U.S. patent application number 17/554182 was filed with the patent office on 2022-05-19 for concatemeric peptide epitope rnas.
This patent application is currently assigned to ModernaTX, Inc.. The applicant listed for this patent is ModernaTX, Inc.. Invention is credited to Giuseppe Ciaramella, Eric Yi-Chun Huang, Nicholas Valiante, Tal Zaks.
Application Number | 20220152178 17/554182 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-19 |
United States Patent
Application |
20220152178 |
Kind Code |
A1 |
Ciaramella; Giuseppe ; et
al. |
May 19, 2022 |
CONCATEMERIC PEPTIDE EPITOPE RNAS
Abstract
The invention relates to concatemeric peptide epitope RNAs, as
well as methods and compositions thereof. mRNA vaccines are also
provided according to the invention, including cancer vaccines.
Inventors: |
Ciaramella; Giuseppe;
(Sudbury, MA) ; Huang; Eric Yi-Chun; (Boston,
MA) ; Valiante; Nicholas; (Cambridge, MA) ;
Zaks; Tal; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ModernaTX, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
ModernaTX, Inc.
Cambridge
MA
|
Appl. No.: |
17/554182 |
Filed: |
December 17, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15748773 |
Jan 30, 2018 |
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PCT/US2016/044918 |
Jul 29, 2016 |
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17554182 |
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62247367 |
Oct 28, 2015 |
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62245145 |
Oct 22, 2015 |
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62199204 |
Jul 30, 2015 |
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International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 45/06 20060101 A61K045/06; A61K 47/54 20060101
A61K047/54; A61K 47/68 20060101 A61K047/68; A61P 35/00 20060101
A61P035/00; A61K 9/127 20060101 A61K009/127; A61K 47/26 20060101
A61K047/26; C12N 15/62 20060101 C12N015/62 |
Claims
1-77. (canceled)
78. A method of treating cancer in a subject, the method
comprising: administering to a subject having cancer (i) a lipid
delivery vehicle comprising a personalized cancer vaccine and (ii)
an immune checkpoint inhibitor, wherein the personalized cancer
vaccine comprises an mRNA comprising an open reading frame encoding
a concatemeric cancer antigen comprising 2-100 peptide epitopes
arranged to minimize pseudo-epitopes.
79. The method of claim 78, wherein at least 30% of the peptide
epitopes in the mRNA are MHC class I epitopes.
80. The method of claim 78, wherein each peptide epitope encoded by
the mRNA comprises 31 amino acids and includes a centrally located
mutation encoded by a SNP with 15 flanking amino acids on each side
of the SNP-encoded mutation.
81. The method of claim 78, wherein the peptide epitopes encoded by
the mRNA are T cell epitopes and/or B cell epitopes.
82. The method of claim 81, wherein the T cell epitope comprises
between 8-11 amino acids.
83. The method of claim 81, wherein the B cell epitope comprises
between 13-17 amino acids.
84. The method of claim 78, wherein the lipid delivery vehicle is a
lipid nanoparticle comprising an ionizable cationic lipid, a
non-cationic lipid, a sterol, and a PEG-modified lipid.
85. The method of claim 84, wherein the lipid nanoparticle carrier
comprises a molar ratio of about 20-60% ionizable cationic lipid;
5-25% non-cationic lipid; 25-55% sterol; and 0.5-15% PEG-modified
lipid.
86. The method of claim 78, wherein the lipid delivery vehicle is a
liposome.
87. The method of claim 78, wherein the lipid delivery vehicle is a
lipoplex.
88. The method of claim 78, wherein the lipid delivery vehicle
comprising the personalized cancer vaccine and the immune
checkpoint inhibitor are administered in separate compositions.
89. The method of claim 78, wherein the immune checkpoint inhibitor
binds to PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO,
KIR, or LAG3.
90. The method of claim 89, wherein the immune checkpoint inhibitor
is an antibody.
91. The method of claim 90, wherein the immune checkpoint inhibitor
binds to PD-1.
92. The method of claim 78, wherein the immune checkpoint inhibitor
is Pembrolizumab.
93. The method of claim 78, wherein at least 30% of the epitopes
encoded by the mRNA are MHC class II epitopes.
94. The method of claim 78, wherein one or more of the 2-100
peptide epitopes are interspersed by a linker.
95. The method of claim 94, wherein the linker is a single amino
acid linker.
96. The method of claim 78, wherein the 2-100 peptide epitopes
comprising the concatemeric cancer antigen are directly linked to
each other.
97. The method of claim 78, wherein each peptide epitope encoded by
the mRNA comprises 25-35 amino acids and includes a centrally
located mutation encoded by a single nucleotide polymorphism
(SNP).
98. The method of claim 78, wherein at least 50% of the peptide
epitopes encoded by the mRNA have a predicted binding affinity of
IC >500 nM for HLA-A, HLA-B and/or DRB1.
Description
RELATED APPLICATIONS
[0001] This application is a national stage filing under 35 U.S.C.
.sctn. 371 of international application number PCT/US2016/044918,
filed Jul. 29, 2016, which was published under PCT Article 21(2) in
English, which claims priority under 35 U.S.C. .sctn. 119(e) to
U.S. provisional application No. 62/199,204, filed Jul. 30, 2015,
U.S. provisional application No. 62/247,367, filed Oct. 28, 2015
and U.S. provisional application No. 62/245,145, filed Oct. 22,
2015, the contents of each of which are incorporated herein by
reference in their entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0002] The instant application contains a sequence listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 10, 2021, is named M137870004US07-SUBSEQ-JXV and is 322,333
bytes in size.
BACKGROUND OF INVENTION
[0003] Cancer vaccines include preventive or prophylactic vaccines,
which are intended to prevent cancer from developing in healthy
people; and therapeutic vaccines, which are intended to treat an
existing cancer by strengthening the body's natural defenses
against the cancer. Cancer preventive vaccines may, for instance,
target infectious agents that cause or contribute to the
development of cancer in order to prevent infectious diseases from
causing cancer. Gardasil.RTM. and Cervarix.RTM., are two examples
of commercially available prophylactic vaccines. Each vaccine
protects against HPV infection. Other preventive cancer vaccines
may target host proteins or fragments that are predicted to
increase the likelihood of an individual developing cancer in the
future.
[0004] Most commercial or developing vaccines are based on whole
microorganisms, protein antigens, peptides, polysaccharides or
deoxyribonucleic acid (DNA) vaccines and their combinations. DNA
vaccination is one technique used to stimulate humoral and cellular
immune responses to antigens. The direct injection of genetically
engineered DNA (e.g., naked plasmid DNA) into a living host results
in a small number of its cells directly producing an antigen,
resulting in a protective immunological response. With this
technique, however, comes potential problems of DNA integration
into the vaccine's genome, including the possibility of insertional
mutagenesis, which could lead to the activation of oncogenes or the
inhibition of tumor suppressor genes.
SUMMARY OF INVENTION
[0005] The methods of the invention enable the production of highly
antigenic RNA cancer vaccines, including mRNAs encoding
concatemeric cancer peptide epitopes. The peptide epitopes are
designed to be processed intracellulary and presented to the immune
system in an efficient manner. The mRNA cancer vaccines described
herein are useful for generating a desired immune response by
selecting appropriate T or B cell epitopes which are able to be
presented more effectively on MHC-I or MHC-II molecules (depending
on whether they are T or B-cell epitope, respectively). In some
embodiments T cell epitopes are preferred.
[0006] The concatemeric vaccines may be personalized cancer
vaccines. For instance, the vaccines may include RNA encoding for
one or more cancer antigens specific for each subject.
[0007] An mRNA cancer vaccine is provided in some aspects of the
invention. The vaccine includes an mRNA having an open reading
frame encoding a concatemeric cancer antigen comprised of 2-100
peptide epitopes, a pharmaceutically acceptable carrier or
excipient and wherein the mRNA cancer vaccine further comprises one
or more of:
[0008] a) the of 2-100 peptide epitopes are interspersed by
cleavage sensitive sites;
[0009] b) the mRNA encoding each peptide epitope is linked directly
to one another without a linker;
[0010] c) the mRNA encoding each peptide epitope is linked to one
another with a single nucleotide linker;
[0011] d) each peptide epitope comprises a 25-35 amino acids and
includes a centrally located SNP mutation;
[0012] e) at least 30% of the peptide epitopes have a highest
affinity for class I MHC molecules from the subject;
[0013] f) at least 30% of the peptide epitopes have a highest
affinity for class II MHC molecules from the subject;
[0014] g) at least 50% of the peptide epitopes have a predicted
binding affinity of IC >500 nM for HLA-A, HLA-B and/or DRB1;
[0015] h) the mRNA encodes 20 peptide epitopes;
[0016] i) 50% of the peptide epitopes have a binding affinity for
class I MHC and 50% of the peptide epitopes have a binding affinity
for class II MHC; and/or
[0017] j) the mRNA encoding the peptide epitopes is arranged such
that the peptide epitopes are ordered to minimize
pseudo-epitopes.
[0018] In some embodiments each peptide epitope comprises 31 amino
acids and includes a centrally located SNP mutation with 15
flanking amino acids on each side of the SNP mutation.
[0019] In some aspects the invention is an mRNA cancer vaccine
having a mRNA with an open reading frame encoding a concatemeric
cancer antigen comprised of peptide epitopes interspersed by
cleavage sensitive sites and a pharmaceutically acceptable carrier
or excipient. In some embodiments, the concatemeric cancer antigen
comprises between 2-100 peptide epitopes interspersed by cleavage
sensitive sites.
[0020] In some aspects the mRNA cancer vaccine is a mRNA having an
open reading frame encoding the following peptide
(Z.sub.1).sub.m--(Y.sub.1).sub.p--(X.sub.1--(Y.sub.1).sub.p--X.sub.2--(Y.-
sub.1).sub.p--X.sub.3-- . . .
--(Y.sub.1).sub.p--X.sub.n)--(Y.sub.1).sub.p--(Z.sub.2).sub.o
wherein Z.sub.1 and Z.sub.2 are targeting sequences, independent of
one another, wherein X is a cancer peptide epitope, each cancer
peptide epitope independent of one another, wherein Y is a cleavage
sensitive site, wherein m is 0-1, wherein n is 4-100, wherein p is
0-5 and wherein o is 0-1.
[0021] An mRNA cancer vaccine comprising an mRNA having an open
reading frame encoding a concatemeric antigen and a recall antigen
is provided in other aspects of the invention. In some embodiments
the recall antigen is an infectious disease antigen. In other
embodiments the recall antigen is an mRNA having an open reading
frame encoding the recall antigen. In other embodiments the recall
antigen is a peptide epitope in the concatemeric antigen. In yet
other embodiments the recall antigen is an influenza antigen.
[0022] The vaccine in some embodiments is a personalized cancer
vaccine and the concatemeric antigen comprises subject specific
cancer peptide epitopes. The subject specific cancer peptide
epitopes may be representative of an exome of a tumor sample of the
subject or a transcriptome of a tumor sample of the subject.
[0023] In some embodiments the concatemeric antigen encodes 5-10
cancer peptide epitopes. In yet other embodiments the concatemeric
antigen encodes 25-100 cancer peptide epitopes. In yet other
embodiments the concatemeric antigen encodes 10-1,000 cancer
peptide epitopes.
[0024] The concatemeric antigen encodes peptide epitopes of 10-50
amino acids in length in some embodiments. In other embodiments the
concatemeric antigen encodes peptide epitopes of 15-20 amino acids
in length. In other embodiments the concatemeric antigen encodes
peptide epitopes of 20-50, 25-100, 100-200, 200-300, 300-400, or
400-500 amino acids in length.
[0025] In some embodiments, the peptide epitopes comprise at least
one MHC class I epitope and at least one MHC class II epitope. In
some embodiments, at least 10% of the epitopes are MHC class I
epitopes. In some embodiments, at least 20% of the epitopes are MHC
class I epitopes. In some embodiments, at least 30% of the epitopes
are MHC class I epitopes. In some embodiments, at least 40% of the
epitopes are MHC class I epitopes. In some embodiments, at least
50%, 60%, 70%, 80%, 90% or 100% of the epitopes are MHC class I
epitopes. In some embodiments, at least 10% of the epitopes are MHC
class II epitopes. In some embodiments, at least 20% of the
epitopes are MHC class II epitopes. In some embodiments, at least
30% of the epitopes are MHC class II epitopes. In some embodiments,
at least 40% of the epitopes are MHC class II epitopes. In some
embodiments, at least 50%, 60%, 70%, 80%, 90% or 100% of the
epitopes are MHC class II epitopes. In some embodiments, the ratio
of MHC class I epitopes to MHC class II epitopes is a ratio
selected from about 10%:about 90%; about 20%:about 80%; about
30%:about 70%; about 40%:about 60%; about 50%:about 50%; about
60%:about 40%; about 70%:about 30%; about 80%:about 20%; about
90%:about 10% MHC class I:MHC class II epitopes. In some
embodiments, the ratio of MHC class II epitopes to MHC class I
epitopes is a ratio selected from about 10%:about 90%; about
20%:about 80%; about 30%:about 70%; about 40%:about 60%; about
50%:about 50%; about 60%:about 40%; about 70%:about 30%; about
80%:about 20%; about 90%:about 10% MHC class II:MHC class I
epitopes. In some embodiments, at least one of the peptide epitopes
of the cancer vaccine is a B cell epitope. In some embodiments, the
T cell epitope of the cancer vaccine comprises between 8-11 amino
acids. In some embodiments, the B cell epitope of the cancer
vaccine comprises between 13-17 amino acids.
[0026] The vaccines described herein may be formulated in a lipid
nanoparticle having a mean diameter of 50-200 nm.
[0027] In some embodiments, the mRNA cancer vaccine further
comprises one or more additional mRNAs having an open reading frame
encoding an antigen.
[0028] In some embodiments, the peptide epitopes of the mRNA cancer
vaccine are T cell epitopes and/or B cell epitopes. In other
embodiments, the peptide epitopes of the mRNA cancer vaccine
comprise a combination of T cell epitopes and B cell epitopes. In
yet another embodiment, at least 1 of the peptide epitopes of the
mRNA cancer vaccine is a T cell epitope. In other embodiments, at
least 1 of the peptide epitopes of the mRNA cancer vaccine is a B
cell epitope.
[0029] In some embodiments, the T cell epitope of the mRNA cancer
vaccine comprises between 8-11 amino acids. In other embodiments,
the B cell epitope of the mRNA cancer vaccine comprises between
13-17 amino acids.
[0030] In some embodiments, the mRNA of the mRNA cancer vaccine
further comprises one or more ubiquitination signals. In some
embodiments, the ubiquitination signal of the mRNA cancer vaccine
is located at the C-terminus of the mRNA.
[0031] In some embodiments, the cleavage sensitive sites of the
mRNA cancer vaccine are protease cleavage sites. In other
embodiments, the protease cleavage site of the mRNA cancer vaccine
is a cleavage site for a serine protease, a threonine protease, a
cysteine protease, an aspartate protease, a glutamic acid protease,
or a metalloprotease.
[0032] In some embodiments, the protease cleavage site of the mRNA
cancer vaccine is for a cysteine protease. In some embodiments, the
cysteine protease is cathepsin B.
[0033] In some embodiments, the protease cleavage site comprises
the amino acid sequence GFLG (SEQ ID NO: 1), KVSR (SEQ ID NO: 2),
TVGLR (SEQ ID NO: 3), PMGLP (SEQ ID NO: 4), or PMGAP (SEQ ID NO:
5).
[0034] In some embodiments, mRNA of the mRNA cancer vaccine further
comprises a 5' terminal cap. In some embodiments, the 5' terminal
cap of the mRNA cancer vaccine is 7mG(5')ppp(5')NlmpNp.
[0035] In some embodiments, the mRNA of the mRNA cancer vaccine
further comprises an endosomal targeting sequence. In some
embodiments, the endosomal targeting sequence of the mRNA cancer
vaccine comprises at least a portion of the transmembrane domain of
lysosome associated membrane protein (LAMP-1). In other
embodiments, the endosomal targeting sequence of the mRNA cancer
vaccine comprises at least a portion of the transmembrane domain of
invariant chain (Ii).
[0036] In some embodiments, the mRNA cancer vaccine does not
comprise a stabilization agent.
[0037] In some embodiments, the mRNA of the mRNA cancer vaccine is
formulated in a lipid nanoparticle carrier. In further embodiments,
the lipid nanoparticle carrier comprises a molar ratio of about
20-60% cationic lipid: 5-25% non-cationic lipid: 25-55% sterol; and
0.5-15% PEG-modified lipid. In other embodiments, the cationic
lipid is selected from the group consisting of for example,
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some
embodiments the lipid nanoparticle has a polydispersity value of
less than 0.4. In other embodiments the lipid nanoparticle has a
net neutral charge at a neutral pH value.
[0038] In some embodiments, the mRNA of the mRNA cancer vaccine
includes at least one chemical modification. In some embodiments,
the chemical modification is selected from the group consisting of
pseudouridine, N1-methylpseudouridine, 2-thiouridine,
4'-thiouridine, 5-methylcytosine,
2-thio-1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,
2-thio-dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,
4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,
4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,
5-methyluridine, 5-methoxyuridine, and 2'-O-methyl uridine.
[0039] In some embodiments, the mRNA of the mRNA cancer vaccine
further includes an open reading frame encoding one or more
traditional cancer antigens. In some embodiments, the mRNA of the
mRNA cancer vaccine further includes an mRNA having an open reading
frame encoding one or more traditional cancer antigens.
[0040] In other aspects the invention is an mRNA vaccine,
comprising: a mRNA having an open reading frame encoding a
concatemeric antigen comprised of 2-100 peptide epitopes
interspersed by linkers and a pharmaceutically acceptable carrier
or excipient, wherein each peptide epitope comprises an antigenic
region and a MHC stabilizing region. In some embodiments the
peptide epitopes are cancer peptide epitopes. In other embodiments
the MHC stabilizing region is 5-10 amino acids in length. In yet
other embodiments the antigenic region is 5-100 amino acids in
length.
[0041] A method for vaccinating a subject by administering to a
subject having cancer a mRNA vaccine having an open reading frame
encoding a concatemeric antigen comprised of 2-100 cancer peptide
epitopes interspersed by linkers and a pharmaceutically acceptable
carrier or excipient, wherein each peptide epitope comprises an
antigenic region and a MHC stabilizing region in order to vaccinate
the subject is provided in other aspects of the invention. In some
embodiments the MHC stabilizing region is designed based on the
subject's MHC.
[0042] In some embodiments, the mRNA vaccine further encodes one or
more traditional cancer antigens. In some embodiments, the one or
more traditional cancer antigens are encoded by the same mRNA that
encode the set of neoepitopes. In some embodiments, the one or more
traditional cancer antigens are encoded by a different mRNA than
the mRNA which encodes the set of neoepitopes.
[0043] In some embodiments, the mRNA vaccine is administered in
combination with a cancer therapeutic agent. In some embodiments,
the cancer therapeutic agent is a traditional cancer vaccine.
[0044] In other aspects the invention is a method for preparing a
mRNA cancer vaccine, by isolating a sample from a subject,
identifying a plurality of cancer antigens in the sample,
determining T-cell epitopes from the plurality of cancer antigens,
preparing a mRNA cancer vaccine having an open reading frame
encoding a concatemeric antigen, wherein the concatemeric antigen
is comprised of 2-100 of the T-cell epitopes interspersed by
linkers. In some embodiments the method further involves
determining binding strength of the T-cell epitopes to a MHC of a
subject. In other embodiments the method further involves
determining a T-cell receptor face (TCR face) for each epitope and
selecting epitopes having a TCR face with low similarity to
endogenous proteins.
[0045] A method for vaccinating a subject by administering to a
subject a mRNA cancer vaccine having an open reading frame encoding
a concatemeric antigen comprised of 2-100 T-cell epitopes from the
plurality of cancer antigens of a subject, interspersed by linkers,
wherein the T-cell epitopes have been optimized for binding
strength to a MHC of the subject is provided in aspects of the
invention.
[0046] In other aspects an mRNA vaccine, comprising: a mRNA having
an open reading frame encoding a concatemeric antigen comprised of
2-100 T-cell epitopes from the plurality of cancer antigens of a
subject, interspersed by linkers, wherein the T-cell epitopes have
been optimized for binding strength to a MHC of the subject is
provided. In some embodiments a TCR face for each epitope has a low
similarity to endogenous proteins.
[0047] Another aspect of the present disclosure includes a method
for administering any one of the mRNA cancer vaccines as described
above, in an effective amount to a subject in need thereof. In some
embodiments, the administration is orally, intranasally,
intravenously, intradermally, intramuscularly, or
intraperitoneally. In some embodiments, the administration is a
single administration. In some embodiments, the subject is
administered the vaccine more than once.
[0048] A method of eliciting an immune response in a subject
against a cancer antigen is provided in aspects of the invention.
The method involves administering to the subject a RNA vaccine
comprising at least one RNA polynucleotide having an open reading
frame encoding at least one antigenic polypeptide or an immunogenic
fragment thereof, thereby inducing in the subject an immune
response specific to the antigenic polypeptide or an immunogenic
fragment thereof, wherein the anti-antigenic polypeptide antibody
titer in the subject is increased following vaccination relative to
anti-antigenic polypeptide antibody titer in a subject vaccinated
with a prophylactically effective dose of a traditional vaccine
against the cancer. An "anti-antigenic polypeptide antibody" is a
serum antibody the binds specifically to the antigenic
polypeptide.
[0049] A prophylactically effective dose is a therapeutically
effective dose that prevents advancement of cancer at a clinically
acceptable level. In some embodiments the therapeutically effective
dose is a dose listed in a package insert for the vaccine. A
traditional vaccine, as used herein, refers to a vaccine other than
the mRNA vaccines of the invention. For instance, a traditional
vaccine includes but is not limited to live microorganism vaccines,
killed microorganism vaccines, subunit vaccines, protein antigen
vaccines, DNA vaccines, etc. In exemplary embodiments, a
traditional vaccine is a vaccine that has achieved regulatory
approval and/or is registered by a national drug regulatory body,
for example the Food and Drug Administration (FDA) in the United
States or the European Medicines Agency (EMA.)
[0050] In some embodiments the anti-antigenic polypeptide antibody
titer in the subject is increased 1 log to 10 log following
vaccination relative to anti-antigenic polypeptide antibody titer
in a subject vaccinated with a prophylactically effective dose of a
traditional vaccine against the cancer.
[0051] In some embodiments the anti-antigenic polypeptide antibody
titer in the subject is increased 1 log following vaccination
relative to anti-antigenic polypeptide antibody titer in a subject
vaccinated with a prophylactically effective dose of a traditional
vaccine against the cancer.
[0052] In some embodiments the anti-antigenic polypeptide antibody
titer in the subject is increased 2 log following vaccination
relative to anti-antigenic polypeptide antibody titer in a subject
vaccinated with a prophylactically effective dose of a traditional
vaccine against the cancer.
[0053] In some embodiments the anti-antigenic polypeptide antibody
titer in the subject is increased 3 log following vaccination
relative to anti-antigenic polypeptide antibody titer in a subject
vaccinated with a prophylactically effective dose of a traditional
vaccine against the cancer.
[0054] In some embodiments the anti-antigenic polypeptide antibody
titer in the subject is increased 5 log following vaccination
relative to anti-antigenic polypeptide antibody titer in a subject
vaccinated with a prophylactically effective dose of a traditional
vaccine against the or cancer.
[0055] In some embodiments the anti-antigenic polypeptide antibody
titer in the subject is increased 10 log following vaccination
relative to anti-antigenic polypeptide antibody titer in a subject
vaccinated with a prophylactically effective dose of a traditional
vaccine against the or cancer.
[0056] A method of eliciting an immune response in a subject
against a cancer antigen is provided in other aspects of the
invention. The method involves administering to the subject a RNA
vaccine comprising at least one RNA polynucleotide having an open
reading frame encoding at least one antigenic polypeptide or an
immunogenic fragment thereof, thereby inducing in the subject an
immune response specific to antigenic polypeptide or an immunogenic
fragment thereof, wherein the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine against the cancer antigen at 2 times to 100
times the dosage level relative to the RNA vaccine.
[0057] In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at twice the dosage level relative to the RNA
vaccine.
[0058] In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at three times the dosage level relative to the
RNA vaccine.
[0059] In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 4 times the dosage level relative to the RNA
vaccine.
[0060] In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 5 times the dosage level relative to the RNA
vaccine.
[0061] In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 10 times the dosage level relative to the
RNA vaccine.
[0062] In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 50 times the dosage level relative to the
RNA vaccine.
[0063] In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 100 times the dosage level relative to the
RNA vaccine.
[0064] In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 10 times to 1000 times the dosage level
relative to the RNA vaccine.
[0065] In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 100 times to 1000 times the dosage level
relative to the RNA vaccine.
[0066] In other embodiments the immune response is assessed by
determining antibody titer in the subject.
[0067] In other aspects the invention is a method of eliciting an
immune response in a subject against a by administering to the
subject a RNA vaccine comprising at least one RNA polynucleotide
having an open reading frame encoding at least one cancer antigenic
polypeptide or an immunogenic fragment thereof, thereby inducing in
the subject an immune response specific to the antigenic
polypeptide or an immunogenic fragment thereof, wherein the immune
response in the subject is induced 2 days to 10 weeks earlier
relative to an immune response induced in a subject vaccinated with
a prophylactically effective dose of a traditional vaccine against
the cancer antign. In some embodiments the immune response in the
subject is induced in a subject vaccinated with a prophylactically
effective dose of a traditional vaccine at 2 times to 100 times the
dosage level relative to the RNA vaccine.
[0068] In some embodiments the immune response in the subject is
induced 2 days earlier relative to an immune response induced in a
subject vaccinated with a prophylactically effective dose of a
traditional vaccine.
[0069] In some embodiments the immune response in the subject is
induced 3 days earlier relative to an immune response induced in a
subject vaccinated a prophylactically effective dose of a
traditional vaccine.
[0070] In some embodiments the immune response in the subject is
induced 1 week earlier relative to an immune response induced in a
subject vaccinated with a prophylactically effective dose of a
traditional vaccine.
[0071] In some embodiments the immune response in the subject is
induced 2 weeks earlier relative to an immune response induced in a
subject vaccinated with a prophylactically effective dose of a
traditional vaccine.
[0072] In some embodiments the immune response in the subject is
induced 3 weeks earlier relative to an immune response induced in a
subject vaccinated with a prophylactically effective dose of a
traditional vaccine.
[0073] In some embodiments the immune response in the subject is
induced 5 weeks earlier relative to an immune response induced in a
subject vaccinated with a prophylactically effective dose of a
traditional vaccine.
[0074] In some embodiments the immune response in the subject is
induced 10 weeks earlier relative to an immune response induced in
a subject vaccinated with a prophylactically effective dose of a
traditional vaccine.
[0075] A method of eliciting an immune response in a subject
against an cancer by administering to the subject a cancer RNA
vaccine having an open reading frame encoding a first antigenic
polypeptide, wherein the RNA polynucleotide does not include a
stabilization element, and wherein an adjuvant is not coformulated
or co-administered with the vaccine.
[0076] In some aspects the invention is a method for vaccinating a
subject, by administering to a subject having cancer an mRNA
vaccine comprising one or more polynucleotides having an open
reading frame encoding cancer peptide epitopes, wherein the mRNA
vaccine encodes 5-100 peptide epitopes as single antigens or as a
concatemer or a combination thereof in order to vaccinate the
subject and further comprising administering to the subject having
cancer an immune checkpoint modulator. In some embodiments the
immune checkpoint modulator is an inhibitory checkpoint
polypeptide. The inhibitory checkpoint polypeptide is an anti-CTLA4
or anti-PD1 antibody in some embodiments. In other embodiments the
inhibitory checkpoint polypeptide is an antibody or fragment
thereof that specifically binds to a molecule selected from the
group consisting of PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA,
CTLA-4, IDO, KIR and LAG3.
[0077] In other aspects the invention is an mRNA cancer vaccine,
having an mRNA having an open reading frame encoding a concatemeric
cancer antigen comprised of 2-100 peptide epitopes and a
pharmaceutically acceptable carrier or excipient, wherein the mRNA
is preparable by ligating 2-100 individual mRNAs encoding each of
the 2-100 peptide epitopes.
[0078] In yet other aspects the invention is a method of producing
an mRNA encoding a concatemeric cancer antigen comprising between
1000 and 3000 nucleotides, the method by
[0079] (a) binding a first polynucleotide comprising an open
reading frame encoding the concatemeric cancer antigen and a second
polynucleotide comprising a 5'-UTR to a polynucleotide conjugated
to a solid support;
[0080] (b) ligating the 3'-terminus of the second polynucleotide to
the 5'-terminus of the first polynucleotide under suitable
conditions, wherein the suitable conditions comprise a DNA Ligase,
thereby producing a first ligation product;
[0081] (c) ligating the 5' terminus of a third polynucleotide
comprising a 3'-UTR to the 3'-terminus of the first ligation
product under suitable conditions, wherein the suitable conditions
comprise an RNA Ligase, thereby producing a second ligation
product; and
[0082] (d) releasing the second ligation product from the solid
support,
[0083] thereby producing an mRNA encoding the concatemeric cancer
antigen comprising between 1000 and 3000 nucleotides.
[0084] A kit for preparing an mRNA cancer vaccine is provided in
other aspects of the invention. The kit has one or more containers
housing one or more polynucleotides comprising a 5'-ORF, one or
more polynucleotides comprising a 3'-ORF, one or more
polynucleotides comprising a poly(A) tail, a ligase enzyme, and
instructions for ligating one or more polynucleotides comprising an
ORF encoding a patient specific epitope to the one or more
polynucleotides comprising the a 5'-ORF, 3'-ORF, and poly(A)
tail.
[0085] A method for treating a subject with a personalized mRNA
cancer vaccine, by isolating a sample from a subject, identifying a
set of neoepitopes by analyzing a patient transcriptome and/or a
patient exome from the sample to produce a patient specific
mutanome, selecting a set of neoepitopes for the vaccine from the
mutanome based on MHC binding strength, MHC binding diversity,
predicted degree of immunogenicity, low self reactivity, and/or T
cell reactivity, preparing the mRNA vaccine to encode the set of
neoepitopes and administering the mRNA vaccine to the subject
within two months of isolating the sample from the subject is
provided in other aspects of the invention. In some embodiments the
mRNA vaccine is administered to the subject within one month of
isolating the sample from the subject.
[0086] In other aspects the invention is a method of identifying a
set of neoepitopes for use in a personalized mRNA cancer vaccine
having one or more polynucleotides that encode the set of
neoepitopes by
[0087] a. identifying a patient specific mutanome by analyzing a
patient transcriptome and a patient exome,
[0088] b. selecting a subset of 15-500 neoepitopes from the
mutanome using a weighted value for the neoepitopes based on at
least three of: an assessment of gene or transcript-level
expression in patient RNA-seq; variant call confidence score;
RNA-seq allele-specific expression; conservative vs.
non-conservative amino acid substitution; position of point
mutation (Centering Score for increased TCR engagement); position
of point mutation (Anchoring Score for differential HLA binding);
Selfness: <100% core epitope homology with patient WES data;
HLA-A and -B IC50 for 8mers-11mers; HLA-DRB1 IC50 for
15mers-20mers; promiscuity Score (i.e. number of patient HLAs
predicted to bind); HLA-C IC50 for 8mers-11mers; HLA-DRB3-5 IC50
for 15mers-20mers; HLA-DQB1/A1 IC50 for 15mers-20mers; HLA-DPB1/A1
IC50 for 15mers-20mers; Class I vs Class II proportion; Diversity
of patient HLA-A, -B and DRB1 allotypes covered; proportion of
point mutation vs complex epitopes (e.g. frameshifts); and/or
pseudo-epitope HLA binding scores, and
[0089] c. selecting the set of neoepitopes for use in a
personalized mRNA cancer vaccine from the subset based on the
highest weighted value, wherein the set of neoepitopes comprise
15-40 neoepitopes.
[0090] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways.
BRIEF DESCRIPTION OF DRAWINGS
[0091] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component may be
labeled in every drawing. In the drawings:
[0092] FIGS. 1A-1D show the results of an assay to demonstrate a
mRNA vaccine antigen specific CD8 response.
[0093] FIG. 2 shows the results of an assay to demonstrate a mRNA
vaccine induced antigen specific effector/memory CD8 T cell
response.
[0094] FIG. 3 is a schematic depicting a multi-factorial
consideration of antigen design of mRNA-based neoepitopes. The
sequences, from top to bottom, correspond to SEQ ID NOs: 1130 and
1131.
[0095] FIG. 4 is a table depicting a multi-factorial consideration
of antigen design of mRNA-based neoepitopes.
[0096] FIG. 5 depicts the results of a validation of FACS-based
assay of mRNA encoded epitopes in MCF7 (HLA*201). Specific
MHC1/mut.MART1peptide presentation by anti-mut.MART1TCRmer was
detected on MCF7 cells. The sequences, from top to bottom,
correspond to SEQ ID NOs: 1123-1127.
[0097] FIGS. 6A and 6B are schematics of an exemplary peptide
epitopes. The polypeptide of FIG. 6A includes two or more epitopes.
The epitopes can be of the same sequence or different sequence and
can be all T-cell epitopes, all B-cell epitopes or a combination of
both. The schematic of FIG. 6B shows the peptide epitope with
various end units for enhancing MHC processing of the peptides.
[0098] FIG. 7 depicts exemplary T cell response elicited with mRNA
encoding concatamers of 20 epitopes. mRNA concatamers induced both
class I and class II T cell responses.
[0099] FIG. 8 depicts exemplary T cell response elicited with mRNA
encoding concatamers with and without RP.
[0100] FIG. 9A depicts exemplary T cell response elicited with mRNA
encoding concatamers with epitopes in differing positions. CA80 and
CA81 encode the same 20 epitopes known to elicit T cell responses.
They include 5 class II epitopes, 10 murine class I epitopes, a
murine positive control (SIINFEKL (SEQ ID NO: 6), derived from
ovalbumin), and 4 human (HLA-A2) epitopes (not shown). CA80 and
CA81 differ only in the relative positions of the different
epitopes. FIG. 9B depicts exemplary correlation between
interferon-gamma spot forming units (SFUs) and CD8+ INFN-g+
responses.
[0101] FIG. 10 is a schematic of an exemplary mRNA component of
mRNA-4379.
[0102] FIG. 11 is a schematic of an exemplary general molecular
sequence of mRNA-4379, in which the patient specific coding region
is depicted by reference as (N). The sequences shown are
TABLE-US-00001 (SEQ ID NO: 1128)
ggggaaauaagagagaaaagaagaguaagaagaaauauaagagccaccau g, and (SEQ ID
NO: 1129) ugauaauaggcuggagccucgguggccaugcuucuugccccuugggccuc
cccccagccccuccuccccuuccugcacccguacccccguggucuuugaa
uaaagucugagugggcggcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaucuag.
[0103] FIG. 12 is a block diagram of an exemplary computer system
on which some embodiments may be implemented.
DETAILED DESCRIPTION
[0104] The generation of antigens that elicit a desired immune
response (e.g. T-cell responses) against targeted polypeptide
sequences in vaccine development remains a challenging task. The
invention involves technology that overcome hurdles associated with
vaccine development. Through the use of the technology of the
invention, it is possible to tailor the desired immune response by
selecting appropriate T or B cell epitopes which, by virtue of the
fact that they are processed intra-cellularly, are able to be
presented more effectively on MHC-1 or MHC-2 molecules (depending
on whether they are T or B-cell epitope, respectively). In
particular, the invention involves the generation of concatemers of
epitopes (particularly T cell epitopes) preferably interspersed
with cleavage sites by proteases that are abundant in Antigen
Presenting Cells (APCs). These methods mimic antigen processing and
may lead to a more effective antigen presentation than can be
achieved with peptide antigens.
[0105] The fact that the peptide epitopes of the invention are
expressed from RNA as intracellular peptides provides advantages
over prior art peptides that are delivered as exogenous peptides or
as DNA. The RNA is delivered intra-cellularly and expresses the
epitopes in proximity to the appropriate cellular machinery for
processing the epitopes such that they will be recognized by the
appropriate immune cells. Additionally, a targeting sequence will
allow more specificity in the delivery of the peptide epitopes. For
instance, the invention may involve the addition of a C-terminus
Ubiquin Ligase targeting protein (FBox Protein) to target the
polypeptide processing to the proteasome and more closely mimic the
MHC processing. The constructs of the invention also may include
linkers such as proteolytic cleavage sites optimized for APCs.
These proteolytic sites provide an advantage because they enhance
the processing of the peptides in APCs.
[0106] Thus, the invention relates to mRNA vaccines. mRNA vaccines
are described in International Patent Application No.
PCT/US2015/027400, filed on Apr. 23, 2015, herein incorporated by
reference in its entirety.
[0107] The mRNA cancer vaccines provide unique therapeutic
alternatives to peptide based or DNA vaccines. When the mRNA cancer
vaccine is delivered to a cell, the mRNA will be processed into a
polypeptide by the intracellular machinery which can then process
the polypeptide into immunosensitive fragments capable of
stimulating an immune response against the tumor.
[0108] The concatemeric cancer vaccines may be personalized cancer
vaccines. For instance, the vaccines may include RNA encoding for
one or more cancer antigens specific for each subject, referred to
as neoepitopes. Antigens that are expressed in or by tumor cells
are referred to as "tumor associated antigens". A particular tumor
associated antigen may or may not also be expressed in
non-cancerous cells. Many tumor mutations are well known in the
art. Tumor associated antigens that are not expressed or rarely
expressed in non-cancerous cells, or whose expression in
non-cancerous cells is sufficiently reduced in comparison to that
in cancerous cells and that induce an immune response induced upon
vaccination, are referred to as neoepitopes. Neoepitopes are
completely foreign to the body and thus would not produce an immune
response against healthy tissue or be masked by the protective
components of the immune system. In some embodiments personalized
vaccines based on neoepitopes are desirable because such vaccine
formulations will maximize specificity against a patients specific
tumor. Mutation-derived neoepitopes can arise from point mutations,
non-synonymous mutations leading to different amino acids in the
protein; read-through mutations in which a stop codon is modified
or deleted, leading to translation of a longer protein with a novel
tumor-specific sequence at the C-terminus; splice site mutations
that lead to the inclusion of an intron in the mature mRNA and thus
a unique tumor-specific protein sequence; chromosomal
rearrangements that give rise to a chimeric protein with
tumor-specific sequences at the junction of 2 proteins (i.e., gene
fusion); frameshift mutations or deletions that lead to a new open
reading frame with a novel tumor-specific protein sequence; and
translocations.
[0109] Methods for generating personalized cancer vaccines
generally involve identification of mutations, e.g., using deep
nucleic acid or protein sequencing techniques, identification of
neoepitopes, e.g., using application of validated peptide-MHC
binding prediction algorithms or other analytical techniques to
generate a set of candidate T cell epitopes that may bind to
patient HLA alleles and are based on mutations present in tumors,
optional demonstration of antigen-specific T cells against selected
neoepitopes or demonstration that a candidate neoepitope is bound
to HLA proteins on the tumor surface and development of the
vaccine. The concatemeric vaccines of the invention may include
multiple copies of a single neoepitope, multiple different
neoepitopes based on a single type of mutation, i.e. point
mutation, multiple different neoepitopes based on a variety of
mutation types, neoepitopes and other antigens, such as tumor
associated antigens or recall antigens.
[0110] Examples of techniques for identifying mutations include but
are not limited to dynamic allele-specific hybridization (DASH),
microplate array diagonal gel electrophoresis (MADGE),
pyrosequencing, oligonucleotide-specific ligation, the TaqMan
system as well as various DNA "chip" technologies i.e. Affymetrix
SNP chips, and methods based on the generation of small signal
molecules by invasive cleavage followed by mass spectrometry or
immobilized padlock probes and rolling-circle amplification.
[0111] The deep nucleic acid or protein sequencing techniques are
known in the art. Any type of sequence analysis method can be used.
For instance nucleic acid sequencing may be performed on whole
tumor genomes, tumor exomes (protein-encoding DNA) or tumor
transcriptomes. Real-time single molecule sequencing-by-synthesis
technologies rely on the detection of fluorescent nucleotides as
they are incorporated into a nascent strand of DNA that is
complementary to the template being sequenced. Other rapid high
throughput sequencing methods also exist. Protein sequencing may be
performed on tumor proteomes. Additionally, protein mass
spectrometry may be used to identify or validate the presence of
mutated peptides bound to MHC proteins on tumor cells. Peptides can
be acid-eluted from tumor cells or from HLA molecules that are
immunoprecipitated from tumor, and then identified using mass
spectrometry. The results of the sequencing may be compared with
known control sets or with sequencing analysis performed on normal
tissue of the patient.
[0112] Accordingly, the present invention relates to methods for
identifying and/or detecting neoepitopes of an antigen, such as
T-cell epitopes. Specifically, the invention provides methods of
identifying and/or detecting tumor specific neoepitopes that are
useful in inducing a tumor specific immune response in a subject.
Optionally, these neoepitopes bind to class I HLA proteins with a
greater affinity than the wild-type peptide and/or are capable of
activating anti-tumor CD8 T-cells. Identical mutations in any
particular gene are rarely found across tumors.
[0113] Proteins of MHC class I are present on the surface of almost
all cells of the body, including most tumor cells. The proteins of
MHC class I are loaded with antigens that usually originate from
endogenous proteins or from pathogens present inside cells, and are
then presented to cytotoxic T-lymphocytes (CTLs). T-Cell receptors
are capable of recognizing and binding peptides complexed with the
molecules of MHC class I. Each cytotoxic T-lymphocyte expresses a
unique T-cell receptor which is capable of binding specific
MHC/peptide complexes.
