptide can comprise at least 110 amino acid residues of a domain I of the
exotoxin. A
carrier can comprise at least 120 amino acid residues of a domain I of the
exotoxin. A carrier can
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comprise at least 130 amino acid residues of a domain I of the exotoxin. A
carrier can comprise
at least 140 amino acid residues of a domain I of the exotoxin. A carrier can
comprise at least
150 amino acid residues of a domain I of the exotoxin. A carrier can comprise
at least 50
contiguous amino acid residues of the domain I of the exotoxin. A carrier can
comprise at least
60 contiguous amino acid residues of the domain I of the exotoxin. A carrier
can comprise at
least 75 contiguous amino acid residues of the domain I of the exotoxin. A
carrier can comprise
at least 100 contiguous amino acid residues of the domain I of the exotoxin. A
carrier can
comprise at least 150 contiguous amino acid residues of the domain I of the
exotoxin.
[0298] The methods and compositions of the present disclosure contemplate
carriers that
can comprise on or more modifications at the N-terminal. Such a modification
can comprise at
least one N-terminal methionine residue. The at least one N-terminal
methionine residue can be
part of an N-cap as described herein. A carrier comprising an N-cap can
further comprise one or
more amino acid variations in the first 5-10 amino acid residues compared to a
reference
sequences. Thus, one or more of the first N-terminal amino acid residues of
the amino acid
sequence set forth in SEQ ID NO: 1 can be substituted with other amino acid
residues, as long as
the consensus sequence that can define a functional Cholix is not altered. In
addition to such
amino acid variations, a carrier described herein comprising an N-cap can
further comprise an N-
terminal methionine residue. An N-cap can also only comprise an addition of an
N-terminal
methionine residue. Exemplary carriers of the present disclosure that comprise
such N-cap (e.g.,
an additional N-terminal methionine) are set forth in any one of SEQ ID NO: 5,
SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, SEQ ID NO: 125. As
described herein, functional variants of such carrier can comprise an amino
acid sequence having
at least 80% sequence identity to an amino acid sequence set forth in any one
of SEQ ID NO: 5,
SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, SEQ
ID
NO: 125, or 80% sequence identity to a functional fragment thereof
[0299] Generally, and as further described herein, a "Cholix" (also
referred to herein as
Cholix toxin or Cholix exotoxin) can encompass a variety of functional
variants (e.g., a
functional genus), wherein the functional variants can comprise one or more
variations is their
amino acid sequence relative to SEQ ID NO: 1 as disclosed herein. Thus, in the
present
disclosure, the Cholix toxin having the amino acid sequence set forth in SEQ
ID NO: 1 is used as
the reference sequence when referred to Cholix. However, as described herein,
the present
disclosure is not limited to the Cholix having the amino acid sequence set
forth in SEQ ID NO: 1
but instead encompasses all Cholix variants that fall within the functional
genus of Cholix. For
example, a first Cholix domain I polypeptide (e.g., a first carrier) can
comprise the amino acid
sequence set forth in SEQ ID NO: 4, and a second Cholix domain I polypeptide
(e.g., a second
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carrier) can comprise the amino acid sequence set forth in SEQ ID NO: 5,
wherein both the first
polypeptide and the second polypeptide are capable of carrying out the same
functions, e.g.,
transcytosis across an epithelial cell, and interact with the same receptors,
such as ribophilin 1,
5EC24, CK-8, TMEM132, GRP75, ERGIC-53, and/or perlecan. As described herein, a
first
carrier and a second carrier can be produced in the same expression system
(e.g., a bacterial
expression system such as E. coli or a mammalian expression system such as a
CHO cell). In
other cases, and as described herein, a first carrier and a second carrier are
produced in a
different expression system (e.g., a bacterial or a mammalian expression
system).
[0300] A carrier of the present disclosure can comprise properties that
allow interactions
with endogenous receptors and/or accessing an endogenous transport and
transcytosis system.
Thus, a carrier of the present disclosure that is derived from Cholix domain I
and comprises an
amino acid sequence set forth in any one of SEQ ID NO: 4 ¨ SEQ ID NO: 125 or
SEQ ID NO:
148 ¨ SEQ ID NO: 152 can interact with one or more endogenous receptors. Such
endogenous
receptors can include TMEM132A, GPR75, ERGIC-53, and/or perlecan, and any
combination
thereof. Such interaction(s) can provide for (e.g., apical-to-basal)
transcytosis across an epithelial
cell and/or transport to the interior of an epithelial cell. These
interactions allow rapid and
efficient delivery. These interactions further provide transport mechanisms
that may not alter the
carrier of the cell that a carrier is delivered into or transported across.
For example, carriers
described herein do not show any chemical modifications upon release from the
basal membrane
of an epithelial cell, suggesting that the carriers of the present disclosure
may harness one or
more endogenous transport system to deliver cargo into and/or across
epithelial cells. Receptors
that a carrier and/or a delivery construct comprising a carrier can interact
with include, but are
not limited to, any one of ribophilin 1, 5EC24, CK-8, TMEM132, GRP75, ERGIC-
53, or
perlecan, or any combination thereof. For example, the interaction of a
carrier and/or a delivery
construct comprising a carrier with ERGIC-53 (also referred to as LAMN1) can
be an integral
part of the endocytosis and/or transcytosis process as this receptor is the
only interacting protein
that may subverte in its cellular distribution following luminal application
of a carrier. Moreover,
and as demonstrated herein, ERGIC-53 (LAMN1) has been implicated in an
indirect retrograde
pathway from the Golgi to the ER, suggesting that this can be a pathway
described as both
efficient and rapid.
[0301] The present disclosure provides methods and compositions comprising
carriers that
allow rapid and efficient transport and delivery of cargo across cells such as
epithelial cells. A
carrier as described herein can transport cargo across an epithelial cell with
a transport rate of
about 10-10 cm/sec to about 102 cm/sec. A carrier can transport cargo across
an epithelial cell
with a transport rate of about 10-9 cm/sec to about 10-3cm/sec. A carrier can
transport cargo
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across an epithelial cell with a transport rate of about 10-8 cm/sec to about
10-4 cm/sec. A carrier
can transport cargo across an epithelial cell with a transport rate of about
10-7 cm/sec to about 10-
cm/sec. A carrier can transport cargo across an epithelial cell with a
transport rate of about 10-6
cm/sec. A carrier can transport cargo across an epithelial cell with a
transport rate of at least 10-8
cm/sec. A carrier can transport cargo across an epithelial cell with a
transport rate of at least 10-7
cm/sec. A carrier can transport cargo across an epithelial cell with a
transport rate of at least 10-6
cm/sec. A carrier can transport cargo across an epithelial cell with a
transport rate of at least 10-5
cm/sec. A carrier can transport cargo across an epithelial cell with a
transport rate of at least 10-4
cm/sec. A carrier can transport cargo across an epithelial cell with a
transport rate of at least 10-3
cm/sec. A carrier can transport cargo across an epithelial cell with a
transport rate of at least 10-2
cm/sec.
Delivery Constructs
[0302] The methods and compositions of the present disclosure provide
carrier molecules
that rapidly and efficiently transport cargo into and/or across epithelial
cells. Delivery and/or
transport of cargo can be achieved by coupling the cargo to a carrier as
described herein. Such a
construct can be referred to herein as a "delivery construct." As described
herein, the present
disclosure contemplates carriers that can comprise a small molecule, a
polypeptide, an aptamer, a
fragment thereof, or any combination thereof As described herein, a carrier
can be derived from
an exotoxin. The exotoxin can be Cholix or PE. A carrier can be coupled
directly or indirectly to
the cargo. A carrier can be covalently or non-covalently coupled to the cargo.
Thus, a delivery
construct can further comprise a spacer that links the carrier to the cargo.
The spacer can be any
molecule that links the carrier to the cargo and can comprise oligomeric or
polymeric spacers
(e.g., polyethylene glycol, etc.), and amino acids. Moreover, a delivery
construct comprising a
carrier coupled to a cargo and, optionally, a spacer and/or another functional
moiety, can be
produced synthetically or recombinantly (e.g., in E. coli or a CHO cell).
[0303] As disclosed herein, the terms "delivery constructs", "delivery
constructs", "toxin-
derived delivery constructs", "chimeric constructs", "proteins" and "fusion
proteins" can be used
interchangeably and can refer to constructs comprising at least one delivery
or carrier domain
(e.g., a Cholix or PE domain I derived carrier, a small molecule, an aptamer,
or any combination
thereof) and at least one heterologous cargo molecule such as a therapeutic
cargo molecule. The
term "heterologous cargo" can be referred to as unrelated to these exotoxins.
As further
described herein, toxicity (e.g., intoxication of enterocytes) of the
bacterial carrier (e.g., Cholix
or PE) may not be a necessary requirement for efficient transport of the
carrier across intact
epithelial layers such as the gut epithelium. Instead, it is demonstrated
herein that a carrier that is
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derived from a domain I (e.g., a truncated version of a domain I) of an
exotoxin such as Cholix
and PE is sufficient for rapid and efficient transcytosis across epithelial
cell (e.g., polarized
epithelial cells of a gut).
[0304]
Generally, a delivery construct (e.g., an isolated delivery construct)
comprises a
carrier that provides rapid and efficient delivery and/or transport of a cargo
to a certain location,
wherein the location can be an organ, a tissue, a cell, or a cellular
compartment. The cargo
molecule can be directly or indirectly coupled to the carrier. The cargo that
is coupled to the
carrier can be a heterologous cargo (e.g., not derived from the carrier
itself). Thus, a delivery
construct described herein can comprise a carrier coupled to a heterologous
cargo. The carrier
can comprise certain functions that allow repaid and efficient transport of
cargo to a location,
e.g., a location within an epithelial cell or a location(s) within a
basolateral compartment. A
carrier contemplated herein can be derived from an exotoxin. The exotoxin can
be Cholix or PE.
A carrier that is derived from a Cholix can comprise an amino acid sequence
having at least 80%
sequence identity to SEQ ID NO: 1, or at least 80% sequence identity to a
functional fragment
thereof. It is noted that a Cholix (also referred to herein as Cholix toxin or
Cholix exotoxin) can
encompass a variety of functional variants (e.g., a functional genus), wherein
the functional
variants can comprise one or more variations is their amino acid sequence
relative to SEQ ID
NO: 1 as disclosed herein. Thus, in the present disclosure, the Cholix toxin
having the amino
acid sequence set forth in SEQ ID NO: 1 is used as the reference sequence when
referred to
Cholix. However, as described herein, the present disclosure is not limited to
the Cholix having
the amino acid sequence set forth in SEQ ID NO: 1 but instead encompasses all
Cholix variants
that fall within the functional genus of Cholix. For example, a first Cholix
domain I polypeptide
(e.g., a first carrier) can comprise the amino acid sequence set forth in SEQ
ID NO: 4, and a
second Cholix domain I polypeptide (e.g., a second carrier) can comprise the
amino acid
sequence set forth in SEQ ID NO: 5, wherein both the first polypeptide and the
second
polypeptide are capable of carrying out the same functions, e.g., transcytosis
across an epithelial
cell, and interact with the same receptors, such as ribophilin 1, 5EC24 (can
also be referred to as
COPII coat complex component), cytokeratin-8 (CK-8), transmembrane protein 132
(TMEM132), glucose regulated protein 75 (GRP75), endoplasmatic reticulum Golgi
intermediate compartment 53 (ERGIC-53, the number 53 may refer to its
molecular weight of
approximately 53 kDa), and/or perlecan (also referred to as basement membrane-
specific heparan sulfate proteoglycan core protein or HSPG). As described
herein, a first carrier
and a second carrier can be produced in the same expression system (e.g., a
bacterial expression
system such as E. coli or a mammalian expression system such as a CHO cell).
As described
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herein, a first carrier and a second carrier can be produced in a different
expression system (e.g.,
a bacterial or a mammalian expression system).
[0305] Importantly, the delivery constructs contemplated herein can provide
advantages
over conventional delivery modalities. Such advantages can include, but are
not limited to: a) aid
in the production of the delivery construct; b) aid in the refolding of the
chimera construct; c) aid
in the formulation of the delivery construct; d) aid in reducing the
sensitivity of the cargo to
proteolytic destruction; e) improve the stability of the delivery construct
during storage; f) in
embodiments wherein the bacterial carrier elements of domain I are coupled to
the heterologous
(e.g., a biologically active) cargo without a spacer, or with a non-cleavable
spacer, the bacterial
carrier elements of domain I can function to retain the chimera to selected
locations in the body
following transcytosis that results in greater exposure of a biologically
active (or diagnostic)
cargo to specific cells to provide improved pharmacodynamics; g) in
embodiments wherein the
bacterial carrier elements of domain I are coupled to a heterologous (e.g.,
biologically active)
cargo with a spacer that is cleavable by an enzyme present at a basolateral
membrane of an
epithelial cell, or an enzyme present in the plasma of the subject, such
cleavage will allow the
heterologous (e.g., biologically active) cargo to be released from the
remainder of the construct
soon after transcytosis across the epithelial membrane; h) the direct delivery
of the heterologous
cargo to the interior of an epithelial cell such as an intracellular vesicle
or compartment or the
cytosol or a supranuclear region of an epithelial cell; i) the direct delivery
of the heterologous
(e.g., biologically active) cargo to the submucosal-GI space and hepatic-
portal system can reduce
the systemic toxicity observed when the cargo is administered by parenteral
routes, as well as
enabling access to the submucosal target biology that would be difficult to
target via non-oral or
GI routes; j) by using endogenous transport and delivery mechanisms, the
delivery constructs
disclosed herein do not damage the epithelial layer; k) once transported
across the GI epithelium,
the delivery construct or the biologically active cargo will exhibit an
extended serum half-life
compared to the biologically active cargo in its non-fused state; 1) oral
administration of the
delivery construct can deliver an increased effective concentration of the
delivered biologically
active cargo to the liver of the subject than is observed in the subject's
plasma; and m) the ability
to deliver the biologically active cargo to a subject without using a needle
to puncture the skin of
the subject, thus improving such subjects' quality of life by avoiding pain or
potential
complications associated therewith, in addition to improved patient/care-giver
convenience and
compliance.
[0306] The present disclosure provides methods and compositions for
delivery and transport
of cargo molecules across an epithelial cell (e.g., via transcytosis) and/or
into the interior of an
epithelial cell. The methods and compositions disclosed herein can comprise a
delivery
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construct, wherein the delivery construct comprises a carrier coupled to a
heterologous cargo
(e.g., via a spacer). The transport and delivery processes described herein
using the carriers of
the present disclosure can comprise endocytosis on the apical side of an
epithelial cell.
Depending on whether the carrier is configured to deliver cargo into or across
an epithelial cell,
the transport processes can comprise the release of the delivery construct on
the basal side.
Furthermore, various mechanisms can be involved in transporting cargo to those
various
locations. For example, delivery of cargo to an intracellular vesicle or
compartment or the
cytosol of an epithelial cell can comprise releasing a delivery construct
comprising a carrier
coupled to that cargo from a vesicle into the an intracellular vesicle or
compartment or the
cytosol. As another example, transcytosis of a delivery construct can include
vesicular
transcytosis and, as such, can comprise encapsulating the delivery construct
in a vesicle during
transcytosis such that the delivery construct may or may not be in contact
with the intracellular
cytosol.
[0307] The methods and compositions of the present disclosure can comprise
delivering a
cargo to a certain location such that the cargo remains at that location for a
certain amount of
time. For example, a cargo molecule can be retained at an intracellular or
basolateral location
that has been targeted using the compositions described herein. Retention can
cause the cargo
molecule to elicit a certain response or biological effect (e.g., a
therapeutic effect). Thus, the
present disclosure provides methods and compositions that allow delivery of
cargo to a location
within across an epithelial cell such the delivery construct (and the cargo)
is retained at that
location for a specific amount of time. Such retention can be modulated, e.g.,
by allowing the
cargo to be cleaved from the carrier, or by allowing the carrier to reversibly
or irreversibly bind a
certain protein (e.g., a receptor) that is present at that location.
[0308] The delivery constructs of the present disclosure can comprise a
carrier, wherein the
carrier can be configured to target a certain location inside or across an
epithelial cell. Such a
location can be an organ, a tissue, a cell, or a cellular compartment. By
targeting such locations,
the methods and compositions described herein can be used for various
applications, e.g., those
that include delivery of cargo across an intact epithelial membrane in vitro
or in vivo.
[0309] As described herein, a carrier can be coupled to a heterologous
cargo in any way
described herein. A delivery construct comprises a carrier that is coupled to
a heterologous cargo
via a spacer. The spacer can comprise any moiety recited herein, and can
comprise any one of
the amino acid sequences set forth in SEQ ID NO: 166 ¨ SEQ ID NO: 213. The
spacer can be a
cleavable spacer. The spacer can be a non-cleavable spacer. A spacer can
comprise the amino
acid sequence set forth in SEQ ID NO: 210, or a fragment or derivative
thereof.
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[0310] Generally, and as described herein, any carrier disclosed herein
(e.g., those listed in
TABLE 2 and TABLE 3) can be combined with any one of the cargo molecules
described
herein (e.g., those listed in TABLE 11 and TABLE 12), and, optionally, with
any spacer
described herein (e.g., those listed in TABLES 7-10 and those having an amino
acid sequence
set forth in SEQ ID NO: 207 ¨ SEQ ID NO: 213) to form a delivery construct.
Thus, a carrier
described herein can be derived from an exotoxin. A carrier can be derived
from a domain I of an
exotoxin. The exotoxin can be Cholix or PE. A delivery construct contemplated
herein can
comprise a carrier derived from Cholix, wherein the carrier can comprise an
amino acid
sequence having at least 80% sequence identity to an amino acid sequence set
forth in any one of
SEQ ID NO: 1 ¨ SEQ ID NO: 125, coupled to a heterologous cargo. A carrier can
comprise an
amino acid sequence having at least 90% sequence identity to an amino acid
sequence set forth
in any one of SEQ ID NO: 1 ¨ SEQ ID NO: 125. A carrier can comprise an amino
acid sequence
having at least 95% sequence identity to an amino acid sequence set forth in
any one of SEQ ID
NO: 1 ¨ SEQ ID NO: 125. A carrier can comprise an amino acid sequence having
at least 99%
sequence identity to an amino acid sequence set forth in any one of SEQ ID NO:
1 ¨ SEQ ID
NO: 125. The exotoxin that a carrier can be derived from can be PE. Thus, a
delivery construct
comprises a carrier can comprise an amino acid sequence having at least 80%
sequence identity
to an amino acid sequence set forth in SEQ ID NO: 137, or a functional
fragment thereof,
coupled to a heterologous cargo.
[0311] Exemplary delivery constructs as described in the present disclosure
are shown
below in TABLE 4.
TABLE 4¨ Amino Acid Sequences of Exemplary Delivery Constructs
SEQ ID NO Amino acid sequence
SEQ ID NO: 153 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKIS
VDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRP
EHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVP
MDAIYNYITQQNCTLGDNWEGGSYETVAGTPKVITVKQGIEQ
KPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLT
DDLSCAYQAQNIVSLEVATRILFSHLDSVFTLNLDEQEPEVAE
RLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQT
SAGAQAGGGGSGGGGSGGGGSFPTIPLSRLEDNAMLRAHRL
HQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNR
EETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASD
SNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTN
SHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCG
SEQ ID NO: 154 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
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GVIHLD IT TENGTKTY SYNRKEGEFAINWLVPIGED SPA S IKI S
VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRP
EHNIAISWP S V S YKAAQKEGSRHKRWAHWHTGLALCWLVP
MDAIYNYITQQNCTLGDNWF GGSYETVAGTPKVITVKQGIEQ
KPVEQRIHF SKGNAMSALAAHRVCGVPLETLARSRKPRDLT
DDLSCAYQAQNIVSLEVATRILF SHLD SVF TLNLDEQEPEVAE
RL SDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQT
SAGAQAGGGGSGGGGSGGGGSMHS S ALLC CLVLLT GVRA SP
GQ GT Q SENSC THF'PGNLPNMLRDLRDAF SRVKTFFQMKDQL
DNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDP
DIKAHVNSLGENLKTLRLRLRRCHRFLPCENK SKAVEQVKN
AFNKLQEKGIYKAM SEED IF INYIEAYMTMKIRN
SEQ ID NO: 155 MVEEALNIF'DECRSPC S LTPEP GKP IQ SKL SIP SDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLD IT TENGTKTY SYNRKEGEFAINWLVPIGED SPA S IKI S
VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRP
EHNIAISWP S V S YKAAQKEGSRHKRWAHWHTGLALCWLVP
MDAIYNYITQQNCTLGDNWF GGSYETVAGTPKVITVKQGIEQ
KPVEQRIHF SKGGGGSGGGGSGGGGSMAALQK S VS SFLMGT
LAT S CLLLLALLVQ GGAAAP IS SHCRLDK SNF QQPYITNRTFM
LAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFT
LEEVLFPQ SDRF QPYMQEVVPFLARL SNRL S TCHIEGDDLHIQ
RNVQKLKD TVKKLGE S GEIKAIGELDLLFM S LRNAC I
SEQ ID NO: 156 MVEEALNIF'DECRSPC S LTPEP GKP IQ SKL SIP SDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLD IT TENGTKTY SYNRKEGEFAINWLVPIGED SPA S IKI S
VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRP
EHNIAISWP S V S YKAAQKEGSRHKRWAHWHTGLALCWLVP
MDAIYNYITQQNCTLGDNWF GGSYETVAGTPKVITVKQGIEQ
KPVEQRIHF SKMRS SKNVIKEFMRFKVRMEGTVNGHEFEIEG
EGEGRPYEGHNTVKLKVTKGGPLPF AWD IL SP QF QYGSKVY
VKHPAD IPDYKKL S FPEGFKWERVMNFED GGVVTVTQD S SL
QDGCFIYKVKFIGVNFP SD GPVMQKKTMGWEA S TERLYPRD
GVLKGEIHKALKLKDGGHYLVEFK SIYMAKKPVQLPGYYYV
D SKLD IT SHNEDYTIVEQYERTEGRHELFL
SEQ ID NO: 157 VEDELNIF'DECR SP C SLTPEPGKPIQ SKL SIP SDVVLDEGVLYY
SMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFG
VIHLD IT TENGTKTY S YNRKEGEF AINWLVPIGED SPA S IKI S V
DELDQQRNIIEVPKLY SIDLDNQTLEQWKTQ GNV SF SVTRPE
HNIAISWP S V S YKAAQKEGSRHKRWAHWHT GLALCWLVPM
DAIYNYITQQNC TLGDNWF GGSYETVAGTPKVITVKQGIEQK
PVEQRIHF SKGNAMSALAAHRVCGVPLETLARSRKPRDLTD
DLSCAYQAQNIVSLF VATRILF SHLD SVF TLNLDEQEPEVAER
LSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQT S
AGAQAADIL SLF CPDADK SCVASNNDQANINIESRSGRSYLPE
NRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNH
VAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYA
RIKEGTGEYGLPTRAERDARGVMLRVYIPRA SLERFYRTNTP
LENAEEHITQVIGHSLPLRNEAFTGPESAGGEDATVIGWDMAI
HAVAIP S TIP GNAYEELAIDEEAVAKEQ S I S TKPPYKERKDEL
KMRS SKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEG
HNTVKLKVTKGGPLPF AWD IL SP QF QYGSKVYVKHPADIPD
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YKKLSFPEGFKWERVMNFEDGGVVTVTQD S SLQDGCFIYKV
KFIGVNFP SD GPVMQKKTMGWEA S TERLYPRDGVLKGEIHK
ALKLKDGGHYLVEFK SIYMAKKPVQLPGYYYVD SKLDIT SH
NEDYTIVEQYERTEGRHHLFL
SEQ ID NO: 158 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLD IT TENGTKTY SYNRKEGEFAINWLVPIGED SPA S IKI S
VDEGGGGSGGGGSGGGGSFPTIPLSRLEDNAMLRAHRLHQL
AFDTYQEFEEAYIPKEQKYSFLQNPQTSLCF SE SIP TP SNREET
QQK SNLELLRISLLLIQ SWLEPVQFLRS VF AN SLVYGA SD SNV
YDLLKDLEEGIQTLMGRLEDGSPRTGQIF'KQ TY SKFD TN SHN
DDALLKNYGLLYCFRKDMDKVETFLRIVQ CRS VEGS C GE
SEQ ID NO: 159 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLD IT TENGTKTY SYNRKEGEFAINWLVPIGED SPA S IKI S
VDELD Q QRNIIEVPKLY S ID GGGGS GGGGS GGGGSFP TIPL SR
LEDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQT
SLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRS
VF AN SLVYGA SD SNVYDLLKDLEEGIQTLMGRLEDGSPRTG
QIF'K Q TY SKFD TN SHNDDALLKNYGLLYCFRKDMDKVETFL
RIVQ CRS VEGS C GF
SEQ ID NO: 160 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLD IT TENGTKTY SYNRKEGEFAINWLVPIGED SPA S IKI S
VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRP
EHNIAISWP S V S YKGGGGS GGGGS GGGGS FP TIPL SRLFDNA
MLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQT SLCF S
ESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFAN
SLVYGA SD SNVYDLLKDLEEGIQ TLMGRLEDGSPRTGQIF'KQ
TYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQC
RSVEGSCGF
SEQ ID NO: 161 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLD IT TENGTKTY SYNRKEGEFAINWLVPIGED SPA S IKI S
VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRP
EHNIAISWP S V S YKAAQKEGSRHKRWAHWHTGLGGGGS GG
GGSGGGGSFPTIPL SRLFDNAMLRAHRLHQLAFDTYQEFEEA
YIPKEQKYSFLQNPQT SLCF SESIPTPSNREETQQKSNLELLRIS
LLLIQ SWLEP VQFLR S VF AN SLVYGA SD SNVYDLLKDLEEGI
QTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGL
LYCFRKDMDKVETFLRIVQ CRS VEGS C GF
SEQ ID NO: 162 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLD IT TENGTKTY SYNRKEGEFAINWLVPIGED SPA S IKI S
VDELD Q QRNIIEVPKLY S IDLDNQ TLEQWKT Q GNV SF SVTRP
EHNIAISWP S V S YKAAQKEGSRHKRWAHWHTGLALCWLVP
MDAIYNYITQQNCTLGDNWF GGSYETVAGTPKGGGGSGGG
GS GGGGSFP TIPL SRLFDNAMLRAHRLHQLAFDTYQEFEEAYI
PKEQKYSFLQNPQT SLCF SESIPTPSNREETQQKSNLELLRISL
LLIQ SWLEP VQFLRS VFAN S LVYGA SD SNVYDLLKDLEEGIQ
TLMGRLED GSPRTGQIF'KQ TY SKFD TN SHNDDALLKNYGLL
YCFRKDMDKVETFLRIVQ CRS VEGS C GE
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SEQ ID NO: 163 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKIS
VDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRP
EHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVP
MDAIYNYITQQNCTLGDNWEGGSYETVAGTPKVITVKQGGG
GSGGGGSGGGGSFPTIPLSRLEDNAMLRAHRLHQLAFDTYQE
FEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLE
LLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDL
EEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKN
YGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF
SEQ ID NO: 164 MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLY
YSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPF
GVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKIS
VDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRP
EHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVP
MDAIYNYITQQNCTLGDNWEGGSYETVAGTPKVITVKQGIEQ
KPVEQRIHFSKGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLR
AHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIP
TPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLV
YGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYS
KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSV
EGSCGF
SEQ ID NO: 165 GVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQ
DAPFGVIRLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPA
SIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFS
VTRPEHNIAISWPSVSYKAGGGGSGGGGSGGGGSFPTIPLSRL
FDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTS
LCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSV
FANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQI
FKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRI
VQCRSVEGSCGF
[0312] The methods and compositions of the present disclosure can comprise
a delivery
construct comprising an amino acid sequence having at least 80% sequence
identity to an amino
acid sequence set forth in any one of SEQ ID NO: 153 ¨ SEQ ID NO: 165, or
having at least
80% sequence identity to a functional fragment thereof. A delivery construct
can comprise an
amino acid sequence having at least 90% sequence identity to an amino acid
sequence set forth
in any one of SEQ ID NO: 153 ¨ SEQ ID NO: 165, or having at least 90% sequence
identity to a
functional fragment thereof A delivery construct can comprise an amino acid
sequence having at
least 95% sequence identity to an amino acid sequence set forth in any one of
SEQ ID NO: 153 ¨
SEQ ID NO: 165, or having at least 95% sequence identity to a functional
fragment thereof A
delivery construct can comprise an amino acid sequence having at least 99%
sequence identity to
an amino acid sequence set forth in any one of SEQ ID NO: 153 ¨ SEQ ID NO:
165, or having at
least 99% sequence identity to a functional fragment thereof A delivery
construct can comprise
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an amino acid sequence having 100% sequence identity to an amino acid sequence
set forth in
any one of SEQ ID NO: 153 ¨ SEQ ID NO: 165, or having 100% sequence identity
to a
functional fragment thereof
[0313] Exemplary combinations of various carriers, spacers, and
heterologous cargos that
can form a delivery construct as described herein are shown below in TABLE 5.
TABLE 5¨ Exemplary Delivery Constructs
Carrier (SEQ ID NO) Spacer (SEQ ID NO) Biologically Active
Cargo
(SEQ ID NO)
SEQ ID NO: 4-5 No Spacer SEQ ID NOs: 214-220
SEQ ID NO: 4-5 SEQ ID NOs: 187-206 SEQ ID NOs: 214-220
SEQ ID NO: 4-5 SEQ ID NOs: 207-211 SEQ ID NOs: 214-220
SEQ ID NO: 137 No Spacer SEQ ID NOs: 214-220
SEQ ID NO: 137 SEQ ID NOs: 187-206 SEQ ID NOs: 214-220
SEQ ID NO: 137 SEQ ID NOs: 207-211 SEQ ID NOs: 214-220
[0314] A delivery construct of the present disclosure can interact with one
or more specific
proteins, enzyme, or receptors during transport and/or delivery across an
epithelial cell and/or
into the interior of an epithelial cell (e.g., a polarized gut epithelial
cell). The one or more
receptors can be endogenous receptors. Thus, the delivery constructs of the
present disclosure
can use endogenous receptor systems that provide for rapid efficient transport
and delivery of
cargo across an epithelial cell or an intact epithelium (e.g., a monolayer of
Caco-2 cells and/or an
intact gut epithelium of a subject), and/or to the interior of an epithelial
cell of an epithelium.
