U.S. patent application number 16/207655 was filed with the patent office on 2019-04-25 for cholix toxin-derived fusion molecules for oral delivery of biologically active cargo.
The applicant listed for this patent is Applied Molecular Transport Inc.. Invention is credited to Tahir Mahmood, Randall J. Mrsny.
Application Number | 20190117744 16/207655 |
Document ID | / |
Family ID | 54393149 |
Filed Date | 2019-04-25 |
United States Patent
Application |
20190117744 |
Kind Code |
A1 |
Mrsny; Randall J. ; et
al. |
April 25, 2019 |
CHOLIX TOXIN-DERIVED FUSION MOLECULES FOR ORAL DELIVERY OF
BIOLOGICALLY ACTIVE CARGO
Abstract
The present disclosure relates to pharmaceutical compositions
comprising a non-naturally occurring fusion molecule and one or
more pharmaceutically acceptable carriers, formulated for oral
delivery to a subject, and designed to provide for improved,
effective therapies for treatment of, e.g., inflammatory diseases,
autoimmune diseases, cancer, metabolic disorders, and growth
deficiency disorders.
Inventors: |
Mrsny; Randall J.; (Los
Altos Hills, CA) ; Mahmood; Tahir; (Burlingame,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Applied Molecular Transport Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
54393149 |
Appl. No.: |
16/207655 |
Filed: |
December 3, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15309177 |
Nov 4, 2016 |
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PCT/US2015/029795 |
May 7, 2015 |
|
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16207655 |
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61990054 |
May 7, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 5/06 20180101; C07K
2319/30 20130101; C12Y 304/21068 20130101; A61P 37/06 20180101;
A61P 5/00 20180101; A61K 38/164 20130101; A61K 38/50 20130101; C07K
2319/21 20130101; C07K 2319/32 20130101; A61P 5/08 20180101; A61P
7/00 20180101; A61P 25/28 20180101; A61P 1/00 20180101; A61P 3/04
20180101; A61P 3/10 20180101; C12Y 402/02001 20130101; C12Y
204/02036 20130101; A61P 7/06 20180101; A61P 35/00 20180101; A61K
38/482 20130101; A61K 38/51 20130101; A61P 1/16 20180101; A61P
29/00 20180101; A61K 38/166 20130101; A61P 17/06 20180101; A61K
38/2066 20130101; A61K 38/20 20130101; A61P 21/04 20180101; C07K
14/5428 20130101; C07K 2317/76 20130101; A61P 1/04 20180101; A61P
31/04 20180101; C07K 16/241 20130101; A61K 38/1793 20130101; C07K
2319/55 20130101; A61P 3/06 20180101; A61P 21/00 20180101; A61P
3/00 20180101; A61K 38/27 20130101; C07K 2319/50 20130101; C07K
2319/60 20130101; A61P 7/04 20180101; A61P 17/00 20180101; A61P
19/02 20180101; C12Y 304/21073 20130101; C12Y 305/04004 20130101;
A61P 35/02 20180101; A61K 38/49 20130101; C07K 2319/00 20130101;
A61K 38/45 20130101; A61K 38/26 20130101; A61P 43/00 20180101; A61P
5/14 20180101; A61P 25/00 20180101; C07K 2319/90 20130101 |
International
Class: |
A61K 38/45 20060101
A61K038/45; A61K 38/27 20060101 A61K038/27; A61K 38/51 20060101
A61K038/51; A61K 38/50 20060101 A61K038/50; A61K 38/49 20060101
A61K038/49; A61K 38/48 20060101 A61K038/48; A61K 38/26 20060101
A61K038/26; A61K 38/20 20060101 A61K038/20; A61K 38/16 20060101
A61K038/16; C07K 14/54 20060101 C07K014/54; C07K 16/24 20060101
C07K016/24; A61K 38/17 20060101 A61K038/17 |
Claims
1-77. (canceled)
78. A method for delivering an anti-TNF-.alpha. antibody or
antibody fragment to a subject, the method comprising orally
delivering to the subject a pharmaceutical composition comprising
(i) a carrier consisting of the amino acid sequence set forth in
SEQ ID NO: 1 or a fragment thereof, wherein the fragment of the
amino acid sequence set forth in SEQ ID NO: 1 consists of the amino
acid sequence set forth in any one of SEQ ID NOS: 3, 42, 52, 70, or
80, and wherein the carrier is conjugated to (ii) the
anti-TNF-.alpha. antibody or antibody fragment consisting of at
least 90% sequence identity to the amino acid sequence set forth in
any one of SEQ ID NOS: 88-91 or a combination thereof.
79. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 90% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 88-89
or a combination thereof.
80. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 90% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 90-91
or a combination thereof.
81. The method of claim 78, wherein the fragment of the amino acid
sequence set forth in SEQ ID NO: 1 consists of the amino acid
sequence set forth in SEQ ID NO: 52.
82. The method of claim 78, wherein the fragment of the amino acid
sequence set forth in SEQ ID NO: 1 consists of the amino acid
sequence set forth in SEQ ID NO: 3.
83. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 92% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 88-89
or a combination thereof.
84. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 92% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 90-91
or a combination thereof.
85. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 95% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 88-89
or a combination thereof.
86. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 95% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 90-91
or a combination thereof.
87. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 99% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 88-89
or a combination thereof.
88. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 99% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 90-91
or a combination thereof.
89. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 97% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 88-89
or a combination thereof.
89. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 97% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 90-91
or a combination thereof.
90. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 93% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 88-89
or a combination thereof.
91. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of at least 93% sequence identity to
the amino acid sequence set forth in any one of SEQ ID NOS: 90-91
or a combination thereof.
92. The method of claim 78, wherein the carrier is covalently
coupled to the anti-TNF-.alpha. antibody or antibody fragment.
93. The method of claim 78, wherein the carrier is non-covalently
coupled to the anti-TNF-.alpha. antibody or antibody fragment.
94. The method of claim 78, wherein the anti-TNF-.alpha. antibody
or antibody fragment consists of the amino acid sequence set forth
in any one of SEQ ID NOS: 88-91 or a combination thereof.
95. The method of claim 78, wherein the carrier is conjugated to
the anti-TNF-.alpha. antibody or antibody fragment by a linker that
consists of the amino acid sequence set forth in any one of SEQ ID
NOS: 96-99.
96. The method of claim 78, wherein the carrier is conjugated to
the anti-TNF-.alpha. antibody or antibody fragment by a linker that
consists of the amino acid sequence set forth in any one of SEQ ID
NOS: 100-120.
97. The method of claim 78, wherein the carrier is conjugated to
the anti-TNF-.alpha. antibody or antibody fragment by a linker that
consists of the amino acid sequence set forth in SEQ ID NO:
121.
98. The method of claim 78, wherein the carrier consists of the
amino acid sequence set forth in SEQ ID NO: 3, conjugated to the
anti-TNF-.alpha. antibody or antibody fragment consisting of the
amino acid sequence set forth in any one of SEQ ID NOS: 88-91 or a
combination thereof.
99. The method of claim 78, wherein the carrier consists of the
amino acid sequence set forth in SEQ ID NO: 3, wherein the
anti-TNF-.alpha. antibody or antibody fragment consists of the
amino acid sequence set forth in any one of SEQ ID NOS: 88-91 or a
combination thereof, and wherein the carrier is conjugated to the
anti-TNF-.alpha. antibody by a linker that consists of the amino
acid sequence set forth in any one of SEQ ID NOS: 96-121.
100. The method of claim 78, wherein the carrier consists of the
amino acid sequence of SEQ ID NO: 52, conjugated to the
anti-TNF-.alpha. antibody or antibody fragment consisting of the
amino acid sequence set forth in any one of SEQ ID NOS: 88-91 or a
combination thereof.
101. The method of claim 78, wherein the carrier consists of the
amino acid sequence set forth in SEQ ID NO: 52, wherein the
anti-TNF-.alpha. antibody or antibody fragment consists of the
amino acid sequence set forth in any one of SEQ ID NOS: 88-91 or a
combination thereof, and wherein the carrier is conjugated to the
anti-TNF-.alpha. antibody by a linker that consists of the amino
acid sequence set forth in any one of SEQ ID NOS: 96-121.
Description
RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/309,177, filed Nov. 4, 2016, which is a U.S. National Phase
Application under 35 U.S.C. .sctn. 371 of International Application
No. PCT/US2015/029795, filed May 7, 2015, which claims the benefit
of U.S. Provisional Application No. 61/990,054, filed on May 7,
2014, the disclosure of which is herein incorporated by reference
in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been filed electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 15, 2015, is named 40566_702_831_SL.txt and is 307
kilobytes in size.
TECHNICAL FIELD
[0003] Oral delivery of biologically active polypeptides (referring
to a polymer composed of amino acid residues; typically also
defined as proteins or peptides) has been a long-standing goal of
the pharmaceutical industry. Unfortunately, the numerous physical,
physiological, and biological barriers of the gastrointestinal (GI)
tract are designed to inhibit uptake of proteins and peptides until
they can be sufficiently degraded for absorption through amino acid
and di- or tri-peptide transporters; and/or to traffic the proteins
and peptides intracellularly to destructive lysosome compartments
after endosomal uptake at the luminal surface. As such, the
feasibility of polypeptide uptake from the intestine in a manner
similar to that achievable with, e.g., small molecules, has been
limited and low oral bioavailability continues to be a problem for
most polypeptides and proteins.
[0004] While there have been some promising results from clinical
studies evaluating various biologically active polypeptides for the
treatment of diseases such as cancer, inflammatory diseases, immune
diseases, growth deficiency disorders, etc., and several DNA-based
therapeutics have been FDA approved for such uses, these
therapeutics often fail to really reach their optimum potential, as
there is often marginal or inadequate overall efficacy due to
inherent limitations such as short biological half-life which
prevents the delivery of optimal therapeutically effective dosages,
and/or detrimental side effects and toxicities observed at the
therapeutically effective doses. Moreover, many such therapeutics
require multiple dosing regimens, necessitating continuous
administration intravenously or by frequent subcutaneous
injections, which are burdensome on the patients and
caregivers.
[0005] Future clinical studies directed toward evaluating the
promising biologically active polypeptides could benefit greatly
from new methods and/or pharmaceutical compositions that could be
used to orally administer such polypeptides to a human subject.
DISCLOSURE OF THE INVENTION
[0006] The present disclosure relates to pharmaceutical
compositions comprising novel, non-naturally occurring fusion
molecules and one or more pharmaceutically acceptable carriers,
formulated for oral delivery, and designed to provide for improved,
effective therapies for treatment of, e.g., inflammatory diseases
and/or autoimmune diseases and/or cancers.
[0007] The present disclosure is based in part on the inventors'
unique insight that oral delivery of a pharmaceutical composition
comprising a fusion molecule which comprises a modified Cholix
toxin coupled to a biologically active cargo may, among other
things, provide the following advantages: a) in embodiments wherein
the modified Cholix toxin is coupled to the biologically active
cargo without a linker, or with a non-cleavable linker, the
anchoring effect of the modified Cholix toxin by its receptor(s) at
the surface of, e.g., immune cells that also express the receptor
for the biologically active cargo, can allow for greater exposure
of the biologically active cargo at the surface of the targeted
cells and provide a synergistic effect by binding to both the
Cholix receptor and the biologically active cargo receptor; b) in
embodiments wherein the modified Cholix toxin is coupled to the
biologically active cargo with a linker 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 biologically active cargo to be released from the
remainder of the fusion molecule soon after transcytosis across the
epithelial membrane c) the direct delivery of the biologically
active cargo to the submucosal-GI space and hepatic-portal system
may reduce the systemic toxicity observed when the cargo are
administered by parenteral routes, as well as enabling access to
the submucosal target biology that was difficult to target via
non-oral or GI routes; d) once transported across the GI
epithelium, the fusion molecules of the disclosure will exhibit
extended half-life in serum, that is, the biologically active cargo
of the fusion molecules will exhibit an extended serum half-life
compared to the biologically active cargo in its non-fused state;
e) oral administration of the fusion molecule 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; and f) 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.
[0008] Thus, in one aspect, the present disclosure relates to
pharmaceutical compositions comprising a non-naturally occurring
fusion molecule and one or more pharmaceutically acceptable
carriers, formulated for oral delivery, wherein the fusion molecule
comprises a modified Cholix toxin coupled to a biologically active
cargo to be delivered to a subject, wherein the Cholix toxin is
non-toxic.
[0009] In one aspect, the present disclosure relates to
pharmaceutical compositions comprising a non-naturally occurring
fusion molecule and one or more pharmaceutically acceptable
carriers, formulated for oral delivery, wherein the fusion molecule
comprises a modified Cholix toxin coupled to a biologically active
cargo to be delivered to a subject, wherein the Cholix toxin is
non-toxic, and wherein the fusion molecule has the ability to
activate the receptor for the biologically active cargo, or to
enable the catalytic process of a catalytically-active
material.
[0010] In various embodiments, the fusion molecules of the
pharmaceutical compositions comprise a modified Cholix toxin
truncated at an amino acid residue within Cholix toxin domain II.
In various embodiments, the fusion molecules comprise a truncated
Cholix toxin having the amino acid sequence set forth in, e.g., SEQ
ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,
SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:
21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:
30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ
ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:
39, SEQ ID NO: 40 or SEQ ID NO: 41.
[0011] In various embodiments, the fusion molecules of the
pharmaceutical compositions comprise a modified Cholix toxin
truncated at an amino acid residue within Cholix toxin domain Ib.
In various embodiments, the fusion molecules comprise a truncated
Cholix toxin having the amino acid sequence set forth in, e.g., SEQ
ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:
46, SEQ ID NO: 47, SEQ ID NO:48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ
ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO:
55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ
ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ
ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO:
73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ
ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80.
[0012] In various embodiments, the fusion molecules of the
pharmaceutical compositions comprise a modified Cholix toxin
wherein domain III has been truncated or mutated. In various
embodiments, the fusion molecules comprise a mutated Cholix toxin
having the amino acid sequence set forth in SEQ ID NO: 81 wherein
the amino acid residue E581 of SEQ ID NO: 1 has been deleted
(designated herein as "Cholix .DELTA.E581").
[0013] In various embodiments, the fusion molecules of the
pharmaceutical compositions comprise a modified Cholix toxin
wherein domain Ia has been mutated.
[0014] In various embodiments, the biologically active cargo is
selected from e.g., a macromolecule, small molecule, peptide,
polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA, antisense
molecule, antibody, DNA, plasmid, vaccine, polymer nanoparticle, or
catalytically-active material.
[0015] In various embodiments, the biologically active cargo is an
enzyme selected from hyaluronidase, streptokinase, tissue
plasminogen activator, urokinase, or PGE-adenosine deaminase.
[0016] In various embodiments, the biologically active cargo is a
polypeptide that is a modulator of inflammation in the GI tract
selected from, e.g., interleukin-10, interleukin-19,
interleukin-20, interleukin-22, interleukin-24, or interleukin-26.
In various embodiments, the biologically active polypeptide is
interleukin-10 having the amino acid sequence set forth is SEQ ID
NO: 82. In various embodiments, the biologically active polypeptide
is interleukin-19 having the amino acid sequence set forth is SEQ
ID NO: 83. In various embodiments, the biologically active
polypeptide is interleukin-20 having the amino acid sequence set
forth is SEQ ID NO: 84. In various embodiments, the biologically
active polypeptide is interleukin-22 having the amino acid sequence
set forth is SEQ ID NO: 85. In various embodiments, the
biologically active polypeptide is interleukin-24 having the amino
acid sequence set forth is SEQ ID NO: 86. In various embodiments,
the biologically active polypeptide is interleukin-26 having the
amino acid sequence set forth is SEQ ID NO: 87. In various
embodiments, the biologically active cargo is a modulator of
inflammation in the GI tract that is a small molecule. In various
embodiments, the biologically active cargo is a modulator of
inflammation in the GI tract that is an antisense or siRNA
molecule.
[0017] In various embodiments, the biologically active cargo is a
TNFSF inhibitor that is an antibody, or a fragment thereof, or an
artificial construct comprising an antibody or fragment thereof, or
an artificial construct designed to mimic the binding of an
antibody or fragment thereof to its antigen. In various
embodiments, the biologically active cargo is a TNFSF inhibitor
that is a soluble TNFSF receptor fusion protein. In various
embodiments, the biologically active cargo is a TNFSF inhibitor
that is a small molecule. In various embodiments, the biologically
active cargo is a TNFSF inhibitor that is an antisense or siRNA
molecule.
[0018] In various embodiments, the biologically active cargo is an
antibody comprising the heavy chain variable region amino acid
sequence set forth in SEQ ID NO: 88 and light chain variable region
amino acid sequence set forth in SEQ ID NO: 89. In various
embodiments, the biologically active cargo is an antibody
comprising the heavy chain variable region amino acid sequence set
forth in SEQ ID NO: 90 and light chain variable region amino acid
sequences set forth in SEQ ID NO: 91. In various embodiments, the
biologically active cargo is a soluble TNFSF receptor fusion
protein dimer comprising the amino acid sequence set forth in SEQ
ID NO: 92.
[0019] In one aspect, the present disclosure relates to
pharmaceutical compositions comprising novel, non-naturally
occurring fusion molecules and one or more pharmaceutically
acceptable carriers, formulated for oral delivery, and designed to
provide for improved, effective therapies for treatment of
metabolic disorders, e.g., Type 1 Diabetes and Type 2 Diabetes.
Oral delivery of biologically active polypeptides (referring to a
polymer composed of amino acid residues; typically also defined as
proteins or peptides) has been a long-standing goal of the
pharmaceutical industry. Unfortunately, the numerous physical,
physiological, and biological barriers of the gastrointestinal (GI)
tract are designed to inhibit uptake of proteins and peptides until
they can be sufficiently degraded for absorption through amino acid
and di- or tri-peptide transporters; and/or to traffic the proteins
and peptides intracellularly to destructive lysosome compartments
after endosomal uptake at the luminal surface. As such, the
feasibility of polypeptide uptake from the intestine in a manner
similar to that achievable with, e.g., small molecules, has been
limited and low oral bioavailability continues to be a problem for
most polypeptides and proteins.
[0020] In various embodiments, the present disclosure relates to
pharmaceutical compositions comprising a non-naturally occurring
fusion molecule and one or more pharmaceutically acceptable
carriers, formulated for oral delivery, wherein the fusion molecule
comprises a modified Cholix toxin coupled to a glucose-lowering
agent to be delivered to a subject.
[0021] In various embodiments, the present disclosure is based in
part on that oral delivery of a pharmaceutical composition
comprising a fusion molecule which comprises a modified Cholix
toxin coupled to a glucose-lowering agent may, among other things,
provide the following advantages: a) in embodiments wherein the
modified Cholix toxin is coupled to the glucose-lowering agent
without a linker, the anchoring effect of the modified Cholix toxin
by its receptor(s) at the surface of cells that also express the
receptor for the glucose-lowering agent, can allow for greater
exposure of the glucose-lowering agent at the surface of the
targeted cells; b) in embodiments wherein the modified Cholix toxin
is coupled to the glucose-lowering agent with a linker that is
cleavable by an enzyme present at a basal-lateral membrane of an
epithelial cell, or an enzyme present in the plasma of the subject,
such cleavage will allow the glucose-lowering agent to be released
from the remainder of the fusion molecule soon after transcytosis
across the epithelial membrane; c) the direct delivery of the
glucose-lowering agent to the submucosal-GI space and
hepatic-portal system may reduce the systemic toxicity observed
when the glucose-lowering agents are administered by parenteral
routes, as well as enabling access to the submucosal target biology
that was difficult to target via non-oral or GI routes; d) the
direct delivery of the glucose-lowering agent to the submucosal-GI
space and hepatic-portal system may provide for improved dosing
regimens, including less frequent insulin injections; and e) the
ability to deliver the glucose-lowering agent 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.
[0022] In various embodiments, the glucose-lowering agent is
selected from e.g., a macromolecule, small molecule, peptide,
polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA, antisense
molecule, antibody, DNA, plasmid, vaccine, polymer nanoparticle, or
catalytically-active material. In various embodiments, the
glucose-lowering agent is an incretin or incretin mimetic. In
various embodiments, the glucose-lowering agent is a GLP-1. In
various embodiments, the glucose-lowering agent is a GLP-1 agonist.
In various embodiments, the glucose-lowering agent is an exendin.
In various embodiments, the glucose-lowering agent is a glucose
inhibitory protein receptor (GIPR) agonist.
[0023] In various embodiments, the glucose-lowering agent is a
GLP-1 agonist that is a peptide. In various embodiments, the
glucose-lowering agent is a GLP-1 agonist that is a small molecule.
In various embodiments, the glucose-lowering agent is a GLP-1
agonist that is an antisense or siRNA molecule. In various
embodiments, the glucose-lowering agent is a GLP-1 agonist that is
an antibody, or a fragment thereof, or an artificial construct
comprising an antibody or fragment thereof, or an artificial
construct designed to mimic the binding of an antibody or fragment
thereof to its antigen.
[0024] In various embodiments, the biologically active cargo is a
glucose-lowering agent that is a GLP-1 agonist peptide comprising
the amino acid sequence set forth in SEQ ID NO: 93. In various
embodiments, the biologically active cargo is a glucose-lowering
agent that is a GLP-1 agonist peptide comprising the amino acid
sequence set forth in SEQ ID NO: 94.
[0025] In one aspect, the present disclosure relates to
pharmaceutical compositions comprising novel, non-naturally
occurring fusion molecules and one or more pharmaceutically
acceptable carriers, formulated for oral delivery, and designed to
provide for improved, effective therapies for treatment of growth
hormone deficiency, and like disorders.
[0026] In various embodiments, the present disclosure relates to
pharmaceutical compositions comprising a non-naturally occurring
fusion molecule and one or more pharmaceutically acceptable
carriers, formulated for oral delivery, wherein the fusion molecule
comprises a modified Cholix toxin coupled to a growth hormone (GH)
to be delivered to a subject.
[0027] In various embodiments, the present disclosure is based in
part on the inventors' unique insight that oral delivery of a
pharmaceutical composition comprising a fusion molecule which
comprises a modified Cholix toxin coupled to a growth hormone may,
among other things, provide the following advantages: a) in
embodiments wherein the modified Cholix toxin is coupled to the
growth hormone with a linker that is cleavable by an enzyme present
at a basolateral membrane surface of an epithelial cell, or an
enzyme present in the plasma of the subject, such cleavage will
allow the growth hormone to be released from the remainder of the
fusion molecule soon after transcytosis across the epithelial
membrane; b) the direct delivery of the growth hormone to the
submucosal-GI space and hepatic-portal system may reduce systemic
toxicities observed when the growth hormones are administered by
parenteral routes, as well as enabling access to the submucosal
target biology that was difficult to target via non-oral or GI
routes (e,g, provide a more efficient induction of IGF-1 relative
to systemic delivery via subcutaneous (sc) injection); c) the
direct delivery of the growth hormone to the submucosal-GI space
and hepatic-portal system may provide for improved dosing regimens;
d) oral delivery will achieve a brief pulse of growth hormone to
the liver that is more consistent with serum level observed in
growing children, and this pulse profile is not achievable by sc
injection; and e) the ability to deliver the growth hormone 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.
[0028] In various embodiments, the growth hormone is selected from
e.g., a macromolecule, small molecule, peptide, polypeptide,
nucleic acid, mRNA, miRNA, shRNA, siRNA, antisense molecule,
antibody, DNA, plasmid, vaccine, polymer nanoparticle, or
catalytically-active material. In various embodiments, the growth
hormone is human growth hormone (or a variant thereof), growth
hormone 2, or growth hormone-releasing hormone. In various
embodiments, the growth hormone is human growth hormone
(somatotropin) comprising the amino acid sequence set forth in SEQ
ID NO: 95.
[0029] In various embodiments, the fusion molecules comprise a
modified Cholix toxin directly coupled to a biologically active
cargo. In various embodiments, the biologically active cargo is
directly coupled to the C-terminus of the Cholix toxin.
[0030] In various embodiments, the fusion molecules comprise a
modified Cholix toxin chemically coupled to a biologically active
cargo.
[0031] In various embodiments, the fusion molecules comprise a
Cholix toxin coupled to a biologically active cargo by a
non-cleavable linker. In various embodiments, the non-cleavable
linker comprises the amino acid sequence of, e.g., SEQ ID NO: 96,
SEQ ID NO: 97, SEQ ID NO: 98 or SEQ ID NO: 99.
[0032] In various embodiments, the fusion molecules comprise a
Cholix toxin coupled to a biologically active cargo by a cleavable
linker. In various embodiments, the linker is cleavable by an
enzyme that is present at a basolateral membrane of a polarized
epithelial cell of the subject. In various embodiments, the linker
is cleavable by an enzyme that is present in the plasma of said
subject. In various embodiments, the cleavable linker comprises the
amino acid sequence of, e.g., SEQ ID NO: 100, SEQ ID NO: 101, SEQ
ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID
NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:
110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:
114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:
118, SEQ ID NO: 119, or SEQ ID NO: 120.
[0033] In various embodiments, the fusion molecules comprise a
Cholix toxin coupled to a biologically active cargo by a cleavable
linker, wherein the cleavable linker comprises an amino acid
sequence that is known to be a substrate for tobacco etch virus
(TEV) protease. In various embodiments, the cleavable linker
comprises the amino acid sequence of, e.g., SEQ ID NO: 121.
[0034] In various embodiments, the fusion molecule comprises the
amino acid sequence set forth in SEQ ID NO: 122. (this is
Cholix.sup.415-TEV-IL-10)
[0035] In various embodiments, the fusion molecule comprises the
amino acid sequence set forth in SEQ ID NO: 123. (this is
Cholix.sup.415-(G.sub.4S).sub.3-IL-10)
[0036] In another aspect, the present disclosure provides a method
of treating an inflammatory disease in a subject, comprising orally
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, ischaemic colitis, diversion colitis,
Behcet's syndrome or indeterminate colitis.
[0037] In another aspect, the present disclosure provides a method
of treating an autoimmune disease in a subject, comprising orally
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.
[0038] In another aspect, the present disclosure provides a method
of treating a cancer in a subject, comprising orally 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.
[0039] In another aspect, the present disclosure provides a method
of treating a subject having a metabolic disorder, said method
comprising orally administering a fusion molecule 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.
[0040] In another aspect, 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 fusion molecule of the present disclosure in an
amount sufficient to treat said disease.
[0041] In another aspect, the present disclosure provides a method
of treating a subject having a GH deficient growth disorder, said
method comprising orally administering a fusion molecule 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.
[0042] In another aspect, the present disclosure relates to the use
of a non-naturally occurring fusion molecule of the present
invention for the preparation of a medicament for treatment,
prophylaxis and/or prevention of an inflammatory disease in a
subject in need thereof.
[0043] In another aspect, the present disclosure relates to the use
of a non-naturally occurring fusion molecule of the present
invention for the preparation of a medicament for treatment,
prophylaxis and/or prevention of an autoimmune disease in a subject
in need thereof.
[0044] In another aspect, the present disclosure relates to the use
of a non-naturally occurring fusion molecule of the present
invention for the preparation of a medicament for treatment,
prophylaxis and/or prevention of a cancer in a subject in need
thereof.
[0045] In another aspect, the present disclosure relates to the use
of a non-naturally occurring fusion molecule of the present
invention for the preparation of a medicament for treatment,
prophylaxis and/or prevention of a metabolic disorder in a subject
in need thereof.
[0046] In another aspect, the present disclosure relates to the use
of a non-naturally occurring fusion molecule of the present
invention for the preparation of a medicament for treatment,
prophylaxis and/or prevention of a fatty liver disease in a subject
in need thereof.
[0047] In another aspect, the present disclosure relates to the use
of a non-naturally occurring fusion molecule of the present
invention for the preparation of a medicament for treatment,
prophylaxis and/or prevention of GH deficient growth disorder in a
subject in need thereof.
[0048] In other aspects, the present disclosure provides
polynucleotides that encode the non-naturally occurring modified
Cholix toxin-biologically active cargo fusion molecules of the
present disclosure; vectors comprising polynucleotides encoding
non-naturally occurring modified Cholix toxin-biologically active
cargo fusion molecules of the disclosure; optionally,
operably-linked to control sequences recognized by a host cell
transformed with the vector; host cells comprising vectors
comprising polynucleotides encoding non-naturally occurring
modified Cholix toxin-biologically active cargo fusion molecules of
the disclosure; a process for producing a non-naturally occurring
modified Cholix toxin-biologically active cargo fusion molecule of
the disclosure comprising culturing host cells comprising vectors
comprising polynucleotides encoding non-naturally occurring
modified Cholix toxin-biologically active cargo fusion molecules of
the disclosure such that the polynucleotide is expressed; and,
optionally, recovering the non-naturally occurring modified Cholix
toxin-biologically active cargo fusion molecule from the host cell
culture medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1 depicts the genetic constructions of two exemplary
Cholix toxin-IL-10 fusion molecules evaluated herein. The
N-terminus of a human IL-10 monomer sequence was genetically
attached to the C-terminus of a modified Cholix toxin
(Cholix.sup.415) using a stable non-cleavable linker sequence
((G.sub.4S).sub.3) or a linker sequence that is a known substrate
for the tobacco etch virus (TEV) protease. Each construct also
contains an N-terminal Methionine (M).
[0050] FIG. 2 is a ribbon diagram representation of an exemplary
"dimer Cholix toxin-IL-10" fusion molecule after refolding that
would be driven by IL-10 dimerization. The first 415 amino acids of
Cholix toxin (SEQ ID NO: 1) are connected through a 16 amino acid
linker (not shown) to connect with the human IL-10 sequence. IL-10
dimerization is envisaged to result in purple Cholix.sup.415/blue
hlL-10 and orange Cholix.sup.415/green organization shown.
[0051] FIG. 3 is a coomassie stained SDS PAGE of
Cholix.sup.415-TEV-IL-10 (depicted as "C") and
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 (depicted as "N") following
induction and expression from inclusion bodies. The expressed
fusion molecules demonstrate the anticipated molecular size of 66
kDa that was comparable to the calculated mass of 66380.78 and
65958.25 Daltons, respectively. SeeBlue.RTM. Plus2 Prestained MW
standards are shown.