[0114] Using computer algorithms, it is possible to predict
potential neoepitopes such as T-cell epitopes, i.e. peptide
sequences, which are bound by the MHC molecules of class I or class
II in the form of a peptide-presenting complex and then, in this
form, recognized by the T-cell receptors of T-lymphocytes. Examples
of programs useful for identifying peptides which will bind to MHC
include for instance: Lonza Epibase, SYFPEITHI (Rammensee et al.,
Immunogenetics, 50 (1999), 213-219) and HLA_BIND (Parker et al., J.
Immunol., 152 (1994), 163-175).
[0115] Once putative neoepitopes are selected, they can be further
tested using in vitro and/or in vivo assays. Conventional in vitro
lab assays, such as Elispot assays may be used with an isolate from
each patient, to refine the list of neoepitopes selected based on
the algorithm's predictions.
[0116] In some embodiments the mRNA cancer vaccines and vaccination
methods include epitopes or antigens based on specific mutations
(neoepitopes) and those expressed by cancer-germline genes
(antigens common to tumors found in multiple patients, referred to
herein as "traditional cancer antigens" or "shared cancer
antigens"). In some embodiments, a traditional antigen is one that
is known to be found in cancers or tumors generally or in a
specific type of cancer or tumor. In some embodiments, a
traditional cancer antigen is a non-mutated tumor antigen. In some
embodiments, a traditional cancer antigen is a mutated tumor
antigen.
[0117] In some embodiments, the vaccines may further include RNA
encoding for one or more non-mutated tumor antigens. In some
embodiments, the vaccines may further include RNA encoding for one
or more mutated tumor antigens.
[0118] Antigens that are expressed in or by tumor cells are
referred to as "tumor associated antigens". A particular tumor
associated antigen may or may not also be expressed in
non-cancerous cells.
[0119] Many tumor antigens are known in the art. In some
embodiments, the cancer or tumor antigen is one of the following
antigens: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40,
CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5,
AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062,
BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbB1,
ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP,
Fibronectin, Folate Receptor, Ganglioside GM3, GD2,
glucocorticoid-induced tumor necrosis factor receptor (GITR),
gp100, gpA33, GPNMB, ICOS, IGF1R, Integrin av, Integrin
.alpha.v.beta., LAG-3, Lewis Y, Mesothelin, c-MET, MN Carbonic
anhydrase IX, MUC1, MUC16, Nectin-4, NKGD2, NOTCH, OX40, OX40L,
PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, Syndecan-1,
TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2,
VEGFR-3, and variants thereof.
[0120] In some embodiments, the mRNA cancer vaccines and
vaccination methods include an mRNA encoding a concatemeric cancer
antigen comprised of one or more neoepitopes and one or more
traditional, cancer antigens. In some embodiments, the mRNA encodes
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, or more traditional, cancer antigens in addition to the encoded
neoepitopes.
[0121] The mRNA cancer vaccines of the invention are compositions,
including pharmaceutical compositions. The invention also
encompasses methods for the selection, design, preparation,
manufacture, formulation, and/or use of mRNA cancer vaccines. Also
provided are systems, processes, devices and kits for the
selection, design and/or utilization of the mRNA cancer vaccines
described herein.
[0122] In some embodiments the mRNA cancer vaccine of the invention
is a poly-epitopic RNA. Poly-epitopes consist of strings of
epitopes on the same mRNA. The RNA sequences that code for the
peptide epitopes may be interspersed by sequences that code for
amino acid sequences recognized by proteolytic enzymes.
[0123] Thus, in some embodiments an mRNA cancer vaccine is an mRNA
having an open reading frame encoding a propeptide. A propeptide,
as used herein, refers to a peptide sequence which includes
multiple peptide epitopes linked together either directly or
through a linker such as a cleavage sensitive site. An exemplary
propeptide has the following peptide sequence:
(Z.sub.1).sub.m--(Y.sub.1).sub.p--(X.sub.1--(Y.sub.1).sub.p--X.sub.2--(Y-
.sub.1).sub.p--X.sub.3-- . . .
--(Y.sub.1).sub.p--X.sub.n)--(Y.sub.1).sub.p--(Z.sub.2).sub.o
[0124] Z.sub.1 and Z.sub.2 are each targeting sequences, where
m=0-1. The mRNA may include a polynucleotide sequence encoding the
targeting sequence at either the N or C terminus or both ends of
the central peptide epitope region. If a polypeptide has two or
more targeting sequences, those targeting sequences may be the same
or different targeting sequences. Alternatively the polypeptide
encoded by the RNA may not include a targeting sequence.
[0125] X refers to a peptide epitope. Each peptide epitope
designated by an X may represent a unique peptide epitope in the
peptide or it may refer to a copy of an epitope. Thus, the peptide
encoded by the mRNA may be composed of multiple peptide epitopes
each having a unique sequence. Alternatively the peptide may
include 1 or more copies of the same peptide epitope. In some
embodiments a peptide may have 3 or more, 4 or more, 5 or more, 6
or more, 7 or more, 8 or more, 9 or more, or 10 or more copies of
the same peptide epitope. In other embodiments the peptide may
include peptide epitopes that all have the same amino acid
sequence.
[0126] Y is a linker, optionally a cleavage sensitive site, wherein
p is 0-5. Each peptide epitope may optionally have one or more
linkers, optionally cleavage sensitive sites adjacent to the N
and/or C terminal end. In a concatemeric peptide, two or more of
the peptide epitopes may have a cleavage sensitive site between
them. Alternatively two or more of the peptide epitopes may be
connected directly to one another or through a linker that is not a
cleavage sensitive site. The targeting sequence may also be
connected to the peptide epitope through a cleavage sensitive site
or it may be connected directly to the peptide epitope through a
linker that is not a cleavage sensitive site.
[0127] A concatemeric peptide as used herein is series of at least
two peptide epitopes linked together to form the propeptide. In
some embodiments a concatemeric peptide is composed of 3 or more, 4
or more, 5 or more 6 or more 7 or more, 8 or more, 9 or more
peptide epitopes. In other embodiments the concatemeric peptide is
composed of 1000 or less, 900 or less, 500 or less, 100 or less, 75
or less, 50 or less, 40 or less, 30 or less, 20 or less or 100 or
less peptide epitopes. In yet other embodiments, a concatemeric
peptide has 3-100, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100,
35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100,
75-100, 80-100, 90-100, 5-50, 10-50, 15-50, 20-50, 25-50, 30-50,
35-50, 40-50, 45-50, 100-150, 100-200, 100-300, 100-400, 100-500,
50-500, 50-800, 50-1,000, or 100-1,000 peptide epitopes.
[0128] In some embodiments the concatemeric antigen encodes 5-10
cancer peptide epitopes. In yet other embodiments the concatemeric
antigen encodes 25-100 cancer peptide epitopes. In some embodiments
the mRNA cancer vaccines and vaccination methods include epitopes
or antigens based on specific mutations (neoepitopes) and those
expressed by cancer-germline genes (antigens common to tumors found
in multiple patients). In some embodiments, the mRNA cancer
vaccines and vaccination methods include one or more traditional
epitopes or antigens, e.g., one or more epitopes or antigens found
in a traditional cancer vaccine.
[0129] An epitope, also known as an antigenic determinant, as used
herein is a portion of an antigen that is recognized by the immune
system in the appropriate context, specifically by antibodies, B
cells, or T cells. Epitopes include B cell epitopes and T cell
epitopes. B-cell epitopes are peptide sequences which are required
for recognition by specific antibody producing B-cells. B cell
epitopes refer to a specific region of the antigen that is
recognized by an antibody. The portion of an antibody that binds to
the epitope is called a paratope. An epitope may be a
conformational epitope or a linear epitope, based on the structure
and interaction with the paratope. A linear, or continuous, epitope
is defined by the primary amino acid sequence of a particular
region of a protein. The sequences that interact with the antibody
are situated next to each other sequentially on the protein, and
the epitope can usually be mimicked by a single peptide.
Conformational epitopes are epitopes that are defined by the
conformational structure of the native protein. These epitopes may
be continuous or discontinuous, i.e. components of the epitope can
be situated on disparate parts of the protein, which are brought
close to each other in the folded native protein structure.
[0130] T-cell epitopes are peptide sequences which, in association
with proteins on APC, are required for recognition by specific
T-cells. T cell epitopes are processed intracellularly and
presented on the surface of APCs, where they are bound to MHC
molecules including MHC class II and MHC class I.
[0131] The present disclosure, in some aspects, relates to a
process of developing T or B cell concatemeric epitopes or
concatemeric epitopes composed of both B and T cell epitopes.
Several tools exist for identifying various peptide epitopes. For
instance, epitopes can be identified using a free or commercial
database (Lonza Epibase, antitope for example). Such tools are
useful for predicting the most immunogenic epitopes within a target
antigen protein. The selected peptides may then be synthesized and
screened in human HLA panels, and the most immunogenic sequences
are used to construct the mRNAs encoding the concatemeric antigens.
One strategy for mapping epitopes of Cytotoxic T-Cells based on
generating equimolar mixtures of the four C-terminal peptides for
each nominal 11-mer across a protein. This strategy would produce a
library antigen containing all the possible active CTL epitopes
[0132] The peptide epitope may be any length that is reasonable for
an epitope. In some embodiments the peptide epitope is 9-30 amino
acids. In other embodiments the length is 9-22, 9-29, 9-28, 9-27,
9-26, 9-25, 9-24, 9-23, 9-21, 9-20, 9-19, 9-18, 10-22, 10-21,
10-20, 11-22, 22-21, 11-20, 12-22, 12-21, 12-20, 13-22, 13-21,
13-20, 14-19, 15-18, or 16-17 amino acids.
[0133] In some embodiments, the optimal length of a peptide epitope
may be obtained through the following procedure: synthesizing a V5
tag concatemer-test protease site, introducing it into DC cells
(for example, using an RNA Squeeze procedure, lysing the cells, and
then running an anti-V5 Western blot to assess the cleavage at
protease sites.
[0134] The RNA Squeeze technique is an intracellular delivery
method by which a variety of materials can be delivered to a broad
range of live cells. Cells are subjected to microfluidic
construction, which causes rapid mechanical deformation. The
deformation results in temporary membrane disruption and the
newly-formed transient pores. Material is then passively diffused
into the cell cytosol via the transient pores. The technique can be
used in a variety of cell types, including primary fibroblasts,
embryonic stem cells, and a host of immune cells, and has been
shown to have relatively high viability in most applications and
does not damage sensitive materials, such as quantum dots or
proteins, through its actions. Sharei et al., PNAS (2013);
110(6):2082-7.
[0135] In some embodiments the concatemeric antigen may include a
recall antigen, also sometimes referred to as a memory antigen. A
recall antigen is an antigen that has previously been encountered
by an individual and for which there are pre-existent memory
lymphocytes. In some embodiments the recall antigen may be an
infectious disease antigen that the individual has likely
encountered such as an influenza antigen. The recall antigen helps
promote a more robust immune response.
[0136] The neoepitopes may be designed to optimally bind to MHC in
order to promote a robust immune response. In some embodiments each
peptide epitope comprises an antigenic region and a MHC stabilizing
region. An MHC stabilizing region is a sequence which stabilizes
the peptide in the MHC. The MHC stabilizing region may be 5-10,
5-15, 8-10, 1-5, 3-7, or 3-8 amino acids in length. In yet other
embodiments the antigenic region is 5-100 amino acids in length.
The peptides interact with the molecules of MHC class I by
competitive affinity binding within the endoplasmic reticulum,
before they are presented on the cell surface. The affinity of an
individual peptide is directly linked to its amino acid sequence
and the presence of specific binding motifs in defined positions
within the amino acid sequence. The peptide being presented in the
MHC is held by the floor of the peptide-binding groove, in the
central region of the .alpha.1/.alpha.2 heterodimer (a molecule
composed of two nonidentical subunits). The sequence of residues,
of the peptide-binding groove's floor determines which particular
peptide residues it binds.
[0137] Optimal binding regions may be identified by a computer
assisted comparison of the affinity of a binding site (MHC pocket)
for a particular amino acid at each amino acid in the binding site
for each of the target epitopes to identify an ideal binder for all
of the examined antigens. The MHC stabilization regions of the
epitopes may be identified using amino acid prediction matrices of
data points for a binding site. An amino acid prediction matrix is
a table having a first and a second axis defining data points.
Prediction matrices can be generated as shown in Singh, H. and
Raghava, G. P. S. (2001), "ProPred: prediction of HLA-DR binding
sites." Bioinformatics, 17(12), 1236-37).
[0138] In some embodiments the MHC stabilizing region is designed
based on the subject's particular MHC. In that way the MHC
stabilizing region can be optimized for each patient.
[0139] In some instances each epitope of a concatemeric antigen may
include a MHC stabilizing region. All of the MHC stabilizing
regions within the epitopes may be the same or they may be
different. The MHC stabilizing regions may be at the N terminal
portion of the peptide or the C terminal portion of the peptide.
Alternatively the MHC stabilizing regions may be in the central
region of the peptide. The neoepitopes in some embodiments are 13
residues or less in length and usually consist of between about 8
and about 11 residues, particularly 9 or 10 residues. In other
embodiments the neoepitopes may be designed to be longer. For
instance, the neoepitopes may have extensions of 2-5 amino acids
toward the N- and C-terminus of each corresponding gene product.
The use of a longer peptide may allow endogenous processing by
patient cells and may lead to more effective antigen presentation
and induction of T cell responses.
[0140] The neoepitopes selected for inclusion in the concatemeric
antigen typically will be high affinity binding peptides. In some
aspect the neoepitopes binds an HLA protein with greater affinity
than a wild-type peptide. The neoepitope has an IC50 of at least
less than 5000 nM, at least less than 500 nM, at least less than
250 nM, at least less than 200 nM, at least less than 150 nM, at
least less than 100 nM, at least less than 50 nM or less in some
embodiments. The neoepitopes in the concatemeric construct may be
the same or different, e.g., they vary by length, amino acid
sequence or both. Typically, peptides with predicted IC50<50 nM,
are generally considered medium to high affinity binding peptides
and will be selected for testing their affinity empirically using
biochemical assays of HLA-binding. Finally, it will be determined
whether the human immune system can mount effective immune
responses against these mutated tumor antigens and thus effectively
kill tumor but not normal cells.
[0141] Neoepitopes having the desired activity may be modified as
necessary to provide certain desired attributes, e.g. improved
pharmacological characteristics, while increasing or at least
retaining substantially all of the biological activity of the
unmodified peptide to bind the desired MHC molecule and activate
the appropriate T cell or B cell. For instance, the neoepitopes may
be subject to various changes, such as substitutions, either
conservative or non-conservative, where such changes might provide
for certain advantages in their use, such as improved MHC binding.
By conservative substitutions is meant replacing an amino acid
residue with another which is biologically and/or chemically
similar, e.g., one hydrophobic residue for another, or one polar
residue for another. The substitutions include combinations such as
Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys,
Arg; and Phe, Tyr. The effect of single amino acid substitutions
may also be probed using D-amino acids. Such modifications may be
made using well known peptide synthesis procedures, as described in
e.g., Merrifield, Science 232:341-347 (1986), Barany &
Merrifield, The Peptides, Gross & Meienhofer, eds. (N.Y.,
Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid
Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed.
(1984).
[0142] The neoepitopes can also be modified by extending or
decreasing the compound's amino acid sequence, e.g., by the
addition or deletion of amino acids. The peptides, polypeptides or
analogs can also be modified by altering the order or composition
of certain residues, it being readily appreciated that certain
amino acid residues essential for biological activity, e.g., those
at critical contact sites or conserved residues, may generally not
be altered without an adverse effect on biological activity.
[0143] Typically, a series of peptides with single amino acid
substitutions are employed to determine the effect of electrostatic
charge, hydrophobicity, etc. on binding. For instance, a series of
positively charged (e.g., Lys or Arg) or negatively charged (e.g.,
Glu) amino acid substitutions are made along the length of the
peptide revealing different patterns of sensitivity towards various
MHC molecules and T cell or B cell receptors. In addition, multiple
substitutions using small, relatively neutral moieties such as Ala,
Gly, Pro, or similar residues may be employed. The substitutions
may be homo-oligomers or hetero-oligomers. The number and types of
residues which are substituted or added depend on the spacing
necessary between essential contact points and certain functional
attributes which are sought (e.g., hydrophobicity versus
hydrophilicity). Increased binding affinity for an MHC molecule or
T cell receptor may also be achieved by such substitutions,
compared to the affinity of the parent peptide. In any event, such
substitutions should employ amino acid residues or other molecular
fragments chosen to avoid, for example, steric and charge
interference which might disrupt binding.
[0144] The neoepitopes may also comprise isosteres of two or more
residues in the neoepitopes. An isostere as defined here is a
sequence of two or more residues that can be substituted for a
second sequence because the steric conformation of the first
sequence fits a binding site specific for the second sequence. The
term specifically includes peptide backbone modifications well
known to those skilled in the art. Such modifications include
modifications of the amide nitrogen, the .alpha.-carbon, amide
carbonyl, complete replacement of the amide bond, extensions,
deletions or backbone crosslinks. See, generally, Spatola,
Chemistry and Biochemistry of Amino Acids, Peptides and Proteins,
Vol. VII (Weinstein ed., 1983).
[0145] In other aspects the invention is a method for preparing a
mRNA cancer vaccine, by isolating a sample from a subject,
identifying a plurality of cancer antigens in the sample,
determining T-cell epitopes from the plurality of cancer antigens,
preparing a mRNA cancer vaccine having an open reading frame
encoding a concatemeric antigen, wherein the concatemeric antigen
is comprised of 2-100 of the T-cell epitopes interspersed by
linkers. In some embodiments the method further involves
determining binding strength of the T-cell epitopes to a MHC of a
subject. In other embodiments the method further involves
determining a T-cell receptor face (TCR face) for each epitope and
selecting epitopes having a TCR face with low similarity to
endogenous proteins. The T-cell epitopes may have been optimized
for binding strength to a MHC of the subject is provided. In some
embodiments a TCR face for each epitope has a low similarity to
endogenous proteins.
[0146] For instance a technology referred to as JanusMatrix
(Epivax), which examines cross-reactive T cell epitopes from both
HLA binding and TCR-facing sides to allow comparison across large
genome sequence databases can be used to identify epitopes having a
desirable TCR face and binding strength to MHC. A suite of
algorithms can be used alone or together with the JanusMatrix to
optimize epitope selection. For example EpiMatrix takes overlapping
9-mer frames derived from the conserved target protein sequences
and scores them for potential binding affinity against a panel of
Class I or Class II HLA alleles; each frame-by-allele assessment
that scores highly and is predicted to bind is a putative T cell
epitope. ClustiMer takes EpiMatrix output and identifies clusters
of 9-mers that contain large numbers of putative T cell epitopes.
BlastiMer automates the process of submitting the previously
identified sequences to BLAST to determine if any share
similarities with the human genome; any such similar sequences
would be likely to be tolerated or to elicit an unwanted autoimmune
response. EpiAssembler takes the conserved, immunogenic sequences
identified by Conservatrix and EpiMatrix and knits them together to
form highly immunogenic consensus sequences. JanusMatrix can be
used to screen out sequences which could potentially elicit an
undesired autoimmune or regulatory T cell response due to homology
with sequences encoded by the human genome. VaccineCAD can be used
to link candidate epitopes into a string-of-beads design while
minimizing nonspecific junctional epitopes that may be created in
the linking process.
[0147] In addition to peptide epitopes, the concatemeric antigen
may have one or more targeting sequences. A targeting sequence, as
used herein, refers to a peptide sequence that facilitates uptake
of the peptide into intracellular compartments such as endosomes
for processing and/or presentation within MHC class I or II
determinants.
[0148] The targeting sequence may be present at the N-terminus
and/or C-terminus of an epitope of the concatemeric antigen, either
directly adjacent thereto or separated by a linker of a cleavage
sensitive site. Targeting sequences have a variety of lengths, for
instance 4-50 amino acids in length.
[0149] The targeting sequence may be, for instance, an endosomal
targeting sequence. An endosomal targeting sequence is a sequence
derived from an endosomal or lysosomal protein known to reside in
MHC class II Ag processing compartments, such as invariant chain,
lysosome-associated membrane proteins (LAMP1,4 LAMP2), and
dendritic cell (DC)-LAMP or a sequence having at least 80% sequence
identity thereto. Additionally, an exemplary nucleic acid encoding
a MHC class I signal peptide fragment (78 bp, secretion signal
(sec)) and the transmembrane and cytosolic domains including the
stop-codon (MHC class I trafficking signal (MITD), 168 bp) both
amplified from activated PBMC, may be used (sec sense, 5'-aag ctt
agc ggc cgc acc atg cgg gtc acg gcg ccc cga acc-3' (SEQ ID NO: 7);
sec antisense, 5'-ctg cag gga gcc ggc cca ggt ctc ggt cag-3' (SEQ
ID NO: 8); MITD sense, 5'-gga tcc atc gtg ggc att gtt get ggc ctg
get-3' (SEQ ID NO: 9); and MITD antisense, 5'-gaa ttc agt ctc gag
tca agc tgt gag aga cac atc aga gcc-3' (SEQ ID NO: 10).
[0150] MHC Class I presentation is typically an inefficient process
(only 1 peptide of 10,000 degraded molecules is actually
presented). Priming of CD8 T cells with APCs provides insufficient
densities of surface peptide/MHC I complexes results in weak
responders exhibiting impaired cytokine secretion and a decreased
memory pool. The methods of the invention are capable of increasing
the efficiency of MHC Class I presentation. MHC class I targeting
sequences include MHC Class I trafficking signal (MITD) and PEST
sequences (increase antigen-specific CD8 T cell responses
presumably by targeting proteins for rapid degradation).
[0151] In some embodiments the RNA vaccines can be combined with
agents for promoting the production of antigen presenting cells
(APCs), for instance, by converting non-APCs into Pseudo-APCs.
Antigen presentation is a key step in the initiation, amplification
and duration of an immune response. In this process fragments of
antigens are presented through the Major Histocompatibility Complex
(MHC) or Human Leukocyte Antigens (HLA) to T cells driving an
antigen-specific immune response. For immune prophylaxis and
therapy, enhancing this response is important for improved
efficacy. The RNA vaccines of the invention may be designed or
enhanced to drive efficient antigen presentation. One method for
enhancing APC processing and presentation, is to provide better
targeting of the RNA vaccines to antigen presenting cells (APC).
Another approach involves activating the APC cells with
immune-stimulatory formulations and/or components.
[0152] Alternatively, methods for reprograming non-APC into
becoming APC may be used with the RNA vaccines of the invention.
Importantly, most cells that take up mRNA formulations and are
targets of their therapeutic actions are not APC. Therefore,
designing a way to convert these cells into APC would be beneficial
for efficacy. Methods and approaches for delivering RNA vaccines,
e.g., mRNA vaccines to cells while also promoting the shift of a
non-APC to an APC are provided herein. In some embodiments a mRNA
encoding an APC reprograming molecule is included in the RNA
vaccine or coadministered with the RNA vaccine.
[0153] An APC reprograming molecule, as used herein, is a molecule
that promotes a transition in a non APC cell to an APC-like
phenotype. An APC-like phenotype is property that enables MHC class
II processing. Thus, an APC cell having an APC-like phenotype is a
cell having one or more exogenous molecules (APC reprograming
molecule) which has enhanced MHC class II processing capabilities
in comparison to the same cell not having the one or more exogenous
molecules. In some embodiments an APC reprograming molecule is a
CIITA (a central regulator of MHC Class II expression); a chaperone
protein such as CLIP, HLA-DO, HLA-DM etc. (enhancers of loading of
antigen fragments into MHC Class II) and/or a costimulatory
molecule like CD40, CD80, CD86 etc. (enhancers of T cell antigen
recognition and T cell activation).
[0154] A CIITA protein is a transactivator that enhances activation
of transcription of MHC Class II genes (Steimle et al., 1993, Cell
75:135-146) by interacting with a conserved set of DNA binding
proteins that associate with the class II promoter region. The
transcriptional activation function of CIITA has been mapped to an
amino terminal acidic domain (amino acids 26-137). A nucleic acid
molecule encoding a protein that interacts with CIITA, termed
CIITA-interacting protein 104 (also referred to herein as CIP104).
Both CITTA and CIP104 have been shown to enhance transcription from
MHC class II promoters and thus are useful as APC reprograming
molecule of the invention. In some embodiments the APC reprograming
molecule are full length CIITA, CIP104 or other related molecules
or active fragments thereof, such as amino acids 26-137 of CIITA,
or amino acids having at least 80% sequence identity thereto and
maintaining the ability to enhance activation of transcription of
MHC Class II genes.
[0155] In preferred embodiments the APC reprograming molecule is
delivered to a subject in the form of an mRNA encoding the APC
reprograming molecule. As such the RNA vaccines of the invention
may include an mRNA encoding an APC reprograming molecule. In some
embodiments the mRNA in monocistronic. In other embodiments it is
polycistronic. In some embodiments the mRNA encoding the one or
more antigens is in a separate formulation from the mRNA encoding
the APC reprograming molecule. In other embodiments the mRNA
encoding the one or more antigens is in the same formulation as the
mRNA encoding the APC reprograming molecule. In some embodiments
the mRNA encoding the one or more antigens is administered to a
subject at the same time as the mRNA encoding the APC reprograming
molecule. In other embodiments the mRNA encoding the one or more
antigens is administered to a subject at a different time than the
mRNA encoding the APC reprograming molecule. For instance, the mRNA
encoding the APC reprograming molecule may be administered prior to
the mRNA encoding the one or more antigens. The mRNA encoding the
APC reprograming molecule may be administered immediately prior to,
at least 1 hour prior to, at least 1 day prior to, at least one
week prior to, or at least one month prior to the mRNA encoding the
antigens. Alternatively, the mRNA encoding the APC reprograming
molecule may be administered after the mRNA encoding the one or
more antigens. The mRNA encoding the APC reprograming molecule may
be administered immediately after, at least 1 hour after, at least
1 day after, at least one week after, or at least one month after
the mRNA encoding the antigens.
[0156] In other embodiments, the targeting sequence is a
ubiquitination signal that is attached at either or both ends of
the encoded peptide. In other embodiments, the targeting sequence
is a ubiquitination signal that is attached at an internal site of
the encoded peptide and/or to either end. Thus, the RNA may include
a nucleic acid sequence encoding a ubiquitination signal at either
or both ends of the nucleotides encoding the concatemeric peptide.
Ubiquitination, a post-translational modification, is the process
of attaching ubiquitin to a substrate target protein. A
ubiquitination signal is a peptide sequence which enables the
targeting and processing of a peptide to one or more proteasomes.
By targeting and processing the peptide through the use of a
ubiquitination signal the intracellular processing of the peptide
can more closely recapitulate antigen processing in Antigen
Presenting Cells (APCs).
[0157] Ubiquitin is an 8.5 kDa regulatory protein that is found in
nearly all tissues of eukaryotic organisms. In the human genome,
there are four genes that produce ubiquitin: UBB, UBC, UBA52, and
RPS27A. UBA52 and RPS27A code for a single copy of ubiquitin fused
to the ribosomal proteins L40 and S27a, respectively. The UBB and
UBC genes code for polyubiquitin precursor proteins. There are
three steps to ubiquitination, performed by three enzymes.
Ubiquitin-activating enzymes, also called E1 enzymes, modify the
ubiquitin so that it is in a reactive state. The E1 binds to both
ATP and ubiquitin, catalyzing the acyl-adenylation of ubiquitin's
C-terminal. Then, the ubiquitin is transferred to an active site
cysteine residue, releasing AMP. Ultimately, a thioester linkage is
formed between the ubiquitin's C-terminal carboxyl group and the E1
cysteine sulfhydryl group. In the human genome, UBA1 and UBA6 are
the two genes that code for the E1 enzymes.
[0158] The activated ubiquitin is then subjected to E2
ubiquitin-conjugating enzymes, which transfer the ubiquitin from E1
to the active site cysteine of the E2 via a
trans(thio)esterification reaction. The E2 binds to both the
activated ubiquitin and the E1 enzyme. Humans have 35 different E2
enzymes, characterized by their highly conserved structure, which
is known as the ubiquitin-conjugating catalytic (UBC) fold. The E3
ubiquitin ligases facilitate the final step of the ubiquitination
cascade. Generally, they create an isopeptide bond between a lysine
of the target protein and the C-terminal glycine of ubiquitin.
There are hundreds of E3 ligases; some also activate the E2
enzymes. E3 enzymes function as the substrate recognition modules
of the system and interact with both the E2 and the substrate. The
enzymes possess one of two domains: the homologous to the E6-AP
carboxyl terminus (HECT) domain or the really interesting new gene
(RING) domain (or the closely related, U-box domain). HECT domain
E3 enzymes transiently bind ubiquitin when an obligate thioester
intermediate is formed with the active-site cysteine of the E3,
whereas RING domain E3 enzymes catalyze the direct transfer from
the E2 enzyme to the substrate.
[0159] The number of ubiquitins added to the antigen can enhance
the efficacy of the processing step. For instance, in
polyubiquitination, additional ubiquitin molecules are added after
the first has been attached to the peptide. The resulting ubiquitin
chain is created by the linking of the glycine residue of the
ubiquitin molecule to a lysine of the ubiquitin bound to the
peptide. Each ubiquitin contains seven lysine residues and an
N-terminal that can serve as sites for ubiquitination. When four or
more ubiquitin molecules are attached to a lysine residue on the
peptide antigen, the 26S proteasome recognizes the complex,
internalizes it, and degrades the protein into small peptides.
[0160] Ubiquitin wild type has the following sequence (Homo
sapiens):
TABLE-US-00002 (SEQ ID NO: 11)
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQL
EDGRTLSDYNIQKESTLHLVLRLRGG
[0161] The epitopes are connected in some embodiments by a cleavage
sensitive site. A cleavage sensitive site is a peptide which is
susceptible to cleavage by an enzyme or protease. These sites are
also called protease cleavage sites. Preferably the protease is an
intracellular enzyme. In some preferred embodiments the protease is
a protease found in an Antigen Presenting Cell (APC). Thus,
protease cleavage sites correspond to high abundance (highly
expressed) proteases in APCs. A cleavage sensitive site that is
sensitive to an APC enzyme is referred to as an APC cleavage
sensitive site. Proteases expressed in APCs include but are not
limited to Cysteine proteases, such as: Cathepsin B, Cathepsin H,
Cathepsin L, Cathepsin S, Cathepsin F, Cathepsin Z, Cathepsin V,
Cathepsin O, Cathepsin C, and Cathepsin K, and Aspartic proteases
such as Cathepsin D, Cathepsin E, and Asparaginyl
endopeptidase.
[0162] The following are exemplary APC cleavage sensitive
sites:
Cathepsin B: cleavage on the caboxyl side of Arg-Arg bonds
Cathepsin D has the following preferential cleavage sequences:
TABLE-US-00003 P6 P5 P4 P3 P2 P1 .dwnarw. P1' P2' P3' P4' Xaa Xaa
Xaa Xaa hy- hy- .dwnarw. hy- Xaa Xaa Xaa dro dro dro Xaa Xaa Xaa
Xaa Glu hy- .dwnarw. hy- Xaa Xaa Xaa, dro dro
where Xaa=any amino acid residue, hydro=Ala, Val, Leu, Ile, Phe,
Trp, or Tyr, and .dwnarw.=cleavage site
Cathepsin H: Arg-.dwnarw.-NHMec; Bz-Arg-.dwnarw.-NhNap;
Bz-Arg-.dwnarw.NHMec; Bz-Phe-Cal-Arg-.dwnarw.-NHMec;
Pro-Gly-.dwnarw.-Phe
Cathepsin S and F: Xaa-Xaa-Val-Val-Arg-Xaa-Xaa
[0163] where Xaa=any amino acid residue
Cathepsin V: Z-Phe-Arg-NHMec; Z-Leu-Arg-NHMec; Z-Val-Arg-NHMec
Cathepsin O: Z-Phe-Arg-NHMec and Z-Arg-Arg-NHMec
[0164] Cathepsin C has the following preferential cleavage
sequences:
TABLE-US-00004 P2 P1 .dwnarw. P1' P2' P3' P4' not Arg not Pro
.dwnarw. not Pro Xaa Xaa Xaa not Lys not Pro .dwnarw. not Pro Xaa
Xaa Xaa,
where Xaa=any amino acid residue and .dwnarw.=cleavage site
Cathepsin E: Arg-X, Glu-X, and Arg-Arg
[0165] Asparaginyl endopeptidase: after asparagine residues
Cathepsin L has the following preferential cleavage sequences:
TABLE-US-00005 P6 P5 P4 P3 P2 P1 .dwnarw. P1' P2' P3' P4' Xaa Xaa
Xaa hydrophobic Phe Arg .dwnarw. Xaa Xaa Xaa Xaa Xaa Xaa Xaa
aromatic Phe Arg .dwnarw. Xaa Xaa Xaa Xaa Xaa Xaa Xaa hydrophobic
Arg Arg .dwnarw. Xaa Xaa Xaa Xaa Xaa Xaa Xaa aromatic Arg Arg
.dwnarw. Xaa Xaa Xaa Xaa, .dwnarw.