Delivery constructs comprising a carrier comprising an amino acid sequence set
forth in any one
of SEQ ID NO: 30 ¨ SEQ ID NO: 125 can enable delivery and transport of a
heterologous (e.g.,
a therapeutically or biologically active) cargo to the interior of an
epithelial cell, e.g., to the basal
side of an epithelial cell, and/or a supranuclear region (e.g., the
endoplasmatic reticulum, the
Golgi apparatus, and/or an endosome) of an epithelial cell. The interior of an
epithelial cell can
be an intracellular vesicle or compartment or the cytosol of the epithelial
cell. A cargo (e.g., a
heterologous cargo) can be delivered to the basal side of the epithelial cell
(e.g., a location or
compartment at the basal side). A heterologous cargo can be delivered to a
supranuclear region
of the epithelial cell. Transport of a delivery construct to the interior of
an epithelial cell can
comprise releasing the delivery construct from a vesicle that formed during
endocytosis of the
delivery construct on the apical surface of the epithelial cell. Delivery
and/or transport to a
location in the interior of a cell can comprise retaining the delivery
construct in a vesicle and/or
releasing the delivery construct from that vesicle, such that the delivery
construct can be in
contact with the cytosol of the epithelial cell (e.g., the construct may or
may not be in contact
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with the cytosol of the epithelial cell during transcytosis due to
encapsulation in the vesicle).
Thus, a carrier comprising a truncated version of Cholix domain I can be
released from a vesicle,
e.g., those comprising an amino acid sequence set forth in any one of SEQ ID
NO: 30 ¨ SEQ ID
NO: 107, or a functional fragment or derivative thereof.
[0315] Delivery constructs of the present disclosure comprising a carrier
comprising an
amino acid sequence set forth in any one of SEQ ID NO: 4 ¨ SEQ ID NO: 29, SEQ
ID NO: 129
¨ SEQ ID NO: 133, or SEQ ID NO: 141 ¨ SEQ ID NO: 145 can enable delivery and
transport of
a heterologous (e.g., a therapeutically or biologically active) cargo across
an epithelial cell.
Transport across an epithelial cell (e.g., a polarized gut epithelial cell)
can occur via transcytosis.
The transcytosis mechanism utilized by the herein described delivery
constructs is an
endogenous trafficking system including a variety of distinct receptors that
the delivery construct
interacts with. A carrier of a delivery construct can comprise the structural
elements that allow
these receptor interactions. The receptors that a carrier as disclosed herein
can interact with
include ribophilin 1, 5EC24, CK-8, TMEM132, GRP75, ERGIC-53, and perlecan, or
any
combination thereof. A carrier as described herein may not or may not
significantly interact with
clathrin or GPR78, or a combination thereof.
[0316] Using an endogenous system including those receptors can have
several advantages
over other transport mechanisms. Using an endogenous transport system can
include the
following advantages: (i) an intact layer of epithelial cells such as a
monolayer or an epithelium
in vivo can be crossed without damaging or disrupting the cells or monolayer
structure; (ii) rapid
and efficient delivery and transport can be achieved; (iii) the interaction of
distinct domains or
regions of an exotoxin derived construct with specific receptors allows
modulation of these
interaction in a way that allows to specifically target certain regions or
compartments within a
cell or within a subject. For example, delivery and transport (e.g., of a
heterologous cargo) to the
interior of an epithelial cell can be provided by using certain truncated
versions of an exotoxin
domain I, such as those having an amino acid sequence set forth in any one of
SEQ ID NO: 30 ¨
SEQ ID NO: 125, or functional fragment thereof The epithelial cell can be
located in the gut of
a subject (e.g., a rodent or a human). In various embodiments, delivery and
transport (e.g., of a
heterologous cargo) across an epithelial layer via transcytosis (e.g., by
using an endogenous
transcytosis system) can be provided by using certain truncated versions or
derivatives of an
exotoxin domain I, such as those having an amino acid sequence set forth in
any one SEQ ID
NO: 4 ¨ SEQ ID NO: 29, SEQ ID NO: 129 ¨ SEQ ID NO: 133, or SEQ ID NO: 141 ¨
SEQ ID
NO: 145. The ability of the herein described delivery constructs to rapidly
and efficiently deliver
therapeutically active and/or diagnostic cargo to those locations enables new
options for
treatment, prevention, and/or diagnosis of various diseases (e.g.,
inflammatory disease,
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autoimmune diseases, hormone-deficiency diseases, obesity and metabolic
disorders, and
cancer).
[0317] Delivery constructs of the present disclosure, in addition to a
carrier, a cargo, and,
optionally, a spacer, can further comprise one or more functional moieties. A
functional moiety
can be a detectable agent, an affinity handle (e.g., a clickable functional
groups such as an azide),
a barcode (e.g., a nucleic acid barcode), cell-penetrating agents, or other
functional moieties that
modulate the pharmacokinetic (PK) and/or pharmacodynamic (PD) profile of the
delivery
construct. A delivery construct can comprise a cell-penetrating agent. The
cell-penetrating agent
can be a peptide. The cell-penetrating agent can comprise polycations,
polyorganic acids,
endosomal releasing polymers, poly(2-propylacrylic acid), poly(2-ethylacrylic
acid), Tat peptide,
Arg patch, a knotted peptide, CysTAT, S19-TAT, R8 (SEQ ID NO: 73), pAntp, Pas-
TAT, Pas-
R8 (SEQ ID NO: 76), Pas-FHV, Pas-pAntP, F2R4 (SEQ ID NO: 79), B55, aurin, IMT-
P8, BR2,
OMOTAG1, OMOTAG2, pVEC, SynB3, DPV1047, C105Y, Transpotan, MTS, hLF, PFVYLI
(SEQ ID NO: 93), maurocalcine, imperatoxin, hadrucalin, hemicalcin, opicalcin-
1, opicalcin-2,
midkin(62-104), MCoTI-II, or a chlorotoxin. A cell-penetrating agent can be
coupled to a
delivery construct as described herein via the N- or the C-terminus. A cell-
penetrating agent can
provide access to a variety of cell types. A cell-penetrating agent can
provide additional
functionality, e.g., for therapeutic cargo delivery, once the delivery
construct has crossed and
epithelial layer (e.g., an epithelium of a subject).
[0318] The methods and compositions of the present disclosure contemplate
delivery
constructs that can form a multimer. A multimer comprising multiple delivery
constructs can be
formed in solution. A multimer can be formed by multimerization of the carrier
and/or the
heterologous cargo. The multimer can be a heteromer or a homomer. The homomer
can be a
homodimer. The homodimer can be formed by dimerization of the heterologous
cargo. For
example, a delivery construct comprising the amino acid sequence set forth in
SEQ ID NO: 217
can form a dimer. Dimerization of such a delivery construct can be due to
dimerization of the
cargo, e.g., IL-10 (e.g., SEQ ID NO: 217) in this case.
Insertion Site for Attachment of the Heterologous Cargo
[0319] The methods and compositions of the present disclosure can comprise
a delivery
construct comprising a carrier coupled to a cargo, such as a heterologous
cargo. A heterologous
(e.g., biologically active) cargo re can be attached to the carrier (e.g., a
small molecule, a
polypeptide, an aptamer, or a nucleic acid) by any method known by one of
skill in the art
without limitation. The heterologous cargo can be introduced into any portion
of the carrier that
does not disrupt the endocytosis and/or transcytosis activity of the carrier.
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[0320] The present disclosure provides delivery constructs that comprise a
polypeptide
carrier. Thus, a heterologous cargo can be directly coupled to the N-terminus
or C-terminus of
such a polypeptide carrier (e.g., a domain I or a truncated version thereof,
e.g., SEQ ID NO: 4 ¨
SEQ ID NO: 125). A heterologous cargo can be couple to the carrier via a side
chain of an amino
acid of the carrier receptor binding domain. A heterologous cargo can be
coupled to the carrier
with a cleavable spacer such that cleavage at the cleavable spacer(s)
separates the heterologous
cargo from the remainder of the delivery construct. A heterologous cargo can
be also a
polypeptide that comprises a short leader peptide that remains attached to the
polypeptide
following cleavage of the cleavable spacer. For example, the heterologous
cargo can comprise a
short leader peptide of greater than 1 amino acid, greater than 5 amino acids,
greater than 10
amino acids, greater than 15 amino acids, greater than 20 amino acids, greater
than 25 amino
acids, greater than 30 amino acids, greater than 50 amino acids, or greater
than 100 amino acids.
A biological active cargo can comprise a short leader peptide of less than 100
amino acids, less
than 50 amino acids, less than 30 amino acids, less than 25 amino acids, less
than 20 amino
acids, less than 15 amino acids, less than 10 amino acids, or less than 5
amino acids. A biological
active cargo can comprise a short leader peptide of between 1- 100 amino
acids, between 5-10
amino acids, between 10 to 50 amino acids, or between 20 to 80 amino acids.
[0321] As described herein, the present disclosure provides methods and
compositions
comprising carrier that are derived from a domain I of an exotoxin, wherein
the exotoxin can be
Cholix or PE. In native Cholix (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) and PE
(e.g., SEQ ID
NO: 135) the domain lb loop is not essential for any known activity of the
toxin, including cell
binding, translocation, ER retention or ADP ribosylation activity.
Accordingly, domain lb can be
deleted entirely, or modified to contain a heterologous cargo, e.g., a
biologically active
cargo. Thus, the heterologous cargo (e.g., biologically active cargo) can be
inserted into Cholix
or PE carrier domain lb. A heterologous cargo (e.g., biologically active
cargo), for example, can
be inserted into a Cholix derived carrier domain lb between the cysteines at
positions 395 and
402 that are not cross-linked. This can be accomplished by reducing the
disulfide linkage
between the cysteines, by deleting one or both of the cysteines entirely from
the lb domain, by
mutating one or both of the cysteines to other residues, for example, serine,
or by other similar
techniques. Alternatively, the biologically active cargo can be inserted into
the domain lb loop
between the cysteines at positions 395 and 402. In such embodiments, the
disulfide linkage
between the cysteines can be used to constrain the biologically active cargo
domain.
[0322] The methods and compositions described herein can comprise delivery
constructs
that are produced such that a heterologous cargo is expressed together with a
carrier (and,
optionally, a spacer) as a fusion protein (e.g., the delivery construct). In
such cases, the
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heterologous cargo can be inserted into the delivery construct by any method
known to one of
skill in the art without limitation. For example, amino acids corresponding to
the heterologous
cargo can be directly inserted into the receptor binding domain, with or
without deletion of
native amino acid sequences. Alternatively, a heterologous cargo may not be
expressed together
with a carrier (and, optionally, a spacer) as a fusion protein, the
heterologous cargo can be
coupled to the carrier by any suitable method known by one of skill in the
art, without limitation,
including peptide conjugation chemistry and/or click chemistry.
Spacers
[0323] The methods and compositions of the present disclosure can comprise
delivery
constructs comprising a carrier coupled to a cargo (e.g., a heterologous
cargo), wherein the
carrier is capable of delivering the heterologous cargo into and/or across an
epithelial cell in vitro
(e.g., an epithelial cell monolayer) or in vivo (e.g., a gut epithelium of a
subject). Such a carrier
can be coupled to a cargo in any way described herein. The carrier can be
directly or indirectly
coupled the cargo. The carrier can also be covalently or non-covalently
coupled to the cargo.
[0324] The present disclosure provides delivery constructs comprising a
carrier coupled to a
heterologous cargo via a spacer. A spacer can comprise any moiety recited
herein. A spacer can
be any molecule that links the carrier to the cargo and can comprise
oligomeric or polymeric
spacers (e.g., polyethylene glycol, etc.), other small molecule spacer (e.g.,
those derived from
dicarbonic acids such as succinic acid, aspartic acid, etc.) and amino acids
(including short
peptide sequences etc.). Thus, a "spacer," as described herein, generally
refers to a chemical
moiety that can be attached to or coupled to a molecule of the present
disclosure. A spacer can be
located between a first molecule and a second molecule. A spacer can connect,
attach, link, or
couple a first molecule (e.g., a polypeptide, small molecule, nucleic acid,
etc.) to a second
molecule (e.g., a polypeptide, small molecule, nucleic acid, etc.). A spacer
can reduce steric
hindrance between the first molecule and the second molecule. A spacer can be
an amino acid
sequence coupled to the C-terminus of a peptide or polypeptide. The amino acid
sequence of a
spacer as disclosed herein can be between 1-100 amino acid residues long. A
spacer can be
between 5-75 amino acid residues long. A spacer can be between 5-50 amino acid
residues long.
In some cases, a spacer is between 5-25 amino acid residues long. A carrier
can comprise any
one of the amino acid sequences set forth in SEQ ID NO: 4 ¨ SEQ ID NO: 125 is
coupled to a
spacer at its C-terminus (and the spacer can be further coupled to a
heterologous cargo via its C-
terminus). The spacer can be an amino acid spacer. The spacer can comprise any
of the amino
acid sequences set forth in SEQ ID NO: 166 ¨ SEQ ID NO: 213. The spacer can
comprise a
portion of a domain II, a domain lb, or a domain III of an exotoxin, or any
combination thereof
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For example, a delivery construct as described herein can comprise a carrier
comprising an
amino acid sequence set forth in any one of SEQ ID NO: 4 ¨ SEQ ID NO: 125,
coupled to a
spacer, wherein the spacer comprises amino acid residues 1-82 of SEQ ID NO:
126.
[0325] The present disclosure provides delivery constructs that comprise
one or more spacer
which are further described herein. A spacer can comprise an amino acid
sequence. A spacer can
comprise at most 82 amino acid residues of any one SEQ ID NO: 126. Thus, a
spacer can
comprise the first 82 amino acid residues of the amino acid sequence set forth
in SEQ ID NO:
126. The amino acid residues of the Cholix domain II can be contiguous amino
acid residues
(e.g., residues 1-82 of SEQ ID NO: 126).
[0326] Other spacer that can be used in combination with the herein
described methods and
compositions comprise those comprising an amino acid sequences set forth in
SEQ ID NO: 207
¨ SEQ ID NO: 211. A spacer comprises the amino acid sequence set forth in SEQ
ID NO: 210,
or a fragment or derivative thereof
[0327] The methods and compositions of the present disclosure can comprise
spacer that
can comprise a portion of a domain II, a domain lb, and/or a domain III of an
exotoxin. For
example, a carrier comprising an amino acid sequence set forth in any one of
SEQ ID NO: 4 ¨
SEQ ID NO: 125 can further be coupled (e.g., via the C-terminus) to a spacer,
wherein the spacer
comprises from about 80 to about 90 amino acid residues from any one of SEQ ID
NO: 126 ¨
SEQ ID NO: 128 and/or SEQ ID NO: 138 ¨ SEQ ID NO: 140. A spacer can comprise
at most 85
amino acid residues of any one of SEQ ID NO: 126¨ SEQ ID NO: 128 and/or SEQ ID
NO: 137
¨ SEQ ID NO: 139. A spacer can comprise at most 82 amino acid residues of any
one of SEQ ID
NO: 126 ¨ SEQ ID NO: 128 and/or SEQ ID NO: 137 ¨ SEQ ID NO: 139 A spacer can
comprise
at most 80 amino acid residues of any one of SEQ ID NO: 126 ¨ SEQ ID NO: 128
and/or SEQ
ID NO: 137¨ SEQ ID NO: 139. A spacer can comprise at most 50 amino acid
residues of any
one of SEQ ID NO: 126¨ SEQ ID NO: 128 and/or SEQ ID NO: 137¨ SEQ ID NO: 139. A
spacer can comprise at most 25 amino acid residues of any one of SEQ ID NO:
126 ¨ SEQ ID
NO: 128 and/or SEQ ID NO: 137 ¨ SEQ ID NO: 139.
[0328] A spacer can comprise at most 82 amino acid residues of any one SEQ
ID NO: 126.
A spacer can comprise the first 82 amino acid residues of the amino acid
sequence set forth in
SEQ ID NO: 126. The amino acid residues of the Cholix domain II can be
contiguous amino
acid residues (e.g., residues 1-82 of SEQ ID NO: 126).
[0329] A spacer of the present disclosure can be a cleavable spacer. A
spacer of the present
disclosure can be a non-cleavable spacer.
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Cleavable Spacers
[0330] The presently described methods and compositions allow for a
heterologous cargo
(e.g., a biologically or therapeutically active cargo) to be delivered to a
location inside or across
an epithelial cell, wherein the heterologous cargo can be coupled to the
carrier forming a
delivery construct as described herein. Such a delivery construct can further
comprise a spacer
that can indirectly couple a carrier to a cargo (e.g., a heterologous cargo).
A spacer as described
herein can be a cleavable spacer. The number of cleavable spacers present in a
delivery construct
depends, at least in part, on the location of the heterologous cargo in
relation to the delivery
construct and the nature of the heterologous cargo. When the heterologous
cargo can be
separated from the remainder of the delivery construct with cleavage at a
single spacer, the
delivery constructs can comprise a single cleavable spacer. Further, where the
heterologous
cargo is, e.g., a dimer or other multimer, each subunit of the biologically
active cargo can be
separated from the remainder of the delivery construct and/or the other
subunits of the
biologically active cargo by cleavage at the cleavable spacer.
[0331] A cleavable spacer can be cleaved by a cleaving enzyme that is
present at or near the
basolateral membrane of an epithelial cell. By selecting the cleavable spacer
to be cleaved by
such enzymes, the biologically active cargo can be liberated from the
remainder of the delivery
construct following transcytosis across the mucous membrane and release from
the epithelial cell
into the cellular matrix on the basolateral side of the membrane. Further,
cleaving enzymes could
be used that are present inside the epithelial cell, such that the cleavable
spacer is cleaved prior
to release of the delivery construct from the basolateral membrane, so long as
the cleaving
enzyme does not cleave the delivery construct before the delivery construct
enters the trafficking
pathway in the polarized epithelial cell that results in release of the
delivery construct and
biologically active cargo from the basolateral membrane of the cell.
[0332] A carrier of the present disclosure can be cleaved by an enzyme. The
enzyme that is
present at a basolateral membrane of a polarized epithelial cell can be
selected from, e.g.,
Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin AT, Subtilisin All,
Thrombin I, or Urokinase
I. TABLE 6 presents these enzymes together with an amino acid sequence that is
recognized and
cleaved by the particular peptidase.
TABLE 6 - Peptidases Present Near Basolateral Mucous Membranes or in Latter
Aspects
of the Transcytosis Pathway
Peptidase Amino Acid Sequence Cleaved SEQ ID NO
Cathepsin GI AAPF SEQ ID NO: 166
Chymotrypsin I GGF SEQ ID NO: 167
Elastase I AAPV SEQ ID NO: 168
Subtilisin AT GGL SEQ ID NO: 169
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Subtilisin All AAL SEQ ID NO: 170
Thrombin I FVR SEQ ID NO: 171
Urokinase I VGR SEQ ID NO: 172
Furin RQPR SEQ ID NO: 173
[0333] A cleavable spacer can exhibit a greater propensity for cleavage
than the remainder
of the delivery construct. As one skilled in the art is aware, many peptide
and polypeptide
sequences can be cleaved by peptidases and proteases. The cleavable spacer can
be selected so
that it will be preferentially cleaved relative to other amino acid sequences
present in the delivery
construct during administration of the delivery construct. A carrier of a
delivery construct can be
substantially (e.g., about 99%, about 95%, about 90%, about 85%, about 80, or
about 75%) intact
following delivery of the delivery construct to the bloodstream of the
subject. A cargo of a
delivery construct can be substantially (e.g., about 99%, about 95%, about
90%, about 85%,
about 80, or about 75%) intact following delivery of the delivery construct to
the bloodstream of
the subject. A cleavable spacer can be substantially (e.g., about 99%, about
95%, about 90%,
about 85%, about 80, or about 75%) cleaved following delivery of the delivery
construct to the
bloodstream of the subject.
[0334] A cleaving enzyme found in the plasma of the subject can be used to
cleave the
cleavable spacer. Any cleaving enzyme known by one of skill in the art to be
present in the
plasma of the subject can be used to cleave the cleavable spacer. Uses of such
enzymes to cleave
the cleavable spacers is less preferred than use of cleaving enzymes found
near the basolateral
membrane of a polarized epithelial cell because it is believed that more
efficient cleavage will
occur in near the basolateral membrane. However, if the skilled artisan
determines that cleavage
mediated by a plasma enzyme is sufficiently efficient to allow cleavage of a
sufficient fraction of
the delivery constructs to avoid adverse effects; such plasma cleaving enzymes
can be used to
cleave the delivery constructs. Accordingly, the cleavable spacer can be
cleaved with an enzyme
that is selected from the group consisting of caspase-1, caspase-3, proprotein
convertase 1,
proprotein convertase 2, proprotein convertase 4, proprotein convertase 4 PACE
4, prolyl
oligopeptidase, endothelin cleaving enzyme, dipeptidyl-peptidase IV, signal
peptidase,
neprilysin, renin, and esterase (see, e.g., U.S. Pat. No. 6,673,574,
incorporated by reference in its
entirety herein). TABLE 7 presents these enzymes together with an amino acid
sequence(s)
recognized by the particular peptidase. The peptidase cleaves a peptide
comprising these
sequences at the N-terminal side of the amino acid identified with an
asterisk.
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TABLE 7¨ Exemplary Plasma Peptidases
Peptidase Amino Acid Sequence Cleaved SEQ ID NO
Caspase-1 Tyr-Val-Ala-Asp-Xaa* SEQ ID NO: 174
Caspase-3 Asp-Xaa-Xaa-Asp-Xaa* SEQ ID NO: 175
Proprotein convertase 1 Arg-(Xaa)õ-Arg-Xaa*; SEQ ID NO: 176
n = 0, 2, 4 or 6
Proprotein convertase 2 Lys-(Xaa)õ-Arg-Xaa*; SEQ ID NO: 177
n = 0, 2, 4 or 6
Proprotein convertase 4 Glu-Arg-Thr-Lys-Arg-Xaa* SEQ ID NO: 178
Proprotein convertase 4 Arg-Val-Arg-Arg-Xaa* SEQ ID NO: 179
PACE 4 Decanoyl-Arg-Val-Arg-Arg- SEQ ID NO: 180
Xaa*
Prolyloligopeptidase Pro-Xaa*-Trp-Val-Pro-Xaa SEQ ID NO: 181
Endothelin cleaving enzyme
in combination with
dipeptidyl-peptidase IV
Signal peptidase Trp-Val*-Ala-Xaa SEQ ID NO: 182
Neprilysin in combination Xaa-Phe*-Xaa-Xaa SEQ ID NO: 183
with Dipeptideyl-peptidase Xaa-Tyr*-Xaa-Xaa SEQ ID NO: 184
IV Xaa-Trp*-Xaa-Xaa SEQ ID NO: 185
Renin in combination with Asp-Arg-Tyr-Ile-Pro-Phe-His- SEQ ID NO: 186
dipeptidyl-peptidase IV Leu*-Leu-(Val, Ala or Pro)-Tyr-
(Ser, Pro, or Ala)
[0335] Thus, a cleavable spacer can be any cleavable spacer known by one of
skill in the art
to be cleavable by an enzyme that is present at the basolateral membrane of an
epithelial cell. A
cleavable spacer can comprise a peptide. A cleavable spacer can comprise a
nucleic acid, such as
RNA or DNA. Furthermore, a cleavable spacer can comprise a carbohydrate, such
as a
disaccharide or a trisaccharide.
[0336] Alternatively, a cleavable spacer can be any cleavable spacer known
by one of skill
in the art to be cleavable by an enzyme that is present in the plasma of the
subject to whom the
delivery construct is administered. Such a cleavable spacer can comprise a
peptide. Such a
cleavable spacer can comprise a nucleic acid, such as RNA or DNA. Such a
cleavable spacer
comprises a carbohydrate, such as a disaccharide or a trisaccharide.
[0337] A cleavable spacer can be selected from, or can be derived from the
exemplary list
presented in TABLE 8.
TABLE 8¨ Amino Acid Sequences of Exemplary Spacers
Peptidase Spacer amino acid sequence SEQ ID NO
Subtilisin A SGGGGSGKAGSRGLT SEQ ID NO: 187
Furin SGGGGSGGGGLRQPR SEQ ID NO: 188
PC5a SGGGGSGKKVERFRY SEQ ID NO: 189
Thrombin SGGGGSGGGGLMTPR SEQ ID NO: 190
Pro-matripase SGGGGSGKAGSF SEG SEQ ID NO: 191
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Cathepsin G SGGGGSGKAGSAAPF SEQ ID NO: 192
TEV GGGGSGGGENLYFQS SEQ ID NO: 193
[0338] Moreover, a cleavable spacer can be a spacer that comprises an amino
acid sequence
that is a known substrate for the tobacco etch virus (TEV) protease.
Accordingly, a cleavable
spacer can comprise the amino acid sequence set in forth in, e.g.,
GGGGSGGGENLYFQS (SEQ
ID NO: 193).
[0339] The novel delivery constructs of the present disclosure can comprise
a peptide
sequence (or like domain), which serves to inhibit, interfere with, or block
the ability of the
biologically active cargo to bind to receptors at the surface of epithelial
cells. Accordingly,
depending upon the biologically cargo to be delivered, the peptide sequence
(or like domain)
which serves to inhibit, interfere with, or block the ability of the
biologically active cargo to bind
to its receptor at the surface of epithelial cells will be directed
specifically to the receptor to
which the biologically active binds.
[0340] Various biologically active cargos can bind to GM-1-gangliosides
found on the
surfaces of mammalian cells. Accordingly, a cleavable spacer of the present
disclosure can
comprise a peptide sequence, which serves to inhibit, interfere with, or block
the ability of the
biologically active cargo to bind GM-1 at the surface of epithelial cells.
U.S. Patent No.
8,877,161 teach a number of peptides that interfere with the binding of
ligands to GM-1.
TABLE 9 presents several examples of peptide sequences, which can be
incorporated in whole,
or in part, into the cleavable spacers to be used in the preparation of the
delivery constructs of the
present disclosure.
TABLE 9¨ Exemplary GM-1 Binding Peptides
Peptide sequence SEQ ID NO
VSWKTWFPNLAV SEQ ID NO: 194
YSPFHKWFPSMH SEQ ID NO: 195
IPQVWRDWFKLP SEQ ID NO: 196
FPAWFTKLYPRT SEQ ID NO: 197
QINTAKWWKTHF SEQ ID NO: 198
DASKALRSSGMP SEQ ID NO: 199
WKTWFP SEQ ID NO: 200
[0341] A cleavable spacer used in the preparation of the delivery
constructs of the present
disclosure can comprise the amino acid sequence of, e.g., SEQ ID NO: 185, SEQ
ID NO: 186,
SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189 and SEQ ID NO: 190, or variants
or
fragments thereof, as depicted in TABLE 10 below.
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TABLE 10¨ Exemplary Cleavable GM-1 Binding Peptide-Containing Spacers
Spacer amino acid sequence SEQ ID NO
SGGGGWKTWFPRGLT SEQ ID NO: 201
SGGGGWKTWFPRQPR SEQ ID NO: 202
SGGGGWKTWFPRFRY SEQ ID NO: 203
SGGGGWKTWFPMTPR SEQ ID NO: 204
SGGGGWKTWFPFSEG SEQ ID NO: 205
SGGGGWKTWFPAAPF SEQ ID NO: 206
Non-cleavable Spacers
[0342] A delivery construct of the present disclosure can comprise a
carrier coupled to a
heterologous cargo (e.g., a biologically active cargo), wherein the cargo can
be separated from
the carrier by a spacer consisting of one or more amino acids (e.g., up to 25
amino acids). The
spacer can be a peptide spacer or any other molecular entity that may be used
to couple to link a
first and a second molecule. Generally, a spacer will have no specific
biological activity other
than to join the proteins or to preserve some minimum distance or other
spatial relationship
between them. However, the constituent amino acids of the spacer can be
selected to influence
some property of the molecule such as the folding, net charge, or
hydrophobicity.
[0343] The spacer can be capable of forming covalent bonds to both the
delivery construct
and to the (e.g., biologically active) cargo. Suitable spacers are well known
to those of skill in
the art and include, but are not limited to, straight or branched-chain carbon
spacers, heterocyclic
carbon spacers, or peptide spacers. The spacer(s) can be joined to the
constituent amino acids of
the delivery construct and/or the biologically active cargo through their side
groups (e.g.,
through a disulfide linkage to cysteine). The spacers can be joined to the
alpha carbon amino
and/or carboxyl groups of the terminal amino acids of the delivery construct
and/or the (e.g.,
biologically active) cargo.
[0344] A bifunctional spacer having one functional group capable of
reacting with a group
on the bacterial carrier and another group reactive on the biologically active
cargo can be used to
form the desired conjugate. Alternatively, derivatization can involve chemical
treatment of the
targeting moiety. Procedures for generation of, for example, free sulfhydryl
groups on
polypeptides, such as antibodies or antibody fragments, are known (See U.S.
Pat. No.
4,659,839).
[0345] Many procedures and spacer molecules for attachment of various
compounds
including radionuclide metal chelates, toxins and drugs to proteins such as
antibodies are known.
See, for example, European Patent Application No. 188,256; U.S. Pat. Nos.