[0052] FIG. 4 is bar graph depicting the results of a flow
cytometry assay using a mouse macrophage-derived J774.2 cell line
treated with an exemplary Cholix toxin-IL-10 fusion molecules of
the present disclosure at two concentrations. % proliferation was
measured at 48 hours post treatment. Values represent
n=4.+-.standard deviation. The data shows that "dimer
Cholix.sup.415-(G.sub.4S).sub.3-IL-10" fusion molecule demonstrates
biologically active IL-10.
[0053] FIG. 5 is a line graph depicting the results of an assay
wherein the dimer Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion
molecule was tested for effects on the barrier properties of Caco-2
cell monolayers in vitro. Fluorescein-labeled 70 kDa dextran and
varying concentrations of dimer
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule was added to
the apical surface of these monolayers and the cumulative amount of
florescence detected in the basal compartment monitored over time
by collecting 150 .mu.L volumes with replacement. Cumulative Basal
Dextran levels (pmol) are plotted vs time. Each line represents the
average (n=4) of basal fluorescence values measured at 0, 15, 30,
45, 60, 90, 120, 180, and 240 min.
[0054] FIG. 6 is a line graph depicting the results of an assay
wherein the dimer Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion
molecule was tested for effects on the barrier properties of Caco-2
cell monolayers in vitro. Fluorescein-labeled 70 kDa dextran and
varying concentrations of dimer
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule was added to
the apical surface of these monolayers and the cumulative amount of
florescence detected in the basal compartment monitored over
time.
[0055] FIG. 7A and FIG. 7B are line graphs depicting the results an
ELISA assay evaluating the ability of the dimer
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule to move
across Caco-2 cell monolayers. The cumulative amount of dimer
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule reaching the
basal compartment over time following an apical addition at various
concentrations denoted in the legend. Each line represents the
average (n=4) of basal IL-10 levels measured at 0, 15, 30, 45, 60,
90, 120, 180, and 240 min. Cumulative IL-10 transported over time
graphed over a range of 6A=8000 fmol IL-10 expanded and 6B=1000
fmol IL-10.
MODE(S) FOR CARRYING OUT THE INVENTION
[0056] 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 and 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., Sambrook et al. Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates
(1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990),
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.
Definitions
[0057] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. In various embodiments, "peptides", "polypeptides", and
"proteins" are chains of amino acids whose alpha carbons are linked
through peptide bonds. 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
.alpha.-amino group on an amino acid at the amino terminal of a
peptide or to the .alpha.-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 as opposed to an amide bond.
[0058] 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) may 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 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:
[0059] 1) Alanine (A), Serine (S), and Threonine (T)
[0060] 2) Aspartic acid (D) and Glutamic acid (E)
[0061] 3) Asparagine (N) and Glutamine (Q)
[0062] 4) Arginine (R) and Lysine (K)
[0063] 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine
(V)
[0064] 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (VV)
[0065] 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 may 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/cysteine (+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).
[0066] 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 may 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.
[0067] 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 embodiments, 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.
[0068] The following hydrophilicity values have been assigned to
these amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0.+-0.1); glutamate (+3.0.+-0.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-0.1); 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.
[0069] Exemplary amino acid substitutions are set forth in Table
1.
TABLE-US-00001 TABLE 1 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
Ile Leu, Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile,
Ile Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric 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 Val Ile, Met, Leu, Phe, Leu Ala, Norleucine
[0070] A skilled artisan will be able to determine suitable
variants of polypeptides as set forth herein using well-known
techniques. In various embodiments, one skilled in the art may
identify suitable areas of the molecule that may be changed without
destroying activity by targeting regions not believed to be
important for activity. In other embodiments, the skilled artisan
can identify residues and portions of the molecules that are
conserved among similar polypeptides. In further embodiments, even
areas that may be important for biological activity or for
structure may be subject to conservative amino acid substitutions
without destroying the biological activity or without adversely
affecting the polypeptide structure.
[0071] 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 may opt for chemically
similar amino acid substitutions for such predicted important amino
acid residues.
[0072] 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 may predict the alignment of
amino acid residues of a polypeptide with respect to its
three-dimensional structure. In various embodiments, one skilled in
the art may choose to not make radical changes to amino acid
residues predicted to be on the surface of the polypeptide, since
such residues may be involved in important interactions with other
molecules. Moreover, one skilled in the art may 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 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.
[0073] 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 linker sequence).
[0074] The terms "polypeptide variant" and "polypeptide mutant" as
used herein refers 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 polypeptide sequence. In various embodiments,
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.
[0075] 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.
[0076] 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%.
[0077] 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%.
[0078] 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, and 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 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.
[0079] 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.
[0080] "Polynucleotide" refers to a polymer composed of nucleotide
units.
[0081] 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-O-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."
[0082] 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"; 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."
[0083] "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.
[0084] "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 ed., NY; and Ausubel et al.,
eds., Current Edition, Current Protocols in Molecular Biology,
Greene Publishing Associates and Wiley Interscience, NY.
[0085] Generally, highly stringent hybridization and wash
conditions are selected to be about 5.degree. 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.degree. C., with the
hybridization being carried out overnight. An example of highly
stringent wash conditions is 0.15 M NaCl at 72.degree. C. for about
15 minutes. An example of stringent wash conditions is a
0.2.times.SSC wash at 65.degree. 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.times.SSC at 45.degree. C.
for 15 minutes. An exemplary low stringency wash for a duplex of,
e.g., more than about 100 nucleotides, is 4-6.times.SSC at
40.degree. C. for 15 minutes. In general, a signal to noise ratio
of 2.times. (or higher) than that observed for an unrelated probe
in the particular hybridization assay indicates detection of a
specific hybridization.
[0086] "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 may 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.
[0087] "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.
[0088] A "vector" is a polynucleotide that can be used to introduce
another nucleic acid 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 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.
[0089] 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.
[0090] 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 an insect cell) or a hybridoma. Typically, a host cell is
a cultured cell that can be transformed or transfected with a
polypeptide-encoding nucleic acid, 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
nucleic acid to be expressed. A host cell also can be a cell that
comprises the nucleic acid but does not express it 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 may 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.
[0091] The term "isolated molecule" (where the molecule is, for
example, a polypeptide 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 may be rendered substantially free of naturally associated
components by isolation, using purification techniques well known
in the art. Molecule purity or homogeneity may be assayed by a
number of means well known in the art. For example, the purity of a
polypeptide sample may 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 may be provided by using HPLC or other
means well known in the art for purification.
[0092] 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 may 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 may 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 may be provided by using HPLC
or other means well known in the art for purification.
[0093] "Linker" refers to a molecule that joins two other
molecules, either covalently, or through ionic, van der Waals 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 linker" refers to a
linker that can be degraded or otherwise severed to separate the
two components connected by the cleavable linker. Cleavable linkers
are generally cleaved by enzymes, typically peptidases, proteases,
nucleases, lipases, and the like. Cleavable linkers may 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 fusion molecules across a polarized
epithelial membrane.
[0094] "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
[0095] "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.
[0096] 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.
Modified Cholix Toxin Polypeptides
[0097] Mature Cholix toxin (Jorgensen, R. et al., J Biol Chem
283(16):10671-10678 (2008)) as used herein is a 70.7 kD, 634
residue protein, whose sequence is set forth in SEQ ID NO: 1:
TABLE-US-00002 (SEQ ID NO: 1)
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTIND
EQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTK
TYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSID
LDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAH
WHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQ
GIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSC
AYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENN
PGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKS
CVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALT
REGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEV
AHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLEN
AEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIP
GNAYEELAIDEEAVAKEQSISTKPPYKERKDELK
[0098] In various embodiments, the Cholix toxin has an amino acid
sequence that shares an observed homology of, e.g., at least about
75%, at least about 80%, at least about 85%, at least about 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with
the sequence of SEQ ID NO: 1.
[0099] An exemplary nucleic acid encoding the mature Cholix toxin
is set forth in SEQ ID NO: 2:
TABLE-US-00003 (SEQ ID NO: 2)
ATGGTCGAAGAAGCTTTAAACATCTTTGATGAATGCCGTTCGCCATGTTC
GTTGACCCCGGAACCGGGTAAGCCGATTCAATCAAAACTGTCTATCCCTA
GTGATGTTGTTCTGGATGAAGGTGTTCTGTATTACTCGATGACGATTAAT
GATGAGCAGAATGATATTAAGGATGAGGACAAAGGCGAGTCCATTATCAC
TATTGGTGAATTTGCCACAGTACGCGCGACTAGACATTATGTTAATCAAG
ATGCGCCTTTTGGTGTCATCCATTTAGATATTACGACAGAAAATGGTACA
AAAACGTACTCTTATAACCGCAAAGAGGGTGAATTTGCAATCAATTGGTT
AGTGCCTATTGGTGAAGATTCTCCTGCAAGCATCAAAATCTCCGTTGATG
AGCTCGATCAGCAACGCAATATCATCGAGGTGCCTAAACTGTATAGTATT
GATCTCGATAACCAAACGTTAGAGCAGTGGAAAACCCAAGGTAATGTTTC
TTTTTCGGTAACGCGTCCTGAACATAATATCGCTATCTCTTGGCCAAGCG
TGAGTTACAAAGCAGCGCAGAAAGAGGGTTCACGCCATAAGCGTTGGGCT
CATTGGCATACAGGCTTAGCACTGTGTTGGCTTGTGCCAATGGATGCTAT
CTATAACTATATCACCCAGCAAAATTGTACTTTAGGGGATAATTGGTTTG
GTGGCTCTTATGAGACTGTTGCAGGCACTCCGAAGGTGATTACGGTTAAG
CAAGGGATTGAACAAAAGCCAGTTGAGCAGCGCATCCATTTCTCCAAGGG
GAATGCGATGAGCGCACTTGCTGCTCATCGCGTCTGTGGTGTGCCATTAG
AAACTTTGGCGCGCAGTCGCAAACCTCGTGATCTGACGGATGATTTATCA
TGTGCCTATCAAGCGCAGAATATCGTGAGTTTATTTGTCGCGACGCGTAT
CCTGTTCTCTCATCTGGATAGCGTATTTACTCTGAATCTTGACGAACAAG
AACCAGAGGTGGCTGAACGTCTAAGTGATCTTCGCCGTATCAATGAAAAT
AACCCGGGCATGGTTACACAGGTTTTAACCGTTGCTCGTCAGATCTATAA
CGATTATGTCACTCACCATCCGGGCTTAACTCCTGAGCAAACCAGTGCGG
GTGCACAAGCTGCCGATATCCTCTCTTTATTTTGCCCAGATGCTGATAAG
TCTTGTGTGGCTTCAAACAACGATCAAGCCAATATCAACATCGAGTCTCG
TTCTGGCCGTTCATATTTGCCTGAAAACCGTGCGGTAATCACCCCTCAAG
GCGTCACAAATTGGACTTACCAGGAACTCGAAGCAACACATCAAGCTCTG
ACTCGTGAGGGTTATGTGTTCGTGGGTTACCATGGTACGAATCATGTCGC
TGCGCAAACCATCGTGAATCGCATTGCCCCTGTTCCGCGCGGCAACAACA
CTGAAAACGAGGAAAAGTGGGGCGGGTTATATGTTGCAACTCACGCTGAA
GTTGCCCATGGTTATGCTCGCATCAAAGAAGGGACAGGGGAGTATGGCCT
TCCGACCCGTGCTGAGCGCGACGCTCGTGGGGTAATGCTGCGCGTGTATA
TCCCTCGTGCTTCATTAGAACGTTTTTATCGCACGAATACACCTTTGGAA
AATGCTGAGGAGCATATCACGCAAGTGATTGGTCATTCTTTGCCATTACG
CAATGAAGCATTTACTGGTCCAGAAAGTGCGGGCGGGGAAGACGAAACTG
TCATTGGCTGGGATATGGCGATTCATGCAGTTGCGATCCCTTCGACTATC
CCAGGGAACGCTTACGAAGAATTGGCGATTGATGAGGAGGCTGTTGCAAA
AGAGCAATCGATTAGCACAAAACCACCTTATAAAGAGCGCAAAGATGAAC TTAAG
[0100] In various embodiments, the Cholix toxin contains an nucleic
acid sequence that shares an observed homology of, e.g., at least
about 75%, at least about 80%, at least about 85%, at least about
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%
with the sequence of SEQ ID NO: 2.
[0101] In various embodiments, the modified Cholix toxin used in
the preparation of the fusion molecules is a truncated Cholix
toxin, wherein the fusion molecule has the ability to activate the
receptor for the biologically active cargo. A truncated Cholix
toxin as described herein will be identified by reference to the
amino acid residues comprising the truncated Cholix toxin, e.g., a
truncated Cholix toxin consisting of amino acid residues 1-386 of
SEQ ID NO: 1 will be identified as Cholix.sup.386.
[0102] In various embodiments, the modified Cholix toxin used in
the preparation of the fusion molecule is mutated Cholix toxin. As
described herein, a mutated Cholix toxin wherein the mutation
involves an amino acid residue deletion will be identified by
reference to the amino acid residue being deleted, e.g., a mutated
Cholix toxin wherein amino acid E581 of SEQ ID NO: 1 has been
deleted, the will be identified as "Cholix .DELTA.E581". A mutated
Cholix toxin wherein the mutation involves 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 "S30A" indicates that the "S" (serine, in standard
single letter code) residue at position 30 in SEQ ID NO: 1 has been
substituted with an "A" (alanine, in standard single letter code)
even if the residue appears in a truncated Cholix toxin, and the
modified toxin will be identified as "Cholix.sup.S30A".
[0103] Cholix toxin Domain Ia (amino acids 1-265 of SEQ ID NO: 1)
is a "receptor binding domain" that functions as a ligand for a
cell surface receptor and mediates binding of the fusion molecule
to a cell, e.g., Domain Ia will bind to a cell surface receptor
that is present on the apical membrane of an epithelial cell, with
sufficient affinity to allow endocytosis of the fusion molecule.
Domain 1a 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. For example, the receptor binding domain can
bind to .alpha.2-MR. Conservative or nonconservative substitutions
can be made to the amino acid sequence of domain Ia, as long as the
ability to mediate binding of the fusion molecule to a cell is not
substantially eliminated. In various embodiments, the fusion
molecules comprise a Cholix toxin comprising a mutated domain
Ia.
[0104] In various embodiments, domain Ia comprises an antigen
presenting cell (APC) receptor binding domain. In various
embodiments, the APC receptor binding domain is the cell
recognition domain of Cholix domain Ia or a portion of Cholix
domain Ia sufficient to engage with a cell surface receptor on
APCs.
[0105] In various embodiments, the APC receptor binding domain
binds to a receptor identified as present on a dendritic cell or
other APC. Examples of cell surface receptors on APCs can include,
but are not limited to, DEC-205 (CD205), CD207, CD209, CD11a,
CD11b, CD11c, CD36, CD14, CD50, CD54, CD58, CD68, CD80, CD83, CD86,
CD102, CD3, CD14, CD19, Clec9a, CMFR-44, dectin-1, dectin-2, FLT3,
HLA-DR, LOX-1, MHC II, BDCA-1, DC-SIGN, Toll-like receptors
(TLR)-2, -3, -4, and -7, and .alpha.2-macroglobulin receptor
(".alpha.2-MR"). In various embodiments, the APC receptor binding
domain is .alpha.2-MR.
[0106] Cholix toxin Domain II (amino acids 266-386 of SEQ ID NO: 1)
is a "transcytosis domain" that mediates transcytosis from a lumen
bordering the apical surface of a mucous membrane to the
basolateral side of a mucous membrane. As referred to herein,
"transcytosis" refers to the trafficking of the fusion molecule
through a polarized epithelial cell. Such trafficking permits the
release of the biologically active cargo from the basolateral
membrane of the polarized epithelial cell. The fusion molecules of
the present disclosure may comprise a modified Cholix toxin
comprising the entire amino acid sequence of Domain II, or may
comprise portions of Domain II, so long as transcytosis acitivity
is not substantially eliminated. Further, conservative or
nonconservative substitutions can be made to the amino acid
sequence of the transcytosis domain, as long as transcytosis
activity is not substantially eliminated. 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
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 modified Cholix toxin comprising the entire
amino acid sequence of Domain II.
[0107] In various embodiments, the non-naturally occurring fusion
molecules comprise a modified Cholix toxin truncated at an amino
acid residue within Cholix toxin domain II, wherein the fusion
molecule has the ability to activate the receptor for the
biologically active cargo. In one embodiment, the truncated Cholix
toxin is Cholix.sup.386 (SEQ ID NO: 3). In one embodiment, the
truncated Cholix toxin is Cholix.sup.385 (SEQ ID NO: 4). In one
embodiment, the truncated Cholix toxin is Cholix.sup.384 (SEQ ID
NO: 5). In one embodiment, the truncated Cholix toxin is
Cholix.sup.383 (SEQ ID NO: 6). In one embodiment, the truncated
Cholix toxin is Cholix.sup.382 (SEQ ID NO: 7). In one embodiment,
the truncated Cholix toxin is Cholix.sup.381 (SEQ ID NO: 8). In one
embodiment, the truncated Cholix toxin is Cholix.sup.380 (SEQ ID
NO: 9). In one embodiment, the truncated Cholix toxin is
Cholix.sup.379 (SEQ ID NO: 10). In one embodiment, the truncated
Cholix toxin is Cholix.sup.378 (SEQ ID NO: 11). In one embodiment,
the truncated Cholix toxin is Cholix.sup.377 (SEQ ID NO: 12). In
one embodiment, the truncated Cholix toxin is Cholix.sup.376 (SEQ
ID NO: 13). In one embodiment, the truncated Cholix toxin is
Cholix.sup.375 (SEQ ID NO: 14). In one embodiment, the truncated
Cholix toxin is Cholix.sup.374 (SEQ ID NO: 15). In one embodiment,
the truncated Cholix toxin is Cholix.sup.373 (SEQ ID NO: 16). In
one embodiment, the truncated Cholix toxin is Cholix.sup.372 (SEQ
ID NO: 17). In one embodiment, the truncated Cholix toxin is
Cholix.sup.371 (SEQ ID NO: 18). In one embodiment, the truncated
Cholix toxin is Cholix.sup.370 (SEQ ID NO: 19). In one embodiment,
the truncated Cholix toxin is Cholix.sup.369 (SEQ ID NO: 20). In
one embodiment, the truncated Cholix toxin is Cholix.sup.368 (SEQ
ID NO: 21). In one embodiment, the truncated Cholix toxin is
Cholix.sup.367 (SEQ ID NO: 22). In one embodiment, the truncated
Cholix toxin is Cholix.sup.366 (SEQ ID NO: 23). In one embodiment,
the truncated Cholix toxin is Cholix.sup.365 (SEQ ID NO: 24). In
one embodiment, the truncated Cholix toxin is Cholix.sup.364 (SEQ
ID NO: 25). In one embodiment, the truncated Cholix toxin is
Cholix.sup.363 (SEQ ID NO: 26). In one embodiment, the truncated
Cholix toxin is Cholix.sup.362 (SEQ ID NO: 27). In one embodiment,
the truncated Cholix toxin is Cholix.sup.361 (SEQ ID NO: 28). In
one embodiment, the truncated Cholix toxin is Cholix.sup.360 (SEQ
ID NO: 29). In one embodiment, the truncated Cholix toxin is
Cholix.sup.359 (SEQ ID NO: 30). In one embodiment, the truncated
Cholix toxin is Cholix.sup.358 (SEQ ID NO: 31). In one embodiment,
the truncated Cholix toxin is Cholix.sup.357 (SEQ ID NO: 32). In
one embodiment, the truncated Cholix toxin is Cholix.sup.356 (SEQ
ID NO: 33). In one embodiment, the truncated Cholix toxin is
Cholix.sup.355 (SEQ ID NO: 34). In one embodiment, the truncated
Cholix toxin is Cholix.sup.354 (SEQ ID NO: 35). In one embodiment,
the truncated Cholix toxin is Cholix.sup.353 (SEQ ID NO: 36). In
one embodiment, the truncated Cholix toxin is Cholix.sup.352 (SEQ
ID NO: 37). In one embodiment, the truncated Cholix toxin is
Cholix.sup.351 (SEQ ID NO: 38). In one embodiment, the truncated
Cholix toxin is Cholix.sup.350 (SEQ ID NO: 39). In one embodiment,
the truncated Cholix toxin is Cholix.sup.349 (SEQ ID NO: 40). In
one embodiment, the truncated Cholix toxin is Cholix.sup.348 (SEQ
ID NO: 41).
[0108] Cholix toxin Domain Ib (amino acids 387-425 of SEQ ID NO: 1)
is not essential for any known activity of Cholix, including cell
binding, translocation, ER retention or ADP ribosylation activity.
In various embodiments, the non-naturally occurring fusion
molecules comprise a modified Cholix toxin truncated at an amino
acid residue within Cholix toxin domain Ib, wherein the fusion
molecule has the ability to activate the receptor for the
biologically active cargo. In one embodiment, the truncated Cholix
toxin is Cholix.sup.425 (SEQ ID NO: 42). In one embodiment, the
truncated Cholix toxin is Cholix.sup.424 (SEQ ID NO: 43). In one
embodiment, the truncated Cholix toxin is Cholix.sup.423 (SEQ ID
NO: 44). In one embodiment, the truncated Cholix toxin is
Cholix.sup.422 (SEQ ID NO: 45). In one embodiment, the truncated
Cholix toxin is Cholix.sup.421 (SEQ ID NO: 46). In one embodiment,
the truncated Cholix toxin is Cholix.sup.420 (SEQ ID NO: 47). In
one embodiment, the truncated Cholix toxin is Cholix.sup.419 (SEQ
ID NO: 48). In one embodiment, the truncated Cholix toxin is
Cholix.sup.418 (SEQ ID NO: 49). In one embodiment, the truncated
Cholix toxin is Cholix.sup.417 (SEQ ID NO: 50). In one embodiment,
the truncated Cholix toxin is Cholix.sup.416 (SEQ ID NO: 51). In
one embodiment, the truncated Cholix toxin is Cholix.sup.415 (SEQ
ID NO: 52). In one embodiment, the truncated Cholix toxin is
Cholix.sup.414 (SEQ ID NO: 53). In one embodiment, the truncated
Cholix toxin is Cholix.sup.413 (SEQ ID NO: 54). In one embodiment,
the truncated Cholix toxin is Cholix.sup.412 (SEQ ID NO: 55). In
one embodiment, the truncated Cholix toxin is Cholix.sup.411 (SEQ
ID NO: 56). In one embodiment, the truncated Cholix toxin is
Cholix.sup.410 (SEQ ID NO: 57). In one embodiment, the truncated
Cholix toxin is Cholix.sup.409 (SEQ ID NO: 58). In one embodiment,
the truncated Cholix toxin is Cholix.sup.408 (SEQ ID NO: 59). In
one embodiment, the truncated Cholix toxin is Cholix.sup.407 (SEQ
ID NO: 60). In one embodiment, the truncated Cholix toxin is
Cholix.sup.406 (SEQ ID NO: 61). In one embodiment, the truncated
Cholix toxin is Cholix.sup.405 (SEQ ID NO: 62). In one embodiment,
the truncated Cholix toxin is Cholix.sup.404 (SEQ ID NO: 63). In
one embodiment, the truncated Cholix toxin is Cholix.sup.403 (SEQ
ID NO: 64). In one embodiment, the truncated Cholix toxin is
Cholix.sup.402 (SEQ ID NO: 65). In one embodiment, the truncated
Cholix toxin is Cholix.sup.401 (SEQ ID NO: 66). In one embodiment,
the truncated Cholix toxin is Cholix.sup.400 (SEQ ID NO: 67). In
one embodiment, the truncated Cholix toxin is Cholix.sup.399 (SEQ
ID NO: 68). In one embodiment, the truncated Cholix toxin is
Cholix.sup.398 (SEQ ID NO: 69). In one embodiment, the truncated
Cholix toxin is Cholix.sup.397 (SEQ ID NO: 70). In one embodiment,
the truncated Cholix toxin is Cholix.sup.396 (SEQ ID NO: 71). In
one embodiment, the truncated Cholix toxin is Cholix.sup.395 (SEQ
ID NO: 72). In one embodiment, the truncated Cholix toxin is
Cholix.sup.394 (SEQ ID NO: 73). In one embodiment, the truncated
Cholix toxin is Cholix.sup.393 (SEQ ID NO: 74). In one embodiment,
the truncated Cholix toxin is Cholix.sup.392 (SEQ ID NO: 75). In
one embodiment, the truncated Cholix toxin is Cholix.sup.391 (SEQ
ID NO: 76). In one embodiment, the truncated Cholix toxin is
Cholix.sup.390 (SEQ ID NO: 77). In one embodiment, the truncated
Cholix toxin is Cholix.sup.389 (SEQ ID NO: 78). In one embodiment,
the truncated Cholix toxin is Cholix.sup.388 (SEQ ID NO: 79). In
one embodiment, the truncated Cholix toxin is Cholix.sup.387 (SEQ
ID NO: 80).
[0109] Cholix toxin Domain III (amino acids 426-634 of SEQ ID NO:
1) is 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 Cholix that "lacks endogenous ADP ribosylation
activity" or a "detoxified Cholix" refers to any Cholix described
herein (including modified variants) that does not comprise Cholix
domain III or which has been modified within domain III in a manner
which detoxifies the molecule. For example, deletion of the
glutamic acid (Glu) residue at amino acid position 581 of SEQ ID
NO: 1 detoxifies the molecule. This detoxified Cholix is referred
to as "Cholix .DELTA.E581". In various embodiments, the portion of
Cholix 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, Cholix
domain III is itself highly immunogenic and can be used in fusion
molecules where a robust humoral immune response is desired.
[0110] As used herein, "a detoxified Cholix sequence" may be a full
length sequence or portion(s) of the full length sequence.
Generally, a detoxified Cholix sequence has one or more domains or
portions of domains with certain biological activities of a
detoxified Cholix, such as a cell recognition domain, a
translocation domain, or an endoplasmic reticulum retention domain.
For example, a detoxified Cholix sequence may include only domain
II and detoxified domain III. In another example, a detoxified
Cholix sequence may include only domain Ia, domain II, and
detoxified domain III. In another example, a detoxified Cholix
sequence may include all of domains Ia, Ib, II, and detoxified III.
Therefore, a detoxified Cholix sequence may be a contiguous
sequence of the native Cholix, or it can be a sequence comprised of
non-contiguous subsequences of the native Cholix that lacks ADP
ribosylation activity. In one embodiment of the present disclosure,
the non-naturally occurring fusion molecule comprises a mutated
modified Cholix toxin, designated herein as Cholix toxin
.DELTA.E581, having the amino acid sequence set forth in SEQ ID NO:
81.
Biologically Active Cargo
[0111] In addition to the modified Cholix toxin polypeptide, the
fusion molecules of the present disclosure further comprise a
biologically active cargo for delivery to a subject. 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.
[0112] In various embodiments, the biologically active cargo is 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. In certain diagnostic embodiments, the
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.
[0113] In various embodiments, the biologically active cargo of the
fusion molecule 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 embodiments, 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. In such embodiments, the biologically
active cargo may or may not be present in the blood, lymph, or
other biological fluid at detectable concentrations, yet may still
accumulate at sufficient concentrations at its site of action to
exert a biological effect.
[0114] In various embodiments, the biologically active cargo is 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. In other embodiments, 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.
[0115] In various embodiments, the biologically active cargo to be
delivered is 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-.beta., tumor
necrosis factor, lymphotoxin, Fas, granulocyte colony stimulating
factor, granulocyte macrophage colony stimulating factor,
interferon-.alpha., interferon-.beta., interferon-.gamma., growth
factors and protein hormones such as erythropoietin, angiogenin,
hepatocyte growth factor, fibroblast growth factor, keratinocyte
growth factor, nerve growth factor, tumor growth factor-.alpha.,
thrombopoietin, thyroid stimulating factor, thyroid releasing
hormone, neurotrophin, epidermal growth factor, VEGF, ciliary
neurotrophic factor, LDL, somatomedin, insulin growth factor,
insulin-like growth factor I and II, chemokines such as ENA-78,
ELC, GRO-.alpha., GRO-.beta., GRO-.gamma., HRG, LEF, IP-10, MCP-1,
MCP-2, MCP-3, MCP-4, MIP-1-.alpha., MIP-1-.beta., 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; .alpha.-chemokine
receptors, e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7;
and .beta.-chemokine receptors, e.g., CCR1, CCR2, CCR3, CCR4, CCR5,
CCR6, CCR7.
[0116] 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., levamisole, diethyl dithiocarbamate, enkephalins, endorphins;
antimicrobial compounds such as antibiotics, e.g., .beta.-lactam,
penicillin, cephalosporins, carbapenims and monobactams,
.beta.-lactamase inhibitors, aminoglycosides, macrolides,
tetracyclins, spectinomycin; antimalarials, amebicides;
antiprotazoals; antifungals, e.g., amphotericin .beta., 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 HCl; antihypertensive direct
vasodilators, e.g., diazoxide, hydralazine HCl; 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; sypathomimetic drugs, e.g., albuterol,
bitolterol mesylate, dobutamine HCl, dopamine HCl, ephedrine So,
epinephrine, fenfluramine HCl, isoproterenol HCl, methoxamine HCl,
norepinephrine bitartrate, phenylephrine HCl, ritodrine HCl;
cholinomimetic drugs, e.g., acetylcholine CI; anticholinesterases,
e.g., edrophonium CI; cholinesterase reactivators; adrenergic
blocking drugs, e.g., acebutolol HCl, atenolol, esmolol HCl,
labetalol HCl, metoprolol, nadolol, phentolamine mesylate,
propanolol HCl; antimuscarinic drugs, e.g., anisotropine
methylbromide, atropine SO.sub.4, clinidium Br, glycopyrrolate,
ipratropium Br, scopolamine HBr; neuromuscular blocking drugs;
depolarizing drugs, e.g., atracurium besylate, hexafluorenium Br,
metocurine iodide, succinylcholine CI, tubocurarine CI, 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.sup.+ channel toxins; antiparkinson drugs, e.g.,
amaltidine HCl, benztropine mesylate, carbidopa; diuretic drugs,
e.g., dichlorphenamide, methazolamide, bendroflumethiazide,
polythiazide; antimigraine drugs, e.g, carboprost tromethamine
mesylate, methysergide maleate.