where Xaa=any amino acid residue, hydrophobic=Ala, Val, Leu, Ile,
Phe, Trp, or Tyr, aromatic=Phe, Trp, His, or Tyr, and
.dwnarw.=cleavage site
[0166] In some preferred embodiments the cleavage sensitive site is
a cathepsin B or S sensitive sites. Exemplary cathepsin B sensitive
sites include but are not limited to:
TABLE-US-00006 (SEQ ID NO: 12)
EGAMVAATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 13)
AMVAATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 14)
GGGGGGGGAGAAGGGGGGENYDDPHK, (SEQ ID NO: 15)
MVAATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 16)
QLLCGAAIGTHEDDKYR, (SEQ ID NO: 17) FSHHFEDADNIYIFLELCSRKS, (SEQ ID
NO: 18) YXLVGAGAIGCELLK, (SEQ ID NO: 19)
IPESCSFGYHAGGWGKPPVDETGKPL, (SEQ ID NO: 20)
VAATQGAAAAAGSGAGTGGGTASGGTEGGSASEGAK, (SEQ ID NO: 21)
SEADIEGPLPAKDIHLDLPSNN, (SEQ ID NO: 22) HFNALGGWGELQNSVK, (SEQ ID
NO: 23) FAQALGLTEAVK, (SEQ ID NO: 24) TSVLAAANPIESQWNPK, (SEQ ID
NO: 25) QLLQANPILESFGNAK, (SEQ ID NO: 26) TSILAAANPISGHYDR, (SEQ ID
NO: 27) IXXANPLLEAFGNAK, (SEQ ID NO: 28) LYGAQFHPEVGLTENGK, (SEQ ID
NO: 29) PQGQAPPLSQAQGHPGIQTPQR, (SEQ ID NO: 30)
AAASAAAASAASGSPGPGEGSAGGEKR, (SEQ ID NO: 31) IXXXFLGASLKDEVLK, (SEQ
ID NO: 32) LTISPDYAYGATGHPGIIPPH, (SEQ ID NO: 33)
LTISPDYAYGATGHPGIIPPHA, (SEQ ID NO: 34) ILISLATGHREEGGENLDQ, (SEQ
ID NO: 35) LSELTQQLAQATGKPPQYIAVHVVPDQ, (SEQ ID NO: 36)
LSELTQQLAQATGKPPQYIAVHVVPDQL, (SEQ ID NO: 37) DATNVGDEGGFAPNILENK,
(SEQ ID NO: 38) ILAQATSDLVNAIK, (SEQ ID NO: 39) VXXVXQHAVGIVVNK,
(SEQ ID NO: 40) GSLAEAVGSPPPAATPTPTPPTR, (SEQ ID NO: 41)
SXGLPVGAVINCADNTGAK, (SEQ ID NO: 42) YCFSEMAPVCAVVGGILAQEIVK, (SEQ
ID NO: 43) HVYGYSMAYGPAQHAISTEK, (SEQ ID NO: 44)
LWQLSKPRPGCSVLGPLPLL, (SEQ ID NO: 45) MILIQDGSQNTNVDKPLR, (SEQ ID
NO: 46) TYSMVVVPLYDTLGPGAIRYII, (SEQ ID NO: 47)
HFAMMHGGTGFAGIDSSSPEVK, (SEQ ID NO: 48) GXLKPGMVVTFAPVNVTTEVK, (SEQ
ID NO: 49) FNALFAQGNYSEAAK, (SEQ ID NO: 50)
GPIHIGGPPGFASSSGKPGPTVIK, (SEQ ID NO: 51) GFGFVTFDDHDPVDK, (SEQ ID
NO: 52) DQGSCGSCWAFGAVEAISDR, (SEQ ID NO: 53)
GXNFGFGDSRGGGGNFGPGPG, (SEQ ID NO: 54) HDLFDSGFGGGAGVETGGK, (SEQ ID
NO: 55) CYLFGGLANDSEDPK, (SEQ ID NO: 56) TTEDSVMLNGFGTVVNALGK, (SEQ
ID NO: 57) LTEGLHGFHVHEFGDNTAGC, (SEQ ID NO: 58) GYAFIEYEHER, (SEQ
ID NO: 59) MFIGGLSWDTSKK, (SEQ ID NO: 60) MFIGGLSWDTTKK, (SEQ ID
NO: 61) SMGFIGHYLDQK, (SEQ ID NO: 62) SMGFIGHYLDQK, (SEQ ID NO: 63)
ALXGGIGFIHHNCTPEFQANE, (SEQ ID NO: 64) NLQSTFSGFGFINSENVFK, (SEQ ID
NO: 65) GFCFITYTDEEPVKK, (SEQ ID NO: 66) MPMFIVNTNVPR, (SEQ ID NO:
67) VSEIFVELQGFLAAEQDIR, (SEQ ID NO: 68) GFCFLEYEDHK, (SEQ ID NO:
69) QAVSMFLGAVEEAKK, (SEQ ID NO: 70) KPXKPMQFLGDEETVRK, (SEQ ID NO:
71) GAAEPHTIAAFLGGAAAQEVIK, (SEQ ID NO: 72) MIPCDFLIPVQTQHPIR, (SEQ
ID NO: 73) QGAPTSFLPPEASQLKPDR, (SEQ ID NO: 74) STGGAPTFNVTVTK,
(SEQ ID NO: 75) MVYMFQYDSTHGK, (SEQ ID NO: 76)
HFPMTHGNTGFSGIESSSPEVK, (SEQ ID NO: 77) AVAFSPVTELKK, (SEQ ID NO:
78) GFGFVTFSSMAEVDAAMAARPH, (SEQ ID NO: 79) TCGFDFTGAVEDISK, (SEQ
ID NO: 80) EYSGLSDGYGFTTDLFGR, (SEQ ID NO: 81)
GQHVXGSPFQFTVGPLGEGGAHK, (SEQ ID NO: 82) GFGFVDFNSEEDAK, (SEQ ID
NO: 83) FXFVEFEDPR, (SEQ ID NO: 84) FXFVEFEDPR, (SEQ ID NO: 85)
IELFVGGELIDPADDRK, (SEQ ID NO: 86) MFVGGLSWDTSKK, (SEQ ID NO: 87)
AFSAFVGQMHQQGILK, (SEQ ID NO: 88) GILFVGSGVSGGEEGAR, (SEQ ID NO:
89) IIAFVGSPVEDNEKDLVK, (SEQ ID NO: 90) DYAFVHFEDR, (SEQ ID NO: 91)
GYAFVHFETQEAADK, (SEQ ID NO: 92) GYGFVHFETQEAAER, (SEQ ID NO: 93)
NYGFVHIEDK, (SEQ ID NO: 94) ITLPVDFVTADKFDENAK, (SEQ ID NO: 95)
GFGFVTFDDHDPVDK, (SEQ ID NO: 96) LPNFGFVVFDDSEPVQK, (SEQ ID NO: 97)
GFGFVYFQNHDAADK, (SEQ ID NO: 98) YQFWDTQPVPK, (SEQ ID NO: 99)
QLLCGAAIGTHEDDKYR, (SEQ ID NO: 100) QLLCGAAIGTHEDDKYR, (SEQ ID NO:
101) PPAGGGGGAGGAGGGPPPGPPGAGDR, (SEQ ID NO: 102)
FGGSFAGSFGGAGGHAPGVAR, (SEQ ID NO: 103) CNPIISGLYQGAGGPGPGGFGAQGPK,
(SEQ ID NO: 104) PGLNLPPPIGGAGPPLGLPKPK, (SEQ ID NO: 105)
QPXVDGFLVGGASLKPEFVDIINAK, (SEQ ID NO: 106) VTGDHIPTPQDLPQR, (SEQ
ID NO: 107) YGGELVPHFPAR, (SEQ ID NO: 108) YQGAGGPGPGGFGAQGPK, (SEQ
ID NO: 109) EYFGGFGEVESIELPMDNK, (SEQ ID NO: 110) ALVLGGFAHMDTETK,
(SEQ ID NO: 111) VSHVSTGGGASLELLEGK, (SEQ ID NO: 112)
AEGGGGGGRPGAPAAGDGK, (SEQ ID NO: 113) RGGGGGGSGGIGYPYPR, (SEQ ID
NO: 114) NMGGPYGGGNYGPGGSGGSGGYG, (SEQ ID NO: 115)
GTGGVDTAATGGVFDISNLDR, (SEQ ID NO: 116) HFNALGGWGELQNSVK, (SEQ ID
NO: 117) PESCSFGYHAGGWGKPPVDETGKPL, (SEQ ID NO: 118)
SSLPNFCGIFNHLER, (SEQ ID NO: 119) AMALXGGIGFIHHNCTPEF, (SEQ ID NO:
120) AMALXGGIGFIHHNCTPEFQANE, (SEQ ID NO: 121)
EWIKPIMFSGGIGSMEADHISK, (SEQ ID NO: 122) GDGPVQGIINFEQK, (SEQ ID
NO: 123) EMAPVCAVVGGILAQEIVK, (SEQ ID NO: 124) LAFHGILLHGLEDR, (SEQ
ID NO: 125) MGVVAGILVQNVLK, (SEQ ID NO: 126) FTASAGIQVVGDDLTVTNPK,
(SEQ ID NO: 127) TPYQIACGISQGLADNTVIAK, (SEQ ID NO: 128)
YPIEHGIVTNWDDMEK, (SEQ ID NO: 129) VASGIPAGWXGLDCGPESSKK, (SEQ ID
NO: 130) LFVGGLDWSTTQETLR, (SEQ ID NO: 131) HGGSLGLGLAAMGTAR, (SEQ
ID NO: 132) IFVGGLSANTVVEDVK, (SEQ ID NO: 133) LFIGGLSFETTDDSLR,
(SEQ ID NO: 134) LFIGGLSFETTDESLR, (SEQ ID NO: 135)
LFIGGLSFETTEESLR, (SEQ ID NO: 136) MFXGGLSWDTSKK, (SEQ ID NO: 137)
MFXGGLSWDTSKK, (SEQ ID NO: 138) MFXGGLSWDTSKK, (SEQ ID NO: 139)
DAVSGMGVIVHIIEK, (SEQ ID NO: 140) GGNFGFGDSR, (SEQ ID NO: 141)
GTTGSGAGSGGPGGLTSAAPAGGDKK, (SEQ ID NO: 142)
IISGLYQGAGGPGPGGFGAQGPK, (SEQ ID NO: 143) IISGLYQGAGGPGPGGFGAQGPK,
(SEQ ID NO: 144) GGGLLIGGQAWDWANQGEDERV, (SEQ ID NO: 145)
GNFGGSFAGSFGGAGGHAPGVAR, (SEQ ID NO: 146) NFGGSFAGSFGGAGGHAPGVAR,
(SEQ ID NO: 147) NFGGSFAGSFGGAGGHAPGVAR, (SEQ ID NO: 148)
NFGGSFAGSFGGAGGHAPGVAR, (SEQ ID NO: 149)
SAADTKPGTTGSGAGSGGPGGLTSAAPAGGDKK, (SEQ ID NO: 150)
AATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 151)
GSSGGSGAKPSDAASEAAR, (SEQ ID NO: 152) IQFHFHWGSLDGQGSEHTVDK, (SEQ
ID NO: 153) MILIQDGSQNTNVDKPLR, (SEQ ID NO: 154) KGTFTDDLHK, (SEQ
ID NO: 155) QQSHFAMMHGGTGFAGIDSSSPEVK, (SEQ ID NO: 156)
VAVLISGTGSNLQALIDSTR, (SEQ ID NO: 157) FLAAGTHLGGTNLDFQ, (SEQ ID
NO: 158) LVLGTHTSDEQNHL, (SEQ ID NO: 159) TGGVDTAATGGVFDISNLDR,
(SEQ ID NO: 160) TGGVDTAAVGGVFDVSNADR, (SEQ ID NO: 161)
AVXIVAAGVGEFEAGISK, (SEQ ID NO: 162) EILTLLQGVHQGAGFQDIPK, (SEQ ID
NO: 163) MKPLMGVIYVPLTDKEK, (SEQ ID NO: 164) ECISXHVGQAGVQIGNACWE,
(SEQ ID NO: 165) HFNALGGWGELQNSVK, (SEQ ID NO: 166)
ESCSFGYHAGGWGKPPVDETGKPL, (SEQ ID NO: 167) AGYVTHLMK, (SEQ ID NO:
168) TMFSSEVQFGHAGACANQASETAVAK, (SEQ ID NO: 169)
MPFPVNHGASSEDTLLK, (SEQ ID NO: 170) FFLHHLIAEIHTAEIRAT, (SEQ ID NO:
171) NXSAXQVLIEHIGNLDR, (SEQ ID NO: 172) GGYVLHIGTIYGDLK, (SEQ ID
NO: 173) DXHLGGEDFDNR, (SEQ ID NO: 174) GILGPPPPSFHLGGPAVGPR, (SEQ
ID NO: 175) PTPPPTLHLVPEPAAPPPP, (SEQ ID NO: 176)
YGPQYGHPPPPPPPPEYGPHADSPV, (SEQ ID NO: 177) KHSGPNSADSANDGFVR, (SEQ
ID NO: 178) RPELLTHSTTEVTQPR,
(SEQ ID NO: 179) LXGHVGFDSLPDQLVNK, (SEQ ID NO: 180)
AASATQTIAAAQHAASTPK, (SEQ ID NO: 181) CLTQSGIAGGYKPF, (SEQ ID NO:
182) ELAQIAGRPTEDEDEKEK, (SEQ ID NO: 183) AITIAGVPQSVTECVK, (SEQ ID
NO: 184) GLCAIAQAESLR, (SEQ ID NO: 185) KPTALIGVAAIGGAFSEQILK, (SEQ
ID NO: 186) DYMNVQCHACIGGTNVGEDIR, (SEQ ID NO: 187)
NTQNFQSLHNIGSVVQHSEGKPL, (SEQ ID NO: 188) LKPPTLIHGQAPSAGLPSQKPK,
(SEQ ID NO: 189) VLIIGGGDGGVLR, (SEQ ID NO: 190) GCITIIGGGDTATCCAK,
(SEQ ID NO: 191) GRPSETGIIGIIDPECR, (SEQ ID NO: 192)
EAFGWHAIIVDGHSVEELCK, (SEQ ID NO: 193) LAAAILGGVDQIHIKPG, (SEQ ID
NO: 194) LYSILGTTLKDEGK, (SEQ ID NO: 195) MILIQDGSQNTNVDKPLR, (SEQ
ID NO: 196) LAMQEFMILPVGAANFR, (SEQ ID NO: 197) VPYLIAGIQHSCQDIGAK,
(SEQ ID NO: 198) TVAGGVHISGLHTESAPR, (SEQ ID NO: 199)
VAVLISGTGSNLQALIDSTR, (SEQ ID NO: 200) GITAIGGTSTISSEGTQHSYSEEEK,
(SEQ ID NO: 201) AGVSISVVHGNLSEEAAK, (SEQ ID NO: 202)
HVTQAHVQTGITAAPPPHPGAPHPPQ, (SEQ ID NO: 203) AGLFLPGSVGITDPCESGNFR,
(SEQ ID NO: 204) AFAHITGGGLLENIPR, (SEQ ID NO: 205) ILAQITGTEHLK,
(SEQ ID NO: 206) TFXNITPAEVGVLVGK, (SEQ ID NO: 207)
HSSGIVADLSEQSLK, (SEQ ID NO: 208) EDGNEEDKENQGDETQGQQPPQR, (SEQ ID
NO: 209) PGPSGITIPGKPGAQGVPGPPG, (SEQ ID NO: 210)
GLTKPAALAAAPAKPGGAGGSK, (SEQ ID NO: 211) LGAQLADLHLDNK, (SEQ ID NO:
212) SLVASLAEPDFVVTDFAK, (SEQ ID NO: 213) MSLPLLAGGVADDINTNKK, (SEQ
ID NO: 214) QPYAVSELAGHQTSAESWGTGR, (SEQ ID NO: 215)
VTVAGLAGKDPVQC, (SEQ ID NO: 216) IITLAGPTNAIFK, (SEQ ID NO: 217)
STHGLAILGPENPK, (SEQ ID NO: 218) ASAELALGENSEVLK, (SEQ ID NO: 219)
ILISLATGHREEGGENLDQ, (SEQ ID NO: 220) AMSRPFGVALLFGGVDEK, (SEQ ID
NO: 221) LQATAHAQAQLGCPVIIHPGR, (SEQ ID NO: 222) ILAGLGFDPEMQNRPT,
(SEQ ID NO: 223) PERPQQLPHGLGGIGMGLGPGGQPIDANHLNK, (SEQ ID NO: 224)
QLMQLIGPAGLGGLGGLGALTGPG, (SEQ ID NO: 225) HFNALGGWGELQNSVK, (SEQ
ID NO: 226) MGAGLGHGMDR, (SEQ ID NO: 227) THMTAIVGMALGHRPIPNQPPT,
(SEQ ID NO: 228) PHGLGGIGMGLGPGGQPIDANHLNK, (SEQ ID NO: 229)
ASQGDSISSQLGPIHPPPR, (SEQ ID NO: 230) VWQLGSSSPNFTLEGHEK, (SEQ ID
NO: 231) YVATLGVEVHPL, (SEQ ID NO: 232) KLIADYSPDDIFN, (SEQ ID NO:
233) TXGLIFVVDSNDR, (SEQ ID NO: 234) VPEFQFLIGDEAATHLK, (SEQ ID NO:
235) CNINLLPLPDPIPSGLME, (SEQ ID NO: 236) LITEMVALNPDFKPPADYKPPA,
(SEQ ID NO: 237) NQVALNPQNTVFDAK, (SEQ ID NO: 238) GLLKPGLNVVLEGPK,
(SEQ ID NO: 239) GVNLPGAAVDLPAVSEK, (SEQ ID NO: 240)
ISXGLPVGAVINCADNTGAK, (SEQ ID NO: 241) GQVCLPVISAENWK, (SEQ ID NO:
242) EILTLLQGVHQGAGFQDIPK, (SEQ ID NO: 243) NNQFQALLQYADPVSAQHAK,
(SEQ ID NO: 244) LFIGGLSFETTDDSLR, (SEQ ID NO: 245) AIQLSGAEQLEALK,
(SEQ ID NO: 246) DVSIEDSVISLSGDHCIIGR, (SEQ ID NO: 247)
EYLLSGDISEAEHCLK, (SEQ ID NO: 248) VVISSDGQFALSGSWDGTLR, (SEQ ID
NO: 249) VHEQLAALSQGPISKPK, (SEQ ID NO: 250)
LVXLXXETALLSSGFSLEDPQTH, (SEQ ID NO: 251) GPDGLTAFEATDNQAIK, (SEQ
ID NO: 252) ALYWLSGLTCTEQNFISK, (SEQ ID NO: 253) IITLTGPTNAIFK,
(SEQ ID NO: 254) LATQLTGPVMPVR, (SEQ ID NO: 255) FPSLLTHNENMVAK,
(SEQ ID NO: 256) LEXLXTINXGLTSIANLPK, (SEQ ID NO: 257)
ALLLLLVGGVDQSPR, (SEQ ID NO: 258) GKPVGLVGVTELSDAQKK, (SEQ ID NO:
259) VNVAGLVLAGSADFK, (SEQ ID NO: 260) QGYIGAALVLGGVDVTGPH, (SEQ ID
NO: 261) LYTLVLTDPDAPSR, (SEQ ID NO: 262) AQIHDLVLVGGSTR,
(SEQ ID NO: 263) LNHVAAGLVSPSLKSDTSSK, (SEQ ID NO: 264)
IEVGLVVGNSQVAFEK, (SEQ ID NO: 265) GYHQSASEHGLVVIAPDTSPR, (SEQ ID
NO: 266) GYHQSASEHGLVVIAPDTSPR, (SEQ ID NO: 267)
QDHPWLLSQNLVVKPDQLIK, (SEQ ID NO: 268) MGLAMGGGGGASFDR, (SEQ ID NO:
269) QLPHGLGGIGMGLGPGGQPIDANHLNK, (SEQ ID NO: 270)
VVVLMGSTSDLGHCEK, (SEQ ID NO: 271) MALIQMGSVEEAVQA, (SEQ ID NO:
272) TTGFGMIYDSLDYAK, (SEQ ID NO: 273) WLLAEMLGDLSDSQLK, (SEQ ID
NO: 274) QAQYLGMSCDGPFKPDH, (SEQ ID NO: 275) AHSSMVGVNLPQK, (SEQ ID
NO: 276) SGPVVAMVWEGLNVVK, (SEQ ID NO: 277)
VNTQNFQSLHNIGSVVQHSEGKPL, (SEQ ID NO: 278) LYVSNLGIGHTR, (SEQ ID
NO: 279) VYVGNLGNNGNKTELER, (SEQ ID NO: 280) IVDLLQMLEMNMAIAFPA,
(SEQ ID NO: 281) VLAQNSGFDLQETLVK, (SEQ ID NO: 282)
QQSHFPMTHGNTGFSGIESSSPEVK, (SEQ ID NO: 283) ILIANTGMDTDKIK, (SEQ ID
NO: 284) NNTVTPGGKPNK, (SEQ ID NO: 285) VVNVANVGAVPSGQDNIHR, (SEQ
ID NO: 286) MPFPVNHGASSEDTLLK, (SEQ ID NO: 287) RPKDPGHPY, (SEQ ID
NO: 288) ELDIMEPKVPDDIYK, (SEQ ID NO: 289) AETSQQEASEGGDPASPALSLS,
(SEQ ID NO: 290) LLAAQNPLSQADRPHQ, (SEQ ID NO: 291)
PDNFXFGQSGAGNNWAK, (SEQ ID NO: 292) MIAGQVLDINLAAEPK, (SEQ ID NO:
293) IILNSHSPAGSAAISQQDFHPK, (SEQ ID NO: 294)
GAVAVSAAPGSAAPAAGSAPAAAEEK, (SEQ ID NO: 295)
SAAGAAGSAGGSSGAAGAAGGGAGAGTRPGDGGTASAGAAGPGAATK, (SEQ ID NO: 296)
FTASAGIQVVGDDLTVTNPK, (SEQ ID NO: 297) FGIVTSSAGTGTTEDTEAKK, (SEQ
ID NO: 298) SLYQSAGVAPESFEYIEAHGTGTK, (SEQ ID NO: 299)
VSEIDEMFEARKM, (SEQ ID NO: 300) FGGSFAGSFGGAGGHAPG, (SEQ ID NO:
301) FGGSFAGSFGGAGGHAPGVAR, (SEQ ID NO: 302) FGGSFAGSFGGAGGHAPGVAR,
(SEQ ID NO: 303) FGGSFAGSFGGAGGHAPGVAR, (SEQ ID NO: 304)
AADTKPGTTGSGAGSGGPGGLTSAAPAGGDKK, (SEQ ID NO: 305)
ATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 306)
EIELIGSGGFGQVFK, (SEQ ID NO: 307) KPGTTGSGAGSGGPGGLTSAAPAGGDKK,
(SEQ ID NO: 308) LYANXVXSGGTTMYPGIADR, (SEQ ID NO: 309) RSGKYDLDFK,
(SEQ ID NO: 310) HDGYGSHGPLLPLPSR, (SEQ ID NO: 311) SLFSSIGEVESAK,
(SEQ ID NO: 312) LQSIGTENTEENR, (SEQ ID NO: 313)
SLVASLAEPDFVVTDFAK, (SEQ ID NO: 314) AEPMGEKPVGSLAGIGEVLGK, (SEQ ID
NO: 315) VQEAINSLGGSVFPK, (SEQ ID NO: 316)
SAAAASAASGSPGPGEGSAGGEKR, (SEQ ID NO: 317) QTIDNSQGAYQEAFDISKK,
(SEQ ID NO: 318) FGIVTSSAGTGTTEDTEAK, (SEQ ID NO: 319)
FGIVTSSAGTGTTEDTEAKK, (SEQ ID NO: 320) XSSFDLDYDFQR, (SEQ ID NO:
321) FQAGTSKPLHSSGINVNAAPF, (SEQ ID NO: 322) HIGGPPGFASSSGKPGPTVIK,
(SEQ ID NO: 323) XSSGPGASSGTSGDHGELVVR, (SEQ ID NO: 324)
ELVSSSSSGSDSDSEVDKK, (SEQ ID NO: 325) MDSTEPPYSQKR, (SEQ ID NO:
326) VVVLMGSTSDLGHCEK, (SEQ ID NO: 327) ILDSVGIEADDDRLNK, (SEQ ID
NO: 328) STQPISSVGKPASVIK, (SEQ ID NO: 329) ALQSVGQIVGEVLK, (SEQ ID
NO: 330) VSSLAEGSVTSVGSVNPAENFR, (SEQ ID NO: 331) TGSISSSVSVPAKPER,
(SEQ ID NO: 332) YXXXXXXYSQSYGGYENQK, (SEQ ID NO: 333)
IYWGTATTGKPHV, (SEQ ID NO: 334) MVQTAVVPVKK, (SEQ ID NO: 335)
MMLGTEGGEGFVVK, (SEQ ID NO: 336) XTFIAIKPDGVQR, (SEQ ID NO: 337)
VSHVSTGGGASLELLEGK, (SEQ ID NO: 338)
AAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 339)
TIGNSCGTIGLIHAVANNQDK, (SEQ ID NO: 340) TGEEIFGTIGMRPNAK, (SEQ ID
NO: 341) TTQFSCTLGEKFEETTADGR, (SEQ ID NO: 342) GCTATLGNFAK, (SEQ
ID NO: 343) YVATLGVEVHPL, (SEQ ID NO: 344) LAATNALLNSLEFTK, (SEQ ID
NO: 345) GPGASSGTSGDHGELVVR, (SEQ ID NO: 346)
STTTGHLIYK, (SEQ ID NO: 347) ALSAADTKPGTTGSGAGSGGPGGLTSAAPAGGDKK,
(SEQ ID NO: 348) STTTGHLIYK, (SEQ ID NO: 349) VTIIGPATVGGIKPGCFK,
(SEQ ID NO: 350) TVVFSHPPIGTVGLTEDEAIHK, (SEQ ID NO: 351)
GSPTSLGTWGSWIGPDHDK, (SEQ ID NO: 352) GSPTSLGTWGSWIGPDHDKF, (SEQ ID
NO: 353) IHFPLATYAPVISAEK, (SEQ ID NO: 354) ANPQVGVAFPHIK, (SEQ ID
NO: 355) TCTTVAFTQVNSEDK, (SEQ ID NO: 356) VLTGVAGEDAECHAAK, (SEQ
ID NO: 357) NIPPYFVALVPQEEELDDQK, (SEQ ID NO: 358)
GQETAVAPSLVAPALNKPK, (SEQ ID NO: 359) QGQETAVAPSLVAPALNKPK, (SEQ ID
NO: 360) GFVTFSSMAEVDAAMAARPH, (SEQ ID NO: 361) VDYYTTTPALVFGKPVR,
(SEQ ID NO: 362) VDYYTTTPALVFGKPVR, (SEQ ID NO: 363)
ASQPXVDGFLVGGASLKPEFVDIINAK, (SEQ ID NO: 364) TIIGPATVGGIKPGCFK,
(SEQ ID NO: 365) GGVDTAAVGGVFDVSNADR, (SEQ ID NO: 366) TTVHAITATQK,
(SEQ ID NO: 367) EEVRPQDTVSVIGGVAGGSK, (SEQ ID NO: 368)
QVIGTGSFFPK, (SEQ ID NO: 369) ASGNYATVISHNPETK, (SEQ ID NO: 370)
MKPLMGVIYVPLTDKEK, (SEQ ID NO: 371) FSVCVLGDQQHCDEAK, (SEQ ID NO:
372) ENAFCNLAAIVPDSVGRHSPA, (SEQ ID NO: 373) AYVGNLPFNTVQGDIDAIFK,
(SEQ ID NO: 374) TLTTVQGIADDYDKK, (SEQ ID NO: 375)
CISXHVGQAGVQIGNACWE, (SEQ ID NO: 376) THALQWPSLTVQWLPEVTKPEGK, (SEQ
ID NO: 377) ASVPAGGAVAVSAAPGSAAPAAGSAPAAAEEK, (SEQ ID NO: 378)
YEEVSVSGFEEFHR, (SEQ ID NO: 379) CMTTVSWDGDKLQCVQK, (SEQ ID NO:
380) MHGGGPTVTAGLPLPK, (SEQ ID NO: 381) LALVTGGEIASTFDHPELVK, (SEQ
ID NO: 382) LEGTLLKPNMVTPGHACTQK, (SEQ ID NO: 383) XVVESAYEVIK,
(SEQ ID NO: 384) ILAQVVGDVDTSLPR, (SEQ ID NO: 385)
CFSEMAPVCAVVGGILAQEIVK, (SEQ ID NO: 386) ETEDTFXADLVVGLCTGQIK, (SEQ
ID NO: 387) EGPAVVGQFIQDVK, (SEQ ID NO: 388) MLISGYALNCVVGSQGMPK,
(SEQ ID NO: 389) HWPFMVVNDAGRPK, (SEQ ID NO: 390) SGPVVAMVWEGLNVVK,
(SEQ ID NO: 391) ALQDEWDAVMLHSFTLRQ, (SEQ ID NO: 392)
EYFSWEGAFQHVGK, (SEQ ID NO: 393) ATVASGIPAGWMGLDCGPESSK, (SEQ ID
NO: 394) ATVASGIPAGWMGLDCGPESSKK, (SEQ ID NO: 395)
DCAFYDPTHAWSGGLDHQLK, (SEQ ID NO: 396) QFQALLQYADPVSAQHAK, (SEQ ID
NO: 397) EQPQHPLHVTYAGAAVDELGK, (SEQ ID NO: 398) TFSYAGFEMQPK, (SEQ
ID NO: 399) GYIWNYGAIPQTWEDPGHNDK, (SEQ ID NO: 400)
DYTGYNNYYGYGDYSNQQSGYGK, (SEQ ID NO: 401) QSGYGGQTKPIFR, (SEQ ID
NO: 402) VPLIESGTAGYLGQVTTIKK, (SEQ ID NO: 403)
GILGYTEHQVVSSDFNSDTH, (SEQ ID NO: 404) GILGYTEHQVVSSDFNSDTHSS, (SEQ
ID NO: 405) QTCVXHYTGMLEDGKK, (SEQ ID NO: 406, QTCVXHYTGMLEDGKKFDS,
and (SEQ ID NO: 407) AXYVTHLMK.
[0167] Exemplary cathepsin S sensitive sites include but are not
limited to:
TABLE-US-00007 (SEQ ID NO: 408) KVSVR, (SEQ ID NO: 3) TVGLR, (SEQ
ID NO: 4) PMGLP, (SEQ ID NO: 5) PMGAP, (SEQ ID NO: 409)
MDLAAAAEPGAGSQHLEVR, (SEQ ID NO: 410)
EGAMVAATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 411)
GTSFDAAATSGGSASSEK, (SEQ ID NO: 412)
AMVAATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 413)
GILAADESTGSIAK, (SEQ ID NO: 414) PAAPALSAADTKPGTTGSGAGSGGPGGLT,
(SEQ ID NO: 415) MVAATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID
NO: 416) SSIQATTAAGSGHPTSCC, (SEQ ID NO: 417) NEAIQAAHDAVAQEGQCR,
(SEQ ID NO: 418) QFGLPAEAVEAANKGDVEAFAK, (SEQ ID NO: 419)
LVIPNTLAVNAAQDSTDLVAK, (SEQ ID NO: 420) EALAAMNAAQVKPLGK, (SEQ ID
NO: 421) APRPPVSAASGRPQDDTDSSR, (SEQ ID NO: 422)
GDPQEAKPQEAAVAPEKPPASDETK, (SEQ ID NO: 423) EGDMIVCAAYAHELPK, (SEQ
ID NO: 424) GILAADESTGSIAK, (SEQ ID NO: 425) PEEACSFILSADFPALVVK,
(SEQ ID NO: 426) GWNAYIDNLMADGTCQDAAIVGYK, (SEQ ID NO: 427)
YLAADKDGNVTCER, (SEQ ID NO: 428) LPVDFVTADKFDENAK, (SEQ ID NO: 429)
TXEAEAAHGTVTR, (SEQ ID NO: 430) TVFAEHISDECK, (SEQ ID NO: 431)
TVFAEHISDECKR, (SEQ ID NO: 432) DLEAEHVEVEDTTLNR, (SEQ ID NO: 433)
CAEIAHNVSSK, (SEQ ID NO: 434) EAAAAGGGVGAGAGGGCGPGGADSSKPR, (SEQ ID
NO: 435) YXLVGAGAIGCELLK, (SEQ ID NO: 436) LIYAGKILNDDTALK, (SEQ ID
NO: 437) FGDNTAGCTSAGPHFNPLSR, (SEQ ID NO: 438) IITLAGPTNAIFK, (SEQ
ID NO: 439) EIVHXQAGQCGNQIGAK, (SEQ ID NO: 440)
AICAGPTALLAHEIGFGSK, (SEQ ID NO: 441) SHEHSPSDLEAHFVPLVK, (SEQ ID
NO: 442) ELQAHGADELLK, (SEQ ID NO: 443) SWADLVNAHVVPGSGVVK, (SEQ ID
NO: 444) TFIAIKPDGVQR, (SEQ ID NO: 445) GYIWNYGAIPQTWEDPGHNDK, (SEQ
ID NO: 446) ATATXXAKPQITNPK, (SEQ ID NO: 447) TTETAQHAQGAKPQVQPQK,
(SEQ ID NO: 448) VASYLLAALGGNSSPSAK, (SEQ ID NO: 449)
IALPAPRGSGTASD, (SEQ ID NO: 450) QIGNVAALPGIVHR, (SEQ ID NO: 451)
DGTVLCELINALYPEGQAPVK, (SEQ ID NO: 452) DGTVLCELINALYPEGQAPVKK,
(SEQ ID NO: 453) DFTVSAMHGDMDQK, (SEQ ID NO: 454) LVTDCVAAMNPDAVLR,
(SEQ ID NO: 455) HELQANCYEEVKDR, (SEQ ID NO: 456)
CSLQAAAILDANDAHQTETSSSQVK, (SEQ ID NO: 457) SGLGRPQLQGAPAAEPMAVP,
(SEQ ID NO: 458) QETAVAPSLVAPALNKPK, (SEQ ID NO: 459)
AQXAAPASVPAQAPK, (SEQ ID NO: 460) GETIFVTAPHEATAGIIGVNR, (SEQ ID
NO: 461) EYSSELNAPSQESDSHPR, (SEQ ID NO: 462) DQVTAQEIFQDNHEDGPTAK,
(SEQ ID NO: 463) LHEEEIQELQAQIQEQHVQ, (SEQ ID NO: 464)
QQQRPLEAQPSAPGHSVK, (SEQ ID NO: 465) AAHTANFLLNASGSTSTPAPSR, (SEQ
ID NO: 466) IXXXFLGASLKDEVLK, (SEQ ID NO: 467)
NVEEADAAMAASPHAVDGNTVELK, (SEQ ID NO: 468) ALLVTASQCQQPAENK, (SEQ
ID NO: 469) QSSWGMMGMLASQQNQSGPSGNNQNQGNMQR, (SEQ ID NO: 470)
LPPGFSASSTVEKPSK, (SEQ ID NO: 471) AAVPSGASTGIYEALE, (SEQ ID NO:
472) LNCQVIGASVDSHFCH, (SEQ ID NO: 473) GANQYTFHLEATENPGALIK, (SEQ
ID NO: 474) LSELTQQLAQATGKPPQYIAVH, (SEQ ID NO: 475)
AGEQEGAMVAATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 476)
ISSIQATTAAGSGHPTSCC, (SEQ ID NO: 477) GLGATTHPTAAVK, (SEQ ID NO:
478) IEPPPLDAVIEAEHTLR, (SEQ ID NO: 479) SXGLPVGAVINCADNTGAK, (SEQ
ID NO: 480) VNVANVGAVPSGQDNIHR, (SEQ ID NO: 481) QFLECAQNQGDIK,
(SEQ ID NO: 482) GGLTDEAALSCCSDADPSTK, (SEQ ID NO: 483)
ILSCGEVIHVK, (SEQ ID NO: 484) AIVDCGFEHPSEVQ, (SEQ ID NO: 485)
HYYEVSCHDQGLCR, (SEQ ID NO: 486) MLVQCMQDQEHPSIR, (SEQ ID NO: 487)
DVVICPDASLEDAKK, (SEQ ID NO: 488)
SGYAFVDCPDEHWAMK, (SEQ ID NO: 489) FVLCPECENPETDLHVNPK, (SEQ ID NO:
490) IAILTCPFEPPKPK, (SEQ ID NO: 491) IAILTCPFEPPKPK, (SEQ ID NO:
492) AATEQYHQVLCPGPSQDDPLHPLNK, (SEQ ID NO: 493) AIVICPTDEDLKDR,
(SEQ ID NO: 494) ATAHAQAQLGCPVIIHPGR, (SEQ ID NO: 495)
IIPGXMCQGGDFTR, (SEQ ID NO: 496) IIPGXMCQGGDFTR, (SEQ ID NO: 497)
PALYWLSGLTCTEQNFISK, (SEQ ID NO: 498) MTVGCVAGDEESYEVFK, (SEQ ID
NO: 499) ATVAFCDAQSTQEIHEK, (SEQ ID NO: 500) NYGILADATEQVGQHK, (SEQ
ID NO: 501) LQDCEGLIVR, (SEQ ID NO: 502) QISAGYXPVXDCHTAHIACK, (SEQ
ID NO: 503) QISAGYXPVXDCHTAHIACK, (SEQ ID NO: 504)
TGEPCCDWVGDEGAGHFVK, (SEQ ID NO: 505) DATNVGDEGGFAPNILENK, (SEQ ID
NO: 506) NILDFPQHVSPSK, (SEQ ID NO: 507) DHVVSDFSEHGSLK, (SEQ ID
NO: 508) ADNELSPECLDGAQHFLK, (SEQ ID NO: 509) IQTLGYFPVGDGDFPHQK,
(SEQ ID NO: 510) DXQEXXXFLLDGLHEDLNR, (SEQ ID NO: 511)
MILIQDGSQNTNVDKPLR, (SEQ ID NO: 512) FTISDHPQPIDPLLK, (SEQ ID NO:
513) VNPTXFFDIAVDGEPLGR, (SEQ ID NO: 514) YEDICPSTHNMDVPNIK, (SEQ
ID NO: 515) NGSIYNPEVLDITEETLHSR, (SEQ ID NO: 516) QHIVNDMNPGNLH,
(SEQ ID NO: 517) MLLDSEQHPCQLK, (SEQ ID NO: 518) EGLMLDSHEELYK,
(SEQ ID NO: 519) TAGDTHLGGEDFDNR, (SEQ ID NO: 520)
PGGLLLGDVAPNFEANTTVGR, (SEQ ID NO: 521) NDGATILSMMDVDHQIAK, (SEQ ID
NO: 522) GEEGLTLNLEDVQPHDLGK, (SEQ ID NO: 523) TIDNSQGAYQEAFDISKK,
(SEQ ID NO: 524) IVGFFDDSFSEAHSEFLK, (SEQ ID NO: 525)
QEEASGVALGEAPDHSYESLR, (SEQ ID NO: 526) DPVQEAWAEDVDLR, (SEQ ID NO:
527) PMIYICGECHTENEIK, (SEQ ID NO: 528) HMSEFMECNLNELVK, (SEQ ID
NO: 529) MGYAEEAPYDAIHVG, (SEQ ID NO: 530) MADQLTEEQIAEFK, (SEQ ID
NO: 531) YLAEFATGNDRK, (SEQ ID NO: 532) LAELEEFINGPNNAHIQ, (SEQ ID
NO: 533) XEFTDHLVK, (SEQ ID NO: 534) FCVGFLEGGKDSCQGDSGGPVVC, (SEQ
ID NO: 535) LLAEGHPDPDAELQR, (SEQ ID NO: 536) GLTEGLHGFH, (SEQ ID
NO: 537) GLTEGLHGFH, (SEQ ID NO: 538) FVHWYVGEGMEEGEFSEAR, (SEQ ID
NO: 539) MLLHEGQHPAQLR, (SEQ ID NO: 540) YHGYTFANLGEHEFVEEK, (SEQ
ID NO: 541) TVFAEHISDECK, (SEQ ID NO: 542) VILEEHSTCENEVSK, (SEQ ID
NO: 543) LLTEIHGGAGGPSGR, (SEQ ID NO: 544) AHLMEIQVNGGTVAEK, (SEQ
ID NO: 545) ISWLDANTLAEKDEFEHK, (SEQ ID NO: 546)
ISWLDANTLAEKDEFEHK, (SEQ ID NO: 547) XEKFEDENFILK, (SEQ ID NO: 548)
SAVEAGSEVSEKPGQEAPVLPK, (SEQ ID NO: 549) SAVEAGSEVSEKPGQEAPVLPK,
(SEQ ID NO: 550) ILNEKPTTDEPEK, (SEQ ID NO: 551) YLAEKYEWDVAEAR,
(SEQ ID NO: 552) CLELFXELAEDKENY, (SEQ ID NO: 553)
CLELFXELAEDKENYK, (SEQ ID NO: 554) MEELHNQEVQK, (SEQ ID NO: 555)
GVNVAGVSLQELNPEMGTDNDSENWK, (SEQ ID NO: 556) ASDIAMTELPPTHPIR, (SEQ
ID NO: 557) VVVAENFDEIVNNENK, (SEQ ID NO: 558) IXEGCEEPATHNALAK,
(SEQ ID NO: 559) VTEQGAELSNEER, (SEQ ID NO: 560) AVTEQGHELSNEER,
(SEQ ID NO: 561) QVDQEEPHVEEQQQQTPAENK, (SEQ ID NO: 562)
QVDQEEPHVEEQQQQTPAENK, (SEQ ID NO: 563) VVFEQTKVIADNVK, (SEQ ID NO:
564) NIFVGENILEESENLHNADQPLR, (SEQ ID NO: 565) LFIHESIHDEVVNR, (SEQ
ID NO: 566) VTNGIEEPLEESSHEPEPEPESETK, (SEQ ID NO: 567)
GIVEESVTGVHR, (SEQ ID NO: 568) QCPSVVSLLSESYNPHVR, (SEQ ID NO: 569)
ASLQETHFDSTQTK, (SEQ ID NO: 570) TFGETHPFTK, (SEQ ID NO: 571)
VMLGETNPADSKPGTIR,
(SEQ ID NO: 572) VMLGETNPADSKPGTIR, (SEQ ID NO: 573)
VMLGETNPADSKPGTIR, (SEQ ID NO: 574) GADFLVTEVENGGSLGSK, (SEQ ID NO:
575) LPTEAYISVEEVHDDGTPTSK, (SEQ ID NO: 576) LPTEAYISVEEVHDDGTPTSK,
(SEQ ID NO: 577) MEEVPHDCPGADSAQAGR, (SEQ ID NO: 578)
VDENCVGFDHTVKPV, (SEQ ID NO: 579) VHVVPDQLMAFGGSSEPCALC, (SEQ ID
NO: 580) IWCFGPDGTGPNILT, (SEQ ID NO: 581) YVXFGPHAGK, (SEQ ID NO:
582) EFAGFQCQIQFGPHNEQK, (SEQ ID NO: 583) KPXKPMQFLGDEETVRK, (SEQ
ID NO: 584) MVYMFQYDSTHGK, (SEQ ID NO: 585) EELGFRPEYSASQLK, (SEQ
ID NO: 586) HLEFSHDQYR, (SEQ ID NO: 587) TCGFDFTGAVEDISK, (SEQ ID
NO: 588) GFGFVDFNSEEDAK, (SEQ ID NO: 589) GFGFVDFNSEEDAK, (SEQ ID
NO: 590) NYGFVHIEDK, (SEQ ID NO: 591) GFGFVTFDDHDPVDK, (SEQ ID NO:
592) LPNFGFVVFDDSEPVQK, (SEQ ID NO: 593) QLLCGAAIGTHEDDK, (SEQ ID
NO: 594) QLLCGAAIGTHEDDKYR, (SEQ ID NO: 595) MTNGFSGADLTEICQR, (SEQ
ID NO: 596) VQGEVMEGADNQGAGEQGRPVR, (SEQ ID NO: 597)
MGGHGYGGAGDASSGFHGGHF, (SEQ ID NO: 598)
LGNVLGGLISGAGGGGGGGGGGGGGGGGGGGGTAMR, (SEQ ID NO: 599)
FGGSFAGSFGGAGGHAPGVAR, (SEQ ID NO: 600) FGGSFAGSFGGAGGHAPGVAR, (SEQ
ID NO: 601) VLVVGAGGIGCELLK, (SEQ ID NO: 602) VTADHGPAVSGAHNTIICAR,
(SEQ ID NO: 603) CEALAGAPLDNAPK, (SEQ ID NO: 604) CEALAGAPLDNAPK,
(SEQ ID NO: 605) STGGAPTFNVTVTK, (SEQ ID NO: 606) KGCDVVVIPAGVPR,
(SEQ ID NO: 607) FSPAGVEGCPALPHK, (SEQ ID NO: 608) HSSLAGCQIINYR,
(SEQ ID NO: 609) SSEVGYDAMAGDFVNMVEK, (SEQ ID NO: 610)
SIEDSVISLSGDHCIIGR, (SEQ ID NO: 611) SIEDSVISLSGDHCIIGR, (SEQ ID
NO: 612) VTGDHIPTPQDLPQR, (SEQ ID NO: 613) VTGDHIPTPQDLPQR, (SEQ ID
NO: 614) NGDTFLGGEDFDQALLR, (SEQ ID NO: 615) IVYICCGEDHTAALTK, (SEQ
ID NO: 616) MVDGNVSGEFTDLVPEK, (SEQ ID NO: 617) MAAQGEPQVQFK, (SEQ
ID NO: 618) QALAVHLALQGESSSEHFLK, (SEQ ID NO: 619)
AFYNNVLGEYEEYITK, (SEQ ID NO: 620) LLNQMDGFDTLHR, (SEQ ID NO: 621)
GLTEGLHGFHVHEFG, (SEQ ID NO: 622) GLTEGLHGFHVHEFGDNTAGCT, (SEQ ID
NO: 623) GLTEGLHGFHVHEFGDNTAGCT, (SEQ ID NO: 624)
GLTEGLHGFHVHEFGDNTAGCT, (SEQ ID NO: 625) GLTEGLHGFHVHEFGDNTAGCT,
(SEQ ID NO: 626) GLTEGLHGFHVHEFGDNTAGCT, (SEQ ID NO: 627)
AADSYFSLLQGFINSLDESTQESK, (SEQ ID NO: 628) INPYLLGTMAGGAADCSFWER,
(SEQ ID NO: 629) QHDLFDSGFGGGAGVETGGK, (SEQ ID NO: 630)
TTHFVEGGDAGNREDQINR, (SEQ ID NO: 631) TTHFVEGGDAGNREDQINR, (SEQ ID
NO: 632) SQPIAQQPLQGGDHSGNYGYK, (SEQ ID NO: 633)