4,671,958,
4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071; and
Borlinghaus et al.
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(1987) Cancer Res. 47: 4071-4075.
[0346] A cargo (e.g., a biologically active cargo) to be delivered to a
location (e.g., a
location within a subject such as a human) can be coupled to the carrier using
one or more non-
cleavable peptide spacers comprising, e.g., the amino acid sequence GTGGS (SEQ
ID NO: 207),
GGGGS (SEQ ID NO: 208), GGGGSGGGGS (SEQ ID NO: 209), GGGGSGGGGSGGGGS
(SEQ ID NO: 210), or GGGGSGGG (SEQ ID NO: 211), wherein the carrier targets
said cargo
(e.g., biologically active cargo) to specific cells, including but not limited
to, cells of the immune
system such as macrophages, antigen-presenting cells and dendritic cells
(e.g., upon transporting
the cargo across an epithelial cell). Generally, a non-cleavable spacer can
comprise one or more
of (GGGGS)x (SEQ ID NO: 212), wherein x = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. A
non-cleavable
spacer can comprise one or more of (GS)( SEQ ID NO: 213), wherein x = 1, 2, 3,
4, 5, 6, 7, 8, 9,
or 10.
Production of Delivery Constructs
[0347] The delivery constructs of the present disclosure can be produced
using a variety of
methods. The selection of a production method can depend on the molecular
structure of the
delivery construct and/or its components (e.g., the carrier, cargo, and/or
spacer). Thus, for some
delivery constructs organic synthetic methods may be advantageous for
producing such delivery
construct. A delivery construct of the present disclosure can be a
polypeptide. Such polypeptides
can be produced, for example, using recombinant DNA methodology. Generally,
this involves
creating a DNA sequence that encodes the delivery construct, placing the DNA
in an expression
cassette under the control of a particular promoter, expressing the molecule
in a host, isolating
the expressed molecule and, if required, folding of the molecule into an
active conformational
form.
[0348] DNA encoding the delivery constructs described herein can be
prepared by any
suitable method, including, for example, cloning and restriction of
appropriate sequences or
direct chemical synthesis by methods such as the phosphotriester method of
Narang et al. (1979)
Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979)
Meth. Enzymol.
68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981)
Tetra. Lett., 22:
1859-1862); the solid support method of U.S. Pat. No. 4,458,066, and the like.
[0349] Chemical synthesis produces a single stranded oligonucleotide. This
can be
converted into double stranded DNA by hybridization with a complementary
sequence or by
polymerization with a DNA polymerase using the single strand as a template.
One of skill would
recognize that while chemical synthesis of DNA is limited to sequences of
about 100 bases,
longer sequences can be obtained by the ligation of shorter sequences.
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[0350] Alternatively, subsequences can be cloned and the appropriate
subsequences cleaved
using appropriate restriction enzymes. The fragments can then be ligated to
produce the desired
DNA sequence. A DNA encoding a delivery constructs of the present disclosure
can be cloned
using DNA amplification methods such as polymerase chain reaction (PCR). Thus,
for example,
the gene for the biologically active cargo is PCR amplified, using a sense
primer containing the
restriction site for, e.g., NdeI and an antisense primer containing the
restriction site for Hind'''.
This can produce a nucleic acid encoding the biologically active cargo
sequence and having
terminal restriction sites. A delivery construct having "complementary"
restriction sites can
similarly be cloned and then ligated to the biologically active cargo and/or
to a spacer attached to
the biologically active cargo. Ligation of the nucleic acid sequences and
insertion into a vector
produces a vector encoding the biologically active cargo joined to the
bacterial carrier receptor
binding domain. In various embodiments, DNA encoding delivery constructs of
the present
disclosure is artificially synthesized by, for example, solid-phase DNA
synthesis.
[0351] The production methods described herein can be used to produce the
delivery
constructs of the present disclosure, or (functional) variants thereof For
example, a "Cholix"
(also referred to herein as Cholix toxin or Cholix exotoxin) can encompass a
variety of
functional variants (e.g., a functional genus), wherein the functional
variants can comprise one or
more variations is their amino acid sequence relative to SEQ ID NO: 1 as
disclosed herein. Thus,
in the present disclosure, the Cholix toxin having the amino acid sequence set
forth in SEQ ID
NO: 1 is used as the reference sequence when referred to Cholix. However, as
described herein,
the present disclosure is not limited to the Cholix having the amino acid
sequence set forth in
SEQ ID NO: 1 but instead encompasses all Cholix variants that fall within the
functional genus
of Cholix. For example, a variant of the Cholix exotoxin with the amino acid
sequence set forth
in SEQ ID NI: 1 can be a Cholix exotoxin which amino acid sequence is set
forth in SEQ ID
NO: 2, wherein both variants are capable of carrying out the same functions,
e.g., transcytosis
across an epithelial cell, and interact with the same receptors, such as
ribophilin 1, 5EC24, CK-8,
TMEM132, GRP75, ERGIC-53, and/or perlecan.
[0352] Moreover, the production method of a polypeptide can affect, to some
degree, the
amino acid sequence of such polypeptide (e.g., due to post-translational
modifications). For
example, a first carrier and a second carrier are produced in the same
expression system (e.g., a
bacterial expression system such as E. coli or a mammalian expression system
such as a CHO
cell). In other cases, and as described herein, a first carrier and a second
carrier are produced in a
different expression system (e.g., a bacterial or a mammalian expression
system). Bacterial
expression systems include E. coli, and mammalian expression systems include
CHO cells, for
example. A bacterially produced polypeptide can comprise an N-cap, wherein the
N-cap can
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comprise one more modifications at the N-terminal of the polypeptide. An N-cap
can comprise
an N-terminal methionine residue. Examples of Cholix domain I derived carrier
polypeptides
that can be bacterially produced and that comprise such N-terminal methionine
include those
comprising the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, and SEQ ID NO: 135.
Cargo
[0353] The present disclosure provides methods and compositions that allow
for the rapid
and efficient delivery of cargo into and/or across epithelial cells (e.g.,
polarized epithelial cells)
in vitro (e.g., a Caco-2 cell monolayer) and in vivo (e.g., a gut epithelium
of a subject). Such
rapid and efficient delivery can be achieved by coupling the cargo (e.g., a
biologically active
cargo) to a carrier to form a delivery construct. Such delivery constructs can
be modified to
target certain locations within an epithelial cell or to transport cargo
across an epithelial cell such
as an intact epithelial membrane.
[0354] In various embodiments, the compositions and methods of the present
disclosure
provide efficient transport and delivery of various cargo molecules to
different locations (e.g.,
organs, tissues, or cells) of a subject (e.g., a rodent or a human). The
delivery constructs of the
present disclosure can allow for delivery into epithelial cells and/or for
rapid transcytosis (e.g.,
vesicular transcytosis) across an epithelial cell layer such as a gut
epithelium of a subject. The
presently described delivery mechanisms allow for transport and delivery of
various cargo
molecules. The herein described delivery constructs can be coupled to at least
one, at least two,
at least three, at least five or at least 10 cargo molecules. The cargo can be
a heterologous cargo,
e.g., heterologous to the carrier. For example, a delivery construct described
herein comprises a
Cholix domain I derived carrier (e.g., those having the amino acid sequences
set forth in SEQ ID
NO: 1 ¨ SEQ ID NO: 125) coupled to a heterologous cargo, wherein the
heterologous cargo is a
non-Cholix derived cargo molecule (e.g., is not derived or does not contain
fragments of a
Cholix toxin domain I, II, lb, and III).
[0355] A heterologous cargo can be a biologically active cargo. A
biologically active cargo
can include therapeutic and/or diagnostic molecules. Thus, the delivery
constructs of the present
disclosure can be used to deliver a biologically active cargo to a subject
(e.g., a rodent or a
human). A "biologically active cargo" as used herein includes, but is not
limited to: a
macromolecule, small molecule, peptide, polypeptide, nucleic acid, mRNA,
miRNA, shRNA,
siRNA, antisense molecule, antibody, DNA, plasmid, vaccine, polymer
nanoparticle, or
catalytically-active material.
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[0356] A biologically active cargo of the present disclosure can be a
macromolecule that
can perform a desirable biological activity when introduced to the bloodstream
of the subject.
For example, the biologically active cargo can have receptor binding activity,
enzymatic activity,
messenger activity (i.e., act as a hormone, cytokine, neurotransmitter, or
other signaling
molecule), luminescent or other detectable activity, or regulatory activity,
or any combination
thereof. For certain diagnostic purposes, a biologically active cargo can be
conjugated to or can
itself be a pharmaceutically acceptable gamma-emitting moiety, including but
not limited to,
indium and technetium, magnetic particles, radiopaque materials such as air or
barium and
fluorescent compounds.
[0357] A heterologous cargo as described herein can be a biologically
active cargo. A
biologically active cargo that is part of a delivery construct can exert its
effects in biological
compartments of the subject other than the subject's blood. For example, in
various
embodiments, the biologically active cargo can exert its effects in the
lymphatic system. In other
cases, the biologically active cargo can exert its effects in an organ or
tissue, such as, for
example, the subject's liver, heart, lungs, pancreas, kidney, brain, bone
marrow, etc. As such, the
biologically active cargo can or cannot be present in the blood, lymph, or
other biological fluid at
detectable concentrations, yet can still accumulate at sufficient
concentrations at its site of action
to exert a biological effect.
[0358] A biologically active cargo can be a protein that comprises more
than one
polypeptide subunit. For example, the protein can be a dimer, trimer, or
higher order multimer.
In various embodiments, two or more subunits of the protein can be connected
with a covalent
bond, such as, for example, a disulfide bond. The subunits of the protein can
be held together
with non-covalent interactions. One of skill in the art can routinely identify
such proteins and
determine whether the subunits are properly associated using, for example, an
immunoassay.
[0359] A biologically active cargo to be delivered to a certain location
(e.g., in a subject)
can be selected from, e.g., cytokines and cytokine receptors such as
Interleukin-1 (IL-1), IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
IL-15, IL-16, IL-17,
IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28,
IL-29, IL-30,
lymphokine inhibitory factor, macrophage colony stimulating factor, platelet
derived growth
factor, stem cell factor, tumor growth factor-0, tumor necrosis factor,
lymphotoxin, Fas,
granulocyte colony stimulating factor, granulocyte macrophage colony
stimulating factor,
interferon-a, interferon-0, interferon-y, growth factors and protein hormones
such as
erythropoietin, angiogenin, hepatocyte growth factor, fibroblast growth
factor, keratinocyte
growth factor, nerve growth factor, tumor growth factor-a, thrombopoietin,
thyroid stimulating
factor, thyroid releasing hormone, neurotrophin, epidermal growth factor,
VEGF, ciliary
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neurotrophic factor, LDL, somatomedin, insulin growth factor, insulin-like
growth factor I and
II, chemokines such as ENA-78, ELC, GRO-a, GRO-0, GRO-y, HRG, LEF, IP-10, MCP-
1,
MCP-2, MCP-3, MCP-4, MIP-1-a, MIP-113, MG, MDC, NT-3, NT-4, SCF, LIF, leptin,
RANTES, lymphotactin, eotaxin-1, eotaxin-2, TARC, TECK, WAP-1, WAP-2, GCP-1,
GCP-2;
a-chemokine receptors, e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7;
and 13-chemokine receptors, e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7.
[0360] An illustrative, but not limiting, list of suitable biologically
active cargo to be used in
the constructs and methods of the present invention is provided in TABLE 11.
TABLE 11 ¨ Exemplary Biologically Active Cargo
Cargo RefSeq (NCBI/Uniprot)
Interleukins
IL-10 NP 000563
IL-19 NP 037503
IL-20 NP 061194
IL-22 NP 065386
IL-24 NP 006841
IL-26 NP 060872
TNFSF Ligands
Tumor necrosis factor-a ("TNF-a") NP 000585.2
lymphotoxin-a ("LT-a") NP 000586.2
lymphotoxin-I3 ("LT-I3") NP 002332.1
CD30 ligand NP 001235.1
CD40 ligand NP 000065.1
CD70 ligand NP 001243.1
0X40 ligand NP 001284491.1
41BB ligand NP 001552.2
Apol ligand (or FasL or CD95L) NP 000630.1
Apo2 ligand (or TRAIL, AIM-1 or NP 001177871.1
AGP-1)
Apo3 ligand (or TWEAK) NP 003800.1
APRIL NP 001185551.1
LIGHT NP 003798.2
OPG ligand (or RANK ligand) NP 003692.1
BlyS (or THANK) NP 001139117.1
BCMA NP 001183.2
TACT NP 036584.1
TNFSFRs
TNFR1 NP 001056.1
TNFR2 NP 001057.1
lymphotoxin-I3R NP 001257916.1
CD40 NP 001241.1
CD95 (or FAS or APO-1) NP 000034.1
OPG NP 002537.3
RANK NP 001257878.1
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CD30 NP 001234.3
CD27 NP 001233.1
0X40 (or CD134) NP 003318.1
41BB NP 001552.2
NGFR NP 002498.1
BCMA NP 001183.2
TAC1 NP 036584.1
EDA2R NP 001186616.1
TROY NP 001191387.1
DR6 NP 055267.1
DR5 (or TRAILR2) NP 003833.4
DR4 NP 003835.3
DR3 NP 001034753.1
HVEM NP 001284534.1
LTOR NP 001257916.1
GITR NP 004186.1
DcR3 NP 003814
Fn14 (or TWEAKR) NP 057723.1
BAFF NP 443177.1
Glucose metabolism-related proteins
Glucagon proprotein NP 002045.1
Glucagon peptide NP 002045.1 (aa 53-81)
Glucagon-like peptide 1 NP 002045.1 (aa 98-128)
Glucagon-like peptide 2 NP 002045.1 (aa 146-178)
Glicentin P01275 (aa 21-89)
Glicentin-related polypeptide P01275 (aa 21-50)
Gastric inhibitory polypeptide NP 004114.1
preprotein
Gastric inhibitory polypeptide NP 004114.1 (aa 52-93)
Dipeptidyl peptidase 4 P27487
Glucose transporter member 4 NP 001033.1
Preproglucagon AAA52567.1
Insulin receptor substrate 1 NP 005535.1
Insulin P01308
Apolipoprotein A-II P02652
Solute carrier family 2, facilitated P11166
glucose transporter member 1
Glycogen synthase 1 P13807
Glycogen synthase 2 P54840
Tyrosine-protein phosphatase non- P18031
receptor type 1
RAC-alpha serine/threonine-protein P31749
kinase
Peroxi some proliferator-activated P37231
receptor gamma
Hexokinase 3 P52790
Phosphatidylinosito1-3,4,5- P60484
triphosphate 3-phosphatase and dual-
specificty protein
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Pyruvate dehydrogenase kinase 1 Q15118
Calcium-binding and coiled-coil Q9P1Z2
domain-containing protein 1
Max-like protein X Q9UH92
Fructose-bisphosphate aldolase A P04075
Glucagon-like peptide 1 receptor P43220
Glucagon-like peptide 2 receptor 095838
Gastric inhibitory polypeptide P48546
receptor
Insulin-like growth factor 1 receptor P08069.1
Insulin-like growth factor 2 receptor P11717.3
Insulin Receptor P06213
GLP-1 agonist-Exenatide DB01276
GLP-1 agonist-Liraglutide DB06655
Growth Hormone Related Proteins
Somatotropin P01241
Synthetic Human Growth Hormone AAA72260.1
Synthetic Human Growth Hormone CAA01435
Partial
Synthetic Human Growth Hormone CAA00380
Partial
Human Growth Hormone 2 P01242
Somatoliberin P01286.1
Appetite-regulating Hormone Q9UBU3
Leptin P41159
Growth Hormone Receptor Proteins
Growth Hormone Receptor P10912
Growth Hormone-Releasing Q02643
Hormone Receptor
Growth Hormone Secretagogue Q92847
Receptor
Growth Hormone-Releasing P78470
Hormone Receptor form a
Growth Hormone Receptor E9PCN7
[0361] A biologically active cargo as described herein can be a hormone.
Such a hormone
can be a growth hormone. The hormone can be a human growth hormone having, for
example,
the amino acid sequence set forth in SEQ ID NO: 214.
[0362] A biologically active cargo can be a molecule affects and/or
interacts with a
metabolism of a subject. Thus, a biologically active cargo as described herein
can be a drug that
can be used to prevent, treat and/or diagnose a metabolic disease or
condition. Thus, a
biologically active cargo as described herein can be a glucagon-like peptide
(GLP). The GLP can
be GLP-1 having the amino acid sequence set forth in SEQ ID NO: 215. A hormone
that can be
used in combination with the methods and compositions described herein can be
insulin (with c-
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peptide element removed from mature protein), or a derivative thereof An
Insulin peptide can
comprise the amino acid sequence set forth in SEQ ID NO: 216.
[0363] A biologically active cargo can be an interleukin. Specifically,
interleukins that can
be used with the methods and compositions described herein can include IL-10
and IL-22,
having the amino acid sequence set forth in SEQ ID NO: 217 and SEQ ID NO: 218,
respectively.
[0364] The biologically active cargo disclosed herein can modulate the
spatial orientation of
a delivery construct. For example, a cargo molecule can induce multimerization
of two or more
delivery constructs. Such multimers can be homomers or heteromers. The
multimer can be a
homodimer. For example, a delivery construct comprising the amino acid
sequence set forth in
SEQ ID NO: 217 can form a dimer. Such dimerization can be induced by IL-10 (as
IL-10 can
form a natural dimer and thus promote dimerization of a delivery construct
comprising an IL-10
as cargo).
[0365] A biologically active cargo can also comprise toxin, such as
endotoxins, enterotoxins
or exotoxins. For example, a biologically active cargo can be an ExtB
polypeptide (which will
form a natural pentamer) having the amino acid sequence set forth in SEQ ID
NO: 219.
[0366] Other examples of biologically active cargo that can be delivered
according to the
present disclosure include, but are not limited to, antineoplastic compounds,
such as
nitrosoureas, e.g., carmustine, lomustine, semustine, strepzotocin;
methylhydrazines, e.g.,
procarbazine, dacarbazine; steroid hormones, e.g., glucocorticoids, estrogens,
progestins,
androgens, tetrahydrodesoxycaricosterone; immunoactive compounds such as
immunosuppressives, e.g., pyrimethamine, trimethopterin, penicillamine,
cyclosporine,
azathioprine; and immunostimulants, e.g., levami sole, diethyl
dithiocarbamate, enkephalins,
endorphins; antimicrobial compounds such as antibiotics, e.g., 13-lactam,
penicillin,
cephalosporins, carbapenims and monobactams, 13-lactamase inhibitors,
aminoglycosides,
macrolides, tetracyclins, spectinomycin; antimalarial s, amebicides;
antiprotazoals; antifungals,
e.g., amphotericin (3, antivirals, e.g., acyclovir, idoxuridine, ribavirin,
trifluridine, vidarbine,
gancyclovir; parasiticides; antihalmintics; radiopharmaceutics;
gastrointestinal drugs;
hematologic compounds; immunoglobulins; blood clotting proteins, e.g.,
antihemophilic factor,
factor IX complex; anticoagulants, e.g., dicumarol, heparin Na; fibrolysin
inhibitors, e.g.,
tranexamic acid; cardiovascular drugs; peripheral anti-adrenergic drugs;
centrally acting
antihypertensive drugs, e.g., methyldopa, methyldopa HC1; antihypertensive
direct vasodilators,
e.g., diazoxide, hydralazine HCI; drugs affecting renin-angiotensin system;
peripheral
vasodilators, e.g., phentolamine; anti-anginal drugs; cardiac glycosides;
inodilators, e.g.,
amrinone, milrinone, enoximone, fenoximone, imazodan, sulmazole;
antidysrhythmics; calcium
entry blockers; drugs affecting blood lipids, e.g., ranitidine, bosentan,
rezulin; respiratory drugs;
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sypathomimetic drugs, e.g., albuterol, bitolterol mesylate, dobutamine HC1,
dopamine HC1,
ephedrine So, epinephrine, fenfluramine HCl, isoproterenol HCl, methoxamine
HCl,
norepinephrine bitartrate, phenylephrine HCl, ritodrine HCl; cholinomimetic
drugs, e.g.,
acetylcholine Cl; anticholinesterases, e.g., edrophonium Cl; cholinesterase
reactivators;
adrenergic blocking drugs, e.g., acebutolol HC1, atenolol, esmolol HC1,
labetalol HC1,
metoprolol, nadolol, phentolamine mesylate, propanolol HC1; antimuscarinic
drugs, e.g.,
anisotropine methylbromide, atropine SO4, clinidium Br, glycopyrrolate,
ipratropium Br,
scopolamine HBr; neuromuscular blocking drugs; depolarizing drugs, e.g.,
atracurium besyl ate,
hexafluorenium Br, metocurine iodide, succinylcholine Cl, tubocurarine Cl,
vecuronium Br;
centrally acting muscle relaxants, e.g., baclofen; neurotransmitters and
neurotransmitter agents,
e.g., acetylcholine, adenosine, adenosine triphosphate; amino acid
neurotransmitters, e.g.,
excitatory amino acids, GABA, glycine; biogenic amine neurotransmitters, e.g.,
dopamine,
epinephrine, histamine, norepinephrine, octopamine, serotonin, tyramine;
neuropeptides, nitric
oxide, K+ channel toxins; antiparkinson drugs, e.g., amaltidine HC1,
benztropine mesylate,
carbidopa; diuretic drugs, e.g., dichlorphenamide, methazolamide,
bendroflumethiazide,
polythiazide; antimigraine drugs, e.g, carboprost tromethamine mesylate,
methysergide maleate.
[0367] Still other examples of biologically active cargo that can be
delivered according to
the present disclosure include, but are not limited to, hormones such as
pituitary hormones, e.g.,
chorionic gonadotropin, cosyntropin, menotropins, somatotropin, iorticotropin,
protirelin,
thyrotropin, vasopressin, lypressin; adrenal hormones, e.g., beclomethasone
dipropionate,
betamethasone, dexarnethasone, triamcinolone; pancreatic hormones, e.g.,
glucagon, insulin;
parathyroid hormone, e.g., dihydrochysterol; thyroid hormones, e.g.,
calcitonin etidronate
disodium, levothyroxine Na, liothyronine Na, liotrix, thyroglobulin,
teriparatide acetate;
antithyroid drugs; estrogenic hormones; progestins and antagonists; hormonal
contraceptives;
testicular hormones; gastrointestinal hormones, e.g., cholecystokinin,
enteroglycan, galanin,
gastric inhibitory polypeptide, epidermal growth factor-urogastrone, gastric
inhibitory
polypeptide, gastrin-releasing peptide, gastrins, pentagastrin, tetragastrin,
motilin, peptide YY,
secretin, vasoactive intestinal peptide, or sincalide.
[0368] Still other examples of biologically active cargo that can be
delivered according to
the present disclosure include, but are not limited to, enzymes such as
hyaluronidase,
streptokinase, tissue plasminogen activator, urokinase, PGE-adenosine
deaminase; intravenous
anesthetics such as droperidol, etomidate, fetanyl citrate/droperidol,
hexobarbital, ketamine HC1,
methohexital Na, thiamylal Na, thiopental Na; antiepileptics, e.g.,
carbamazepine, clonazepam,
divalproex Na, ethosuximide, mephenyloin, paramethadione, phenyloin,
primidone. In various
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embodiments, the biologically active cargo is an enzyme selected from
hyaluronidase,
streptokinase, tissue plasminogen activator, urokinase, PGE-adenosine
deaminase.
[0369] A biologically active cargo as described herein can also include a
therapeutic and/or
diagnostic antibody, an antibody fragment, a diabody, a minibody, or a single-
chain variable
fragment (e.g., scFv), or a combination thereof For example, a biologically
active cargo as
described herein can be an anti-tumor necrosis factor alpha (anti-TNFa) agent.
An anti-TNFa
agent is an anti-TNFa antibody or a functional fragment thereof An Anti-TNFa
antibody can be
adalimumab (Abbvie HUMIRA , Drug Bank DB 00051) or infliximab (Centocor
REMICADE , Drug Bank DB 00065), or functional fragment (e.g., a binding
fragment thereof).
[0370] Yet other examples of biologically active cargo that can be
delivered according to
the present disclosure include, but are not limited to, chemotherapeutics,
such as chemotherapy
or anti-tumor agents which are effective against various types of human
cancers, including
leukemia, lymphomas, carcinomas, sarcomas, myelomas etc., such as, for
example, doxorubicin,
mitomycin, cisplatin, daunorubicin, bleomycin, actinomycin D, and
neocarzinostatin.
[0371] Yet other examples of biologically active cargo that can be
delivered according to
the present disclosure include inhibitors of regulatory T cells (Tregs) such
as Tregs that express
CD4, CD25 and Foxp3, and Tregs such as Trl, Th3, CD8+CD28-, Qa-1 restricted T
cells, and
IL-17 Treg cells. Such Treg inhibitors have been extensively studied and
described in the art
(see, e.g., Casares et al, Journal of Immunology, 185(9): 5150-5159, 2010, and
references cited
therein).
[0372] TABLE 12 shows exemplary amino acid sequences of various
heterologous cargos
that can be used in combination with the herein disclosed methods and
compositions. For
example, any of the heterologous cargo molecules shown in TABLE 12 below can
be combined
with any carrier disclosed herein, e.g., those carrier listed in TABLE 2
and/or TABLE 3 above.
TABLE 12¨ Exemplary Heterologous Cargo Amino Acid Sequences
SEQ ID NO Sequence
SEQ ID NO: 214 FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQ
NPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLR
SVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIF
KQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCR
SVEGSCGF
SEQ ID NO: 215 MKIILWLCVFGLFLATLFPISWQMPVESGLSSEDSASSESFASKIKR
HGEGTF T SDLSKQMEEEAVRLFIEWLKNGGP S SGAPPP SG
SEQ ID NO: 216 MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVC
GERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ
KRGIVEQCCTSICSLYQLENYCN
SEQ ID NO: 217 MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDL
RDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQ
FYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCE
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NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIR
SEQ ID NO: 218 MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKS
NFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERC
YLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHI
EGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI
SEQ ID NO: 219 MKKNIAFLLASMFVF SIATNAYASTQSNKKDLCEHYRQIAKESCK
KGFLGVRDGTAGACFGAQIMVAAKGC
SEQ ID NO: 220 MRS SKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPE
GFKWERVMNFEDGGVVTVTQDSSLQDGCFIYKVKFIGVNEPSDG
PVMQKKTMGWEASTERLYPRDGVLKGEIHKALKLKDGGHYLVE
FKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
HLFL
[0373] A cargo molecule of the present disclosure can comprise an amino
acid sequence
having at least 80% sequence identity to any one of the amino acid sequences
set forth in SEQ
ID NO: 214 ¨ SEQ ID NO: 220, at least 80% sequence identity to a functional
fragment thereof,
and/or any combination of thereof. A cargo molecule of the present disclosure
can comprise an
amino acid sequence having at least 90% sequence identity to any one of the
amino acid
sequences set forth in SEQ ID NO: 214 ¨ SEQ ID NO: 220, at least 80% sequence
identity to a
functional fragment thereof, and/or any combination of thereof. A cargo
molecule of the present
disclosure can comprise an amino acid sequence having at least 95% sequence
identity to any
one of the amino acid sequences set forth in SEQ ID NO: 214 ¨ SEQ ID NO: 220,
at least 80%
sequence identity to a functional fragment thereof, and/or any combination of
thereof A cargo
molecule of the present disclosure can comprise an amino acid sequence having
at least 99%
sequence identity to any one of the amino acid sequences set forth in SEQ ID
NO: 214 ¨ SEQ ID
NO: 220, at least 80% sequence identity to a functional fragment thereof,
and/or any
combination of thereof. A cargo molecule of the present disclosure can
comprise any one of the
amino acid sequences set forth in SEQ ID NO: 214 ¨ SEQ ID NO: 220, a
functional fragment
thereof, and/or any combination of thereof.
[0374] Generally, a cargo described and disclosed herein can be retained at
a location that
has been targeted using the compositions described herein. Retention can cause
the cargo
molecule to elicit a certain response or biological effect (e.g., a
therapeutic effect). The delivery
of a molecule (e.g., a heterologous cargo) to a location (e.g., an
intracellular compartment or a
supranuclear region) can refer to the retention of the molecule at that
location. Retention of a
molecule at a certain intracellular or extracellular region or compartment can
be for a certain
amount of time, e.g., at least 2 minutes, at least 5 minutes, at least 10
minutes, at least 15
minutes, at 30 minutes, or at least 60 minutes. Retention of a molecule can
depend on various
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factors such as the location where the molecule is retained and/or the types
of molecular
interactions that occur between the molecule (e.g., a carrier, a delivery
construct, and/or a
heterologous cargo). For example, delivery of a heterologous cargo to a
basolateral compartment
via transcytosis across a polarized epithelial cell can comprise retaining the
heterologous cargo at
the basolateral location for a time sufficient to elicit a certain effect,
such as a therapeutic effect
in case of a therapeutic and/or biologically active cargo. A delivery
construct can be configured
to release a cargo at a specific location, e.g., by using pH-dependent and/or
enzyme-dependent
spacer. Upon release of a cargo from a carrier, the cargo molecule can elicit
a certain effect
and/or response. For example, and in the case of biologically and/or
therapeutically active
cargos, such cargos can elicit their therapeutic effects in vitro or in vivo
upon release from the
carrier. A cargo may also be capable of eliciting a response when still bound
to the carrier. This
may depend on the cargo and/or the delivery construct.
[0375] A heterologous cargo can be a detectable agent such as a fluorescent
molecule or a
radioactive moiety. A detectable agent as described herein can be used to
detect the molecule
that the detectable agent is coupled to in various locations, e.g., inside a
subject or inside a cell.