[0117] 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.
[0118] 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 HCl, methohexital Na,
thiamylal Na, thiopental Na; antiepileptics, e.g., carbamazepine,
clonazepam, divalproex Na, ethosuximide, mephenyloin,
paramethadione, phenyloin, primidone. In various embodiments, the
biologically active cargo is an enzyme selected from hyaluronidase,
streptokinase, tissue plasminogen activator, urokinase,
PGE-adenosine deaminase.
[0119] 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.
Modulators of Inflammation (Interleukin-10 and Related
Cytokines)
[0120] Interleukin-10 (IL-10) is an important immunoregulatory
cytokine produced by many cell populations and whose main
biological function seems to be the limitation and termination of
inflammatory responses and the regulation of differentiation and
proliferation of several immune cells such as T cells, B cells,
natural killer cells, antigen-presenting cells, mast cells, and
granulocytes. More recent data suggests that IL-10 also mediates
immunostimulatory properties that help to eliminate infectious and
noninfectious particles with limited inflammation; Asadullah et
al., Pharmacol Rev, 55:241-269, 2003. Moreover, numerous
investigations suggest a major impact of IL-10 in inflammatory,
malignant, and autoimmune diseases, and IL-10 overexpression was
found in certain tumors such as melanoma, basal cell and squamous
cell carcinoma and several lymphomas; Id. Five new human molecules
structurally related to IL-10 have been discovered, IL-19
(Gallagher et al., Genes Immun., 1:442-450, 2000); IL-20 (Blumberg
et al., Cell, 104:9-19, 2001), IL-22 (Dumoutier et al., Genes
Immun., 1:488-494, 2000), IL-24 (Jiang et al., Oncogene,
11:2477-2486, 1995) and IL-26 (Knappe et al., J. Virol.,
74:3881-3887, 2000) and data suggests that immune cells are a major
source of the new IL-10 family members; Wolk et al., J. Immunol.,
168:5397-5402, 2002.
[0121] While there were some promising results from IL-10 delivery
on the course of several inflammatory diseases in experimental
models, several clinical studies evaluating IL-10 as a therapeutic
agent for the treatment of inflammatory and/or immune disorders
remain somewhat disappointing, with much of the data conflicting;
Asadullah et al., Pharmacol Rev, 55:241-269, 2003. Overall, the
data suggests that IL-10 is safe and generally well tolerated,
however, the ultimate local IL-10 concentration in the intestine
after systemic administration with standard doses is too low,
resulting in only marginal efficacy. Id. Unfortunately, the ability
to sufficiently increase the doses is limited due to side effects
(e.g., anemia, headache), and there are concerns higher doses of
systemically administered IL-10 may be detrimental rather than
helpful in certain indications, e.g., Crohn's; Herfarth et al, Gut,
50(2): 146-147, 2002.
[0122] In various embodiments, the biologically active cargo is a
polypeptide that has been determined to be a modulator of
inflammation in the GI tract selected from, e.g., interleukin-10,
interleukin-19, interleukin-20, interleukin-22, interleukin-24, or
interleukin-26.
[0123] Interleukin-10 (IL-10) was first identified as a product of
the type 2 helper T cell and later shown to be produced by other
cell types including B cells and macrophages (Moore et al., Annu
Rev Immunol, 19:683-765, 2001). It also inhibits the synthesis of
several cytokines produced from type 1 helper T cells, such as
.gamma.-interferon, IL-2, and tumor necrosis factor-.alpha.
(TNF-.alpha.) (Fiorentino et al., J Immunol, 146:3444-3451, 1991).
The ability of IL-10 to inhibit cell-mediated immune response
modulators and suppress antigen-presenting cell-dependent T cell
responses demonstrates IL-10 has immunosuppressive properties. This
cytokine also inhibits monocyte/macrophage production of other
cytokines such as IL-1, IL-6, IL-8, granulocyte-macrophage
colony-stimulating factor (GM-CSF), granulocyte colony-stimulating
factor (G-CSF), and TNF-.alpha..
[0124] The IL-10 protein forms a functional dimer that becomes
biologically inactive upon disruption of the non-covalent
interactions connecting its two monomer subunits. The N-terminus
does not appear to be directly involved with IL-10 receptor
activation. Thus, in one aspect of the disclosure, a fusion
molecule is constructed via conjugation through the N-terminus of
the IL-10 protein to the C-terminus of a modified Cholix toxin
using a cleavable linker. Such a construction may result in a
solution dimer as a result of IL-10 interactions.
[0125] In various embodiments, the biologically active cargo is
human interleukin-10 having the amino acid sequence set forth in
SEQ ID NO: 82:
TABLE-US-00004 (SEQ ID NO: 82)
MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSR
VKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAEN
QDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQ
EKGIYKAMSEFDIFINYIEAYMTMKIRN
or a fragment or variant thereof.
[0126] In various embodiments, the biologically active cargo
contains an amino acid sequence that shares an observed homology
of, e.g., at least about 75%, at least about 80%, at least about
85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
at least about 99% with the sequence of SEQ ID NO: 82.
[0127] IL-19 a cytokine that belongs to the IL-10 cytokine
subfamily. This cytokine is found to be preferentially expressed in
monocytes. It can bind the IL-20 receptor complex and lead to the
activation of the signal transducer and activator of transcription
3 (STAT3) (Yamamoto-Furusho J K, et al. Hum Immunol,
72(11):1029-32, 2011). In various embodiments, the biologically
active cargo is human interleukin-19 having the amino acid sequence
set forth in SEQ ID NO: 83:
TABLE-US-00005 (SEQ ID NO: 83)
MKLQCVSLWLLGTILILCSVDNHGLRRCLISTDMHHIEESFQEIKRAIQA
KDTFPNVTILSTLETLQIIKPLDVCCVTKNLLAFYVDRVFKDHQEPNPKI
LRKISSIANSFLYMQKTLRQCQEQRQCHCRQEATNATRVIHDNYDQLEVH
AAAIKSLGELDVFLAWINKNHEVMSSA
or a fragment or variant thereof.
[0128] In various embodiments, the biologically active cargo
contains an amino acid sequence that shares an observed homology
of, e.g., at least about 75%, at least about 80%, at least about
85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
at least about 99% with the sequence of SEQ ID NO: 83.
[0129] IL-20 is a cytokine structurally related to interleukin 10
(IL-10). This cytokine has been shown to transduce its signal
through signal transducer and activator of transcription 3 (STAT3)
in keratinocytes. A specific receptor for this cytokine is found to
be expressed in skin and upregulated dramatically in psoriatic
skin, suggesting a role for this protein in epidermal function and
psoriasis (Yamamoto-Furusho J K, et al. Immunol Lett, 149(1-2):50-3
2013). In various embodiments, the biologically active cargo is
human interleukin-20 having the amino acid sequence set forth in
SEQ ID NO: 84:
TABLE-US-00006 (SEQ ID NO: 84)
MKASSLAFSLLSAAFYLLWTPSTGLKTLNLGSCVIATNLQEIRNGFSEIR
GSVQAKDGNIDIRILRRTESLQDTKPANRCCLLRHLLRLYLDRVFKNYQT
PDHYTLRKISSLANSFLTIKKDLRLCHAHMTCHCGEEAMKKYSQILSHFE
KLEPQAAVVKALGELDILLQVVMEETE
or a fragment or variant thereof.
[0130] In various embodiments, the biologically active cargo
contains an amino acid sequence that shares an observed homology
of, e.g., at least about 75%, at least about 80%, at least about
85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
at least about 99% with the sequence of SEQ ID NO: 84.
[0131] IL-22 is a cytokine structurally related to interleukin 10
(IL-10). IL-22 secreting CD4(+) T (Th22) cells and IL-22 are
involved in the pathogenesis of autoimmune disease, and may play an
important role in the pathogenesis of NMO and MS (Xu et al., J
Neuroimmunol., August 15; 261(1-2):87-91, 2013). In various
embodiments, the biologically active cargo is human interleukin-22
having the amino acid sequence set forth in SEQ ID NO: 85:
TABLE-US-00007 (SEQ ID NO: 85)
MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQP
YITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTL
EEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKD
TVKKLGESGEIKAIGELDLLFMSLRNACI
or a fragment or variant thereof.
[0132] In various embodiments, the biologically active cargo
contains an amino acid sequence that shares an observed homology
of, e.g., at least about 75%, at least about 80%, at least about
85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
at least about 99% with the sequence of SEQ ID NO: 85.
[0133] IL-24 is a cytokine structurally related to interleukin 10
(IL-10) which can induce apoptosis selectively in various cancer
cells. Overexpression of this gene leads to elevated expression of
several GADD family genes, which correlates with the induction of
apoptosis. The phosphorylation of mitogen-activated protein kinase
14 (MAPK7/P38), and heat shock 27 kDa protein 1 (HSPB2/HSP27) are
found to be induced by this gene in melanoma cells, but not in
normal immortal melanocytes (Lin B W, et al., J Korean Med Sci,
28(6):833-9, 2013). In various embodiments, the biologically active
cargo is human interleukin-24 having the amino acid sequence set
forth in SEQ ID NO: 86:
TABLE-US-00008 (SEQ ID NO: 86)
MNFQQRLQSLVVTLASRPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSG
AQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQ
NVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLKSFSTLANNFVLIV
SQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLT WMQKFYKL
or a fragment or variant thereof.
[0134] In various embodiments, the biologically active cargo
contains an amino acid sequence that shares an observed homology
of, e.g., at least about 75%, at least about 80%, at least about
85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
at least about 99% with the sequence of SEQ ID NO: 86.
[0135] IL-26 was identified by its overexpression specifically in
herpesvirus saimiri-transformed T cells. The encoded protein is a
member of the IL-10 family of cytokines. It is a secreted protein
and may function as a homodimer. This protein is thought to
contribute to the transformed phenotype of T cells after infection
by herpesvirus saimiri (Corvaisier M, et al. PLoS Biol,
10(9):e1001395, 2012). In various embodiments, the biologically
active cargo is human interleukin-26 having the amino acid sequence
set forth in SEQ ID NO: 87:
TABLE-US-00009 (SEQ ID NO: 87)
MLVNFILRCGLLLVTLSLAIAKHKQSSFTKSCYPRGTLSQAVDALYIKAA
WLKATIPEDRIKNIRLLKKKTKKQFMKNCQFQEQLLSFFMEDVFGQLQLQ
GCKKIRFVEDFHSLRQKLSHCISCASSAREMKSITRMKRIFYRIGNKGIY
KAISELDILLSWIKKLLESSQ
or a fragment or variant thereof.
[0136] In various embodiments, the biologically active cargo
contains an amino acid sequence that shares an observed homology
of, e.g., at least about 75%, at least about 80%, at least about
85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
at least about 99% with the sequence of SEQ ID NO: 87.
[0137] Importantly, the non-naturally occurring fusion molecules
which lack a cleavable linker can be advantageous in that the
anchoring effect of the modified Cholix toxin by its receptor(s) at
the surface of, e.g., immune cells that also express the receptor
for the IL-10 (but in considerably lower quantity) can allow for
greater exposure of the IL-10 at the surface of the targeted cells,
and provide a synergistic effect via the binding of the Cholix to
its receptor and the binding of IL-10 to the IL-10R.
Tumor Necrosis Factor Super Family
[0138] Tumor necrosis factor is a rapidly growing superfamily of
cytokines (hereinafter "TNFSF") that interact with a corresponding
superfamily of receptors (hereinafter "TNFSFR"). Since the
discovery of tumor necrosis factor-alpha ("TNF-.alpha.") about 25
years ago, the TNFSF has grown to a large family of related
proteins consisting of over 20 members that signal through over 30
receptors (see, e.g., "Therapeutic Targets of the TNF Superfamily",
edited by Iqbal S. Grewal, Landes Bioscience/Springer
Science+Business Media, LLC dual imprint/Springer series: Advances
in Experimental Medicine and Biology, 2009). Members of TNFSF have
wide tissue distribution and TNFSF ligand-receptor interactions are
involved in numerous biological processes, ranging from
hematopoiesis to pleiotropic cellular responses, including
activation, proliferation, differentiation, and apoptosis. TNFSF
ligand-receptor interactions have also been implicated in
tumorigenesis, transplant rejection, septic shock, viral
replication, bone resorption and autoimmunity. The particular
response depends upon the receptor that is signaling, the cell
type, and the concurrent signals received by the cell.
[0139] Because a number of TNFSF members are expressed on tumor
cells, antibody based therapies are being developed to target these
molecules and some are currently undergoing clinical trials (e.g.,
TNF-.alpha. for human use in the treatment of sarcomas and
melanomas (Eggermont et al., Lancet Oncol, 4:429-437, 2003; Lans et
al., Clin Cancer Res, 7:784-790, 2001). In addition, many of these
molecules are also being exploited as targets for antibody-drug
conjugates (e.g., CD30 and CD70), or exploited for
radioimmunotherapy (e.g., the BLyS receptors TACI and BR3)
(Buchsbaum et al., J Nucl Med, 44:434-436, 2003).
[0140] Similarly, because a number of TNFSF members have been
implicated in both innate and adaptive immune responses such as
defense against pathogens, inflammatory response and autoimmunity,
approaches to target many of TNFSF receptors and ligands for
treatment of autoimmunity and other inflammatory diseases are being
exploited. Indeed, a number of biologic TNF blocking therapies
(hereinafter "TNF inhibitors") including humanized/human monoclonal
antibodies (e.g., infliximab (REMICADE.RTM.) or adalimumab
(HUMIRA.RTM.)) or recombinant fusion proteins of IgG and soluble
TNFSF receptors (e.g., etanercept (ENBREL.RTM.)) have been
developed and are now being used in humans to inhibit the
inflammation associated with Crohn's disease and rheumatoid
arthritis (Mitoma et al., Arthritis Rheum, 58:1248-1257, 2008;
Shealy et al., Handb Exp Pharmacol, 181:101-129, 2008). Thus, the
potential to deliver such agents locally including, but not limited
to, intestinal and pulmonary mucosa, would provide added benefits
for efficacy and safety.
[0141] Although these various TNF inhibitors have been approved for
human therapies and are being successfully used in human patients,
there remains a number of toxicities associated with these TNF
inhibitors, e.g., hepatotoxicity, thromboembolic complications, and
increased risk of development of tuberculosis and lymphoma (Gardam
et al., Lancet Infect Dis, 3:148-155, 2003). Moreover, while
effective in halting progression of disease, these agents are very
expensive, generally administered intravenously or subcutaneously,
and do not cure the diseases. The continued examination of signal
transduction of TNFSF members is needed to develop approaches for
tissue specific interventions, which could allow targeted therapies
to have fewer side effects.
[0142] In various embodiments, the biologically active cargo is a
TNF inhibitor that is an isolated antibody or an antibody fragment.
Isolated antibodies and antibody fragments useful in the constructs
and methods of the present invention include, without limitation,
monoclonal Abs (mAbs), polyclonal Abs, Ab fragments (e.g., Fab,
Fab', F(ab')2, Fv, Fc, etc.), chimeric Abs, mini-Abs or domain Abs
(dAbs), dual specific Abs, bispecific Abs, heteroconjugate Abs,
single chain Abs (SCA), single chain variable region fragments
(ScFv), fusion proteins comprising an Ab portion or multiple Ab
portions, humanized Abs, fully human Abs, and any other modified
configuration of the immunoglobulin (Ig) molecule that comprises an
antigen recognition site of the required specificity.
[0143] Anti-TNF-.alpha. Antibodies. The FDA approved
anti-TNF-.alpha. antibody, Adalimumab (Abbvie HUMIRA.RTM.; DrugBank
DB 00051) has been used to treat humans. In various embodiments of
the present invention, the biologically active cargo is a human
antibody or antigen-binding fragment comprising the heavy chain
variable region sequence set forth in SEQ ID NO: 88:
TABLE-US-00010 (SEQ ID NO: 88)
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGK
GLEWVSAITWNSGHIDYADSVERGFTISRDNAKNSLYLQMNSLRAE
DTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
and the light chain variable region sequence set forth in SEQ ID
NO:89:
TABLE-US-00011 (SEQ ID NO: 89)
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
or an antigen-binding or an immunologically functional
immunoglobulin fragment thereof.
[0144] In various embodiments, the invention provides antibodies,
comprising a heavy chain and a light chain, wherein the heavy chain
comprises a heavy chain variable region, and wherein the heavy
chain variable region comprises a sequence that has at least about
75%, at least about 80%, at least about 85%, at least about 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%
identity to the amino acid sequence as set forth in SEQ ID NO:88;
and wherein the light chain comprises a light chain variable
region, and wherein the light chain variable region comprises a
sequence that has at least about 80%, at least about 85%, at least
about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least
about 99% identity to the amino acid sequence as set forth in any
of SEQ ID NO:89; wherein the antibody binds specifically to human
TNF-.alpha..
[0145] The FDA approved anti-TNF-.alpha. antibody, Infliximab
(Centocor REMICADE.RTM.; DrugBank DB 00065) has been used to treat
humans. In various embodiments of the present invention, the
biologically active cargo is a human antibody or antigen-binding
fragment comprising the heavy chain variable region sequence set
forth in SEQ ID NO: 90:
TABLE-US-00012 (SEQ ID NO: 90)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQA
PGKGLEWVAIISFDGSNKSSADSVKGRFTUSRRNSKNALFLQM
NSLRAEDTAVFYCARDRGVSAGGNYYYYGMDVWGQGTTVTVSS
and the light chain variable region sequence set forth in SEQ ID
NO:91:
TABLE-US-00013 (SEQ ID NO: 91)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA
PRLLIYDASNRATGIPARFSGSGSGTRFTLTISSLEPEDFAVYYC
QQRSNWPPFTFGPGTKVDIL
or an antigen-binding or an immunologically functional
immunoglobulin fragment thereof.
[0146] In various embodiments, the invention provides antibodies,
comprising a heavy chain and a light chain, wherein the heavy chain
comprises a heavy chain variable region, and wherein the heavy
chain variable region comprises a sequence that has at least about
75%, at least about 80%, at least about 85%, at least about 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%
identity to the amino acid sequence as set forth in SEQ ID NO:90;
and wherein the light chain comprises a light chain variable
region, and wherein the light chain variable region comprises a
sequence that has at least about 80%, at least about 85%, at least
about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least
about 99% identity to the amino acid sequence as set forth in any
of SEQ ID NO:91; wherein the antibody binds specifically to human
TNF-.alpha..
[0147] Antibodies to several other TNFSF ligands or TNFSFRs have
been described in the literature, and evaluated as therapeutic
candidates in the treatment or prevention of a variety of
inflammatory diseases, autoimmune diseases and cancer. Nucleotide
and amino acid sequences of antibodies to the designated TNFSF
polypeptides or TNFSFRs are readily available from publicly
available databases. A comprehensive review of such antibodies as
well as additional TNF inhibitors is provided in "Therapeutic
Targets of the TNF Superfamily", edited by Iqbal S. Grewal, Landes
Bioscience/Springer Science+Business Media, LLC dual
imprint/Springer series: Advances in Experimental Medicine and
Biology, 2009, which is hereby incorporated by reference in its
entirety for the purpose of teaching such TNF inhibitors.
[0148] In various embodiments, the biologically active cargo is a
TNFSF inhibitor that comprises a soluble receptor or soluble
co-ligand. The terms "soluble receptor", "soluble cytokine
receptor" (SCR) and "immunoadhesin" are used interchangeably to
refer to soluble chimeric molecules comprising the extracellular
domain of a receptor, e.g., a receptor of a TNFSF member and an Ig
sequence, which retains the binding specificity of the receptor and
is capable of binding to the TNFSF member. In various embodiments,
a TNFSFSCR comprises a fusion of a TNFSFR amino acid sequence (or a
portion thereof) from a TNFSF member extracellular domain capable
of binding the TNFSF member (in some embodiments, an amino acid
sequence that substantially retains the binding specificity of the
TNFSFR) and an Ig sequence. Two distinct types of TNFSFR are known
to exist: Type I TNFSFR (TNFSFRI) and Type II TNFSFR (TNFSFRII). In
various embodiments, the TNFSF receptor is a human TNFSF receptor
sequence, and the fusion is with an Ig constant domain sequence. In
other embodiments, the Ig constant domain sequence is an Ig heavy
chain constant domain sequence. In other embodiments, the
association of two TNF receptor-Ig heavy chain fusions (e.g., via
covalent linkage by disulfide bond(s)) results in a homodimeric
Ig-like structure.
[0149] An example of a commercially available soluble receptor
useful in the present invention is ENBREL.RTM. (etanercept).
ENBREL.RTM. consists of recombinant human TNFR-p75-Fc dimeric
fusion protein consisting of the extracellular ligand-binding
portion of the human 75 kilodalton (p75) tumor necrosis factor
receptor (TNFR) linked to the Fc portion of human IgG1. The Fc
component of etanercept contains the CH2 domain, the CH3 domain and
hinge region, but not the CH1 domain of IgG1. Etanercept is
produced by recombinant DNA technology in a Chinese hamster ovary
(CHO) mammalian cell expression system. It consists of 934 amino
acids. The product is made by encoding the DNA of the soluble
portion of human TNFR-p75 with the Fc portion of IgG. In various
embodiments of the present invention, the biologically active cargo
is a TNF inhibitor that is dimeric fusion protein comprising the
sequence set forth in SEQ ID NO: 92:
TABLE-US-00014 (SEQ ID NO: 92)
LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTS
DTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCR
PGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSN
TTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVS
TRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
or a fragment or variant thereof.
[0150] In various embodiments, the biologically active cargo
contains an amino acid sequence that shares an observed homology of
at least about 75%, at least about 80%, at least about 85%, at
least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at
least about 99% identity to the sequence of SEQ ID NO: 92.
[0151] An illustrative, but not limiting list of suitable TNFSF
ligands and TNFSFRs from which a TNF inhibitor will be derived and
used as a biologically active cargo in the constructs and methods
of the present invention is provided in Table 2.
TABLE-US-00015 TABLE 2 RefSeq (protein) TNFSF Ligands Tumor
necrosis factor-.alpha. ("TNF-.alpha.") NP_000585.2
lymphotoxin-.alpha. ("LT-.alpha.") NP_000586.2 lymphotoxin-.beta.
("LT-.beta.") NP_002332.1 CD30 ligand NP_001235.1 CD40 ligand
NP_000065.1 CD70 ligand NP_001243.1 OX40 ligand NP_001284491.1 41BB
ligand NP_001552.2 Apo1 ligand (or FasL or CD95L) NP_000630.1 Apo2
ligand (or TRAIL, AIM-1 or AGP-1) NP_001177871.1 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 TACI NP_036584.1 TNFSFRs TNFR1 NP_001056.1 TNFR2
NP_001057.1 lymphotoxin-.beta.R NP_001257916.1 CD40 NP_001241.1
CD95 (or FAS orAPO-1) NP_000034.1 OPG NP_002537.3 RANK
NP_001257878.1 CD30 NP_001234.3 CD27 NP_001233.1 OX40 (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 LT.beta.R NP_001257916.1 GITR
NP_004186.1 DcR3 NP_003814 Fn14 (or TWEAKR) NP_057723.1 BAFF
NP_443177.1
Glucose-Lowering Agents
[0152] In various embodiments, the biologically active cargo is a
glucose-lowering agent. In various embodiments, the
glucose-lowering agent is a peptide that comprises about 5, about
6, about 7, about 8, about 9, about 10, about 11, about 12, about
13, about 14, about 15, about 16, about 17, about 18, about 19,
about 20, about 25, about 30, about 35, about 40, about 45, about
50, about 55, about 60, about 65, about 70, about 75, about 80,
about 85, about 90, about 95, about 100, about 150, about 200,
about 250, about 300, about 400, about 500, about 600, about 700,
about 800, about 900 or about 1000 amino acids.
[0153] An illustrative, but not limiting, list of suitable glucose
metabolism-related proteins to be used as the glucose-lowering
agent in the fusion molecules of the present disclosure, or from
which the glucose-lowering agents contemplated for use as a
glucose-lowering agent could be derived, is provided in Table
3.
TABLE-US-00016 TABLE 3 Glucose metabolism-related proteins RefSeq
(NCBI/Uniprot) 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, faciliated
P11166 glucose transporter member 1 Glycogen synthase 1 P13807
Glycogen synthase 2 P54840 Tyrosin-protein phosphatase non- P18031
receptor type 1 RAC-alpha serinel threonine- P31749 protein kinase
Peroxisome proliferator-activated P37231 receptor gamma Hexokinase
3 P52790 Phosphatidylinositol-3,4,5- P60484 triphosphate
3-phosphatase and dual-specificty protein 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 O95838 Gastric
inhibitory polypeptide P48546 receptor Insulin-like growth factor 1
P08069.1 receptor Insulin-like growth factor 2 P11717.3 receptor
Insulin Receptor P06213 GLP-1 agonist-Exenatide DB01276 GLP-1
agonist-Liraglutide DB06655
[0154] Glucagon-like peptide-1 (GLP-1), a member of the
pro-glucagon incretin family synthesized in intestinal L-cells by
tissue-specific post-translational processing of the glucagon
precursor preproglucagon, is a potent glucose-lowering agent
implicated in the control of appetite and satiety. GLP-1 acts
through GLP-1 receptor (GLP-1R), which is widely distributed in
tissues, including brain, pancreas, intestine, lung, stomach, and
kidney. The effects of GLP-1 appear to be both insulinotropic and
insulinomimetic, depending on the ambient glucose concentration.
Due to their ability to increase insulin secretion from the
pancreas, increase insulin-sensitivity in both alpha cells and beta
cells, and decrease glucagon secretion from the pancreas, GLP-1 and
its analogs have attracted considerable attention as a therapeutic
strategy for diabetes.
[0155] Several clinical trials have studied the addition of GLP-1
agonists in conjunction with ongoing insulin therapy and several
GLP-1 agonists have been approved for treatment of T2D, including,
e.g., exenatide (tradename Byetta.RTM., Amylin/Astrazeneca);
liraglutide (tradename Victoza.RTM., Novo Nordisk A/S);
lixisenatide (tradename Lyxumia.RTM., Sanofi); albiglutide
(tradename Tanzeum.RTM., GlaxoSmithKline); dulaglutide (tradename
Trulicity.RTM., Eli Lilly). While proven efficacious, the major
drawback associated with the clinical use of GLP-1 agonists is the
short biological half-life, necessitating continuous administration
intravenously or by frequent subcutaneous injections, and all GLP-1
drugs approved to date are subcutaneous administered on a twice
daily or once weekly basis. Moreover, there are safety concerns
associated with the use of these GLP-1 agonists, namely,
pancreatitis and pancreatic neoplasia, hypoglycemia, and renal
impairment. Other reported side effects include gastrointestinal
disorders, such as dyspepsia, decreased appetite, nausea, vomiting,
abdominal pain, diarrhea, dizziness, headache, and feeling jittery.
As such, there continues to be extensive research directed to
preparing analogs of the natural GLP-1 that are longer lasting, as
well as development of sustained release and other related
technologies in order to lower the frequency of injections for the
T2D patients.
[0156] In various embodiments, the biologically active cargo is
GLP-1 agonist having the amino acid sequence set forth in SEQ ID
NO: 93:
TABLE-US-00017 (SEQ ID NO: 93)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
or a fragment or variant thereof.
[0157] In various embodiments, the biologically active cargo
contains an amino acid sequence that shares an observed homology
of, e.g., at least about 75%, at least about 80%, at least about
85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
at least about 99% with the sequence of SEQ ID NO: 93.
[0158] In various embodiments, the biologically active cargo is
GLP-1 agonist having the amino acid sequence set forth in SEQ ID
NO: 94:
TABLE-US-00018 (SEQ ID NO: 94)
HAEGTFTSDVSSYLEGQAAKEEFIIAWLVKGRG
or a fragment or variant thereof.
[0159] In various embodiments, the biologically active cargo
contains an amino acid sequence that shares an observed homology
of, e.g., at least about 75%, at least about 80%, at least about
85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
at least about 99% with the sequence of SEQ ID NO: 94.
Human Growth Hormone
[0160] Growth Hormone (GH) (also known as somatropin or
somatotropin) is the master hormone in the human body, and is
synthesized and secreted by the endocrinal system. This hormone
controls essential functions like: growth and replication of cells
in various organs of the body. Some of the essential functions of
GH include: controlling muscle growth, improving bone
mineralization and strength, reducing fat deposition, and
sustaining good energy levels. The production and secretion of the
growth hormone is controlled by Growth Hormone Releasing Hormone
(GHRH), which is secreted by the hypothalamus. The GHRH stimulates
the pituitary gland to produce GH, which is directly released into
the blood stream. The GH in turn stimulates the liver to produce
Insulin-like Growth Factor (IGF-1) which stimulates the
proliferation of chondrocytes (cartilaginous cells), promotes
differentiation of myoblasts and enhances protein synthesis, which
in turn, helps in the growth of other muscles and tissue cells.
[0161] In the US, synthetically produced human growth hormone (HGH)
has been used in the pediatric population to treat short stature
due to growth hormone deficiency (GHD), Turner syndrome (TS),
Noonan syndrome, Prader-Willi syndrome, short stature
homeobox-containing gene (SHOX) deficiency, chronic renal
insufficiency, idiopathic short stature and children small for
gestational age. In adults, HGH has been used to treat short bowel
syndrome, a condition in which nutrients are not properly absorbed
due to sever intestinal disease or the surgical removal of a large
portion of the small intestine, GH deficiency due to rare pituitary
tumors or their treatment, and muscle-wasting disease associated
with HIV/AIDS.