GTDGTDNPLSGGDQYQNITVHR, (SEQ ID NO: 634) GCITXIGGGDTATCCAK, (SEQ ID
NO: 635) WGSGGGGGGGGGGGGGGGGGGGGGGGGGGGRKSSSAAA, (SEQ ID NO: 636)
LAAGSLAAPGGGGGSAGGARP, (SEQ ID NO: 637) GSXXXGGGSYNDFGNY, (SEQ ID
NO: 638) VNAANXSLLGGGGVDGCIHR, (SEQ ID NO: 639)
FCVGFLEGGKDSCQGDSGGPVVC, (SEQ ID NO: 640) LVDGQIFCLHGGLSPSIDTLDHIR,
(SEQ ID NO: 641) MFXGGLSWDTSKK, (SEQ ID NO: 642)
DPQELLEGGNQGEGDPQAEGR, (SEQ ID NO: 643) NMGGPYGGGNYGPGGSGGSGGYGGR,
(SEQ ID NO: 644) RGGPGGPGGPGGPMGR, (SEQ ID NO: 645)
SVLDDWFPLQGGQGQVHLR, (SEQ ID NO: 646) IIMEYLGGGSALDLLR, (SEQ ID NO:
647) SHFAMMHGGTGFAGIDSSSPEVK, (SEQ ID NO: 648)
QGFQLTHSLGGGTGSGMGTLLI, (SEQ ID NO: 649) MADYLISGGTSYVPDDGLT, (SEQ
ID NO: 650) VTVAGGVHISGLH, (SEQ ID NO: 651) VTVAGGVHISGLHT, (SEQ ID
NO: 652) VTVAGGVHISGLHTE, (SEQ ID NO: 653) YAVSELAGHQTSAESWGTGR,
(SEQ ID NO: 654) TFQGHTNEVNAIK, (SEQ ID NO: 655)
GDGPVQGIINFEQK,
(SEQ ID NO: 656) VTIIGPATVGGIKPGCFK, (SEQ ID NO: 657)
FSLPGMEHVYGIPEHADNLR, (SEQ ID NO: 658) IFVGGIPHNCGETELR, (SEQ ID
NO: 659) LPPSGAVPVTGIPPHVVK, (SEQ ID NO: 660) MDGIVPDIAVGTK, (SEQ
ID NO: 661) RGIWHNDNK, (SEQ ID NO: 662) GKPEIEGKPESEGEPGSETR, (SEQ
ID NO: 663) YDINAHACVTGKPISQGGIHGR, (SEQ ID NO: 664)
ELTQQLAQATGKPPQYIAVH, (SEQ ID NO: 665) NPKPFLNGLTGKPVMVK, (SEQ ID
NO: 666) CPSILGGLAPEKDQPK, (SEQ ID NO: 667) VASGIPAGWXGLDCGPESSKK,
(SEQ ID NO: 668) QVLQGLDYLHSK, (SEQ ID NO: 669) GALEGLPRPPPPVK,
(SEQ ID NO: 670) LFIGGLSFETTDESLR, (SEQ ID NO: 671)
VFVGGLSPDTSEEQIK, (SEQ ID NO: 672) MFXGGLSWDTSKK, (SEQ ID NO: 673)
NVIIWGNHSSTQYPDVNHAK, (SEQ ID NO: 674) LLSGLAEGLGGNIEQLVAR, (SEQ ID
NO: 675) LVINGNPITIFQER, (SEQ ID NO: 676) SAAMLGNSEDHTALSR, (SEQ ID
NO: 677) IFQGNVHNFEK, (SEQ ID NO: 678) NNPPTLEGNYSKPLK, (SEQ ID NO:
679) MVGPAVIVDKK, (SEQ ID NO: 680) MMLGPEGGEGFVVK, (SEQ ID NO: 681)
SIYEALGGPHDPNVAK, (SEQ ID NO: 682) TFQGPNCPATCGR, (SEQ ID NO: 683)
IMGPNYTPGKK, (SEQ ID NO: 684) MVIITGPPEAQFK, (SEQ ID NO: 685)
AFGLTDDQVSGPPSAPAEDR, (SEQ ID NO: 686) TVQGPPTSDDIFER, (SEQ ID NO:
687) FVIGGPQGDAGLTGR, (SEQ ID NO: 688) IITLXGPTNAIFK, (SEQ ID NO:
689) KPPTLIHGQAPSAGLPSQKPK, (SEQ ID NO: 690) RGQGGYPGKPR, (SEQ ID
NO: 691) RPDNFXFGQSGAGNNWAK, (SEQ ID NO: 692) GLLALSSALSGQSHLAIK,
(SEQ ID NO: 693) ALPPVLTTVNGQSPPEHSAPAK, (SEQ ID NO: 694)
QSGYGGQTKPIFR, (SEQ ID NO: 695) LSGQTNIHLSK, (SEQ ID NO: 696)
VVLMSHLGRPDGVPMPDK, (SEQ ID NO: 697) VVLMSHLGRPDGVPMPDKY, (SEQ ID
NO: 698) QQSIAGSADSKPIDVSR, (SEQ ID NO: 699) VTLGPVPEIGGSEAPAPQNK,
(SEQ ID NO: 700) NFGGSFAGSFGGAGGHAPGVAR, (SEQ ID NO: 701)
MMDYLQGSGETPQTDVR, (SEQ ID NO: 702) DSVWGSGGGQQSVNHLVK, (SEQ ID NO:
703) PQVAIICGSGLGGLTDK, (SEQ ID NO: 704)
PTSSEQGGLEGSGSAAGEGKPALSEEER, (SEQ ID NO: 705)
TVEQLLTGSPTSPTVEPEKPTR, (SEQ ID NO: 706) GCLEGSQGTQALHK, (SEQ ID
NO: 707) LLAVSAPALQGSRPGETEENVR, (SEQ ID NO: 708)
IXXGSSGAQGSGGGSTSAHY, (SEQ ID NO: 709) VAFTGSTEVGHLIQK, (SEQ ID NO:
710) VVVLMGSTSDLGHCEK, (SEQ ID NO: 711) MVELLGSYTEDNASQAR, (SEQ ID
NO: 712) IYWGTATTGKPHVA, (SEQ ID NO: 713) IVGFCWGGTAVHHLM, (SEQ ID
NO: 714) GVVPLAGTDGETTTQGLDGLSER, (SEQ ID NO: 715)
GXVXFXGTDHIDQWNK, (SEQ ID NO: 716) SVSGTDVQEECR, (SEQ ID NO: 717)
MMLGTEGGEGFVVK, (SEQ ID NO: 718) IAFHQDGSLAGTGGLDAFGR, (SEQ ID NO:
719) LNFSHGTHEYHAETIK, (SEQ ID NO: 720) LVLGTHTSDEQNHLV, (SEQ ID
NO: 721) ALHWLVLGTHTSDEQNHLVVAR, (SEQ ID NO: 722) VLSGTIHAGQPVK,
(SEQ ID NO: 723) IITITGTQDQIQNAQY, (SEQ ID NO: 724) GGTSDVEVNEK,
(SEQ ID NO: 725) VLTGVAGEDAECHAAK, (SEQ ID NO: 726)
TGGVDTAAVGGVFDVSNADR, (SEQ ID NO: 727) FIVDGWHEMDAENPLH, (SEQ ID
NO: 728) TMFSSEVQFGHAGACANQASETAVAK, (SEQ ID NO: 729)
PIYDVLQMVGHANRPLQDDEGR, (SEQ ID NO: 730) EWAHATIIPK, (SEQ ID NO:
731) KHEANNPQLK, (SEQ ID NO: 732) MVNHFIAEFK, (SEQ ID NO: 733)
LVXHFVEEFK, (SEQ ID NO: 734) MPFPVNHGASSEDTLLK, (SEQ ID NO: 735)
NXCWELYCLEHGIQPDGQMPSDK, (SEQ ID NO: 736) NXCWELYCLEHGIQPDGQMPSDK,
(SEQ ID NO: 737) VHAGPFANIAHGNSSIIADR, (SEQ ID NO: 738)
INQVFHGSCITEGNELTK, (SEQ ID NO: 739)
FELQHGTEEQQEEVR, (SEQ ID NO: 740) EQQEAIEHIDEVQNEIDR, (SEQ ID NO:
741) AVEALAAALAHISGATSVDQR, (SEQ ID NO: 742) RHLAPTGNAPASR, (SEQ ID
NO: 743) LLTDFCTHLPNLPDSTAK, (SEQ ID NO: 744) VDEFVTHNLSFDEINK,
(SEQ ID NO: 745) ATLELTHNWGTEDDETQSY, (SEQ ID NO: 746)
EEFTAFLHPEEYDYMK, (SEQ ID NO: 747) QXFHPEQLITGK, (SEQ ID NO: 748)
PVTHNLPTVAHPSQAPSPNQPTK, (SEQ ID NO: 749) AXXXXXQHQAGQAPHLG, (SEQ
ID NO: 750) CNFTDGALVQHQEWDGK, (SEQ ID NO: 751) GVLHQFSGTETNK, (SEQ
ID NO: 752) QIGAVVSHQSSVIPDR, (SEQ ID NO: 753) IEPNEVTHSGDTGVETDGR,
(SEQ ID NO: 754) HYAHTDCPGHADYVK, (SEQ ID NO: 755) TICSHVQNMIK,
(SEQ ID NO: 756) LLGHWEEAAHDLA, (SEQ ID NO: 757) TYTIANQFPLNK, (SEQ
ID NO: 758) NPTXFFDIAVDGEPLGR, (SEQ ID NO: 759) LVSIGAEEIVDGNAK,
(SEQ ID NO: 760) TTDGVYEGVAIGGDRYPGSTF, (SEQ ID NO: 761)
THINIVVIGHVDSGK, (SEQ ID NO: 762) DNDFCGTDMTIGTDSALHR, (SEQ ID NO:
763) VLXNMEIGTSLFDEEGAK, (SEQ ID NO: 764) VCTLAIIDPGDSDIIR, (SEQ ID
NO: 765) GCITIIGGGDTATCCAK, (SEQ ID NO: 766) TFNQVEIKPEMIGH, (SEQ
ID NO: 767) CQLEINFNTLQTK, (SEQ ID NO: 768) HLEINPDHPIVE, (SEQ ID
NO: 769) HLEINPDHSIIETLR, (SEQ ID NO: 770) VPYLIAGIQHSCQDIGAK, (SEQ
ID NO: 771) VLSIQSHVIR, (SEQ ID NO: 772) ELGITALHIK, (SEQ ID NO:
773) LVAIVDPHIK, (SEQ ID NO: 774) TLTIVDTGIGMTK, (SEQ ID NO: 775)
LVAIVDVIDQNR, (SEQ ID NO: 776) QIILEKEETEELKR, (SEQ ID NO: 777)
XKHPDADSLY, (SEQ ID NO: 778) CIGKPGGSLDNSEQK, (SEQ ID NO: 779)
HHIYLEGTLLKPNMVTPGHACTQK, (SEQ ID NO: 780) LTQQLAQATGKPPQYIAVH,
(SEQ ID NO: 781) SSPPELPDVMKPQDSGSSANEQAVQ, (SEQ ID NO: 782)
LQELEKYPGIQTR, (SEQ ID NO: 783) WIGLDLSNGKPR, (SEQ ID NO: 784)
MPFLELDTNLPANR, (SEQ ID NO: 785) ETALLSSGFSLEDPQTHANR, (SEQ ID NO:
786) EAFSLFDKDGDGTITTK, (SEQ ID NO: 787) YELGRPAANTK, (SEQ ID NO:
788) GNPICSLHDQGAGGNGNVLK, (SEQ ID NO: 789) VILHLKEDQTEYLEER, (SEQ
ID NO: 790) IQQLCEDIIQLKPDVVITEK, (SEQ ID NO: 791)
IQQLCEDIIQLKPDVVITEK, (SEQ ID NO: 792) TLNNDIMLIK, (SEQ ID NO: 793)
NQVALNPQNTVFDAK, (SEQ ID NO: 794) NQVALNPQNTVFDAK, (SEQ ID NO: 795)
STATLAWGVNLPAHTVIIK, (SEQ ID NO: 796) EXLELPEDEEEKK, (SEQ ID NO:
797) GVNLPGAAVDLPAVSEK, (SEQ ID NO: 798) RLPPAAGDEP, (SEQ ID NO:
799) LDLPPYETF, (SEQ ID NO: 800) DGDSVMVLPTIPEEEAKK, (SEQ ID NO:
801) EIVHLQAGQCGNQIGAK, (SEQ ID NO: 802) DVSIEDSVISLSGDHCIIGR, (SEQ
ID NO: 803) SSAPGPLELDLTGDLESFKK, (SEQ ID NO: 804) FLEMCNDLLAR,
(SEQ ID NO: 805) TTGFGMIYDSLDYAK, (SEQ ID NO: 806) XMNPTNTVFDAK,
(SEQ ID NO: 807) EDAMAMVDHCLK, (SEQ ID NO: 808) ANXVXSGGXTMYPGIADR,
(SEQ ID NO: 809) ANXVXSGGXTMYPGIADR, (SEQ ID NO: 810)
ALQDLENAASGDAAVHQR, (SEQ ID NO: 811) DPVTNLNNAFEVAEK, (SEQ ID NO:
812) XNAGPNTNGSQFF, (SEQ ID NO: 813) NYSVFYYEIQNAPEQACH, (SEQ ID
NO: 814) ELISNASDALDKIR, (SEQ ID NO: 815) YYFNHITNASQWERPSGNSSSGGK,
(SEQ ID NO: 816) TNDWEDHLAVK, (SEQ ID NO: 817) AFHNEAQVNPERK, (SEQ
ID NO: 818) NCLTNFHGMDLTR, (SEQ ID NO: 819) TNVANFPGHSGPIT, (SEQ ID
NO: 820) ILNNGHAFNVEFDDSQDK, (SEQ ID NO: 821) IEQLQNHENEDIYK, (SEQ
ID NO: 822) PVFVHAGPFANIAHGNSSIIADR,
(SEQ ID NO: 823) VWYVSNIDGTHIAK, (SEQ ID NO: 824)
CDEVMQLLLENLGNENVHR, (SEQ ID NO: 825) QDQRPLHPVANPHAEISTK, (SEQ ID
NO: 826) XNPLDAGAAEPI, (SEQ ID NO: 827) LIPQLVANVTNPNSTEHMK, (SEQ
ID NO: 828) SAAMLGNSEDHTALSR, (SEQ ID NO: 829)
NYQQNYQNSESGEKNEGSESAPEGQAQQR, (SEQ ID NO: 830) LGEMWNNTAADDKQPYEK,
(SEQ ID NO: 831) IMQNTDPHSQEYVEHLK, (SEQ ID NO: 832)
ILIANTGMDTDKIK, (SEQ ID NO: 833) AWVWNTHADFADECPKPELL, (SEQ ID NO:
834) DHASIQMNVAEVDKVTGR, (SEQ ID NO: 835) ALANVNIGSLIC, (SEQ ID NO:
836) EHGXXTNWDDMEK, (SEQ ID NO: 837) SAAQAAAQTNSNAAGK, (SEQ ID NO:
838) EETFEAAMLGQAEEVVQER, (SEQ ID NO: 839) PPYDEQTQAFIDAAQEAR, (SEQ
ID NO: 840) LEQGQAIDDLMPAQK, (SEQ ID NO: 841) SLHQAIEGDTSGDFLK,
(SEQ ID NO: 842) QLQQAQAAGAEQEVEK, (SEQ ID NO: 843)
YLEVVLNTLQQASQAQVDK, (SEQ ID NO: 844) YLEVVLNTLQQASQAQVDK, (SEQ ID
NO: 845) FLSELTQQLAQATGKPPQYI, (SEQ ID NO: 846)
FLSELTQQLAQATGKPPQYIA, (SEQ ID NO: 847) FLSELTQQLAQATGKPPQYIAVH,
(SEQ ID NO: 848) MTSMGQATWSDPHK, (SEQ ID NO: 849)
EELGLIEQAYDNPHEALSR, (SEQ ID NO: 850) SLGTIQQCCDAIDHLCR, (SEQ ID
NO: 851) AAAAAAQQQQQCGGGGATKPAVSGK, (SEQ ID NO: 852)
NSCNQCNEPRPEDSR, (SEQ ID NO: 853) VLIAFAQYLQQCPFEDHVK, (SEQ ID NO:
854) DSLLQDGEFSMDLR, (SEQ ID NO: 855) YFLGSIVNFSQDPDVHFK, (SEQ ID
NO: 856) VFSWLQQEGHLSEEEMAR, (SEQ ID NO: 857) VMSQEIQEQLHK, (SEQ ID
NO: 858) KQEPVKPEEGR, (SEQ ID NO: 859) LWYCDLQQESSGIAGILK, (SEQ ID
NO: 860) KQEYDESGPSIVHR, (SEQ ID NO: 861) ETEAICFFVQQFTDMEHNR, (SEQ
ID NO: 862) VTEQGAELSNEER, (SEQ ID NO: 863) AYMGNVLQGGEGQAPTR, (SEQ
ID NO: 864) AVTEQGHELSNEER, (SEQ ID NO: 865) VAHTFVVDVAQGTQVTGR,
(SEQ ID NO: 866) VGQGYPHDPPK, (SEQ ID NO: 867) IYAVEASTMAQHAEVLVK,
(SEQ ID NO: 868) TLAIYFEVVNQHNAPIPQGGR, (SEQ ID NO: 869)
ELAQIAGRPTEDEDEKEK, (SEQ ID NO: 870) MDEMATTQISKDELDELK, (SEQ ID
NO: 871) YPHLGQKPGGSDFLR, (SEQ ID NO: 872) TMLELLNQLDGFQPNTQVK,
(SEQ ID NO: 873) ILLELLNQMDGFDQNVNVK, (SEQ ID NO: 874)
LLNQMDGFDTLHR, (SEQ ID NO: 875) FQESAEAILGQNAAYLGELK, (SEQ ID NO:
876) HPCFIIAEIGQNHQGDLDVAK, (SEQ ID NO: 877)
LLQDHPWLLSQNLVVKPDQLIK, (SEQ ID NO: 878) ALPAVQQNNLDEDLIRK, (SEQ ID
NO: 879) ALGQNPTNAEVLK, (SEQ ID NO: 880) NYQQNYQNSESGEK, (SEQ ID
NO: 881) NYQQNYQNSESGEKNEGSESAPEGQAQQR, (SEQ ID NO: 882)
CGAPSATQPATAETQHIADQVR, (SEQ ID NO: 883)
QAAAAAAQQQQQCGGGGATKPAVSGK, (SEQ ID NO: 884) IDVTDFLSMTQQDSHAPLR,
(SEQ ID NO: 885) IGSCTQQDVELHVQK, (SEQ ID NO: 886) LFPLNQQDVPDKFK,
(SEQ ID NO: 887) IGQQPQQPGAPPQQDYTK, (SEQ ID NO: 888)
HQAAAAAAQQQQQCGGGGATKPAVSGK, (SEQ ID NO: 889) MFTQQQPQELAR, (SEQ ID
NO: 890) LQQQQRPEDAEDGAEGGGK, (SEQ ID NO: 891)
LQQQQRPEDAEDGAEGGGKR, (SEQ ID NO: 892) SSEADMECLNQRPPENPDTDK, (SEQ
ID NO: 893) SSEADMECLNQRPPENPDTDKNVQ, (SEQ ID NO: 894)
NVNPESQLIQQSEQSESETAGSTK, (SEQ ID NO: 895) PDNFXFGQSGAGNNWAK, (SEQ
ID NO: 896) SQTCEFNMIEQSGPPHEPR, (SEQ ID NO: 897)
SAVLPPEDMSQSGPSGSHPQGPR, (SEQ ID NO: 898) IEFLQSHENQEIYQK, (SEQ ID
NO: 899) NTVSQSISGDPEIDKK, (SEQ ID NO: 900) LLIHQSLAGGIIGVK, (SEQ
ID NO: 901) MVXYLANLTQSQIALNEK, (SEQ ID NO: 902)
PPKPEPFQFGQSSQKPPVAGGK, (SEQ ID NO: 903) NGNYCVLQMDQSYKPDENEVR,
(SEQ ID NO: 904) ILVGDVGQTVDDPYATFVK, (SEQ ID NO: 905)
ADDVDLEQVANETHGHVG, (SEQ ID NO: 906) ADDVDLEQVANETHGHVGA,
(SEQ ID NO: 907) SINFLHQVCHDQTPTTK, (SEQ ID NO: 908)
CTTVAFTQVNSEDKGALAK, (SEQ ID NO: 909) QQLQQVPGLLHR, (SEQ ID NO:
910) SQQYPAARPAEP, (SEQ ID NO: 911) DFCIQVGRNIIHGSDSVK, (SEQ ID NO:
912) VLMSHLGRPDGVPMPDKY, (SEQ ID NO: 913) VLMSHLGRPDGVPMPDKYS, (SEQ
ID NO: 914) AQVARPGGDTIFGK, (SEQ ID NO: 915) AQVARPGGDTIFGK, (SEQ
ID NO: 916) FMSVQRPGPYDRPGTAR, (SEQ ID NO: 917) VLVERSAAETVTK, (SEQ
ID NO: 918) FLPSARSSPASSPE, (SEQ ID NO: 919) RPELGSEGLGSAAHGSQPDLR,
(SEQ ID NO: 920) MPDQGMTSADDFFQGTK, (SEQ ID NO: 921)
DVPAPSTSADKVESLDVDSEAK, (SEQ ID NO: 922) QVCLPVISAENWKPATK, (SEQ ID
NO: 923) GFGSGDDPYSSAEPHVSGVK, (SEQ ID NO: 924)
EFGDNTAGCTSAGPHFNPLSR, (SEQ ID NO: 925) TYFSCTSAHTSTGDGTAMITR, (SEQ
ID NO: 926) TYSLGSALRPSTSR, (SEQ ID NO: 927) VSDQELQSANASVDDSR,
(SEQ ID NO: 928) APGSAAPAAGSAPAAAEEK, (SEQ ID NO: 929)
APGSAAPAAGSAPAAAEEK, (SEQ ID NO: 930) APGSAAPAAGSAPAAAEEK, (SEQ ID
NO: 931) APGSAAPAAGSAPAAAEEKK, (SEQ ID NO: 932) NEGSESAPEGQAQQR,
(SEQ ID NO: 933) QVEPLDPPAGSAPGEHVFVK, (SEQ ID NO: 934)
PTGEAGPSCSSASDKLPR, (SEQ ID NO: 935) YYTSASGDEMVSLK, (SEQ ID NO:
936) NQQGAHSALSSASTSSHNLQ, (SEQ ID NO: 937) EALLSSAVDHGSDEVK, (SEQ
ID NO: 938) DYMVEIDILASCDHPNIVK, (SEQ ID NO: 939) MESCGIHETTF, (SEQ
ID NO: 940) QLSSCLPNIVPK, (SEQ ID NO: 941) LIXSDGHEFIVK, (SEQ ID
NO: 942) EIVDGGVILESDPQQVVHR, (SEQ ID NO: 943) SLEDALSSDTSGHFR,
(SEQ ID NO: 944) VGVEAHVDIHSDVPKGANSF, (SEQ ID NO: 945)
VILGSEAAQQHPEEVR, (SEQ ID NO: 946) XSEDKGALAK, (SEQ ID NO: 947)
GGTSXXSSEGTQHSYSEEEK, (SEQ ID NO: 948) CALGGTSELSSEGTQHSYSEEEKY,
(SEQ ID NO: 949) MDPNIVGSEHYDVAR, (SEQ ID NO: 950)
SPAPSSVPLGSEKPSNVSQDR, (SEQ ID NO: 951) MTQAGVEELESENKIPATQK, (SEQ
ID NO: 952) MLLDSEQHPCQLK, (SEQ ID NO: 953)
GLGNVLGGLISGAGGGGGGGGGGGGGGGGGGGGTAMR, (SEQ ID NO: 954)
IMDDLTEVLCSGAGGVHSGGSGDGAGSGGPGAQNHVK, (SEQ ID NO: 955)
ATQGAAAAAGSGAGTGGGTASGGTEGGSAESEGAK, (SEQ ID NO: 956)
LEPAPLDSLCSGASAEEPTSHR, (SEQ ID NO: 957) VIGSGCNLDSAR, (SEQ ID NO:
958) WXLNSGDGAFYGPK, (SEQ ID NO: 959) FFDMAYQGFASGDGDKDAWAVR, (SEQ
ID NO: 960) VSIEDSVISLSGDHCIIGR, (SEQ ID NO: 961) EYLLSGDISEAEHCLK,
(SEQ ID NO: 962) DDGLFSGDPNWFPK, (SEQ ID NO: 963)
WQHDLFDSGFGGGAGVETGGK, (SEQ ID NO: 964) DSVWGSGGGQQSVNHLVK, (SEQ ID
NO: 965) PEGPNEAEVTSGKPEQEVPDAEEEK, (SEQ ID NO: 966) VQSGNINAAK,
(SEQ ID NO: 967) YQYGGLNSGRPVTPPR, (SEQ ID NO: 968)
VLQATVVAVGSGSKGKGGEIQPVSVK, (SEQ ID NO: 969) GILFVGSGVSGGEEGAR,
(SEQ ID NO: 970) IEFLQSHENQEIYQK, (SEQ ID NO: 971)
LDEVITSHGAIEPDKDNVR, (SEQ ID NO: 972) EHPVIESHPDNALEDLR, (SEQ ID
NO: 973) LIQSHPESAEDLQEK, (SEQ ID NO: 974) TIVITSHPGQIVK, (SEQ ID
NO: 975) IEWLESHQDADIEDFK, (SEQ ID NO: 976) GYPHLCSICDLPVHSNK, (SEQ
ID NO: 977) SEPCALCSLHSIGKIGGAQNR, (SEQ ID NO: 978) LQSIGTENTEENR,
(SEQ ID NO: 979) LFIHESIHDEVVNR, (SEQ ID NO: 980)
VTFNINNSIPPTFDGEEEPSQGQK, (SEQ ID NO: 981) NLNTLCWAIGSISGAMHEEDEKR,
(SEQ ID NO: 982) EASATNSPCTSKPATPAPSEK, (SEQ ID NO: 983)
PPNPNCYVCASKPEVTVR, (SEQ ID NO: 984) ICSKPVVLPK, (SEQ ID NO: 985)
QFHFHWGSLDGQGSEHTVDK, (SEQ ID NO: 986) QFHFHWGSLDGQGSEHTVDKK, (SEQ
ID NO: 987) GNPICSLHDQGAGGNGNVLK, (SEQ ID NO: 988)
EANFTVSSMHGDMPQK, (SEQ ID NO: 989) NQLTSNPENTVFDAK, (SEQ ID NO:
990)
QVLVGSYCVFSNQGGLVHPK, (SEQ ID NO: 991) DLQSNVEHLTEK, (SEQ ID NO:
992) EEMQSNVEVVHTYR, (SEQ ID NO: 993) APVQPQQSPAAAPGGTDEKPSGK, (SEQ
ID NO: 994) APVQPQQSPAAAPGGTDEKPSGK, (SEQ ID NO: 995)
NDGPVTIELESPAPGTATSDPK, (SEQ ID NO: 996) INSLFLTDLYSPEYPGPSHR, (SEQ
ID NO: 997) NGSLDSPGKQDTEEDEEEDEKDK, (SEQ ID NO: 998)
SAAAASAASGSPGPGEGSAGGEKR, (SEQ ID NO: 999)
SAAAASAASGSPGPGEGSAGGEKR, (SEQ ID NO: 1000) NADTDLVSWLSPHDPNSVVTK,
(SEQ ID NO: 1001) LSPPYSSPQEFAQDVGR, (SEQ ID NO: 1002)
IIAFVGSPVEDNEKDLVK, (SEQ ID NO: 1003) MESQEPTESSQNGK, (SEQ ID NO:
1004) AXASQLDCNFLK, (SEQ ID NO: 1005) SQGDSISSQLGPIHPPPR, (SEQ ID
NO: 1006) LGGLLKPTVASQNQNLPVAK, (SEQ ID NO: 1007)
SSWGMMGMLASQQNQSGPSGNNQNQGNMQR, (SEQ ID NO: 1008) DEYLINSQTTEHIVK,
(SEQ ID NO: 1009) YQLGLAYGYNSQYDEAVAQFSK, (SEQ ID NO: 1010)
GLLLLSVVVTSRPEAFQPH, (SEQ ID NO: 1011) RPASVSSSAAVEHEQR, (SEQ ID
NO: 1012) FGIVTSSAGTGTTEDTEAK, (SEQ ID NO: 1013)
FGIVTSSAGTGTTEDTEAKK, (SEQ ID NO: 1014) STASAPAAVNSSASADKPLSNMK,
(SEQ ID NO: 1015) EALLSSAVDHGSDEVK, (SEQ ID NO: 1016)
VSWLEYESSFSNEEAQK, (SEQ ID NO: 1017) IXXGSSGAQGSGGGSTSAHY, (SEQ ID
NO: 1018) HIGGPPGFASSSGKPGPTVIK, (SEQ ID NO: 1019)
FEMYEPSELESSHLTDQDNEIR, (SEQ ID NO: 1020) SPDDDLGSSNWEAADLGNEER,
(SEQ ID NO: 1021) GDSQVSSNPTSSPPGEAPAPVSVDSEPS, (SEQ ID NO: 1022)
FVNGQPRPLESSQVKYLR, (SEQ ID NO: 1023) KPLTSSSAAPQRPISTQR, (SEQ ID
NO: 1024) IHIGGPPGFASSSGKPGPTVIK, (SEQ ID NO: 1025)
ELVSSSSSGSDSDSEVDKK, (SEQ ID NO: 1026) LLDSSTVTHLFK, (SEQ ID NO:
1027) PPPAAPPPSSSSVPEAGGPPIKK, (SEQ ID NO: 1028)
YVELFLNSTAGASGGAYEHR, (SEQ ID NO: 1029) SHELSDFGLESTAGEIPVVAIR,
(SEQ ID NO: 1030) ECEEEAINIQSTAPEEEHESPR, (SEQ ID NO: 1031)
EGTGSTATSSSSTAGAAGK, (SEQ ID NO: 1032) PLHSIISSTESVQGSTSK, (SEQ ID
NO: 1033) VAFTGSTEVGHLIQK, (SEQ ID NO: 1034) LALVTGGEIASTFDHPELVK,
(SEQ ID NO: 1035) ATIELCSTHANDASALR, (SEQ ID NO: 1036)
VHITLSTHECAGLSER, (SEQ ID NO: 1037) EEEEPQAPQESTPAPPKK, (SEQ ID NO:
1038) SITILSTPEGTSAACK, (SEQ ID NO: 1039) ETLASSDSFASTQPTHSWK, (SEQ
ID NO: 1040) VVVLMGSTSDLGHCEK, (SEQ ID NO: 1041)
VLLSNLSSTSHVPEVDPGSAELQK, (SEQ ID NO: 1042) LFDSTTLEHQK, (SEQ ID
NO: 1043) TQLEGLQSTVTGHVER, (SEQ ID NO: 1044)
GSESGGSAVDSVAGEHSVSGR, (SEQ ID NO: 1045) YEILQSVDDAAIVIK, (SEQ ID
NO: 1046) NDLSICGTLHSVDQYLNIK, (SEQ ID NO: 1047) ILDSVGIEADDDR,
(SEQ ID NO: 1048) ILDSVGIEADDDRLNK, (SEQ ID NO: 1049)
IYVASVHQDLSDDDIK, (SEQ ID NO: 1050) ELQSVKPQEAPK, (SEQ ID NO: 1051)
HYTEGAELVDSVLDVVRK, (SEQ ID NO: 1052) LAEGSVTSVGSVNPAENFR, (SEQ ID
NO: 1053) GSPTSLGTWGSWIGPDHDK, (SEQ ID NO: 1054) VLNSYWVGEDSTYK,
(SEQ ID NO: 1055) SLGTADVHFER, (SEQ ID NO: 1056) MAGTAFDFENMK, (SEQ
ID NO: 1057) VLATAFDTTLGGR, (SEQ ID NO: 1058) VELFLNSTAGASGGAYEHR,
(SEQ ID NO: 1059) APPPSGSAVSTAPQQKPIGK, (SEQ ID NO: 1060)
SQIFSTASDNQPTVTIK, (SEQ ID NO: 1061) IYWGTATTGKPHVA, (SEQ ID NO:
1062) MMLGTEGGEGFVVK, (SEQ ID NO: 1063) FGAVWTGDNTAEWDHLK, (SEQ ID
NO: 1064) VSHVSTGGGASLELL, (SEQ ID NO: 1065) VSHVSTGGGASLELLE, (SEQ
ID NO: 1066) VSHVSTGGGASLELLEGK, (SEQ ID NO: 1067)
ILISLATGHREEGGENLDQAR, (SEQ ID NO: 1068) TLDQCIQTGVDNPGHPFIK, (SEQ
ID NO: 1069) SGFTLDDVIQTGVDNPGHPY, (SEQ ID NO: 1070)
DLTTGYDDSQPDKK, (SEQ ID NO: 1071) FFFGTHETAFLGPK, (SEQ ID NO: 1072)
FPSLLTHNENMVAK, (SEQ ID NO: 1073) YEDICPSTHNMDVPNIK,
(SEQ ID NO: 1074) DYALHWLVLGTHTSDEQNHLVVAR, (SEQ ID NO: 1075)
FGTINIVHPK, (SEQ ID NO: 1076) SMVNTKPEKTEEDSEEVR, (SEQ ID NO: 1077)
VTLLTPAGATGSGGGTSGDSSKGEDKQDR, (SEQ ID NO: 1078)
PGETLTEILETPATSEQEAEHQR, (SEQ ID NO: 1079) NSVQTPVENSTNSQHQVK, (SEQ
ID NO: 1080) AXXITPVPGGVGPMTV, (SEQ ID NO: 1081)
STVLTPMFVETQASQGTLQTR, (SEQ ID NO: 1082) TFTTQETITNAETAK, (SEQ ID
NO: 1083) SPVSTRPLPSASQK, (SEQ ID NO: 1084) TNEQWQMSLGTSEDHQHFT,
(SEQ ID NO: 1085) QEIIXQLDVTTSEYEKEK, (SEQ ID NO: 1086)
LLAFLLAELGTSGSIDGNNQLVIK, (SEQ ID NO: 1087) LXNMEIGTSLFDEEGAK, (SEQ
ID NO: 1088) AEKPAETPVATSPTATDSTSGDSSR, (SEQ ID NO: 1089)
LLETTDRPDGHQNNLR, (SEQ ID NO: 1090) AQTITSEXXSTTTTTHITK, (SEQ ID
NO: 1091) ADAVGMSTVPEVIVAR, (SEQ ID NO: 1092) IHFPLATYAPVISAEK,
(SEQ ID NO: 1093) DTXVXXDTYNCDLHFK, (SEQ ID NO: 1094)
VVIGMDVAASEFFR, (SEQ ID NO: 1095) GXXXXXIGLXVADLAESIMK, (SEQ ID NO:
1096) ANPQVGVAFPHIK, (SEQ ID NO: 1097) PQEAKPQEAAVAPEKPPASDETK,
(SEQ ID NO: 1098) HFSVEGQLEFR, (SEQ ID NO: 1099) VATLGVEVHPLVFH,
(SEQ ID NO: 1100) HWPFQVINDGDKPK, (SEQ ID NO: 1101)
LPVPAFNVINGGSHAGNK, (SEQ ID NO: 1102) EVANGIESLGVKPDLPPPPSK, (SEQ
ID NO: 1103) TYYDVLGVKPNATQEELKK, (SEQ ID NO: 1104)
ETVAVKPTENNEEEFTSK, (SEQ ID NO: 1105) SLLVNPEGPTLMR, (SEQ ID NO:
1106) NWMNSLGVNPHVNHLY, (SEQ ID NO: 1107) HGLLVPNNTTDQELQHIR, (SEQ
ID NO: 1108) QELEFLEVQEEYIKDEQK, (SEQ ID NO: 1109)
LEGTLLKPNMVTPGHACTQK, (SEQ ID NO: 1110) FVNVVPTFGKK, (SEQ ID NO:
1111) EDLVFIFWAPESAPLK, (SEQ ID NO: 1112) AIYIDASCLTWEGQQFQGK, (SEQ
ID NO: 1113) EQPQHPLHVTYAGAAVDELGK, (SEQ ID NO: 1114)
SPDGHLFQVEYAQEAVKK, (SEQ ID NO: 1115) NYKPPAQK, (SEQ ID NO: 1116)
VYNYNHLMPTR, (SEQ ID NO: 1117) LAEAELEYNPEHVSR, (SEQ ID NO: 1118)
MPYQYPALTPEQK, (SEQ ID NO: 1119) TSSANNPNLMYQDECDRR, (SEQ ID NO:
1120) VGINYQPPTVVPGGDLAK, (SEQ ID NO: 1121) YMACCXLYRGDVVPK, and
(SEQ ID NO: 1122) SYCYVSKEELK.
[0168] Other cathepsin sensitive sites are known to the skilled
artisan or can easily be determined experimentally using digestion
assays with no more than routine experimentation.
[0169] The mRNA cancer vaccines of the present invention comprise
one or more polynucleotides, e.g., polynucleotide constructs, which
encode one or more wild type or engineered antigens, including the
concatemeric antigens. Exemplary polynucleotides, e.g.,
polynucleotide constructs, include antigen-encoding mRNAs. In
exemplary aspect, polynucleotides of the invention, e.g.,
antigen-encoding mRNAs, may include at least one chemical
modification.
[0170] The polynucleotides (e.g., antigen-encoding polynucleotides)
can include various substitutions and/or insertions. As used herein
in a polynucleotide, the terms "chemical modification" or, as
appropriate, "chemically modified" refer to modification with
respect to adenosine (A), guanosine (G), uridine (U), thymidine (T)
or cytidine (C) ribo- or deoxyribnucleosides in one or more of
their position, pattern, percent or population. Generally, herein,
these terms are not intended to refer to the ribonucleotide
modifications in naturally occurring 5'-terminal mRNA cap moieties.
In a polypeptide, the term "modification" refers to a modification
as compared to the canonical set of 20 amino acids.
[0171] The skilled artisan will appreciate that, except where
otherwise noted, polynucleotide sequences set forth in the instant
application will recite "T"s in a representative DNA sequence but
where the sequence represents RNA, the "T"s would be substituted
for "U"s.
[0172] The modifications may be various distinct modifications. In
some embodiments, the regions may contain one, two, or more
(optionally different) nucleoside or nucleotide modifications. In
some embodiments, a modified polynucleotide, introduced to a cell
or organism may exhibit reduced degradation in the cell or
organism, as compared to an unmodified polynucleotide. In some
embodiments, a modified polynucleotide, introduced into a call or
organism, may exhibit reduced immunogenicity in the cell or
organism (e.g., a reduced innate response.)
[0173] Modifications of the polynucleotides of the mRNA cancer
vaccines include, but are not limited to those listed in detail
below. The polynucleotide may comprise modifications which are
naturally occurring, non-naturally occurring or the polynucleotide
can comprise both naturally and non-naturally occurring
modifications.
[0174] The polynucleotides of the mRNA cancer vaccines of the
invention can include any useful modification, such as to the
sugar, the nucleobase, or the internucleoside linkage (e.g. to a
linking phosphate/to a phosphodiester linkage/to the phosphodiester
backbone). One or more atoms of a pyrimidine nucleobase may be
replaced or substituted with optionally substituted amino,
optionally substituted thiol, optionally substituted alkyl (e.g.,
methyl or ethyl), or halo (e.g., chloro or fluoro). In certain
embodiments, modifications (e.g., one or more modifications) are
present in each of the sugar and the internucleoside linkage.
Modifications according to the present invention may be
modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids
(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),
peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or
hybrids thereof). Additional modifications are described
herein.
[0175] Non-natural modified nucleotides may be introduced to
polynucleotides, e.g., of the mRNA cancer vaccines, or nucleic
acids during synthesis or post-synthesis of the chains to achieve
desired functions or properties. The modifications may be on
internucleotide lineage, the purine or pyrimidine bases, or sugar.
The modification may be introduced at the terminal of a chain or
anywhere else in the chain; with chemical synthesis or with a
polymerase enzyme. Any of the regions of the polynucleotides may be
chemically modified.
[0176] The present disclosure provides for modified nucleosides and
nucleotides. As described herein "nucleoside" is defined as a
compound containing a sugar molecule (e.g., a pentose or ribose) or
a derivative thereof in combination with an organic base (e.g., a
purine or pyrimidine) or a derivative thereof (also referred to
herein as "nucleobase"). As described herein, "nucleotide" is
defined as a nucleoside including a phosphate group. The modified
nucleotides may by synthesized by any useful method, as described
herein (e.g., chemically, enzymatically, or recombinantly to
include one or more modified or non-natural nucleosides). The
polynucleotides may comprise a region or regions of linked
nucleosides. Such regions may have variable backbone linkages. The
linkages may be standard phosphodiester linkages, in which case the
polynucleotides would comprise regions of nucleotides.
[0177] The modified nucleotide base pairing encompasses not only
the standard adenosine-thymine, adenosine-uracil, or
guanosine-cytosine base pairs, but also base pairs formed between
nucleotides and/or modified nucleotides comprising non-standard or
modified bases, wherein the arrangement of hydrogen bond donors and
hydrogen bond acceptors permits hydrogen bonding between a
non-standard base and a standard base or between two complementary
non-standard base structures. One example of such non-standard base
pairing is the base pairing between the modified nucleotide inosine
and adenine, cytosine or uracil. Any combination of base/sugar or
linker may be incorporated into the polynucleotides of the
invention.