A detectable agent can also have additional features and functions, such as
therapeutic or other
biological properties. For example, a radionuclide coupled to a carrier as
described herein can
allow the detection of the carrier but can also have therapeutic properties,
e.g., as a therapeutic
radionuclide. Generally, a carrier can conjugated to, linked to, or fused with
detectable agents,
such as a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle,
a metal-containing
nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a metal,
a radioisotope, a
dye, radionuclide chelator, or another suitable material that may be used in
imaging.
[0376] A delivery construct can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 detectable
agents. Non-limiting examples of radioisotopes that may be used as detectable
agents include
alpha emitters, beta emitters, positron emitters, and gamma emitters. The
metal or radioisotope
may be selected from the group consisting of actinium, americium, bismuth,
cadmium, cesium,
cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium,
polonium, radium,
ruthenium, samarium, strontium, technetium, thallium, and yttrium. The metal
may be actinium,
bismuth, lead, radium, strontium, samarium, or yttrium. The radioisotope may
be actinium-225
or lead-212. The near-infrared dyes that may be used in combination with the
herein described
chimeric binding agents may not be easily quenched by biological tissues and
fluids. The
fluorophore may be a fluorescent agent emitting electromagnetic radiation at a
wavelength
between 650 nm and 4000 nm, such emissions being used to detect such agent.
Non-limiting
examples of fluorescent dyes that may be used as a conjugating molecule in the
present
disclosure include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-
800,
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VivoTag-680, Cy5.5, or indocyanine green (ICG). Near infrared dyes may include
cyanine dyes
(e.g., Cy7, Cy5.5, and Cy5). Additional non-limiting examples of fluorescent
dyes for use as a
conjugating molecule in the present disclosure may include acradine orange or
yellow, Alexa
Fluors (e.g., Alexa Fluor 790, 750, 700, 680, 660, and 647) and any derivative
thereof, 7-
actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO dye and any
derivative thereof,
auramine-rhodamine stain and any derivative thereof, bensantrhone, bimane, 9-
10-
bis(phenylethynyl)anthracene, 5,12 - bis(phenylethynyl)naththacene,
bisbenzimide, brainbow,
calcein, carbodyfluorescein and any derivative thereof, 1-chloro-9,10-
bis(phenylethynyl)anthracene and any derivative thereof, DAPI, Di0C6, DyLight
Fluors and any
derivative thereof, epicocconone, ethidium bromide, FlAsH-EDT2, Fluo dye and
any derivative
thereof, FluoProbe and any derivative thereof, Fluorescein and any derivative
thereof, Fura and
any derivative thereof, GelGreen and any derivative thereof, GelRed and any
derivative thereof,
fluorescent proteins and any derivative thereof, m isoform proteins and any
derivative thereof
such as mCherry, hetamethine dye and any derivative thereof, hoeschst stain,
iminocoumarin,
indian yellow, indo-1 and any derivative thereof, laurdan, lucifer yellow and
any derivative
thereof, luciferin and any derivative thereof, luciferase and any derivative
thereof, mercocyanine
and any derivative thereof, nile dyes and any derivative thereof, perylene,
phloxine, phyco dye
and any derivative thereof, propium iodide, pyranine, rhodamine and any
derivative thereof,
ribogreen, RoGFP, rubrene, stilbene and any derivative thereof, sulforhodamine
and any
derivative thereof, SYBR and any derivative thereof, synapto-pHluorin,
tetraphenyl butadiene,
tetrasodium tris, Texas Red, Titan Yellow, TSQ, umbelliferone, violanthrone,
yellow fluroescent
protein and YOYO-1. Other Suitable fluorescent dyes may include, but are not
limited to,
fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC,
naphthofluorescein,
4', 5'-dichloro-2',7' -dimethoxyfluorescein, 6-carboxyfluorescein or FAM,
etc.), carbocyanine,
merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin,
rhodamine dyes (e.g.,
carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-
rhodamine
(ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red,
tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g.,
methoxycoumarin,
dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.),
Oregon Green
Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.), Texas
Red, Texas
Red-X, SPECTRUM RED, SPECTRUM GREEN, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5,
CY-
5.5, etc.), ALEXA FLUOR dyes (e.g., ALEXA FLUOR 350, ALEXA FLUOR 488, ALEXA
FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA
FLUOR 633, ALEXA FLUOR 660, ALEXA FLUOR 680, etc.), BODIPY dyes (e.g., BODIPY
FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568,
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BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY
650/665,
etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), and the like. Additional
suitable detectable
agents are described in PCT/US14/56177. Non-limiting examples of radioisotopes
include alpha
emitters, beta emitters, positron emitters, and gamma emitters. The metal or
radioisotope may be
selected from the group consisting of actinium, americium, bismuth, cadmium,
cesium, cobalt,
europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium,
radium,
ruthenium, samarium, strontium, technetium, thallium, and yttrium. The metal
may be actinium,
bismuth, lead, radium, strontium, samarium, or yttrium. The radioisotope may
be actinium-225
or lead-212. Additionally, the following radionuclides may be used for
diagnosis and/or therapy:
carbon (e.g., or
14C), nitrogen (e.g., 13N), fluorine (e.g., 18F), gallium (e.g., 67Ga or
68Ga),
copper (e.g., 64Cu or 67Cu), zirconium (e.g., 89Zr), lutetium (e.g., 177Lu).
[0377] A delivery construct as disclosed herein can be conjugated to,
coupled to, or fused to
a radiosensitizer or photosensitizer. Examples of radiosensitizers may include
but are not limited
to: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin,
oxaliplatin, gemcitabine,
etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives
(e.g., halogenated
purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of
photosensitizers may
include but are not limited to: fluorescent molecules or beads that generate
heat when
illuminated, nanoparticles, porphyrins and porphyrin derivatives (e.g.,
chlorins, bacteriochlorins,
isobacteriochlorins, phthalocyanines, and naphthalocyanines),
metalloporphyrins,
metallophthalocyanines, angelicins, chalcogenapyrrillium dyes, chlorophylls,
coumarins, flavins
and related compounds such as alloxazine and riboflavin, fullerenes,
pheophorbides,
pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins,
texaphyrins,
purpurins, porphycenes, phenothiaziniums, methylene blue derivatives,
naphthalimides, nile blue
derivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins, and
cercosporins),
psoralens, quinones, retinoids, rhodamines, thiophenes, verdins, xanthene dyes
(e.g., eosins,
erythrosins, rose bengals), dimeric and oligomeric forms of porphyrins, and
prodrugs such as 5-
aminolevulinic acid. Advantageously, this approach may allow for highly
specific targeting of
diseased cells (e.g., cancer cells) using both a therapeutic agent (e.g.,
drug) and electromagnetic
energy (e.g., radiation or light) concurrently. The proteins of the present
disclosure can be
conjugated to, coupled to, fused with, or covalently or non-covalently coupled
to the agent, e.g.,
directly or via a spacer.
[0378] A radionuclide may be attached to a carrier or delivery construct as
described herein
using a chelator. Exemplary chelator moieties may include 2,2',2"-(3-(4-(3-(1-
(4-(1,2,4,5-
tetrazin-3-yl)pheny1)-1-oxo-5,8,11,14,17,20,23-heptaoxa-2-azapentacosan-25-
yl)thioureido)benzy1)-1,4,7-triazonane-2,5,8-triy1)triacetic acid; 2,2',2"-(3-
(4-(3-(1-(4-(1,2,4,5-
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tetrazin-3-yl)pheny1)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-
azaheptatriacontan-37-
yl)thioureido)benzy1)-1,4,7-triazonane-2,5,8-triy1)triacetic acid; 2,2'-(7-(4-
(3-(1-(4-(1,2,4,5-
tetrazin-3-yl)pheny1)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-
azaheptatriacontan-37-
yl)thioureido)benzy1)-1,4,7-triazonane-1,4-diy1)diacetic acid; 2,2',2"-(3-(4-
(3-(1-(4-(1,2,4,5-
tetrazin-3 -yl)pheny1)-3,7-di ox o-11,14,17,20,23,26,29-heptaox a-2, 8-di
azahentri acontan-31-
yl)thiourei do)b enzy1)-1,4,7-triazonane-2,5,8-triy1)triacetic acid; 2,2',2"-
(3-(4-(3-(1-(4-(1,2,4,5-
tetrazin-3-yl)pheny1)-3,7-dioxo-11,14,17,20,23,26,29,32,35,38,41-undecaoxa-2,8-
diazatritetracontan-43-yl)thioureido)benzy1)-1,4,7-triazonane-2,5,8-
triyptriacetic acid; 2,2',2"-(3-
(4-(3-(25,28-dioxo-2846-(6-(pyridin-2-y1)-1,2,4,5-tetrazin-3-yl)pyridin-3-
yl)amino)-
3,6,9,12,15,18,21-heptaoxa-24-azaoctacosyl)thioureido)benzy1)-1,4,7-triazonane-
2,5,8-
triy1)triacetic acid; 2,2',2"-(3-(4-(3-(37,40-dioxo-4046-(6-(pyridin-2-y1)-
1,2,4,5-tetrazin-3-
yl)pyridin-3-yl)amino)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-
azatetracontyl)thioureido)benzy1)-1,4,7-triazonane-2,5,8-triy1)triacetic acid;
2,2',2"-(3-(4-(1-(4-
(6-methy1-1,2,4,5-tetrazin-3-yl)pheny1)-3-oxo-6,9,12,15,18,21,24-heptaoxa-2-
azaheptacosan-27-
amido)benzy1)-1,4,7-triazonane-2,5,8-triy1)triacetic acid; 2,2',2"-(2-(4-(1-(4-
(6-methy1-1,2,4,5-
tetrazin-3-yl)phenoxy)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-
36-
amido)benzy1)-1,4,7-triazonane-1,4,7-triy1)triacetic acid; 2,2',2"-(3-(4-(3-(5-
amino-6-((4-(6-
methy1-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-
triazonane-
2,5,8-triy1)triacetic acid; 2,2'-(7-(4-(3-(5-amino-6-((4-6-methy1-1,2,4,5-
tetrazin-3-
yl)benzyl)amino)-6-oxohexyl)thioureido)benzy1)-1,4,7-triazonane-1,4-
diy1)diacetic acid; 2,2',2"-
(3-(4-(3-(5-amino-645-amino-644-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-
6-
oxohexyl)amino)-6-oxohexyl)thioureido)benzy1)-1,4,7-triazonane-2,5,8-
triy1)triacetic acid; and
2,2',2"-(3-(4-(3-(5-amino-645-amino-645-amino-644-(6-methyl-1,2,4,5-tetrazin-3-
yl)benzyl)amino)-6-oxohexyl)amino)-6-oxohexyl)amino)-6-
oxohexyl)thioureido)benzy1)-1,4,7-
triazonane-2,5,8-triy1)triacetic acid.
Uses
[0379] The
present disclosure provides methods and compositions for transport and/or
delivery of a cargo molecule to certain location(s) within a cell (e.g., a
supranuclear location) or
across a cell (e.g., epithelial cell), either in vitro or in vivo (e.g., in a
rodent or a human). Such
cargo can be directed to a set of location(s) by coupling it to a carrier
molecule. Such carrier
molecule can interact with unique receptors both on the cell surface and
intracellularly for the
targeted delivery of the cargo. Various such carrier, cargos, and uses thereof
are described
herein.
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[0380] Contemplated herein are delivery constructs that can be used to
deliver a cargo to a
location within a cell (e.g., epithelial cell) or across a cell (e.g.,
epithelial cell). Such carriers can
be a small molecule, a polypeptide, an aptamer, an antibody, a nucleic acid a
fragment of any of
the above, or a combination of any of the above. The delivery constructs
described herein can be
used for various applications, including but not limited to, therapeutic,
preventative, and/or
diagnostic applications. Such therapeutic, preventative, and/or diagnostic
applications can be
provided if, for example, therapeutically active cargo molecules are coupled
to carriers described
herein that enable targeted delivery to various locations (e.g., in a subject
such as a human).
Pharmaceutical Compositions and Delivery Methods
[0381] The pharmaceutical compositions of the present disclosure relate to
compositions for
administration to a human subject. The pharmaceutical compositions comprise
the non-naturally
occurring delivery constructs recited herein, alone or in combination. The
pharmaceutical
compositions can comprise additional molecules capable of altering the
characteristics of the
non-naturally occurring delivery constructs, for example, stabilizing,
modulating and/or
activating their function. The composition may, e.g., be in solid or liquid
form and can be, inter
alia, in the form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an)
aerosol(s). The
pharmaceutical composition of the present disclosure may, optionally and
additionally, comprise
a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier"
refers to a non-toxic
solid, semisolid or liquid filler, diluent, encapsulating material and any of
the standard
pharmaceutical carriers, vehicles, buffers, and excipients, such as a
phosphate buffered saline
solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water
or water/oil
emulsion, and various types of wetting agents and/or adjuvants.
[0382] The pharmaceutical compositions are generally formulated
appropriately for the
immediate use intended for the delivery construct. For example, if the
delivery construct is not to
be administered immediately, the delivery construct can be formulated in a
composition suitable
for storage. One such composition is a lyophilized preparation of the delivery
construct together
with a suitable stabilizer. Alternatively, the delivery construct composition
can be formulated for
storage in a solution with one or more suitable stabilizers. Any such
stabilizer known to one of
skill in the art without limitation can be used. For example, stabilizers
suitable for lyophilized
preparations include, but are not limited to, sugars, salts, surfactants,
proteins, chaotropic agents,
lipids, and amino acids. Stabilizers suitable for liquid preparations include,
but are not limited to,
sugars, salts, surfactants, proteins, chaotropic agents, lipids, and amino
acids. Specific stabilizers
than can be used in the compositions include, but are not limited to,
trehalose, serum albumin,
phosphatidylcholine, lecithin, and arginine. Other compounds, compositions,
and methods for
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stabilizing a lyophilized or liquid preparation of the delivery constructs can
be found, for
example, in U.S. Pat. Nos. 6,573,237, 6,525,102, 6,391,296, 6,255,284,
6,133,229, 6,007,791,
5,997,856, and 5,917,021.
[0383] In various embodiments, the pharmaceutical compositions of the
present disclosure
are formulated for oral delivery. The pharmaceutical compositions formulated
for oral
administration take advantage of the bacterial toxin's ability to mediate
transcytosis across the
gastrointestinal (GI) epithelium and/or delivery to the interior of a cell of
the GI epithelium (e.g.,
gut). It is anticipated that oral administration of these pharmaceutical
compositions will result in
absorption of the delivery construct through polarized epithelial cells of the
digestive mucosa,
e.g., the intestinal mucosa, followed by release of the biologically active
cargo at the basolateral
side of the mucous membrane. In various embodiments, the epithelial cell is
selected from the
group consisting of nasal epithelial cells, oral epithelial cells, intestinal
epithelial cells, rectal
epithelial cells, vaginal epithelial cells, and pulmonary epithelial cells.
Pharmaceutical
compositions of the disclosure can include the addition of a transcytosis
enhancer to facilitate
transfer of the fusion protein across the GI epithelium. Such enhancers are
known in the art. See
Xia et al., (2000) J. Pharmacol. Experiment. Therap., 295:594-600; and Xia et
al. (2001)
Pharmaceutical Res., 18(2):191-195, each incorporated by reference in its
entirety herein.
[0384] Without being bound to any theory, it is assumed that once
transported across the GI
epithelium, the delivery constructs of the disclosure will exhibit extended
half-life in serum, that
is, the biologically active cargo of the delivery constructs will exhibit an
extended serum half-life
compared to the biologically active cargo in its non-fused state. As such, the
oral formulations of
the pharmaceutical compositions of the present disclosure are prepared so that
they are suitable
for transport to the GI epithelium and protection of the delivery construct in
the stomach. Such
formulations can include carrier and dispersant components and can be in any
suitable form,
including aerosols (for oral or pulmonary delivery), syrups, elixirs, tablets,
including chewable
tablets, hard or soft capsules, troches, lozenges, aqueous or oily
suspensions, emulsions, cachets
or pellets granulates, and dispersible powders. In various embodiments, the
pharmaceutical
compositions are employed in solid dosage forms, e.g., tablets, capsules, or
the like, suitable for
simple oral administration of precise dosages.
[0385] The oral formulation can comprise a delivery construct and one or
more compounds
that can protect the delivery construct while it is in the stomach. For
example, the protective
compound should be able to prevent acid and/or enzymatic hydrolysis of the
delivery construct.
In various embodiments, the oral formulation comprises a delivery construct
and one or more
compounds that can facilitate transit of the construct from the stomach to the
small intestine. The
one or more compounds that can protect the delivery construct from degradation
in the stomach
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can also facilitate transit of the construct from the stomach to the small
intestine. For example,
inclusion of sodium bicarbonate can be useful for facilitating the rapid
movement of intra-gastric
delivered materials from the stomach to the duodenum as described in Mrsny et
al., Vaccine
17:1425-1433, 1999. Other methods for formulating compositions so that the
delivery constructs
can pass through the stomach and contact polarized epithelial membranes in the
small intestine
include, but are not limited to, enteric-coating technologies as described in
DeYoung, Int J
Pancreatol, 5 Supp1:31-6, 1989 and the methods provided in U.S. Pat. Nos.
6,613,332, 6,174,529,
6,086,918, 5,922,680, and 5,807,832, each incorporated by reference in its
entirety herein.
[0386] Pharmaceutical compositions intended for oral use can be prepared
according to any
method known to the art for the manufacture of pharmaceutical compositions and
such
compositions can contain one or more agents selected from the group consisting
of sweetening
agents in order to provide a pharmaceutically elegant and palatable
preparation. For example, to
prepare orally deliverable tablets, the delivery construct is mixed with at
least one
pharmaceutical excipient, and the solid formulation is compressed to form a
tablet according to
known methods, for delivery to the gastrointestinal tract. The tablet
composition is typically
formulated with additives, e.g. a saccharide or cellulose carrier, a binder
such as starch paste or
methyl cellulose, a filler, a disintegrator, or other additives typically
usually used in the
manufacture of medical preparations. To prepare orally deliverable capsules,
DHEA is mixed
with at least one pharmaceutical excipient, and the solid formulation is
placed in a capsular
container suitable for delivery to the gastrointestinal tract. Compositions
comprising delivery
constructs can be prepared as described generally in Remington's
Pharmaceutical Sciences, 18th
Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which is herein
incorporated
by reference.
[0387] The pharmaceutical compositions can be formulated as orally
deliverable tablets
containing delivery constructs in admixture with non-toxic pharmaceutically
acceptable
excipients, which are suitable for manufacture of tablets. These excipients
can be inert diluents,
such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or
sodium phosphate;
granulating and disintegrating agents, for example, maize starch, gelatin or
acacia, and
lubricating agents, for example, magnesium stearate, stearic acid, or talc.
The tablets can be
uncoated or they can be coated with known techniques to delay disintegration
and absorption in
the gastrointestinal track and thereby provide a sustained action over a
longer period of time. For
example, a time delay material such as glyceryl monostearate or glyceryl
distearate alone or with
a wax can be employed.
[0388] The pharmaceutical compositions can be formulated as hard gelatin
capsules wherein
the delivery construct is mixed with an inert solid diluent, for example,
calcium carbonate,
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calcium phosphate, or kaolin or as soft gelatin capsules wherein the delivery
construct is mixed
with an aqueous or an oil medium, for example, arachis oil, peanut oil, liquid
paraffin or olive
oil.
[0389] Aqueous suspensions can contain a delivery construct in the
admixture with
excipients suitable for the manufacture of aqueous suspensions. Such
excipients are suspending
agents, for example, sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum
tragacanth and gum
acacia; dispersing or wetting agents can be a naturally occurring phosphatide,
for example,
lecithin, or condensation products of an alkylene oxide with fatty acids, for
example,
polyoxyethylene stearate, or condensation products of ethylene oxide with long
chain aliphatic
alcohols, for example, heptadecylethyloxycetanol, or condensation products of
ethylene oxide
with partial esters derived from fatty acids and a hexitol such as
polyoxyethylene sorbitol
monooleate, or condensation products of ethylene oxide with partial esters
derived from fatty
acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate.
The aqueous
suspensions can also contain one or more preservatives for example, ethyl or n-
propyl p-
hydroxybenzoate, one or more coloring agents, one or more flavoring agents and
one or more
sweetening agents such as sucrose or saccharin.
[0390] Oily suspensions can be formulated by suspending the delivery
construct in a
vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil,
or in a mineral oil
such as liquid paraffin. The oil suspensions can contain a thickening agent,
for example,
beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set
forth above, and
flavoring agents can be added to provide a palatable oral preparation. These
compositions can be
preserved by the addition of an antioxidant such as ascorbic acid.
[0391] The pharmaceutical compositions can be in the form of oil-in-water
emulsions. The
oil phase can be a vegetable oil, for example, olive oil or arachis oil, or a
mineral oil for
example, gum acacia or gum tragacanth, naturally-occurring phosphatides, for
example soybean
lecithin, and esters or partial esters derived from fatty acids and hexitol
anhydrides, for example,
sorbitan monooleate, and condensation products of the same partial esters with
ethylene oxide,
for example, polyoxyethylene sorbitan monooleate. The emulsions can also
contain sweetening
and flavoring agents.
[0392] The pharmaceutical composition can be in the form of a tablet or
capsule, and the
tablet or capsule can be coated or encapsulated to protect a therapeutically
or biologically active
cargo from enzymatic action in the stomach and to ensure that there is
sufficient biologically
active cargo to be absorbed by the subject to produce an effective response.
Such coating or
encapsulation methods include, e.g., encapsulation in nanoparticles composed
of polymers with a
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hydrophobic backbone and hydrophilic branches as drug carriers, encapsulation
in
microparticles, insertion into liposomes in emulsions, and conjugation to
other molecules. In
some cases, the capsule or tablet releases the delivery construct in a pH-
dependent manner.
Capsules or tablets used for administering a delivery construct as described
herein can comprise
one or more enteric coatings.
[0393] Examples of nanoparticles include mucoadhesive nanoparticles coated
with chitosan
and Carbopol (Takeuchi et al., Adv. Drug Deliv. Rev. 47(1):39-54, 2001) and
nanoparticles
containing charged combination polyesters, poly(2-sulfobutyl-vinyl alcohol)
and poly(D,L-
lactic-co-glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50(1):147-160,
2000).
[0394] Encapsulated or coated tablets can be used that release a
biologically active cargo in
a layer-by-layer manner, thereby releasing biologically active cargo over a
pre-determined time
frame while moving along the gastrointestinal tract. In addition, tablets
comprising the
biologically active cargo can be placed within a larger tablet, thereby
protecting the inner tablet
from environmental and processing conditions, such as temperature, chemical
agents (e.g.,
solvents), pH, and moisture. The outer tablet and coatings further serve to
protect the
biologically active cargo in the gastric environment.
[0395] Pharmaceutical compositions described herein can be formulated for
oral delivery
using polyester microspheres, zein microspheres, proteinoid microspheres,
polycyanoacrylate
microspheres, and lipid-based systems (see, for example, DiBase and Morrel,
Oral Delivery of
Microencapsulated Proteins, in Protein Delivery: Physical Systems, Sanders and
Hendren (eds.),
pages 255-288 (Plenum Press 1997)).
[0396] Surface-active agents or surfactants promote absorption of
polypeptides through
mucosal membrane or lining. Useful surface-active agents or surfactants
include fatty acids and
salts thereof, bile salts, phospholipid, or an alkyl saccharide. Examples of
fatty acids and salts
thereof include sodium, potassium and lysine salts of caprylate (CO, caprate
(C10), laurate (C12)
and myristate (C14). Examples of bile salts include cholic acid,
chenodeoxycholic acid,
glycocholic acid, taurocholic acid, glycochenodeoxycholic acid,
taurochenodeoxycholic acid,
deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic
acid, and
ursodeoxycholic acid. Examples of phospholipids include single-chain
phospholipids, such as
lysophosphatidylcholine, lysophosphatidylglycerol,
lysophosphatidylethanolamine,
lysophosphatidylinositol and lysophosphatidylserine; or double-chain
phospholipids, such as
diacylphosphatidylcholines, diacylphosphatidylglycerols,
diacylphosphatidylethanolamines,
diacylphosphatidylinositols and diacylphosphatidylserines. Examples of alkyl
saccharides
include alkyl glucosides or alkyl maltosides, such as decyl glucoside and
dodecyl maltoside.
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[0397] The present disclosure relates to methods and compositions that
allow orally
administering the pharmaceutical compositions of the disclosure. Without
intending to be bound
to any particular theory or mechanism of action, it is believed that oral
administration of the
delivery constructs results in absorption of the delivery construct through
polarized epithelial
cells of the digestive mucosa, e.g., the intestinal mucosa, followed by
cleavage of the delivery
construct and release of the biologically active cargo at the basolateral side
of the mucous
membrane. Thus, when the biologically active cargo exerts a biological
activity in the liver, such
as, for example, activities mediated by IL-10 binding to its cognate receptor,
the biologically
active cargo is believed to exert an effect in excess of what would be
expected based on the
plasma concentrations observed in the subject, i.e., oral administration of
the delivery construct
can deliver a higher effective concentration of the delivered biologically
active cargo to the liver
of the subject than is observed in the subject's plasma.
[0398] The present disclosure relates to methods of orally administering
the pharmaceutical
compositions of the disclosure. Such methods can include, but are not limited
to, steps of orally
administering the compositions by the patient or a caregiver. Such
administration steps can
include administration on intervals such as once or twice per day depending on
the delivery
construct, disease or patient condition or individual patient. Such methods
also include the
administration of various dosages of the individual delivery construct. For
instance, the initial
dosage of a pharmaceutical composition can be at a higher level to induce a
desired effect, such
as reduction in blood glucose levels. Subsequent dosages can then be decreased
once a desired
effect is achieved. Such changes or modifications to administration protocols
can be performed
by the attending physician or health care worker.
[0399] These pharmaceutical compositions can be administered to the subject
at a suitable
dose. The dosage regimen can be determined by the attending physician based
upon specific
clinical factors. As is well known in the medical arts, dosages for any one
patient depend upon
many factors, including the patient's size, body surface area, age, the
particular compound to be
administered, sex, time and route of administration, general health, and other
drugs being
administered concurrently. The therapeutically effective amount for a given
situation will readily
be determined by routine experimentation and is within the skills and judgment
of the ordinary
clinician or physician. The skilled person knows that the effective amount of
a pharmaceutical
composition administered to an individual will, inter alia, depend on the
nature of the
biologically active cargo. The length of treatment needed to observe changes
and the interval
following treatment for responses to occur vary depending on the desired
effect. The particular
amounts can be determined by conventional tests, which are well known to the
person skilled in
the art.
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[0400] The amount of biologically active cargo is an amount effective to
accomplish the
purpose of the particular active agent. The amount in the composition
typically is a
pharmacologically, biologically, therapeutically, or chemically effective
amount. However, the
amount can be less than a pharmacologically, biologically, therapeutically, or
chemically
effective amount when the composition is used in a dosage unit form, such as a
capsule, a tablet
or a liquid, because the dosage unit form can contain a multiplicity of
carrier/biologically or
chemically active agent compositions or can contain a divided
pharmacologically, biologically,
therapeutically, or chemically effective amount. The total effective amounts
can then be
administered in cumulative units containing, in total, pharmacologically,
biologically,
therapeutically or chemically active amounts of biologically active cargo.
[0401] As used herein, the terms "co-administration", "co-administered" and
"in
combination with", referring to the delivery constructs of the disclosure and
one or more other
therapeutic agents, is intended to mean, and does refer to and include the
following:
simultaneous administration of such combination of delivery constructs of the
disclosure and
therapeutic agent(s) to a patient in need of treatment, when such components
are formulated
together into a single dosage form which releases said components at
substantially the same time
to said patient; substantially simultaneous administration of such combination
of delivery
constructs of the disclosure and therapeutic agent(s) to a patient in need of
treatment, when such
components are formulated apart from each other into separate dosage forms
which are taken at
substantially the same time by said patient, whereupon said components are
released at
substantially the same time to said patient; sequential administration of such
combination of
delivery constructs of the disclosure and therapeutic agent(s) to a patient in
need of treatment,
when such components are formulated apart from each other into separate dosage
forms which
are taken at consecutive times by said patient with a significant time
interval between each
administration, whereupon said components are released at substantially
different times to said
patient; and sequential administration of such combination of delivery
constructs of the
disclosure and therapeutic agent(s) to a patient in need of treatment, when
such components are
formulated together into a single dosage form which releases said components
in a controlled
manner whereupon they are released in a concurrent, consecutive, and/or
overlapping manner at
the same and/or different times to said patient, where each part can be
administered by either the
same or a different route.
[0402] A combination therapy can comprise administering the isolated
delivery construct
composition and the second agent composition simultaneously, either in the
same
pharmaceutical composition or in separate pharmaceutical compositions. In
various
embodiments, isolated delivery construct composition and the second agent
composition are
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administered sequentially, i.e., the isolated delivery construct composition
is administered either
prior to or after the administration of the second agent composition.
[0403] An administration of the isolated delivery construct composition and
the second
agent composition can be concurrent, i.e., the administration period of the
isolated delivery
construct composition and the second agent composition overlap with each
other.
[0404] An administration of the isolated delivery construct composition and
the second
agent composition can be non-concurrent. For example, in various embodiments,
the
administration of the isolated delivery construct composition is terminated
before the second
agent composition is administered. The administration second agent composition
can be
terminated before the isolated delivery construct composition is administered.