[0162] Growth hormone deficiency (GHD) is a rare disorder that
includes a group of different pathologies characterized by the
inadequate secretion of growth hormone (GH) from the anterior
pituitary gland, a small gland located at the base of the brain
that is responsible for the production of several hormones. GHD may
occur by itself or in combination with other pituitary hormone
deficiencies. GHD may be present from birth (congenital) or
acquired as a result of trauma, infiltrations, tumor or radiation
therapy. There is a third category that has no known cause
(idiopathic). Childhood-onset GHD may be all three: congenital,
acquired, or idiopathic. It results in growth retardation, short
stature, and maturation delays reflected by the delay of
lengthening of the bones of the extremities that is inappropriate
to the chronological age of the child. Adult-onset GHD is most
often acquired from a pituitary tumor or trauma to the brain but
may also be idiopathic. It is characterized by a number of variable
symptoms including reduced energy levels, altered body composition,
osteoporosis (reduced bone mineral density), reduced muscle
strength, lipid abnormalities such as increased LDL or cholesterol
levels, insulin resistance, and impaired cardiac function. Adult
GHD has been estimated to affect 1 in 100,000 people annually,
while its incidence rate is approximately 2 cases per 100,000
population when childhood-onset GHD patients are considered. About
15-20% of the cases represent the transition of childhood GHD into
adulthood (Stochholm K et al., Eur J Endocrinol., 155:61-71,
2006).
[0163] Turner (or Ullrich-Turner) syndrome (TS) is a chromosomal
abnormality characterized by the absence of the entire chromosome X
or a deletion within that chromosome and that affects development
in females. The most common feature of Turner syndrome is short
stature, which becomes evident by about age 5. This condition
occurs in about 1 in 2,500 newborn girls worldwide, but it is much
more common among pregnancies that do not survive to term
(miscarriages and stillbirths). As a chromosomal condition, there
is no cure for Turner syndrome.
[0164] Recombinant DNA-derived human growth hormone is the only
drug approved specifically for treatment of GHD and TS. As of 2005,
various recombinant human growth hormones (also referred to as
somatropin [rDNA origin] for injection) available in the United
States (and their manufacturers) included NUTROPIN.RTM.
(Genentech), HUMATROPEO (Lilly), GENOTROPIN.RTM. (Pfizer),
NORDITROPIN.RTM. (Novo), and SAIZEN.RTM. (Merck Serono). In 2006,
the U.S. Food and Drug Administration (FDA) approved a version of
rHGH called OMNITROPEO (Sandoz). A sustained-release form of human
growth hormone, NUTROPIN DEPOT.RTM. (Genentech/Alkermes) was
approved by the FDA in 1999, allowing for fewer injections (every 2
or 4 weeks instead of daily); however, the product was discontinued
by Genentech/Alkermes in 2004 for financial reasons. Additional
approved recombinant HGH products include SEROSTIM.RTM. (EMD
Serono), TEV-TROPIN.RTM. (Teva) and ZORBITIVEO (Merck Serono) for
short bowel syndrome.
[0165] While proven to be the most effective, spontaneous and
trusted treatment option for the management of growth disorders
such as GHD, these injectable rHGH's have some significant
limitations including, e.g, 1) complications associated with
prolonged use and high dosages which are severe and irreversible,
and include, e.g, the probability of developing diabetes,
cardiovascular disorders and colon cancer. Other common side
effects include: joint pain, generalized edema, severe headache,
hypoglycemia, wrist pain (carpel tunnel syndrome), increased level
of LDL in the blood increasing the possibility of developing
atherosclerosis, etc.; 2) HGH injections are not available over the
counter, nevertheless, due to rigid FDA norms, black-marketing is
rampant. The procurement of the HGH injections without medical
prescription is considered illegal and is punishable by law, with
imprisonment and fine; and 3) the cost of the treatment is
exorbitant. Depending upon the pharmaceutical company the cost of
HGH injections for a month of treatment, typically range from
between $800 to $3000. Finally, conventional methods using rHGH
typically involve multi-dose regimens in which the HGH is
administered via subcutaneous injection. The inconvenience, pain
and social stigma associated with such methods can be considerable.
Management of the pediatric population to treat short stature due
to growth hormone deficiency (GHD), Turner syndrome (TS) and
related disorders, with these highly invasive and repetitive
therapies can be especially difficult.
[0166] Full length human HGH consists of 191 amino acids. HGH
produced using molecular biological techniques may have an amino
acid sequence identical to naturally occurring HGH. Alternatively,
the HGH used may be an HGH analog comprising one or more variations
in amino acid sequence with respect to the native hormone. These
amino acid variations may provide enhanced biological activity or
some other biological or logistical advantages. In various
embodiments, the recombinant HGH comprises the amino acid sequence
set forth in Genbank Accession No. P01241. The HGH amino acid
sequence (without the 26 aa signal sequence of P01241) is set forth
in SEQ ID NO: 95:
TABLE-US-00019 (SEQ ID NO: 95)
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTS
LCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLV
YGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHND
DALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF
[0167] HGH of the present disclosure refers to HGH from any source
which has the sequence of SEQ ID NO: 95, including isolated,
purified and/or recombinant HGH produced from any source or
chemically synthesizes, for example using solid phase synthesis.
Also included herein are conserved amino acid substitutions of
native HGH. For example, conservative amino acid changes may be
made, which although they alter the primary sequence of the protein
or peptide, do not normally alter its function. Conservative amino
acid substitutions typically include substitutions within the
following groups: glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid; asparagine, glutamine; serine,
threonine; lysine, arginine; and phenylalanine, tyrosine. In
various embodiments, the HGH has an amino acid sequence that shares
an observed homology of, e.g., at least about 75%, at least about
80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or at least about 99% with the sequence of SEQ
ID NO: 95.
[0168] In various embodiments, the HGH contemplated for use in the
fusion molecules of the present disclosure include human growth
hormone variants and mutants which have been extensively described
in the art (see, e.g. U.S. Pat. No. 8,637,646 (Wells et al) and
references cited therein, and US 20110130331 (Guyon et al), each
incorporated by reference in its entirety herein for the specific
purpose of providing such growth hormone variants and mutants).
[0169] In various embodiments, the HGH contemplated for use in the
fusion molecules of the present disclosure include, e.g.,
NUTROPIN.RTM. (Genentech), HUMATROPE.RTM. (Lilly), GENOTROPIN.RTM.
(Pfizer), NORDITROPIN.RTM. (Novo), SAIZEN.RTM. (Merck Serono),
OMNITROPE.RTM. (Sandoz), SEROSTIM.RTM. (EMD Serono),
TEV-TROPIN.RTM. (Teva) and ZORBITIVE.RTM. (Merck Serono).
[0170] An illustrative, but not limiting, list of suitable growth
hormone proteins to be used as the growth hormone in the fusion
molecules of the present disclosure, or from which the growth
hormones contemplated for use as a growth hormone could be derived,
is provided in Table 4.
TABLE-US-00020 TABLE 4 RefSeq (NCBI/Uniprot) 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 Hormone Q02643
Receptor Growth Hormone Secretagogue Q92847 Receptor Growth
Hormone-Releasing Hormone P78470 Receptor form a Growth Hormone
Receptor E9PCN7
Insertion Site for Attachment of the Biologically Active Cargo
[0171] The biologically active cargo of the fusion molecule can be
attached to the remainder of the fusion molecule by any method
known by one of skill in the art without limitation. The
biologically active cargo can be introduced into any portion of the
fusion molecule that does not disrupt the cell-binding or
transcytosis activity of the modified Cholix toxin. In various
embodiments, the biologically active cargo is directly coupled to
the N-terminus or C-terminus of the modified Cholix toxin. In
various embodiments, the biologically active cargo can be connected
with a side chain of an amino acid of the modified Cholix toxin. In
various embodiments, the biologically active cargo is coupled to
the modified Cholix with a non-cleavable peptide linker. In various
embodiments, the biologically active cargo is coupled to the
modified Cholix toxin with a cleavable linker such that cleavage at
the cleavable linker(s) separates the biologically active cargo
from the remainder of the fusion molecule. In various embodiments,
the biologically active cargo is a polypeptide that may also
comprise a short leader peptide that remains attached to the
polypeptide following cleavage of the cleavable linker. For
example, the biological active 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. In some cases, 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. In some cases, 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. In native Cholix toxin, the domain Ib
loop spans amino acids 387 to 425, and is structurally
characterized by a disulfide bond between two cysteines at
positions 395 and 402. This domain Ib portion of Cholix toxin is
not essential for any known activity of Cholix toxin, including
cell binding, translocation, ER retention or ADP ribosylation
activity. Accordingly, domain Ib can be deleted entirely, or
modified to contain a biologically active cargo. Thus, in various
embodiments, the biologically active cargo can be inserted into
Cholix toxin domain Ib. If desirable, the biologically active cargo
can be inserted into Cholix toxin domain Ib between the cysteines
at positions 395 and 402 that are not crosslinked. This can be
accomplished by reducing the disulfide linkage between the
cysteines, by deleting one or both of the cysteines entirely from
the Ib 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 Ib 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.
[0172] In embodiments where the biologically active cargo is
expressed together with another portion of the fusion molecule as a
fusion protein, the biologically active cargo can be can be
inserted into the fusion molecule by any method known to one of
skill in the art without limitation. For example, amino acids
corresponding to the biologically active cargo can be directly
inserted into the fusion molecule, with or without deletion of
native amino acid sequences. In various embodiments, all or part of
the Ib domain of Cholix toxin can be deleted and replaced with the
biologically active cargo. In various embodiments, the cysteine
residues of the Ib loop are deleted so that the biologically active
cargo remains unconstrained. In other embodiments, the cysteine
residues of the Ib loop are linked with a disulfide bond and
constrain the biologically active cargo.
[0173] In embodiments where the biologically active cargo is not
expressed together with the remainder of the fusion molecule as a
fusion protein, the biologically active cargo can be connected with
the remainder of the fusion molecule by any suitable method known
by one of skill in the art, without limitation. More specifically,
the exemplary methods described above for connecting a receptor
binding domain to the remainder of the molecule are equally
applicable for connecting the biologically active cargo to the
remainder of the molecule.
Production of Fusion Proteins
[0174] In various embodiments, the non-naturally occurring fusion
molecule is synthesized using recombinant DNA methodology.
Generally this involves creating a DNA sequence that encodes the
fusion molecule, 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, renaturing
the molecule.
[0175] DNA encoding the fusion molecules (e.g.
Cholix.sup.415-IL-10) 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.
[0176] 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 may be
obtained by the ligation of shorter sequences.
[0177] 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.
[0178] In various embodiments, DNA encoding fusion molecules of the
present disclosure can be cloned using DNA amplification methods
such as polymerase chain reaction (PCR). Thus, for example, the
gene for the IL-10 is PCR amplified, using a sense primer
containing the restriction site for, e.g., NdeI and an antisense
primer containing the restriction site for HindIII. This can
produce a nucleic acid encoding the mature IL-10 sequence and
having terminal restriction sites. A modified Cholix toxin having
"complementary" restriction sites can similarly be cloned and then
ligated to the IL-10 and/or to a linker attached to the IL-10.
Ligation of the nucleic acid sequences and insertion into a vector
produces a vector encoding the IL-10 joined to the modified Cholix
toxin.
Non-Cleavable Linkers
[0179] In various embodiments, the modified Cholix toxin and
biologically active cargo can be separated by a peptide spacer
consisting of one or more amino acids (e.g., up to 25 amino acids).
Generally the 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. In various
embodiments, 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.
[0180] In various embodiments, the linker is capable of forming
covalent bonds to both the Cholix toxin and to the biologically
active cargo. Suitable linkers are well known to those of skill in
the art and include, but are not limited to, straight or
branched-chain carbon linkers, heterocyclic carbon linkers, or
peptide linkers. In various embodiments, the linker(s) can be
joined to the constituent amino acids of the Cholix toxin and/or
the biologically active cargo through their side groups (e.g.,
through a disulfide linkage to cysteine). In various embodiments,
the linkers are joined to the alpha carbon amino and/or carboxyl
groups of the terminal amino acids of the Cholix toxin and/or the
biologically active cargo.
[0181] A bifunctional linker having one functional group reactive
with a group on the Cholix toxin 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).
[0182] Many procedures and linker 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. (1987) Cancer Res. 47:
4071-4075.
[0183] In various embodiments, the biologically active cargo to be
delivered to the subject is coupled to the modified Cholix toxin
using one or more non-cleavable peptide linkers comprising, e.g.,
the amino acid sequence GGGGS (SEQ ID NO: 96), GGGGSGGGGS (SEQ ID
NO: 97), GGGGSGGGGSGGGGS (SEQ ID NO: 98), or GGGGSGGG (SEQ ID NO:
99), wherein the modified Cholix toxin targets said 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.
Cleavable Linkers
[0184] In various embodiments, the biologically active cargo to be
delivered to the subject is coupled to the modified Cholix toxin
using one or more cleavable linkers. The number of cleavable
linkers present in the fusion molecule depends, at least in part,
on the location of the biologically active cargo in relation to the
modified Cholix toxin and the nature of the biologically active
cargo. When the biologically active cargo can be separated from the
remainder of the fusion molecule with cleavage at a single linker,
the fusion molecules can comprise a single cleavable linker.
Further, where the biologically active cargo is, e.g., a dimer or
other multimer, each subunit of the biologically active cargo can
be separated from the remainder of the fusion molecule and/or the
other subunits of the biologically active cargo by cleavage at the
cleavable linker.
[0185] In various embodiments, the cleavable linkers are cleavable
by a cleaving enzyme that is present at or near the basolateral
membrane of an epithelial cell. By selecting the cleavable linker
to be cleaved by such enzymes, the biologically active cargo can be
liberated from the remainder of the fusion molecule 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 linker
is cleaved prior to release of the fusion molecule from the
basolateral membrane, so long as the cleaving enzyme does not
cleave the fusion molecule before the fusion molecule enters the
trafficking pathway in the polarized epithelial cell that results
in release of the fusion molecule and biologically active cargo
from the basolateral membrane of the cell.
[0186] In various embodiments, the enzyme that is present at a
basolateral membrane of a polarized epithelial cell is selected
from, e.g., Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin
AI, Subtilisin AII, Thrombin I, or Urokinase I. Table 5 presents
these enzymes together with an amino acid sequence that is
recognized and cleaved by the particular peptidase.
TABLE-US-00021 TABLE 5 Peptidases Present Near Basolateral Mucous
Membranes or in Latter Aspects of the Transcytosis Pathway
Peptidase Amino Acid Sequence Cleaved Cathepsin GI AAPF (SEQ ID NO:
100) Chymotrypsin I GGF (SEQ ID NO: 101) Elastase I AAPV (SEQ ID
NO: 102) Subtilisin AI GGL (SEQ ID NO: 103) Subtilisin AII AAL (SEQ
ID NO: 104) Thrombin I FVR (SEQ ID NO: 105) Urokinase I VGR (SEQ ID
NO: 106) Furin RKPR (SEQ ID NO: 107)
[0187] In various embodiments, the cleavable linker exhibits 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.
In various embodiments, the cleavable linker is selected to be
preferentially cleaved relative to other amino acid sequences
present in the delivery construct during administration of the
delivery construct. In various embodiments, the receptor binding
domain is 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. In various
embodiments, the translocation domain is 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. In various embodiments, the
macromolecule is 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. In
various embodiments, the cleavable linker is 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.
[0188] In other embodiments, the cleavable linker is cleaved by a
cleaving enzyme found in the plasma of the subject. 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 linker. Uses of
such enzymes to cleave the cleavable linkers 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, in various embodiments, the
cleavable linker 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
6 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.
TABLE-US-00022 TABLE 6 Plasma Peptidases Peptidase Amino Acid
Sequence Cleaved Caspase-1 Tyr-Val-Ala-Asp-Xaa* (SEQ ID NO: 108)
Caspase-3 Asp-Xaa-Xaa-Asp-Xaa* (SEQ ID NO: 109) Proprotein
convertase 1 Arg-(Xaa).sub.n-Arg-Xaa*; n = 0, 2, 4 or 6 (SEQ ID NO:
110) Proprotein convertase 2 Lys-(Xaa).sub.n-Arg-Xaa*; n = 0, 2, 4,
or 6 (SEQ ID NO: 111) Proprotein convertase 4
Glu-Arg-Thr-Lys-Arg-Xaa* (SEQ ID NO: 112) Proprotein convertase 4
PACE 4 Arg-Val-Arg-Arg-Xaa* (SEQ ID NO: 113)
Decanoyl-Arg-Val-Arg-Arg-Xaa* (SEQ ID NO: 114) Prolyloligopeptidase
Endothelin Pro-Xaa*-Trp-Val-Pro-Xaa cleaving enzyme in combination
(SEQ ID NO: 115) with dipeptidyl-peptidase IV Signal peptidase
Trp-Val*-Ala-Xaa (SEQ ID NO: 116) Neprilysin in combination with
Xaa-Phe*-Xaa-Xaa dipeptidyl-peptidase IV (SEQ ID NO: 117)
Xaa-Tyr*-Xaa-Xaa (SEQ ID NO: 118) Xaa-Trp*-Xaa-Xaa (SEQ ID NO: 119)
Renin Asp-Arg-Tyr-Ile-Pro-Phe-His-Leu*-Leu in combination with
(Val, Ala or Pro)-Tyr-(Ser, Pro, or Ala) dipeptidyl-peptidase IV
(SEQ ID NO: 120)
[0189] Thus, in various embodiments, the cleavable linker can be
any cleavable linker 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. In various embodiments, the cleavable linker
comprises a peptide. In other embodiments, the cleavable linker
comprises a nucleic acid, such as RNA or DNA. In still other
embodiments, the cleavable linker comprises a carbohydrate, such as
a disaccharide or a trisaccharide.
[0190] Alternatively, in various embodiments, the cleavable linker
can be any cleavable linker 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. In various
embodiments, the cleavable linker comprises a peptide. In other
embodiments, the cleavable linker comprises a nucleic acid, such as
RNA or DNA. In still other embodiments, the cleavable linker
comprises a carbohydrate, such as a disaccharide or a
trisaccharide.
[0191] In various embodiments, the peptidases exhibit much higher
(e.g., 100%, 200%, or more increase in activity relative to the
apical side) on the baso-lateral side (also referred to as
basolateral). Thus, in various embodiments, the cleavable linker is
cleavable by an enzyme that exhibits 50% higher activity on the
basolateral side of the membrane than on the apical side of the
membrane. In various embodiments, the cleavable linker is cleavable
by an enzyme that exhibits 100% higher activity on the basolateral
side of the membrane than on the apical side of the membrane. In
various embodiments, the cleavable linker is cleavable by an enzyme
that exhibits 200% higher activity on the basolateral side of the
membrane than on the apical side of the membrane. In various
embodiments, the cleavable linker is cleavable by an enzyme that
exhibits 500% higher activity on the basolateral side of the
membrane than on the apical side of the membrane. In various
embodiments, the cleavable linker is cleavable by an enzyme that
exhibits 1,000% higher activity on the basolateral side of the
membrane than on the apical side of the membrane.
[0192] In various embodiments, the fusion molecule comprises a
cleavable linker having an amino acid sequence selected from, e.g.,
SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ
ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 or SEQ ID NO: 107 and is
cleavable by an enzyme that exhibits higher activity on the
basolateral side of a polarized epithelial cell than it does on the
apical side of the polarized epithelial cell, and/or is cleavable
by an enzyme that exhibits higher activity in the plasma than it
does on the apical side of a polarized epithelial cell.
[0193] In various embodiments, the cleavable linker can be a
cleavable linker that is cleaved following a change in the
environment of the fusion molecule. For example, the cleavable
linker can be a cleavable linker that is pH sensitive and is
cleaved by a change in pH that is experienced when the fusion
molecule is released from the basolateral membrane of a polarized
epithelial cell. For instance, the intestinal lumen is strongly
alkaline, while plasma is essentially neutral. Thus, a cleavable
linker can be a moiety that is cleaved upon a shift from alkaline
to neutral pH. The change in the environment of the fusion molecule
that cleaves the cleavable linker can be any environmental change
that that is experienced when the fusion molecule is released from
the basolateral membrane of a polarized epithelial cell known by
one of skill in the art, without limitation.
[0194] In various embodiments, the cleavable linker is cleaved by a
cleaving enzyme found in the plasma of the subject. 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 linker.
Accordingly, in various embodiments, the cleavable linker can be
cleaved with an enzyme that is selected from e.g., SEQ ID NO: 108,
SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ
ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID
NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.
[0195] In various embodiment, the cleavable linker is a linker that
contains an amino acid sequence that is a known substrate for the
tobacco etch virus (TEV) protease. Accordingly, in various
embodiments, the cleavable linker comprises the amino acid sequence
set in forth in, e.g., GGGGSGGGENLYFQS (SEQ ID NO: 121).
Chemical Conjugation of the Cargo to the Modified Cholix Toxin
[0196] In various embodiments, the biologically active cargo to be
delivered to the subject is chemically conjugated to the modified
Cholix toxin. Means of chemically conjugating molecules are well
known to those of skill.
[0197] The procedure for conjugating two molecules varies according
to the chemical structure of the agent. Polypeptides typically
contain variety of functional groups; e.g., carboxylic acid (COOH)
or free amine (--NH.sub.2) groups, that are available for reaction
with a suitable functional group on the other peptide, or on a
linker to join the molecules thereto.
[0198] Alternatively, the antibody and/or the biologically active
cargo can be derivatized to expose or attach additional reactive
functional groups. The derivatization can involve attachment of any
of a number of linker molecules such as those available from Pierce
Chemical Company, Rockford III.
[0199] In various embodiments, isolated modified Cholix toxins are
prepared by bacterial fermentation and purified by established
methods. The purified modified Cholix toxin is then modified at its
C-terminus to allow direct chemical coupling through a free
sulfhydryl residue located near the C-terminus of the protein. The
C-terminal modification includes a cysteine-constrained loop
harboring the consensus cleavage sequence for the highly selective
protease from the tobacco etch virus (TEV), a second cysteine, and
a hexa-histadine (His.sub.6) tag. The second Cys is included to
form a disulphide bridge with the Cys ultimately used for coupling.
Adding the His.sub.6 sequence to the protein simplifies the
purification and the TEV cleavage sequence provides a mechanism to
selectively remove the terminal Cys residue following mild
reduction. TEV cleavage and mild reduction with 0.1 mM
dithiotheitol following expression and isolation of the ntCholix
constructs allows for the direct chemical coupling of a
biologically active cargo via a maleimide-based reaction as a
generic mechanism of cargo attachment. Following TEV protease
cleavage, reduction, and cargo coupling through a maleimide
reaction with the free sulfhydryl, removal of the freed C-terminal
sequence was achieved by a second Ni.sup.2+ column chromatography
step.
[0200] In various embodiments, the fusion molecule comprises
particles which are decorated covalently with the modified Cholix
toxin, and wherein the biologically active cargo is integrated into
the particles. In various embodiments, the particles can be smaller
than .about.150 nm in diameter, smaller than .about.100 nm, or
smaller than .about.50 nm.
[0201] In various embodiments, the fusion molecule comprises a
biologically active cargo coupled non-covalently to the modified
Cholix toxin. This fusion molecule could ferry, e.g., a
non-covalently associated IL-10 across the epithelium such as a
surface element of the IL-10 receptor (Josephson, K., Logsdon,
N.J., Walter, M. R., Immunity 15: 35-46, 2001, incorporated by
reference in its entirety herein).
Pharmaceutical Compositions and Delivery Methods
[0202] The pharmaceutical compositions of the present disclosure
relate to compositions for administration to a human subject. The
pharmaceutical compositions comprise the non-naturally occurring
fusion molecules recited herein, alone or in combination. The
pharmaceutical compositions may comprise additional molecules
capable of altering the characteristics of the non-naturally
occurring fusion molecules, for example, stabilizing, modulating
and/or activating their function. The composition may, e.g., be in
solid or liquid form and may 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.
[0203] The pharmaceutical compositions are generally formulated
appropriately for the immediate use intended for the fusion
molecule. For example, if the fusion molecule is not to be
administered immediately, the fusion molecule can be formulated in
a composition suitable for storage. One such composition is a
lyophilized preparation of the fusion molecule together with a
suitable stabilizer. Alternatively, the fusion molecule 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 stabilizing a lyophilized or liquid
preparation of the fusion molecules may 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.
[0204] 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 modified Cholix toxin's ability to mediate
transcytosis across the gastrointestinal (GI) epithelium. It is
anticipated that oral administration of these pharmaceutical
compositions will result in absorption of the fusion molecule
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 may 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.
[0205] It is anticipated that once transported across the GI
epithelium, the fusion molecules of the disclosure will exhibit
extended half-life in serum, that is, the biologically active cargo
of the fusion molecules 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 fusion
molecule in the stomach. Such formulations may include carrier and
dispersant components and may 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.
[0206] In various embodiments, the oral formulation comprises a
fusion molecule and one or more compounds that can protect the
fusion molecule while it is in the stomach. For example, the
protective compound should be able to prevent acid and/or enzymatic
hydrolysis of the fusion molecule. In various embodiments, the oral
formulation comprises a fusion molecule and one or more compounds
that can facilitate transit of the construct from the stomach to
the small intestine. In various embodiments, the one or more
compounds that can protect the fusion molecule from degradation in
the stomach 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 fusion molecules
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 Suppl: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.
[0207] Pharmaceutical compositions intended for oral use may be
prepared according to any method known to the art for the
manufacture of pharmaceutical compositions and such compositions
may 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 fusion molecule 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 fusion molecules may 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.
[0208] In various embodiments, the pharmaceutical compositions are
formulated as orally deliverable tablets containing fusion
molecules in admixture with non-toxic pharmaceutically acceptable
excipients which are suitable for manufacture of tablets. These
excipients may 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 may be uncoated or
they may 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 may be employed.
[0209] In various embodiments, the pharmaceutical compositions are
formulated as hard gelatin capsules wherein the fusion molecule is
mixed with an inert solid diluent, for example, calcium carbonate,
calcium phosphate, or kaolin or as soft gelatin capsules wherein
the fusion molecule is mixed with an aqueous or an oil medium, for
example, arachis oil, peanut oil, liquid paraffin or olive oil.
[0210] In various embodiments, aqueous suspensions may contain a
fusion molecule 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 may 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 may
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.
[0211] In various embodiments, oily suspensions may be formulated
by suspending the fusion molecule 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 may contain a
thickening agent, for example, beeswax, hard paraffin or cetyl
alcohol. Sweetening agents, such as those set forth above, and
flavoring agents may be added to provide a palatable oral
preparation. These compositions may be preserved by the addition of
an antioxidant such as ascorbic acid.
[0212] In various embodiments, the pharmaceutical compositions may
be in the form of oil-in-water emulsions. The oil phase may be a
vegetable oil, for example, olive oil or arachis oil, or a mineral
oil for example, gum acacia or gum tragacanth, naturally-occurring
phosphotides, 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 may also contain sweetening and
flavoring agents.
[0213] In various embodiments wherein the pharmaceutical
composition is in the form of a tablet or capsule, the tablet or
capsule is coated or encapsulated to protect the 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 hydrophobic backbone and hydrophilic
branches as drug carriers, encapsulation in microparticles,
insertion into liposomes in emulsions, and conjugation to other
molecules. 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).
[0214] Encapsulated or coated tablets can be used that release the
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.
[0215] In various embodiments, pharmaceutical compositions may 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)).
[0216] 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 (C.sub.8), caprate (C.sub.10), laurate (C.sub.12) and
myristate (C.sub.14). 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.
[0217] In another aspect, the present disclosure relates to methods
of 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 fusion molecules results in absorption of the fusion molecule
through polarized epithelial cells of the digestive mucosa, e.g.,
the intestinal mucosa, followed by cleavage of the fusion molecule
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 fusion molecule 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.
[0218] In another aspect, the present disclosure relates to methods
of orally administering the pharmaceutical compositions of the
disclosure. Such methods may include, but are not limited to, steps
of orally administering the compositions by the patient or a
caregiver. Such administration steps may include administration on
intervals such as once or twice per day depending on the fusion
molecule, disease or patient condition or individual patient. Such
methods also include the administration of various dosages of the
individual fusion molecule. For instance, the initial dosage of a
pharmaceutical composition may be at a higher level to induce a
desired effect, such as reduction in blood glucose levels.
Subsequent dosages may then be decreased once a desired effect is
achieved. These changes or modifications to administration
protocols may be done by the attending physician or health care
worker.
[0219] These pharmaceutical compositions can be administered to the
subject at a suitable dose. The dosage regimen will be determined
by the attending physician and 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 may be determined by
conventional tests which are well known to the person skilled in
the art.
[0220] 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 may contain a
multiplicity of carrier/biologically or chemically active agent
compositions or may 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.
[0221] In various embodiments, an amount of fusion molecule
administered to the subject is at most 0.001 pg, at most 1 pg, at
most 2 pg, at most 3 pg, at most 4 pg, at most 5 pg, at most 10 pg,
at most 50 pg, at most 100 pg, at most 1 .mu.g, at most 2 .mu.g, at
most 3 .mu.g, at most 4 .mu.g, at most 5 .mu.g, at most 10 .mu.g,
at most 50 .mu.g, at most 100 .mu.g, at most 1 mg, at most 2 mg, at
most 3 mg, at most 4 mg, at most 5 mg, at most 10 mg, at most 50
mg, at most 100 mg, or at most 1 g.
[0222] In various embodiments, an amount of fusion molecule
administered to the subject is at least 0.001 pg, at least 1 pg, at
least 2 pg, at least 3 pg, at least 4 pg, at least 5 pg, at least
10 pg, at least 50 pg, at least 100 pg, at least 1 .mu.g, at least
2 .mu.g, at least 3 .mu.g, at least 4 .mu.g, at least 5 .mu.g, at
least 10 .mu.g, at least 50 .mu.g, at least 100 .mu.g, at least 1
mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at
least 10 mg, at least 50 mg, at least 100 mg, or at least 1 g.
[0223] In various embodiments, an amount of fusion molecule
administered to the subject is from 0.001 pg and about 1 g, from 1
pg to 10 pg, from 50 pg to 100 pg, from 1 .mu.g to 5 .mu.g, from 10
.mu.g to 20 .mu.g, from 10 .mu.g to 500 mg, from 10 .mu.g to 100
mg, from 10 .mu.g to 1000 .mu.g, from 10 .mu.g to 250 .mu.g, from
10 .mu.g to 100 .mu.g, from 10 .mu.g to 50 .mu.g, from 1 mg to 5
mg, or from 10 mg to 100 mg.