[0178] Modifications of the polynucleotides of the mRNA cancer
vaccines which are useful in the present invention include, but are
not limited to the following:
2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;
2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl
carbamoyladenosine; N6-glycinylcarbamoyladenosine;
N6-isopentenyladenosine; N6-methyladenosine;
N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine;
1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine
(phosphate); 2-methyladenosine; 2-methylthio-N6
isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl
carbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine
(phosphate); Isopentenyladenosine;
N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine;
N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine;
N6,N6-dimethyladenosine; N6-acetyladenosine;
N6-hydroxynorvalylcarbamoyladenosine;
N6-methyl-N6-threonylcarbamoyladeno sine; 2-methyladenosine;
2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;
N1-methyl-adenosine; N6,N6 (dimethyl)adenine;
N6-cis-hydroxy-isopentenyl-adenosine; .alpha.-thio-adenosine; 2
(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6
(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;
2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;
2-(propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP;
2'-Deoxy-2'-a-aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6
(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine;
6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8
(alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine;
8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine;
8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine;
8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza
adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine;
7-deaza-8-aza-adenosine; 7-methyladenine; 1-Deazaadenosine TP;
2'Fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2-Amino-ATP;
2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a-Ethynyladenosine TP;
2-aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP;
2'-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;
2'-b-Ethynyladenosine TP; 2-Bromoadenosine TP;
2'-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;
2'-Deoxy-2', 2'-difluoroadenosine TP;
2'-Deoxy-2'-a-mercaptoadenosine TP;
2'-Deoxy-2'-a-thiomethoxyadenosine TP; 2'-Deoxy-2'-b-aminoadenosine
TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'-Deoxy-2'-b-bromoadenosine
TP; 2'-Deoxy-2'-b-chloroadenosine TP; 2'-Deoxy-2'-b-fluoroadenosine
TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'-Deoxy-2'-b-mercaptoadenosine
TP; 2'-Deoxy-2'-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;
2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;
2-methylthio-adenine; 2-Trifluoromethyladenosine TP;
3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;
3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;
3-Deazaadenosine TP; 4'-Azidoadenosine TP; 4'-Carbocyclic adenosine
TP; 4'-Ethynyladenosine TP; 5'-Homo-adenosine TP; 8-Aza-ATP;
8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP;
9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine;
7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;
2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;
2-thiocytidine; 3-methylcytidine; 5-formylcytidine;
5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;
2'-O-methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine;
5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O-dimethylcytidine;
N4-acetyl-2'-O-methylcytidine; N4-methylcytidine;
N4,N4-Dimethyl-2'-OMe-Cytidine TP; 4-methylcytidine;
5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine;
.alpha.-thio-cytidine; 2-(thio)cytosine; 2'-Amino-2'-deoxy-CTP;
2'-Azido-2'-deoxy-CTP; 2'-Deoxy-2'-a-aminocytidine TP;
2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3
(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine;
3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5 (halo)cytosine; 5
(methyl)cytosine; 5 (propynyl)cytosine; 5
(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;
5-(halo)cytosine; 5-(propynyl)cytosine;
5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine;
5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine;
deaza cytosine; N4 (acetyl)cytosine;
1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;
2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;
2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;
4-methoxy-pseudoisocytidine;
4-thio-1-methyl-1-deaza-pseudoisocytidine;
4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;
5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;
Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine
TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP;
2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl-cytidine TP;
2'O-methyl-N4-Bz-cytidine TP; 2'-a-Ethynylcytidine TP;
2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP;
2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine
TP; 2'-Deoxy-2'-a-mercaptocytidine TP;
2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine
TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP;
2'-Deoxy-2'-b-chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP;
2'-Deoxy-2'-b-iodocytidine TP; 2'-Deoxy-2'-b-mercaptocytidine TP;
2'-Deoxy-2'-b-thiomethoxycytidine TP;
2'-O-Methyl-5-(1-propynyl)cytidine TP; 3'-Ethynylcytidine TP;
4'-Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine
TP; 5-(1-Propynyl)ara-cytidine TP;
5-(2-Chloro-phenyl)-2-thiocytidine TP;
5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP;
5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP;
5'-Homo-cytidine TP; 5-Methoxycytidine TP;
5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP;
N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine;
N2,2'-O-dimethylguanosine; N2-methylguanosine; Wyosine;
1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine;
2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine;
2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine;
7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine;
N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine;
N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;
N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;
8-oxo-guanosine; N1-methyl-guanosine; .alpha.-thio-guanosine; 2
(propyl)guanine; 2-(alkyl)guanine; 2'-Amino-2'-deoxy-GTP;
2'-Azido-2'-deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP;
2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl)guanine;
6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7
(alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine;
7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8
(alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8
(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine;
8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;
8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza
guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;
1-methyl-6-thio-guanosine; 6-methoxy-guanosine;
6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;
6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;
7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;
N2-methyl-6-thio-guanosine; 1-Me-GTP;
2'Fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine
TP; 2'-a-Ethynylguanosine TP; 2'-a-Trifluoromethylguanosine TP;
2'-b-Ethynylguano sine TP; 2'-b-Trifluoromethylguanosine TP;
2'-Deoxy-2',2'-difluoroguanosine TP;
2'-Deoxy-2'-a-mercaptoguanosine TP;
2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b-aminoguanosine
TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine
TP; 2'-Deoxy-2'-b-chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine
TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'-Deoxy-2'-b-mercaptoguanosine
TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP;
4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP;
5'-Homo-guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP;
N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;
1,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine;
2'-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;
Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine;
deaza thymidine; deoxy-thymidine; 2'-O-methyluridine;
2-thiouridine; 3-methyluridine; 5-carboxymethyluridine;
5-hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine;
5-taurinomethyluridine; Dihydrouridine; Pseudouridine;
(3-(3-amino-3-carboxypropyl)uridine;
1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;
1-methylpseduouridine; 1-methyl-pseudouridine; 2'-O-methyluridine;
2'-O-methylpseudouridine; 2'-O-methyluridine;
2-thio-2'-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine;
3,2'-O-dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4-thiouridine;
5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine
methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydro-uridine;
5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine;
5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;
5-carboxyhydroxymethyluridine methyl ester;
5-carboxymethylaminomethyl-2'-O-methyluridine;
5-carboxymethylaminomethyl-2-thiouridine;
5-carboxymethylaminomethyl-2-thiouridine;
5-carboxymethylaminomethyluridine;
5-carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP;
5-methoxycarbonylmethyl-2'-O-methyluridine;
5-methoxycarbonylmethyl-2-thiouridine;
5-methoxycarbonylmethyluridine; 5-methyluridine, 5-methoxyuridine;
5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine;
5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine;
5-Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP; 5-Oxyacetic
acid-methyl ester-Uridine TP; N1-methyl-pseudo-uridine; uridine
5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester;
3-(3-Amino-3-carboxypropyl)-Uridine TP;
5-(iso-Pentenylaminomethyl)-2-thiouridine TP;
5-(iso-Pentenylaminomethyl)-2'-O-methyluridine TP;
5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;
.alpha.-thio-uridine; 1
(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1
(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1
(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminocarbonylethylenyl)-pseudouracil; 1 substituted
2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1
substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;
1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;
1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;
1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP;
1-Methyl-pseudo-UTP; 2 (thio)pseudouracil; 2' deoxy uridine; 2'
fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2' methyl,
2'amino, 2'azido, 2'fluro-guanosine; 2'-Amino-2'-deoxy-UTP;
2'-Azido-2'-deoxy-UTP; 2'-Azido-deoxyuridine TP;
2'-O-methylpseudouridine; 2' deoxy uridine; 2' fluorouridine;
2'-Deoxy-2'-a-aminouridine TP; 2'-Deoxy-2'-a-azidouridine TP;
2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4
(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil;
4-thiouracil; 5 (1,3-diazole-1-alkyl)uracil; 5
(2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5
(dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5
(methoxycarbonylmethyl)-2-(thio)uracil; 5
(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5
(methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5
(methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4
(dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5
(propynyl)uracil; 5 (trifluoromethyl)uracil;
5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil;
5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil;
5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil;
5-(allylamino)uracil; 5-(cyanoalkyl)uracil;
5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;
5-(guanidiniumalkyl)uracil; 5-(halo)uracil;
5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;
5-(methoxycarbonylmethyl)-2-(thio)uracil;
5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil;
5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;
5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4
(dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil;
5-(methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil;
5-(methylaminomethyl)-2,4(dithio)uracil;
5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;
5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;
5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil;
6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3
(methyl)uracil; P seudo-UTP-1-2-ethanoic acid; Pseudouracil;
4-Thio-pseudo-UTP; 1-carboxymethyl-pseudouridine;
1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine;
1-taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine;
1-taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine;
2-thio-1-methyl-1-deaza-pseudouridine;
2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;
2-thio-dihydropseudouridine; 2-thio-dihydrouridine;
2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;
4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;
4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;
(.+-.)1-(2-Hydroxypropyl)pseudouridine TP;
(2R)-1-(2-Hydroxypropyl)pseudouridine TP;
(2S)-1-(2-Hydroxypropyl)pseudouridine TP;
(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine
TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP;
(Z)-5-(2-Bromo-vinyl)uridine TP;
1-(2,2,2-Trifluoroethyl)-pseudo-UTP;
1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;
1-(2,2-Diethoxyethyl)pseudouridine TP;
1-(2,4,6-Trimethylbenzyl)pseudouridine TP;
1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;
1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;
1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;
1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine
TP; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;
1-(3,4-Dimethoxybenzyl)pseudouridine TP;
1-(3-Amino-3-carboxypropyl)pseudo-UTP;
1-(3-Amino-propyl)pseudo-UTP;
1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;
1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;
1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;
1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine
TP; 1-(4-Chlorobenzyl)pseudouridine TP;
1-(4-Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine
TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP;
1-(4-Methoxybenzyl)pseudouridine TP;
1-(4-Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxy-phenyl)pseudo-UTP;
1-(4-Methylbenzyl)pseudouridine TP; 1-(4-Methyl-benzyl)pseudo-UTP;
1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP;
1(4-Nitro-phenyl)pseudo-UTP; 1-(4-Thiomethoxybenzyl)pseudouridine
TP; 1-(4-Trifluoromethoxybenzyl)pseudouridine TP;
1-(4-Trifluoromethylbenzyl)pseudouridine TP;
1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;
1,6-Dimethyl-pseudo-UTP;
1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouri-
dine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine
TP; 1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;
1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;
1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;
1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;
1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;
1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;
1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;
1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;
1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;
1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;
1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;
1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;
1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;
1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;
1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine
TP; 1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;
1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;
1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine
TP; 1-Methoxymethylpseudouridine TP;
1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;
1-Methyl-6-(4-morpholino)-pseudo-UTP;
1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substituted
phenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;
1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;
1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;
1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;
1-Methyl-6-ethoxy-pseudo-UTP;
1-Methyl-6-ethylcarboxylate-pseudo-UTP;
1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;
1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;
1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;
1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;
1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;
1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;
1-Methyl-6-trifluoromethoxy-pseudo-UTP;
1-Methyl-6-trifluoromethyl-pseudo-UTP;
1-Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP;
1-Phenyl-pseudo-UTP; 1-Pivaloylpseudouridine TP;
1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;
1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP;
1-tert-Butyl-pseudo-UTP; 1-Thiomethoxymethylpseudouridine TP;
1-Thiomorpholinomethylpseudouridine TP;
1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;
1-Vinylpseudouridine TP; 2,2'-anhydro-uridine TP;
2'-bromo-deoxyuridine TP; 2'-F-5-Methyl-2'-deoxy-UTP;
2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-Ethynyluridine TP;
2'-a-Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP;
2'-b-Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP;
2'-Deoxy-2'-a-mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine
TP; 2'-Deoxy-2'-b-aminouridine TP; 2'-Deoxy-2'-b-azidouridine TP;
2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP;
2'-Deoxy-2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP;
2'-Deoxy-2'-b-mercaptouridine TP; 2'-Deoxy-2'-b-thiomethoxyuridine
TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine;
2'-O-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo-UTP;
4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine
TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP;
5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5'-Homo-uridine TP;
5-iodo-2'-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;
5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine
TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;
6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;
6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;
6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;
6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP;
6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP;
6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;
6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP;
6-Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP;
6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo-UTP;
6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP;
6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP;
6-Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP;
Alpha-thio-pseudo-UTP; Pseudouridine 1-(4-methylbenzenesulfonic
acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine
TP 1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP
1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;
Pseudouridine TP
1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic
acid; Pseudouridine TP
1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine
TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP
1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid
diethyl ester; Pseudo-UTP-N1-3-propionic acid;
Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;
Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;
Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;
Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;
undermodified hydroxywybutosine; 4-demethylwyosine;
2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;
2,4,5-(trimethyl)phenyl; 2 ` methyl, 2` amino, 2' azido,
2'fluro-cytidine; 2' methyl, 2' amino, 2' azido, 2'fluro-adenine;
2'methyl, 2' amino, 2' azido, 2'fluro-uridine;
2'-amino-2'-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl;
2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose;
2'-fluoro-modified bases; 2'-O-methyl-ribose;
2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;
2-pyridinone; 3 nitropyrrole;
3-(methyl)-7-(propynyl)isocarbostyrilyl;
3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;
4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl;
5 nitroindole; 5 substituted pyrimidines;
5-(methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine;
6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine;
6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(aza)indolyl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,
propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;
Aminoindolyl; Anthracenyl;
bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;
Isocarbostyrilyl; Isoguanisine; N2-substituted purines;
N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated
derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;
Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines;
O-alkylated derivative;
ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin
TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;
Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;
pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,
2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;
Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted
1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine;
Xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine;
7-deaza-2-amino-purine; pyridin-4-one ribonucleoside;
2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP;
2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine
TP; 2'-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; and
N6-(19-Amino-pentaoxanonadecyl)adenosine TP.
[0179] In some embodiments, an mRNA of the invention includes a
combination of one or more of the aforementioned modified
nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned
modified nucleobases.)
[0180] In some embodiments, the modified nucleobase is
pseudouridine (.psi.), N1-methylpseudouridine (m.sup.1.psi.),
2-thiouridine, 4'-thiouridine, 5-methylcytosine,
2-thio-1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,
2-thio-dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,
4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,
4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,
5-methyluridine, 5-methoxyuridine, or 2'-O-methyl uridine. In some
embodiments, an mRNA of the invention includes a combination of one
or more of the aforementioned modified nucleobases (e.g., a
combination of 2, 3 or 4 of the aforementioned modified
nucleobases.)
[0181] In some embodiments, the modified nucleobase is
1-methyl-pseudouridine (m.sup.1.psi.), 5-methoxy-uridine
(mo.sup.5U), 5-methyl-cytidine (m.sup.5C), pseudouridine (.psi.),
.alpha.-thio-guanosine, or .alpha.-thio-adenosine. In some
embodiments, an mRNA of the invention includes a combination of one
or more of the aforementioned modified nucleobases (e.g., a
combination of 2, 3 or 4 of the aforementioned modified
nucleobases.)
[0182] In some embodiments, the mRNA comprises pseudouridine
(.psi.) and 5-methyl-cytidine (m.sup.5C). In some embodiments, the
mRNA comprises 1-methyl-pseudouridine (m.sup.1.psi.). In some
embodiments, the mRNA comprises 1-methyl-pseudouridine
(m.sup.1.psi.) and 5-methyl-cytidine (m.sup.5C). In some
embodiments, the mRNA comprises 2-thiouridine (s.sup.2U). In some
embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine
(m.sup.5C). In some embodiments, the mRNA comprises
5-methoxy-uridine (mo.sup.5U). In some embodiments, the mRNA
comprises 5-methoxy-uridine (mo.sup.5U) and 5-methyl-cytidine
(m.sup.5C). In some embodiments, the mRNA comprises 2'-O-methyl
uridine. In some embodiments, the mRNA comprises 2'-O-methyl
uridine and 5-methyl-cytidine (m.sup.5C). In some embodiments, the
mRNA comprises N6-methyl-adenosine (m.sup.6A). In some embodiments,
the mRNA comprises N6-methyl-adenosine (m.sup.6A) and
5-methyl-cytidine (m.sup.5C).
[0183] In certain embodiments, an mRNA of the invention is
uniformly modified (i.e., fully modified, modified through-out the
entire sequence) for a particular modification. For example, an
mRNA can be uniformly modified with 5-methyl-cytidine (m.sup.5C),
meaning that all cytosine residues in the mRNA sequence are
replaced with 5-methyl-cytidine (m.sup.5C). Similarly, mRNAs of the
invention can be uniformly modified for any type of nucleoside
residue present in the sequence by replacement with a modified
residue such as those set forth above.
[0184] In some embodiments, the modified nucleobase is a modified
cytosine. Exemplary nucleobases and nucleosides having a modified
cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine
(m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine),
5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine,
2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine.
[0185] In some embodiments, the modified nucleobase is a modified
uridine. Exemplary nucleobases and nucleosides having a modified
uridine include 5-cyano uridine or 4'-thio uridine.
[0186] In some embodiments, the modified nucleobase is a modified
adenine. Exemplary nucleobases and nucleosides having a modified
adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A),
2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and
2,6-Diaminopurine.
[0187] In some embodiments, the modified nucleobase is a modified
guanine. Exemplary nucleobases and nucleosides having a modified
guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG),
methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine
(preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1),
7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G),
8-oxo-guanosine, 7-methyl-8-oxo-guanosine.
[0188] The mRNA cancer vaccines are nucleic acid molecules,
specifically polynucleotides which, in some embodiments, encode one
or more peptides or polypeptides of interest. Such peptides or
polypeptides serve as an antigen or antigenic molecule. The term
"nucleic acid," in its broadest sense, includes any compound and/or
substance that comprise a polymer of nucleotides. These polymers
are often referred to as polynucleotides.
[0189] Exemplary nucleic acids or polynucleotides of the invention
include, but are not limited to, ribonucleic acids (RNAs),
deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol
nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic
acids (LNAs, including LNA having a .beta.-D-ribo configuration,
.alpha.-LNA having an .alpha.-L-ribo configuration (a diastereomer
of LNA), 2'-amino-LNA having a 2'-amino functionalization, and
2'-amino-.alpha.-LNA having a 2'-amino functionalization), ethylene
nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids
or combinations thereof.
[0190] In one embodiment, the polynucleotides of the present
invention is or functions as a messenger RNA (mRNA). As used
herein, the term "messenger RNA" (mRNA) refers to any
polynucleotide which encodes at least one peptide or polypeptide of
interest and which is capable of being translated to produce the
encoded peptide polypeptide of interest in vitro, in vivo, in situ
or ex vivo.
[0191] Traditionally, the basic components of an mRNA molecule
include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a
poly-A tail. The polynucleotides of the present invention may
function as mRNA but are distinguished from wild-type mRNA in their
functional and/or structural design features which serve to
overcome existing problems of effective polypeptide production
using nucleic-acid based therapeutics. It is to be understood that
the antigens of the mRNA cancer vaccines of the present invention
may be encoded by in vitro translated (IVT) polynucleotides. A
"primary construct" refers to a polynucleotide which encodes one or
more polypeptides of interest and which retains sufficient
structural and/or chemical features to allow the polypeptide of
interest encoded therein to be translated.
[0192] An "in vitro transcription template (IVT)," as used herein,
refers to deoxyribonucleic acid (DNA) suitable for use in an IVT
reaction for the production of messenger RNA (mRNA). In some
embodiments, an IVT template encodes a 5' untranslated region,
contains an open reading frame, and encodes a 3' untranslated
region and a polyA tail. The particular nucleotide sequence
composition and length of an IVT template will depend on the mRNA
of interest encoded by the template.
[0193] A "5' untranslated region (UTR)" refers to a region of an
mRNA that is directly upstream (i.e., 5') from the start codon
(i.e., the first codon of an mRNA transcript translated by a
ribosome) that does not encode a protein or peptide.
[0194] A "3' untranslated region (UTR)" refers to a region of an
mRNA that is directly downstream (i.e., 3') from the stop codon
(i.e., the codon of an mRNA transcript that signals a termination
of translation) that does not encode a protein or peptide.
[0195] An "open reading frame" is a continuous stretch of DNA
beginning with a start codon (e.g., methionine (ATG)), and ending
with a stop codon (e.g., TAA, TAG or TGA) and encodes a protein or
peptide.
[0196] A "polyA tail" is a region of mRNA that is downstream, e.g.,
directly downstream (i.e., 3'), from the 3' UTR that contains
multiple, consecutive adenosine monophosphates. A polyA tail may
contain 10 to 300 adenosine monophosphates. For example, a polyA
tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,
260, 270, 280, 290 or 300 adenosine monophosphates. In some
embodiments, a polyA tail contains 50 to 250 adenosine
monophosphates. In a relevant biological setting (e.g., in cells,
in vivo, etc.) the poly(A) tail functions to protect mRNA from
enzymatic degradation, e.g., in the cytoplasm, and aids in
transcription termination, export of the mRNA from the nucleus, and
translation.
[0197] In some embodiments, the polynucleotide includes from about
200 to about 3,000 nucleotides (e.g., from 200 to 500, from 200 to
1,000, from 200 to 1,500, from 200 to 3,000, from 500 to 1,000,
from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 1,000
to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,500 to
3,000, and from 2,000 to 3,000).
[0198] In other aspects, the invention relates to a method for
preparing an mRNA cancer vaccine by IVT methods. In vitro
transcription (IVT) methods permit template-directed synthesis of
RNA molecules of almost any sequence. The size of the RNA molecules
that can be synthesized using IVT methods range from short
oligonucleotides to long nucleic acid polymers of several thousand
bases. IVT methods permit synthesis of large quantities of RNA
transcript (e.g., from microgram to milligram quantities) (Beckert
et al., Synthesis of RNA by in vitro transcription, Methods Mol
Biol. 703:29-41(2011); Rio et al. RNA: A Laboratory Manual. Cold
Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220.;
Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed.
Washington D.C.: ASM Press, 2007. 262-299). Generally, IVT utilizes
a DNA template featuring a promoter sequence upstream of a sequence
of interest. The promoter sequence is most commonly of
bacteriophage origin (ex. the T7, T3 or SP6 promoter sequence) but
many other promotor sequences can be tolerated including those
designed de novo. Transcription of the DNA template is typically
best achieved by using the RNA polymerase corresponding to the
specific bacteriophage promoter sequence. Exemplary RNA polymerases
include, but are not limited to T7 RNA polymerase, T3 RNA
polymerase, or SP6 RNA polymerase, among others. IVT is generally
initiated at a dsDNA but can proceed on a single strand.
[0199] It will be appreciated that mRNA vaccines of the present
disclosure, e.g., mRNAs encoding the concatameric antigen, may be
made using any appropriate synthesis method. For example, in some
embodiments, mRNA vaccines of the present disclosure are made using
IVT from a single bottom strand DNA as a template and complementary
oligonucleotide that serves as promotor. The single bottom strand
DNA may act as a DNA template for in vitro transcription of RNA,
and may be obtained from, for example, a plasmid, a PCR product, or
chemical synthesis. In some embodiments, the single bottom strand
DNA is linearized from a circular template. The single bottom
strand DNA template generally includes a promoter sequence, e.g., a
bacteriophage promoter sequence, to facilitate IVT. Methods of
making RNA using a single bottom strand DNA and a top strand
promoter complementary oligonucleotide are known in the art. An
exemplary method includes, but is not limited to, annealing the DNA
bottom strand template with the top strand promoter complementary
oligonucleotide (e.g., T7 promoter complementary oligonucleotide,
T3 promoter complementary oligonucleotide, or SP6 promoter
complementary oligonucleotide), followed by IVT using an RNA
polymerase corresponding to the promoter sequence, e.g., aT7 RNA
polymerase, a T3 RNA polymerase, or an SP6 RNA polymerase.
[0200] IVT methods can also be performed using a double-stranded
DNA template. For example, in some embodiments, the double-stranded
DNA template is made by extending a complementary oligonucleotide
to generate a complementary DNA strand using strand extension
techniques available in the art. In some embodiments, a single
bottom strand DNA template containing a promoter sequence and
sequence encoding one or more epitopes of interest is annealed to a
top strand promoter complementary oligonucleotide and subjected to
a PCR-like process to extend the top strand to generate a
double-stranded DNA template. Alternatively or additionally, a top
strand DNA containing a sequence complementary to the bottom strand
promoter sequence and complementary to the sequence encoding one or
more epitopes of interest is annealed to a bottom strand promoter
oligonucleotide and subjected to a PCR-like process to extend the
bottom strand to generate a double-stranded DNA template. In some
embodiments, the number of PCR-like cycles ranges from 1 to 20
cycles, e.g., 3 to 10 cycles. In some embodiments, a
double-stranded DNA template is synthesized wholly or in part by
chemical synthesis methods. The double-stranded DNA template can be
subjected to in vitro transcription as described herein.
[0201] In another aspect, mRNA vaccines of the present disclosure,
e.g., mRNAs encoding the concatameric antigen, may be made using
two DNA strands that are complementary across an overlapping
portion of their sequence, leaving single-stranded overhangs (i.e.,
sticky ends) when the complementary portions are annealed. These
single-stranded overhangs can be made double-stranded by extending
using the other strand as a template, thereby generating
double-stranded DNA. In some cases, this primer extension method
can permit larger ORFs to be incorporated into the template DNA
sequence, e.g., as compared to sizes incorporated into the template
DNA sequences obtained by top strand DNA synthesis methods. In the
primer extension method, a portion of the 3'-end of a first strand
(in the 5''-3' direction) is complementary to a portion the 3'-end
of a second strand (in the 3'-5' direction). In some such
embodiments, the single first strand DNA may include a sequence of
a promoter (e.g., T7, T3, or SP6), optionally a 5'-UTR, and some or
all of an ORF (e.g., a portion of the 5'-end of the ORF). In some
embodiments, the single second strand DNA may include complementary
sequences for some or all of an ORF (e.g., a portion complementary
to the 3'-end of the ORF), and optionally a 3'-UTR, a stop
sequence, and/or a poly(A) tail. Methods of making RNA using two
synthetic DNA strands may include annealing the two strands with
overlapping complementary portions, followed by primer extension
using one or more PCR-like cycles to extend the strands to generate
a double-stranded DNA template. In some embodiments, the number of
PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles.
Such double-stranded DNA can be subjected to in vitro transcription
as described herein.
[0202] In another aspect, mRNA vaccines of the present disclosure,
e.g., mRNAs encoding the concatameric antigen, may be made using
synthetic double-stranded linear DNA molecules, such as
gBlocks.RTM. (Integrated DNA Technologies, Coralville, Iowa), as
the double-stranded DNA template. An advantage to such synthetic
double-stranded linear DNA molecules is that they provide a longer
template from which to generate mRNAs. For example, gBlocks.RTM.
can range in size from 45-1000 (e.g., 125-750 nucleotides). In some
embodiments, a synthetic double-stranded linear DNA template
includes a full length 5'-UTR, a full length 3'-UTR, or both. A
full length 5'-UTR may be up to 100 nucleotides in length, e.g.,
about 40-60 nucleotides. A full length 3'-UTR may be up to 300
nucleotides in length, e.g., about 100-150 nucleotides.
[0203] To facilitate generation of longer constructs, two or more
double-stranded linear DNA molecules and/or gene fragments that are
designed with overlapping sequences on the 3' strands may be
assembled together using methods known in art. For example, the
Gibson Assembly.TM. Method (Synthetic Genomics, Inc., La Jolla,
Calif.) may be performed with the use of a mesophilic exonuclease
that cleaves bases from the 5'-end of the double-stranded DNA
fragments, followed by annealing of the newly formed complementary
single-stranded 3'-ends, polymerase-dependent extension to fill in
any single-stranded gaps, and finally, covalent joining of the DNA
segments by a DNA ligase.
[0204] In another aspect, mRNA vaccines of the present disclosure,
e.g., mRNAs encoding the concatameric antigen, may be made using
chemical synthesis of the RNA. Methods, for instance, involve
annealing a first polynucleotide comprising an open reading frame
encoding the polypeptide and a second polynucleotide comprising a
5'-UTR to a complementary polynucleotide conjugated to a solid
support. The 3'-terminus of the second polynucleotide is then
ligated to the 5'-terminus of the first polynucleotide under
suitable conditions. Suitable conditions include the use of a DNA
Ligase. The ligation reaction produces a first ligation product.
The 5' terminus of a third polynucleotide comprising a 3'-UTR is
then ligated to the 3'-terminus of the first ligation product under
suitable conditions. Suitable conditions for the second ligation
reaction include an RNA Ligase. A second ligation product is
produced in the second ligation reaction. The second ligation
product is released from the solid support to produce an mRNA
encoding a polypeptide of interest. In some embodiments the mRNA is
between 30 and 1000 nucleotides.
[0205] An mRNA encoding a polypeptide of interest may also be
prepared by binding a first polynucleotide comprising an open
reading frame encoding the polypeptide to a second polynucleotide
comprising 3'-UTR to a complementary polynucleotide conjugated to a
solid support. The 5'-terminus of the second polynucleotide is
ligated to the 3'-terminus of the first polynucleotide under
suitable conditions. The suitable conditions include a DNA Ligase.
The method produces a first ligation product. A third
polynucleotide comprising a 5'-UTR is ligated to the first ligation
product under suitable conditions to produce a second ligation
product. The suitable conditions include an RNA Ligase, such as T4
RNA. The second ligation product is released from the solid support
to produce an mRNA encoding a polypeptide of interest.
[0206] In some embodiments the first polynucleotide features a
5'-triphosphate and a 3'-OH. In other embodiments the second
polynucleotide comprises a 3'-OH. In yet other embodiments, the
third polynucleotide comprises a 5'-triphosphate and a 3'-OH. The
second polynucleotide may also include a 5'-cap structure. The
method may also involve the further step of ligating a fourth
polynucleotide comprising a poly-A region at the 3'-terminus of the
third polynucleotide. The fourth polynucleotide may comprise a
5'-triphosphate.
[0207] The method may or may not comprise reverse phase
purification. The method may also include a washing step wherein
the solid support is washed to remove unreacted polynucleotides.
The solid support may be, for instance, a capture resin. In some
embodiments the method involves dT purification.
[0208] In accordance with the present disclosure, template DNA
encoding the mRNA vaccines of the present disclosure includes an
open reading frame (ORF) encoding one or more cancer epitopes. In
some embodiments, the template DNA includes an ORF of up to 1000
nucleotides, e.g., about 10-350, 30-300 nucleotides or about 50-250
nucleotides. In some embodiments, the template DNA includes an ORF
of about 150 nucleotides. In some embodiments, the template DNA
includes an ORF of about 200 nucleotides.
[0209] In some embodiments, IVT transcripts are purified from the
components of the IVT reaction mixture after the reaction takes
place. For example, the crude IVT mix may be treated with
RNase-free DNase to digest the original template. The mRNA can be
purified using methods known in the art, including but not limited
to, precipitation using an organic solvent or column based
purification method. Commercial kits are available to purify RNA,
e.g., MEGACLEAR.TM. Kit (Ambion, Austin, Tex.). The mRNA can be
quantified using methods known in the art, including but not
limited to, commercially available instruments, e.g., NanoDrop.
Purified mRNA can be analyzed, for example, by agarose gel
electrophoresis to confirm the RNA is the proper size and/or to
confirm that no degradation of the RNA has occurred.
[0210] The template DNA may include one or more stabilizing
elements, including, but not limited to untranslated regions (UTR)
at their 5'-end (5'UTR) and/or at their 3'-end (3'UTR), in addition
to other structural features, such as a 5'-cap structure or a
3'-poly(A) tail. In some embodiments, the template DNA includes a
5'-UTR of about 1-30 nucleotides, e.g., about 5-25 nucleotides or
about 10-20 nucleotides. In some embodiments, the template DNA
includes a 5'-UTR of 13 nucleotides. In some embodiments, the
template DNA does not include a 5'-UTR. In some embodiments, the
template DNA includes a 3'-UTR of about 1-60 nucleotides, e.g.,
10-50 nucleotides. In some embodiments, the template DNA includes a
3'-UTR of 40 nucleotides. In some embodiments, the template DNA
does not include a 3'-UTR. In some embodiments, the template DNA
includes a 3'-poly(A) tail of 1-150 nucleotides, e.g., 10-100
nucleotides, e.g., 30 nucleotides. Such stabilizing elements may be
included in the DNA for transcription in the IVT reaction, or may
be synthesized separately and added to the resulting RNA generated
from the IVT reaction.
[0211] A 3'-poly(A) tail may be added to an RNA of the present
disclosure. Methods for poly(A) tail addition are well known in the
art. Such methods include, but are not limited to poly(A)
polymerase catalysis or periodate treatment. Alternatively or
additionally, a poly(A) tail can be synthesized separately and then
added to the RNA using any appropriate technique, such as click
chemistry, orthoclick chemistry, solulink, or other bioconjugate
chemistries known to those in the art.
[0212] A 7-methyl guanosine (m7G) cap may be added to an RNA of the
present disclosure. Methods for m7G cap addition are well known in
the art. Examples include, but are not limited to,
co-transcriptional incorporation of anti-reverse cap analog (ARCA)
using RNA polymerase, such as T7 polymerase. Commercial kits are
available for T7 ARCA mRNA generation, such as the HiScribe.TM. T7
ARCA mRNA kit (New England BioLabs).
[0213] According to the present disclosure, two regions or parts of
a chimeric polynucleotide may be joined or ligated, for example,
using triphosphate chemistry. In some embodiments, a first region
or part of 100 nucleotides or less is chemically synthesized with a
5'-monophosphate and terminal 3'-desOH or blocked OH. If the region
is longer than 80 nucleotides, it may be synthesized as two or more
strands that will subsequently be chemically linked by ligation. If
the first region or part is synthesized as a non-positionally
modified region or part using IVT, conversion to the
5'-monophosphate with subsequent capping of the 3'-terminus may
follow. Monophosphate protecting groups may be selected from any of
those known in the art. A second region or part of the chimeric
polynucleotide may be synthesized using either chemical synthesis
or IVT methods, e.g., as described herein. IVT methods may include
use of an RNA polymerase that can utilize a primer with a modified
cap. Alternatively, a cap may be chemically synthesized and coupled
to the IVT region or part.
[0214] It is noted that for ligation methods, ligation with DNA T4
ligase followed by DNAse treatment (to eliminate the DNA splint
required for DNA T4 Ligase activity) should readily prevent the
undesirable formation of concatenation products.
[0215] The entire chimeric polynucleotide need not be manufactured
with a phosphate-sugar backbone. If one of the regions or parts
encodes a polypeptide, then it is preferable that such region or
part comprise a phosphate-sugar backbone.
[0216] Ligation may be performed using any appropriate technique,
such as enzymatic ligation, click chemistry, orthoclick chemistry,
solulink, or other bioconjugate chemistries known to those in the
art. In some embodiments, the ligation is directed by a
complementary oligonucleotide splint. In some embodiments, the
ligation is performed without a complementary oligonucleotide
splint.
[0217] In other aspects, the invention relates to kits for
preparing an mRNA cancer vaccine by IVT methods. In personalized
cancer vaccines, it is important to identify patient specific
mutations and vaccinate the patient with one or more neoepitopes.
In such vaccines, the antigen(s) encoded by the ORFs of an mRNA
will be specific to the patient. The 5'- and 3'-ends of RNAs
encoding the antigen(s) may be more broadly applicable, as they
include untranslated regions and stabilizing regions that are
common to many RNAs. Among other things, the present disclosure
provides kits that include one or parts of a chimeric
polynucleotide, such as one or more 5'- and/or 3'-regions of RNA,
which may be combined with an ORF encoding a patient-specific
epitope. For example, a kit may include a polynucleotide containing
one or more of a 5'-ORF, a 3'-ORF, and a poly(A) tail. In some
embodiments, each polynucleotide component is in an individual
container. In other embodiments, more than one polynucleotide
component is present together in a single container. In some
embodiments, the kit includes a ligase enzyme. In some embodiments,
provided kits include instructions for use. In some embodiments,
the instructions include an instruction to ligate the epitope
encoding ORF to one or more other components from the kit, e.g.,
5'-ORF, a 3'-ORF, and/or a poly(A) tail.
[0218] Methods for generating personalized cancer vaccines
according to the invention involve identification of mutations
using techniques such as deep nucleic acid or protein sequencing
methods as described herein of tissue samples. In some embodiments
an initial identification of mutations in a patient's transcriptome
is performed. The data from the patient's transcriptome is compared
with sequence information from the patients exome in order to
identify patient specific and tumor specific mutations that are
expressed. The comparison produces a dataset of putative
neoepitopes, referred to as a mutanome. The mutanome may include
approximately 100-10,000 candidate mutations per patients. The
mutanome is subject to a data probing analysis using a set of
inquiries or algorithms to identify an optimal mutation set for
generation of a neoantigen vaccine. In some embodiments an mRNA
neoantigen vaccine is designed and manufactured. The patient is
then treated with the vaccine.
[0219] The neoantigen vaccine may be a polycistronic vaccine
including multiple neoepitopes or one or more single RNA vaccines
or a combination thereof.
[0220] In some embodiments the entire method from the initiation of
the mutation identification process to the start of patient
treatment is achieved in less than 2 months. In other embodiments
the whole process is achieved in 7 weeks or less, 6 weeks or less,
5 weeks or less, 4 weeks or less, 3 weeks or less, 2 weeks or less
or less than 1 week. In some embodiments the whole method is
performed in less than 30 days.
[0221] The mutation identification process may involve both
transcriptome and exome analysis or only transcriptome or exome
analysis. In some embodiments transcriptome analysis is performed
first and exome analysis is performed second. The analysis is
performed on a biological or tissue sample. In some embodiments a
biological or tissue sample is a blood or serum sample. In other
embodiments the sample is a tissue bank sample or EBV
transformation of B-cells.
[0222] Once an mRNA vaccine is synthesized, it is administered to
the patient. In some embodiments the vaccine is administered on a
schedule for up to two months, up to three months, up to four
month, up to five months, up to six months, up to seven months, up
to eight months, up to nine months, up to ten months, up to eleven
months, up to 1 year, up to 11/2 years, up to two years, up to
three years, or up to four years. The schedule may be the same or
varied. In some embodiments the schedule is weekly for the first 3
weeks and then monthly thereafter.
[0223] The vaccine may be administered by any route. In some
embodiments the vaccine is administered by an IM or IV route.
[0224] At any point in the treatment the patient may be examined to
determine whether the mutations in the vaccine are still
appropriate. Based on that analysis the vaccine may be adjusted or
reconfigured to include one or more different mutations or to
remove one or more mutations.
[0225] It has been recognized and appreciated that, by analyzing
certain properties of cancer associated mutations, optimal
neoepitopes may be assessed and/or selected for inclusion in an
mRNA vaccine. For example, at a given time, one or more of several
properties may be assessed and weighted in order to select a set of
neoepitopes for inclusion in a vaccine. A property of a neoepitope
or set of neoepitopes may include, for instance, an assessment of
gene or transcript-level expression in patient RNA-seq or other
nucleic acid analysis, tissue-specific expression in available
databases, known oncogenes/tumor suppressors, variant call
confidence score, RNA-seq allele-specific expression, conservative
vs. non-conservative AA substitution, position of point mutation
(Centering Score for increased TCR engagement), position of point
mutation (Anchoring Score for differential HLA binding), Selfness:
<100% core epitope homology with patient WES data, HLA-A and -B
IC50 for 8mers-11mers, HLA-DRB1 IC50 for 15mers-20mers, promiscuity
Score (i.e. number of patient HLAs predicted to bind), HLA-C IC50
for 8mers-11mers, HLA-DRB3-5 IC50 for 15mers-20mers, HLA-DQB1/A1
IC50 for 15mers-20mers, HLA-DPB1/A1 IC50 for 15mers-20mers, Class I
vs Class II proportion, Diversity of patient HLA-A, -B and DRB1
allotypes covered, proportion of point mutation vs complex epitopes
(e.g. frameshifts), and/or pseudo-epitope HLA binding scores.
[0226] In some embodiments, the properties of cancer associated
mutations used to identify optimal neoepitopes are properties
related to the type of mutation, abundance of mutation in patient
sample, immunogenicity, lack of self-reactivity, and nature of
peptide composition.
[0227] The type of mutation should be determined and considered as
a factor in determining whether a putative epitope should be
included in a vaccine. The type of mutation may vary. In some
instances it may be desirable to include multiple different types
of mutations in a single vaccine. In other instances a single type
of mutation may be more desirable. A value for particular mutation
can be weighted and calculated.
[0228] The abundance of the mutation in patient sample may also be
scored and factored into the decision of whether a putative epitope
should be included in a vaccine. Highly abundant mutations may
promote a more robust immune response.
[0229] The consideration of the immunogenicity is an important
component in the selection of optimal neoepitopes for inclusion in
a vaccine. Immunogenicity may be assessed for instance, by
analyzing the MHC binding capacity of a neoepitope, HLA
promiscuity, mutation position, predicted T cell reactivity, actual
T cell reactivity, structure leading to particular conformations
and resultant solvent exposure, and representation of specific
amino acids. Known algorithms such as the NetMHC prediction
algorithm can be used to predict capacity of a peptide to bind to
common HLA-A and -B alleles. Structural assessment of a MHC bound
peptide may also be conducted by in silico 3-dimensional analysis
and/or protein docking programs. Use of a predicted epitope
structure when bound to a MHC molecule, such as acquired from a
Rosetta algorithm, may be used to evaluate the degree of solvent
exposure of an amino acid residues of an epitope when the epitope
is bound to a MHC molecule. T cell reactivity may be assessed
experimentally with epitopes and T cells in vitro. Alternatively T
cell reactivity may be assessed using T cell response/sequence
datasets.
[0230] An important component of a neoepitope included in a
vaccine, is a lack of self-reactivity. The putative neoepitopes may
be screened to confirm that the epitope is restricted to tumor
tissue, for instance, arising as a result of genetic change within
malignant cells. Ideally, the epitope should not be present in
normal tissue of the patient and thus, self-similar epitopes are
filtered out of the dataset.
[0231] The nature of peptide composition may also be considered in
the epitope design. For instance a score can be provided for each
putative epitope on the value of conserved versus non-conserved
amino acids found in the epitope.
[0232] In some embodiments, the analysis performed by the tools
described herein may include comparing different sets of properties
acquired at different times from a patient, i.e. prior to and
following a therapeutic intervention, from different tissue
samples, from different patients having similar tumors, etc. In
some embodiments, an average of peak values from one set of
properties may be compared with an average of peak values from
another set of properties. For example, an average value for HLA
binding may be compared between two different sets of
distributions. The two sets of distributions may be determined for
time durations separated by days, months, or years, for
instance.
[0233] Moreover, the inventors have recognized and appreciated that
such data on properties of cancer mutations may be collected and
analyzed using the algorithms described herein. The data is useful
for identifying neoepitopes and sets of neoepitopes for the
development of personalized cancer vaccines.
[0234] A neoepitope characterization system in accordance with the
techniques described herein may take any suitable form, as
embodiments are not limited in this respect. An illustrative
implementation of a computer system 900 that may be used in
connection with some embodiments is shown in FIG. 12. One or more
computer systems such as computer system 900 may be used to
implement any of the functionality described above. The computer
system 900 may include one or more processors 910 and one or more
computer-readable storage media (i.e., tangible, non-transitory
computer-readable media), e.g., volatile storage 920 and one or
more non-volatile storage media 930, which may be formed of any
suitable data storage media. The processor 910 may control writing
data to and reading data from the volatile storage 920 and the
non-volatile storage device 930 in any suitable manner, as
embodiments are not limited in this respect. To perform any of the
functionality described herein, the processor 910 may execute one
or more instructions stored in one or more computer-readable
storage media (e.g., volatile storage 920 and/or non-volatile
storage 930), which may serve as tangible, non-transitory
computer-readable media storing instructions for execution by the
processor 910.