Methods of Treatment
[0405] The pharmaceutical compositions formulated for oral delivery can be
used to treat
certain classes of diseases or medical conditions that are particularly
amenable for oral
formulation and delivery. Such classes of diseases or conditions include,
e.g., viral disease or
infections, cancer, a metabolic disease, obesity, autoimmune diseases,
inflammatory diseases,
allergy, graft-vs-host disease, systemic microbial infection, anemia,
cardiovascular disease,
psychosis, genetic diseases, neurodegenerative diseases, disorders of
hematopoietic cells,
diseases of the endocrine system or reproductive systems, gastrointestinal
diseases. In many
chronic diseases, oral formulations of the delivery constructs of the
disclosure are particularly
useful because they allow long-term patient care and therapy via home oral
administration
without reliance on injectable treatment or drug protocols.
[0406] A pharmaceutical composition of the present disclosure can comprise
any of the
delivery constructs described herein, which includes any combination of
carrier, cargo, and/or
spacer described herein. Specifically, the delivery constructs described
herein allow for oral
administration, which can be followed by transport of the delivery construct
across or into a cell
of an epithelium of a subject. A delivery construct that has been transported
across such an
epithelial layer can subsequently reach various parts and/or organs and/or
tissues within the
subject. A delivery construct, and in various cases the cargo that a delivery
construct comprises,
can elicit an effect upon reaching a submucosal compartment. For example, a
biologically active
cargo can be a cargo capable of eliciting an immune response, and thus a
delivery construct can
present such cargo to immune cell once it has reached a submucosal
compartment.
[0407] The present disclosure relates to methods for treatment, prophylaxis
and/or
prevention of an inflammatory disease in a subject, comprising administering a
pharmaceutical
composition of the present disclosure to the subject. "Inflammatory diseases"
include all diseases
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associated with acute or chronic inflammation. Acute inflammation is the
initial response of the
body to harmful stimuli and results from an increased movement of plasma and
leukocytes (such
as e.g. granulocytes) from the blood into the injured tissues. A number of
biochemical events
propagates and matures the inflammatory response, involving the local vascular
system, the
immune system, and various cells within the injured tissue. Prolonged
inflammation is referred
to as chronic inflammation, which leads to a progressive shift in the type of
cells present at the
site of inflammation and is characterized by simultaneous destruction and
healing of the tissue
from the inflammatory process. Inflammatory diseases can be caused by e.g.
burns, chemical
irritants, frostbite, toxins, infection by pathogens, physical injury, immune
reactions due to
hypersensitivity, ionizing radiation, or foreign bodies, such as e.g.
splinters, dirt and debris.
Examples of inflammatory diseases are well known in the art.
[0408] An inflammatory disease can be selected from the group consisting of
inflammatory
bowel disease, psoriasis and bacterial sepsis. The term "inflammatory bowel
disease", as used
herein, refers to a group of inflammatory conditions of the colon and small
intestine including,
for example, Crohn's disease, ulcerative colitis, collagenous colitis,
lymphocytic colitis,
ischemic colitis, diversion colitis, Behcet's syndrome and indeterminate
colitis. Delivery
constructs that can be used to prevent and/or treat such inflammatory disease
include those
comprising the amino acid sequence set forth in SEQ ID NO: 154 and/or SEQ ID
NO: 155.
[0409] "Crohn's disease", in accordance with the present disclosure, is a T-
helper Type 1
(Thl) inflammatory bowel disease, which has an immune response pattern that
includes an
increased production of interleukin-12, tumor necrosis factor (TNF), and
interferon-y
(Romagnani. Inflamm Bowel Dis 1999; 5:285-94), and which can have a
devastating impact on
the lifestyle of a patient afflicted therewith. Common symptoms of Crohn's
disease include
diarrhea, cramping, abdominal pain, fever, and even rectal bleeding. Crohn's
disease and
complications associated with it often results in the patient requiring
surgery, often more than
once. There is no known cure for Crohn's disease, and long-term, effective
treatment options are
limited. The goals of treatment are to control inflammation, correct
nutritional deficiencies, and
relieve symptoms like abdominal pain, diarrhea, and rectal to bleeding. While
treatment can help
control the disease by lowering the number of times a person experiences a
recurrence, there is
no cure. Treatment can include drugs, nutrition supplements, surgery, or a
combination of these
options. Common treatments which can be administered for treatment include
anti-inflammation
drugs, including sulfasalazine, cortisone or steroids, including prednisone,
immune system
suppressors, such as 6-mercaptopurine or azathioprine, and antibiotics.
[0410] "Psoriasis", in accordance with the present disclosure, is a
disease, which affects the
skin and joints. It commonly causes red scaly patches to appear on the skin.
The scaly patches
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caused by psoriasis, called psoriatic plaques, are areas of inflammation and
excessive skin
production. Skin rapidly accumulates at these sites and takes a silvery-white
appearance. Plaques
frequently occur on the skin of the elbows and knees, but can affect any area
including the scalp
and genitals. Psoriasis is hypothesized to be immune-mediated and is not
contagious. The
disorder is a chronic recurring condition, which varies in severity from minor
localized patches
to complete body coverage. Fingernails and toenails are frequently affected
(psoriatic nail
dystrophy) -- and can be seen as an isolated finding. Psoriasis can also cause
inflammation of the
joints, which is known as psoriatic arthritis. Ten to fifteen percent of
people with psoriasis have
psoriatic arthritis.
[0411] The term "bacterial sepsis", as used herein, refers to life-
threatening conditions
resulting from the circulation of bacteria in the blood stream. Sepsis results
in generalized
systemic production of pro-inflammatory cytokines that results in tissue
damage and ultimately
septic shock due to failure of the microcirculation.
[0412] The present disclosure relates to methods for treatment, prophylaxis
and/or
prevention of an autoimmune disease in a subject, comprising administering a
pharmaceutical
composition of the present disclosure to the subject. An autoimmune disease,
as pertains to the
present disclosure, is a disease or disorder arising from and directed against
an individual's own
tissues or a co-segregate or manifestation thereof or resulting condition
therefrom. In various
embodiments. the autoimmune disease is selected from the group consisting of
systemic lupus
erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia,
thrombocytopenia purpura, Grave's disease, Sjogren's disease, dermatomyositis,
Hashimoto's
disease, polymyositis, inflammatory bowel disease, multiple sclerosis (MS),
diabetes mellitus,
rheumatoid arthritis, and scleroderma. Exemplary delivery constructs that can
be used to treat
those disease can include those comprising any carrier set forth in SEQ ID NO:
4 ¨ SEQ ID NO:
125 coupled to, for example, an anti-TNFa antibody, or a functional binding
fragment thereof.
[0413] "Rheumatoid arthritis", in accordance with the present disclosure,
is an autoimmune
disorder that causes the body's immune system to attack the bone joints
(Muller B et al.,
Springer Semin. Immunopathol., 20:181-96, 1998). Rheumatoid arthritis is a
chronic, systemic
inflammatory disorder that can affect many tissues and organs, but principally
attacks synovial
joints. The process produces an inflammatory response of the synovium
(synovitis) secondary to
hyperplasia of synovial cells, excess synovial fluid, and the development of
pannus in the
synovium. The pathology of the disease process often leads to the destruction
of articular
cartilage and ankylosis of the joints. Rheumatoid arthritis can also produce
diffuse inflammation
in the lungs, pericardium, pleura, and sclera, and also nodular lesions, most
common in
subcutaneous tissue under the skin.
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[0414] The present disclosure relates to methods and compositions for
treatment,
prophylaxis and/or prevention of a cancer in a subject, comprising
administering a
pharmaceutical composition of the present disclosure to the subject. Cancers
to be treated
include, but are not limited to, non-Hodgkin's lymphomas, Hodgkin's lymphoma,
chronic
lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia,
multiple myeloma,
carcinomas of the pancreas, colon, gastric intestine, prostate, bladder,
kidney ovary, cervix,
breast, lung, nasopharynx, malignant melanoma and rituximab resistant NHL and
leukemia.
[0415] The therapeutically effective amount of a pharmaceutical composition
of the present
disclosure will be administered in combination with one or more other
therapeutic agents. Such
therapeutic agents can be accepted in the art as a standard treatment for a
particular disease state
as described herein, such as inflammatory disease, autoimmune disease, or
cancer. Exemplary
therapeutic agents contemplated include, but are not limited to, cytokines,
growth factors,
steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutics,
radiotherapeutics, or
other active and ancillary agents.
[0416] The present disclosure relates to methods for treatment, prophylaxis
and/or
prevention of a metabolic disorder in a subject, comprising administering a
pharmaceutical
composition of the present disclosure to the subject. In various embodiments,
the metabolic
disorder is selected from the group consisting of: diabetes, obesity, diabetes
as a consequence of
obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X,
insulin resistance,
impaired glucose tolerance (IGT), diabetic dyslipidemia, and hyperlipidemia.
[0417] The present disclosure relates to methods for treatment, prophylaxis
and/or
prevention of a fatty liver disease (e.g., nonalcoholic fatty liver disease
(NAFLD); nonalcoholic
steatohepatitis (NASH)), a gastrointestinal disease, or a neurodegenerative
disease in a subject,
comprising administering a pharmaceutical composition of the present
disclosure to the subject.
[0418] The present disclosure relates to methods and compositions for
treatment,
prophylaxis and/or prevention of a GH deficient growth disorder in a subject,
said method
comprising administering a pharmaceutical composition of the present
disclosure to the subject.
In various embodiments, the disorder is selected from the group consisting of:
growth hormone
deficiency (GHD), Turner syndrome (TS), Noonan syndrome, Prader-Willi
syndrome, short
stature homeobox-containing gene (SHOX) deficiency, chronic renal
insufficiency, and
idiopathic short stature short bowel syndrome, GH deficiency due to rare
pituitary tumors or
their treatment, and muscle-wasting disease associated with HIV/AIDS.
[0419] A subject of the present disclosure can be a human or a rodent. The
subject can be a
human. A subject can be affected by one or more of the following: inflammatory
bowel disease,
psoriasis, bacterial sepsis, systemic lupus erythematosus (SLE), pemphigus
vulgaris, myasthenia
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gravis, hemolytic anemia, thrombocytopenia purpura, Grave's disease, Sjogren's
disease,
dermatomyositis, Hashimoto's disease, polymyositis, inflammatory bowel
disease, multiple
sclerosis (MS), diabetes mellitus, rheumatoid arthritis, scleroderma, non-
Hodgkin's lymphomas,
Hodgkin's lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, acute
lymphoblastic
leukemia, multiple myeloma, carcinomas of the bladder, kidney ovary, cervix,
breast, lung,
nasopharynx, malignant melanoma, rituximab resistant NHL or leukemia,
diabetes, obesity,
diabetes as a consequence of obesity, hyperglycemia, dyslipidemia,
hypertriglyceridemia,
syndrome X, insulin resistance, impaired glucose tolerance (IGT), diabetic
dyslipidemia,
hyperlipidemia, growth hormone deficiency (GHD), Turner syndrome (TS), Noonan
syndrome,
Prader-Willi syndrome, short stature homeobox-containing gene (SHOX)
deficiency, chronic
renal insufficiency, or idiopathic short stature short bowel syndrome.
[0420] The methods and compositions described herein can also be used to
diagnose disease
or condition. Diagnosing a disease or condition can include invasive and non-
invasive diagnostic
modalities. Specifically, the compositions described herein can be used to non-
invasively
diagnose a disease, e.g., by measuring the expression of a certain marker
(e.g., a biomarker) or
antigen. Such diagnoses can be conducted by coupling a cargo to a carrier,
wherein the cargo can
have a binding affinity for a certain marker (e.g., a biomolecule
representative which presence or
concentration in a certain organ, tissue, or cell is representative of a
certain disease or condition).
Diagnoses as described herein can further comprise monitoring a response to a
treatment (e.g.,
the treatment of a subject). For example, if response to a treatment
correlates with a reduction of
a certain marker (e.g., a biomarker), the delivery constructs of the present
disclosure can be used
to measure such marker at a certain location (e.g., a certain immune cell
population in a
submucosal compartment). In addition to non-invasive diagnoses, the methods
and compositions
described herein can be used to provide biologically and/or therapeutically
relevant information,
e.g., upon a biopsy sample has been taken from a subject, which can be
followed by
immunohistochemistry, e.g., the detection of accumulation of a delivery
constructs in a certain
tissue etc.
[0421] Non-invasive diagnosis can comprise molecular and/or nuclear
imaging. For
example, a delivery construct can comprise cargo that is labeled with a
fluorescent and/or
radioactive compound such that the location and/or concentration of such a
delivery construct
can be determined in a subject after administration. Any moiety with
diagnostic applicability as
described herein can be used to provide diagnostic and/or theranostic
(therapeutic and
diagnostic) agents.
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Polynucleotides Encoding Delivery constructs
[0422] The methods and compositions of the present disclosure provides
polynucleotides
comprising a nucleotide sequence encoding non-naturally occurring delivery
constructs and/or
hybrid delivery construct polypeptides. These polynucleotides are useful, for
example, for
making the delivery constructs and/or hybrid delivery construct polypeptides.
The disclosure
provides an expression system that comprises a recombinant polynucleotide
sequence encoding a
bacterial carrier receptor binding domain, and a polyspacer insertion site for
a polynucleotide
sequence encoding a biologically active cargo. The polyspacer insertion site
can be anywhere in
the polynucleotide sequence so long as the polyspacer insertion does not
disrupt the delivery
construct of the bacterial toxin. The expression system can comprise a
polynucleotide sequence
that encodes a cleavable spacer so that cleavage at the cleavable spacer
separates a biologically
active cargo encoded by a nucleic acid inserted into the polyspacer insertion
site from the
remainder of the encoded delivery construct. Thus, in embodiments where the
polyspacer
insertion site is at an end of the encoded construct, the polynucleotide
comprises one nucleotide
sequence encoding a cleavable spacer between the polyspacer insertion site and
the remainder of
the polynucleotide. In embodiments where the polyspacer insertion site is not
at the end of the
encoded construct, the polyspacer insertion site can be flanked by nucleotide
sequences that each
encode a cleavable spacer.
[0423] Various in vitro methods that can be used to prepare a
polynucleotide encoding a
delivery construct useful in the delivery constructs of the disclosure
include, but are not limited
to, reverse transcription, the polymerase chain reaction (PCR), the ligase
chain reaction (LCR),
the transcription-based amplification system (TAS), the self-sustained
sequence replication
system (3 SR) and the QP replicase amplification system (QB). Any such
technique known by
one of skill in the art to be useful in construction of recombinant nucleic
acids can be used. For
example, a polynucleotide encoding the protein or a portion thereof can be
isolated by
polymerase chain reaction of cDNA using primers based on the DNA sequence of a
delivery
construct or a nucleotide encoding the receptor binding domain.
[0424] Guidance for using these cloning and in vitro amplification
methodologies are
described in, for example, U.S. Pat. No. 4,683,195; Mullis et al., 1987, Cold
Spring Harbor
Symp. Quant. Biol. 51:263; and Erlich, ed., 1989, PCR Technology, Stockton
Press, NY.
Polynucleotides encoding a delivery construct, or a portion thereof, also can
be isolated by
screening genomic of cDNA libraries using probes selected from the sequences
of the desired
polynucleotide under stringent, moderately stringent, or highly stringent
hybridization
conditions.
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[0425] Further, the polynucleotides can also encode a secretory sequence at
the amino
terminus of the encoded delivery construct. Such constructs are useful for
producing the delivery
constructs in mammalian cells as they simplify isolation of the delivery
construct and/or hybrid
delivery construct polypeptides.
[0426] Furthermore, the polynucleotides of the disclosure also encompass
derivative
versions of polynucleotides encoding a delivery construct. Such derivatives
can be made by any
method known by one of skill in the art without limitation. For example,
derivatives can be made
by site-specific mutagenesis, including substitution, insertion, or deletion
of one, two, three, five,
ten or more nucleotides, of polynucleotides encoding the delivery construct.
Alternatively,
derivatives can be made by random mutagenesis. One method for randomly
mutagenizing a
nucleic acid comprises amplifying the nucleic acid in a PCR reaction in the
presence of 0.1 mM
MnC12 and unbalanced nucleotide concentrations. These conditions increase the
inaccuracy
incorporation rate of the polymerase used in the PCR reaction and result in
random mutagenesis
of the amplified nucleic acid.
[0427] Accordingly, the disclosure provides a polynucleotide that can
encode one or more
delivery constructs. A delivery construct comprises a bacterial carrier and a
biologically active
cargo to be delivered to a subject; and, optionally, a non-cleavable or
cleavable spacer. Cleavage
at the cleavable spacer can separate the biologically active cargo from the
remainder of the
delivery construct. The cleavable spacer can be cleaved by an enzyme that is
present at a
basolateral membrane of a polarized epithelial cell of the subject or in the
plasma of the subject.
[0428] The polynucleotide can hybridize under stringent hybridization
conditions to any
polynucleotide of this disclosure. The polynucleotide can hybridize under
stringent conditions to
a nucleic acid that encodes any delivery construct of the disclosure.
[0429] The disclosure provides expression vectors for expressing the
delivery constructs
and/or hybrid delivery construct polypeptides. Generally, expression vectors
are recombinant
polynucleotide molecules comprising expression control sequences operatively
linked to a
nucleotide sequence encoding a polypeptide. Expression vectors can readily be
adapted for
function in prokaryotes or eukaryotes by inclusion of appropriate promoters,
replication
sequences, selectable markers, etc. to result in stable transcription and
translation or mRNA.
Techniques for construction of expression vectors and expression of genes in
cells comprising
the expression vectors are well known in the art. See, e.g., Sambrook et al.,
2001, Molecular
Cloning--A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, Cold
Spring
Harbor, N.Y., and Ausubel et al., eds., Current Edition, Current Protocols in
Molecular Biology,
Greene Publishing Associates and Wiley Interscience, NY.
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[0430] Useful promoters for use in expression vectors include, but are not
limited to, a
metallothionein promoter, a constitutive adenovirus major late promoter, a
dexamethasone-
inducible MMTV promoter, a SV40 promoter, a MRP pol III promoter, a
constitutive MPSV
promoter, a tetracycline-inducible CMV promoter (such as the human immediate-
early CMV
promoter), and a constitutive CMV promoter.
[0431] The expression vectors should contain expression and replication
signals compatible
with the cell in which the delivery constructs are expressed. Expression
vectors useful for
expressing delivery constructs include viral vectors such as retroviruses,
adenoviruses and
adeno-associated viruses, plasmid vectors, cosmids, and the like. Viral and
plasmid vectors are
preferred for transfecting the expression vectors into mammalian cells. For
example, the
expression vector pcDNA1 (Invitrogen, San Diego, Calif.), in which the
expression control
sequence comprises the CMV promoter, provides good rates of transfection and
expression into
such cells.
[0432] The expression vectors can be introduced into the cell for
expression of the delivery
constructs by any method known to one of skill in the art without limitation.
Such methods
include, but are not limited to, e.g., direct uptake of the molecule by a cell
from solution;
facilitated uptake through lipofection using, e.g., liposomes or
immunoliposomes; particle-
mediated transfection; etc. See, e.g., U.S. Pat. No. 5,272,065; Goeddel et
al., eds, 1990, Methods
in Enzymology, vol. 185, Academic Press, Inc., CA; Krieger, 1990, Gene
Transfer and
Expression--A Laboratory Manual, Stockton Press, NY; Sambrook et al., 1989,
Molecular
Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, NY; and Ausubel
et al., eds.,
Current Edition, Current Protocols in Molecular Biology, Greene Publishing
Associates and
Wiley Interscience, NY.
[0433] The expression vectors can also contain a purification moiety that
simplifies
isolation of the delivery construct and/or hybrid delivery construct
polypeptides. For example, a
polyhistidine moiety of, e.g., six histidine residues, can be incorporated at
the amino terminal
end of the protein. The polyhistidine moiety allows convenient isolation of
the protein in a single
step by nickel-chelate chromatography. In various embodiments, the
purification moiety can be
cleaved from the remainder of the delivery construct following purification.
In other
embodiments, the moiety does not interfere with the function of the functional
domains of the
delivery construct and thus need not be cleaved.
[0434] The present disclosure provides a cell that can comprise an
expression vector for
expression of the delivery constructs and/or hybrid delivery construct
polypeptides, or portions
thereof. The cell can be selected for its ability to express high
concentrations of the delivery
construct to facilitate purification of the protein. In various embodiments,
the cell is a
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prokaryotic cell, for example, E. coli. As described in the examples, the
delivery constructs are
properly folded and comprise the appropriate disulfide linkages when expressed
in E. coli. The
cell is a eukaryotic cell. Useful eukaryotic cells include yeast and mammalian
cells. Any
mammalian cell known by one of skill in the art to be useful for expressing a
recombinant
polypeptide, without limitation, can be used to express the delivery
constructs. For example,
Chinese hamster ovary (CHO) cells can be used to express the delivery
constructs. The delivery
constructs and/or hybrid delivery construct polypeptides of the disclosure can
be produced by
recombination, as described below. However, the delivery constructs can also
be produced by
chemical synthesis using methods known to those of skill in the art.
[0435] The delivery constructs of the present disclosure can be produced
using a variety of
methods. The selection of a production method can depend on the molecular
structure of the
delivery construct and/or its components (e.g., the carrier, cargo, and/or
spacer). Thus, for some
delivery constructs organic synthetic methods may be advantageous for
producing such delivery
construct. A delivery construct of the present disclosure can be a
polypeptide. Such polypeptides
can be produced, for example, using recombinant DNA methodology. Generally,
this involves
creating a DNA sequence that encodes the delivery construct, placing the DNA
in an expression
cassette under the control of a particular promoter, expressing the molecule
in a host, isolating
the expressed molecule and, if required, folding of the molecule into an
active conformational
form.
[0436] DNA encoding the delivery constructs described herein can be
prepared by any
suitable method, including, for example, cloning and restriction of
appropriate sequences or
direct chemical synthesis by methods such as the phosphotriester method of
Narang et al. (1979)
Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979)
Meth. Enzymol.
68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981)
Tetra. Lett., 22:
1859-1862); the solid support method of U.S. Pat. No. 4,458,066, and the like.
[0437] Chemical synthesis produces a single stranded oligonucleotide. This
can be
converted into double stranded DNA by hybridization with a complementary
sequence or by
polymerization with a DNA polymerase using the single strand as a template.
One of skill would
recognize that while chemical synthesis of DNA is limited to sequences of
about 100 bases,
longer sequences can be obtained by the ligation of shorter sequences.
[0438] Alternatively, subsequences can be cloned and the appropriate
subsequences cleaved
using appropriate restriction enzymes. The fragments can then be ligated to
produce the desired
DNA sequence. A DNA encoding a delivery constructs of the present disclosure
can be cloned
using DNA amplification methods such as polymerase chain reaction (PCR). Thus,
for example,
the gene for the biologically active cargo is PCR amplified, using a sense
primer containing the
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restriction site for, e.g., NdeI and an antisense primer containing the
restriction site for Hind'''.
This can produce a nucleic acid encoding the biologically active cargo
sequence and having
terminal restriction sites. A delivery construct having "complementary"
restriction sites can
similarly be cloned and then ligated to the biologically active cargo and/or
to a spacer attached to
the biologically active cargo. Ligation of the nucleic acid sequences and
insertion into a vector
produces a vector encoding the biologically active cargo joined to the
bacterial carrier receptor
binding domain. In various embodiments, DNA encoding delivery constructs of
the present
disclosure is artificially synthesized by, for example, solid-phase DNA
synthesis.
[0439] The production methods described herein can be used to produce the
delivery
constructs of the present disclosure, or (functional) variants thereof For
example, a "Cholix"
(also referred to herein as Cholix toxin or Cholix exotoxin) can encompass a
variety of
functional variants (e.g., a functional genus), wherein the functional
variants can comprise one or
more variations is their amino acid sequence relative to SEQ ID NO: 1 as
disclosed herein. Thus,
in the present disclosure, the Cholix toxin having the amino acid sequence set
forth in SEQ ID
NO: 1 is used as the reference sequence when referred to Cholix. However, as
described herein,
the present disclosure is not limited to the Cholix having the amino acid
sequence set forth in
SEQ ID NO: 1 but instead encompasses all Cholix variants that fall within the
functional genus
of Cholix. For example, a variant of the Cholix exotoxin with the amino acid
sequence set forth
in SEQ ID NI: 1 can be a Cholix exotoxin which amino acid sequence is set
forth in SEQ ID
NO: 2, wherein both variants are capable of carrying out the same functions,
e.g., transcytosis
across an epithelial cell, and interact with the same receptors, such as
ribophilin 1, 5EC24, CK-8,
TMEM132, GRP75, ERGIC-53, and/or perlecan.
[0440] Moreover, the production method of a polypeptide can affect, to some
degree, the
amino acid sequence of such polypeptide (e.g., due to post-translational
modifications). For
example, a first carrier and a second carrier are produced in the same
expression system (e.g., a
bacterial expression system such as E. coli or a mammalian expression system
such as a CHO
cell). In other cases, and as described herein, a first carrier and a second
carrier are produced in a
different expression system (e.g., a bacterial or a mammalian expression
system). Bacterial
expression systems include E. coli, and mammalian expression systems include
CHO cells, for
example. A bacterially produced polypeptide can comprise an N-cap, wherein the
N-cap can
comprise one more modifications at the N-terminal of the polypeptide. An N-cap
can comprise
an N-terminal methionine residue. Examples of Cholix domain I derived carrier
polypeptides
that can be bacterially produced and that comprise such N-terminal methionine
include those
comprising the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO: 107, and SEQ ID NO: 135.
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Experimental Methods
[0441] Transcytosis Testing. The transcytosis function of the isolated
delivery constructs
can be tested as a function of the delivery construct's ability to pass
through an epithelial
membrane. Because transcytosis first requires binding to the epithelial cell,
these assays can also
be used to assess the function of the delivery construct of the delivery
construct.
[0442] The delivery construct's transcytosis activity can be tested by any
method known by
one of skill in the art, without limitation. In various embodiments,
transcytosis activity can be
tested by assessing the ability of a delivery construct to enter a non-
polarized cell to which it
binds. In case of Cholix derived carrier, and without intending to be bound to
any particular
theory or mechanism of action, it is described herein that the transcytosis
function that allows a
delivery construct to pass through a polarized epithelial cell and the
function to enter non-
polarized cells resides in the same domain, i.e. the domain described herein
as domain I. Thus,
the delivery construct's ability to enter the cell can be assessed, for
example, by detecting the
physical presence of the construct in the interior of the cell. For example,
the delivery construct
can be labeled with, for example, a fluorescent marker, and the delivery
construct exposed to the
cell. Then, the cells can be washed, removing any delivery construct that has
not entered the cell,
and the amount of label remaining determined. Detecting the label in this
traction indicates that
the delivery construct has entered the cell.
[0443] The delivery construct's transcytosis ability can be tested by
assessing the delivery
construct's ability to pass through a polarized epithelial cell. For example,
the delivery construct
can be labeled with, for example, a fluorescent marker (e.g., RFP) and
contacted to the apical
membranes of a layer of epithelial cells. Fluorescence detected on the
basolateral side of the
membrane formed by the epithelial cells indicates that the transcytosis domain
is functioning
properly.
[0444] In vivo transcytosis can be tested using male Wistar rats. Male
Wistar rats can be
housed 3-5 per cage with a 12/12 h light/dark cycle and can be 225-275 g
(approximately 6-8
weeks old) when placed on study. Experiments can be conducted during the light
phase using a
non-recovery protocol that uses continuous isoflurane anesthesia. A 4-5 cm
midline abdominal
incision that exposes mid-jejunum regions can be conducted. Stock solutions at
3.86x105M of
test articles can be prepared in phosphate buffered saline (PBS), with 50 [IL
(per 250 g rat) being
administered by intraluminal injection (ILI) using a 29-gauge needle. The
injection site
mesentery can then be marked with a permanent marker. At study termination, a
3-5 mm region
that captured the marked intestine segment can be isolated and processed for
microscopic
assessment. In vivo experiments are performed in accordance with the U.K.
Animals (Scientific
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Procedures) Act of 1986, the European Communities Council Directive of 1986
(86/609/EEC),
and the University of Bath's ethical review procedures.
[0445] Cleavable Spacer Cleavage Testing. The function of the cleavable
spacer can
generally be tested in a cleavage assay. Any suitable cleavage assay known by
one of skill in the
art, without limitation, can be used to test the cleavable spacers. Both cell-
based and cell-free
assays can be used to test the ability of an enzyme to cleave the cleavable
spacers.
[0446] An exemplary cell-free assay for testing cleavage of cleavable
spacers comprises
preparing extracts of polarized epithelial cells and exposing a labeled
delivery construct bearing
a cleavable spacer to the fraction of the extract that corresponds to membrane-
associated
enzymes. In such assays, the label can be attached to either the biologically
active cargo to be
delivered or to the remainder of the delivery construct. Among these enzymes
are cleavage
enzymes found near the basolateral membrane of a polarized epithelial cell, as
described above.
Cleavage can be detected, for example, by binding the delivery construct with,
for example, an
antibody and washing off unbound molecules. If label is attached to the
biologically active cargo
to be delivered, then little or no label should be observed on the molecule
bound to the
antibodies. Alternatively, the binding agent used in the assay can be specific
for the biologically
active cargo, and the remainder of the construct can be labeled. In either
case, cleavage can be
assessed.
[0447] Cleavage can also be tested using cell-based assays that test
cleavage by polarized
epithelial cells assembled on semi-permeable membranes. For example, a labeled
delivery
construct, or portion of a delivery construct comprising the cleavable spacer,
can be contacted to
either the apical or basolateral side of a monolayer of suitable epithelial
cells, such as, for
example, Caco-2 cells, under conditions that permit cleavage of the spacer.
Cleavage can be
detected by detecting the presence or absence of the label using a reagent
that specifically binds
the delivery construct, or portion thereof. For example, an antibody specific
for the delivery
construct can be used to bind a delivery construct comprising a label distal
to the cleavable
spacer in relation to the portion of the delivery construct bound by the
antibody. Cleavage can
then be assessed by detecting the presence of the label on molecules bound to
the antibody. If
cleavage has occurred, little or no label should be observed on the molecules
bound to the
antibody. By performing such experiments, enzymes that preferentially cleave
at the basolateral
membrane rather than the apical membrane can be identified, and, further, the
ability of such
enzymes to cleave the cleavable spacer in a delivery construct can be
confirmed.