[0224] The volume of a composition comprising the fusion molecule
that is administered will generally depend on the concentration of
fusion molecule and the formulation of the composition. In various
embodiments, a unit dose of the fusion molecule composition is from
0.001 .mu.l to 1 ml, from 1 .mu.l to 100 .mu.l, from 50 .mu.l to
500 .mu.l, from 0.01 ml to 1 ml, from 1 ml to 100 ml, from 0.05 ml
to 1 ml. For example, the unit dose of the fusion molecule
composition can be about 0.5 ml.
[0225] In some embodiments, a unit dose of the fusion molecule
composition is at most about 0.001 .mu.l, at most 1 .mu.l, at most
10 .mu.l, at most 50 .mu.l, at most 200 .mu.l, at most 0.01 ml, at
most 0.05 ml, at most 0.1 ml, at most 0.2 ml, at most 0.5 ml, or at
most 1 ml.
[0226] In some a unit dose of the fusion molecule composition is at
least 0.001 .mu.l, at least 1 .mu.l, at least 10 .mu.l, at least 50
.mu.l, at least 200 .mu.l, at least 0.01 ml, at least 0.05 ml, at
least 0.1 ml, at least 0.2 ml, at least 0.5 ml, or at least 1
ml.
[0227] The fusion molecule compositions can be prepared in dosage
forms containing between 1 and 50 doses (e.g., 0.5 ml to 25 ml),
more usually between 1 and 10 doses (e.g., 0.5 ml to 5 ml).
[0228] The fusion molecule compositions of the disclosure can be
administered in one dose or in multiple doses. A dose can be
followed by one or more doses spaced by about 1 to about 6 hours,
by about 6 to about 12 hours, by about 12 to about 24 hours, by
about 1 day to about 3 days, by about 1 day to about 1 week, by
about 1 week to about 2 weeks, by about 2 weeks to about 1 month,
by about 4 to about 8 weeks, by about 1 to about 3 months, or by
about 1 to about 6 months.
[0229] In various embodiments, the pharmaceutical compositions
comprising the fusion molecules may be, though not necessarily,
administered daily, in an effective amount to ameliorate a symptom.
Generally, the total daily dosage can be administered at an amount
of at least about 0.001 pg, at least about 0.1 mg, at least about 1
mg, at least about 10 mg, at least about 50 mg, at least about 100
mg, at least about 150 mg, at least about 200 mg, at least about
250 mg, at least about 300 mg, at least about 350 mg, at least
about 400 mg, at least about 450 mg, at least about 500 mg per day,
or at least about 1000 mg per day. For example, the dosage can be
formulated for oral administration in capsules or tablets, such
that 4 capsules or tablets, each containing 50 mg fusion molecule.
Capsules or tablets for oral delivery can conveniently contain up
to a full daily oral dose, e.g., 200 mg or more per day.
[0230] In various embodiments, the pharmaceutical compositions
comprising the fusion molecules may be, though not necessarily,
administered daily, in an effective amount to ameliorate a symptom.
Generally, the total daily dosage can be administered at an amount
of at most 50 mg per day, at most 100 mg per day, at most 150 mg
per day, at most 200 mg per day, at most 250 mg per day, at most
300 mg per day, at most 350 mg per day, at most 400 mg per day, at
most 450 mg per day, at most 500 mg per day, or at most 1000 mg per
day.
[0231] As used herein, the terms "co-administration",
"co-administered" and "in combination with", referring to the
fusion molecules 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 fusion molecules 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
fusion molecules 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 fusion
molecules 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 fusion molecules 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 may be administered by either the
same or a different route.
[0232] In various embodiments, the pharmaceutical compositions
comprising the fusion molecules may be co-administered with a
second component, wherein the second component is a hormone, toxin,
or bioactive agent which is capable of binding to the GM-1
(monosialotetrahexosylganglioside) receptor (Hakomori, Advances in
Exp. Medicine and Biology, 174:333-339, 1984). In various
embodiments, the second component is SV40 virus, polyoma virus, or
a toxin such as cholera toxin, or exotoxin A from Pseudomonas
aeruginosa (PE).
[0233] As used herein, the terms "cholera toxin" or "CT" refer to
the eponymous virulence agent of Vibrio cholerae bacterium, which
can cause acute, life-threatening massive watery diarrhea. CT is a
protein complex composed of a single A subunit organized with a
pentamer of B subunits that binds to cell surfaceG.sub.M1
ganglioside structures at the apical surface of epithelia. CT is
secreted by V. cholera following horizontal gene transfer with
virulent strains of V. cholerae carrying a variant of lysogenic
bacteriophage called CTXf or CTX.sub..phi.. Recent cholera
outbreaks, however, have suggested that strains of some serogroups
(non-01, non-0139) do not express CT but rather use other virulence
factors. Detailed analyses of non-01, non-0139 environmental and
clinical data suggested the presence of a novel putative secreted
exotoxin with some similarity to PE. The sequence of CT is known
and has been described (Mekalanos J. J. et al Nature 306, page 551
(1983)).
[0234] As used herein the terms "exotoxin A from Pseudomonas
aeruginosa", "Pseudomonas exotoxin A" or "PE" refer to an extremely
active monomeric protein (molecular weight 66 kD), secreted by
Pseudomonas aeruginosa, which inhibits protein synthesis in
eukaryotic cells. The 613-residue sequence of PE is well known in
the art and is set forth, for example, in U.S. Pat. No. 5,602,095.
Domain Ia (amino acids 1-252) mediates cell binding. Domain II
(amino acids 253-364) is responsible for translocation into the
cytosol and domain III (amino acids 400-613) mediates ADP
ribosylation of elongation factor 2. The function of domain Ib
(amino acids 365-399) remains undefined, although it has been known
a large part of it, amino acids 365-380, can be deleted without
loss of cytotoxicity. See Siegall et al., J Biol Chem, 264:14256-61
(1989).
[0235] Certain cytotoxic fragments of PE are known in the art and
are often referenced by the molecular weight of the fragment, which
designates for the person of skill in the art the particular
composition of the PE fragment. For example, PE40 was one of the
first fragments that was studied and used as the toxic portion of
immunotoxins. The term designates a truncated form of PE in which
domain Ia, the domain responsible for non-specific binding. See,
e.g., Pai et al., Proc. Nat'l Acad. Sci. USA, 88:3358-3362 (1991);
and Kondo et al., J. Biol. Chem., 263:9470-9475 (1988). Elimination
of non-specific binding, however, can also be achieved by mutating
certain residues of domain Ia. U.S. Pat. No. 5,512,658, for
instance, discloses that a mutated PE in which domain Ia is present
but in which the basic residues of domain Ia at positions 57, 246,
247, and 249 are replaced with acidic residues (glutamic acid, or
"E")) exhibits greatly diminished non-specific cytotoxicity. This
mutant form of PE is sometimes referred to as "PE.DELTA.E."
[0236] In various embodiments, the combination therapy comprises
administering the isolated fusion molecule composition and the
second agent composition simultaneously, either in the same
pharmaceutical composition or in separate pharmaceutical
compositions. In various embodiments, isolated fusion molecule
composition and the second agent composition are administered
sequentially, i.e., the isolated fusion molecule composition is
administered either prior to or after the administration of the
second agent composition.
[0237] In various embodiments, the administrations of the isolated
fusion molecule composition and the second agent composition are
concurrent, i.e., the administration period of the isolated fusion
molecule composition and the second agent composition overlap with
each other.
[0238] In various embodiments, the administrations of the isolated
fusion molecule composition and the second agent composition are
non-concurrent. For example, in various embodiments, the
administration of the isolated fusion molecule composition is
terminated before the second agent composition is administered. In
various embodiments, the administration second agent composition is
terminated before the isolated fusion molecule composition is
administered. In various embodiments, the administrations of the
fusion molecule of the invention, whether alone or in combination
with a therapeutic agent, can be archived with a meal, e.g. prior
to the meal, during the meal or after the meal.
[0239] In some embodiments, the administration of the fusion
molecule of the invention, whether alone or in combination with a
therapeutic agent, can be archived prior to a meal. In various
embodiments, the fusion molecule of the invention, whether alone or
in combination with a therapeutic agent, can be administered more
than 12 hours, more than 11 hours, more than 10 hours, more than 9
hours, more than 8 hours, more than 7 hours, more than 6 hours,
more than 5 hours, more than 4 hours, more than 3 hours, more than
2 hours, more than 1 hour, more than 50 minutes, more than 40
minutes, more than 30 minutes, more than 20 minutes, more than 10
minutes, more than 5 minutes, or more than 1 minute prior to the
meal. In various embodiments, the fusion molecule of the invention,
whether alone or in combination with a therapeutic agent, can be
administered less than 12 hours, less than 11 hours, less than 10
hours, less than 9 hours, less than 8 hours, less than 7 hours,
less than 6 hours, less than 5 hours, less than 4 hours, less than
3 hours, less than 2 hours, less than 1 hour, less than 50 minutes,
less than 40 minutes, less than 30 minutes, less than 20 minutes,
less than 10 minutes, less than 5 minutes, or less than 1 minute
prior to the meal. In various embodiments, the fusion molecule of
the invention, whether alone or in combination with a therapeutic
agent, can be administered between about 1 minute to about 10
minutes, between about 5 minutes to about 30 minutes, between about
20 minutes to about 60 minutes, between about 1 hour to about 3
hours, between about 2 hours to about 10 hours, or between about 5
hours to about 12 hour prior to the meal.
[0240] In some embodiments, the administration of the fusion
molecule of the invention, whether alone or in combination with a
therapeutic agent, can be archived after a meal. In various
embodiments, the fusion molecule of the invention, whether alone or
in combination with a therapeutic agent, can be administered more
than 12 hours, more than 11 hours, more than 10 hours, more than 9
hours, more than 8 hours, more than 7 hours, more than 6 hours,
more than 5 hours, more than 4 hours, more than 3 hours, more than
2 hours, more than 1 hour, more than 50 minutes, more than 40
minutes, more than 30 minutes, more than 20 minutes, more than 10
minutes, more than 5 minutes, or more than 1 minute after the meal.
In some embodiments, the fusion molecule of the invention, whether
alone or in combination with a therapeutic agent, can be
administered less than 12 hours, less than 11 hours, less than 10
hours, less than 9 hours, less than 8 hours, less than 7 hours,
less than 6 hours, less than 5 hours, less than 4 hours, less than
3 hours, less than 2 hours, less than 1 hour, less than 50 minutes,
less than 40 minutes, less than 30 minutes, less than 20 minutes,
less than 10 minutes, less than 5 minutes, or less than 1 minute
after the meal. In various embodiments, the fusion molecule of the
invention, whether alone or in combination with a therapeutic
agent, can be administered less than 12 hours, less than 11 hours,
less than 10 hours, less than 9 hours, less than 8 hours, less than
7 hours, less than 6 hours, less than 5 hours, less than 4 hours,
less than 3 hours, less than 2 hours, less than 1 hour, less than
50 minutes, less than 40 minutes, less than 30 minutes, less than
20 minutes, less than 10 minutes, less than 5 minutes, or less than
1 minute prior to the meal. In various embodiments, the fusion
molecule of the invention, whether alone or in combination with a
therapeutic agent, can be administered between about 1 minute to
about 10 minutes, between about 5 minutes to about 30 minutes,
between about 20 minutes to about 60 minutes, between about 1 hour
to about 3 hours, between about 2 hours to about 10 hours, or
between about 5 hours to about 12 hour after the meal.
Methods of Use
[0241] In another aspect, the pharmaceutical compositions
formulated for oral delivery are 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 diseases, 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 fusion molecules 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.
[0242] In various embodiments of the present disclosure,
pharmaceutical compositions comprising the fusion molecules of the
disclosure are provided for use in treating and/or preventing
inflammatory diseases. "Inflammatory diseases" include all diseases
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.
[0243] In various embodiments, the inflammatory disease is 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,
ischaemic colitis, diversion colitis, Behcet's syndrome and
indeterminate colitis.
[0244] "Crohn's disease", in accordance with the present
disclosure, is a T-helper Type 1 (Th 1) inflammatory bowel disease,
which has an immune response pattern that includes an increased
production of interleukin-12, tumour necrosis factor (TNF), and
interferon-.gamma. (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 may include drugs, nutrition supplements, surgery,
or a combination of these options. Common treatments which may 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.
[0245] "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 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 localised
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.
[0246] 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 proinflammatory cytokines that results in
tissue damage and ultimately septic shock due to failure of the
microcirculation.
[0247] Another aspect of the present disclosure relates to methods
for treatment, prophylaxis and/or prevention of an autoimmune
disease, comprising administering to said patient a therapeutically
effective amount (either as monotherapy or in a combination therapy
regimen) of a fusion molecule described herein, in pharmaceutically
acceptable carrier.
[0248] 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.
[0249] "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 may 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.
[0250] In various embodiments of the present disclosure,
pharmaceutical compositions comprising the fusion molecules of the
disclosure are provided for use in the treatment, prophylaxis
and/or prevention of a cancer, comprising administering to said
patient a therapeutically effective amount (either as monotherapy
or in a combination therapy regimen) of a fusion molecule described
herein, in pharmaceutically acceptable carrier. 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.
[0251] In various embodiments, the therapeutically effective amount
of a fusion molecule described herein will be administered in
combination with one or more other therapeutic agents. Such
therapeutic agents may 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.
[0252] In various embodiments, the present disclosure provides a
method of treating a subject having a metabolic disorder, said
method comprising orally administering a fusion molecule 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.
[0253] In another aspect, 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 fusion molecule of the present disclosure in an
amount sufficient to treat said disease.
[0254] In another aspect, the present disclosure relates to the use
of a non-naturally occurring fusion molecule of the present
disclosure for the preparation of a medicament for treatment,
prophylaxis and/or prevention of GH deficient growth disorders in a
subject in need thereof.
[0255] In another aspect, the present disclosure provides a method
of treating a subject having a GH deficient growth disorder, said
method comprising orally administering a fusion molecule 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.
Polynucleotides Encoding Fusion Molecules
[0256] In another aspect, the disclosure provides polynucleotides
comprising a nucleotide sequence encoding the non-naturally
occurring fusion molecules. These polynucleotides are useful, for
example, for making the fusion molecules. In yet another aspect,
the disclosure provides an expression system that comprises a
recombinant polynucleotide sequence encoding a modified Cholix
toxin, and a polylinker insertion site for a polynucleotide
sequence encoding a biologically active cargo. The polylinker
insertion site can be anywhere in the polynucleotide sequence so
long as the polylinker insertion does not disrupt the receptor
binding domain or the transcytosis domain of the modified Cholix
toxin. In various embodiments, the expression system may comprise a
polynucleotide sequence that encodes a cleavable linker so that
cleavage at the cleavable linker separates a biologically active
cargo encoded by a nucleic acid inserted into the polylinker
insertion site from the remainder of the encoded fusion molecule.
Thus, in embodiments where the polylinker insertion site is at an
end of the encoded construct, the polynucleotide comprises one
nucleotide sequence encoding a cleavable linker between the
polylinker insertion site and the remainder of the polynucleotide.
In embodiments where the polylinker insertion site is not at the
end of the encoded construct, the polylinker insertion site can be
flanked by nucleotide sequences that each encode a cleavable
linker.
[0257] Various in vitro methods that can be used to prepare a
polynucleotide encoding a modified Cholix toxin useful in the
fusion molecules 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 (3SR)
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 modified Cholix toxin or a nucleotide
encoding, e.g., a receptor binding domain.
[0258] 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 fusion molecule or a portion
thereof also can be isolated by screening genomic or cDNA libraries
with probes selected from the sequences of the desired
polynucleotide under stringent, moderately stringent, or highly
stringent hybridization conditions.
[0259] Construction of nucleic acids encoding the fusion molecules
of the disclosure can be facilitated by introducing an insertion
site for a nucleic acid encoding the biologically active cargo into
the construct. In various embodiments, an insertion site for the
biologically active cargo can be introduced between the nucleotides
encoding the cysteine residues of domain Ib of the modified Cholix
toxin. In other embodiments, the insertion site can be introduced
anywhere in the nucleic acid encoding the construct so long as the
insertion does not disrupt the functional domains encoded thereby.
In various embodiments, the insertion site can be in the ER
retention domain.
[0260] Further, the polynucleotides can also encode a secretory
sequence at the amino terminus of the encoded fusion molecule. Such
constructs are useful for producing the fusion molecules in
mammalian cells as they simplify isolation of the immunogen.
[0261] Furthermore, the polynucleotides of the disclosure also
encompass derivative versions of polynucleotides encoding a fusion
molecule. 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 fusion molecule.
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 MnCl.sub.2 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.
[0262] Accordingly, in various embodiments, the disclosure provides
a polynucleotide that encodes a fusion molecule. The fusion
molecule comprises a modified Cholix toxin and a biologically
active cargo to be delivered to a subject; and, optionally, a
non-cleavable or cleavable linker. Cleavage at the cleavable linker
can separate the biologically active cargo from the remainder of
the fusion molecule. The cleavable linker 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.
[0263] In various embodiments, the polynucleotide hybridizes under
stringent hybridization conditions to any polynucleotide of this
disclosure. In further embodiments, the polynucleotide hybridizes
under stringent conditions to a nucleic acid that encodes any
fusion molecule of the disclosure.
[0264] In still another aspect, the disclosure provides expression
vectors for expressing the fusion molecules. 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.
[0265] 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.
[0266] The expression vectors should contain expression and
replication signals compatible with the cell in which the fusion
molecules are expressed. Expression vectors useful for expressing
fusion molecules 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.
[0267] The expression vectors can be introduced into the cell for
expression of the fusion molecules 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.
[0268] The expression vectors can also contain a purification
moiety that simplifies isolation of the fusion molecule. 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 fusion molecule following purification. In other
embodiments, the moiety does not interfere with the function of the
functional domains of the fusion molecule and thus need not be
cleaved.
[0269] In yet another aspect, the disclosure provides a cell
comprising an expression vector for expression of the fusion
molecules, or portions thereof. The cell is selected for its
ability to express high concentrations of the fusion molecule to
facilitate purification of the protein. In various embodiments, the
cell is a prokaryotic cell, for example, E. coli. As described in
the examples, the fusion molecules are properly folded and comprise
the appropriate disulfide linkages when expressed in E. coli.
[0270] In other embodiments, 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 fusion molecules. For example, Chinese hamster ovary (CHO)
cells can be used to express the fusion molecules.
[0271] The fusion molecules of the disclosure can be produced by
recombination, as described below. However, the fusion molecules
may also be produced by chemical synthesis using methods known to
those of skill in the art.
[0272] Methods for expressing and purifying the fusion molecules of
the disclosure are described extensively in the examples below.
Generally, the methods rely on introduction of an expression vector
encoding the fusion molecule to a cell that can express the fusion
molecule from the vector. The fusion molecule can then be purified
for administration to a subject.
Transcytosis Testing
[0273] The function of the transcytosis domain can be tested as a
function of the fusion molecule's ability to pass through an
epithelial membrane. Because transcytosis first requires binding to
the cell, these assays can also be used to assess the function of
the cell recognition domain.
[0274] The fusion molecule'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 fusion molecule to enter a non-polarized
cell to which it binds. Without intending to be bound to any
particular theory or mechanism of action, it is believed that the
same property that allows a transcytosis domain to pass through a
polarized epithelial cell also allows molecules bearing the
transcytosis domain to enter non-polarized cells. Thus, the fusion
molecule'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 fusion molecule can be labeled with,
for example, a fluorescent marker, and the fusion molecule exposed
to the cell. Then, the cells can be washed, removing any fusion
molecule that has not entered the cell, and the amount of label
remaining determined. Detecting the label in this traction
indicates that the fusion molecule has entered the cell.
[0275] In other embodiments, the fusion molecule's transcytosis
ability can be tested by assessing the fusion molecule's ability to
pass through a polarized epithelial cell. For example, the fusion
molecule can be labeled with, for example, a fluorescent marker 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.
Cleavable Linker Cleavage Testing
[0276] The function of the cleavable linker 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 linkers. Both cell-based and cell-free assays can be used
to test the ability of an enzyme to cleave the cleavable
linkers.
[0277] An exemplary cell-free assay for testing cleavage of
cleavable linkers comprises preparing extracts of polarized
epithelial cells and exposing a labeled fusion molecule bearing a
cleavable linker 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 fusion molecule. 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 fusion molecule 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.
[0278] Cleavage can also be tested using cell-based assays that
test cleavage by polarized epithelial cells assembled into
membranes. For example, a labeled fusion molecule, or portion of a
fusion molecule comprising the cleavable linker, can be contacted
to either the apical or basolateral side of a monolayer of suitable
epithelial cells, such as, for example, Coco-2 cells, under
conditions that permit cleavage of the linker. Cleavage can be
detected by detecting the presence or absence of the label using a
reagent that specifically binds the fusion molecule, or portion
thereof. For example, an antibody specific for the fusion molecule
can be used to bind a fusion molecule comprising a label distal to
the cleavable linker in relation to the portion of the fusion
molecule 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 linker
in a fusion molecule can be confirmed.
[0279] 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 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.
[0280] 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.
Exemplary Cholix Toxin-Biologically Active Cargo Fusion
Molecules
[0281] Embodiments of the present disclosure include, but are not
limited to, the fusion molecules described in Table 7.
TABLE-US-00023 TABLE 7 Biologically Modified Cholix Toxin Cleavable
Linker Active Cargo (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) SEQ ID NO:
3 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 4 SEQ
ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 5 SEQ ID NOs:
96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 6 SEQ ID NOs: 96-121
SEQ ID NOs: 82-95 No Linker SEQ ID NO: 7 SEQ ID NOs: 96-121 SEQ ID
NOs: 82-95 No Linker SEQ ID NO: 8 SEQ ID NOs: 96-121 SEQ ID NOs:
82-95 No Linker SEQ ID NO: 9 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95
No Linker SEQ ID NO: 10 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No
Linker SEQ ID NO: 11 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker
SEQ ID NO: 12 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID
NO: 13 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 14
SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 15 SEQ ID
NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 16 SEQ ID NOs:
96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 17 SEQ ID NOs: 96-121
SEQ ID NOs: 82-95 No Linker SEQ ID NO: 18 SEQ ID NOs: 96-121 SEQ ID
NOs: 82-95 No Linker SEQ ID NO: 19 SEQ ID NOs: 96-121 SEQ ID NOs:
82-95 No Linker SEQ ID NO: 20 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95
No Linker SEQ ID NO: 21 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No
Linker SEQ ID NO: 22 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker
SEQ ID NO: 23 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID
NO: 24 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 25
SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 26 SEQ ID
NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 27 SEQ ID NOs:
96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 28 SEQ ID NOs: 96-121
SEQ ID NOs: 82-95 No Linker SEQ ID NO: 29 SEQ ID NOs: 96-121 SEQ ID
NOs: 82-95 No Linker SEQ ID NO: 30 SEQ ID NOs: 96-121 SEQ ID NOs:
82-95 No Linker SEQ ID NO: 31 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95
No Linker SEQ ID NO: 32 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No
Linker SEQ ID NO: 33 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker
SEQ ID NO: 34 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID
NO: 35 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 36
SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 37 SEQ ID
NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 38 SEQ ID NOs:
96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 39 SEQ ID NOs: 96-121
SEQ ID NOs: 82-95 No Linker SEQ ID NO: 40 SEQ ID NOs: 96-121 SEQ ID
NOs: 82-95 No Linker SEQ ID NO: 41 SEQ ID NOs: 96-121 SEQ ID NOs:
82-95 No Linker SEQ ID NO: 42 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95
No Linker SEQ ID NO: 43 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No
Linker SEQ ID NO: 44 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker
SEQ ID NO: 45 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID
NO: 46 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 47
SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 48 SEQ ID
NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 49 SEQ ID NOs:
96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 50 SEQ ID NOs: 96-121
SEQ ID NOs: 82-95 No Linker SEQ ID NO: 51 SEQ ID NOs: 96-121 SEQ ID
NOs: 82-95 No Linker SEQ ID NO: 52 SEQ ID NOs: 96-121 SEQ ID NOs:
82-95 No Linker SEQ ID NO: 53 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95
No Linker SEQ ID NO: 54 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No
Linker SEQ ID NO: 55 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker
SEQ ID NO: 56 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID
NO: 57 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 58
SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 59 SEQ ID
NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 60 SEQ ID NOs:
96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 61 SEQ ID NOs: 96-121
SEQ ID NOs: 82-95 No Linker SEQ ID NO: 62 SEQ ID NOs: 96-121 SEQ ID
NOs: 82-95 No Linker SEQ ID NO: 63 SEQ ID NOs: 96-121 SEQ ID NOs:
82-95 No Linker SEQ ID NO: 64 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95
No Linker SEQ ID NO: 65 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No
Linker SEQ ID NO: 66 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker
SEQ ID NO: 67 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID
NO: 68 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 69
SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 70 SEQ ID
NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 71 SEQ ID NOs:
96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 72 SEQ ID NOs: 96-121
SEQ ID NOs: 82-95 No Linker SEQ ID NO: 73 SEQ ID NOs: 96-121 SEQ ID
NOs: 82-95 No Linker SEQ ID NO: 74 SEQ ID NOs: 96-121 SEQ ID NOs:
82-95 No Linker SEQ ID NO: 75 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95
No Linker SEQ ID NO: 76 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No
Linker SEQ ID NO: 77 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker
SEQ ID NO: 78 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID
NO: 79 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 80
SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 81 SEQ ID
NOs: 96-121 SEQ ID NOs: 82-95 No Linker
[0282] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
80 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 82.
[0283] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
70 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 82.
[0284] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
42 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 82.
[0285] In various embodiments, the fusion molecule comprises the
amino acid sequence set forth in SEQ ID NO: 114.
[0286] In various embodiments, the fusion molecule comprises the
amino acid sequence set forth in SEQ ID NO: 115
[0287] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
52 and a biologically active cargo that is an antibody comprising a
heavy chain variable having the amino acid sequence of SEQ ID NO:
88 and a light chain variable having the amino acid sequence of SEQ
ID NO: 89.
[0288] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
52 and a biologically active cargo that is an antibody comprising a
heavy chain variable having the amino acid sequence of SEQ ID NO:
90 and a light chain variable having the amino acid sequence of SEQ
ID NO: 91
[0289] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
52 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 92.
[0290] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
52 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 93.
[0291] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
52 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 94.
[0292] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
52 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 95.
[0293] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
80 and a biologically active cargo that is an antibody comprising a
heavy chain variable having the amino acid sequence of SEQ ID NO:
88 and a light chain variable having the amino acid sequence of SEQ
ID NO: 89.
[0294] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
80 and a biologically active cargo that is an antibody comprising a
heavy chain variable having the amino acid sequence of SEQ ID NO:
90 and a light chain variable having the amino acid sequence of SEQ
ID NO: 91
[0295] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
80 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 92.
[0296] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
80 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 93.
[0297] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
80 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 94. In various embodiments, the fusion molecule
comprises a modified Cholix toxin having the amino acid sequence of
SEQ ID NO: 80 and a biologically active cargo having the amino acid
sequence of SEQ ID NO: 95.
[0298] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
70 and a biologically active cargo that is an antibody comprising a
heavy chain variable having the amino acid sequence of SEQ ID NO:
88 and a light chain variable having the amino acid sequence of SEQ
ID NO: 89.
[0299] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
70 and a biologically active cargo that is an antibody comprising a
heavy chain variable having the amino acid sequence of SEQ ID NO:
90 and a light chain variable having the amino acid sequence of SEQ
ID NO: 91
[0300] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
70 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 92.
[0301] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
70 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 93.
[0302] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
70 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 94.
[0303] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
70 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 95.
[0304] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
42 and a biologically active cargo that is an antibody comprising a
heavy chain variable having the amino acid sequence of SEQ ID NO:
88 and a light chain variable having the amino acid sequence of SEQ
ID NO: 89.
[0305] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
42 and a biologically active cargo that is an antibody comprising a
heavy chain variable having the amino acid sequence of SEQ ID NO:
90 and a light chain variable having the amino acid sequence of SEQ
ID NO: 91.
[0306] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
42 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 92.
[0307] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
42 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 93.
[0308] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
42 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 94.
[0309] In various embodiments, the fusion molecule comprises a
modified Cholix toxin having the amino acid sequence of SEQ ID NO:
42 and a biologically active cargo having the amino acid sequence
of SEQ ID NO: 95.
[0310] The following examples merely illustrate the disclosure, and
are not intended to limit the disclosure in any way.
Example 1
[0311] In this Example, the preparation of a non-naturally
occurring fusion molecule as a single amino acid sequence and
comprising a modified Cholix toxin sequence, a cleavable linker
sequence, and a biologically active cargo, is generally
described.
[0312] Seven exemplary fusion molecule expression vectors for
delivering the polypeptides interleukin-10 (SEQ ID NO: 82),
interleukin-19 (SEQ ID NO: 83), interleukin-20 (SEQ ID NO: 84),
interleukin-22 (SEQ ID NO: 85), interleukin-24 (SEQ ID NO: 86), or
interleukin-26 (SEQ ID NO: 87) are constructed as generally
described below. First, the polypeptide genes are amplified by PCR,
incorporating restriction enzymes pairs of NdeI and EcoRI, PstI and
PstI, AgeI and EcoRI, or PstI and EcoRI sites at two ends of the
PCR products. After restriction enzyme digestion, the PCR products
are cloned into an appropriate plasmid for cellular expression,
which is digested with the corresponding restriction enzyme pairs.
The resulting constructs comprise a modified Cholix toxin
comprising an amino acid sequence encoding amino acids 1-386 of SEQ
ID NO: 1 (Cholix.sup.386) and the respective polypeptides, and are
also tagged with a 6-His motif at the N-terminus of the polypeptide
to facilitate purification. The final plasmids are verified by
restriction enzyme digestions and DNA sequencing.
[0313] Also prepared was a non-naturally occurring fusion molecule
comprising a Cholix.sup.415 (SEQ ID NO: 52), a cleavable linker
sequence having the amino acid sequence set forth in SEQ ID NO:
121, and a biologically active cargo that is a IL-10 polypeptide
consisting of amino acid residues 20-178 of SEQ ID NO: 82 (this
fusion molecule is designated "Cholix.sup.415-TEV-IL-10", see FIG.