[0235] The above-described embodiments can be implemented in any of
numerous ways. For example, the embodiments may be implemented
using hardware, software or a combination thereof. When implemented
in software, the software code can be executed on any suitable
processor or collection of processors, whether provided in a single
computer or distributed among multiple computers. It should be
appreciated that any component or collection of components that
perform the functions described above can be generically considered
as one or more controllers that control the above-discussed
functions. The one or more controllers can be implemented in
numerous ways, such as with dedicated hardware, or with general
purpose hardware (e.g., one or more processors) that is programmed
using microcode or software to perform the functions recited
above.
[0236] In this respect, it should be appreciated that one
implementation comprises at least one computer-readable storage
medium (i.e., at least one tangible, non-transitory
computer-readable medium), such as a computer memory (e.g., hard
drive, flash memory, processor working memory, etc.), a floppy
disk, an optical disk, a magnetic tape, or other tangible,
non-transitory computer-readable medium, encoded with a computer
program (i.e., a plurality of instructions), which, when executed
on one or more processors, performs above-discussed functions. The
computer-readable storage medium can be transportable such that the
program stored thereon can be loaded onto any computer resource to
implement techniques discussed herein. In addition, it should be
appreciated that the reference to a computer program which, when
executed, performs above-discussed functions, is not limited to an
application program running on a host computer. Rather, the term
"computer program" is used herein in a generic sense to reference
any type of computer code (e.g., software or microcode) that can be
employed to program one or more processors to implement
above-techniques.
[0237] The mRNA cancer vaccines of the invention can be used as
therapeutic or prophylactic agents. They are provided for use in
medicine and/or for the priming of immune effector cells, e.g.,
stimulate/transfect PBMCs ex vivo and re-infuse the activated
cells. For example, a vaccine described herein can be administered
to a subject, wherein the polynucleotides are translated in vivo to
produce an antigen. Provided are compositions, methods, kits, and
reagents for diagnosis, treatment or prevention of a disease or
condition in humans and other mammals. The active therapeutic
agents of the invention include the mRNA cancer vaccines, cells
containing mRNA cancer vaccines or polypeptides translated from the
polynucleotides contained in the mRNA cancer vaccines.
[0238] The mRNA cancer vaccines may be induced for translation of a
polypeptide (e.g., antigen or immunogen) in a cell, tissue or
organism. Such translation can be in vivo, ex vivo, in culture, or
in vitro. The cell, tissue or organism is contacted with an
effective amount of a composition containing a mRNA cancer vaccine
which contains a polynucleotide that has at least one a
translatable region encoding the antigen or concatemeric
antigen.
[0239] An "effective amount" of the mRNA cancer vaccine is provided
based, at least in part, on the target tissue, target cell type,
means of administration, physical characteristics of the
polynucleotide (e.g., size, and extent of modified nucleosides) and
other components of the mRNA cancer vaccine, and other
determinants. In general, an effective amount of the mRNA cancer
vaccine composition provides an induced or boosted immune response
as a function of antigen production in the cell, preferably more
efficient than a composition containing a corresponding unmodified
polynucleotide encoding the same antigen or a peptide antigen.
Increased antigen production may be demonstrated by increased cell
transfection (i.e., the percentage of cells transfected with the
mRNA cancer vaccine), increased protein translation from the
polynucleotide, decreased nucleic acid degradation (as
demonstrated, e.g., by increased duration of protein translation
from a modified polynucleotide), or altered antigen specific immune
response of the host cell.
[0240] In some embodiments, polynucleotides of the mRNA cancer
vaccines and their encoded polypeptides in accordance with the
present invention may be used for treatment of cancer.
[0241] mRNA cancer vaccines may be administered prophylactically or
therapeutically as part of an active immunization scheme to healthy
individuals or early in cancer or late stage and/or metastatic
cancer. In one embodiment, the effective amount of the
polynucleotides of the mRNA cancer vaccines of the invention
provided to a cell, a tissue or a subject may be enough for immune
activation, and in particular antigen specific immune
activation.
[0242] The polynucleotides of the mRNA cancer vaccines may be
administered with other therapeutic compounds. As a non-limiting
example, the prophylactic or therapeutic compound may be an
adjuvant or a booster. As used herein, when referring to a
composition, such as a vaccine, the term "booster" refers to an
extra administration of the composition. A booster (or booster
vaccine) may be given after an earlier administration of the
composition. The time of administration between the initial
administration of the composition and the booster may be, but is
not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5
minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15
minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes,
55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14
hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours,
21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4
days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2
months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months,
9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3
years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years,
17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35
years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years,
70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more
than 99 years.
[0243] In some embodiments, the polynucleotides of the mRNA cancer
vaccines may be administered with an anti-cancer therapeutic agent,
including but not limited to, a traditional cancer vaccine. The
mRNA cancer vaccine and anti-cancer therapeutic can be combined to
enhance immune therapeutic responses even further. The mRNA cancer
vaccine and other therapeutic agent may be administered
simultaneously or sequentially. When the other therapeutic agents
are administered simultaneously they can be administered in the
same or separate formulations, but are administered at the same
time. The other therapeutic agents are administered sequentially
with one another and with the mRNA cancer vaccine, when the
administration of the other therapeutic agents and the mRNA cancer
vaccine is temporally separated. The separation in time between the
administration of these compounds may be a matter of minutes or it
may be longer, e.g. hours, days, weeks, months. Other therapeutic
agents include but are not limited to anti-cancer therapeutic,
adjuvants, cytokines, antibodies, antigens, etc.
[0244] In one embodiment, the polynucleotides may be administered
intramuscularly or intradermally similarly to the administration of
vaccines known in the art.
[0245] The mRNA cancer vaccines may be utilized in various settings
depending on the severity of the cancer or the degree or level of
unmet medical need. As a non-limiting example, the mRNA cancer
vaccines may be utilized to treat any stage of cancer. The mRNA
cancer vaccines have superior properties in that they produce much
larger antibody titers, T cell responses and produce responses
early than commercially available anti-cancer vaccines. While not
wishing to be bound by theory, the inventors hypothesize that the
mRNA cancer vaccines, as mRNAs, are better designed to produce the
appropriate protein conformation on translation as the mRNA cancer
vaccines co-opt natural cellular machinery. Unlike traditional
vaccines which are manufactured ex vivo and may trigger unwanted
cellular responses, the mRNA cancer vaccines are presented to the
cellular system in a more native fashion.
[0246] A non-limiting list of cancers that the mRNA cancer vaccines
may treat is presented below. Peptide epitopes may be derived from
any antigen of these cancers or tumors. Such epitopes are referred
to as cancer or tumor antigens. Cancer cells may differentially
express cell surface molecules during different phases of tumor
progression. For example, a cancer cell may express a cell surface
antigen in a benign state, yet down-regulate that particular cell
surface antigen upon metastasis. As such, it is envisioned that the
tumor or cancer antigen may encompass antigens produced during any
stage of cancer progression. The methods of the invention may be
adjusted to accommodate for these changes. For instance, several
different mRNA vaccines may be generated for a particular patient.
For instance a first vaccine may be used at the start of the
treatment. At a later time point, a new mRNA vaccine may be
generated and administered to the patient to account for different
antigens being expressed.
[0247] In some embodiments, the tumor antigen is one of the
following antigens: CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38,
CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5,
AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062,
BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbB1,
ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP,
Fibronectin, Folate Receptor, Ganglioside GM3, GD2,
glucocorticoid-induced tumor necrosis factor receptor (GITR),
gp1OO, gpA33, GPNMB, ICOS, IGF1R, Integrin av, Integrin
.alpha.v.beta., LAG-3, Lewis Y, Mesothelin, c-MET, MN Carbonic
anhydrase IX, MUC1, MUC16, Nectin-4, NKGD2, NOTCH, OX40, OX40L,
PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, Syndecan-1,
TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2,
VEGFR-3, and variants thereof.
[0248] Cancers or tumors include but are not limited to neoplasms,
malignant tumors, metastases, or any disease or disorder
characterized by uncontrolled cell growth such that it would be
considered cancerous. The cancer may be a primary or metastatic
cancer. Specific cancers that can be treated according to the
present invention include, but are not limited to, those listed
below (for a review of such disorders, see Fishman et al., 1985,
Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia). Cancers
include, but are not limited to, biliary tract cancer; bladder
cancer; brain cancer including glioblastomas and medulloblastomas;
breast cancer; cervical cancer; choriocarcinoma; colon cancer;
endometrial cancer; esophageal cancer; gastric cancer;
hematological neoplasms including acute lymphocytic and myelogenous
leukemia; multiple myeloma; AIDS-associated leukemias and adult
T-cell leukemia lymphoma; intraepithelial neoplasms including
Bowen's disease and Paget's disease; liver cancer; lung cancer;
lymphomas including Hodgkin's disease and lymphocytic lymphomas;
neuroblastomas; oral cancer including squamous cell carcinoma;
ovarian cancer including those arising from epithelial cells,
stromal cells, germ cells and mesenchymal cells; pancreatic cancer;
prostate cancer; rectal cancer; sarcomas including leiomyosarcoma,
rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin
cancer including melanoma, Kaposi's sarcoma, basocellular cancer,
and squamous cell cancer; testicular cancer including germinal
tumors such as seminoma, non-seminoma, teratomas, choriocarcinomas;
stromal tumors and germ cell tumors; thyroid cancer including
thyroid adenocarcinoma and medullar carcinoma; and renal cancer
including adenocarcinoma and Wilms' tumor. Commonly encountered
cancers include breast, prostate, lung, ovarian, colorectal, and
brain cancer.
[0249] The polynucleotides contained in the mRNA cancer vaccines of
the invention, their regions or parts or subregions may be codon
optimized. Codon optimization methods are known in the art and may
be useful in efforts to achieve one or more of several goals. These
goals include to match codon frequencies in target and host
organisms to ensure proper folding, bias GC content to increase
mRNA stability or reduce secondary structures, minimize tandem
repeat codons or base runs that may impair gene construction or
expression, customize transcriptional and translational control
regions, insert or remove protein trafficking sequences, remove/add
post translation modification sites in encoded protein (e.g.
glycosylation sites), add, remove or shuffle protein domains,
insert or delete restriction sites, modify ribosome binding sites
and mRNA degradation sites, to adjust translational rates to allow
the various domains of the protein to fold properly, or to reduce
or eliminate problem secondary structures within the
polynucleotide. Codon optimization tools, algorithms and services
are known in the art, non-limiting examples include services from
GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or
proprietary methods. In one embodiment, the ORF sequence is
optimized using optimization algorithms.
[0250] In some embodiments, a codon optimized sequence shares less
than 95% sequence identity to a naturally-occurring or wild-type
sequence (e.g., a naturally-occurring or wild-type mRNA sequence
encoding a polypeptide or protein of interest (e.g., an antigenic
protein or polypeptide. In some embodiments, a codon optimized
sequence shares less than 90% sequence identity to a
naturally-occurring or wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding a
polypeptide or protein of interest (e.g., an antigenic protein or
polypeptide. In some embodiments, a codon optimized sequence shares
less than 85% sequence identity to a naturally-occurring or
wild-type sequence (e.g., a naturally-occurring or wild-type mRNA
sequence encoding a polypeptide or protein of interest (e.g., an
antigenic protein or polypeptide. In some embodiments, a codon
optimized sequence shares less than 80% sequence identity to a
naturally-occurring or wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding a
polypeptide or protein of interest (e.g., an antigenic protein or
polypeptide. In some embodiments, a codon optimized sequence shares
less than 75% sequence identity to a naturally-occurring or
wild-type sequence (e.g., a naturally-occurring or wild-type mRNA
sequence encoding a polypeptide or protein of interest (e.g., an
antigenic protein or polypeptide.
[0251] In some embodiments, a codon optimized sequence shares
between 65% and 85% (e.g., between about 67% and about 85% or
between about 67% and about 80%) sequence identity to a
naturally-occurring or wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding a
polypeptide or protein of interest (e.g., an antigenic protein or
polypeptide. In some embodiments, a codon optimized sequence shares
between 65% and 75 or about 80% sequence identity to a
naturally-occurring or wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding a
polypeptide or protein of interest (e.g., an antigenic protein or
polypeptide.
[0252] In some embodiments a codon optimized RNA may, for instance,
be one in which the levels of G/C are enhanced. The G/C-content of
nucleic acid molecules may influence the stability of the RNA. RNA
having an increased amount of guanine (G) and/or cytosine (C)
residues may be functionally more stable than nucleic acids
containing a large amount of adenine (A) and thymine (T) or uracil
(U) nucleotides. WO02/098443 discloses a pharmaceutical composition
containing an mRNA stabilized by sequence modifications in the
translated region. Due to the degeneracy of the genetic code, the
modifications work by substituting existing codons for those that
promote greater RNA stability without changing the resulting amino
acid. The approach is limited to coding regions of the RNA.
[0253] In one embodiment, the polynucleotides of the present
invention (e.g., antigen-encoding polynucleotides featured in the
mRNA cancer vaccines of the invention) may be quantified in a
biological sample or when derived from one or more bodily fluid. As
used herein "bodily fluids" include peripheral blood, serum,
plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva,
bone marrow, synovial fluid, aqueous humor, amniotic fluid,
cerumen, breast milk, broncheoalveolar lavage fluid, semen,
prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat,
fecal matter, hair, tears, cyst fluid, pleural and peritoneal
fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial
fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal
secretion, stool water, pancreatic juice, lavage fluids from sinus
cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and
umbilical cord blood. Alternatively, the biological sample may be
retrieved from an organ selected from the group consisting of lung,
heart, pancreas, stomach, intestine, bladder, kidney, ovary,
testis, skin, colon, breast, prostate, brain, esophagus, liver, and
placenta.
[0254] In some instances the polypeptide encoded is larger than 25
amino acids and smaller than about 50 amino acids. Thus,
polypeptides include gene products, naturally occurring
polypeptides, synthetic polypeptides, homologs, orthologs,
paralogs, fragments and other equivalents, variants, and analogs of
the foregoing. A polypeptide may be a single molecule or may be a
multi-molecular complex such as a dimer, trimer or tetramer. They
may also comprise single chain or multichain polypeptides such as
antibodies or insulin and may be associated or linked. Most
commonly disulfide linkages are found in multichain polypeptides.
The term polypeptide may also apply to amino acid polymers in which
one or more amino acid residues are an artificial chemical analogue
of a corresponding naturally occurring amino acid.
[0255] The term "polypeptide variant" refers to molecules which
differ in their amino acid sequence from a native or reference
sequence. The amino acid sequence variants may possess
substitutions, deletions, and/or insertions at certain positions
within the amino acid sequence, as compared to a native or
reference sequence. Ordinarily, variants will possess at least
about 50% identity (homology) to a native or reference sequence,
and preferably, they will be at least about 80%, more preferably at
least about 90% identical (homologous) to a native or reference
sequence.
[0256] In some embodiments "variant mimics" are provided. As used
herein, the term "variant mimic" is one which contains one or more
amino acids which would mimic an activated sequence. For example,
glutamate may serve as a mimic for phosphoro-threonine and/or
phosphoro-serine. Alternatively, variant mimics may result in
deactivation or in an inactivated product containing the mimic,
e.g., phenylalanine may act as an inactivating substitution for
tyrosine; or alanine may act as an inactivating substitution for
serine.
[0257] "Homology" as it applies to amino acid sequences is defined
as the percentage of residues in the candidate amino acid sequence
that are identical with the residues in the amino acid sequence of
a second sequence after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent homology.
Methods and computer programs for the alignment are well known in
the art. It is understood that homology depends on a calculation of
percent identity but may differ in value due to gaps and penalties
introduced in the calculation. By "homologs" as it applies to
polypeptide sequences means the corresponding sequence of other
species having substantial identity to a second sequence of a
second species.
[0258] "Analogs" is meant to include polypeptide variants which
differ by one or more amino acid alterations, e.g., substitutions,
additions or deletions of amino acid residues that still maintain
one or more of the properties of the parent or starting
polypeptide.
[0259] The present invention contemplates several types of
compositions which are polypeptide based including variants and
derivatives. These include substitutional, insertional, deletion
and covalent variants and derivatives. The term "derivative" is
used synonymously with the term "variant" but generally refers to a
molecule that has been modified and/or changed in any way relative
to a reference molecule or starting molecule.
[0260] As such, polynucleotides encoding peptides or polypeptides
containing substitutions, insertions and/or additions, deletions
and covalent modifications with respect to reference sequences, in
particular the polypeptide sequences disclosed herein, are included
within the scope of this invention. For example, sequence tags or
amino acids, such as one or more lysines, can be added to the
peptide sequences of the invention (e.g., at the N-terminal or
C-terminal ends). Sequence tags can be used for peptide
purification or localization. Lysines can be used to increase
peptide solubility or to allow for biotinylation. Alternatively,
amino acid residues located at the carboxy and amino terminal
regions of the amino acid sequence of a peptide or protein may
optionally be deleted providing for truncated sequences. Certain
amino acids (e.g., C-terminal or N-terminal residues) may
alternatively be deleted depending on the use of the sequence, as
for example, expression of the sequence as part of a larger
sequence which is soluble, or linked to a solid support.
[0261] "Substitutional variants" when referring to polypeptides are
those that have at least one amino acid residue in a native or
starting sequence removed and a different amino acid inserted in
its place at the same position. The substitutions may be single,
where only one amino acid in the molecule has been substituted, or
they may be multiple, where two or more amino acids have been
substituted in the same molecule.
[0262] As used herein the term "conservative amino acid
substitution" refers to the substitution of an amino acid that is
normally present in the sequence with a different amino acid of
similar size, charge, or polarity. Examples of conservative
substitutions include the substitution of a non-polar (hydrophobic)
residue such as isoleucine, valine and leucine for another
non-polar residue. Likewise, examples of conservative substitutions
include the substitution of one polar (hydrophilic) residue for
another such as between arginine and lysine, between glutamine and
asparagine, and between glycine and serine. Additionally, the
substitution of a basic residue such as lysine, arginine or
histidine for another, or the substitution of one acidic residue
such as aspartic acid or glutamic acid for another acidic residue
are additional examples of conservative substitutions. Examples of
non-conservative substitutions include the substitution of a
non-polar (hydrophobic) amino acid residue such as isoleucine,
valine, leucine, alanine, methionine for a polar (hydrophilic)
residue such as cysteine, glutamine, glutamic acid or lysine and/or
a polar residue for a non-polar residue.
[0263] "Features" when referring to polypeptide or polynucleotide
are defined as distinct amino acid sequence-based or
nucleotide-based components of a molecule respectively. Features of
the polypeptides encoded by the polynucleotides of the present
invention include surface manifestations, local conformational
shape, folds, loops, half-loops, domains, half-domains, sites,
termini or any combination thereof.
[0264] As used herein when referring to polypeptides the term
"domain" refers to a motif of a polypeptide having one or more
identifiable structural or functional characteristics or properties
(e.g., binding capacity, serving as a site for protein-protein
interactions).
[0265] As used herein when referring to polypeptides the terms
"site" as it pertains to amino acid based embodiments is used
synonymously with "amino acid residue" and "amino acid side chain."
As used herein when referring to polynucleotides the terms "site"
as it pertains to nucleotide based embodiments is used synonymously
with "nucleotide." A site represents a position within a peptide or
polypeptide or polynucleotide that may be modified, manipulated,
altered, derivatized or varied within the polypeptide or
polynucleotide based molecules of the present invention.
[0266] As used herein the terms "termini" or "terminus" when
referring to polypeptides or polynucleotides refers to an extremity
of a polypeptide or polynucleotide respectively. Such extremity is
not limited only to the first or final site of the polypeptide or
polynucleotide but may include additional amino acids or
nucleotides in the terminal regions. The polypeptide based
molecules of the present invention may be characterized as having
both an N-terminus (terminated by an amino acid with a free amino
group (NH2)) and a C-terminus (terminated by an amino acid with a
free carboxyl group (COOH)). Proteins of the invention are in some
cases made up of multiple polypeptide chains brought together by
disulfide bonds or by non-covalent forces (multimers, oligomers).
These sorts of proteins will have multiple N- and C-termini.
Alternatively, the termini of the polypeptides may be modified such
that they begin or end, as the case may be, with a non-polypeptide
based moiety such as an organic conjugate.
[0267] As recognized by those skilled in the art, protein
fragments, functional protein domains, and homologous proteins are
also considered to be within the scope of polypeptides of interest
of this invention. For example, provided herein is any protein
fragment (meaning a polypeptide sequence at least one amino acid
residue shorter than a reference polypeptide sequence but otherwise
identical) of a reference protein 10, 20, 30, 40, 50, 60, 70, 80,
90, 100 or greater than 100 amino acids in length. In another
example, any protein that includes a stretch of about 20, about 30,
about 40, about 50, or about 100 amino acids which are about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,
or about 100% identical to any of the sequences described herein
can be utilized in accordance with the invention. In certain
embodiments, a polypeptide to be utilized in accordance with the
invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as
shown in any of the sequences provided or referenced herein.
[0268] Reference molecules (polypeptides or polynucleotides) may
share a certain identity with the designed molecules (polypeptides
or polynucleotides). The term "identity" as known in the art,
refers to a relationship between the sequences of two or more
peptides, polypeptides or polynucleotides, as determined by
comparing the sequences. In the art, identity also means the degree
of sequence relatedness between them as determined by the number of
matches between strings of two or more amino acid residues or
nucleosides. Identity measures the percent of identical matches
between the smaller of two or more sequences with gap alignments
(if any) addressed by a particular mathematical model or computer
program (i.e., "algorithms"). Identity of related peptides can be
readily calculated by known methods. Generally, variants of a
particular polynucleotide or polypeptide of the invention will have
at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100%
sequence identity to that particular reference polynucleotide or
polypeptide as determined by sequence alignment programs and
parameters described herein and known to those skilled in the art.
Such tools for alignment include those of the BLAST suite (Stephen
F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs", Nucleic Acids Res.
25:3389-3402.) A general global alignment technique based on
dynamic programming is the Needleman-Wunsch algorithm. More
recently a Fast Optimal Global Sequence Alignment Algorithm
(FOGSAA) has been developed that purportedly produces global
alignment of nucleotide and protein sequences faster than other
optimal global alignment methods, including the Needleman-Wunsch
algorithm. Other tools are described herein, specifically in the
definition of "Identity" below.
[0269] As used herein, the term "homology" refers to the overall
relatedness between polymeric molecules, e.g. between nucleic acid
molecules (e.g. DNA molecules and/or RNA molecules) and/or between
polypeptide molecules. In some embodiments, polymeric molecules are
considered to be "homologous" to one another if their sequences are
at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or 99% identical or similar. The term
"homologous" necessarily refers to a comparison between at least
two sequences (polynucleotide or polypeptide sequences). In
accordance with the invention, two polynucleotide sequences are
considered to be homologous if the polypeptides they encode are at
least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least
one stretch of at least about 20 amino acids. In some embodiments,
homologous polynucleotide sequences are characterized by the
ability to encode a stretch of at least 4-5 uniquely specified
amino acids. For polynucleotide sequences less than 60 nucleotides
in length, homology is determined by the ability to encode a
stretch of at least 4-5 uniquely specified amino acids. In
accordance with the invention, two protein sequences are considered
to be homologous if the proteins are at least about 50%, 60%, 70%,
80%, or 90% identical for at least one stretch of at least about 20
amino acids.
[0270] As used herein, the term "identity" refers to the overall
relatedness between polymeric molecules, e.g., between
polynucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of the percent
identity of two polynucleotide sequences, for example, can be
performed by aligning the two sequences for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second nucleic acid sequences for optimal alignment and
non-identical sequences can be disregarded for comparison
purposes). In certain embodiments, the length of a sequence aligned
for comparison purposes is at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, or 100% of the length of the reference sequence. The
nucleotides at corresponding nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which needs
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. For example, the percent identity between two nucleotide
sequences can be determined using methods such as those described
in Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin,
A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994;
and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,
M Stockton Press, New York, 1991; each of which is incorporated
herein by reference. For example, the percent identity between two
nucleotide sequences can be determined using the algorithm of
Meyers and Miller (CABIOS, 1989, 4:11-17), which has been
incorporated into the ALIGN program (version 2.0) using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. The percent identity between two nucleotide sequences can,
alternatively, be determined using the GAP program in the GCG
software package using an NWSgapdna.CMP matrix. Methods commonly
employed to determine percent identity between sequences include,
but are not limited to those disclosed in Carillo, H., and Lipman,
D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by
reference. Techniques for determining identity are codified in
publicly available computer programs. Exemplary computer software
to determine homology between two sequences include, but are not
limited to, GCG program package, Devereux, J., et al., Nucleic
Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA
Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
[0271] In some preferred embodiments of the invention the mRNA
cancer vaccines are administered with a T cell activator such as be
an immune checkpoint modulator. Immune checkpoint modulators
include both stimulatory checkpoint molecules and inhibitory
checkpoint molecules i.e., an anti-CTLA4 and anti-PD1 antibody.
[0272] Stimulatory checkpoint inhibitors function by promoting the
checkpoint process. Several stimulatory checkpoint molecules are
members of the tumor necrosis factor (TNF) receptor
superfamily-CD27, CD40, OX40, GITR and CD137, while others belong
to the B7-CD28 superfamily-CD28 and ICOS. OX40 (CD134), is involved
in the expansion of effector and memory T cells. Anti-OX40
monoclonal antibodies have been shown to be effective in treating
advanced cancer. MEDI0562 is a humanized OX40 agonist. GITR,
Glucocorticoid-Induced TNFR family Related gene, is involved in T
cell expansion Several antibodies to GITR have been shown to
promote an anti-tumor responses. ICOS, Inducible T-cell
costimulator, is important in T cell effector function. CD27
supports antigen-specific expansion of naive T cells and is
involved in the generation of T and B cell memory. Several
agonistic anti-CD27 antibodies are in development. CD122 is the
Interleukin-2 receptor beta sub-unit. NKTR-214 is a CD122-biased
immune-stimulatory cytokine.
[0273] Inhibitory checkpoint molecules include but are not limited
to PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR
and LAG3. CTLA-4, PD-1 and its ligands are members of the CD28-B7
family of co-signaling molecules that play important roles
throughout all stages of T-cell function and other cell functions.
CTLA-4, Cytotoxic T-Lymphocyte-Associated protein 4 (CD152), is
involved in controlling T cell proliferation.
[0274] The PD-1 receptor is expressed on the surface of activated T
cells (and B cells) and, under normal circumstances, binds to its
ligands (PD-L1 and PD-L2) that are expressed on the surface of
antigen-presenting cells, such as dendritic cells or macrophages.
This interaction sends a signal into the T cell and inhibits it.
Cancer cells take advantage of this system by driving high levels
of expression of PD-L1 on their surface. This allows them to gain
control of the PD-1 pathway and switch off T cells expressing PD-1
that may enter the tumor microenvironment, thus suppressing the
anticancer immune response. Pembrolizumab (formerly MK-3475 and
lambrolizumab, trade name Keytruda) is a human antibody used in
cancer immunotherapy. It targets the PD-1 receptor.
[0275] IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic
enzyme, which suppresses T and NK cells, generates and activates
Tregs and myeloid-derived suppressor cells, and promotes tumor
angiogenesis. TIM-3, T-cell Immunoglobulin domain and Mucin domain
3, acts as a negative regulator of Th1/Tc1 function by triggering
cell death upon interaction with its ligand, galectin-9. VISTA,
V-domain Ig suppressor of T cell activation.
[0276] The checkpoint inhibitor is a molecule such as a monoclonal
antibody, a humanized antibody, a fully human antibody, a fusion
protein or a combination thereof or a small molecule. For instance,
the checkpoint inhibitor inhibits a checkpoint protein which may be
CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9,
LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7
family ligands or a combination thereof. Ligands of checkpoint
proteins include but are not limited to CTLA-4, PDL1, PDL2, PD1,
B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160,
CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands. In some
embodiments the anti-PD-1 antibody is BMS-936558 (nivolumab). In
other embodiments the anti-CTLA-4 antibody is ipilimumab (trade
name Yervoy, formerly known as MDX-010 and MDX-101).
[0277] In some preferred embodiments the cancer therapeutic agents,
including the checkpoint modulators, are delivered in the form of
mRNA encoding the cancer therapeutic agents, e.g anti-PD1,
cytokines, chemokines or stimulatory receptors/ligands (e.g.
OX40.
[0278] In some embodiments the cancer therapeutic agent is a
targeted therapy. The targeted therapy may be a BRAF inhibitor such
as vemurafenib (PLX4032) or dabrafenib. The BRAF inhibitor may be
PLX 4032, PLX 4720, PLX 4734, GDC-0879, PLX 4032, PLX-4720, PLX
4734 and Sorafenib Tosylate. BRAF is a human gene that makes a
protein called B-Raf, also referred to as proto-oncogene B-Raf and
v-Raf murine sarcoma viral oncogene homolog B1. The B-Raf protein
is involved in sending signals inside cells, which are involved in
directing cell growth. Vemurafenib, a BRAF inhibitor, was approved
by FDA for treatment of late-stage melanoma.
[0279] The T-cell therapeutic agent in other embodiments is OX40L.
OX40 is a member of the tumor necrosis factor/nerve growth factor
receptor (TNFR/NGFR) family. OX40 may play a role in T-cell
activation as well as regulation of differentiation, proliferation
or apoptosis of normal and malignant lymphoid cells.
[0280] In other embodiments the cancer therapeutic agent is a
cytokine. In yet other embodiments the cancer therapeutic agent is
a vaccine comprising a population based tumor specific antigen.
[0281] The present invention provides pharmaceutical compositions
including mRNA cancer vaccines and mRNA cancer vaccine compositions
and/or complexes optionally in combination with one or more
pharmaceutically acceptable excipients.
[0282] The vaccines may be formulated or administered alone or in
conjunction with one or more other components. For instance the
vaccine compositions of the invention may comprise other components
including, but not limited to, adjuvants. Optionally the vaccine is
free of adjuvants.
[0283] The present invention provides mRNA cancer vaccines and mRNA
cancer vaccine pharmaceutical compositions and complexes optionally
in combination with one or more pharmaceutically acceptable
excipients. Pharmaceutical compositions may optionally comprise one
or more additional active substances, e.g. therapeutically and/or
prophylactically active substances. Pharmaceutical compositions of
the present invention may be sterile and/or pyrogen-free. General
considerations in the formulation and/or manufacture of
pharmaceutical agents may be found, for example, in Remington: The
Science and Practice of Pharmacy 21st ed., Lippincott Williams
& Wilkins, 2005 (incorporated herein by reference in its
entirety).
[0284] In some embodiments, compositions are administered to
humans, human patients or subjects. For the purposes of the present
disclosure, the phrase "active ingredient" generally refers to the
mRNA cancer vaccines or the polynucleotides contained therein,
e.g., antigen-encoding polynucleotides, for example, mRNAs, to be
delivered as described herein.
[0285] Formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of bringing the active ingredient into association
with an excipient and/or one or more other accessory ingredients,
and then, if necessary and/or desirable, dividing, shaping and/or
packaging the product into a desired single- or multi-dose
unit.
[0286] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
invention will vary, depending upon the identity, size, and/or
condition of the subject treated and further depending upon the
route by which the composition is to be administered. By way of
example, the composition may comprise between 0.1% and 100%, e.g.,
between 0.5 and 50%, between 1-30%, between 5-80%, at least 80%
(w/w) active ingredient.
[0287] The mRNA cancer vaccines of the invention can be formulated
using one or more excipients to: (1) increase stability; (2)
increase cell transfection; (3) permit the sustained or delayed
release (e.g., from a depot formulation); (4) alter the
biodistribution (e.g., target to specific tissues or cell types);
(5) increase the translation of encoded protein in vivo; and/or (6)
alter the release profile of encoded protein (antigen) in vivo. In
addition to traditional excipients such as any and all solvents,
dispersion media, diluents, or other liquid vehicles, dispersion or
suspension aids, surface active agents, isotonic agents, thickening
or emulsifying agents, preservatives, excipients of the present
invention can include, without limitation, lipidoids, liposomes,
lipid nanoparticles, polymers, lipoplexes, core-shell
nanoparticles, peptides, proteins, cells transfected with mRNA
cancer vaccines (e.g., for transplantation into a subject),
hyaluronidase, nanoparticle mimics and combinations thereof.
[0288] Stabilizing Elements
[0289] Naturally-occurring eukaryotic mRNA molecules have been
found to contain stabilizing elements, including, but not limited
to untranslated regions (UTR) at their 5'-end (5'UTR) and/or at
their 3'-end (3'UTR), in addition to other structural features,
such as a 5'-cap structure or a 3'-poly(A) tail. Both the 5'UTR and
the 3'UTR are typically transcribed from the genomic DNA and are
elements of the premature mRNA. Characteristic structural features
of mature mRNA, such as the 5'-cap and the 3'-poly(A) tail are
usually added to the transcribed (premature) mRNA during mRNA
processing. The 3'-poly(A) tail is typically a stretch of adenine
nucleotides added to the 3'-end of the transcribed mRNA. It can
comprise up to about 400 adenine nucleotides. In some embodiments
the length of the 3'-poly(A) tail may be an essential element with
respect to the stability of the individual mRNA.
[0290] In some embodiments the RNA vaccine may include one or more
stabilizing elements. Stabilizing elements may include for instance
a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa
protein has been identified. It is associated with the histone
stem-loop at the 3'-end of the histone messages in both the nucleus
and the cytoplasm. Its expression level is regulated by the cell
cycle; it is peaks during the S-phase, when histone mRNA levels are
also elevated. The protein has been shown to be essential for
efficient 3'-end processing of histone pre-mRNA by the U7 snRNP.
SLBP continues to be associated with the stem-loop after
processing, and then stimulates the translation of mature histone
mRNAs into histone proteins in the cytoplasm. The RNA binding
domain of SLBP is conserved through metazoa and protozoa; its
binding to the histone stem-loop depends on the structure of the
loop. The minimum binding site includes at least three nucleotides
5' and two nucleotides 3' relative to the stem-loop.
[0291] In some embodiments, the RNA vaccines include a coding
region, at least one histone stem-loop, and optionally, a poly(A)
sequence or polyadenylation signal. The poly(A) sequence or
polyadenylation signal generally should enhance the expression
level of the encoded protein. The encoded protein, in some
embodiments, is not a histone protein, a reporter protein (e.g.
Luciferase, GFP, EGFP, .beta.-Galactosidase, EGFP), or a marker or
selection protein (e.g. alpha-Globin, Galactokinase and
Xanthine:guanine phosphoribosyl transferase (GPT)).
[0292] In some embodiments, the combination of a poly(A) sequence
or polyadenylation signal and at least one histone stem-loop, even
though both represent alternative mechanisms in nature, acts
synergistically to increase the protein expression beyond the level
observed with either of the individual elements. It has been found
that the synergistic effect of the combination of poly(A) and at
least one histone stem-loop does not depend on the order of the
elements or the length of the poly(A) sequence.
[0293] In some embodiments, the RNA vaccine does not comprise a
histone downstream element (HDE). "Histone downstream element"
(HDE) includes a purine-rich polynucleotide stretch of
approximately 15 to 20 nucleotides 3' of naturally occurring
stem-loops, representing the binding site for the U7 snRNA, which
is involved in processing of histone pre-mRNA into mature histone
mRNA. Ideally, the inventive nucleic acid does not include an
intron.
[0294] In some embodiments, the RNA vaccine may or may not contain
a enhancer and/or promoter sequence, which may be modified or
unmodified or which may be activated or inactivated. In some
embodiments, the histone stem-loop is generally derived from
histone genes, and includes an intramolecular base pairing of two
neighbored partially or entirely reverse complementary sequences
separated by a spacer, consisting of a short sequence, which forms
the loop of the structure. The unpaired loop region is typically
unable to base pair with either of the stem loop elements. It
occurs more often in RNA, as is a key component of many RNA
secondary structures, but may be present in single-stranded DNA as
well. Stability of the stem-loop structure generally depends on the
length, number of mismatches or bulges, and base composition of the
paired region. In some embodiments, wobble base pairing
(non-Watson-Crick base pairing) may result. In some embodiments,
the at least one histone stem-loop sequence comprises a length of
15 to 45 nucleotides.
[0295] In other embodiments the RNA vaccine may have one or more
AU-rich sequences removed. These sequences, sometimes referred to
as AURES are destabilizing sequences found in the 3'UTR. The AURES
may be removed from the RNA vaccines. Alternatively the AURES may
remain in the RNA vaccine.
[0296] In one embodiment, the mRNA cancer vaccines may be
formulated in a lipid-polycation complex. The formation of the
lipid-polycation complex may be accomplished by methods known in
the art and/or as described in U.S. Pub. No. 20120178702, herein
incorporated by reference in its entirety. As a non-limiting
example, the polycation may include a cationic peptide or a
polypeptide such as, but not limited to, polylysine, polyornithine
and/or polyarginine and the cationic peptides described in
International Pub. No. WO2012013326 or US Patent Pub. No.
US20130142818; each of which is herein incorporated by reference in
its entirety. In another embodiment, the mRNA cancer vaccines may
be formulated in a lipid-polycation complex which may further
include a non-cationic lipid such as, but not limited to,
cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
[0297] The liposome formulation may be influenced by, but not
limited to, the selection of the cationic lipid component, the
degree of cationic lipid saturation, the nature of the PEGylation,
ratio of all components and biophysical parameters such as size. In
one example by Semple et al. (Semple et al. Nature Biotech. 2010
28:172-176; herein incorporated by reference in its entirety), the
liposome formulation was composed of 57.1% cationic lipid, 7.1%
dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4%
PEG-c-DMA. As another example, changing the composition of the
cationic lipid could more effectively deliver siRNA to various
antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200;
herein incorporated by reference in its entirety). In some
embodiments, liposome formulations may comprise from about 35 to
about 45% cationic lipid, from about 40% to about 50% cationic
lipid, from about 50% to about 60% cationic lipid and/or from about
55% to about 65% cationic lipid. In some embodiments, the ratio of
lipid to mRNA in liposomes may be from about 5:1 to about 20:1,
from about 10:1 to about 25:1, from about 15:1 to about 30:1 and/or
at least 30:1.
[0298] In some embodiments, the ratio of PEG in the lipid
nanoparticle (LNP) formulations may be increased or decreased
and/or the carbon chain length of the PEG lipid may be modified
from C14 to C18 to alter the pharmacokinetics and/or
biodistribution of the LNP formulations. As a non-limiting example,
LNP formulations may contain from about 0.5% to about 3.0%, from
about 1.0% to about 3.5%, from about 1.5% to about 4.0%, from about
2.0% to about 4.5%, from about 2.5% to about 5.0% and/or from about
3.0% to about 6.0% of the lipid molar ratio of PEG-c-DOMG
(R-3-[(.omega.-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristy-
loxypropyl-3-amine) (also referred to herein as PEG-DOMG) as
compared to the cationic lipid, DSPC and cholesterol. In another
embodiment the PEG-c-DOMG may be replaced with a PEG lipid such as,
but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol,
methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol)
and/or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene
glycol). The cationic lipid may be selected from any lipid known in
the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA,
C12-200 and DLin-KC2-DMA.
[0299] In one embodiment, the mRNA cancer vaccine formulation
comprising the polynucleotide is a nanoparticle which may comprise
at least one lipid. The lipid may be selected from, but is not
limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA,
DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino
alcohol lipids. In another aspect, the lipid may be a cationic
lipid such as, but not limited to, DLin-DMA, DLin-D-DMA,
DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The
amino alcohol cationic lipid may be the lipids described in and/or
made by the methods described in US Patent Publication No.
US20130150625, herein incorporated by reference in its entirety. As
a non-limiting example, the cationic lipid may be
2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-
-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in US20130150625);
2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methy-
l}propan-1-ol (Compound 2 in US20130150625);
2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propa-
n-1-ol (Compound 3 in US20130150625); and
2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-oc-
tadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 4 in
US20130150625); or any pharmaceutically acceptable salt or
stereoisomer thereof.
[0300] Lipid nanoparticle formulations typically comprise a lipid,
in particular, an ionizable cationic lipid, for example,
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and
further comprise a neutral lipid, a sterol and a molecule capable
of reducing particle aggregation, for example a PEG or PEG-modified
lipid.
[0301] In one embodiment, the lipid nanoparticle formulation
consists essentially of (i) at least one lipid selected from the
group consisting of
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a
neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a
sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or
PEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25%
neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.
[0302] In one embodiment, the formulation includes from about 25%
to about 75% on a molar basis of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g.,
from about 35 to about 65%, from about 45 to about 65%, about 60%,
about 57.5%, about 50% or about 40% on a molar basis.
[0303] In one embodiment, the formulation includes from about 0.5%
to about 15% on a molar basis of the neutral lipid e.g., from about
3 to about 12%, from about 5 to about 10% or about 15%, about 10%,
or about 7.5% on a molar basis. Exemplary neutral lipids include,
but are not limited to, DSPC, POPC, DPPC, DOPE and SM. In one
embodiment, the formulation includes from about 5% to about 50% on
a molar basis of the sterol (e.g., about 15 to about 45%, about 20
to about 40%, about 40%, about 38.5%, about 35%, or about 31% on a
molar basis. An exemplary sterol is cholesterol. In one embodiment,
the formulation includes from about 0.5% to about 20% on a molar
basis of the PEG or PEG-modified lipid (e.g., about 0.5 to about
10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%,
about 3.5%, or about 5% on a molar basis. In one embodiment, the
PEG or PEG modified lipid comprises a PEG molecule of an average
molecular weight of 2,000 Da. In other embodiments, the PEG or PEG
modified lipid comprises a PEG molecule of an average molecular
weight of less than 2,000, for example around 1,500 Da, around
1,000 Da, or around 500 Da. Exemplary PEG-modified lipids include,
but are not limited to, PEG-distearoyl glycerol (PEG-DMG) (also
referred herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed
in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the
contents of which are herein incorporated by reference in its
entirety)
[0304] In one embodiment, the formulations of the inventions
include 25-75% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15%
of the neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG
or PEG-modified lipid on a molar basis.