[0448] Further, cleavage can also be tested using a fluorescence reporter
assay as described
in U.S. Pat. No. 6,759,207. Briefly, in such assays, the fluorescence reporter
is contacted to the
basolateral side of a monolayer of suitable epithelial cells under conditions
that allow the
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cleaving enzyme to cleave the reporter. Cleavage of the reporter changes the
structure of the
fluorescence reporter, changing it from a non-fluorescent configuration to a
fluorescent
configuration. The amount of fluorescence observed indicates the activity of
the cleaving
enzyme present at the basolateral membrane.
[0449] Further, cleavage can also be tested using an intra-molecularly
quenched molecular
probe, such as those described in U.S. Pat. No. 6,592,847. Such probes
generally comprise a
fluorescent moiety that emits photons when excited with light of appropriate
wavelength and a
quencher moiety that absorbs such photons when in close proximity to the
fluorescent moiety.
Cleavage of the probe separates the quenching moiety from the fluorescent
moiety, such that
fluorescence can be detected, thereby indicating that cleavage has occurred.
Thus, such probes
can be used to identify and assess cleavage by particular cleaving enzymes by
contacting the
basolateral side of a monolayer of suitable epithelial cells with the probe
under conditions that
allow the cleaving enzyme to cleave the probe. The amount of fluorescence
observed indicates
the activity of the cleaving enzyme being tested.
[0450] In Vivo Studies. Male Wistar rats, housed in groups of 3-5 per cage
with a 12/12 h
light/dark cycle, were 225-275 g (approximately 6-8 weeks old) when placed on
study after an
overnight fast. All experiments were conducted during the light phase and
carried out using a
non-recovery protocol that used continuous isoflurane anesthesia. A 4-5 cm
midline abdominal
incision was made to expose the small intestine (mid-jejunum to proximal ileum
regions).
Equimolar stock solutions of Exotoxin A (PE)-RPF or Cholix-RFP truncation
chimeras prepared
in phosphate buffered saline were injected intra-lumenally using a 29-gauge
hypodermic needle
in a volume of 200 l.L/kg (or ¨50 tL per 250 g rat). The adjacent mesentery of
each intra-
lumenal injection site was marked with a permanent ink pen. At selected time
internals, the
animal was euthanized and a 3-5 mm region that captured the marked intestine
segment was
isolated. All experiments were performed in accordance with the U.K. Animals
(Scientific
Procedures) Act of 1986, the European Communities Council Directive of 1986
(86/609/EEC),
and the University of Bath's ethical review procedures.
[0451] Microscopy. Isolated tissues were rinse briefly in ice-cold PBS and
then fixed with
4% paraformaldehyde on ice prior to labeling with primary antibodies to
Exotoxin A (PE),
Cholix, or RFP. Tissue distribution of a fluorescent-labeled secondary
antibody that recognized
these primary antibodies was assessed using a Zeiss LSM 510 fluorescence
microscope. DAPI
(4',6-diamidino-2-phenylindole) was used as a nuclear stain.
[0452] Immunohistochemistry. Rehydrate tissue slides in decreasing
concentrations of
ethanol. Slides are immersed in histoclear (x2), 100% ethanol, 90% ethanol,
80% ethanol, 70%
ethanol and PBS for 5 minutes each. Boil slides in 10 mM sodium citrate (pH 6)
for 10 minutes.
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Remove from boil and allow to cool for 20 minutes. Dry slide and add a wax
border around each
tissue section using an ImmEdge hydrophobic pen. Wash tissue sections by
pipetting PBS
directly onto tissue. Perform 3 x 5 minute washes. Permeabilise tissue by
pipetting 0.2% Triton
x-100 in PBS onto sections. Incubate at room temperature for 45 minutes. Wash
3 x 5 minutes
with PBS. Block tissue sections by pipetting 2% BSA and 2% donkey serum in
0.1% Triton x-
100 in PBS onto sections. Incubate for 2 hours at room temperature. Remove
blocking solution
and add primary antibodies. Dilute antibodies to required concentration in
0.05% Triton x-100
and 1% BSA in PBS. Incubate overnight at 4 C. Wash 3 x 5 minutes with PBS.
Incubate with
secondary antibodies. Dilute antibodies to required concentration in 0.05%
Triton x-100 and 1%
BSA in PBS. Incubate for 2 hours at room temperature. Wash 3 x 5 minutes with
PBS. Incubate
with 200 nM DAPI at room temperature for 45 minutes. Wash 3 x 5 minutes with
PBS.
Dehydrate tissue sections by immersing in 70% ethanol, 100% ethanol,
histoclear and 100%
ethanol for 5 minutes each. Place a drop of fluorshield mounting media on each
tissue section
and cover with a glass coverslip. Gently apply pressure to the coverslip to
remove air bubbles.
Allow mounting media to dry for 4 hours. Store slides at 4 C and image using
confocal
fluorescent microscope.
[0453] Evaluation of Cholix Domain I Interacting Proteins. In order to
identify Cholix
and/or PE interacting partners (e.g., receptors, enzymes, etc.) and establish
the vesicular
compartments where they interact with Cholix or PE exotoxins (e.g., a domain I
of those
exotoxins or a truncated version thereof), a series of pull-downs can be
performed to identify
potential interaction partners that can be followed by in sit/co associations
using surface plasmon
resonance, in vitro transcytosis studies using polarized Caco-2 human
intestinal epithelial cells
where genetic knockdown of specific targets can be achieved, and in vivo
transcytosis studies
where Cholix elements and specific receptors can be co-localized in
established vesicular
structures. Without being bound to any theory, it is assumed that a
transcytosis process can
involve elements that are normally restricted within specific vesicular
elements of polarized
intestinal epithelial cells but can be recruited or "hijacked" by, e.g.,
Cholix domain I or truncated
versions thereof, to leave the late endosome and avoid lysosomal degradation
following apical
receptor-mediated endocytosis.
EXAMPLES
[0454] The following examples merely illustrate the disclosure, and are not
intended to limit
the disclosure in any way.
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EXAMPLE 1
Preparation of Cholix and PE Derived Delivery Constructs
[0455] This Example describes the exemplary preparation of delivery
constructs comprising
truncated Cholix carriers (truncation in domains II and/or lb) and truncated
PE carriers
(truncation in domains II and/or lb) conjoined to heterologous cargos. In this
Example, various
non-naturally occurring delivery constructs were prepared as a single amino
acid sequence and
comprising a modified Cholix carrier sequence and/or a modified PE carrier
sequence, a
polyglycine-serine peptide spacer sequence, and a heterologous cargo.
[0456] The following modified Cholix and/or PE carriers were prepared and
used to prepare
Constructs 1-12: 1) a modified Cholix carrier truncated at amino acid residue
425 of SEQ ID
NO: 1 (Cholix425, SEQ ID NO: 129); 2) a modified Cholix carrier truncated at
amino acid residue
415 of SEQ ID NO: 1 (Cholix415, SEQ ID NO: 130); 3) a modified Cholix carrier
truncated at
amino acid residue 397 of SEQ ID NO: 1 (Cholix397, SEQ ID NO: 131); 4) a
modified Cholix
carrier truncated at amino acid residue 386 of SEQ ID NO: 1 (Cholix386, SEQ ID
NO: 132); 5) a
modified Cholix carrier truncated at amino acid residue 291 of SEQ ID NO: 1
(Cholix291, SEQ
ID NO: 133); 6) a modified Cholix carrier truncated at amino acid residue 265
of SEQ ID NO: 1
(Cholix265, SEQ ID NO: 4); 7) a modified PE carrier truncated at amino acid
residue 404 of SEQ
ID NO: 3 (PE404, SEQ ID NO: 140); 8) a modified PE carrier truncated at amino
acid residue 395
of SEQ ID NO: 3 (PE395, SEQ ID NO: 141); 9) a modified PE carrier truncated at
amino acid
residue 375 of SEQ ID NO: 3 (PE375, SEQ ID NO: 142); 10) a modified PE carrier
truncated at
amino acid residue 364 of SEQ ID NO: 3 (PE364, SEQ ID NO: 143); 11) a modified
PE carrier
truncated at amino acid residue 277 of SEQ ID NO: 3 (PE277, SEQ ID NO: 144);
and 12) a
modified PE carrier truncated at amino acid residue 252 of SEQ ID NO: 3
(PE252, SEQ ID NO:
145). In each Construct 1-12, the polyglycine-serine peptide spacer GTGGS (SEQ
ID NO: 201)
was used to conjoin red fluorescent protein (RFP, SEQ ID NO: 220) at the C-
terminus of each
modified toxin. The RFP emulated the presence of a biologically active cargo.
[0457] Codon-optimized genes were obtained from a commercial source and
cloned into the
pET26(+) expression vector that was used to transform BL21(DE3) component E.
coli cells
using the manufacturer's suggested protocol. Clones were selected using
Kanamycin/Agar plates
incubate overnight at 37 C. Protein expression in fermented cultures of
selected clones was
achieved by 1 mM IPTG induction. Pelleted bacteria were lysed to collect
inclusion bodies that
were extensively washed in 50 mM Tris, 20 mM EDTA, 2.5 % Triton X-100, 0.5 M
NaCl, pH 8
prior to solubilization facilitated by sonication in 100 mM Tris, pH 8, 7 M
Guanidine HC1. After
centrifugation to pellet insoluble materials and the addition of
dithiothreitol, proteins in the
supernatant were refolded using a shuffle buffer containing 100 mM Tris, pH 8,
0.5 M L-
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Arginine, 1 M Urea, 2 mM EDTA, 1 mM oxidized glutathione (fresh made), and 1
mM Reduced
glutathione (fresh made) that was dialyzed at 4 C against 25 mM Tris, pH 8,
0.1 M Urea, and 1
mM EDTA. Following 0.451.tm filtration, desired proteins were purified using
ion exchange and
size exclusion chromatography. Final protein samples were analyzed by SDS-
polyacrylamide gel
electrophoresis and stored at -80 C.
EXAMPLE 2
In vivo Experiments Assessing Delivery Functions of Exotoxin Derived Carrier
Molecules
[0458] This Example describes an exemplary in vivo study to evaluate
epithelial trafficking
and delivery functions of the exotoxin derived carrier molecules described
herein.
[0459] The in vivo studies used 7-8 weeks old male adult Wistar rats with
an average
weight of about 250 g. Where required, experiments were performed using a non-
recovery
protocol. Generally, rats were anesthetized using inhaled isoflurane and
euthanized, when
required, by inhaled CO2. Experiments were initiated by making an about 4 cm
long abdominal
incision to access the mid-jejunum region of the small intestine. Upon making
the incision,
approximately 50 !IL of a prepared solution containing the delivery construct
or multiple
delivery constructs in a concentration of about 1-1.5 mg/mL was injected into
the intestinal
lumen of an area devoid of foodstuffs through a 27-gauge needle using a 1 mL
syringe. The
mesentery adjacent to the site of injection was labeled with a marker and the
intestine was
returned to the abdominal cavity, with the incision being closed with clamps
At specific time
points, the injected intestine was retrieved, surgically isolated and flushed
with a 4 C isotonic
PBS solution.
[0460] Washed, excised samples were fixed (4% paraformaldehyde in PBS)
overnight at
4 C before dehydration through graded series of ethanol/water solutions and
overnight
incubation in chloroform. Dehydrated tissues were immersed in wax, sectioned,
and mounted on
polylysine slides and processed for antigen retrieval using sodium citrate.
Afterward, sections
were permeabilized with 0.2% Triton-X100 in PBS prior to thrice washing in PBS
and blocking
with 2% BSA + 2% serum of the animal the secondary antibodies have been
raised. Primary
antibodies were diluted in 1% BSA, 0.1% Triton-X100 in PBS and incubate
overnight at 4 C in
humidified air. Fluorescent secondary antibodies were diluted in 1% BSA, 0.1%
Triton-X100 in
PBS and incubated for 2 hours at room temperature prior to processing for
confocal microscopy.
On occasion, an approximately 1 cm section of intestine at the injection site
was collected for
biochemical studies.
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EXAMPLE 3
Evaluation of Specific Marker Proteins for Interactions with Cholix Derived
Delivery
Constructs During Transcytosis and Intracellular Delivery
[0461] This Example shows an exemplary list of proteins or markers for
specific cell
compartments that were analyzed using immunohistochemical (111C) staining and
immuno-
fluorescence confocal microscopy and when evaluating the delivery constructs
of the present
disclosure.
[0462] The
following TABLE 13 shows exemplary Cell compartment specific protein
markers used herein. For example, Cholix derived delivery constructs
comprising an IL-10 as the
heterologous cargo were followed during experiments using either a monoclonal
antibody (mAb)
against IL-10 and/or a polyclonal antibody (pAb) raised against the Cholix
carrier (e.g., a
truncated domain I).
TABLE 13 ¨ Cell Compartment Specific Protein Markers
Target pAb/mAb Species Host Dilution for
Notes Storage Cat. #
reactivity IHC (P)
Cholix386 pAb Rabbit 1/500 Whole -20 C
carrier antiserum
IL-10 mAb; pAb Human Mouse, 1/25 -20 C
Goat
EEA1 pAb Mouse, Rabbit 1/200 Early -20 C Ab2900
rat, human endosome
Rab7 mAb Mouse, Rabbit 1/100 Late -20 C Ab12677
rat, human endosome 12
Rabll pAb Mouse, Rabbit 1/500 Recycling -20 C Fisher71-
rat, endosome
5300
human,
rabbit, dog
LAMP1 pAb Mouse, Rabbit 1/500 Lysosome -20 C Ab24170
rat, human marker
GM130 pAb Mouse, Rabbit 1/500 Cis-
Golgi -20 C
rat, human
Glantin mAb Rat, Mouse 1/20 Golgi -20 C
Ab37266
human
58K mAb Mouse, Mouse 1/100 Golgi -20 C Nb600-
Golgi rat, human
4512
protein
TGN38 mAb Mouse, Mouse 1/1000 Trans-Golgi -20 C Nb300-
rat, human 575
Calnexin pAb Mouse, Rabbit 1/500 Endoplasma -20 C Ab22595
rat, human tic reticulum
Clathrin mAb Mouse, Mouse 1/500 Clathrin- -20 C Ab2731
rat, human mediated
endocytosis
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EXAMPLE 4
Trans-epithelial in vivo Delivery of Cholix and PE Derived Carriers Truncated
within
Domain II or Domain lb
[0463] This example describes trans-epithelial transport of delivery
constructs comprising
truncated Cholix carriers (e.g., truncation in domains II and/or lb) and
truncated PE carriers (e.g.,
truncation in domain II and/or lb) conjoined to biologically active cargos
across polarized
intestinal epithelium occurred (e.g., via apical-to-basolateral transcytosis).
[0464] Using the purified isolated monomeric forms of the various delivery
constructs
prepared as described in EXAMPLE 1, a series of in vivo transport studies were
performed
using the rat intra-luminal injection model (see additional methods section
below). In this
analysis, a rat is maintained under isoflurane anesthesia for the duration of
this non-recovery
study. During this time, the small intestine is exposed following a midline
abdominal incision
and a mid-jejunum region is selected with the adjacent mesentery being
identified using an
indelible marking pen. At the location, a 50 [IL injection volume was used to
administer ¨51.ig of
Constructs 7-12. The test protein was dissolved in phosphate buffered saline
(PBS) and injected
using a 28-gauge needle. After intra-luminal injection, the small intestine
segment was returned
to the abdominal cavity and retrieved at set periods of time.
[0465] Once retrieved, the tissue segment exposed to Constructs 7-12 was
flushed with
PBS, lightly fixed in paraformaldehyde, and prepared for fluorescence
microscopy (see
additional methods section below) where the delivery construct was detected
using a polyclonal
raised against the PE or Cholix that was recognized using a secondary antibody
labelled with a
red fluorescent dye. The presence or absence of transport for Constructs 7-12
is depicted in FIG.
1 (Constructs 12 (FIG.1A-FIG. 1C), 11 (FIG.1D-FIG. 1F) and 10 (FIG.1G-FIG.
1I)) and FIG.
2 (Constructs 9 (FIG.2A-FIG. 2C), 8 (FIG.2D-FIG. 2F) and 7 (FIG.2G-FIG. 2I)).
[0466] Uptake of each of Constructs 7-12 into a limited population of cells
within the
lamina propria was consistent with that observed previously for full-length
PE. The finding that
efficient trans-epithelial transport of PE across polarized intestinal
epithelium occurred even for
Construct 12 (PE252-RFP, e.g., amino acid with SEQ ID NO: 137 coupled to amino
acid with
SEQ ID NO: 220) was surprising and remarkable, suggesting that not only trans-
epithelial
transport across polarized intestinal epithelial cells, but also targeting to
specific cells within the
submucosal compartment can be achieved by elements found within domain I (SEQ
ID NO:
137) of PE.
[0467] Based upon the surprising findings using the PE Constructs 7-12,
Construct 6
(Cholix265-RFP), which comprises only domain I of Cholix (SEQ ID NO: 4) was
evaluated.
Immunofluorescence assessment of Cholix265-RFP (e.g., amino acid of SEQ ID NO:
4 or 5
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coupled to SEQ ID NO: 220) transport across rat small intestine in vivo
produced a similar
outcome as observed with PE Construct 12. Evaluation using an antibody to both
the Cholix and
the RFP component was used to demonstrate that both elements of the chimera
were being
transported (see e.g., FIG. 3). The observed staining pattern for these two
antibodies was similar,
showing that both elements moved across the epithelium and ended up in the
same population of
cells within the lamina propria. This data suggests that domain I (SEQ ID NO:
4) of Cholix may
be sufficient to transport a protein cargo (26.9 kDa) and deliver it to a
selected cell population
(FIG. 3A ¨FIG. 3F).
EXAMPLE 5
Transport Across Rat Jejunum of Chimeric Cholix-PE Constructs
[0468] In this example, delivery constructs comprising the amino acid
sequences set forth in
SEQ ID NO: 146 (Construct 13) and SEQ ID NO: 146 (Construct 14) were prepared,
and
evaluated as described above in EXAMPLE 2. The chimeric Cholix-PE constructs
of this
example comprise mixed domains I, II, lb, and III from either the Cholix
exotoxin or the PE
exotoxin as described herein.
[0469] The chimeric carrier construct 13 (SEQ ID NO: 146) comprises a
Cholix domain I
derived from the sequence set forth in SEQ ID NO: 1 (amino acid residues 1-265
of SEQ ID NO:
2), a PE translocation domain II derived from the sequence set forth in SEQ ID
NO: 138, a PE
domain lb derived from the sequence set forth in SEQ ID NO: 139, and a non-
toxic PE catalytic
domain III derived from the sequence set forth in SEQ ID NO: 140. The chimeric
carrier
construct 14 (SEQ ID NO: 147) comprises a PE domain I derived from the
sequence set forth in
SEQ ID NO: 137, a Cholix carrier translocation domain II derived from the
sequence set forth in
SEQ ID NO: 126, a Cholix carrier domain lb derived from the sequence set forth
in SEQ ID NO:
127, and a non-toxic Cholix carrier catalytic domain III derived from the
sequence set forth in
SEQ ID NO: 128.
[0470] As shown in FIG. 4 (after 1 minute, FIG.4A ¨ FIG. 4E) and FIG. 5
(after 20
minute, FIG.5A ¨ FIG. 5E) for construct 13 and FIG. 6 (after 1 minute, FIG.6A
¨ FIG. 6E)
and FIG. 7 (after 20 minute, FIG.7A ¨ FIG. 7E) for construct 14, the chimeric
carrier constructs
are capable of transport across rat jejunum and target cells in the lamina
propria in vivo. Thus, it
is demonstrated herein that also chimeric delivery constructs comprising
portions or domains
from two or more different exotoxins (e.g., Cholix and PE) can efficiently
deliver cargo into and
across epithelial cells.
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EXAMPLE 6
Production and First In vivo Transport Studies of Truncated Cholix Domain I
Delivery
Constructs
[0471] This example demonstrates the transcytosis function of truncated
Cholix derived
carrier polypeptides, wherein, importantly, the truncation occurred at various
locations within the
domain I of the Cholix exotoxin.
[0472] The non-cleavable polyglycine-serine peptide spacer GGGGSGGGGSGGGGS
(SEQ
ID NO: 210) was used to couple human growth hormone (HGH) (SEQ ID NO: 214) to
the C-
terminus of various modified Cholix carrier polypeptides to prepare the
following delivery
constructs for evaluation according to the protein production procedure
described in EXAMPLE
1 above (in this bacterially produced): 1) SEQ ID NO: 160, which comprises a
modified Cholix
carrier truncated at amino acid residue 187 of SEQ ID NO: 5; 2) SEQ ID NO:
159, which
comprises a modified Cholix carrier truncated at amino acid residue 151 of SEQ
ID NO: 5; 3)
SEQ ID NO: 158, which comprises a modified Cholix carrier truncated at amino
acid residue
134 of SEQ ID NO: 5; 4) SEQ ID NO: 161, which comprises a modified Cholix
carrier truncated
at amino acid residue 206 of SEQ ID NO: 5; 5) SEQ ID NO: 162, which comprises
a modified
Cholix carrier truncated at amino acid residue 245 of SEQ ID NO: 5; 6) SEQ ID
NO: 163, which
comprises a modified Cholix carrier truncated at amino acid residue 251 of SEQ
ID NO: 5; and
8) SEQ ID NO: 165, which comprises a modified Cholix carrier comprising amino
acid residues
40-187 of SEQ ID NO: 5.
[0473] In order to analyze the produced fusion proteins, constructs were
injected into the
lumen of the small intestine of rats and the injection site was collected 5,
10 or 15 minutes after
injection. The tissue was fixed and sectioned then stained with anti-Cholix
and anti-HGH
antibodies. Fluorescent secondary antibodies were used to visualize the
protein location using
confocal microscopy. As depicted in FIG. 8A-FIG. 8C, the construct comprising
the amino acid
sequence of SEQ ID NO: 165 was visualized in the epithelial cells and limited
to an area near the
membrane in the apical side of the cells at 5, 10 and 15 minutes. There was no
significant
movement of the protein through the cells away from this compartment and no
protein appearing
in the lamina propria. This suggests that the polypeptide carrier with the
amino acid sequence of
SEQ ID NO: 165 is sufficient for uptake into the epithelial cells, e.g., via
its receptor binding site
and/or via interactions with TMEM132 and/or LRP1, but lacks the part of the
sequence for
transcytosis, resulting in accumulation in the epithelial cells. In
comparison, constructs
comprising the amino acid sequences of SEQ ID NO: 161 ¨ SEQ ID NO: 164 were
shown to
rapidly move across the epithelial cells following uptake and transport out of
the cells into the
lamina propria. As depicted in FIG. 9, the Cholix derived construct with SEQ
ID NO: 160 was
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also visualized in the apical compartment of the epithelial cells at 5
minutes. At 10 and 15
minutes, small amounts of the protein moved to the basal side of the cell, but
did not appear in
the lamina propria.
[0474] This suggests that the N-terminal can be involved in the transport
pathway but is not
sufficient for complete transcytosis through the cell. The surprising findings
depicted in FIG.
8A-FIG. 8C and FIG. 9 represent a potential method of specifically delivering
a cargo attached
to these truncations to the epithelial cells, as they are taken up into the
cells but cannot transport
out and so accumulate inside.
EXAMPLE 7
In vitro Apical-to-Basal Transcytosis of Non-toxic full-length
Cholix Constructs
[0475] This example demonstrates apical-to-basolateral transcytosis of a
modified, non-
toxic Cholix (ntChx) across polarized intestinal epithelial cells in vitro.
[0476] In this example, the Cholix construct was rendered non-toxic through
an amino acid
variation of a specific glutamic acid residue (substituted with alanine)
within the enzymatic
pocket for ADP-ribosylation, resulting in the E581A substitution and a
polypeptide with the
amino acid sequence set forth in SEQ ID NO: 3 (ntChx).
[0477] transport at 37 C of non-toxic E581A Cholix (ntChx) was measured
across
confluent sheets (0.6 cm2 filter surface area) of primary human intestinal
epithelium in vitro,
with concentrations of 2.5- 200 i.tg/mL being applied to the apical surface
and the amount of
ntChx in the basal compartment after 2 h being measured by ELISA (FIG. 10A).
The
concentration range tested demonstrated an apparent permeability for apical
ntChx
concentrations of 2.5- 20 i.tg/mL that was roughly 2-fold greater than for
apical concentrations
above 20 i.tg/mL, although all concentrations demonstrated significant
efficiencies for transport
of this ¨70 kDa protein. The break in permeability rates may be consistent
with a receptor-
mediated transport mechanism that saturated at ¨20 i.tg/mL for the 0.6 cm2
filter surface area
systems of primary human intestinal epithelium used for this in vitro study.
Importantly,
transported ntChx did not appear to be significantly modified (e.g.,
chemically modified) in its
apparent size when assessed by Western blot analysis (FIG. 10B).
[0478] A time-course assessing transcytosis of ntChx at concentrations of
5, 10, and 20
i.tg/mL demonstrated that ntChx transcytosis began to reach a linearity after
an approximately
20-25 min lag phase (FIG. 10C). Using these transport values, the permeability
for ntChx at
37 C across primary human intestinal epithelium in vitro was calculated to be
¨1.92 x 10-5
cm/sec. This permeability rate was reduced when the same in vitro transport
protocol was
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performed at 4 C (FIG. 10C). It can be assumed that, at the quantities of
ntChx applied apically,
the apical concentration of ntChx begins to be depleted after ¨ 90 min in this
in vitro model.
Together, these data suggest that ntChx transported via an energy-requiring,
highly-efficient,
receptor-mediated transcytosis process. Such a process can be dependent upon
the amount of
ntChx capable of interacting with apical surface receptors (e.g., low density
lipoprotein receptor-
related protein 1 (LRP1)) involved in endocytosis that results in accessing a
transcytosis pathway
that remains privileged from significant catabolism.
[0479] The apical membrane surface pH of the small intestinal epithelium
can be between 5
and 7 (17). transcytosis across primary human intestinal epithelium in vitro
tested for 20 i.tg/mL
and examined after 120 min was observed to be approximately twice as efficient
when the apical
media was pH 7 compared to pH 5, while the basal pH of 7 or 5 did not seem to
have an effect
(FIG. 11).
[0480] Without being bound to any theory, it was assumed that the greater
variability
observed for outcomes where the apical pH of 5 was tested on ntChx transport
can have been due
to efforts by the epithelium to neutralize this apical compartment during the
course of the study
that can be occurring just at the apical plasma membrane in close proximity to
the site of
receptor-endocytosis of ntChx. In sum, these data suggest that ntChx as
disclosed herein can be
capable of efficient, consistent, and continuous transport across human
intestinal epithelium
through a receptor-mediated process that can not result in significant size
modification to the
transported protein.
EXAMPLE 8
Analysis of the Transcytosis Pathway of Full-length Non-toxic Cholix
Constructs
[0481] This example demonstrates apical-to-basolateral transcytosis of
ntChx (SEQ ID NO:
3) across polarized intestinal epithelial cells in vivo, examining the ability
of ntChx to transport
across an intestinal epithelium in vivo by direct intra-luminal injection
(ILI) into rat jejunum.
[0482] Immunofluorescent microscopic images showed that ntChx, identified
using an anti-
Cholix polyclonal antisera, entered into epithelial cells rapidly and
trafficked through these
epithelial cells in vesicular-like structures that involved clatherin co-
localizations, with the
transcytosis process being completed within minutes (FIG. 12A-FIG. 12C). Time-
dependent
changes in the location of vesicle-like structures positive for ntChx
demonstrated its distribution
within compartments localized above and below the enterocyte nucleus as well
as within a
selected population of cells within the lamina propria. Transcytosis of ntChx
appeared to also
occur through goblet cells. Transport of ntChx between adjacent epithelial
cells, the so-called
paracellular route, was not observed in this experiment.
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[0483] ILI of ntChx into rat jejunum appeared to restrict the exposure to
the upper segments
and tip of the intestinal villi.
EXAMPLE 9
Transcytosis of Non-toxic Cholix Constructs Involves Specific Vesicular
Compartments
[0484] This example demonstrates apical-to-basolateral transcytosis of
ntChx (SEQ ID NO:
3) that involves accessing specific vesicular compartments (e.g., FIG. 13A-
FIG. 13F).
[0485] Transcytosis of ntChx can involve receptor-mediated endocytosis
processes and
subsequent vesicular trafficking that avoids the typical fate for ligands
internalized by this route
of lysosomal degradation. A number of obligate and facilitative intracellular
pathogens are
capable of subverting host cells endocytic and secretory pathways that
restrict their exposure to
the lysosomal through effector proteins capable or manipulating host cells
processes. This
example examined ntChx-containing vesicles undergoing transcytosis to
determine the identity
of associated proteins that can define the trafficking elements and cellular
compartments that can
be involved in transcytosis of Chx and Chx variants as disclosed herein.
[0486] Upon uptake at the apical plasma membrane, ntChx-positive vesicles
were observed
to co-localize with Ras-related protein Rab 5 and early endosomal antigen 1
(EEA1) (FIG. 13A),
demonstrating hallmarks of the early endosome/sorting endosome (EE/SE),
suggesting that this
exotoxin can enter intestinal epithelial cells through a receptor-mediated
endocytosis.
Populations of EEAl-positive vesicles were also observed in the basal
compartment of
enterocytes, but these were less heavily associated with the presence of ntChx
(FIG. 13A). Ras-
related protein Rab 11 a, known to be associated with recycling endosomes,
were observed in
both apical and basal compartments of enterocytes but Rab lla co-localization
with ntChx-
containing vesicles was observed predominantly in the basal compartment (FIG.