1 (SEQ ID NO: 122)), and a non-naturally occurring fusion molecule
comprising a Cholix.sup.415 (SEQ ID NO: 52), a non-cleavable linker
sequence having the amino acid sequence set forth in SEQ ID NO: 98,
and a biologically active cargo that is a IL-10 polypeptide
consisting of amino acid residues 20-178 of SEQ ID NO: 82 (this
fusion molecule is designated
"Cholix.sup.415-(G.sub.4S).sub.3-IL-10", see FIG. 1 (SEQ ID NO:
123)).
[0314] Expression vectors comprising non-cleavable or cleavable
linkers are constructed by introducing sequences encoding the
appropriate amino acid sequence. To do so, oligonucleotides that
encode sequences complementary to appropriate restriction sites and
the amino acid sequence of the desired linker are synthesized, then
ligated into an expression vector prepared as described above
between the modified Cholix sequence and the polypeptide
sequence.
[0315] In various embodiments, the fusion molecules are expressed
as follows: E. coli BL21(DE3) pLysS competent cells (Novagen,
Madison, Ws.) are transformed using a standard heat-shock method in
the presence of the appropriate plasmid to generate fusion molecule
expression cells, selected on ampicillin-containing media, and
isolated and grown in Luria-Bertani broth (Difco; Becton Dickinson,
Franklin Lakes, N.J.) with antibiotic, then induced for protein
expression by the addition of 1 mM
isopropyl-D-thiogalactopyranoside (IPTG) at OD 0.6. Two hours
following IPTG induction, cells are harvested by centrifugation at
5,000 rpm for 10 min. Inclusion bodies are isolated following cell
lysis and proteins are solubilized in the buffer containing 100 mM
Tris-HCl (pH 8.0), 2 mM EDTA, 6 M guanidine HCl, and 65 mM
dithiothreitol. Solubilized fusion molecule is refolded in the
presence of 0.1 M Tris, pH=7.4, 500 mM L-arginine, 0.9 mM GSSG, 2
mM EDTA. The refolded proteins are purified by Q sepharose Ion
Exchange and Superdex 200 Gel Filtration chromatography (Amersham
Biosciences, Inc., Sweden). The purity of proteins is assessed by
SDS-PAGE and analytic HPLC (Agilent, Inc. Palo Alto, Calif.).
[0316] FIG. 2 is a ribbon diagram representation of an exemplary
fusion molecule, e.g., Cholix.sup.415-TEV-IL-10 after refolding
that would be driven by IL-10 dimerization. IL-10 dimerization is
envisaged to result in purple Cholix.sup.415/blue hlL-10 and orange
Cholix.sup.415/green organization shown.
[0317] Cholix.sup.415-TEV-IL-10 and
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 were evaluated to verify the
proper folding with regard to their anticipated molecular size.
Following induction, expressed protein was collected from inclusion
bodies. The extent of Cholix.sup.415-TEV-IL-10 (depicted as "C" on
the gel) expression and Cholix.sup.415-(G.sub.4S).sub.3-IL-10
(depicted as "N" on the gel) expression in inclusion bodies showed
an apparent molecular weight of .about.66 kDa that was comparable
to the calculated mass of 66380.78 and 65958.25 Daltons,
respectively. See FIG. 3. The lack of these proteins in supernatant
media following inclusion body removal for the TEV linker (Cs) and
non-TEV linker (Ns) are shown to demonstrate the extent and
specificity of chimera induction. SeeBlue.RTM. Plus2 Prestained MW
standards are shown.
Example 2
[0318] This example describes in vitro methods to verify the proper
folding of the fusion molecules with regard to their ability to
carry a biologically active cargo across an intact epithelium.
[0319] The J774 mouse macrophage cell line can be used as an IL-10
responsive cell line (O'Farrell A M, et al., EMBO J, 17(4):1006-18,
1998). IL-10 naturally forms a dimer that is required for its
optimal activity. Cholix.sup.415-(G.sub.4S).sub.3-IL-10 expressed
by E coli was collected from inclusion bodies and folded using a
disulphide shuffle exchange buffer system. The resulting material
was purified by ion exchange and size exclusion chromatography that
resulted in the isolation of a protein of .about.130 kDa, the
anticipated size of an IL-10 dimer conjoined to two Cholix.sup.415
molecules (hereinafter "dimer Cholix.sup.415-IL-10" fusion
molecule). The preparation had a protein purity of .about.85-90%
based upon SDS PAGE. Cultures of the J774.2 cell line were treated
for 48 h with dimer Cholix.sup.415-IL-10 fusion molecule at
concentrations of 25 nM and 250 nM. Compared to untreated matched
cells, dimer Cholix.sup.415-IL-10 fusion molecule produced a
dose-dependent decrease in cell number as assessed by flow
cytometry of live/dead cells (see FIG. 4). Values represent
n=4.+-.standard deviation.
[0320] Alternatively, one could co-culture the IL-10 responsive
cells in the basal compartment of the cell monolayers used for
apical to basolateral transcytosis (Rubas W, et al., Pharm Res.
13(1):23-6, 1996).
Example 3
[0321] In this example, dimer Cholix.sup.415-(G.sub.4S).sub.3-IL-10
fusion molecule was evaluated for its effect on the barrier
properties of Caco-2 cell monolayers in vitro. Caco-2 cells (a
human colon cancer derived cell line) with media from the
basolateral compartment being sampled periodically for several
hours (Rubas W, et al., J Pharm Sci., 85(2):165-9, 1996). Caco-2
(ATCC HTB-37.TM.) cells are maintained in 5% CO.sub.2 at 37.degree.
C. in complete media: Dulbecco's modified Eagle's medium F12 (DMEM
F12) supplemented with 10% fetal bovine serum, 2.5 mM glutamine,
100 U of penicillin/ml, and 100 .mu.g of streptomycin/ml (Gibco
BRL, Grand Island, N.Y.). Cells are fed every 2 to 3 days with this
media (designated complete medium) and passaged every 5 to 7 days.
For assays, cells are seeded into 24- or 96-well plates and grown
to confluence.
[0322] Established Caco-2 monolayers used for these studies had
transepithelial electrical resistance (TER) values of between
.about.450-600 .OMEGA.cm.sup.2 (579 .OMEGA.cm.sup.2 average) as
measured using a chopstick Millicell-ERS.RTM. voltmeter
(Millipore). Fluorescein-labeled 70 kDa dextran and varying
concentrations (4.7 nM, 23.6 nM and 236 nM) of dimer
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule were added to
the apical surface of these monolayers and the cumulative amount of
florescence detected in the basal compartment monitored over time
by collecting 150 .mu.L volumes with replacement. As depicted in
FIG. 5 and FIG. 6, in the absence of Caco-2 cells on the filter
support, the dextran rapidly moved from the apical to basal
compartment. By comparison, the extent of 70 kDa dextran transport
was much less across Caco-2 monolayers and the various dimer
Cholix.sup.415-IL-10 fusion molecules failed to have any
dose-dependent effect on the extent of 70 kDa dextran transport
across these Caco-2 monolayers and were not strikingly different
from results obtained with Caco-2 monolayers not exposed to dimer
Cholix.sup.415-IL-10 fusion molecules. The dimer
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule does not
overtly affect the barrier properties of Caco-2 cell monolayers in
vitro.
Example 4
[0323] In this example, an ELISA assay is performed to evaluate the
ability of the dimer Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion
molecule to move across Caco-2 cell monolayers. A549 (ATCC
CCL-185.TM.), L929 (ATCC CRL-2148.TM.), and Caco-2 (ATCC
HTB-37.TM.) cells are maintained in 5% CO.sub.2 at 37.degree. C. in
complete media: Dulbecco's modified Eagle's medium F12 (DMEM F12)
supplemented with 10% fetal bovine serum, 2.5 mM glutamine, 100 U
of penicillin/ml, and 100 .mu.g of streptomycin/ml (Gibco BRL,
Grand Island, N.Y.). Cells are fed every 2 to 3 days with this
media (designated complete medium) and passaged every 5 to 7 days.
For assays, cells are seeded into 24- or 96-well plates and grown
to confluence.
[0324] Caco-2 cells are grown as confluent monolayers on
collagen-coated 0.4-.mu.m pore size polycarbonate membrane
transwell supports (Corning-Costar, Cambridge, Mass.) and used
18-25 days after attaining a trans-epithelial electrical resistance
(TER) of >250 .OMEGA.cm2 as measured using a chopstick
Millicell-ERS.RTM. voltmeter (Millipore). Apical to basolateral
(A.fwdarw.B) transport of dimer
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule across these
monolayer is determined by measuring the amount of transported
protein 4 hr after a 4.7 nM, 23.6 nM and 236 nM application at
37.degree. C. TER measurements and the extent of 10 kDa fluorescent
dextran (measured using an HPLC size exclusion protocol) are used
to verify monolayer barrier properties during the course of the
study. The extent of Cholix transport is determined by titration of
collected media in the cell-based cytotoxicity assay. Transported
dimer Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule is
measured by enzyme linked immunosorbant assay (ELISA) using
anti-IL-10 antibody for capture and the polyclonal sera to Cholix
for detection. As depicted in FIG. 7A and FIG. 7B, dimer
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule moves across
Caco-2 cell monolayers.
Example 5
[0325] In this Example, the preparation of a non-naturally
occurring fusion molecule that lacks a cleavable sequence is
described. These fusions molecules are designed to specifically
target the submucosal/GI space and limit the actions of the
biologically active cargo to that space.
[0326] A plasmid construct is prepared encoding the non-toxic
mutant form of the Cholix toxin, Cholix toxin .DELTA.E581 (SEQ ID
NO: 81). Protein expression is achieved using E. coli DH5a cells
(Invitrogen, Carlsbad, Calif.) following transformation by
heat-shock (1 min at 42.degree. C.) with the appropriate plasmid.
Transformed cells, selected on antibiotic-containing media, are
isolated and grown in Luria-Bertani broth (Difco). Protein
expression is induced by addition of 1 mM
isopropyl-D-thiogalactopyranoside (IPTG). Two hours following IPTG
induction, cells are harvested by centrifugation at 5,000.times.g
for 10 min at 4.degree. C. Inclusion bodies are isolated following
cell lysis and proteins are solubilized in 6 M guanidine HCl and 2
mM EDTA (pH 8.0) plus 65 mM dithiothreitol. Following refolding and
purification, proteins are stored at -5 ml/ml in PBS (pH 7.4)
lacking Ca.sup.2+ and Mg.sup.2+ at -80.degree. C. All proteins used
in these studies are confirmed to be at >90% purity based upon
size exclusion chromatography.
[0327] The Cholix toxin .DELTA.E581 protein is then modified at its
C-terminus to allow direct chemical coupling through a free
sulfhydryl residue located near the C-terminus of the protein. The
C-terminal modification includes a cysteine-constrained loop
harboring the consensus cleavage sequence for the highly selective
protease from the tobacco etch virus (TEV), a second cysteine, and
a hexa-histadine (His.sub.6) tag. The second Cys is included to
form a disulphide bridge with the Cys ultimately used for coupling.
Adding the His.sub.6 sequence to the protein simplifies the
purification and the TEV cleavage sequence provides a mechanism to
selectively remove the terminal Cys residue following mild
reduction. TEV cleavage and mild reduction with 0.1 mM
dithiotheitol following expression and isolation of the ntCholix
constructs allows for the direct chemical coupling of a
biologically active cargo via a maleimide-based reaction as a
generic mechanism of cargo attachment. Following TEV protease
cleavage, reduction, and cargo coupling through a maleimide
reaction with the free sulfhydryl, removal of the freed C-terminal
sequence was achieved by a second Ni.sup.2+ column chromatography
step.
Example 6
[0328] Trans-epithelial transport of Cholix toxin .DELTA.E581-cargo
is assessed using Caco-2 monolayers in vitro. Caco-2 cells (passage
number 25-35) are grown to confluent monolayers as previously
described; Rubas, W. et al., Pharm Res, 10:113-118 (1993). Briefly,
cells are maintained at 37.degree. C. in DMEM/high growth media
enriched with 2 mM L-glutamine, 10% fetal bovine serum, and 100
Units of penicillin/streptomycin in an atmosphere of 5% CO.sub.2
and 90% humidity. Cells are passaged every week at a split ratio of
1:3 in 75 cm.sup.2 flasks and seeded onto prewetted and
collagen-coated permeable (0.4 .mu.m pore size) polycarbonate
(Transwell.TM.) filter supports from Corning Costar (Cambridge,
Mass.) at a density of 63,000 cells/cm.sup.2. Growth media is
replaced every other day. Confluent monolayers, determined by the
acquisition of significant trans-epithelial resistance (TEER)
determine using an volt-ohm-meter (World Precision Instruments,
Sarasota, Fla.), are used 20-26 days post seeding.
[0329] Trans-epithelial transport flux rates are measured in vitro
in the apical (Ap) to basolateral (BI) and the BI to Ap directions
using polarized monolayers of Caco-2 cells to describe mucosal to
serosal and serosal to mucosal flux events, respectively. Just
prior to initiation of a transport study, the transepithelial
resistance (TEER) of each filter is measured; monolayers TEER
reading of <200 .OMEGA.cm2 are excluded from the study. Ap and
BI media is removed from included monolayers and these surfaces are
washed once with phosphate buffered saline (PBS). One set of
monolayers then receives an Ap (donor) application of 100 .mu.L PBS
containing 10 .mu.g Cholix toxin .DELTA.E581-cargo and 10 .mu.g
TRITC-Dextran or 10 .mu.g BSA-cargo and 10 .mu.g TRITC-Dextran.
Receiver (BI) compartments then receive 500 .mu.L PBS to set the
T.sub.0 for the transport study. Both donor and receiver
compartments are sampled after 4 hr of incubation at 37.degree. C.
to determine the amount of material transported across the
monolayer and the amount retained at the apical surface,
respectively.
Example 7
[0330] This example describes the preparation and expression in E.
coli. of a fusion molecule comprising a modified Cholix toxin
comprising a sequence encoding amino acids 1-415 of SEQ ID NO: 1
directly fused at its C-terminus to an IL-10 polypeptide (referred
to as a "Cholix.sup.415-IL-10 fusion molecule"). Protein expression
is achieved using E. coli DH5a cells (Invitrogen, Carlsbad, Calif.)
following transformation by heat-shock (1 min at 42.degree. C.)
with the appropriate plasmid. Transformed cells, selected on
antibiotic-containing media, are isolated and grown in
Luria-Bertani broth (Difco). Protein expression is induced by
addition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG). Two
hours following IPTG induction, cells are harvested by
centrifugation at 5,000.times.g for 10 min at 4.degree. C.
Inclusion bodies are isolated following cell lysis and proteins are
solubilized in 6 M guanidine HCl and 2 mM EDTA (pH 8.0) plus 65 mM
dithiothreitol. Following refolding and purification, proteins are
stored at .about.5 ml/ml in PBS (pH 7.4) lacking Ca.sup.2+ and
Mg.sup.2+ at -80.degree. C. All proteins used in these studies were
confirmed to be at >90% purity based upon size exclusion
chromatography.
[0331] Polystyrene beads (10 nm diameter) containing a covalently
integrated red fluorescent dye with excitation/emission properties
of 468/508 nm and having aldehyde surface functional groups
(XPR-582) are obtained from Duke Scientific (Palo Alto, Calif.).
One hundred .mu.l of XPR-582 beads (at 2% solids) are mixed with
approximately 2.5 nmoles IL-10 or Cholix.sup.415-IL-10 fusion
molecule in a final volume of 200 .mu.l neutral (pH 7.0) phosphate
buffered saline (PBS). After 2 hr of gentle rocking at room
temperature, 20 .mu.l of a 2 mg/ml solution of bovine serum albumin
(BSA; Sigma, St. Louis, Mo.) in PBS is added. Preparations are then
dialyzed by three cycles of dilution with PBS and concentration
using a 100,000 molecular weight cutoff Microcon filter device from
Millipore (Bedford, Mass.). Final preparations of coated beads were
at 1% solids.
Example 8
[0332] In this Example, non-naturally occurring isolated fusion
molecules comprising the modified Cholix toxin sequence of SEQ ID
NO: 52 (Cholix.sup.415), a cleavable linker sequence (SEQ ID NO:
121) or a non-cleavable linker (SEQ ID NO: 98), and a biologically
active cargo that is a TNFSF inhibitor, are prepared as described
in Example 1, and evaluated as described in the Examples above to
confirm proper folding, proper size,
[0333] Six exemplary fusion molecule expression vectors (3 for each
linker) were prepared to test for the ability of the fusion
molecules to transport apical to basal across epithelial cells a
TNFSF inhibitor selected from: 1) a TNF inhibitor that is an
antibody comprising the heavy chain variable region and light chain
variable region sequences of SEQ ID NO: 88 and 89; 2) a TNF
inhibitor that is an antibody comprising the heavy chain variable
region and light chain variable region sequences of SEQ ID NO: 90
and 91; and 3) a TNFSF inhibitor that is a dimer of a soluble human
TNFR-p75 with the Fc portion of IgG comprising the sequence of SEQ
ID NO: 92.
Example 9
[0334] In this Example, non-naturally occurring isolated fusion
molecules comprising the modified Cholix toxin sequence of SEQ ID
NO: 52 (Cholix.sup.415), a cleavable linker sequence (SEQ ID NO:
121) or a non-cleavable linker (SEQ ID NO: 98), and a biologically
active cargo that is a glucose-lowering agent, are prepared as
described in Example 1, and evaluated as described in the Examples
above to confirm proper folding, proper size,
[0335] Four exemplary fusion molecule expression vectors (2 for
each linker) were prepared to test for the ability of the fusion
molecules to transport apical to basal across epithelial cells a
glucose-lowering agent selected from: 1) a GLP-1 agonist comprising
the sequence of SEQ ID NO: 93; and 2) a GLP-1 agonist comprising
the sequence of SEQ ID NO: 94.
Example 10
[0336] In this Example, non-naturally occurring isolated fusion
molecules comprising the modified Cholix toxin sequence of SEQ ID
NO: 52 (Cholix.sup.415), a cleavable linker sequence (SEQ ID NO:
121) or a non-cleavable linker (SEQ ID NO: 98), and a biologically
active cargo that is a human growth hormone, are prepared as
described in Example 1, and evaluated as described in the Examples
above to confirm proper folding, proper size,
[0337] Two exemplary fusion molecule expression vectors (one for
each linker) were prepared to test for the ability of the fusion
molecules to transport apical to basal across epithelial cells a
human growth hormone comprising the sequence of SEQ ID NO: 95.
Example 11
[0338] This example describes histological detection in tissues of
a representative biologically active cargo of the fusion molecules
prepared in Example 1. Following administration of a fusion
molecule, animals are euthanized by CO.sub.2 asphyxiation and
exsanguinated by cardiac puncture. Specific tissues (lymph nodes,
trachea, brain, spleen liver, GI tract) are removed, briefly rinsed
in PBS to remove any residual blood and frozen in OCT. Sections (5
microns thick) are placed onto slides. Slides are fixed in acetone
for 10 min and rinsed with PBS. Slides are incubated with 3%
peroxidase for 5 min. Slides are then blocked with protein for an
additional 5 min. Primary antibody to the respective biologically
active cargo is incubated onto slides for 30 min at a 1:100
dilution followed by PBS washes. Biotin-labeled secondary antibody
is then incubated for approximately 15 minutes followed by PBS
washes. Streptavidin HRP label is incubated onto slides for 15 min
followed by PBS washes. HRP Chromagen is applied for 5 min followed
by several rinses in distilled H.sub.2O. Finally, the slides are
counterstained with hematoxylin for 1 min, coverslipped, and
examined for the presence of the biologically active cargo.
[0339] The fusion molecules of the disclosure offer several
advantages over conventional techniques for local or systemic
delivery of macromolecules to a subject. Foremost among such
advantages is the ability to deliver the biologically active cargo
to a subject without using a needle to puncture the skin of the
subject. Many subjects require repeated, regular doses of
macromolecules. For example, diabetics must inject insulin several
times per day to control blood sugar concentrations. Such subjects'
quality of life would be greatly improved if the delivery of a
macromolecule could be accomplished without injection, by avoiding
pain or potential complications associated therewith.
[0340] In addition, coupling of the biologically active cargo to
the remainder of the fusion molecule with a linker that is cleaved
by an enzyme present at a basolateral membrane of an epithelial
cell allows the biologically active cargo to be liberated from the
fusion molecule and released from the remainder of the fusion
molecule soon after transcytosis across the epithelial membrane.
Such liberation reduces the probability of induction of an immune
response against the biologically active cargo. It also allows the
biologically active cargo to interact with its target free from the
remainder of the fusion molecule.
[0341] In addition, the non-naturally occurring fusion molecules
which lack a cleavable linker can be advantageous in that the
anchoring effect of the modified Cholix toxin by its receptor(s) at
the surface of, e.g., immune cells that also express the receptor
for the biologically active cargo (but in considerably lower
quantity) can allow for greater exposure of the biologically active
cargo at the surface of the targeted cells, and provide a
synergistic effect via the binding of the Cholix to its receptor
and, e.g., binding of IL-10 to the IL-10R.
[0342] Moreover, once transported across the GI epithelium, the
fusion molecules of the disclosure will exhibit extended half-life
in serum, that is, the biologically active cargo of the fusion
molecules will exhibit an extended serum half-life compared to the
biologically active cargo in its non-fused state, and oral
administration of the fusion molecule 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.
[0343] Furthermore, the embodiments of the fusion molecules can be
constructed and expressed in recombinant systems. Recombinant
technology allows one to make a fusion molecule having an insertion
site designed for introduction of any suitable biologically active
cargo. Such insertion sites allow the skilled artisan to quickly
and easily produce fusion molecules for delivery of new
biologically active cargo, should the need to do so arise.
[0344] 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 may 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 may be practiced in the absence of any element(s) not
specifically disclosed herein. Thus, for example, in each instance
herein any of the terms "comprising", "consisting essentially of",
and "consisting of" may be replaced with either of the other two
terms. 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 may 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
[0345] The amino acid sequences listed in the accompanying sequence
listing are shown using standard three letter code for amino acids,
as defined in 37 C.F.R. 1.822.
[0346] SEQ ID NO: 1 is the 634 amino acid sequence of mature Vibrio
cholera Cholix toxin.
[0347] SEQ ID NO: 2 is a nucleic acid sequence encoding the 634
amino acid sequence mature V. cholera Cholix toxin.
[0348] SEQ ID NOs: 3-80 are the amino acid sequences of various
truncated Cholix toxins derived from the mature Cholix toxin
sequence set forth in SEQ ID NO: 1.
[0349] SEQ ID NO: 81 is the amino acid sequence of a mutated Cholix
toxin wherein the amino acid residue E581 of SEQ ID NO: 1 has been
deleted.
[0350] SEQ ID NO: 82 is the amino acid sequence of human
interleukin-10
[0351] SEQ ID NO: 83 is the amino acid sequence of human
interleukin-19 (IL-19).
[0352] SEQ ID NO: 84 is the amino acid sequence of human
interleukin-20 (IL-20).
[0353] SEQ ID NO: 85 is the amino acid sequence of human
interleukin-22 (IL-22).
[0354] SEQ ID NO: 86 is the amino acid sequence of human
interleukin-24 (IL-24).
[0355] SEQ ID NO: 87 is the amino acid sequence of human
interleukin-26 (IL-26).
[0356] SEQ ID NO: 88--heavy chain variable region sequence for an
anti-TNF-alpha antibody.
[0357] SEQ ID NO: 89--light chain variable region sequence for an
anti-TNF-alpha antibody.
[0358] SEQ ID NO: 90--heavy chain variable region sequence for an
anti-TNF-alpha antibody.
[0359] SEQ ID NO: 91--light chain variable region sequence for an
anti-TNF-alpha antibody.
[0360] SEQ ID NO: 92--amino acid sequence of human TNFR-p75-Fc
dimeric fusion protein.
[0361] SEQ ID NO: 93--GLP-1 agonist peptide amino acid sequence
(exenatide)
[0362] SEQ ID NO: 94--GLP-1 agonist peptide amino acid sequence
(Liraglutide)
[0363] SEQ ID NO: 95--amino acid sequence of human growth hormone
(somatotropin)
[0364] SEQ ID NOs: 96-121 are the amino acid sequences of various
peptide linkers
[0365] SEQ ID NO: 122 is the amino acid sequence of a
Cholix.sup.415-TEV-IL-10 fusion molecule.
[0366] SEQ ID NO: 123 is the amino acid sequence of a
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule.