[0305] In one embodiment, the formulations of the inventions
include 35-65% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12%
of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG
or PEG-modified lipid on a molar basis.
[0306] In one embodiment, the formulations of the inventions
include 45-65% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10%
of the neutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG
or PEG-modified lipid on a molar basis.
[0307] In one embodiment, the formulations of the inventions
include about 60% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
7.5% of the neutral lipid, about 31% of the sterol, and about 1.5%
of the PEG or PEG-modified lipid on a molar basis.
[0308] In one embodiment, the formulations of the inventions
include about 50% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
10% of the neutral lipid, about 38.5% of the sterol, and about 1.5%
of the PEG or PEG-modified lipid on a molar basis.
[0309] In one embodiment, the formulations of the inventions
include about 50% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
10% of the neutral lipid, about 35% of the sterol, about 4.5% or
about 5% of the PEG or PEG-modified lipid, and about 0.5% of the
targeting lipid on a molar basis.
[0310] In one embodiment, the formulations of the inventions
include about 40% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
15% of the neutral lipid, about 40% of the sterol, and about 5% of
the PEG or PEG-modified lipid on a molar basis.
[0311] In one embodiment, the formulations of the inventions
include about 57.2% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
7.1% of the neutral lipid, about 34.3% of the sterol, and about
1.4% of the PEG or PEG-modified lipid on a molar basis.
[0312] In one embodiment, the formulations of the inventions
include about 57.5% of a cationic lipid selected from the PEG lipid
is PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. (J.
Controlled Release, 107, 276-287 (2005), the contents of which are
herein incorporated by reference in its entirety), about 7.5% of
the neutral lipid, about 31.5% of the sterol, and about 3.5% of the
PEG or PEG-modified lipid on a molar basis.
[0313] In preferred embodiments, lipid nanoparticle formulation
consists essentially of a lipid mixture in molar ratios of about
20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol:
0.5-15% PEG-modified lipid; more preferably in a molar ratio of
about 20-60% cationic lipid: 5-25% neutral lipid: 25-55%
cholesterol: 0.5-15% PEG-modified lipid.
[0314] In particular embodiments, the molar lipid ratio is
approximately 50/10/38.5/1.5 (mol % cationic lipid/neutral lipid,
e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or
PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid,
e.g., DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5
(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified
lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic
lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,
PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid, e.g.,
DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol %
cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,
e.g., PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic
lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,
PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutral
lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or
PEG-cDMA).
[0315] Exemplary lipid nanoparticle compositions and methods of
making same are described, for example, in Semple et al. (2010)
Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem.
Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy
21, 1570-1578 (the contents of each of which are incorporated
herein by reference in their entirety).
[0316] In one embodiment, the lipid nanoparticle formulations
described herein may comprise a cationic lipid, a PEG lipid and a
structural lipid and optionally comprise a non-cationic lipid. As a
non-limiting example, the lipid nanoparticle may comprise about
40-60% of cationic lipid, about 5-15% of a non-cationic lipid,
about 1-2% of a PEG lipid and about 30-50% of a structural lipid.
As another non-limiting example, the lipid nanoparticle may
comprise about 50% cationic lipid, about 10% non-cationic lipid,
about 1.5% PEG lipid and about 38.5% structural lipid. As yet
another non-limiting example, the lipid nanoparticle may comprise
about 55% cationic lipid, about 10% non-cationic lipid, about 2.5%
PEG lipid and about 32.5% structural lipid. In one embodiment, the
cationic lipid may be any cationic lipid described herein such as,
but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.
[0317] In one embodiment, the lipid nanoparticle formulations
described herein may be 4 component lipid nanoparticles. The lipid
nanoparticle may comprise a cationic lipid, a non-cationic lipid, a
PEG lipid and a structural lipid. As a non-limiting example, the
lipid nanoparticle may comprise about 40-60% of cationic lipid,
about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and
about 30-50% of a structural lipid. As another non-limiting
example, the lipid nanoparticle may comprise about 50% cationic
lipid, about 10% non-cationic lipid, about 1.5% PEG lipid and about
38.5% structural lipid. As yet another non-limiting example, the
lipid nanoparticle may comprise about 55% cationic lipid, about 10%
non-cationic lipid, about 2.5% PEG lipid and about 32.5% structural
lipid. In one embodiment, the cationic lipid may be any cationic
lipid described herein such as, but not limited to, DLin-KC2-DMA,
DLin-MC3-DMA and L319.
[0318] In one embodiment, the lipid nanoparticle formulations
described herein may comprise a cationic lipid, a non-cationic
lipid, a PEG lipid and a structural lipid. As a non-limiting
example, the lipid nanoparticle comprise about 50% of the cationic
lipid DLin-KC2-DMA, about 10% of the non-cationic lipid DSPC, about
1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structural
lipid cholesterol. As a non-limiting example, the lipid
nanoparticle comprise about 50% of the cationic lipid DLin-MC3-DMA,
about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG
lipid PEG-DOMG and about 38.5% of the structural lipid cholesterol.
As a non-limiting example, the lipid nanoparticle comprise about
50% of the cationic lipid DLin-MC3-DMA, about 10% of the
non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and
about 38.5% of the structural lipid cholesterol. As yet another
non-limiting example, the lipid nanoparticle comprise about 55% of
the cationic lipid L319, about 10% of the non-cationic lipid DSPC,
about 2.5% of the PEG lipid PEG-DMG and about 32.5% of the
structural lipid cholesterol.
[0319] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
present disclosure may vary, depending upon the identity, size,
and/or condition of the subject being treated and further depending
upon the route by which the composition is to be administered. For
example, the composition may comprise between 0.1% and 99% (w/w) of
the active ingredient. By way of example, the composition may
comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between
1-30%, between 5-80%, at least 80% (w/w) active ingredient.
[0320] In one embodiment, the mRNA cancer vaccine composition may
comprise the polynucleotide described herein, formulated in a lipid
nanoparticle comprising MC3, Cholesterol, DSPC and PEG2000-DMG, the
buffer trisodium citrate, sucrose and water for injection. As a
non-limiting example, the composition comprises: 2.0 mg/mL of drug
substance (e.g., polynucleotides encoding H10N8 influenza virus),
21.8 mg/mL of MC3, 10.1 mg/mL of cholesterol, 5.4 mg/mL of DSPC,
2.7 mg/mL of PEG2000-DMG, 5.16 mg/mL of trisodium citrate, 71 mg/mL
of sucrose and about 1.0 mL of water for injection.
[0321] The RNA vaccines of the invention can be formulated using
one or more liposomes, lipoplexes, or lipid nanoparticles. In one
embodiment, pharmaceutical compositions of RNA vaccines include
liposomes. Liposomes are artificially-prepared vesicles which may
primarily be composed of a lipid bilayer and may be used as a
delivery vehicle for the administration of nutrients and
pharmaceutical formulations. Liposomes can be of different sizes
such as, but not limited to, a multilamellar vesicle (MLV) which
may be hundreds of nanometers in diameter and may contain a series
of concentric bilayers separated by narrow aqueous compartments, a
small unicellular vesicle (SUV) which may be smaller than 50 nm in
diameter, and a large unilamellar vesicle (LUV) which may be
between 50 and 500 nm in diameter. Liposome design may include, but
is not limited to, opsonins or ligands in order to improve the
attachment of liposomes to unhealthy tissue or to activate events
such as, but not limited to, endocytosis. Liposomes may contain a
low or a high pH in order to improve the delivery of the
pharmaceutical formulations.
[0322] The formation of liposomes may depend on the physicochemical
characteristics such as, but not limited to, the pharmaceutical
formulation entrapped and the liposomal ingredients, the nature of
the medium in which the lipid vesicles are dispersed, the effective
concentration of the entrapped substance and its potential
toxicity, any additional processes involved during the application
and/or delivery of the vesicles, the optimization size,
polydispersity and the shelf-life of the vesicles for the intended
application, and the batch-to-batch reproducibility and possibility
of large-scale production of safe and efficient liposomal
products.
[0323] As a non-limiting example, liposomes such as synthetic
membrane vesicles may be prepared by the methods, apparatus and
devices described in US Patent Publication No. US20130177638,
US20130177637, US20130177636, US20130177635, US20130177634,
US20130177633, US20130183375, US20130183373 and US20130183372, the
contents of each of which are herein incorporated by reference in
its entirety.
[0324] In one embodiment, pharmaceutical compositions described
herein may include, without limitation, liposomes such as those
formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA)
liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.),
1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by
reference in its entirety) and liposomes which may deliver small
molecule drugs such as, but not limited to, DOXIL.RTM. from Janssen
Biotech, Inc. (Horsham, Pa.).
[0325] In one embodiment, pharmaceutical compositions described
herein may include, without limitation, liposomes such as those
formed from the synthesis of stabilized plasmid-lipid particles
(SPLP) or stabilized nucleic acid lipid particle (SNALP) that have
been previously described and shown to be suitable for
oligonucleotide delivery in vitro and in vivo (see Wheeler et al.
Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999
6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et
al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature.
2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;
Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin
Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008
19:125-132; U.S. Patent Publication No US20130122104; all of which
are incorporated herein in their entireties). The original
manufacture method by Wheeler et al. was a detergent dialysis
method, which was later improved by Jeffs et al. and is referred to
as the spontaneous vesicle formation method. The liposome
formulations are composed of 3 to 4 lipid components in addition to
the polynucleotide. As an example a liposome can contain, but is
not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline
(DSPC), 10% PEG-S-DSG, and 15%
1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by
Jeffs et al. As another example, certain liposome formulations may
contain, but are not limited to, 48% cholesterol, 20% DSPC, 2%
PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be
1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA,
or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as
described by Heyes et al.
[0326] In some embodiments, liposome formulations may comprise from
about about 25.0% cholesterol to about 40.0% cholesterol, from
about 30.0% cholesterol to about 45.0% cholesterol, from about
35.0% cholesterol to about 50.0% cholesterol and/or from about
48.5% cholesterol to about 60% cholesterol. In a preferred
embodiment, formulations may comprise a percentage of cholesterol
selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%,
37.0%, 38.5%, 39.0% and 43.5%. In some embodiments, formulations
may comprise from about 5.0% to about 10.0% DSPC and/or from about
7.0% to about 15.0% DSPC.
[0327] In one embodiment, pharmaceutical compositions may include
liposomes which may be formed to deliver polynucleotides which may
encode at least one immunogen (antigen) or any other polypeptide of
interest. The RNA vaccine may be encapsulated by the liposome
and/or it may be contained in an aqueous core which may then be
encapsulated by the liposome (see International Pub. Nos.
WO2012031046, WO2012031043, WO2012030901 and WO2012006378 and US
Patent Publication No. US20130189351, US20130195969 and
US20130202684; the contents of each of which are herein
incorporated by reference in their entirety).
[0328] In another embodiment, liposomes may be formulated for
targeted delivery. As a non-limiting example, the liposome may be
formulated for targeted delivery to the liver. The liposome used
for targeted delivery may include, but is not limited to, the
liposomes described in and methods of making liposomes described in
US Patent Publication No. US20130195967, the contents of which are
herein incorporated by reference in its entirety.
[0329] In another embodiment, the polynucleotide which may encode
an immunogen (antigen) may be formulated in a cationic oil-in-water
emulsion where the emulsion particle comprises an oil core and a
cationic lipid which can interact with the polynucleotide anchoring
the molecule to the emulsion particle (see International Pub. No.
WO2012006380; herein incorporated by reference in its
entirety).
[0330] In one embodiment, the RNA vaccines may be formulated in a
water-in-oil emulsion comprising a continuous hydrophobic phase in
which the hydrophilic phase is dispersed. As a non-limiting
example, the emulsion may be made by the methods described in
International Publication No. WO201087791, the contents of which
are herein incorporated by reference in its entirety.
[0331] In another embodiment, the lipid formulation may include at
least cationic lipid, a lipid which may enhance transfection and a
least one lipid which contains a hydrophilic head group linked to a
lipid moiety (International Pub. No. WO2011076807 and U.S. Pub. No.
20110200582; the contents of each of which is herein incorporated
by reference in their entirety). In another embodiment, the
polynucleotides encoding an immunogen may be formulated in a lipid
vesicle which may have crosslinks between functionalized lipid
bilayers (see U.S. Pub. No. 20120177724, the contents of which is
herein incorporated by reference in its entirety).
[0332] In one embodiment, the polylnucleotides may be formulated in
a lipsome as described in International Patent Publication No.
WO2013086526, the contents of which is herein incorporated by
reference in its entirety. The RNA vaccines may be encapsulated in
a liposome using reverse pH gradients and/or optimized internal
buffer compositions as described in International Patent
Publication No. WO2013086526, the contents of which is herein
incorporated by reference in its entirety.
[0333] In one embodiment, the RNA vaccine pharmaceutical
compositions may be formulated in liposomes such as, but not
limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.),
SMARTICLES.RTM. (Marina Biotech, Bothell, Wash.), neutral DOPC
(1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,
siRNA delivery for ovarian cancer (Landen et al. Cancer Biology
& Therapy 2006 5(12)1708-1713); herein incorporated by
reference in its entirety) and hyaluronan-coated liposomes (Quiet
Therapeutics, Israel).
[0334] In one embodiment, the cationic lipid may be a low molecular
weight cationic lipid such as those described in US Patent
Application No. 20130090372, the contents of which are herein
incorporated by reference in its entirety.
[0335] In one embodiment, the RNA vaccines may be formulated in a
lipid vesicle which may have crosslinks between functionalized
lipid bilayers.
[0336] In one embodiment, the RNA vaccines may be formulated in a
liposome comprising a cationic lipid. The liposome may have a molar
ratio of nitrogen atoms in the cationic lipid to the phophates in
the RNA (N:P ratio) of between 1:1 and 20:1 as described in
International Publication No. WO2013006825, herein incorporated by
reference in its entirety. In another embodiment, the liposome may
have a N:P ratio of greater than 20:1 or less than 1:1.
[0337] In one embodiment, the RNA vaccines may be formulated in a
lipid-polycation complex. The formation of the lipid-polycation
complex may be accomplished by methods known in the art and/or as
described in U.S. Pub. No. 20120178702, herein incorporated by
reference in its entirety. As a non-limiting example, the
polycation may include a cationic peptide or a polypeptide such as,
but not limited to, polylysine, polyornithine and/or polyarginine
and the cationic peptides described in International Pub. No.
WO2012013326 or US Patent Pub. No. US20130142818; each of which is
herein incorporated by reference in its entirety. In another
embodiment, the RNA vaccines may be formulated in a
lipid-polycation complex which may further include a non-cationic
lipid such as, but not limited to, cholesterol or dioleoyl
phosphatidylethanolamine (DOPE).
[0338] In one embodiment, the RNA vaccines may be formulated in an
aminoalcohol lipidoid. Aminoalcohol lipidoids which may be used in
the present invention may be prepared by the methods described in
U.S. Pat. No. 8,450,298, herein incorporated by reference in its
entirety.
[0339] The liposome formulation may be influenced by, but not
limited to, the selection of the cationic lipid component, the
degree of cationic lipid saturation, the nature of the PEGylation,
ratio of all components and biophysical parameters such as size. In
one example by Semple et al. (Semple et al. Nature Biotech. 2010
28:172-176; herein incorporated by reference in its entirety), the
liposome formulation was composed of 57.1% cationic lipid, 7.1%
dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4%
PEG-c-DMA. As another example, changing the composition of the
cationic lipid could more effectively deliver siRNA to various
antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200;
herein incorporated by reference in its entirety). In some
embodiments, liposome formulations may comprise from about 35 to
about 45% cationic lipid, from about 40% to about 50% cationic
lipid, from about 50% to about 60% cationic lipid and/or from about
55% to about 65% cationic lipid. In some embodiments, the ratio of
lipid to mRNA in liposomes may be from about about 5:1 to about
20:1, from about 10:1 to about 25:1, from about 15:1 to about 30:1
and/or at least 30:1.
[0340] In some embodiments, the ratio of PEG in the lipid
nanoparticle (LNP) formulations may be increased or decreased
and/or the carbon chain length of the PEG lipid may be modified
from C14 to C18 to alter the pharmacokinetics and/or
biodistribution of the LNP formulations. As a non-limiting example,
LNP formulations may contain from about 0.5% to about 3.0%, from
about 1.0% to about 3.5%, from about 1.5% to about 4.0%, from about
2.0% to about 4.5%, from about 2.5% to about 5.0% and/or from about
3.0% to about 6.0% of the lipid molar ratio of PEG-c-DOMG
(R-3-[(.omega.-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristy-
loxypropyl-3-amine) (also referred to herein as PEG-DOMG) as
compared to the cationic lipid, DSPC and cholesterol. In another
embodiment the PEG-c-DOMG may be replaced with a PEG lipid such as,
but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol,
methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol)
and/or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene
glycol). The cationic lipid may be selected from any lipid known in
the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA,
C12-200 and DLin-KC2-DMA.
[0341] In one embodiment, the RNA vaccines may be formulated in a
lipid nanoparticle such as those described in International
Publication No. WO2012170930, the contents of which is herein
incorporated by reference in its entirety.
[0342] In one embodiment, the RNA vaccine formulation comprising
the polynucleotide is a nanoparticle which may comprise at least
one lipid. The lipid may be selected from, but is not limited to,
DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA,
DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol
lipids. In another aspect, the lipid may be a cationic lipid such
as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA,
DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol
cationic lipid may be the lipids described in and/or made by the
methods described in US Patent Publication No. US20130150625,
herein incorporated by reference in its entirety. As a non-limiting
example, the cationic lipid may be
2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-
-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in US20130150625);
2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methy-
l}propan-1-01 (Compound 2 in US20130150625);
2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propa-
n-1-01 (Compound 3 in US20130150625); and
2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-oc-
tadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 4 in
US20130150625); or any pharmaceutically acceptable salt or
stereoisomer thereof.
[0343] Lipid nanoparticle formulations typically comprise a lipid,
in particular, an ionizable cationic lipid, for example,
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and
further comprise a neutral lipid, a sterol and a molecule capable
of reducing particle aggregation, for example a PEG or PEG-modified
lipid.
[0344] In one embodiment, the lipid nanoparticle formulation
consists essentially of (i) at least one lipid selected from the
group consisting of
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a
neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a
sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or
PEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25%
neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid. In one embodiment,
the formulation includes from about 25% to about 75% on a molar
basis of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g.,
from about 35 to about 65%, from about 45 to about 65%, about 60%,
about 57.5%, about 50% or about 40% on a molar basis.
[0345] In one embodiment, the formulation includes from about 0.5%
to about 15% on a molar basis of the neutral lipid e.g., from about
3 to about 12%, from about 5 to about 10% or about 15%, about 10%,
or about 7.5% on a molar basis. Exemplary neutral lipids include,
but are not limited to, DSPC, POPC, DPPC, DOPE and SM. In one
embodiment, the formulation includes from about 5% to about 50% on
a molar basis of the sterol (e.g., about 15 to about 45%, about 20
to about 40%, about 40%, about 38.5%, about 35%, or about 31% on a
molar basis. An exemplary sterol is cholesterol. In one embodiment,
the formulation includes from about 0.5% to about 20% on a molar
basis of the PEG or PEG-modified lipid (e.g., about 0.5 to about
10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%,
about 3.5%, or about 5% on a molar basis. In one embodiment, the
PEG or PEG modified lipid comprises a PEG molecule of an average
molecular weight of 2,000 Da. In other embodiments, the PEG or PEG
modified lipid comprises a PEG molecule of an average molecular
weight of less than 2,000, for example around 1,500 Da, around
1,000 Da, or around 500 Da. Exemplary PEG-modified lipids include,
but are not limited to, PEG-distearoyl glycerol (PEG-DMG) (also
referred herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed
in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the
contents of which are herein incorporated by reference in its
entirety)
[0346] In one embodiment, the formulations of the inventions
include 25-75% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15%
of the neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG
or PEG-modified lipid on a molar basis.
[0347] In one embodiment, the formulations of the inventions
include 35-65% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12%
of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG
or PEG-modified lipid on a molar basis.
[0348] In one embodiment, the formulations of the inventions
include 45-65% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10%
of the neutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG
or PEG-modified lipid on a molar basis.
[0349] In one embodiment, the formulations of the inventions
include about 60% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
7.5% of the neutral lipid, about 31% of the sterol, and about 1.5%
of the PEG or PEG-modified lipid on a molar basis.
[0350] In one embodiment, the formulations of the inventions
include about 50% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
10% of the neutral lipid, about 38.5% of the sterol, and about 1.5%
of the PEG or PEG-modified lipid on a molar basis.
[0351] In one embodiment, the formulations of the inventions
include about 50% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
10% of the neutral lipid, about 35% of the sterol, about 4.5% or
about 5% of the PEG or PEG-modified lipid, and about 0.5% of the
targeting lipid on a molar basis.
[0352] In one embodiment, the formulations of the inventions
include about 40% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
15% of the neutral lipid, about 40% of the sterol, and about 5% of
the PEG or PEG-modified lipid on a molar basis.
[0353] In one embodiment, the formulations of the inventions
include about 57.2% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
7.1% of the neutral lipid, about 34.3% of the sterol, and about
1.4% of the PEG or PEG-modified lipid on a molar basis.
[0354] In one embodiment, the formulations of the inventions
include about 57.5% of a cationic lipid selected from the PEG lipid
is PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. (J.
Controlled Release, 107, 276-287 (2005), the contents of which are
herein incorporated by reference in its entirety), about 7.5% of
the neutral lipid, about 31.5% of the sterol, and about 3.5% of the
PEG or PEG-modified lipid on a molar basis.
[0355] In preferred embodiments, lipid nanoparticle formulation
consists essentially of a lipid mixture in molar ratios of about
20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol:
0.5-15% PEG-modified lipid; more preferably in a molar ratio of
about 20-60% cationic lipid: 5-25% neutral lipid: 25-55%
cholesterol: 0.5-15% PEG-modified lipid.
[0356] In particular embodiments, the molar lipid ratio is
approximately 50/10/38.5/1.5 (mol % cationic lipid/neutral lipid,
e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or
PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid,
e.g., DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5
(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified
lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic
lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,
PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid, e.g.,
DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol %
cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,
e.g., PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic
lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,
PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutral
lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or
PEG-cDMA).
[0357] Exemplary lipid nanoparticle compositions and methods of
making same are described, for example, in Semple et al. (2010)
Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem.
Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy
21, 1570-1578 (the contents of each of which are incorporated
herein by reference in their entirety).
[0358] In one embodiment, the lipid nanoparticle formulations
described herein may comprise a cationic lipid, a PEG lipid and a
structural lipid and optionally comprise a non-cationic lipid. As a
non-limiting example, the lipid nanoparticle may comprise about
40-60% of cationic lipid, about 5-15% of a non-cationic lipid,
about 1-2% of a PEG lipid and about 30-50% of a structural lipid.
As another non-limiting example, the lipid nanoparticle may
comprise about 50% cationic lipid, about 10% non-cationic lipid,
about 1.5% PEG lipid and about 38.5% structural lipid. As yet
another non-limiting example, the lipid nanoparticle may comprise
about 55% cationic lipid, about 10% non-cationic lipid, about 2.5%
PEG lipid and about 32.5% structural lipid. In one embodiment, the
cationic lipid may be any cationic lipid described herein such as,
but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.
[0359] In one embodiment, the lipid nanoparticle formulations
described herein may be 4 component lipid nanoparticles. The lipid
nanoparticle may comprise a cationic lipid, a non-cationic lipid, a
PEG lipid and a structural lipid. As a non-limiting example, the
lipid nanoparticle may comprise about 40-60% of cationic lipid,
about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and
about 30-50% of a structural lipid. As another non-limiting
example, the lipid nanoparticle may comprise about 50% cationic
lipid, about 10% non-cationic lipid, about 1.5% PEG lipid and about
38.5% structural lipid. As yet another non-limiting example, the
lipid nanoparticle may comprise about 55% cationic lipid, about 10%
non-cationic lipid, about 2.5% PEG lipid and about 32.5% structural
lipid. In one embodiment, the cationic lipid may be any cationic
lipid described herein such as, but not limited to, DLin-KC2-DMA,
DLin-MC3-DMA and L319.
[0360] In one embodiment, the lipid nanoparticle formulations
described herein may comprise a cationic lipid, a non-cationic
lipid, a PEG lipid and a structural lipid. As a non-limiting
example, the lipid nanoparticle comprise about 50% of the cationic
lipid DLin-KC2-DMA, about 10% of the non-cationic lipid DSPC, about
1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structural
lipid cholesterol. As a non-limiting example, the lipid
nanoparticle comprise about 50% of the cationic lipid DLin-MC3-DMA,
about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG
lipid PEG-DOMG and about 38.5% of the structural lipid cholesterol.
As a non-limiting example, the lipid nanoparticle comprise about
50% of the cationic lipid DLin-MC3-DMA, about 10% of the
non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and
about 38.5% of the structural lipid cholesterol. As yet another
non-limiting example, the lipid nanoparticle comprise about 55% of
the cationic lipid L319, about 10% of the non-cationic lipid DSPC,
about 2.5% of the PEG lipid PEG-DMG and about 32.5% of the
structural lipid cholesterol.
[0361] In one embodiment, the cationic lipid may be selected from,
but not limited to, a cationic lipid described in International
Publication Nos. WO2012040184, WO2011153120, WO2011149733,
WO2011090965, WO2011043913, WO2011022460, WO2012061259,
WO2012054365, WO2012044638, WO2010080724, WO201021865,
WO2008103276, WO2013086373 and WO2013086354, U.S. Pat. Nos.
7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent
Publication No. US20100036115, US20120202871, US20130064894,
US20130129785, US20130150625, US20130178541 and US20130225836; the
contents of each of which are herein incorporated by reference in
their entirety. In another embodiment, the cationic lipid may be
selected from, but not limited to, formula A described in
International Publication Nos. WO2012040184, WO2011153120,
WO2011149733, WO2011090965, WO2011043913, WO2011022460,
WO2012061259, WO2012054365, WO2012044638 and WO2013116126 or US
Patent Publication No. US20130178541 and US20130225836; the
contents of each of which is herein incorporated by reference in
their entirety. In yet another embodiment, the cationic lipid may
be selected from, but not limited to, formula CLI-CLXXIX of
International Publication No. WO2008103276, formula CLI-CLXXIX of
U.S. Pat. No. 7,893,302, formula CLI-CLXXXXII of U.S. Pat. No.
7,404,969 and formula I-VI of US Patent Publication No.
US20100036115, formula I of US Patent Publication No US20130123338;
each of which is herein incorporated by reference in their
entirety. As a non-limiting example, the cationic lipid may be
selected from (20Z,23Z)--N,N-dimethylnonacosa-20,23-dien-10-amine,
(17Z,20Z)--N,N-dimemylhexacosa-17,20-dien-9-amine,
(1Z,19Z)--N5N-dimethylpentacosa-1 6, 19-dien-8-amine,
(13Z,16Z)--N,N-dimethyldocosa-13,16-dien-5-amine,
(12Z,15Z)--N,N-dimethylhenicosa-12,15-dien-4-amine,
(14Z,17Z)--N,N-dimethyltricosa-14,17-dien-6-amine,
(15Z,18Z)--N,N-dimethyltetracosa-15,18-dien-7-amine,
(18Z,21Z)--N,N-dimethylheptacosa-18,21-dien-10-amine,
(15Z,18Z)--N,N-dimethyltetracosa-15,18-dien-5-amine,
(14Z,17Z)--N,N-dimethyltricosa-14,17-dien-4-amine,
(19Z,22Z)--N,N-dimeihyloctaco sa-19,22-dien-9-amine, (18Z,21
Z)--N,N-dimethylheptacosa-18,21-dien-8-amine,
(17Z,20Z)--N,N-dimethylhexacosa-17,20-dien-7-amine,
(16Z,19Z)--N,N-dimethylpentacosa-16,19-dien-6-amine,
(22Z,25Z)--N,N-dimethylhentriaconta-22,25-dien-10-amine, (21
Z,24Z)--N,N-dimethyltriaconta-21,24-dien-9-amine,
(18Z)--N,N-dimetylheptacos-18-en-10-amine,
(17Z)--N,N-dimethylhexacos-17-en-9-amine,
(19Z,22Z)--N,N-dimethyloctacosa-19,22-dien-7-amine,
N,N-dimethylheptacosan-10-amine,
(20Z,23Z)--N-ethyl-N-methylnonacosa-20,23-dien-10-amine,
1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,
(20Z)--N,N-dimethylheptacos-20-en-10-amine, (15Z)--N,N-dimethyl
eptacos-15-en-10-amine, (14Z)--N,N-dimethylnonacos-14-en-10-amine,
(17Z)--N,N-dimethylnonacos-17-en-10-amine,
(24Z)--N,N-dimethyltritriacont-24-en-10-amine,
(20Z)--N,N-dimethylnonacos-20-en-10-amine,
(22Z)--N,N-dimethylhentriacont-22-en-10-amine,
(16Z)--N,N-dimethylpentacos-16-en-8-amine,
(12Z,15Z)--N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,
(13Z,16Z)--N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine,
1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,
N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimeth-
yl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcycIopropyl]methyl}cyclopropyl]nonadecan-
-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,
N,N-dimethyl-[(1R,2S)-2-undecyIcyclopropyl]tetradecan-5-amine,
N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,
1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,
1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,
R--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propa-
n-2-amine,
S--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octy-
loxy)propan-2-amine,
1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrr-
olidine,
(2S)--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-
)-oct-5-en-1-yloxy]propan-2-amine,
1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azet-
idine,
(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-ylo-
xy]propan-2-amine,
(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]pr-
opan-2-amine,
N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-
-amine,
N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-am-
ine;
(2S)--N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(o-
ctyloxy)propan-2-amine,
(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)pro-
pan-2-amine,
(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylprop-
an-2-amine,
1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2--
amine,
1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)pr-
opan-2-amine,
(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpro-
pan-2-amine,
(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amin-
e,
1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,
1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,
(2R)--N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1--
yloxy]propan-2-amine,
(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-di-
en-1-yloxy]propan-2-amine,
N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-
methyl}cyclopropyl]octyl}oxy)propan-2-amine,
N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-am-
ine and (11E,20Z,23Z)--N,N-dimethylnonacosa-11,20,2-trien-10-amine
or a pharmaceutically acceptable salt or stereoisomer thereof.
[0362] In one embodiment, the lipid may be a cleavable lipid such
as those described in International Publication No. WO2012170889,
herein incorporated by reference in its entirety.
[0363] In another embodiment, the lipid may be a cationic lipid
such as, but not limited to, Formula (I) of U.S. Patent Application
No. US20130064894, the contents of which are herein incorporated by
reference in its entirety.
[0364] In one embodiment, the cationic lipid may be synthesized by
methods known in the art and/or as described in International
Publication Nos. WO2012040184, WO2011153120, WO2011149733,
WO2011090965, WO2011043913, WO2011022460, WO2012061259,
WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2013086373
and WO2013086354; the contents of each of which are herein
incorporated by reference in their entirety.
[0365] In another embodiment, the cationic lipid may be a trialkyl
cationic lipid. Non-limiting examples of trialkyl cationic lipids
and methods of making and using the trialkyl cationic lipids are
described in International Patent Publication No. WO2013126803, the
contents of which are herein incorporated by reference in its
entirety.
[0366] In one embodiment, the LNP formulations of the RNA vaccines
may contain PEG-c-DOMG at 3% lipid molar ratio. In another
embodiment, the LNP formulations of the RNA vaccines may contain
PEG-c-DOMG at 1.5% lipid molar ratio.
[0367] In one embodiment, the pharmaceutical compositions of the
RNA vaccines may include at least one of the PEGylated lipids
described in International Publication No. WO2012099755, the
contents of which is herein incorporated by reference in its
entirety.
[0368] In one embodiment, the LNP formulation may contain PEG-DMG
2000
(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene
glycol)-2000). In one embodiment, the LNP formulation may contain
PEG-DMG 2000, a cationic lipid known in the art and at least one
other component. In another embodiment, the LNP formulation may
contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and
cholesterol. As a non-limiting example, the LNP formulation may
contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol. As another
non-limiting example the LNP formulation may contain PEG-DMG 2000,
DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see
e.g., Geall et al., Nonviral delivery of self-amplifying RNA
vaccines, PNAS 2012; PMID: 22908294; herein incorporated by
reference in its entirety).
[0369] In one embodiment, the LNP formulation may be formulated by
the methods described in International Publication Nos.
WO2011127255 or WO2008103276, the contents of each of which is
herein incorporated by reference in their entirety. As a
non-limiting example, the RNA vaccines described herein may be
encapsulated in LNP formulations as described in WO2011127255
and/or WO2008103276; each of which is herein incorporated by
reference in their entirety.
[0370] In one embodiment, the RNA vaccines described herein may be
formulated in a nanoparticle to be delivered by a parenteral route
as described in U.S. Pub. No. US20120207845; the contents of which
are herein incorporated by reference in its entirety.
[0371] In one embodiment, the RNA vaccines may be formulated in a
lipid nanoparticle made by the methods described in US Patent
Publication No US20130156845 or International Publication No
WO2013093648 or WO2012024526, each of which is herein incorporated
by reference in its entirety.
[0372] The lipid nanoparticles described herein may be made in a
sterile environment by the system and/or methods described in US
Patent Publication No. US20130164400, herein incorporated by
reference in its entirety.
[0373] In one embodiment, the LNP formulation may be formulated in
a nanoparticle such as a nucleic acid-lipid particle described in
U.S. Pat. No. 8,492,359, the contents of which are herein
incorporated by reference in its entirety. As a non-limiting
example, the lipid particle may comprise one or more active agents
or therapeutic agents; one or more cationic lipids comprising from
about 50 mol % to about 85 mol % of the total lipid present in the
particle; one or more non-cationic lipids comprising from about 13
mol % to about 49.5 mol % of the total lipid present in the
particle; and one or more conjugated lipids that inhibit
aggregation of particles comprising from about 0.5 mol % to about 2
mol % of the total lipid present in the particle. The nucleic acid
in the nanoparticle may be the polynucleotides described herein
and/or are known in the art.
[0374] In one embodiment, the LNP formulation may be formulated by
the methods described in International Publication Nos.
WO2011127255 or WO2008103276, the contents of each of which are
herein incorporated by reference in their entirety. As a
non-limiting example, modified RNA described herein may be
encapsulated in LNP formulations as described in WO2011127255
and/or WO2008103276; the contents of each of which are herein
incorporated by reference in their entirety.
[0375] In one embodiment, LNP formulations described herein may
comprise a polycationic composition. As a non-limiting example, the
polycationic composition may be selected from formula 1-60 of US
Patent Publication No. US20050222064; the content of which is
herein incorporated by reference in its entirety. In another
embodiment, the LNP formulations comprising a polycationic
composition may be used for the delivery of the modified RNA
described herein in vivo and/or in vitro.
[0376] In one embodiment, the LNP formulations described herein may
additionally comprise a permeability enhancer molecule.
Non-limiting permeability enhancer molecules are described in US
Patent Publication No. US20050222064; the content of which is
herein incorporated by reference in its entirety.
[0377] In one embodiment, the RNA vaccine pharmaceutical
compositions may be formulated in liposomes such as, but not
limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.),
SMARTICLES.RTM. (Marina Biotech, Bothell, Wash.), neutral DOPC
(1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,
siRNA delivery for ovarian cancer (Landen et al. Cancer Biology
& Therapy 2006 5(12)1708-1713); herein incorporated by
reference in its entirety) and hyaluronan-coated liposomes (Quiet
Therapeutics, Israel).
[0378] In one embodiment, the RNA vaccines may be formulated in a
lyophilized gel-phase liposomal composition as described in US
Publication No. US2012060293, herein incorporated by reference in
its entirety.
[0379] In some embodiments the RNA vaccine may be associated with a
cationic or polycationic compounds, including protamine,
nucleoline, spermine or spermidine, or other cationic peptides or
proteins, such as poly-L-lysine (PLL), polyarginine, basic
polypeptides, cell penetrating peptides (CPPs), including
HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides,
Penetratin, VP22 derived or analog peptides, Pestivirus Ems, HSV,
VP22 (Herpes simplex), MAP, KALA or protein transduction domains
(PTDs), PpT620, prolin-rich peptides, arginine-rich peptides,
lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers,
Calcitonin peptide(s), Antennapedia-derived peptides (particularly
from Drosophila antennapedia), pAntp, pIs1, FGF, Lactoferrin,
Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived
peptides, SAP, histones, cationic polysaccharides, for example
chitosan, polybrene, cationic polymers, e.g. polyethyleneimine
(PEI), cationic lipids, e.g. DOTMA:
[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride,
DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC,
DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC,
DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI:
Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP:
dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:
O,O-ditetradecanoyl-N-.alpha.-trimethylammonioacetyl)diethanolamine
chloride, CLIP 1:
rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium
chloride, CLIP6:
rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium,
CLIPS:
rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammo-
-nium, oligofectamine, or cationic or polycationic polymers, e.g.
modified polyaminoacids, such as beta-aminoacid-polymers or
reversed polyamides, etc., modified polyethylenes, such as PVP
(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified
acrylates, such as pDMAEMA (poly(dimethylaminoethyl
methylacrylate)), etc., modified amidoamines such as pAMAM
(poly(amidoamine)), etc., modified polybetaminoester (PBAE), such
as diamine end modified 1,4 butanediol
diacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such
as polypropylamine dendrimers or pAMAM based dendrimers, etc.,
polyimine(s), such as PEI: poly(ethyleneimine),
poly(propyleneimine), etc., polyallylamine, sugar backbone based
polymers, such as cyclodextrin based polymers, dextran based
polymers, chitosan, etc., silan backbone based polymers, such as
PMOXA-PDMS copolymers, etc., blockpolymers consisting of a
combination of one or more cationic blocks (e.g. selected from a
cationic polymer as mentioned above) and of one or more hydrophilic
or hydrophobic blocks (e.g. polyethyleneglycole); etc.
[0380] In other embodiments the RNA vaccine is not associated with
a cationic or polycationic compounds.
[0381] The nanoparticle formulations may comprise a phosphate
conjugate. The phosphate conjugate may increase in vivo circulation
times and/or increase the targeted delivery of the nanoparticle.
Phosphate conjugates for use with the present invention may be made
by the methods described in International Application No.
WO2013033438 or US Patent Publication No. US20130196948, the
contents of each of which are herein incorporated by reference in
its entirety. As a non-limiting example, the phosphate conjugates
may include a compound of any one of the formulas described in
International Application No. WO2013033438, herein incorporated by
reference in its entirety.
[0382] The nanoparticle formulation may comprise a polymer
conjugate. The polymer conjugate may be a water soluble conjugate.
The polymer conjugate may have a structure as described in U.S.
Patent Application No. 20130059360, the contents of which are
herein incorporated by reference in its entirety. In one aspect,
polymer conjugates with the polynucleotides of the present
invention may be made using the methods and/or segmented polymeric
reagents described in U.S. Patent Application No. 20130072709,
herein incorporated by reference in its entirety. In another
aspect, the polymer conjugate may have pendant side groups
comprising ring moieties such as, but not limited to, the polymer
conjugates described in US Patent Publication No. US20130196948,
the contents of which is herein incorporated by reference in its
entirety.
[0383] The nanoparticle formulations may comprise a conjugate to
enhance the delivery of nanoparticles of the present invention in a
subject. Further, the conjugate may inhibit phagocytic clearance of
the nanoparticles in a subject. In one aspect, the conjugate may be
a "self" peptide designed from the human membrane protein CD47
(e.g., the "self" particles described by Rodriguez et al (Science
2013 339, 971-975), herein incorporated by reference in its
entirety). As shown by Rodriguez et al. the self peptides delayed
macrophage-mediated clearance of nanoparticles which enhanced
delivery of the nanoparticles. In another aspect, the conjugate may
be the membrane protein CD47 (e.g., see Rodriguez et al. Science
2013 339, 971-975, herein incorporated by reference in its
entirety). Rodriguez et al. showed that, similarly to "self"
peptides, CD47 can increase the circulating particle ratio in a
subject as compared to scrambled peptides and PEG coated
nanoparticles.
[0384] In one embodiment, the RNA vaccines of the present invention
are formulated in nanoparticles which comprise a conjugate to
enhance the delivery of the nanoparticles of the present invention
in a subject. The conjugate may be the CD47 membrane or the
conjugate may be derived from the CD47 membrane protein, such as
the "self" peptide described previously. In another aspect the
nanoparticle may comprise PEG and a conjugate of CD47 or a
derivative thereof. In yet another aspect, the nanoparticle may
comprise both the "self" peptide described above and the membrane
protein CD47.
[0385] In another aspect, a "self" peptide and/or CD47 protein may
be conjugated to a virus-like particle or pseudovirion, as
described herein for delivery of the RNA vaccines of the present
invention.
[0386] In another embodiment, RNA vaccine pharmaceutical
compositions comprising the polynucleotides of the present
invention and a conjugate which may have a degradable linkage.