13B).
Furthermore, the trans-Golgi network (TGN) can be considered to function as a
secretory
pathway sorting station, directing newly synthesized proteins to different
cellular compartments,
with TGN-38 being identified as indicative. Remarkably, ntChx was not observed
to co-localize
with TGN-38 in enterocytes in this experiment, but did co-localize after
transcytosis in non-
polarized cells with the lamina propria (FIG. 13C). Calnexin is a lectin
chaperone located in the
endoplasmic reticulum (ER) that can be involved in regulating the free
cytosolic Ca2+
concentration. During transcytosis, it was found that ntChx can co-localize
with calnexin
primarily in the apical compartment of enterocytes but with distributions that
included the basal
compartment (FIG. 13D). Furthermore, Ras-related protein Rab 7, a marker late
endosomes and
vesicles being trafficked to lysosomes, was observed to co-localized in both
the apical and basal
portions of enterocytes with ntChx (FIG. 13E). LAMP1 (lysosome-associated
membrane protein
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1), distributed in both late endosomes and lysosomes was observed to similarly
co-localize with
ntChx in both the apical and basal portions of enterocytes, but did not appear
to co-localize with
larger vesicular structures that were consistent in size and number with
lysosomal structures
(FIG. 13F).
[0487] Apical application of ntChx-NPs resulted in a transcytosis process
consistent with
that observed for ntChx alone and without co-localization with LAMP1-positive
structures (FIG.
13F). Once across the intestinal epithelial cell layer, ntChx-NPs was observed
in LAMP1-
positive structures within cells present within the lamina propria (FIG. 13C).
EXAMPLE 10
Comparison of Transcytosis Function of Non-toxic Full-length Cholix Exotoxin
vs. Cholix Domain I Only
[0488] This example demonstrates the in vivo transport of Cholix domain I
(e.g., SEQ ID
NO: 4 or SEQ ID NO: 4) truncated carrier proteins as described in EXAMPLE 6
compared to
non-toxic full-length Cholix as described in EXAMPLE 7.
[0489] Transcytosis of ntChx (SEQ ID NO: 3)/n vitro was monitored by
Western blot and
immunofluorescence microscopy using a polyclonal antibody raised against the
full-length
protein. To test whether this polyclonal antibody can provide equivalent
coverage across all
regions of ntChx for the purpose of detecting truncated forms of the exotoxin,
the capacity for a
truncated form of Cholix to ferry a protein cargo that could be used to assess
transcytosis
equivalence was investigated.
[0490] Genetic chimeras of full-length protein (ntChx) and a version of the
protein
composed of only domain I were prepared, comprising the amino acid sequence
set forth in SEQ
NO: 5, and its C-terminus was used to conjugate red fluorescent protein (RFP,
SEQ ID NO: 220)
via its N-terminus, resulting in the construct having the amino acid sequence
set forth in SEQ ID
NO: 157). RFP alone, having a molecular weight of 25.9 kDa (225 amino acid
residues) was
used as a transcytosis control.
[0491] The data show that the extent of transcytosis of RFP alone was
minimal compared
that of ntChx-RFP and Cholix domain I (M+Cholix1-265 or residues 1-266 of SEQ
ID NO: 5) ¨
RFP (SEQ ID NO: 156) when examined 30 min after ILI in to rat jejunum in vivo
(FIG. 14A).
Epithelial transcytosis patterns were similar and the extent of RFP detectable
in the lamina
propria was also comparable for both ntChx-RFP (FIG. 14B) and Cholix domain I-
RFP (FIG.
14C) (arrows in these figures illustrate the apical side of the epithelium).
These results suggest
that elements of Cholix involved in transcytosis reside within domain I and
that this domain can
be sufficient to ferry a protein cargo replacing domains II and III of Cholix
across the intestinal
epithelium.
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EXAMPLE 11
In vitro Transcytosis and Intracellular Delivery Function of Truncated Cholix
Domain I Carrier Proteins
[0492] This example demonstrates the in vitro apical-basolateral
transcytosis and
intracellular delivery functions of various truncated Cholix domain I carrier
proteins conjugated
to human growth hormone via a spacer as described above in EXAMPLE 6.
[0493] To further explore the function of elements within Cholix domain I
involved in
transcytosis, a series of chimeras that contained a pharmaceutically-relevant
protein, human
growth hormone (HGH, SEQ ID NO: 214), were prepared as genetic constructs.
Each truncated
sequence of the Cholix domain I (SEQ ID NO: 5, bacterially expressed) was
conjugated via the
C-terminus to the N-terminus of HGH through a G45G45G45 spacer sequence (SEQ
ID NO:
210), resulting in the chimeras having the amino acid sequences set forth in
SEQ ID NO: 158,
SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO:
163, and
SEQ ID NO: 164. In the case of the K187 truncation of Cholix domain I with SEQ
ID NO: 5, the
first 39 amino acids were also deleted to produce the E40-K187 fragment of
Cholix domain I
(SEQ ID NO: 5) to yield the construct with the amino acid sequence set forth
in SEQ ID NO:
165 (TABLE 14).
TABLE 14¨ Truncated Cholix Domain I Delivery Constructs for HGH
SEQ ID NO Notation (relative to SEQ ID NO: 4) Mol. Ma Ca
SEQ ID NO: 158 M+Cholix1-133-HGH 38.0
4.70
SEQ ID NO: 159 M+Cholix1-150-HGH 40.0
4.69
SEQ ID NO: 160 M+Cholix1-186-HGH 44.2
4.76
SEQ ID NO: 161 M+Cholix1-205-HGH 46.4
5.04
SEQ ID NO: 162 M+Cholix1-244-HGH 50.7
4.96
SEQ ID NO: 163 M+Cholix1-250-HGH 51.4
5.01
SEQ ID NO: 164 M+Cholix1-265-HGH 53.1
5.12
SEQ ID NO: 165 Chx39-186-HGH 40.0
4.91
[0494] Experiments investigating the transport capabilities of these
chimeras across human
intestinal epithelial monolayers in vitro demonstrated that the delivery
constructs with the amino
acid sequences set forth in SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160,
and SEQ ID
NO: 165 did not transport, unlike SEQ ID NO: 161, as shown in FIG. 15A.
Despite its much
lower molecular weight, HGH alone transported to a lesser extent when compared
to SEQ ID
NO: 164 (Fig. 15A, right part showing presence in basal compartment).
Transcytosis of SEQ ID
NO: 161 and SEQ ID NO: 162 were comparable to that of SEQ ID NO: 164 at the 2
h time point
of assessment in this in vitro model of human small intestine (FIG. 15B).
Surprisingly, the
delivery construct with the amino acid set forth in SEQ ID NO: 161 was
superior in its transport
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capacity compared to SEQ ID NO: 164 as demonstrated by the higher relative
signal of SEQ ID
NO: 161 compared to SEQ ID NO: 164.
[0495] These results suggest that domain I of Cholix is sufficient for
apical-to-basal
transport, and that it can function as a transcytosis element to deliver
various heterologous cargos
across epithelial cells, wherein the heterologous cargo may replace the
domains II, lb, and III of
the Cholix exotoxin. Additionally, it has been demonstrated that elements
within the first 206
amino acid residues of the bacterially expressed Cholix protein (e.g., SEQ ID
NO: 5, or,
alternatively the first 205 amino acid residues of SEQ ID NO: 4) can be
sufficient for the
transcytosis function and thus may be used as an efficient carrier for various
heterologous
cargos. Remarkably, the results suggest that the transcytosis efficiency of
these first 206 amino
acids of SEQ ID NO: 5 may even be greater than that of the entire domain I
(e.g., SEQ ID NO:
5) or the full-length mature Cholix exotoxin (e.g., SEQ ID NO: 1 or SEQ ID NO:
2). Thus, the
herein disclosed truncated Cholix domain I constructs can be used to
efficiently shuttle
heterologous cargo molecules such as therapeutic and/or diagnostic agents
across an epithelial
cell layer (e.g., the gut epithelium) enabling oral administration of
therapeutic and/or diagnostic
agents (e.g., larger polypeptides or proteins such as antibodies) that are
otherwise administered
via parenteral administration routes (e.g., intravenously of subcutaneously).
In addition, these
results show that truncated versions of Cholix domain I may be used to deliver
various
heterologous cargo into epithelial cells using the delivery constructs as
described herein.
EXAMPLE 12
In vivo Transcytosis and Intracellular Delivery Function of Truncated Cholix
Domain I Carrier Proteins
[0496] This example demonstrates the in vivo transport of a Cholix domain I
(e.g., SEQ ID
NO: 4 or SEQ ID NO: 5) truncated protein chimeras across gut epithelial cells
for delivery of
heterologous cargo (in this example: human growth hormone).
[0497] Selected truncation chimeras as shown above in TABLE 14 of EXAMPLE
11 were
examined for their capacity for transcytosis in vivo following ILI into rat
jejunum. The notation
indicating the length and residue at which the truncated occurred of the
truncated Cholix domain
I carriers are relative to SEQ ID NO: 5.
[0498] The data obtained in these studies show that while M+Cholix1-133-
(SEQ ID NO: 10)-
HGH (SEQ ID NO: 158) did not enter into rat epithelium by 15 min (FIG. 16A),
M+Cholix1-150-
(SEQ ID NO: 10)-HGH (SEQ ID NO: 159) underwent endocytosis, but did not
complete
transcytosis as evidenced by a lack of this chimera in the lamina propria
(FIG. 16B). The
construct with SEQ ID NO: 159 was retained in a vesicular pool restricted to
compartments near
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the apical and basal plasma membranes of enterocytes (FIG. 16B). The
truncation mutant
M+Cholix1-186-(SEQ ID NO: 10)-HGH (SEQ ID NO: 160) resulted in similar
outcomes as
observed for the construct with SEQ ID NO: 159 with one difference that the
construct with SEQ
ID NO: 160 appeared to access a supra-nuclear vesicular compartment that was
not accessed by
the construct with SEQ ID NO: 159 (FIG. 16C). Furthermore, removal of the
first 39 amino
acids of the Cholix domain I with SEQ ID NO: 5 (resulting in the construct
M+Cholix39-186-(SEQ
ID NO: 210)-HGH, SEQ ID NO: 165) resulted in a protein that could undergo
endocytosis, but
did not migrate from the apical vesicular compartment to the basal vesicular
compartment of the
enterocyte by 15 min after ILI (FIG. 16D).
[0499] These results suggest that elements of Cholix domain I (SEQ ID NO:
5) between
E134 and D151 are essential to apical endocytosis, that elements within the
first 39 amino acids
of Cholix domain I may be critical for trafficking from the apical vesicular
pool to the basal
vesicular pool, and that elements between K187 and K206 may be critical for
Cholix secretion
from the basolateral surface of enterocytes. Thus, these results demonstrate
that Cholix domain I
and truncated version thereof, e.g., those comprising the first 206 amino acid
residues of Cholix
domain I (SEQ ID NO: 5), can efficiently deliver various cargo across
epithelial cells (e.g.,
across polarized gut epithelial cells of a subject). Moreover, these results
show that truncated
versions of Cholix domain I may be used to deliver various heterologous cargo
into epithelial
cells using the delivery constructs as described herein.
EXAMPLE 13
Evaluation of Functional Peptide Fragments of Cholix Domain I
[0500] This example demonstrates recapitulation of Cholix domain I (SEQ ID
NO: 5)
transcytosis using functional elements, e.g., functional peptide fragments, of
Cholix domain Ito
generate a synthetic polypeptide that is capable of delivery into epithelial
cells and/or across an
epithelial cell layer (e.g., an epithelial cell monolayer and/or a gut
epithelium of a subject) in
vitro (FIG. 17A) and in vivo (FIG. 17B). In addition, FIG. 18, for example,
depicts some
functional amino acid sequences within Cholix Domain I as colored sequence
fragments.
Moreover, FIG. 20 illustrates general 3D structures of Cholix domain I with
the highlighted
functional fragments SEQ ID NO: 148 (FIG. 20A), SEQ ID NO: 151 (FIG. 20B), and
SEQ ID
NO: 152 (FIG. 20C). All amino acid residues and their positions are shown
relative to the
bacterially expressed Cholix domain I sequence set forth in SEQ ID NO: 5.
[0501] To that end, transcytosis experiments using specific peptides
derived from portions
of Cholix domain I (SEQ ID NO: 5) that were identified as critical for apical
endocytosis were
used to model the various steps and aspects such as intracellular trafficking,
and basal membrane
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secretion of transcytosis of full-length ntChx, exemplary peptide fragments of
Cholix domain are
shown below in TABLE 15 (including the molecular mass and isoelectric point
(pI)).
TABLE 15 ¨ Identified Exemplary Functional Peptide Domains Within the Amino
Acid
Sequence of Cholix Domain I (SEQ ID NO: 5)
SEQ ID NO Peptide sequence Designation
Mol. Mass (Da) pI
SEQ ID NO: 134 ELDQQRNIIEVPKLYSID151 Endocytosis (E) 2173.5
4.32
148
SEQ ID NO: iMVEEALNIFDECRSPCSLTP Apical-Basal (T) 4258.9
4.20
149 EPGKPIQSKLSIPSDVVLD39
SEQ ID NO: 151DLDNQTLEQWKTQGNVS Supranuclear (N) 4276.7
5.48
151 FSVTRPEHNIAISWPSVSYK
1
87
SEQ ID NO: KAAQKEGSRHKRWAHW Basal release (R) 2568.9
11.1
187
152 HTGLAL
206
[0502] For example, the peptide 134ELDQQRNIIEVPKLYSID151(SEQ ID NO: 148)
was
the element that differed between M+Cholix1-150-HGH (amino acid sequence set
forth in SEQ ID
NO: 159) and M+Cholix1-133-HGH (amino acid sequence set forth in SEQ ID NO:
158), one
chimera that could undergo endocytosis and one that could not. Another peptide
of interest is
within the first 39 amino acids (1MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLD39,
SEQ ID NO: 149), which was lacking in the M+Cholix39-186 construct (SEQ ID NO:
165) that
lacked the ability to traffic from the apical portion of the epithelial cell
to the basal domain
following endocytosis. The peptide that provided the difference between
M+Cholix1-150-(SEQ ID
NO: 210)-HGH and M+Cholix1-186-(SEQ ID NO: 210)-HGH was
151DLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYK187(SEQ ID NO: 151);
M+Cholix1-150-(SEQ ID NO: 210)-HGH showed the ability to access a supra-
nuclear area of the
cell that was not accessed by M+Cholix1-150-(SEQ ID NO: 210)-HGH. Finally,
M+Cholix1-265-
(SEQ ID NO: 210)-HGH was secreted from the basal surface of intestinal
epithelial cells while
M+Cholix1-186-(SEQ ID NO: 210)-HGH was not; 187KAAQKEGSRHKRWAHWHTGLAL206
(SEQ ID NO: 152) is the peptide that describes the difference in the sequences
of these two
chimeras (see e.g., FIG. 17A).
[0503] A polymer framework containing peptide sequences of amino acids from
positions
1-39, 134-151, 151-178, and 178-206 of Cholix domain I with SEQ ID NO: 5 in
various
combinations was labeled with different of quantum dot forms.
[0504] The in vitro data show that some selected peptide sequences of
Cholix domain I
peptide elements are sufficient to achieve apical to basal transcytosis in
vitro and in vivo (FIG.
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17). For example, transcytosis across polarized intestinal epithelium was
measure in vitro after 2
h as shown in FIG. 17A. The amount of transported material is reported as the
florescence-fold
increase relative to polymer-quantum dot preparation lacking any Chx peptides.
(N=2; mean
S.E) (FIG. 17A). Subsequently, in vivo transcytosis at 15 min of polymer-
quantum dot
preparations labeled with quantum dots was conducted (FIG. 17B and FIG. 17C).
[0505] The data shows that various Cholix sequence variants as disclosed
herein retain
efficient endocytosis following uptake from the lumen but lack ability to
complete transcytosis,
being useful to target apical or apical and basal vesicular structures. From
the data presented, it
appears that Cholix utilizes a receptor-mediated-type endocytosis process that
involves amino
acids 134-151, which provides access to an early endosomal vesicular
compartment in the apical
portion of enterocytes (e.g., gut epithelial cells). Amino acids 151-187 of
the Cholix exotoxin
domain I (e.g., SEQ ID NO: 5) as described herein appear to allow its movement
to a supra-
nuclear compartment consistent with a sorting site in the cell for secretory
events, and thus allow
delivery of various cargos to those locations as well. Movement to the basal
compartment of the
cells becomes more efficient with the presence of amino acids 1-40. Finally,
amino acids 187-
206 provide a mechanism for secretion from the basal membrane that releases
the entire and
intact protein into the lamina propria where it could provide therapeutically
effective
concentrations of therapeutic cargo molecules (e.g., interleukins), present
antigens to immune
cells, and/or allow the cargo to be taken up into systemic circulation for
delivery to other target
organs/tissues in a subject.
EXAMPLE 14
Co-localization of Cholix Domain I Delivery Constructs with Various Marker
Proteins
Demonstrates Trafficking Using Distinct Compartments
[0506] This example demonstrates that Cholix domain I derived delivery
constructs utilize
distinct compartments for trafficking into and across (e.g., via transcytosis)
epithelial cells using
various marker proteins (see e.g., EXAMPLE 3) that indicate Cholix derived
carrier constructs
utilize specific and endogenous trafficking pathways. The study described in
this example was
conducted using the Cholix derived delivery construct having the amino acid
sequence set forth
in SEQ ID NO: 154 (comprising the M+Cholix386 carrier coupled to IL-10 via the
spacer with
the sequence set forth in SEQ ID NO: 210, M = N-term. methionine).
[0507] EEA1 antigen. FIG. 21A shows that the Cholix-IL-10 delivery
constructs (SEQ ID
NO: 154) strongly co-localized with the EEA1 antigen in cellular locations
consistent with
trafficking at both the apical and basal domains of enterocytes, suggesting
the presence of the
Cholix derived delivery constructs in early endosome compartments.
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[0508] Rab7. Moreover, it was shown that the Cholix-IL-10 delivery
constructs (SEQ ID
NO: 154, top right) strongly co-localizes with the Rab7 (top left)
predominantly in the apical
compartment of enterocytes, but with only limited co-localization in cells
within the lamina
propria, suggesting the presence of the Cholix derived delivery constructs in
late endosome
compartments (FIG. 21B, bottom left shows white light image, and bottom right
shows merged
staining with DAPI).
[0509] LAMP!. LAMP1 was identified in large, specific vesicles consistent
mature
lysosomes that were devoid of Cholix-IL-10 delivery constructs (SEQ ID NO:
154,
whitearrows). Cholix-IL-10 chimera, however, also co-localizes with the LAMP1
antigen in
cellular locations other than lysosome-like structures, consistent with
vesicle trafficking at both
the apical and basal domains of enterocytes, suggesting the presence of the
Cholix derived
delivery constructs in lysosomal compartments (FIG. 21C).
[0510] Clathrin. Next, Cholix-IL-10 chimera (SEQ ID NO: 154) also strongly
co-localized
with clathrin-coated vesicles, particularly in areas adjacent to the nucleus
and with Rab 1
predominantly in the basal compartment of enterocytes as well as in selected
cells within the
lamina propria (FIG. 21D).
[0511] Calnexin. Cholix-IL-10 chimera (SEQ ID NO: 154) co-localized with
the
endoplasmic reticulum as demonstrated by calnexin in a pattern adjacent to the
nucleus in
enterocytes and in a large fraction of cells with in the lamina propria (FIG.
21E).
[0512] Endoplasmatic reticulum Golgi intermediate compartment. Cholix-IL-10
chimera (SEQ ID NO: 154) strongly co-localizes with the endoplasmatic
reticulum Golgi
intermediate compartment (ERGIC) and the LAMN1 antigen appeared to re-
distribute in
response to carrier endocytosis and transcytosis, as shown for 1 (FIG. 21F), 5
(FIG. 21G), 10
(FIG. 21H), and 15 minutes after injection (FIG. 211). Transcytosis of the
delivery construct
was demonstrated to consistently traffic in large quantities across
enterocytes. Specific
compartments that strongly co-localized with this transcytosis included early
endosomes and late
endosomes. The Cholix derived carrier appeared to be associated with clathrin-
coated vesicles in
the vicinity of the ER-Golgi network organized adjacent to enterocyte nuclei.
Co-localization of
the Cholix derived carrier was observed with the ER and ERGIC, also described
as LMAN1
(lectin, mannose binding 1), but limited in its association with elements of
the cis-Golgi, Golgi,
and trans-Golgi network. The Cholix derived carrier co-localized with
recycling endosomes near
the basal surface of enterocytes in a manner that might coordinate with ERGIC
re-distribution.
ERGIC-53 can also function as an intracellular cargo receptor involved in the
anterograde
transport of a limited number of glycoprotein ligands in the early exocytic
pathway and is used
by a number of RNA viruses as part of their exocytosis strategy. ELISA-based
binding studies
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demonstrated that Cholix and Cholix-derived delivery constructs can associate
with ERGIC-53
at pH 7.4, but this interaction is significantly stronger at pH 5.5. SPR
studies further supported
this pH-dependent interaction (FIG. 31).
[0513] Giantin. Cholix-IL-10 chimera (SEQ ID NO: 154) did not co-localize
with the low
levels of giantin present in enterocytes (FIG. 21J). Some giantin co-localized
with the chimera
in a subset of cells present in the lamina propria, suggesting that the Cholix
derived carrier does
not locate with the Golgi compartment.
[0514] 58K antigen. The 58K antigen localized in enterocytes at a site
apical to the nucleus
and the Cholix-IL10 chimera shows some co-localization with this antigen in a
manner that
suggests a brief movement through this compartment. No 58K antigen was
observed in cells
within the lamina propria (FIG. 21K).
[0515] TGN38 antigen. Cholix-IL-10 chimera (SEQ ID NO: 154) showed some
level of
co-localization with the TGN38 antigen (top right), which showed a cellular
distribution that was
restricted to the apical side of nuclei in enterocytes and adjacent to the
nucleus in a few cells
within the lamina propria (FIG. 21L, white light and merge images shown bottom
left and
bottom right, respectively).
[0516] Rab 1. Cholix-IL-10 chimera (SEQ ID NO: 154, top right) strongly co-
localized
with Rab 1 (top left) predominantly in the basal compartment of enterocytes
and in selected cells
within the lamina propria (FIG. 21M, white light and merge images shown bottom
left and
bottom right, respectively).
[0517] This data demonstrates that the Cholix derived delivery constructs
of the present
disclosure interact with various endogenous proteins and receptors to harness
an endogenous
transport system for efficient delivery of cargo across and/or into epithelial
cells (e.g., polarized
gut epithelial cells).
EXAMPLE 15
General Protocol for Assessing Cholix Exotoxin Interacting Receptors
[0518] Based on the results shown above in EXAMPLE 14, this example
demonstrates a
protocol for the assessment of Cholix interacting receptors and various
additional information
regarding those interactions. Various Cholix derived delivery constructs
including those having
an amino sequence set forth in SEQ ID NO: 154 (M+Cholix386-GGGGSGGGGSGGGGS
(SEQ
ID NO: 210)-IL-10) and a construct comprising the Cholix domain I set forth in
SEQ ID NO: 5
coupled to IL-10 (SEQ ID NO: 217) or HGH (SEQ ID NO: 153) via the
GGGGSGGGGSGGGGS spacer (SEQ ID NO: 210) were used in this study, e.g., SEQ ID
NO:
164. First, a limited set of candidate proteins as carrier protein receptors
have been identified
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through bead capture and mass spectrometry analysis studies. Then, the
interactions of the
Cholix carrier with these candidate proteins were assessed in vitro (e.g.,
using Caco-2 cell
monolayers) and in vivo (e.g., in the rat jejunum).
[0519] Generally, nano-sized magnetic beads (25 nm or 100 nm diameter) were
decorated
with the non-toxic carrier elements of the Cholix protein using either a
biotin-based or poly-
histidine-based method of interaction (e.g., using 1D SDS-PAGE, described in
e.g., FIG. 22).
These decorated beads were allowed to transport across polarized monolayers in
vitro of the
human colon cancer cell lines Caco2 for set periods of time before gentle cell
disruption and
capture of vesicles containing these magnetic beads.
[0520] After multiple washings, these magnetic bead-enriched vesicles were
solubilized in
lysis buffer and the protein components present were separated by 2-D SDS-PAGE
(FIG. 23).
The pattern of these proteins was compared to the total protein content of
these cells and mass
spectrometry was used to identify specific elements associated with vesicular
structures accessed
by the Cholix derived delivery construct (FIG. 24).
[0521] Comparison of outcomes from repeats of this protocol were used to
identify a limited
set of most promising candidates that were then examined for their content in
Caco-2 cells and in
rat small intestine (FIG. 25). Subsequently, interaction of Cholix with the
identified candidate
proteins was confirmed using Cholix carrier-coated magnetic beads and purified
candidate
protein. Incubation of the Cholix carrier-coated beads with the pure proteins
and subsequent
Western Blots or ELISA enabled detection of Cholix-protein interaction as
exemplary shown in
FIG. 26. This figure shows interaction of Cholix carrier with heparan sulfate
proteoglycan
(HSPG), Dickkopf-related protein 1 (DKK1), the chaperone glucose-regulated
protein 75
(GRP75), and cytokeratin-8 (K8 or CK8).
[0522] Microscopic co-localization of candidate proteins and Cholix derived
delivery
construct was evaluated in rat jejunum in vivo. Here, co-localization of a
delivery construct
comprising a Cholix carrier protein coupled to IL-10 (SEQ ID NO: 154,
M+Cholix386-
GGGGSGGGGSGGGGS (SEQ ID NO: 210)-IL-10) with CK8 was shown in vivo, after rat
jejunum was treated with a luminal application of the construct having the
amino acid sequence
set forth in SEQ ID NO: 154 for 1 minute (FIG. 27A), 5 minutes (FIG. 27B), and
10 minutes
(FIG. 27C). Co-localization in a supra-nuclear region increased over time.
Although transcytosis
of the delivery construct was observed in most epithelial cells, it interacted
with only a discrete
population of cells within the lamina propria.
[0523] To ensure that the receptor distribution was consistent between rat
in vivo studies
and human intestine, information was compared to IHC studies described in the
human atlas.
Here, two of the receptors identified by mass spectrometry and verified in rat
jejunum were
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examined. FIG. 28A and FIG 28B show that the intestinal localization of GRP75
and HSPC is
consistent between rat and human intestine.
[0524] Knock-down in Caco-2 cells using sh-RNA technology were used to
establish stable
cell lines that were used to validate the involvement of these target proteins
in the apical-to-basal
transcytosis of the Cholix carrier protein. These studies were then repeated
using rat jejunum in
vivo to compare to the Caco-2 cell in vitro findings.
[0525] Here, transport of Cholix domain I derived delivery construct in
CRISPR knockout
HSPC stable Caco-2 cells was evaluated. FIG. 29 shows effects of HSPG knockout
by CRISPR
on transport function of the delivery construct (SEQ ID NO: 164) comprising
Cholix domain I
coupled to HGH and HGH alone as internal control of non-selective transport.
Cells were seeded
at 1.5x105 cells/mL in transwelIs. On day 18, transepithelial/transendothelial
electrical resistance
(TEER) was measured and PBS containing 20 ug/mL of the carrier with SEQ ID NO:
164 was
added to the apical chambers. After 3 h, basolateral samples were collected
and concentrated.
The extent of protein transport was analyzed by Western blotting using anti-
HGH antibody. The
results shown in FIG. 29 demonstrate that transcytosis and active, selective
transport of Cholix
derived carrier proteins is HSPG-dependent, as the Cholix carrier showed
significantly less
transcytosis function in HSPC-knock-down cells compared to normal, HSPG-
positive Caco-2
cells.
[0526] Similar studies were conducted for the additional candidate proteins
K8 (FIG. 30A),
HSPC (FIG. 30B), and GRP75 (FIG. 30C, control run shown in FIG. 30D). Stable
cell lines of
Caco-2 cells lacking the expression of specific candidate proteins were used
as monolayers in
vitro to verify their requirement for carrier transcytosis using active and
selective endogenous
transport mechanisms. The specific transport of delivery construct SEQ ID NO:
164 vs non-
selective transport of HGH alone was reduced in HSPG and GRP75 knockouts, but
not the K8
knockout. This suggests that HSPG and GRP75 are required to active transport
and transcytosis
of Cholix derived carrier proteins, and that K8 may not be required for active
transport across an
epithelial cell.
[0527] Rat jejunum ILI studies demonstrated that GRP75 was distributed in
enterocytes in
distinct apical and basal vesicular populations; SEQ ID NO: 164 co-localized
with GRP75 in
vesicles within the apical third of enterocytes not immediately adjacent to
the apical plasma
membrane (Fig. 3D). The construct having the sequence set forth in SEQ ID NO:
164 was
observed in this apical GPR75-positive compartment occurred within 5 min of
luminal
application and was consistent with an early endosomal compartment. At later
times, a portion of
GPR75-positive vesicles observed near the basal membrane were observed that
contained the
constructs with SEQ ID NO: . TMEM132A is highly similar to the rat GRP78
binding protein.
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Without being bound by any theory, it was assumed that the similarities
between GRP78 and
GRP75 may provide a rational for TMEM132A interactions with GRP75 and the
potential for
their co-localization at the time of Cholix endocytosis.
[0528] This data demonstrates that, indeed, Cholix derived carrier and
cargo transport and
delivery is an active and selective process involving distinct receptors. This
may be useful for the
targeted delivery and therapeutic and/or diagnostic molecules across and/or to
the interior of
epithelial cells (e.g., gut epithelial cells) for the treatment and diagnosis
of diseases as described
herein.