TABLE-US-00024 SEQUENCE LISTINGS SEQ ID NO: 1-mature Vibrio cholera
Cholix toxin amino acid sequence
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQAL
TREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTG
EYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESA
GGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK SEQ ID
NO: 2-nucleic acid sequence encoding the mature V. cholera Cholix
toxin
ATGGTCGAAGAAGCTTTAAACATCTTTGATGAATGCCGTTCGCCATGTTCGTTGACCCCGGAACCGG
GTAAGCCGATTCAATCAAAACTGTCTATCCCTAGTGATGTTGTTCTGGATGAAGGTGTTCTGTATTAC
TCGATGACGATTAATGATGAGCAGAATGATATTAAGGATGAGGACAAAGGCGAGTCCATTATCACTAT
TGGTGAATTTGCCACAGTACGCGCGACTAGACATTATGTTAATCAAGATGCGCCTTTTGGTGTCATCC
ATTTAGATATTACGACAGAAAATGGTACAAAAACGTACTCTTATAACCGCAAAGAGGGTGAATTTGCA
ATCAATTGGTTAGTGCCTATTGGTGAAGATTCTCCTGCAAGCATCAAAATCTCCGTTGATGAGCTCGA
TCAGCAACGCAATATCATCGAGGTGCCTAAACTGTATAGTATTGATCTCGATAACCAAACGTTAGAGC
AGTGGAAAACCCAAGGTAATGTTTCTTTTTCGGTAACGCGTCCTGAACATAATATCGCTATCTCTTGG
CCAAGCGTGAGTTACAAAGCAGCGCAGAAAGAGGGTTCACGCCATAAGCGTTGGGCTCATTGGCAT
ACAGGCTTAGCACTGTGTTGGCTTGTGCCAATGGATGCTATCTATAACTATATCACCCAGCAAAATTG
TACTTTAGGGGATAATTGGTTTGGTGGCTCTTATGAGACTGTTGCAGGCACTCCGAAGGTGATTACG
GTTAAGCAAGGGATTGAACAAAAGCCAGTTGAGCAGCGCATCCATTTCTCCAAGGGGAATGCGATGA
GCGCACTTGCTGCTCATCGCGTCTGTGGTGTGCCATTAGAAACTTTGGCGCGCAGTCGCAAACCTC
GTGATCTGACGGATGATTTATCATGTGCCTATCAAGCGCAGAATATCGTGAGTTTATTTGTCGCGACG
CGTATCCTGTTCTCTCATCTGGATAGCGTATTTACTCTGAATCTTGACGAACAAGAACCAGAGGTGGC
TGAACGTCTAAGTGATCTTCGCCGTATCAATGAAAATAACCCGGGCATGGTTACACAGGTTTTAACC
GTTGCTCGTCAGATCTATAACGATTATGTCACTCACCATCCGGGCTTAACTCCTGAGCAAACCAGTG
CGGGTGCACAAGCTGCCGATATCCTCTCTTTATTTTGCCCAGATGCTGATAAGTCTTGTGTGGCTTCA
AACAACGATCAAGCCAATATCAACATCGAGTCTCGTTCTGGCCGTTCATATTTGCCTGAAAACCGTGC
GGTAATCACCCCTCAAGGCGTCACAAATTGGACTTACCAGGAACTCGAAGCAACACATCAAGCTCTG
ACTCGTGAGGGTTATGTGTTCGTGGGTTACCATGGTACGAATCATGTCGCTGCGCAAACCATCGTGA
ATCGCATTGCCCCTGTTCCGCGCGGCAACAACACTGAAAACGAGGAAAAGTGGGGCGGGTTATATG
TTGCAACTCACGCTGAAGTTGCCCATGGTTATGCTCGCATCAAAGAAGGGACAGGGGAGTATGGCC
TTCCGACCCGTGCTGAGCGCGACGCTCGTGGGGTAATGCTGCGCGTGTATATCCCTCGTGCTTCAT
TAGAACGTTTTTATCGCACGAATACACCTTTGGAAAATGCTGAGGAGCATATCACGCAAGTGATTGGT
CATTCTTTGCCATTACGCAATGAAGCATTTACTGGTCCAGAAAGTGCGGGCGGGGAAGACGAAACTG
TCATTGGCTGGGATATGGCGATTCATGCAGTTGCGATCCCTTCGACTATCCCAGGGAACGCTTACGA
AGAATTGGCGATTGATGAGGAGGCTGTTGCAAAAGAGCAATCGATTAGCACAAAACCACCTTATAAA
GAGCGCAAAGATGAACTTAAG SEQ ID NO: 3-modified Vibrio cholera Cholix
toxin amino acid sequence Cholix.sup.386
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ A
SEQ ID NO: 4-modified Vibrio cholera Cholix toxin amino acid
sequence Cholix.sup.385
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ SEQ
ID NO: 5-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.384
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGA SEQ
ID NO: 6-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.383
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAG SEQ
ID NO: 7-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.382
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSA SEQ ID
NO: 8-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.381
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTS SEQ ID
NO: 9-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.380
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQT SEQ ID
NO: 10-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.379
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQ SEQ ID
NO: 11-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.378
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPE SEQ ID NO:
12-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.377
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTP SEQ ID NO:
13-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.376
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLT SEQ ID NO:
14-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.375
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGL SEQ ID NO:
15-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.374
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPG SEQ ID NO:
16-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.373
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHP SEQ ID NO:
17-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.372
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHH SEQ ID NO:
18-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.371
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTH SEQ ID NO:
19-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.379
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVT SEQ ID NO:
20-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.369
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYV SEQ ID NO:
21-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.368
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDY SEQ ID NO:
22-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.367
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYND SEQ ID NO:
23-modified Vibrio cholera Cholix toxin amino acid sequence
Cholix.sup.366
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYN SEQ ID NO: 24-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.365
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIY SEQ ID NO: 25-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.364
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQI SEQ ID NO: 26-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.363
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQ SEQ ID NO: 27-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.362
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVAR SEQ ID NO: 28-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.361
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVA SEQ ID NO: 29-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.360
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTV SEQ ID NO: 30-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.359
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLT SEQ ID NO: 31-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.358
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVL SEQ ID NO: 32-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.357
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQV SEQ ID NO: 33-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.356
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQ SEQ ID NO: 34-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.355
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVT SEQ ID NO: 35-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.354
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMV SEQ ID NO: 36-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.353
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGM SEQ ID NO: 37-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.352
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPG SEQ ID NO: 38-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.351
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNP SEQ ID NO: 39-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.350
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENN SEQ ID NO: 40-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.349
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINEN SEQ ID NO: 41-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.348
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINE SEQ ID NO: 42-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.425
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPEN SEQ ID NO: 43-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.424
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPE SEQ ID NO: 44-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.423
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSGRSYLP SEQ ID NO: 45-modified
Vibrio cholera Cholix toxin amino acid sequence Cholix.sup.422
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSGRSYL SEQ ID NO: 46-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.421
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSGRSY SEQ ID NO: 47-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.420
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSGRS SEQ ID NO: 48-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.419
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSGR SEQ ID NO: 49-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.418
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSG SEQ ID NO: 50-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.417
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRS SEQ ID NO: 51-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.416
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESR SEQ ID NO: 52-modified Vibrio
cholera Cholix toxin amino acid sequence
Cholix.sup.415
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIES SEQ ID NO: 53-modified Vibrio
cholera Cholix toxin amino acid sequence Cholix.sup.414
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIE SEQ ID NO: 54-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.413
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINI SEQ ID NO: 55-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.412
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANIN SEQ ID NO: 56-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.411
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANI SEQ ID NO: 57-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.410
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQAN SEQ ID NO: 58-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.409
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQA SEQ ID NO: 59-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.408
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQ SEQ ID NO: 60-modified Vibrio cholera
Cholix toxin amino acid sequence Cholix.sup.407
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNND SEQ ID NO: 61-modified Vibrio cholera Cholix
toxin amino acid sequence Cholix.sup.406
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNN SEQ ID NO: 62-modified Vibrio cholera Cholix
toxin amino acid sequence Cholix.sup.405
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASN SEQ ID NO: 63-modified Vibrio cholera Cholix
toxin amino acid sequence Cholix.sup.404
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVAS SEQ ID NO: 64-modified Vibrio cholera Cholix
toxin amino acid sequence Cholix.sup.403
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVA SEQ ID NO: 65-modified Vibrio cholera Cholix
toxin amino acid sequence Cholix.sup.402
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCV SEQ ID NO: 66-modified Vibrio cholera Cholix
toxin amino acid sequence Cholix.sup.401
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSC SEQ ID NO: 67-modified Vibrio cholera Cholix toxin
amino acid sequence Cholix.sup.400
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKS SEQ ID NO: 68-modified Vibrio cholera Cholix toxin
amino acid sequence Cholix.sup.399
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADK SEQ ID NO: 69-modified Vibrio cholera Cholix toxin
amino acid sequence Cholix.sup.398
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDAD SEQ ID NO: 70-modified Vibrio cholera Cholix toxin
amino acid sequence Cholix.sup.397
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDA SEQ ID NO: 71-modified Vibrio cholera Cholix toxin
amino acid sequence Cholix.sup.396
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPD SEQ ID NO: 72-modified Vibrio cholera Cholix toxin
amino acid sequence Cholix.sup.395
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCP SEQ ID NO: 73-modified Vibrio cholera Cholix toxin amino
acid sequence Cholix.sup.394
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFC SEQ ID NO: 74-modified Vibrio cholera Cholix toxin amino
acid sequence Cholix.sup.393
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLF SEQ ID NO: 75-modified Vibrio cholera Cholix toxin amino
acid sequence Cholix.sup.392
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSL SEQ ID NO: 76-modified Vibrio cholera Cholix toxin amino
acid sequence Cholix.sup.391
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILS SEQ ID NO: 77-modified Vibrio cholera Cholix toxin amino
acid sequence Cholix.sup.390
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADIL SEQ ID NO: 78-modified Vibrio cholera Cholix toxin amino acid
sequence Cholix.sup.389
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADI SEQ ID NO: 79-modified Vibrio cholera Cholix toxin amino acid
sequence Cholix.sup.388
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ AAD
SEQ ID NO: 80-modified Vibrio cholera Cholix toxin amino acid
sequence Cholix.sup.387
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ AA
SEQ ID NO: 81-modified Vibrio cholera Cholix toxin amino acid
sequence Cholix .DELTA.581
VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITI
GEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVD
ELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHK
RWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVE
QRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLD
SVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ
AADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQAL
TREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTG
EYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESA
GGEDTVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK SEQ ID
NO: 82-human interleukin-10 amino acid sequence
MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD
NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRC
HRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO:
83-human interleukin-19 amino acid sequence
MKLQCVSLWLLGTILILCSVDNHGLRRCLISTDMHHIEESFQEIKRAIQAKDTFPNVTILSTLETLQ
IIKPLDVCCVTKNLLAFYVDRVFKDHQEPNPKILRKISSIANSFLYMQKTLRQCQEQRQCHCRQE
ATNATRVIHDNYDQLEVHAAAIKSLGELDVFLAWINKNHEVMSSA SEQ ID NO: 84-human
interleukin-20 amino acid sequence
MKASSLAFSLLSAAFYLLWTPSTGLKTLNLGSCVIATNLQEIRNGFSEIRGSVQAKDGNIDIRILR
RTESLQDTKPANRCCLLRHLLRLYLDRVFKNYQTPDHYTLRKISSLANSFLTIKKDLRLCHAHMT
CHCGEEAMKKYSQILSHFEKLEPQAAVVKALGELDILLQWMEETE SEQ ID NO: 85-human
interleukin-22 amino acid sequence
MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEAS
LADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSN
RLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI SEQ ID NO:
86-human interleukin-24 amino acid sequence
MNFQQRLQSLWTLASRPFOPPLLATASQMQMVVLPOLGFTLLLWSQVSGAQGQEFHFGPOQ
VKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKN
YHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALT
KALGEVDILLTWMQKFYKL SEQ ID NO: 87-human interleukin-26 amino acid
sequence
MLVNFILRCGLLLVTLSLAIAKHKQSSFTKSCYPRGTLSQAVDALYIKAAWLKATIPEDRIKNIRLL
KKKTKKQFMKNCQFQEQLLSFFMEDVFGQLQLQGCKKIRFVEDFHSLRQKLSHCISCASSARE
MKSITRMKRIFYRIGNKGIYKAISELDILLSWIKKLLESSQ SEQ ID NO: 88-heavy chain
variable region sequence for an anti-TNF-alpha antibody
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYA
DSVERGFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 89-light chain
variable region sequence for an anti-TNF-alpha antibody
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSG
SGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
YACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 90-heavy chain variable region
sequence for an anti-TNF-alpha antibody
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAIISFDGSNKSSAD
SVKGRFTYSRRNSKNALFLQMNSLRAEDTAVFYCARDRGVSAGGNYYYYGMDVWGQGTTVT VSS
SEQ ID NO: 91-light chain variable region sequence for an
anti-TNF-alpha antibody
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG
SGSGTRFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIL SEQ ID NO: 92-amino
acid sequence of human TNFR-p75-Fc dimeric fusion protein
LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYT
QLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGORLCAPLRKCRP
GFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRS
MAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 93-GLP-1 agonist peptide
amino acid sequence (exenatide)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO: 94-GLP-1 agonist
peptide amino acid sequence (Liraglutide)
HAEGTFTSDVSSYLEGQAAKEEFIIAWLVKGRG SEQ ID NO: 95-amino acid sequence
of human growth hormone (somatotropin)
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNRE
ETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLED
GSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF SEQ
ID NO: 96-amino acid sequence of a peptide linker GGGGS SEQ ID NO:
97-amino acid sequence of a peptide linker GGGGSGGGGS SEQ ID NO:
98-amino acid sequence of a peptide linker GGGGSGGGGSGGGGS SEQ ID
NO: 99-amino acid sequence of a peptide linker GGGGSGGG SEQ ID NO:
100-amino acid sequence of a peptide linker AAPF SEQ ID NO:
101-amino acid sequence of a peptide linker GGF SEQ ID NO:
102-amino acid sequence of a peptide linker AAPV SEQ ID NO:
103-amino acid sequence of a peptide linker GGL SEQ ID NO:
104-amino acid sequence of a peptide linker AAL SEQ ID NO:
105-amino acid sequence of a peptide linker FVR SEQ ID NO:
106-amino acid sequence of a peptide linker VGR SEQ ID NO:
107-amino acid sequence of a peptide linker RKPR SEQ ID NO:
108-amino acid sequence of a peptide linker Y V A D Xaa Xaa = any
amino acid SEQ ID NO: 109-amino acid sequence of a peptide linker D
Xaa Xaa D Xaa Xaa = any amino acid SEQ ID NO: 110-amino acid
sequence of a peptide linker R (Xaa).sub.n R Xaa Xaa = any amino
acid n = 0, 2, 4 or 6 SEQ ID NO: 111-amino acid sequence of a
peptide linker K (Xaa).sub.n R Xaa Xaa = any amino acid n = 0, 2, 4
or 6 SEQ ID NO: 112-amino acid sequence of a peptide linker E R T K
R Xaa Xaa = any amino acid SEQ ID NO: 113-amino acid sequence of a
peptide linker R V R R Xaa Xaa = any amino acid SEQ ID NO:
114-amino acid sequence of a peptide linker Decanoyl-R V R R Xaa
Xaa = any amino acid SEQ ID NO: 115-amino acid sequence of a
peptide linker P Xaa W V P Xaa Xaa = any amino acid SEQ ID NO:
116-amino acid sequence of a peptide linker W V A Xaa Xaa = any
amino acid SEQ ID NO: 117-amino acid sequence of a peptide linker
Xaa F Xaa Xaa Xaa = any amino acid SEQ ID NO: 118-amino acid
sequence of a peptide linker Xaa Y Xaa Xaa Xaa = any amino acid n =
0, 2, 4 or 6 SEQ ID NO: 119-amino acid sequence of a peptide linker
Xaa W Xaa Xaa Xaa = any amino acid n = 0, 2, 4 or 6 SEQ ID NO:
120-amino acid sequence of a peptide linker D R W I P F H L L in
combination with (V, A or P)-Y-(S, P or A) SEQ ID NO: 121-amino
acid sequence of a peptide linker GGGGSGGGENLYFQS SEQ ID NO:
122-amino acid sequence of a Cholix.sup.415-TEV-IL-10 fusion
molecule
MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDED
KGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPA
SIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKE
GSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQ
KPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFT
HIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGA
QAADILSLFCPDADKSCVASNSDQANINIESGGGGSGGGENLYFQSPGQGTQSENSCTHFPG
NLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA
ENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEF
DIFINYIEAYMTMKIRN SEQ ID NO: 123-amino acid sequence of a
Cholix.sup.415-(G.sub.4S).sub.3-IL-10 fusion molecule
MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDED
KGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPA
SIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKE
GSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQ
KPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFT
HIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGA
QAADILSLFCPDADKSCVASNSDQANINIESGGGGSGGGGSGGGGSPGQGTQSENSCTHFPG
NLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA
ENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEF
DIFINYIEAYMTMKIRN
Sequence CWU 1
1
1231634PRTvibrio cholerae 1Val Glu Asp Glu Leu Asn Ile Phe Asp Glu
Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile
Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly
Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile
Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe
Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala
Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr
Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile
Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120
125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro
130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln
Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg
Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr
Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala
His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met
Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu
Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235
240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro
245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser
Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr
Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu
Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val
Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe
Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg
Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met
Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360
365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala
370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp
Lys Ser385 390 395 400Cys Val Ala Ser Asn Asn Asp Gln Ala Asn Ile
Asn Ile Glu Ser Arg 405 410 415Ser Gly Arg Ser Tyr Leu Pro Glu Asn
Arg Ala Val Ile Thr Pro Gln 420 425 430Gly Val Thr Asn Trp Thr Tyr
Gln Glu Leu Glu Ala Thr His Gln Ala 435 440 445Leu Thr Arg Glu Gly
Tyr Val Phe Val Gly Tyr His Gly Thr Asn His 450 455 460Val Ala Ala
Gln Thr Ile Val Asn Arg Ile Ala Pro Val Pro Arg Gly465 470 475
480Asn Asn Thr Glu Asn Glu Glu Lys Trp Gly Gly Leu Tyr Val Ala Thr
485 490 495His Ala Glu Val Ala His Gly Tyr Ala Arg Ile Lys Glu Gly
Thr Gly 500 505 510Glu Tyr Gly Leu Pro Thr Arg Ala Glu Arg Asp Ala
Arg Gly Val Met 515 520 525Leu Arg Val Tyr Ile Pro Arg Ala Ser Leu
Glu Arg Phe Tyr Arg Thr 530 535 540Asn Thr Pro Leu Glu Asn Ala Glu
Glu His Ile Thr Gln Val Ile Gly545 550 555 560His Ser Leu Pro Leu
Arg Asn Glu Ala Phe Thr Gly Pro Glu Ser Ala 565 570 575Gly Gly Glu
Asp Glu Thr Val Ile Gly Trp Asp Met Ala Ile His Ala 580 585 590Val
Ala Ile Pro Ser Thr Ile Pro Gly Asn Ala Tyr Glu Glu Leu Ala 595 600
605Ile Asp Glu Glu Ala Val Ala Lys Glu Gln Ser Ile Ser Thr Lys Pro
610 615 620Pro Tyr Lys Glu Arg Lys Asp Glu Leu Lys625
63021905DNAvibrio cholerae 2atggtcgaag aagctttaaa catctttgat
gaatgccgtt cgccatgttc gttgaccccg 60gaaccgggta agccgattca atcaaaactg
tctatcccta gtgatgttgt tctggatgaa 120ggtgttctgt attactcgat
gacgattaat gatgagcaga atgatattaa ggatgaggac 180aaaggcgagt
ccattatcac tattggtgaa tttgccacag tacgcgcgac tagacattat
240gttaatcaag atgcgccttt tggtgtcatc catttagata ttacgacaga
aaatggtaca 300aaaacgtact cttataaccg caaagagggt gaatttgcaa
tcaattggtt agtgcctatt 360ggtgaagatt ctcctgcaag catcaaaatc
tccgttgatg agctcgatca gcaacgcaat 420atcatcgagg tgcctaaact
gtatagtatt gatctcgata accaaacgtt agagcagtgg 480aaaacccaag
gtaatgtttc tttttcggta acgcgtcctg aacataatat cgctatctct
540tggccaagcg tgagttacaa agcagcgcag aaagagggtt cacgccataa
gcgttgggct 600cattggcata caggcttagc actgtgttgg cttgtgccaa
tggatgctat ctataactat 660atcacccagc aaaattgtac tttaggggat
aattggtttg gtggctctta tgagactgtt 720gcaggcactc cgaaggtgat
tacggttaag caagggattg aacaaaagcc agttgagcag 780cgcatccatt
tctccaaggg gaatgcgatg agcgcacttg ctgctcatcg cgtctgtggt
840gtgccattag aaactttggc gcgcagtcgc aaacctcgtg atctgacgga
tgatttatca 900tgtgcctatc aagcgcagaa tatcgtgagt ttatttgtcg
cgacgcgtat cctgttctct 960catctggata gcgtatttac tctgaatctt
gacgaacaag aaccagaggt ggctgaacgt 1020ctaagtgatc ttcgccgtat
caatgaaaat aacccgggca tggttacaca ggttttaacc 1080gttgctcgtc
agatctataa cgattatgtc actcaccatc cgggcttaac tcctgagcaa
1140accagtgcgg gtgcacaagc tgccgatatc ctctctttat tttgcccaga
tgctgataag 1200tcttgtgtgg cttcaaacaa cgatcaagcc aatatcaaca
tcgagtctcg ttctggccgt 1260tcatatttgc ctgaaaaccg tgcggtaatc
acccctcaag gcgtcacaaa ttggacttac 1320caggaactcg aagcaacaca
tcaagctctg actcgtgagg gttatgtgtt cgtgggttac 1380catggtacga
atcatgtcgc tgcgcaaacc atcgtgaatc gcattgcccc tgttccgcgc
1440ggcaacaaca ctgaaaacga ggaaaagtgg ggcgggttat atgttgcaac
tcacgctgaa 1500gttgcccatg gttatgctcg catcaaagaa gggacagggg
agtatggcct tccgacccgt 1560gctgagcgcg acgctcgtgg ggtaatgctg
cgcgtgtata tccctcgtgc ttcattagaa 1620cgtttttatc gcacgaatac
acctttggaa aatgctgagg agcatatcac gcaagtgatt 1680ggtcattctt
tgccattacg caatgaagca tttactggtc cagaaagtgc gggcggggaa
1740gacgaaactg tcattggctg ggatatggcg attcatgcag ttgcgatccc
ttcgactatc 1800ccagggaacg cttacgaaga attggcgatt gatgaggagg
ctgttgcaaa agagcaatcg 1860attagcacaa aaccacctta taaagagcgc
aaagatgaac ttaag 19053386PRTvibrio cholerae 3Val Glu Asp Glu Leu
Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu
Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val
Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp
Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile
Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu
Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala3854385PRTvibrio cholerae
4Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5
10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr
Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375
380Gln3855384PRTvibrio cholerae 5Val Glu Asp Glu Leu Asn Ile Phe
Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys
Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp
Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn
Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly
Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln
Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn
Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 3806383PRTvibrio cholerae 6Val Glu Asp Glu Leu Asn
Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro
Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val
Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu
Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr
Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln
Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro
Glu Gln Thr Ser Ala Gly 370 375 3807382PRTvibrio cholerae 7Val Glu
Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu
Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25
30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile
35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser
Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His
Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp
Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys
Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu
Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp
Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile
Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln
Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170
175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly
180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala
Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile
Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly
Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr
Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val
Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly
Leu Thr Pro Glu Gln Thr Ser Ala 370 375 3808381PRTvibrio cholerae
8Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5
10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr
Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
Gly Leu Thr Pro Glu Gln Thr Ser 370 375 3809380PRTvibrio cholerae
9Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5
10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr
Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
Gly Leu Thr Pro Glu Gln Thr 370 375 38010379PRTvibrio cholerae
10Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1
5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr
Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
Gly Leu Thr Pro Glu Gln 370 37511378PRTvibrio cholerae 11Val Glu
Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu
Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25
30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile
35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser
Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His
Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp
Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys
Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu
Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp
Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile
Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln
Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170
175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly
180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala
Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile
Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly
Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr
Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val
Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly
Leu Thr Pro Glu 370 37512377PRTvibrio cholerae 12Val Glu Asp Glu
Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro
Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp
Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn
Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55
60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65
70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr
Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu
Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro
Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg
Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp
Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val
Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser
Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser
Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro 370
37513376PRTvibrio cholerae 13Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His
His Pro Gly Leu Thr 370 37514375PRTvibrio cholerae 14Val Glu Asp
Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr
Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser
Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40
45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile
50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr
Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile
Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu
Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp
Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln
Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp
Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly
Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala
Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185
190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys
195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln
Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr
Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys
Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe
Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val
Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro
Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln
Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310
315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu
Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn
Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln
Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu 370
37515374PRTvibrio cholerae 15Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly 37016373PRTvibrio cholerae 16Val
Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10
15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro
20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr
Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu
Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg
His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu
Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg
Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly
Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu
Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser
Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr
Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170
175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly
180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala
Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile
Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly
Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr
Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val
Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
37017372PRTvibrio cholerae 17Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His 37018371PRTvibrio cholerae 18Val Glu Asp
Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr
Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser
Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40
45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile
50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr
Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile
Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu
Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp
Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln
Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp
Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly
Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala
Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185
190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys
195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln
Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr
Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys
Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe
Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val
Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro
Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln
Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310
315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu
Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn
Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln
Ile Tyr Asn Asp Tyr 355 360 365Val Thr His 37019370PRTvibrio
cholerae 19Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro
Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu
Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr
Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp
Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg
Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val
Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser
Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val
Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val
Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys
Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr
37020369PRTvibrio cholerae 20Val Glu Asp Glu Leu Asn Ile Phe
Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val21368PRTvibrio cholerae 21Val Glu Asp Glu Leu Asn Ile
Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly
Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu
Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln
Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile
Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn
Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90
95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala
100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser
Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile
Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln
Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe
Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro
Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His
Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp
Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215
220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val
Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile
Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly
Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val
Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu
Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val
Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu
Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330
335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly
340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn
Asp Tyr 355 360 36522367PRTvibrio cholerae 22Val Glu Asp Glu Leu
Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu
Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val
Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp
Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile
Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp 355 360 36523366PRTvibrio cholerae 23Val Glu Asp Glu
Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro
Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp
Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn
Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55
60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65
70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr
Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu
Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro
Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg
Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp
Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val
Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser
Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser
Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn 355 360 36524365PRTvibrio cholerae 24Val Glu Asp Glu Leu
Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu
Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val
Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp
Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile
Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr 355 360 36525364PRTvibrio cholerae 25Val Glu Asp Glu Leu Asn
Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro
Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val
Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu
Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr
Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
355 36026363PRTvibrio cholerae 26Val Glu Asp Glu Leu Asn Ile Phe
Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys
Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp
Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn
Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly
Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln
Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn
Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val
Ala Arg Gln 355 36027362PRTvibrio cholerae 27Val Glu Asp Glu Leu
Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu
Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val
Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp
Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile
Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg 355
36028361PRTvibrio cholerae 28Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala 355 36029360PRTvibrio
cholerae 29Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro
Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu
Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr
Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp
Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg
Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val
Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser
Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val
Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val
Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys
Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu Thr Val 355 36030359PRTvibrio cholerae 30Val Glu Asp Glu Leu
Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu
Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val
Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp
Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile
Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr 35531358PRTvibrio
cholerae 31Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro
Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu
Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr
Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp
Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg
Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val
Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser
Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val
Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val
Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys
Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu 35532357PRTvibrio cholerae 32Val Glu Asp Glu Leu Asn Ile Phe
Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys
Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp
Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn
Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly
Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln
Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn
Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val 35533356PRTvibrio cholerae 33Val Glu Asp Glu
Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro
Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp
Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn
Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55
60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65
70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr
Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu
Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro
Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg
Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp
Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val
Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser
Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser
Arg His Lys Arg Trp
Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro
Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr
Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235
240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro
245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser
Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr
Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu
Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val
Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe
Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg
Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met
Val Thr Gln 35534355PRTvibrio cholerae 34Val Glu Asp Glu Leu Asn
Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro
Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val
Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu
Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr
Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr 35535354PRTvibrio cholerae 35Val Glu
Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu
Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25
30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile
35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser
Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His
Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp
Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys
Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu
Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp
Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile
Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln
Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170
175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly
180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala
Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile
Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly
Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr
Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn Pro Gly 340 345 350Met Val36353PRTvibrio cholerae
36Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1
5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met37352PRTvibrio cholerae
37Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1
5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 35038351PRTvibrio cholerae
38Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1
5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro 340 345 35039350PRTvibrio cholerae 39Val
Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10
15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro
20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr
Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu
Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg
His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu
Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg
Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly
Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu
Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser
Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr
Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170
175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly
180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala
Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile
Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly
Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr
Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn 340 345 35040349PRTvibrio cholerae 40Val Glu Asp
Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr
Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser
Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40
45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile
50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr
Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile
Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu
Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp
Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln
Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp
Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly
Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165
170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu
Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu
Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr
Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly
Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile
Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg
Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala
His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn 340 34541348PRTvibrio cholerae 41Val Glu Asp Glu
Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro
Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp
Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn
Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55
60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65
70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr
Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu
Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro
Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg
Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp
Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val
Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser
Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser
Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu 340
34542425PRTvibrio cholerae 42Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp
Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn Asn Asp Gln Ala
Asn Ile Asn Ile Glu Ser Arg 405 410 415Ser Gly Arg Ser Tyr Leu Pro
Glu Asn 420 42543424PRTvibrio cholerae 43Val Glu Asp Glu Leu Asn
Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro
Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val
Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu
Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr
Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu
Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu
Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn
Asn Asp Gln Ala Asn Ile Asn Ile Glu Ser Arg 405 410 415Ser Gly Arg
Ser Tyr Leu Pro Glu 42044423PRTvibrio cholerae 44Val Glu Asp Glu
Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro
Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp
Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn
Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55
60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65
70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr
Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu
Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro
Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg
Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp
Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val
Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser
Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser
Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu
Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu
Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn
Asn Asp Gln Ala Asn Ile Asn Ile Glu Ser Arg 405 410 415Ser Gly Arg
Ser Tyr Leu Pro 42045422PRTvibrio cholerae 45Val Glu Asp Glu Leu
Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu
Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val
Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp
Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile
Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu
Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu
Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn
Asn Asp Gln Ala Asn Ile Asn Ile Glu Ser Arg 405 410 415Ser Gly Arg
Ser Tyr Leu 42046421PRTvibrio cholerae 46Val Glu Asp Glu Leu Asn
Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro
Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val
Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu
Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr
Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr
Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250
255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu
260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala
Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys
Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr
Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu
Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser
Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr
Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val
Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375
380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys
Ser385 390 395 400Cys Val Ala Ser Asn Asn Asp Gln Ala Asn Ile Asn
Ile Glu Ser Arg 405 410 415Ser Gly Arg Ser Tyr 42047420PRTvibrio
cholerae 47Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro
Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu
Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr
Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp
Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg
Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val
Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser
Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val
Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val
Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys
Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His
His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln
Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390
395 400Cys Val Ala Ser Asn Asn Asp Gln Ala Asn Ile Asn Ile Glu Ser
Arg 405 410 415Ser Gly Arg Ser 42048419PRTvibrio cholerae 48Val Glu
Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu
Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25
30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile
35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser
Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His
Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp
Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys
Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu
Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp
Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile
Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln
Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170
175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly
180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala
Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile
Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly
Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr
Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val
Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly
Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp
Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys
Val Ala Ser Asn Asn Asp Gln Ala Asn Ile Asn Ile Glu Ser Arg 405 410
415Ser Gly Arg49418PRTvibrio cholerae 49Val Glu Asp Glu Leu Asn Ile
Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly
Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu
Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln
Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile
Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn
Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90
95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala
100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser
Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile
Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln
Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe
Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro
Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His
Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp
Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215
220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val
Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile
Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly
Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val
Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu
Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val
Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu
Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330
335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly
340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn
Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr
Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys
Pro Asp Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn Asn Asp
Gln Ala Asn Ile Asn Ile Glu Ser Arg 405 410 415Ser
Gly50417PRTvibrio cholerae 50Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp
Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn Asn Asp Gln Ala
Asn Ile Asn Ile Glu Ser Arg 405 410 415Ser51416PRTvibrio cholerae
51Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1
5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr
Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala
Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390 395
400Cys Val Ala Ser Asn Asn Asp Gln Ala Asn Ile Asn Ile Glu Ser Arg
405 410 41552415PRTvibrio cholerae 52Val Glu Asp Glu Leu Asn Ile
Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly
Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu
Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln
Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile
Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn
Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90
95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala
100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser
Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile
Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln
Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe
Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro
Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly
180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala
Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile
Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly
Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr
Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val
Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly
Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp
Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys
Val Ala Ser Asn Asn Asp Gln Ala Asn Ile Asn Ile Glu Ser 405 410
41553414PRTvibrio cholerae 53Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp
Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn Asn Asp Gln Ala
Asn Ile Asn Ile Glu 405 41054413PRTvibrio cholerae 54Val Glu Asp
Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr
Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser
Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40
45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile
50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr
Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile
Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu
Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp
Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln
Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp
Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly
Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala
Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185
190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys
195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln
Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr
Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys
Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe
Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val
Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro
Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln
Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310
315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu
Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn
Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln
Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro
Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser
Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser
Asn Asn Asp Gln Ala Asn Ile Asn Ile 405 41055412PRTvibrio cholerae
55Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1
5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr
Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala
Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390 395
400Cys Val Ala Ser Asn Asn Asp Gln Ala Asn Ile Asn 405
41056411PRTvibrio cholerae 56Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp
Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn Asn Asp Gln Ala
Asn Ile 405 41057410PRTvibrio cholerae 57Val Glu Asp Glu Leu Asn
Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro
Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val
Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu
Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr
Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu
Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu
Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn
Asn Asp Gln Ala Asn 405 41058409PRTvibrio cholerae 58Val Glu Asp
Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr
Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser
Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40
45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile
50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr
Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile
Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu
Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp
Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln
Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp
Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly
Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165
170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu
Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu
Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr
Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly
Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile
Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg
Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala
His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr
Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala
Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390 395
400Cys Val Ala Ser Asn Asn Asp Gln Ala 40559408PRTvibrio cholerae
59Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1
5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr
Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala
Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390 395
400Cys Val Ala Ser Asn Asn Asp Gln 40560407PRTvibrio cholerae 60Val
Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10
15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro
20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr
Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu
Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg
His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu
Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg
Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly
Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu
Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser
Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr
Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170
175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly
180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala
Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile
Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly
Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr
Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val
Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly
Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp
Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys
Val Ala Ser Asn Asn Asp 40561406PRTvibrio cholerae 61Val Glu Asp
Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr
Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser
Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40
45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile
50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr
Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile
Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu
Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp
Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln
Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp
Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly
Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala
Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185
190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys
195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln
Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr
Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys
Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe
Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val
Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro
Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln
Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310
315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu
Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn
Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln
Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro
Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser
Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser
Asn Asn 40562405PRTvibrio cholerae 62Val Glu Asp Glu Leu Asn Ile
Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly
Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu
Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln
Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile
Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn
Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90
95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala
100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser
Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile
Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln
Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe
Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro
Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His
Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp
Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215
220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val
Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile
Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly
Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val
Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu
Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val
Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu
Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330
335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly
340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn
Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr
Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys
Pro Asp Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser Asn
40563404PRTvibrio cholerae 63Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp
Ala Asp Lys Ser385 390 395 400Cys Val Ala Ser64403PRTvibrio
cholerae 64Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro
Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu
Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr
Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp
Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg
Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val
Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser
Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val
Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val
Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys
Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His
Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His
His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln
Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390
395 400Cys Val Ala65402PRTvibrio cholerae 65Val Glu Asp Glu Leu Asn
Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro
Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val
Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu
Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr
Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu
Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu
Phe Cys Pro Asp Ala Asp Lys Ser385 390 395 400Cys Val66401PRTvibrio
cholerae 66Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro
Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu
Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr
Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp
Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg
Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val
Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser
Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val
Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val
Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys
Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His
His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln
Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys Ser385 390
395 400Cys67400PRTvibrio cholerae 67Val Glu Asp Glu Leu Asn Ile Phe
Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys
Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp
Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn
Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly
Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln
Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn
Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp
Ala Asp Lys Ser385 390 395 40068399PRTvibrio cholerae 68Val Glu Asp
Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr
Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser
Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40
45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile
50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr
Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile
Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu
Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp
Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln
Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp
Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly
Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala
Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185
190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys
195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln
Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr
Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys
Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe
Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val
Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro
Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln
Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310
315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu
Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn
Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln
Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro
Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser
Leu Phe Cys Pro Asp Ala Asp Lys385 390 39569398PRTvibrio cholerae
69Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1
5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile
Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met
Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly
Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg Ala Thr
Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val Ile His
Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn
Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val Pro Ile
Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val Asp Glu
Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr
Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150 155
160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn Ile
165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys
Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr Gly
Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn
Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp Phe
Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys Val
Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu Gln
Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala
Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280
285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala
290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg
Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr
Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro
Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala
Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp385 390
39570397PRTvibrio cholerae 70Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile
His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His
Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg
Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295
300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser
His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln
Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile
Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val
Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly
Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp
Ile Leu Ser Leu Phe Cys Pro Asp Ala385 390 39571396PRTvibrio
cholerae 71Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro
Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu
Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr
Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp
Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg
Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val
Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser
Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val
Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val
Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys
Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His
His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln
Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp385 390
39572395PRTvibrio cholerae 72Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro385
390 39573394PRTvibrio cholerae 73Val Glu Asp Glu Leu Asn Ile Phe
Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys
Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp
Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn
Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly
Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln
Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn
Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys385
39074393PRTvibrio cholerae 74Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe385
39075392PRTvibrio cholerae 75Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser Leu385
39076391PRTvibrio cholerae 76Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu
Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu Ser385
39077390PRTvibrio cholerae 77Val Glu Asp Glu Leu Asn Ile Phe Asp
Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro
Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu
Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp
Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu
Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp
Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly
Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105
110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys
115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu
Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu
Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val
Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val
Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg
Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val
Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys
Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230
235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys
Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met
Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp
Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe
Val Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val
Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu
Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345
350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr
355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala
Gly Ala 370 375 380Gln Ala Ala Asp Ile Leu385 39078389PRTvibrio
cholerae 78Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro
Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu
Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr
Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp
Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg
Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val
Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser
Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val
Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val
Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys
Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His
His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln
Ala Ala Asp Ile38579388PRTvibrio cholerae 79Val Glu Asp Glu Leu Asn
Ile Phe Asp Glu Cys Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro
Gly Lys Pro Ile Gln Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val
Leu Asp Glu Gly Val Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu
Gln Asn Asp Ile Lys Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr
Ile Gly Glu Phe Ala Thr Val Arg Ala Thr Arg His Tyr Val65 70 75
80Asn Gln Asp Ala Pro Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu
85 90 95Asn Gly Thr Lys Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe
Ala 100 105 110Ile Asn Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala
Ser Ile Lys 115 120 125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn
Ile Ile Glu Val Pro 130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn
Gln Thr Leu Glu Gln Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser
Phe Ser Val Thr Arg Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp
Pro Ser Val Ser Tyr Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg
His Lys Arg Trp Ala His Trp His Thr Gly Leu Ala Leu Cys 195 200
205Trp Leu Val Pro Met Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn
210 215 220Cys Thr Leu Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr
Val Ala225 230 235 240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly
Ile Glu Gln Lys Pro 245 250 255Val Glu Gln Arg Ile His Phe Ser Lys
Gly Asn Ala Met Ser Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly
Val Pro Leu Glu Thr Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp
Leu Thr Asp Asp Leu Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile
Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe Ser His305 310 315
320Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val
325 330 335Ala Glu Arg Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn
Pro Gly 340 345 350Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile
Tyr Asn Asp Tyr 355 360 365Val Thr His His Pro Gly Leu Thr Pro Glu
Gln Thr Ser Ala Gly Ala 370 375 380Gln Ala Ala Asp38580387PRTvibrio
cholerae 80Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys Arg Ser Pro
Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys Leu
Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val Leu Tyr Tyr
Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu Asp
Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala Thr Val Arg
Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro Phe Gly Val
Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys Thr Tyr Ser
Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn Trp Leu Val
Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120 125Ile Ser Val
Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro 130 135 140Lys
Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp Lys145 150
155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His Asn
Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala Gln
Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala His Trp His Thr
Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met Asp Ala Ile Tyr
Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu Gly Asp Asn Trp
Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235 240Gly Thr Pro Lys
Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro 245 250 255Val Glu
Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser Ala Leu 260 265
270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg Ser
275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu Ser Cys Ala Tyr
Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile
Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe Thr Leu Asn Leu
Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg Leu Ser Asp Leu
Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met Val Thr Gln Val
Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360 365Val Thr His
His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala 370 375 380Gln
Ala Ala38581633PRTvibrio choleraemature Cholix Toxin deleted at
amio acid residue 581 81Val Glu Asp Glu Leu Asn Ile Phe Asp Glu Cys
Arg Ser Pro Cys Ser1 5 10 15Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln
Ser Lys Leu Ser Ile Pro 20 25 30Ser Asp Val Val Leu Asp Glu Gly Val
Leu Tyr Tyr Ser Met Thr Ile 35 40 45Asn Asp Glu Gln Asn Asp Ile Lys
Asp Glu Asp Lys Gly Glu Ser Ile 50 55 60Ile Thr Ile Gly Glu Phe Ala
Thr Val Arg Ala Thr Arg His Tyr Val65 70 75 80Asn Gln Asp Ala Pro
Phe Gly Val Ile His Leu Asp Ile Thr Thr Glu 85 90 95Asn Gly Thr Lys
Thr Tyr Ser Tyr Asn Arg Lys Glu Gly Glu Phe Ala 100 105 110Ile Asn
Trp Leu Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile Lys 115 120
125Ile Ser Val Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val Pro
130 135 140Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln
Trp Lys145 150 155 160Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg
Pro Glu His Asn Ile 165 170 175Ala Ile Ser Trp Pro Ser Val Ser Tyr
Lys Ala Ala Gln Lys Glu Gly 180 185 190Ser Arg His Lys Arg Trp Ala
His Trp His Thr Gly Leu Ala Leu Cys 195 200 205Trp Leu Val Pro Met
Asp Ala Ile Tyr Asn Tyr Ile Thr Gln Gln Asn 210 215 220Cys Thr Leu
Gly Asp Asn Trp Phe Gly Gly Ser Tyr Glu Thr Val Ala225 230 235
240Gly Thr Pro Lys Val Ile Thr Val Lys Gln Gly Ile Glu Gln Lys Pro
245 250 255Val Glu Gln Arg Ile His Phe Ser Lys Gly Asn Ala Met Ser
Ala Leu 260 265 270Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr
Leu Ala Arg Ser 275 280 285Arg Lys Pro Arg Asp Leu Thr Asp Asp Leu
Ser Cys Ala Tyr Gln Ala 290 295 300Gln Asn Ile Val Ser Leu Phe Val
Ala Thr Arg Ile Leu Phe Ser His305 310 315 320Leu Asp Ser Val Phe
Thr Leu Asn Leu Asp Glu Gln Glu Pro Glu Val 325 330 335Ala Glu Arg
Leu Ser Asp Leu Arg Arg Ile Asn Glu Asn Asn Pro Gly 340 345 350Met
Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp Tyr 355 360
365Val Thr His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly Ala
370 375 380Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp
Lys Ser385 390 395 400Cys Val Ala Ser Asn Asn Asp Gln Ala Asn Ile
Asn Ile Glu Ser Arg 405 410 415Ser Gly Arg Ser Tyr Leu Pro Glu Asn
Arg Ala Val Ile Thr Pro Gln 420 425 430Gly Val Thr Asn Trp Thr Tyr
Gln Glu Leu Glu Ala Thr His Gln Ala 435 440 445Leu Thr Arg Glu Gly
Tyr Val Phe Val Gly Tyr His Gly Thr Asn His 450 455 460Val Ala Ala
Gln Thr Ile Val Asn Arg Ile Ala Pro Val Pro Arg Gly465 470 475
480Asn Asn Thr Glu Asn Glu Glu Lys Trp Gly Gly Leu Tyr Val Ala Thr
485 490 495His Ala Glu Val Ala His Gly Tyr Ala Arg Ile Lys Glu Gly
Thr Gly 500 505 510Glu Tyr Gly Leu Pro Thr Arg Ala Glu Arg Asp Ala
Arg Gly Val Met 515 520 525Leu Arg Val Tyr Ile Pro Arg Ala Ser Leu
Glu Arg Phe Tyr Arg Thr 530 535 540Asn Thr Pro Leu Glu Asn Ala Glu
Glu His Ile Thr Gln Val Ile Gly545 550 555 560His Ser Leu Pro Leu
Arg Asn Glu Ala Phe Thr Gly Pro Glu Ser Ala 565 570 575Gly Gly Glu
Asp Thr Val Ile Gly Trp Asp Met Ala Ile His Ala Val 580 585 590Ala
Ile Pro Ser Thr Ile Pro Gly Asn Ala Tyr Glu Glu Leu Ala Ile 595 600
605Asp Glu Glu Ala Val Ala Lys Glu Gln Ser Ile Ser Thr Lys Pro Pro
610 615 620Tyr Lys Glu Arg Lys Asp Glu Leu Lys625 63082178PRTHomo
sapiens 82Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr
Gly Val1 5 10 15Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser
Cys Thr His 20 25 30Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu
Arg Asp Ala Phe 35 40 45Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp
Gln Leu Asp Asn Leu 50 55 60Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe
Lys Gly Tyr Leu Gly Cys65 70 75 80Gln Ala Leu Ser Glu Met Ile Gln
Phe Tyr Leu Glu Glu Val Met Pro 85 90 95Gln Ala Glu Asn Gln Asp Pro
Asp Ile Lys Ala His Val Asn Ser Leu 100 105 110Gly Glu Asn Leu Lys
Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg 115 120 125Phe Leu Pro
Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn 130 135 140Ala
Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu145 150
155 160Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys
Ile 165 170 175Arg Asn83177PRTHomo sapiens 83Met Lys Leu Gln Cys
Val Ser Leu Trp Leu Leu Gly Thr Ile Leu Ile1 5 10 15Leu Cys Ser Val
Asp Asn His Gly Leu Arg Arg Cys Leu Ile Ser Thr 20 25 30Asp Met His
His Ile Glu Glu Ser Phe Gln Glu Ile Lys Arg Ala Ile 35 40 45Gln Ala
Lys Asp Thr Phe Pro Asn Val Thr Ile Leu Ser Thr Leu Glu 50 55
60Thr Leu Gln Ile Ile Lys Pro Leu Asp Val Cys Cys Val Thr Lys Asn65
70 75 80Leu Leu Ala Phe Tyr Val Asp Arg Val Phe Lys Asp His Gln Glu
Pro 85 90 95Asn Pro Lys Ile Leu Arg Lys Ile Ser Ser Ile Ala Asn Ser
Phe Leu 100 105 110Tyr Met Gln Lys Thr Leu Arg Gln Cys Gln Glu Gln
Arg Gln Cys His 115 120 125Cys Arg Gln Glu Ala Thr Asn Ala Thr Arg
Val Ile His Asp Asn Tyr 130 135 140Asp Gln Leu Glu Val His Ala Ala
Ala Ile Lys Ser Leu Gly Glu Leu145 150 155 160Asp Val Phe Leu Ala
Trp Ile Asn Lys Asn His Glu Val Met Ser Ser 165 170
175Ala84176PRTHomo sapiens 84Met Lys Ala Ser Ser Leu Ala Phe Ser
Leu Leu Ser Ala Ala Phe Tyr1 5 10 15Leu Leu Trp Thr Pro Ser Thr Gly
Leu Lys Thr Leu Asn Leu Gly Ser 20 25 30Cys Val Ile Ala Thr Asn Leu
Gln Glu Ile Arg Asn Gly Phe Ser Glu 35 40 45Ile Arg Gly Ser Val Gln
Ala Lys Asp Gly Asn Ile Asp Ile Arg Ile 50 55 60Leu Arg Arg Thr Glu
Ser Leu Gln Asp Thr Lys Pro Ala Asn Arg Cys65 70 75 80Cys Leu Leu
Arg His Leu Leu Arg Leu Tyr Leu Asp Arg Val Phe Lys 85 90 95Asn Tyr
Gln Thr Pro Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu 100 105
110Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys His Ala
115 120 125His Met Thr Cys His Cys Gly Glu Glu Ala Met Lys Lys Tyr
Ser Gln 130 135 140Ile Leu Ser His Phe Glu Lys Leu Glu Pro Gln Ala
Ala Val Val Lys145 150 155 160Ala Leu Gly Glu Leu Asp Ile Leu Leu
Gln Trp Met Glu Glu Thr Glu 165 170 17585179PRTHomo sapiens 85Met
Ala Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr Leu1 5 10
15Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Gly Gly Ala
20 25 30Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn Phe
Gln 35 40 45Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu Ala Lys Glu
Ala Ser 50 55 60Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu
Lys Leu Phe65 70 75 80His Gly Val Ser Met Ser Glu Arg Cys Tyr Leu
Met Lys Gln Val Leu 85 90 95Asn Phe Thr Leu Glu Glu Val Leu Phe Pro
Gln Ser Asp Arg Phe Gln 100 105 110Pro Tyr Met Gln Glu Val Val Pro
Phe Leu Ala Arg Leu Ser Asn Arg 115 120 125Leu Ser Thr Cys His Ile
Glu Gly Asp Asp Leu His Ile Gln Arg Asn 130 135 140Val Gln Lys Leu
Lys Asp Thr Val Lys Lys Leu Gly Glu Ser Gly Glu145 150 155 160Ile
Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn 165 170
175Ala Cys Ile86207PRTHomo sapiens 86Met Asn Phe Gln Gln Arg Leu
Gln Ser Leu Trp Thr Leu Ala Ser Arg1 5 10 15Pro Phe Cys Pro Pro Leu
Leu Ala Thr Ala Ser Gln Met Gln Met Val 20 25 30Val Leu Pro Cys Leu
Gly Phe Thr Leu Leu Leu Trp Ser Gln Val Ser 35 40 45Gly Ala Gln Gly
Gln Glu Phe His Phe Gly Pro Cys Gln Val Lys Gly 50 55 60Val Val Pro
Gln Lys Leu Trp Glu Ala Phe Trp Ala Val Lys Asp Thr65 70 75 80Met
Gln Ala Gln Asp Asn Ile Thr Ser Ala Arg Leu Leu Gln Gln Glu 85 90
95Val Leu Gln Asn Val Ser Asp Ala Glu Ser Cys Tyr Leu Val His Thr
100 105 110Leu Leu Glu Phe Tyr Leu Lys Thr Val Phe Lys Asn Tyr His
Asn Arg 115 120 125Thr Val Glu Val Arg Thr Leu Lys Ser Phe Ser Thr
Leu Ala Asn Asn 130 135 140Phe Val Leu Ile Val Ser Gln Leu Gln Pro
Ser Gln Glu Asn Glu Met145 150 155 160Phe Ser Ile Arg Asp Ser Ala
His Arg Arg Phe Leu Leu Phe Arg Arg 165 170 175Ala Phe Lys Gln Leu
Asp Val Glu Ala Ala Leu Thr Lys Ala Leu Gly 180 185 190Glu Val Asp
Ile Leu Leu Thr Trp Met Gln Lys Phe Tyr Lys Leu 195 200
20587171PRTHomo sapiens 87Met Leu Val Asn Phe Ile Leu Arg Cys Gly
Leu Leu Leu Val Thr Leu1 5 10 15Ser Leu Ala Ile Ala Lys His Lys Gln
Ser Ser Phe Thr Lys Ser Cys 20 25 30Tyr Pro Arg Gly Thr Leu Ser Gln
Ala Val Asp Ala Leu Tyr Ile Lys 35 40 45Ala Ala Trp Leu Lys Ala Thr
Ile Pro Glu Asp Arg Ile Lys Asn Ile 50 55 60Arg Leu Leu Lys Lys Lys
Thr Lys Lys Gln Phe Met Lys Asn Cys Gln65 70 75 80Phe Gln Glu Gln
Leu Leu Ser Phe Phe Met Glu Asp Val Phe Gly Gln 85 90 95Leu Gln Leu
Gln Gly Cys Lys Lys Ile Arg Phe Val Glu Asp Phe His 100 105 110Ser
Leu Arg Gln Lys Leu Ser His Cys Ile Ser Cys Ala Ser Ser Ala 115 120
125Arg Glu Met Lys Ser Ile Thr Arg Met Lys Arg Ile Phe Tyr Arg Ile
130 135 140Gly Asn Lys Gly Ile Tyr Lys Ala Ile Ser Glu Leu Asp Ile
Leu Leu145 150 155 160Ser Trp Ile Lys Lys Leu Leu Glu Ser Ser Gln
165 17088224PRTArtificial Sequenceheavy chain variable region
sequence for an anti-TNF-alpha antibody 88Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Ala Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile
Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60Glu Arg
Gly Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 22089214PRTArtificial Sequencelight chain variable region
sequence for an anti-TNF-alpha antibody 89Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21090126PRTArtificial Sequenceheavy
chain variable region sequence for an anti-TNF-alpha antibody 90Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Ile Ile Ser Phe Asp Gly Ser Asn Lys Ser Ser Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Tyr Ser Arg Arg Asn Ser Lys Asn
Ala Leu Phe65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Phe Tyr Cys 85 90 95Ala Arg Asp Arg Gly Val Ser Ala Gly Gly
Asn Tyr Tyr Tyr Tyr Gly 100 105 110Met Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120 12591108PRTArtificial Sequencelight
chain variable region sequence for an anti-TNF-alpha antibody 91Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Arg Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser Asn Trp Pro Pro 85 90 95Phe Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Leu 100 10592467PRTArtificial Sequenceamino acid sequence of
human TNFR-p75 -Fc dimeric fusion protein 92Leu Pro Ala Gln Val Ala
Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser1 5 10 15Thr Cys Arg Leu Arg
Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30Ser Lys Cys Ser
Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45Ser Asp Thr
Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60Trp Asn
Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser65 70 75
80Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys
85 90 95Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly
Cys 100 105 110Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe
Gly Val Ala 115 120 125Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys
Lys Pro Cys Ala Pro 130 135 140Gly Thr Phe Ser Asn Thr Thr Ser Ser
Thr Asp Ile Cys Arg Pro His145 150 155 160Gln Ile Cys Asn Val Val
Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 165 170 175Val Cys Thr Ser
Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190His Leu
Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 195 200
205Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly
210 215 220Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys
Ser Cys225 230 235 240Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly 245 250 255Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 260 265 270Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 275 280 285Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr305 310 315
320Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
325 330 335Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 340 345 350Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 355 360 365Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser 370 375 380Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu385 390 395 400Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440
445His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
450 455 460Pro Gly Lys4659339PRTArtificial SequenceGLP-1 agonist
peptide amino acid sequence (exenatide) 93His Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro
Pro Pro Ser 359433PRTArtificialGLP-1 agonist peptide amino acid
sequence (Liraglutide) 94His Ala Glu Gly Thr Phe Thr Ser Asp Val
Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Glu Phe Ile Ile
Ala Trp Leu Val Lys Gly Arg 20 25 30Gly95191PRTArtificial
Sequencehuman growth hormone (somatotropin) 95Phe Pro Thr Ile Pro
Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg1 5 10 15Ala His Arg Leu
His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30Glu Ala Tyr
Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45Gln Thr
Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60Glu
Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu65 70 75
80Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val
85 90 95Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr
Asp 100 105 110Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met
Gly Arg Leu 115 120 125Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe
Lys Gln Thr Tyr Ser 130 135 140Lys Phe Asp Thr Asn Ser His Asn Asp
Asp Ala Leu Leu Lys Asn Tyr145 150 155 160Gly Leu Leu Tyr Cys Phe
Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175Leu Arg Ile Val
Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185
190965PRTArtificial Sequencepeptide 96Gly Gly Gly Gly Ser1
59710PRTArtificial Sequencepeptide 97Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 109815PRTArtificial Sequencepeptide 98Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
15998PRTArtificial Sequencepeptide 99Gly Gly Gly Gly Ser Gly Gly
Gly1 51004PRTArtificial Sequencepeptide 100Ala Ala Pro
Phe11013PRTArtificial Sequencepeptide 101Gly Gly
Phe11024PRTArtificial Sequencepeptide 102Ala Ala Pro
Val11033PRTArtificial Sequencepeptide 103Gly Gly
Leu11043PRTArtificial Sequencepeptide 104Ala Ala
Leu11053PRTArtificial Sequencepeptide 105Phe Val
Arg11063PRTArtificial Sequencepeptide 106Val Gly
Arg11074PRTArtificial Sequencepeptide 107Arg
Lys Pro Arg11085PRTArtificial SequencePeptideVariant5Xaa = Any
Amino Acid 108Tyr Val Ala Asp Xaa1 51095PRTArtificial
SequencePeptideVariant2, 3, 5Xaa = Any Amino Acid 109Asp Xaa Xaa
Asp Xaa1 51105PRTArtificial SequencePeptideVariant2, 5Xaa = Any
Amino Acid 110Arg Xaa Asn Arg Xaa1 51115PRTArtificial
SequencePeptideVariant2, 5Xaa = Any Amino Acid 111Lys Xaa Asn Arg
Xaa1 51126PRTArtificial SequencePeptideVariant6Xaa = Any Amino Acid
112Glu Arg Thr Lys Arg Xaa1 51135PRTArtificial
SequencePeptideVariant5Xaa = Any Amino Acid 113Arg Val Arg Arg Xaa1
51146PRTArtificial SequencePeptideVariant6Xaa = Any Amino Acid
114Asp Arg Val Arg Arg Xaa1 51156PRTArtificial
SequencePeptideVariant2, 6Xaa = Any Amino Acid 115Pro Xaa Trp Val
Pro Xaa1 51164PRTArtificial SequencePeptideVariant4Xaa = Any Amino
Acid 116Trp Val Ala Xaa11174PRTArtificial SequencePeptideVariant1,
3, 4Xaa = Any Amino Acid 117Xaa Phe Xaa Xaa11184PRTArtificial
SequencePeptideVariant1, 3, 4Xaa = Any Amino Acid 118Xaa Tyr Xaa
Xaa11194PRTArtificial SequencePeptideVariant1, 3, 4Xaa = Any Amino
Acid 119Xaa Trp Xaa Xaa112016PRTArtificial SequencePeptide 120Asp
Arg Tyr Ile Pro Phe His Leu Leu Val Ala Pro Tyr Ser Pro Ala1 5 10
1512115PRTArtificial SequencePeptide 121Gly Gly Gly Gly Ser Gly Gly
Gly Glu Asn Leu Tyr Phe Gln Ser1 5 10 15122590PRTArtificial
Sequenceamino acid sequence of a Cholix415-TEV-IL-10 fusion
molecule 122Met Val Glu Glu Ala Leu Asn Ile Phe Asp Glu Cys Arg Ser
Pro Cys1 5 10 15Ser Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys
Leu Ser Ile 20 25 30Pro Gly Asp Val Val Leu Asp Glu Gly Val Leu Tyr
Tyr Ser Met Thr 35 40 45Ile Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu
Asp Lys Gly Glu Ser 50 55 60Ile Ile Thr Ile Gly Glu Phe Ala Thr Val
Arg Ala Thr Arg His Tyr65 70 75 80Val Ser Gln Asp Ala Pro Phe Gly
Val Ile Asn Leu Asp Ile Thr Thr 85 90 95Glu Asn Gly Thr Lys Thr Tyr
Ser Phe Asn Arg Lys Glu Ser Glu Phe 100 105 110Ala Ile Asn Trp Leu
Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile 115 120 125Lys Ile Ser
Ile Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val 130 135 140Pro
Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp145 150
155 160Lys Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His
Asn 165 170 175Ile Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala
Gln Lys Glu 180 185 190Gly Ser Arg His Lys Arg Trp Ala His Trp His
Thr Gly Leu Ala Leu 195 200 205Cys Trp Leu Val Pro Ile Asp Ala Ile
Tyr Asn Tyr Ile Thr Gln Gln 210 215 220Asn Cys Thr Leu Gly Asp Asn
Trp Phe Gly Gly Ser Tyr Glu Thr Val225 230 235 240Ala Gly Thr Pro
Lys Ala Ile Thr Val Lys Gln Gly Ile Glu Gln Lys 245 250 255Pro Val
Glu Gln Arg Ile His Phe Ser Lys Lys Asn Ala Met Glu Ala 260 265
270Leu Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg
275 280 285Ser Arg Lys Pro Arg Asp Leu Pro Asp Asp Leu Ser Cys Ala
Tyr Asn 290 295 300Ala Gln Gln Ile Val Ser Leu Phe Leu Ala Thr Arg
Ile Leu Phe Thr305 310 315 320His Ile Asp Ser Ile Phe Thr Leu Asn
Leu Asp Gly Gln Glu Pro Glu 325 330 335Val Ala Glu Arg Leu Asp Asp
Leu Arg Arg Ile Asn Glu Asn Asn Pro 340 345 350Gly Met Val Ile Gln
Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp 355 360 365Tyr Val Thr
His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly 370 375 380Ala
Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys385 390
395 400Ser Cys Val Ala Ser Asn Ser Asp Gln Ala Asn Ile Asn Ile Glu
Ser 405 410 415Gly Gly Gly Gly Ser Gly Gly Gly Glu Asn Leu Tyr Phe
Gln Ser Pro 420 425 430Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr
His Phe Pro Gly Asn 435 440 445Leu Pro Asn Met Leu Arg Asp Leu Arg
Asp Ala Phe Ser Arg Val Lys 450 455 460Thr Phe Phe Gln Met Lys Asp
Gln Leu Asp Asn Leu Leu Leu Lys Glu465 470 475 480Ser Leu Leu Glu
Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser 485 490 495Glu Met
Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn 500 505
510Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu
515 520 525Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
Pro Cys 530 535 540Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn
Ala Phe Asn Lys545 550 555 560Leu Gln Glu Lys Gly Ile Tyr Lys Ala
Met Ser Glu Phe Asp Ile Phe 565 570 575Ile Asn Tyr Ile Glu Ala Tyr
Met Thr Met Lys Ile Arg Asn 580 585 590123590PRTArtificial
Sequenceamino acid sequence of a Cholix415-(G4S)3-IL-10 fusion
molecule 123Met Val Glu Glu Ala Leu Asn Ile Phe Asp Glu Cys Arg Ser
Pro Cys1 5 10 15Ser Leu Thr Pro Glu Pro Gly Lys Pro Ile Gln Ser Lys
Leu Ser Ile 20 25 30Pro Gly Asp Val Val Leu Asp Glu Gly Val Leu Tyr
Tyr Ser Met Thr 35 40 45Ile Asn Asp Glu Gln Asn Asp Ile Lys Asp Glu
Asp Lys Gly Glu Ser 50 55 60Ile Ile Thr Ile Gly Glu Phe Ala Thr Val
Arg Ala Thr Arg His Tyr65 70 75 80Val Ser Gln Asp Ala Pro Phe Gly
Val Ile Asn Leu Asp Ile Thr Thr 85 90 95Glu Asn Gly Thr Lys Thr Tyr
Ser Phe Asn Arg Lys Glu Ser Glu Phe 100 105 110Ala Ile Asn Trp Leu
Val Pro Ile Gly Glu Asp Ser Pro Ala Ser Ile 115 120 125Lys Ile Ser
Ile Asp Glu Leu Asp Gln Gln Arg Asn Ile Ile Glu Val 130 135 140Pro
Lys Leu Tyr Ser Ile Asp Leu Asp Asn Gln Thr Leu Glu Gln Trp145 150
155 160Lys Thr Gln Gly Asn Val Ser Phe Ser Val Thr Arg Pro Glu His
Asn 165 170 175Ile Ala Ile Ser Trp Pro Ser Val Ser Tyr Lys Ala Ala
Gln Lys Glu 180 185 190Gly Ser Arg His Lys Arg Trp Ala His Trp His
Thr Gly Leu Ala Leu 195 200 205Cys Trp Leu Val Pro Ile Asp Ala Ile
Tyr Asn Tyr Ile Thr Gln Gln 210 215 220Asn Cys Thr Leu Gly Asp Asn
Trp Phe Gly Gly Ser Tyr Glu Thr Val225 230 235 240Ala Gly Thr Pro
Lys Ala Ile Thr Val Lys Gln Gly Ile Glu Gln Lys 245 250 255Pro Val
Glu Gln Arg Ile His Phe Ser Lys Lys Asn Ala Met Glu Ala 260 265
270Leu Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala Arg
275 280 285Ser Arg Lys Pro Arg Asp Leu Pro Asp Asp Leu Ser Cys Ala
Tyr Asn 290 295 300Ala Gln Gln Ile Val Ser Leu Phe Leu Ala Thr Arg
Ile Leu Phe Thr305 310 315 320His Ile Asp Ser Ile Phe Thr Leu Asn
Leu Asp Gly Gln Glu Pro Glu 325 330 335Val Ala Glu Arg Leu Asp Asp
Leu Arg Arg Ile Asn Glu Asn Asn Pro 340 345 350Gly Met Val Ile Gln
Val Leu Thr Val Ala Arg Gln Ile Tyr Asn Asp 355 360 365Tyr Val Thr
His His Pro Gly Leu Thr Pro Glu Gln Thr Ser Ala Gly 370 375 380Ala
Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp Lys385 390
395 400Ser Cys Val Ala Ser Asn Ser Asp Gln Ala Asn Ile Asn Ile Glu
Ser 405 410 415Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Pro 420 425 430Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr
His Phe Pro Gly Asn 435 440 445Leu Pro Asn Met Leu Arg Asp Leu Arg
Asp Ala Phe Ser Arg Val Lys 450 455 460Thr Phe Phe Gln Met Lys Asp
Gln Leu Asp Asn Leu Leu Leu Lys Glu465 470 475 480Ser Leu Leu Glu
Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser 485 490 495Glu Met
Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn 500 505
510Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu
515 520 525Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
Pro Cys 530 535 540Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn
Ala Phe Asn Lys545 550 555 560Leu Gln Glu Lys Gly Ile Tyr Lys Ala
Met Ser Glu Phe Asp Ile Phe 565 570 575Ile Asn Tyr Ile Glu Ala Tyr
Met Thr Met Lys Ile Arg Asn 580 585 590
* * * * *