EXAMPLE 16
Assessment of pH-dependence of Cholix Carrier-GRP75 Interaction
[0529] This example demonstrates the assessment of the pH-dependence of a
Cholix
derived carrier protein (e.g., SEQ ID NO: 4) and one of its interacting
receptors during active
transcytosis, GRP75.
[0530] For this study, Biacore binding interactions were used to examine
the pH-
dependency of Cholix carrier-GRP75 interactions. Cholix carrier proteins were
attached to
magnetic beads using the biotin-streptavidin bioconjugation and incubated with
purified GRP75
protein in buffer solutions with pH 5.5, 6.5, and 7.5, respectively (FIG. 31).
[0531] Binding affinities at those three pH levels were generally in the
low nanomolar
range, however, a significantly higher (approximately 20-fold higher) binding
affinity of the
Cholix carrier to GRP75 was measured at pH 6.5, indicating pH dependency of
this interaction.
EXAMPLE 17
Assessment of Type and Location of Cholix Domain I Interaction Partners
[0532] In this example, the type of proteins and their compartmental
locations in epithelial
cells was examined using a Cholix domain I derived delivery construct (SEQ ID
NO: 5) that was
shown to possess equal, if not higher, transcytosis function than full-length
Cholix exotoxin
(SEQ ID NO: 1). All amino acid residues and their positions are shown relative
to the bacterially
expressed Cholix domain I sequence set forth in SEQ ID NO: 5.
[0533] Interaction partners for Cholix domain I ending at K266 of SEQ ID
NO: 5,
bacterially expressed Cholix carrier comprise an N-terminal methionine) were
captured using a
magnetic bead and isolation procedure followed separated by 2-D gel
electrophoresis separation
and identified using mass spectrometry as described above in EXAMPLE 15.
Importantly, in
this example a number of truncated forms of the full-length Cholix exotoxin
were also prepared
to examine various aspects of these interactions: truncations at amino acid
E134, D151, K187,
L206, K245, or Q251, or L206 conjoined to the N-terminus of human growth
hormone (HGH)
through a G4 S G4 S G4S sequence with this glycine-serine spacer being
identified previously for
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constructing genetic chimeras. In the case of the K187 truncation, we also
deleted the first 39
amino acids to produce the E40-K187 fragment of Chx domain I with SEQ ID NO:
5. As
described above, e.g., in EXAMPLE 10, the construct with SEQ ID NO: 158 failed
to achieve
apical entry into intestinal epithelial cells, the construct with SEQ ID NO:
159 and SEQ ID NO:
160 underwent endocytosis to reach both apical and basal vesicular pools
within enterocytes but
did not to access the lamina propria, and the construct with SEQ ID NO: 165
underwent
endocytosis but failed to migrate from the apical to the basal vesicular
compartment of the
enterocyte, and, finally, the construct with SEQ ID NO: 164 efficiently and
rapidly completes
transcytosis. Thus, the first 206 amino acids of Cholix domain I were
evaluated for elements that
may participate in the events that resulted in apical-to-basal transcytosis.
EXAMPLE 18
Evaluation of Apical Endocytosis of Cholix Domain I Derived Delivery
Donstructs
[0534] First, the transmembrane protein 123A (TMEM132A) was evaluated.
TMEM132A
is a single-pass transmembrane protein that contains cohesin and three tandem
immunoglobulin
domains, thus connecting the extracellular medium with the intracellular
cytoskeleton.
Interactions between TMEM132A and serine/threonine-protein phosphatase 1 (PP1)
have been
demonstrated, providing a mechanism to regulate intracellular cytoskeletal
events through a
RVxF interaction motif
[0535] It was found that Cholix domain I (SEQ ID NO: 5, bacterially
expressed) enriched
the isolation of TMEM132A along with catalytic and regulatory subunits of the
PP1 complex.
ELISA-based studies showed that Cholix domain I with SEQ ID NO: 5 interacted
with the
extracellular domain of TMEM132A; SPR studies showed this interaction to occur
at pH 7.5 and
less at pH 5.5. Knock-down studies in Caco-2 cells, to generate Caco-2TmEm/32A-
cells, showed
that reduction of TMEM132A dramatically reduced the transport of transcytosis
of Cholix
domain I with SEQ ID NO: 5.
[0536] Intraluminal injection (ILI) studies performed in rat jejunum
resulted in co-
localization of the Cholix domain I-HGH delivery construct (SEQ ID NO: 164)
with
TMEM132A that was restricted to the apical plasma membrane of enterocytes. A
time course
examining transcytosis of SEQ ID NO: 164 suggested that TMEM132A remained at
or adjacent
to the apical plasma membrane and did not redistribute significantly to other
regions of these
polarized epithelial cells. While the construct with SEQ ID NO: 158 did not
significantly enter
into enterocytes following apical application, the construct with SEQ ID NO:
159 did enter,
suggesting that a domain of Chx critical for apical cell entry resided with
the 17 amino acids that
discriminated where two carrier constructs. Once internalized, the internal pH
of an early or a
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recycling endosome can drop from neutrality to ¨6Ø Examination of
interactions between the
construct having sequence set forth in SEQ ID NO: 164 and TMEM132A
demonstrated that
these occurred to a much greater extent at pH 7.4 compared to pH 5.5,
consistent with the theory
that release from an internalization receptor, in this case TMEM132A, could be
facilitated by
decreased vesicle pH that could occur following apical Chx uptake.
[0537] Studies examining clathrin distribution in rat enterocytes did not
show co-
localizations with the construct having sequence set forth in SEQ ID NO: 164
at the apical
surface, suggesting that Chx entry may not involve clathrin-mediated uptake.
Strikingly, the
cellular distribution of TMEM132A following the construct having sequence set
forth in SEQ ID
NO: 164 application remained at the apical surface, but was found more
frequently in apical
vesicles following apportion of the construct with SEQ ID NO: 159, suggesting
that the lack of
other elements within Cholix domain I (SEQ ID NO: 5) were necessary for the
handoff to a
receptor (or receptors) required for the next step in its trafficking. These
results suggest that Chx
can engage TMEM132A at the luminal surface and that these interactions are
involved in the
initial endocytosis process from the intestinal lumen and that subsequent
interactions within
early endosomes are required for the transcytosis process to continue.
[0538] Cytokeratin 8 (CK-8) was one of several proteins in this family
identified in the
initial screen as a potential Chx interaction partner; CK-8 was previously
identified as a cell-
surface receptor for the Pet toxin secreted from enteroaggregative E. coli.
ELISA-based binding
studies showed CK-8 to interact with TMEM132A and also Chx. CK-8 distribution
in rat
enterocytes was restricted to the apical surface and at discrete domains in
the apical and basal
compartments. A time course examining transcytosis of the construct having
sequence set forth
in SEQ ID NO: 164 suggested that CK-8 cellular distribution did not change
dramatically
following apical M+Cholix1-265-HGH (SEQ ID NO: 164) application and that some
co-
localizations were observed in the apical compartment of enterocytes. Knock-
down of CK-8 was
performed in Caco-2 cells (Caco-2c7(8-) and evaluated with its impact on
transcytosis of the
construct having sequence set forth in SEQ ID NO: 164. CK-8 knockdown, as
further shown
above in EXAMPLE 15, did not affect the transport of the construct having
sequence set forth in
SEQ ID NO: 164, demonstrating the ability for this in vitro model to determine
selective
function for interaction partners in the transcytosis process of Chx. These
studies point out that
proteins identified in Chx pull-downs that co-localize with cellular
compartments visited by the
construct having sequence set forth in SEQ ID NO: 164 may not be essential for
transcytosis.
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EXAMPLE 19
Early Endosomal Sorting of Cholix Domain I Derived Delivery Constructs
[0539] This example demonstrates that distinct proteins such as GRP75 are
involved in
early endosomal sorting of Cholix and Cholix derived delivery constructs
(e.g., those having an
amino acid sequence set forth in SEQ ID NO: 158 ¨ SEQ ID NO: 165).
[0540] Transcytosis of the delivery construct was demonstrated to
consistently traffic in
large quantities across enterocytes. Specific compartments that strongly co-
localized with this
transcytosis included early endosomes and late endosomes. The Cholix derived
carrier appeared
to be associated with clathrin-coated vesicles in the vicinity of the ER-Golgi
network organized
adjacent to enterocyte nuclei. Co-localization of the Cholix derived carrier
was observed with the
ER and ERGIC, also described as LMAN1 (lectin, mannose binding 1), but limited
in its
association with elements of the cis-Golgi, Golgi, and trans-Golgi network.
The Cholix derived
carrier having SEQ ID NO: 164 co-localized with recycling endosomes near the
basal surface of
enterocytes in a manner that might coordinate with ERGIC re-distribution.
ERGIC-53 can also
function as an intracellular cargo receptor involved in the anterograde
transport of a limited
number of glycoprotein ligands in the early exocytic pathway and is used by a
number of RNA
viruses as part of their exocytosis strategy. ELISA-based binding studies
demonstrated that the
construct having sequence set forth in SEQ ID NO: 164 can associate with ERGIC-
53 at pH 7.4,
but this interaction is significantly stronger at pH 5.5. SPR studies further
supported this pH-
dependent interaction.
[0541] The observed distribution of GPR75 in both apical and basal
vesicular compartments
of enterocytes did not suggest a role for an efficient vectored routing
mechanism of the construct
having sequence set forth in SEQ ID NO: 164 from an apical to a basal
vesicular compartment;
rather, GPR75 could play a role in the local vesicular pools in each location.
Further, we did not
observe any subverted distribution of GPR75 as has been observed for the
actions of other
bacterial effector proteins. Thus, we conjectured that Chx interactions with
GPR75 may provide
some function other that routing vesicles from an apical endosomal compartment
to a basal
endosome compartment and hypothesized that GPR75 interactions could function
to minimize
routing of this bacterial effector protein from vesicles to lysosomes at both
locations.
[0542] Additional studies with TMEM132A demonstrated a greater interaction
affinity with
the construct having sequence set forth in SEQ ID NO: 164 at neutral compared
to an acidic pH
suggesting that once internalized, the construct having sequence set forth in
SEQ ID NO: 164
could find another receptor for trafficking while the internalization receptor
cycled back to the
apical surface of the cell. In this hypothesis, the trafficking receptor would
have a greater
interaction affinity for Chx at an acidic pH relative to neutrality. Indeed,
examination of GPR75
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interactions with the construct having sequence set forth in SEQ ID NO: 164
demonstrated a
higher affinity between these two molecules at pH 5.5 compared to pH 7.4.
[0543] This data demonstrates that the herein described Cholix derived
delivery constructs
efficiently access the enterocytes (e.g., polarized gut epithelial cells) and
interact with proteins
involved in early endosomal sorting, allowing these constructs to avoid
intracellular degradation
pathways that results in the delivery and transport of intact carrier and
cargo, e.g., across
epithelial cell via transcytosis and/or to the interior via endocytosis.
EXAMPLE 20
Intracellular Sorting of Cholix Domain I Derived Delivery Constructs
[0544] This example demonstrates that Cholix domain I derived delivery
constructs interact
with distinct proteins such as ERGIC-53 during intracellular sorting.
[0545] Luminal introduction of the delivery constructs with SEQ ID NO: 164
in the rat ILI
model provided data to support ERGIC-53 as an element subverted by Cholix
constructs of the
present disclosure to achieve efficient transcytosis. Prior to and at times
immediately following
apical application of the construct having sequence set forth in SEQ ID NO:
164, ERGIC-53 was
observed in discrete populations in enterocytes that was focused near the
apical surface of the
cell nucleus, a location where ERGIC is consistently located. Within a few
minutes of luminal
application of the construct having sequence set forth in SEQ ID NO: 164,
ERGIC-53 was
observed to move to areas within enterocytes adjacent to the apical plasma
membrane and to a
basal domain. Thus, Cholix carrier transcytosis and ERGIC-53 redistribution to
the basal area of
enterocytes was coincident.
[0546] Since ERGIC-53 is involved in glycoprotein export from the ER, the
cellular
distribution of an ER resident protein ribophorin 1 (dolichyl-
diphosphooligosaccharide protein
glycosyltransferase subunit 1) that mediates N-glycosylation events was
examined. Importantly,
ribophorin 1 was observed in a pull-down using GRP75. Transcytosis of the
construct having
sequence set forth in SEQ ID NO: 164 was observed following its ILI into rat
jejunum did not
affect the intracellular distribution of ribophorin 1, showing it to co-
localize to a limited extent in
the apical vesicular compartment where ribophorin 1 was present throughout the
time course
during which ERGIC-53 was subverted to the basal compartment. Additionally,
ERGIC-53
interacts with a constellation of proteins, including 5EC24, in its role as a
soluble cargo receptor.
Notably, a pull-down with GRP75 as bait identified 5EC24. Similarly, apical
application of the
construct having sequence set forth in SEQ ID NO: 164 did not induce a gross
alteration of
intracellular compartment organization.
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[0547] The ERGIC is involved in sorting soluble molecules destined for
secretion from the
cell and ERGIC-53 undergoes a process of concentrative sorting that involves
the coat protein
CONT. Since both COPT and COPII are involved in vesicle trafficking at the ER-
Golgi interface,
the potential for these coat proteins to co-localized with the construct
having sequence set forth
in SEQ ID NO: 164 during the transcytosis process was investigated. Rat
enterocytes
demonstrated a COPT distribution beneath the apical plasma membrane and at a
supra-nuclear
site consistent with the Golgi apparatus, a distribution that was also
observed following the
apical application of the construct with SEQ ID NO: 158 that did not enter
these cells, and the
construct with SEQ ID NO: 165 that underwent endocytosis but remained in an
apical vesicular
compartment. Similar to that observed for untreated tissues or those exposed
to the construct
with SEQ ID NO: 158, ERGIC-53 (LMAN1) distribution in enterocytes exposed to
an apical
application of the construct with SEQ ID NO: 159 or the construct with SEQ ID
NO: 165
remained primarily in an apical vesicular compartment, with very little basal
vesicular
compartment distribution in enterocytes (Fig. 5). Movement of ERGIC-53 (LMAN1)
to a basal
vesicular compartment was observed sporadically in enterocytes following
apical application of
the construct with SEQ ID NO: 159, and this movement was much more consistent
in
enterocytes treated apically with the construct with SEQ ID NO: 160 or the
construct with SEQ
ID NO: 164. The distribution of COPT in enterocytes treated apically with the
construct with
SEQ ID NO: 160 was now longer evident in the supra-nuclear region of the cell
but was now
focused to the apical surface and apical vesicular compartment where it co-
localized to some
extent with ERGIC-53 (LMAN1).
[0548] Apical application of the construct with SEQ ID NO: 159 or the
construct with SEQ
ID NO: 165 resulted in the co-localization of HGH with COPT beneath the apical
membrane and
at a supra-nuclear site, with limited co-localization events being observed in
the apical vesicular
pool region of enterocytes. Apical treatment with the construct with SEQ ID
NO: 160 resulted in
extensive co-localization of HGH with COPT beneath the apical membrane and
some co-
localizations within the apical vesicular compartment, and less co-
localization events in the
supra-nuclear region.
[0549] This data demonstrate that Cholix derived delivery constructs such
as those having
amino acid sequences set forth in SEQ ID NO: 158, SEQ ID NO: 159 and SEQ ID
NO: 165 can
be used to delivery various cargo molecules to intracellular compartments of
an epithelial cell.
This may be particularly useful for the targeted delivery of therapeutic
and/or biologically active
molecules capable of eliciting a therapeutic and/or biological effect at those
locations.
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EXAMPLE 21
Basal Secretion of Cholix Domain I Derived Delivery Constructs
[0550] This example demonstrates a mechanism that can be harnessed to
achieve efficient
basal release of Cholix derived delivery constructs, allowing efficient
transport of cargo across
epithelial layers (e.g., a gut epithelium).
[0551] The basement membrane-specific heparan sulfate proteoglycan protein
(HSPG,
HSPG2 or perlecan), an integral component of basement membranes that support
the simple
columnar epithelium of the intestine, was identified in initial capture
studies (see e.g., FIG. 29,
FIG. 30). Perlecan is modulated by tyrosine phosphatase (PTPN6) and interacts
with von
Willebrand factor A domain-containing protein 1 (VWA1), both of which was also
enriched in
pull-down captures using the construct with SEQ ID NO: 164.
[0552] Stable knock-down of perlecan in Caco-2 cells (Caco-2HsPG2-) grown
in a polarized
format significantly reduced the ability of the construct with SEQ ID NO: 164
to complete
transcytosis in vitro. ELISA-based binding studies demonstrated that Chx can
associate with
perlecan. SPR studies showed that Chx interacts with perlecan at pH 5.5 but
not at pH 7.4.
[0553] In enterocytes from untreated animals of following apical exposure
with the
construct with SEQ ID NO: 158, perlecan-positive immunolabeling was observed
in vesicles that
were distributed in the apical, supra-nuclear, and basal compartments; ERGIC-
53 (LAMN1)
distribution was restricted to a location between the apical and supra-nuclear
compartments (Fig.
9D). Apical application of the construct with SEQ ID NO: 164 resulted in a
reduction of relative
perlecan labeling observed in the supra-nuclear compartment and a co-
localization with ERGIC-
53 (LAMN1) in the apical and basal compartments, with an area beneath the
luminal plasma
membrane that positive for ERGIC-53 (LAMN1) but not perlecan. ERGIC-53 (LAMN1)
present
in area beneath the luminal plasma membrane was positive for the construct
with SEQ ID NO:
164, but devoid of perlecan. Vesicles positive for HGH were also positive for
ERGIC-53
(LAMN1) or perlecan or both to varying degrees in the apical and basal
compartments. The
striking co-localization of the construct with SEQ ID NO: 164, ERGIC-53
(LAMN1) and
perlecan in these compartments and suggests that Cholix might traffic to the
basal side of the cell
with either ERGIC-53 (LAMN1) and/or perlecan (and/or possibly other
interacting elements)
where it can be released through an exocytosis process.
[0554] Notably, the Chx transcytosis pathway described herein suggests that
Cholix derived
carrier proteins coupled to a heterologous (non-Cholix derived) cargo do not
result in cellular
disorganization, outside of the subversion of ERGIC-53 (LAMN1), indicating
that the cargo
(e.g., therapeutic) delivery mechanisms described herein do not impair the
epithelial layer or the
cell itself. Thus, highly efficient cargo delivery without impairing the
epithelium of the delivery
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construct itself may be an important feature of the herein described delivery
constructs and
methods. It may allow delivery of various therapeutic and/or diagnostic or
other cargo molecules
in a non-invasive manner, potentially allowing for standards of safety and
therapeutic efficacy.
[0555] This data demonstrates that specific proteins (e.g., ERGIC-53,
ribophilin 1, SEC24,
CK-8, TMEM132A, GPR75, and perlecan) are involved in trafficking a Cholix
domain I derived
delivery construct across an epithelial cell and allow its release from the
basal membrane. The
identity of the distinct interaction partners and pH-dependencies of these
interactions may be
particularly useful for the therapeutic applications as the herein described
trafficking pathway
allows the delivery of Cholix domain I derived carriers coupled to various
cargo molecules to
reach the basolateral compartment (e.g., lamina propria) by using endogenous
trafficking
pathways. However, and demonstrated in EXAMPLE 11 above, specific functional
elements of
Cholix (and PE) domain I allow the delivery of heterologous cargo to distinct
locations within
and/or across the epithelium.
[0556] Thus, the herein described multi-receptor, multi-compartment
pathways used by
Cholix derived delivery constructs to efficiently achieve the transcytosis can
provide a potential
roadmap for the oral delivery of therapeutic molecules (e.g., protein
therapeutics such as
therapeutic antibodies) that can be coupled to a Cholix domain I carrier or a
carrier derived
therefrom.
EXAMPLE 22
Functional Peptide Fragments of Cholix Domain I are Located on the Protein
Surface
[0557] This example shows that portions of the functional sequence elements
of Cholix
domain I that are described herein to promote transcytosis and apical-to-basal
trafficking are
located at the protein surface of domain I of the Cholix exotoxin.
[0558] Protein structure analyses demonstrated that functional elements
required for apical
endocytosis, apical-to-basal trafficking and basal release are in proximity to
each other on the
surface of the Cholix domain I protein.
[0559] A surface model of bacterially expressed Cholix domain I (SEQ ID NO:
5) was used
to highlight selected areas of potential interest in this transcytosis process
due to their projection
from the protein surface (FIG. 32). It is interesting to note that two amino
acids regions between
Ml and G4 are adjacent to surface exposed amino acids D151-A187 and A187-
1_,206.
Specifically,
26
L18 -I (domain X1) and T171-I176
(domain X2) coordinate to form a pocket surrounded by
several negative charges. Similarly, 1(187-E1203 (domain X3) coordinates with
132-E4 (domain X4)
to form a continuous ridge structure (FIG. 32A-FIG. 32D).
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[0560] This data shows that portions of the amino acid sequence of Cholix
domain I that are
distant from each other in terms of amino acid position within the sequence
can, in fact, be in
close proximity to each other in a 3D configuration of the Cholix domain I
protein. This data
suggests that the 3D structure and surface morphology of Cholix domain I
allows interaction
with receptors and receptor elements required to efficient endocytosis and/or
transport the
epithelial cell. This can be useful to design orally administrable
therapeutics comprising such
Cholix domain I derived carrier molecules to provide therapeutically effective
doses basolateral
compartments, the lamina propria, etc. to elicit therapeutic effects.
[0561] All of the articles and methods disclosed and claimed herein can be
made and
executed without undue experimentation in light of the present disclosure.
While the articles and
methods of this disclosure have been described in terms of embodiments, it
will be apparent to
those of skill in the art that variations can be applied to the articles and
methods without
departing from the spirit and scope of the disclosure. All such variations and
equivalents
apparent to those skilled in the art, whether now existing or later developed,
are deemed to be
within the spirit and scope of the disclosure as defined by the appended
claims. All patents,
patent applications, and publications mentioned in the specification are
indicative of the levels of
those of ordinary skill in the art to which the disclosure pertains. All
patents, patent applications,
and publications are herein incorporated by reference in their entirety for
all purposes and to the
same extent as if each individual publication was specifically and
individually indicated to be
incorporated by reference in its entirety for any and all purposes. The
disclosure illustratively
described herein suitably can be practiced in the absence of any element(s)
not specifically
disclosed herein. The terms and expressions which have been employed are used
as terms of
description and not of limitation, and there is no intention that in the use
of such terms and
expressions of excluding any equivalents of the features shown and described
or portions
thereof, but it is recognized that various modifications are possible within
the scope of the
disclosure claimed. Thus, it should be understood that although the present
disclosure has been
specifically disclosed by embodiments and optional features, modification and
variation of the
concepts herein disclosed can be resorted to by those skilled in the art, and
that such
modifications and variations are considered to be within the scope of this
disclosure as defined
by the appended claims.
Sequence Listings
[0562] SEQ ID NO: 1 is a 634 amino acid sequence of mature Vibrio cholera
Cholix toxin.
[0563] SEQ ID NO: 2 is a 634 amino acid sequence of mature Vibrio cholera
Cholix toxin.
[0564] SEQ ID NO: 3 is a non-toxic (nt) variant of the mature V. cholera
Cholix toxin.
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[0565] SEQ ID NO: 4 is a domain I of a Cholix toxin.
[0566] SEQ ID NO: 5 is a domain I of a Cholix toxin comprising an N-
terminal methionine
residue (e.g., due to bacterial expression).
[0567] SEQ ID NO: 6 ¨ SEQ ID NO: 125 are truncated versions of Cholix
domain I.
[0568] SEQ ID NO: 126 is an amino acid sequence of the V. cholera Cholix
carrier
translocation domain (domain II).
[0569] SEQ ID NO: 127 is an amino acid sequence of the V. cholera Cholix
carrier domain
lb.
[0570] SEQ ID NO: 128 is an amino acid sequence of the V. cholera Cholix
carrier catalytic
domain (domain III).
[0571] SEQ ID NO: 129 is the amino acid sequence of Cholix1-425.
[0572] SEQ ID NO: 130 is the amino acid sequence of Cholix1-415.
[0573] SEQ ID NO: 131 is the amino acid sequence of Cholix1-397.
[0574] SEQ ID NO: 132 is the amino acid sequence of Cholix1-386.
[0575] SEQ ID NO: 133 is the amino acid sequence of Cholix1-291.
[0576] SEQ ID NO: 134 is a nucleic acid sequence encoding the mature Vibrio
cholera
Cholix toxin set forth in SEQ ID NO: 2.
[0577] SEQ ID NO: 135 is a nucleic acid sequence encoding the 613 amino
acid sequence
of mature Pseudomonas exotoxin A (PE).
[0578] SEQ ID NO: 136 is a nucleic acid sequence encoding the mature
Pseudomonas
exotoxin A (PE) set forth in SEQ ID NO: 135.
[0579] SEQ ID NO: 137 is an amino acid sequence of the Pseudomonas exotoxin
A (PE)
receptor binding domain (Domain I).
[0580] SEQ ID NO: 138 is an amino acid sequence of the Pseudomonas exotoxin
A (PE)
translocation domain (Domain II).
[0581] SEQ ID NO: 139 is an amino acid sequence of the Pseudomonas exotoxin
A (PE)
Domain lb.
[0582] SEQ ID NO: 140 is an amino acid sequence of the Pseudomonas exotoxin
A (PE)
catalytic domain (Domain III).
[0583] SEQ ID NO: 141 is the amino acid sequence of PE1-404.
[0584] SEQ ID NO: 142 is the amino acid sequence of PE1-395.
[0585] SEQ ID NO: 143 is the amino acid sequence of PE1-375.
[0586] SEQ ID NO: 144 is the amino acid sequence of PE1-364.
[0587] SEQ ID NO: 145 is the amino acid sequence of PE1-277.
[0588] SEQ ID NO: 146 is the amino acid sequence of a hybrid delivery
construct.
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[0589] SEQ ID NO: 147 is the amino acid sequence of a hybrid delivery
construct.
[0590] SEQ ID NO: 148 is the amino acid sequence of a functional peptide
sequence
derived from Cholix domain I (e.g., for endocytosis).
[0591] SEQ ID NO: 149 is the amino acid sequence of a functional peptide
sequence
derived from Cholix domain I (e.g., for apical-to-basal transport).
[0592] SEQ ID NO: 150 is the amino acid sequence of a functional peptide
sequence
derived from Cholix domain I (e.g., for apical-to-basal transport).
[0593] SEQ ID NO: 151 is the amino acid sequence of a functional peptide
sequence
derived from Cholix domain I (e.g., for supranuclear localization).
[0594] SEQ ID NO: 152 is the amino acid sequence of a functional peptide
sequence
derived from Cholix domain I (e.g., for basal release).
[0595] SEQ ID NO: 153 is the amino acid sequence of a Cholix derived
delivery construct
comprising human growth hormone (HGH).
[0596] SEQ ID NO: 154 is the amino acid sequence of a Cholix derived
delivery construct
comprising interleukin-10 (IL-10).
[0597] SEQ ID NO: 155 is the amino acid sequence of a Cholix derived
delivery construct
comprising interleukin-10 (IL-22).
[0598] SEQ ID NO: 156 is the amino acid sequence of a Cholix derived
delivery construct
comprising red fluorescent protein (RFP).
[0599] SEQ ID NO: 157 is the amino acid sequence of a Cholix derived
(comprising non-
toxic mature Cholix) delivery construct comprising red fluorescent protein
(RFP).
[0600] SEQ ID NO: 158 is the amino acid sequence of a truncated Cholix
domain I delivery
construct comprising HGH.
[0601] SEQ ID NO: 159 is the amino acid sequence of a truncated Cholix
domain I delivery
construct comprising HGH.
[0602] SEQ ID NO: 160 is the amino acid sequence of a truncated Cholix
domain I delivery
construct comprising HGH.
[0603] SEQ ID NO: 161 is the amino acid sequence of a truncated Cholix
domain I delivery
construct comprising HGH.
[0604] SEQ ID NO: 162 is the amino acid sequence of a truncated Cholix
domain I delivery
construct comprising HGH.
[0605] SEQ ID NO: 163 is the amino acid sequence of a truncated Cholix
domain I delivery
construct comprising HGH.
[0606] SEQ ID NO: 164 is the amino acid sequence of a Cholix domain I
delivery construct
comprising HGH.
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[0607] SEQ ID NO: 165 is the amino acid sequence of a truncated Cholix
domain I delivery
construct comprising HGH.
[0608] SEQ ID NO: 166 ¨ SEQ ID NO: 186 are the amino acid sequences of
various
peptidase cleavage sites.
[0609] SEQ ID NO: 187 ¨ SEQ ID NO: 193 are the amino acid sequences of
various GM-1
receptor binding peptides.
[0610] SEQ ID NO: 194 ¨ SEQ ID NO: 206 are the amino acid sequences of
various
cleavable spacers.
[0611] SEQ ID NO: 207 ¨ SEQ ID NO: 213 are the amino acid sequences of
various non-
cleavable spacers.
[0612] SEQ ID NO: 214 is the amino acid sequence of a human growth hormone
(HGH).
[0613] SEQ ID NO: 215 is the amino acid sequence of a GLP-1 agonist
peptide.
[0614] SEQ ID NO: 216 is the amino acid sequence of an insulin peptide.
[0615] SEQ ID NO: 217 is the amino acid sequence of a human interleukin-10
(IL-10).
[0616] SEQ ID NO: 218 is the amino acid sequence of a human interleukin-22
(IL-22).
[0617] SEQ ID NO: 219 is the amino acid sequence of an ExtB polypeptide.
[0618] SEQ ID NO: 220 is the amino acid sequence of a Red Fluorescent
Protein (RFP).
[0619] SEQ ID NO: 221 is the amino acid sequence of a Cholix domain I
derived carrier
coupled to a spacer.
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