Non-limiting examples of conjugates include an aromatic moiety
comprising an ionizable hydrogen atom, a spacer moiety, and a
water-soluble polymer. As a non-limiting example, pharmaceutical
compositions comprising a conjugate with a degradable linkage and
methods for delivering such pharmaceutical compositions are
described in US Patent Publication No. US20130184443, the contents
of which are herein incorporated by reference in its entirety.
[0387] The nanoparticle formulations may be a carbohydrate
nanoparticle comprising a carbohydrate carrier and a RNA vaccine.
As a non-limiting example, the carbohydrate carrier may include,
but is not limited to, an anhydride-modified phytoglycogen or
glycogen-type material, phtoglycogen octenyl succinate,
phytoglycogen beta-dextrin, anhydride-modified phytoglycogen
beta-dextrin. (See e.g., International Publication No.
WO2012109121; the contents of which are herein incorporated by
reference in its entirety).
[0388] Nanoparticle formulations of the present invention may be
coated with a surfactant or polymer in order to improve the
delivery of the particle. In one embodiment, the nanoparticle may
be coated with a hydrophilic coating such as, but not limited to,
PEG coatings and/or coatings that have a neutral surface charge.
The hydrophilic coatings may help to deliver nanoparticles with
larger payloads such as, but not limited to, RNA vaccines within
the central nervous system. As a non-limiting example nanoparticles
comprising a hydrophilic coating and methods of making such
nanoparticles are described in US Patent Publication No.
US20130183244, the contents of which are herein incorporated by
reference in its entirety.
[0389] In one embodiment, the lipid nanoparticles of the present
invention may be hydrophilic polymer particles. Non-limiting
examples of hydrophilic polymer particles and methods of making
hydrophilic polymer particles are described in US Patent
Publication No. US20130210991, the contents of which are herein
incorporated by reference in its entirety.
[0390] In another embodiment, the lipid nanoparticles of the
present invention may be hydrophobic polymer particles.
[0391] Lipid nanoparticle formulations may be improved by replacing
the cationic lipid with a biodegradable cationic lipid which is
known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable
cationic lipids, such as, but not limited to, DLinDMA,
DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in
plasma and tissues over time and may be a potential source of
toxicity. The rapid metabolism of the rapidly eliminated lipids can
improve the tolerability and therapeutic index of the lipid
nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10
mg/kg dose in rat. Inclusion of an enzymatically degraded ester
linkage can improve the degradation and metabolism profile of the
cationic component, while still maintaining the activity of the
reLNP formulation. The ester linkage can be internally located
within the lipid chain or it may be terminally located at the
terminal end of the lipid chain. The internal ester linkage may
replace any carbon in the lipid chain.
[0392] In one embodiment, the internal ester linkage may be located
on either side of the saturated carbon.
[0393] In one embodiment, an immune response may be elicited by
delivering a lipid nanoparticle which may include a nanospecies, a
polymer and an immunogen. (U.S. Publication No. 20120189700 and
International Publication No. WO2012099805; each of which is herein
incorporated by reference in their entirety). The polymer may
encapsulate the nanospecies or partially encapsulate the
nanospecies. The immunogen may be a recombinant protein, a modified
RNA and/or a polynucleotide described herein. In one embodiment,
the lipid nanoparticle may be formulated for use in a vaccine such
as, but not limited to, against a pathogen.
[0394] Lipid nanoparticles may be engineered to alter the surface
properties of particles so the lipid nanoparticles may penetrate
the mucosal barrier. Mucus is located on mucosal tissue such as,
but not limited to, oral (e.g., the buccal and esophageal membranes
and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach,
small intestine, large intestine, colon, rectum), nasal,
respiratory (e.g., nasal, pharyngeal, tracheal and bronchial
membranes), genital (e.g., vaginal, cervical and urethral
membranes). Nanoparticles larger than 10-200 nm which are preferred
for higher drug encapsulation efficiency and the ability to provide
the sustained delivery of a wide array of drugs have been thought
to be too large to rapidly diffuse through mucosal barriers. Mucus
is continuously secreted, shed, discarded or digested and recycled
so most of the trapped particles may be removed from the mucosla
tissue within seconds or within a few hours. Large polymeric
nanoparticles (200 nm-500 nm in diameter) which have been coated
densely with a low molecular weight polyethylene glycol (PEG)
diffused through mucus only 4 to 6-fold lower than the same
particles diffusing in water (Lai et al. PNAS 2007 104(5):1482-487;
Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; each of which
is herein incorporated by reference in their entirety). The
transport of nanoparticles may be determined using rates of
permeation and/or fluorescent microscopy techniques including, but
not limited to, fluorescence recovery after photobleaching (FRAP)
and high resolution multiple particle tracking (MPT). As a
non-limiting example, compositions which can penetrate a mucosal
barrier may be made as described in U.S. Pat. No. 8,241,670 or
International Patent Publication No. WO2013110028, the contents of
each of which are herein incorporated by reference in its
entirety.
[0395] The lipid nanoparticle engineered to penetrate mucus may
comprise a polymeric material (i.e. a polymeric core) and/or a
polymer-vitamin conjugate and/or a tri-block co-polymer. The
polymeric material may include, but is not limited to, polyamines,
polyethers, polyamides, polyesters, polycarbamates, polyureas,
polycarbonates, poly(styrenes), polyimides, polysulfones,
polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines,
polyisocyanates, polyacrylates, polymethacrylates,
polyacrylonitriles, and polyarylates. The polymeric material may be
biodegradable and/or biocompatible. Non-limiting examples of
biocompatible polymers are described in International Patent
Publication No. WO2013116804, the contents of which are herein
incorporated by reference in its entirety. The polymeric material
may additionally be irradiated. As a non-limiting example, the
polymeric material may be gamma irradiated (See e.g., International
App. No. WO201282165, herein incorporated by reference in its
entirety). Non-limiting examples of specific polymers include
poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA),
poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic
acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA),
poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)
(PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),
poly(D,L-lactide-co-caprolactone-co-glycolide),
poly(D,L-lactide-co-PEO-co-D,L-lactide),
poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,
polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate
(HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy
acids), polyanhydrides, polyorthoesters, poly(ester amides),
polyamides, poly(ester ethers), polycarbonates, polyalkylenes such
as polyethylene and polypropylene, polyalkylene glycols such as
poly(ethylene glycol) (PEG), polyalkylene oxides (PEO),
polyalkylene terephthalates such as poly(ethylene terephthalate),
polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such
as poly(vinyl acetate), polyvinyl halides such as poly(vinyl
chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene
(PS), polyurethanes, derivatized celluloses such as alkyl
celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, hydroxypropylcellulose,
carboxymethylcellulose, polymers of acrylic acids, such as
poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate),
poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate),
poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate),
poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl
acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),
poly(octadecyl acrylate) and copolymers and mixtures thereof,
polydioxanone and its copolymers, polyhydroxyalkanoates,
polypropylene fumarate, polyoxymethylene, poloxamers,
poly(ortho)esters, poly(butyric acid), poly(valeric acid),
poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene
carbonate, polyvinylpyrrolidone. The lipid nanoparticle may be
coated or associated with a co-polymer such as, but not limited to,
a block co-polymer (such as a branched polyether-polyamide block
copolymer described in International Publication No. WO2013012476,
herein incorporated by reference in its entirety), and
(poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene
glycol)) triblock copolymer (see e.g., US Publication 20120121718
and US Publication 20100003337 and U.S. Pat. No. 8,263,665; each of
which is herein incorporated by reference in their entirety). The
co-polymer may be a polymer that is generally regarded as safe
(GRAS) and the formation of the lipid nanoparticle may be in such a
way that no new chemical entities are created. For example, the
lipid nanoparticle may comprise poloxamers coating PLGA
nanoparticles without forming new chemical entities which are still
able to rapidly penetrate human mucus (Yang et al. Angew. Chem.
Int. Ed. 2011 50:2597-2600; the contents of which are herein
incorporated by reference in its entirety). A non-limiting scalable
method to produce nanoparticles which can penetrate human mucus is
described by Xu et al. (See e.g., J Control Release 2013,
170(2):279-86; the contents of which are herein incorporated by
reference in its entirety).
[0396] The vitamin of the polymer-vitamin conjugate may be vitamin
E. The vitamin portion of the conjugate may be substituted with
other suitable components such as, but not limited to, vitamin A,
vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a
hydrophobic component of other surfactants (e.g., sterol chains,
fatty acids, hydrocarbon chains and alkylene oxide chains).
[0397] The lipid nanoparticle engineered to penetrate mucus may
include surface altering agents such as, but not limited to,
polynucleotides, anionic proteins (e.g., bovine serum albumin),
surfactants (e.g., cationic surfactants such as for example
dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives
(e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin,
polyethylene glycol and poloxamer), mucolytic agents (e.g.,
N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,
acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna,
ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,
gelsolin, thymosin .beta.4 dornase alfa, neltenexine, erdosteine)
and various DNases including rhDNase. The surface altering agent
may be embedded or enmeshed in the particle's surface or disposed
(e.g., by coating, adsorption, covalent linkage, or other process)
on the surface of the lipid nanoparticle. (see e.g., US Publication
20100215580 and US Publication 20080166414 and US20130164343; the
contents of each of which is herein incorporated by reference in
their entirety).
[0398] In one embodiment, the mucus penetrating lipid nanoparticles
may comprise at least one polynucleotide described herein. The
polynucleotide may be encapsulated in the lipid nanoparticle and/or
disposed on the surface of the particle. The polynucleotide may be
covalently coupled to the lipid nanoparticle. Formulations of mucus
penetrating lipid nanoparticles may comprise a plurality of
nanoparticles. Further, the formulations may contain particles
which may interact with the mucus and alter the structural and/or
adhesive properties of the surrounding mucus to decrease
mucoadhesion which may increase the delivery of the mucus
penetrating lipid nanoparticles to the mucosal tissue.
[0399] In another embodiment, the mucus penetrating lipid
nanoparticles may be a hypotonic formulation comprising a mucosal
penetration enhancing coating. The formulation may be hypotonice
for the epithelium to which it is being delivered. Non-limiting
examples of hypotonic formulations may be found in International
Patent Publication No. WO2013110028, the contents of which are
herein incorporated by reference in its entirety.
[0400] In one embodiment, in order to enhance the delivery through
the mucosal barrier the RNA vaccine formulation may comprise or be
a hypotonic solution. Hypotonic solutions were found to increase
the rate at which mucoinert particles such as, but not limited to,
mucus-penetrating particles, were able to reach the vaginal
epithelial surface (See e.g., Ensign et al. Biomaterials 2013
34(28):6922-9; the contents of which is herein incorporated by
reference in its entirety).
[0401] In one embodiment, the RNA vaccine is formulated as a
lipoplex, such as, without limitation, the ATUPLEX.TM. system, the
DACC system, the DBTC system and other siRNA-lipoplex technology
from Silence Therapeutics (London, United Kingdom), STEMFECT.TM.
from STEMGENT.RTM. (Cambridge, Mass.), and polyethylenimine (PEI)
or protamine-based targeted and non-targeted delivery of nucleic
acids acids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg
et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene
Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370;
Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et
al. Microvasc Res 2010 80:286-293Weide et al. J Immunother. 2009
32:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo
Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J.
Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005,
23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6;
104:4095-4100; deFougerolles Hum Gene Ther. 2008 19:125-132; all of
which are incorporated herein by reference in its entirety).
[0402] In one embodiment such formulations may also be constructed
or compositions altered such that they passively or actively are
directed to different cell types in vivo, including but not limited
to hepatocytes, immune cells, tumor cells, endothelial cells,
antigen presenting cells, and leukocytes (Akinc et al. Mol Ther.
2010 18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717;
Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al.,
Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006
13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier
et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol.
Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv.
2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and
Lieberman, Gene Ther. 2011 18:1127-1133; all of which are
incorporated herein by reference in its entirety). One example of
passive targeting of formulations to liver cells includes the
DLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticle
formulations which have been shown to bind to apolipoprotein E and
promote binding and uptake of these formulations into hepatocytes
in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein
incorporated by reference in its entirety). Formulations can also
be selectively targeted through expression of different ligands on
their surface as exemplified by, but not limited by, folate,
transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted
approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011
8:197-206; Musacchio and Torchilin, Front Biosci. 2011
16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et
al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al.,
Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug
Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364;
Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et
al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control
Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007
104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353;
Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat
Biotechnol. 2005 23:709-717; Peer et al., Science. 2008
319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all
of which are incorporated herein by reference in its entirety).
[0403] In one embodiment, the RNA vaccine is formulated as a solid
lipid nanoparticle. A solid lipid nanoparticle (SLN) may be
spherical with an average diameter between 10 to 1000 nm. SLN
possess a solid lipid core matrix that can solubilize lipophilic
molecules and may be stabilized with surfactants and/or
emulsifiers. In a further embodiment, the lipid nanoparticle may be
a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS
Nano, 2008, 2 (8), pp 1696-1702; the contents of which are herein
incorporated by reference in its entirety). As a non-limiting
example, the SLN may be the SLN described in International Patent
Publication No. WO2013105101, the contents of which are herein
incorporated by reference in its entirety. As another non-limiting
example, the SLN may be made by the methods or processes described
in International Patent Publication No. WO2013105101, the contents
of which are herein incorporated by reference in its entirety.
[0404] Liposomes, lipoplexes, or lipid nanoparticles may be used to
improve the efficacy of polynucleotides directed protein production
as these formulations may be able to increase cell transfection by
the RNA vaccine; and/or increase the translation of encoded
protein. One such example involves the use of lipid encapsulation
to enable the effective systemic delivery of polyplex plasmid DNA
(Heyes et al., Mol Ther. 2007 15:713-720; herein incorporated by
reference in its entirety). The liposomes, lipoplexes, or lipid
nanoparticles may also be used to increase the stability of the
polynucleotide.
[0405] In one embodiment, the RNA vaccines of the present invention
can be formulated for controlled release and/or targeted delivery.
As used herein, "controlled release" refers to a pharmaceutical
composition or compound release profile that conforms to a
particular pattern of release to effect a therapeutic outcome. In
one embodiment, the RRNA vaccines may be encapsulated into a
delivery agent described herein and/or known in the art for
controlled release and/or targeted delivery. As used herein, the
term "encapsulate" means to enclose, surround or encase. As it
relates to the formulation of the compounds of the invention,
encapsulation may be substantial, complete or partial. The term
"substantially encapsulated" means that at least greater than 50,
60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than
99.999% of the pharmaceutical composition or compound of the
invention may be enclosed, surrounded or encased within the
delivery agent. "Partially encapsulated" means that less than 10,
10, 20, 30, 40 50 or less of the pharmaceutical composition or
compound of the invention may be enclosed, surrounded or encased
within the delivery agent. Advantageously, encapsulation may be
determined by measuring the escape or the activity of the
pharmaceutical composition or compound of the invention using
fluorescence and/or electron micrograph. For example, at least 1,
5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99,
99.9, 99.99 or greater than 99.99% of the pharmaceutical
composition or compound of the invention are encapsulated in the
delivery agent.
[0406] In one embodiment, the controlled release formulation may
include, but is not limited to, tri-block co-polymers. As a
non-limiting example, the formulation may include two different
types of tri-block co-polymers (International Pub. No. WO2012131104
and WO2012131106; the contents of each of which is herein
incorporated by reference in its entirety).
[0407] In another embodiment, the RNA vaccines may be encapsulated
into a lipid nanoparticle or a rapidly eliminated lipid
nanoparticle and the lipid nanoparticles or a rapidly eliminated
lipid nanoparticle may then be encapsulated into a polymer,
hydrogel and/or surgical sealant described herein and/or known in
the art. As a non-limiting example, the polymer, hydrogel or
surgical sealant may be PLGA, ethylene vinyl acetate (EVAc),
poloxamer, GELSITE.RTM. (Nanotherapeutics, Inc. Alachua, Fla.),
HYLENEX.RTM. (Halozyme Therapeutics, San Diego Calif.), surgical
sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.),
TISSELL.RTM. (Baxter International, Inc Deerfield, Ill.), PEG-based
sealants, and COSEAL.RTM. (Baxter International, Inc Deerfield,
Ill.).
[0408] In another embodiment, the lipid nanoparticle may be
encapsulated into any polymer known in the art which may form a gel
when injected into a subject. As another non-limiting example, the
lipid nanoparticle may be encapsulated into a polymer matrix which
may be biodegradable.
[0409] In one embodiment, the the RNA vaccine formulation for
controlled release and/or targeted delivery may also include at
least one controlled release coating. Controlled release coatings
include, but are not limited to, OPADRY.RTM.,
polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,
hydroxypropyl methylcellulose, hydroxypropyl cellulose,
hydroxyethyl cellulose, EUDRAGIT RL.RTM., EUDRAGIT RS.RTM. and
cellulose derivatives such as ethylcellulose aqueous dispersions
(AQUACOAT.RTM. and SURELEASE.RTM.).
[0410] In one embodiment, the RNA vaccine controlled release and/or
targeted delivery formulation may comprise at least one degradable
polyester which may contain polycationic side chains. Degradeable
polyesters include, but are not limited to, poly(serine ester),
poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and
combinations thereof. In another embodiment, the degradable
polyesters may include a PEG conjugation to form a PEGylated
polymer.
[0411] In one embodiment, the RNA vaccine controlled release and/or
targeted delivery formulation comprising at least one
polynucleotide may comprise at least one PEG and/or PEG related
polymer derivatives as described in U.S. Pat. No. 8,404,222, herein
incorporated by reference in its entirety.
[0412] In another embodiment, the RNA vaccine controlled release
delivery formulation comprising at least one polynucleotide may be
the controlled release polymer system described in US20130130348,
herein incorporated by reference in its entirety.
[0413] In one embodiment, the the RNA vaccines of the present
invention may be encapsulated in a therapeutic nanoparticle,
referred to herein as "therapeutic nanoparticle RRNA vaccines."
Therapeutic nanoparticles may be formulated by methods described
herein and known in the art such as, but not limited to,
International Pub Nos. WO2010005740, WO2010030763, WO2010005721,
WO2010005723, WO2012054923, US Pub. Nos. US20110262491,
US20100104645, US20100087337, US20100068285, US20110274759,
US20100068286, US20120288541, US20130123351 and US20130230567 and
U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and 8,318,211; the
contents of each of which are herein incorporated by reference in
their entirety. In another embodiment, therapeutic polymer
nanoparticles may be identified by the methods described in US Pub
No. US20120140790, the contents of which is herein incorporated by
reference in its entirety.
[0414] In one embodiment, the therapeutic nanoparticle RNA vaccine
may be formulated for sustained release. As used herein, "sustained
release" refers to a pharmaceutical composition or compound that
conforms to a release rate over a specific period of time. The
period of time may include, but is not limited to, hours, days,
weeks, months and years. As a non-limiting example, the sustained
release nanoparticle may comprise a polymer and a therapeutic agent
such as, but not limited to, the the polynucleotides of the present
invention (see International Pub No. 2010075072 and US Pub No.
US20100216804, US20110217377 and US20120201859, each of which is
herein incorporated by reference in their entirety). In another
non-limiting example, the sustained release formulation may
comprise agents which permit persistent bioavailability such as,
but not limited to, crystals, macromolecular gels and/or
particulate suspensions (see US Patent Publication No
US20130150295, the contents of which is herein incorporated by
reference in its entirety).
[0415] In one embodiment, the therapeutic nanoparticle RNA vaccines
may be formulated to be target specific. As a non-limiting example,
the thereapeutic nanoparticles may include a corticosteroid (see
International Pub. No. WO2011084518; herein incorporated by
reference in its entirety). As a non-limiting example, the
therapeutic nanoparticles may be formulated in nanoparticles
described in International Pub No. WO2008121949, WO2010005726,
WO2010005725, WO2011084521 and US Pub No. US20100069426,
US20120004293 and US20100104655, each of which is herein
incorporated by reference in their entirety.
[0416] In one embodiment, the nanoparticles of the present
invention may comprise a polymeric matrix. As a non-limiting
example, the nanoparticle may comprise two or more polymers such
as, but not limited to, polyethylenes, polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates,
polycaprolactones, polyamides, polyacetals, polyethers, polyesters,
poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,
polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines,
polylysine, poly(ethylene imine), poly(serine ester),
poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or
combinations thereof.
[0417] In one embodiment, the therapeutic nanoparticle comprises a
diblock copolymer. In one embodiment, the diblock copolymer may
include PEG in combination with a polymer such as, but not limited
to, polyethylenes, polycarbonates, polyanhydrides,
polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides, polyacetals, polyethers, polyesters, poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines,
polylysine, poly(ethylene imine), poly(serine ester),
poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or
combinations thereof. In another embodiment, the diblock copolymer
may comprise the diblock copolymers described in European Patent
Publication No. the contents of which are herein incorporated by
reference in its entirety. In yet another embodiment, the diblock
copolymer may be a high-X diblock copolymer such as those described
in International Patent Publication No. WO2013120052, the contents
of which are herein incorporated by reference in its entirety.
[0418] As a non-limiting example the therapeutic nanoparticle
comprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293
and U.S. Pat. No. 8,236,330, each of which is herein incorporated
by reference in their entirety). In another non-limiting example,
the therapeutic nanoparticle is a stealth nanoparticle comprising a
diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No.
8,246,968 and International Publication No. WO2012166923, the
contents of each of which are herein incorporated by reference in
its entirety). In yet another non-limiting example, the therapeutic
nanoparticle is a stealth nanoparticle or a target-specific stealth
nanoparticle as described in US Patent Publication No.
US20130172406, the contents of which are herein incorporated by
reference in its entirety.
[0419] In one embodiment, the therapeutic nanoparticle may comprise
a multiblock copolymer (See e.g., U.S. Pat. Nos. 8,263,665 and
8,287,910 and US Patent Pub. No. US20130195987; the contents of
each of which are herein incorporated by reference in its
entirety).
[0420] In yet another non-limiting example, the lipid nanoparticle
comprises the block copolymer PEG-PLGA-PEG (see e.g., the
thermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF-beta1
gene delivery vehicle in Lee et al. Thermosensitive Hydrogel as a
Tgf-.beta.1 Gene Delivery Vehicle Enhances Diabetic Wound Healing.
Pharmaceutical Research, 2003 20(12): 1995-2000; as a controlled
gene delivery system in Li et al. Controlled Gene Delivery System
Based on Thermosensitive Biodegradable Hydrogel. Pharmaceutical
Research 2003 20(6):884-888; and Chang et al., Non-ionic
amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene
delivery efficiency in rat skeletal muscle. J Controlled Release.
2007 118:245-253; each of which is herein incorporated by reference
in its entirety). The RNA vaccines of the present invention may be
formulated in lipid nanoparticles comprising the PEG-PLGA-PEG block
copolymer.
[0421] In one embodiment, the therapeutic nanoparticle may comprise
a multiblock copolymer (See e.g., U.S. Pat. Nos. 8,263,665 and
8,287,910 and US Patent Pub. No. US20130195987; the contents of
each of which are herein incorporated by reference in its
entirety).
[0422] In one embodiment, the block copolymers described herein may
be included in a polyion complex comprising a non-polymeric micelle
and the block copolymer. (See e.g., U.S. Pub. No. 20120076836;
herein incorporated by reference in its entirety).
[0423] In one embodiment, the therapeutic nanoparticle may comprise
at least one acrylic polymer. Acrylic polymers include but are not
limited to, acrylic acid, methacrylic acid, acrylic acid and
methacrylic acid copolymers, methyl methacrylate copolymers,
ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl
methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),
polycyanoacrylates and combinations thereof.
[0424] In one embodiment, the therapeutic nanoparticles may
comprise at least one poly(vinyl ester) polymer. The poly(vinyl
ester) polymer may be a copolymer such as a random copolymer. As a
non-limiting example, the random copolymer may have a structure
such as those described in International Application No.
WO2013032829 or US Patent Publication No US20130121954, the
contents of which are herein incorporated by reference in its
entirety. In one aspect, the poly(vinyl ester) polymers may be
conjugated to the polynucleotides described herein. In another
aspect, the poly(vinyl ester) polymer which may be used in the
present invention may be those described in, herein incorporated by
reference in its entirety.
[0425] In one embodiment, the therapeutic nanoparticle may comprise
at least one diblock copolymer. The diblock copolymer may be, but
it not limited to, a poly(lactic) acid-poly(ethylene)glycol
copolymer (see e.g., International Patent Publication No.
WO2013044219; herein incorporated by reference in its entirety). As
a non-limiting example, the therapeutic nanoparticle may be used to
treat cancer (see International publication No. WO2013044219;
herein incorporated by reference in its entirety).
[0426] In one embodiment, the therapeutic nanoparticles may
comprise at least one cationic polymer described herein and/or
known in the art.
[0427] In one embodiment, the therapeutic nanoparticles may
comprise at least one amine-containing polymer such as, but not
limited to polylysine, polyethylene imine, poly(amidoamine)
dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No.
8,287,849; herein incorporated by reference in its entirety) and
combinations thereof.
[0428] In another embodiment, the nanoparticles described herein
may comprise an amine cationic lipid such as those described in
International Patent Application No. WO2013059496, the contents of
which are herein incorporated by reference in its entirety. In one
aspect the cationic lipids may have an amino-amine or an
amino-amide moiety.
[0429] In one embodiment, the therapeutic nanoparticles may
comprise at least one degradable polyester which may contain
polycationic side chains. Degradeable polyesters include, but are
not limited to, poly(serine ester), poly(L-lactide-co-L-lysine),
poly(4-hydroxy-L-proline ester), and combinations thereof. In
another embodiment, the degradable polyesters may include a PEG
conjugation to form a PEGylated polymer.
[0430] In another embodiment, the therapeutic nanoparticle may
include a conjugation of at least one targeting ligand. The
targeting ligand may be any ligand known in the art such as, but
not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res.
2006 66:6732-6740; herein incorporated by reference in its
entirety).
[0431] In one embodiment, the therapeutic nanoparticle may be
formulated in an aqueous solution which may be used to target
cancer (see International Pub No. WO2011084513 and US Pub No.
US20110294717, each of which is herein incorporated by reference in
their entirety).
[0432] In one embodiment, the therapeutic nanoparticle RNA
vaccines, e.g., therapeutic nanoparticles comprising at least one
RNA vaccine may be formulated using the methods described by
Podobinski et al in U.S. Pat. No. 8,404,799, the contents of which
are herein incorporated by reference in its entirety.
[0433] In one embodiment, the RNA vaccines may be encapsulated in,
linked to and/or associated with synthetic nanocarriers. Synthetic
nanocarriers include, but are not limited to, those described in
International Pub. Nos. WO2010005740, WO2010030763, WO201213501,
WO2012149252, WO2012149255, WO2012149259, WO2012149265,
WO2012149268, WO2012149282, WO2012149301, WO2012149393,
WO2012149405, WO2012149411, WO2012149454 and WO2013019669, and US
Pub. Nos. US20110262491, US20100104645, US20100087337 and
US20120244222, each of which is herein incorporated by reference in
their entirety. The synthetic nanocarriers may be formulated using
methods known in the art and/or described herein. As a non-limiting
example, the synthetic nanocarriers may be formulated by the
methods described in International Pub Nos. WO2010005740,
WO2010030763 and WO201213501 and US Pub. Nos. US20110262491,
US20100104645, US20100087337 and US2012024422, each of which is
herein incorporated by reference in their entirety. In another
embodiment, the synthetic nanocarrier formulations may be
lyophilized by methods described in International Pub. No.
WO2011072218 and U.S. Pat. No. 8,211,473; the content of each of
which is herein incorporated by reference in their entirety. In yet
another embodiment, formulations of the present invention,
including, but not limited to, synthetic nanocarriers, may be
lyophilized or reconstituted by the methods described in US Patent
Publication No. US20130230568, the contents of which are herein
incorporated by reference in its entirety.
[0434] In one embodiment, the synthetic nanocarriers may contain
reactive groups to release the polynucleotides described herein
(see International Pub. No. WO20120952552 and US Pub No.
US20120171229, each of which is herein incorporated by reference in
their entirety).
[0435] In one embodiment, the synthetic nanocarriers may contain an
immunostimulatory agent to enhance the immune response from
delivery of the synthetic nanocarrier. As a non-limiting example,
the synthetic nanocarrier may comprise a Th1 immunostimulatory
agent which may enhance a Th1-based response of the immune system
(see International Pub No. WO2010123569 and US Pub. No.
US20110223201, each of which is herein incorporated by reference in
its entirety).
[0436] In one embodiment, the synthetic nanocarriers may be
formulated for targeted release. In one embodiment, the synthetic
nanocarrier is formulated to release the polynucleotides at a
specified pH and/or after a desired time interval. As a
non-limiting example, the synthetic nanoparticle may be formulated
to release the RNA vaccines after 24 hours and/or at a pH of 4.5
(see International Pub. Nos. WO2010138193 and WO2010138194 and US
Pub Nos. US20110020388 and US20110027217, each of which is herein
incorporated by reference in their entireties).
[0437] In one embodiment, the synthetic nanocarriers may be
formulated for controlled and/or sustained release of the
polynucleotides described herein. As a non-limiting example, the
synthetic nanocarriers for sustained release may be formulated by
methods known in the art, described herein and/or as described in
International Pub No. WO2010138192 and US Pub No. 20100303850, each
of which is herein incorporated by reference in their entirety.
[0438] In one embodiment, the RNA vaccine may be formulated for
controlled and/or sustained release wherein the formulation
comprises at least one polymer that is a crystalline side chain
(CYSC) polymer. CYSC polymers are described in U.S. Pat. No.
8,399,007, herein incorporated by reference in its entirety.
[0439] In one embodiment, the synthetic nanocarrier may be
formulated for use as a vaccine. In one embodiment, the synthetic
nanocarrier may encapsulate at least one polynucleotide which
encode at least one antigen. As a non-limiting example, the
synthetic nanocarrier may include at least one antigen and an
excipient for a vaccine dosage form (see International Pub No.
WO2011150264 and US Pub No. US20110293723, each of which is herein
incorporated by reference in their entirety). As another
non-limiting example, a vaccine dosage form may include at least
two synthetic nanocarriers with the same or different antigens and
an excipient (see International Pub No. WO2011150249 and US Pub No.
US20110293701, each of which is herein incorporated by reference in
their entirety). The vaccine dosage form may be selected by methods
described herein, known in the art and/or described in
International Pub No. WO2011150258 and US Pub No. US20120027806,
each of which is herein incorporated by reference in their
entirety).
[0440] In one embodiment, the synthetic nanocarrier may comprise at
least one polynucleotide which encodes at least one adjuvant. As
non-limiting example, the adjuvant may comprise
dimethyldioctadecylammonium-bromide,
dimethyldioctadecylammonium-chloride,
dimethyldioctadecylammonium-phosphate or
dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or
part of said apolar fraction of a total lipid extract of a
mycobacterium (See e.g, U.S. Pat. No. 8,241,610; herein
incorporated by reference in its entirety). In another embodiment,
the synthetic nanocarrier may comprise at least one polynucleotide
and an adjuvant. As a non-limiting example, the synthetic
nanocarrier comprising and adjuvant may be formulated by the
methods described in International Pub No. WO2011150240 and US Pub
No. US20110293700, each of which is herein incorporated by
reference in its entirety.
[0441] In one embodiment, the synthetic nanocarrier may encapsulate
at least one polynucleotide which encodes a peptide, fragment or
region from a virus. As a non-limiting example, the synthetic
nanocarrier may include, but is not limited to, the nanocarriers
described in International Pub No. WO2012024621, WO201202629,
WO2012024632 and US Pub No. US20120064110, US20120058153 and
US20120058154, each of which is herein incorporated by reference in
their entirety.
[0442] In one embodiment, the synthetic nanocarrier may be coupled
to a polynucleotide which may be able to trigger a humoral and/or
cytotoxic T lymphocyte (CTL) response (See e.g., International
Publication No. WO2013019669, herein incorporated by reference in
its entirety).
[0443] In one embodiment, the RNA vaccine may be encapsulated in,
linked to and/or associated with zwitterionic lipids. Non-limiting
examples of zwitterionic lipids and methods of using zwitterionic
lipids are described in US Patent Publication No. US20130216607,
the contents of which are herein incorporated by reference in its
entirety. In one aspect, the zwitterionic lipids may be used in the
liposomes and lipid nanoparticles described herein.
[0444] In one embodiment, the RNA vaccine may be formulated in
colloid nanocarriers as described in US Patent Publication No.
US20130197100, the contents of which are herein incorporated by
reference in its entirety.
[0445] In one embodiment, the nanoparticle may be optimized for
oral administration. The nanoparticle may comprise at least one
cationic biopolymer such as, but not limited to, chitosan or a
derivative thereof. As a non-limiting example, the nanoparticle may
be formulated by the methods described in U.S. Pub. No.
20120282343; herein incorporated by reference in its entirety.
[0446] In some embodiments, LNPs comprise the lipid KL52 (an
amino-lipid disclosed in U.S. Application Publication No.
2012/0295832 expressly incorporated herein by reference in its
entirety). Activity and/or safety (as measured by examining one or
more of ALT/AST, white blood cell count and cytokine induction) of
LNP administration may be improved by incorporation of such lipids.
LNPs comprising KL52 may be administered intravenously and/or in
one or more doses. In some embodiments, administration of LNPs
comprising KL52 results in equal or improved mRNA and/or protein
expression as compared to LNPs comprising MC3.
[0447] In some embodiments, RNA vaccine may be delivered using
smaller LNPs. Such particles may comprise a diameter from below 0.1
um up to 100 nm such as, but not limited to, less than 0.1 um, less
than 1.0 um, less than 5 um, less than 10 um, less than 15 um, less
than 20 um, less than 25 um, less than 30 um, less than 35 um, less
than 40 um, less than 50 um, less than 55 um, less than 60 um, less
than 65 um, less than 70 um, less than 75 um, less than 80 um, less
than 85 um, less than 90 um, less than 95 um, less than 100 um,
less than 125 um, less than 150 um, less than 175 um, less than 200
um, less than 225 um, less than 250 um, less than 275 um, less than
300 um, less than 325 um, less than 350 um, less than 375 um, less
than 400 um, less than 425 um, less than 450 um, less than 475 um,
less than 500 um, less than 525 um, less than 550 um, less than 575
um, less than 600 um, less than 625 um, less than 650 um, less than
675 um, less than 700 um, less than 725 um, less than 750 um, less
than 775 um, less than 800 um, less than 825 um, less than 850 um,
less than 875 um, less than 900 um, less than 925 um, less than 950
um, less than 975 um.
[0448] In another embodiment, RNA vaccines may be delivered using
smaller LNPs which may comprise a diameter from about 1 nm to about
100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm,
from about 1 nm to about 30 nm, from about 1 nm to about 40 nm,
from about 1 nm to about 50 nm, from about 1 nm to about 60 nm,
from about 1 nm to about 70 nm, from about 1 nm to about 80 nm,
from about 1 nm to about 90 nm, from about 5 nm to about from 100
nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from
about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from
about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from
about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from
about 5 nm to about 90 nm, about 10 to about 50 nM, from about 20
to about 50 nm, from about 30 to about 50 nm, from about 40 to
about 50 nm, from about 20 to about 60 nm, from about 30 to about
60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm,
from about 30 to about 70 nm, from about 40 to about 70 nm, from
about 50 to about 70 nm, from about 60 to about 70 nm, from about
20 to about 80 nm, from about 30 to about 80 nm, from about 40 to
about 80 nm, from about 50 to about 80 nm, from about 60 to about
80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm,
from about 40 to about 90 nm, from about 50 to about 90 nm, from
about 60 to about 90 nm and/or from about 70 to about 90 nm.
[0449] In some embodiments, such LNPs are synthesized using methods
comprising microfluidic mixers. Exemplary microfluidic mixers may
include, but are not limited to a slit interdigitial micromixer
including, but not limited to those manufactured by Microinnova
(Allerheiligen bei Wildon, Austria) and/or a staggered herringbone
micromixer (SHM) (Zhigaltsev, I. V. et al., Bottom-up design and
synthesis of limit size lipid nanoparticle systems with aqueous and
triglyceride cores using millisecond microfluidic mixing have been
published (Langmuir. 2012. 28:3633-40; Belliveau, N. M. et al.,
Microfluidic synthesis of highly potent limit-size lipid
nanoparticles for in vivo delivery of siRNA. Molecular
Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapid
discovery of potent siRNA-containing lipid nanoparticles enabled by
controlled microfluidic formulation. J Am Chem Soc. 2012.
134(16):6948-51; each of which is herein incorporated by reference
in its entirety). In some embodiments, methods of LNP generation
comprising SHM, further comprise the mixing of at least two input
streams wherein mixing occurs by microstructure-induced chaotic
advection (MICA). According to this method, fluid streams flow
through channels present in a herringbone pattern causing
rotational flow and folding the fluids around each other. This
method may also comprise a surface for fluid mixing wherein the
surface changes orientations during fluid cycling. Methods of
generating LNPs using SHM include those disclosed in U.S.
Application Publication Nos. 2004/0262223 and 2012/0276209, each of
which is expressly incorporated herein by reference in their
entirety.
[0450] In one embodiment, the RNA vaccine of the present invention
may be formulated in lipid nanoparticles created using a micromixer
such as, but not limited to, a Slit Interdigital Microstructured
Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM)
or Caterpillar (CPMM) or Impinging-jet (IJMM) from the Institut fur
Mikrotechnik Mainz GmbH, Mainz Germany).
[0451] In one embodiment, the RNA vaccines of the present invention
may be formulated in lipid nanoparticles created using microfluidic
technology (see Whitesides, George M. The Origins and the Future of
Microfluidics. Nature, 2006 442: 368-373; and Abraham et al.
Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; each
of which is herein incorporated by reference in its entirety). As a
non-limiting example, controlled microfluidic formulation includes
a passive method for mixing streams of steady pressure-driven flows
in micro channels at a low Reynolds number (See e.g., Abraham et
al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651;
which is herein incorporated by reference in its entirety).
[0452] In one embodiment, the RNA vaccines of the present invention
may be formulated in lipid nanoparticles created using a micromixer
chip such as, but not limited to, those from Harvard Apparatus
(Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). A
micromixer chip can be used for rapid mixing of two or more fluid
streams with a split and recombine mechanism.
[0453] In one embodiment, the RNA vaccines of the invention may be
formulated for delivery using the drug encapsulating microspheres
described in International Patent Publication No. WO2013063468 or
U.S. Pat. No. 8,440,614, each of which is herein incorporated by
reference in its entirety. The microspheres may comprise a compound
of the formula (I), (II), (III), (IV), (V) or (VI) as described in
International Patent Publication No. WO2013063468, the contents of
which are herein incorporated by reference in its entirety. In
another aspect, the amino acid, peptide, polypeptide, lipids (APPL)
are useful in delivering the RNA vaccines of the invention to cells
(see International Patent Publication No. WO2013063468, the
contents of which is herein incorporated by reference in its
entirety).
[0454] In one embodiment, the RNA vaccines of the invention may be
formulated in lipid nanoparticles having a diameter from about 10
to about 100 nm such as, but not limited to, about 10 to about 20
nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to
about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm,
about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about
30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20
to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm,
about 20 to about 90 nm, about 20 to about 100 nm, about 30 to
about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm,
about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about
90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40
to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm,
about 40 to about 90 nm, about 40 to about 100 nm, about 50 to
about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about
50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70
nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to
about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm,
about 70 to about 100 nm, about 80 to about 90 nm, about 80 to
about 100 nm and/or about 90 to about 100 nm.
[0455] In one embodiment, the lipid nanoparticles may have a
diameter from about 10 to 500 nm.
[0456] In one embodiment, the lipid nanoparticle may have a
diameter greater than 100 nm, greater than 150 nm, greater than 200
nm, greater than 250 nm, greater than 300 nm, greater than 350 nm,
greater than 400 nm, greater than 450 nm, greater than 500 nm,
greater than 550 nm, greater than 600 nm, greater than 650 nm,
greater than 700 nm, greater than 750 nm, greater than 800 nm,
greater than 850 nm, greater than 900 nm, greater than 950 nm or
greater than 1000 nm.
[0457] In one aspect, the lipid nanoparticle may be a limit size
lipid nanoparticle described in International Patent Publication
No. WO2013059922, the contents of which are herein incorporated by
reference in its entirety. The limit size lipid nanoparticle may
comprise a lipid bilayer surrounding an aqueous core or a
hydrophobic core; where the lipid bilayer may comprise a
phospholipid such as, but not limited to,
diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a
ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, a
cerebroside, a C8-C20 fatty acid diacylphophatidylcholine, and
1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). In another aspect
the limit size lipid nanoparticle may comprise a polyethylene
glycol-lipid such as, but not limited to, DLPE-PEG, DMPE-PEG,
DPPC-PEG and DSPE-PEG.
[0458] In one embodiment, the RNA vaccines may be delivered,
localized and/or concentrated in a specific location using the
delivery methods described in International Patent Publication No.
WO2013063530, the contents of which are herein incorporated by
reference in its entirety. As a non-limiting example, a subject may
be administered an empty polymeric particle prior to,
simultaneously with or after delivering the RNA vaccines to the
subject. The empty polymeric particle undergoes a change in volume
once in contact with the subject and becomes lodged, embedded,
immobilized or entrapped at a specific location in the subject.
[0459] In one embodiment, the RNA vaccines may be formulated in an
active substance release system (See e.g., US Patent Publication
No. US20130102545, the contents of which is herein incorporated by
reference in its entirety). The active substance release system may
comprise 1) at least one nanoparticle bonded to an oligonucleotide
inhibitor strand which is hybridized with a catalytically active
nucleic acid and 2) a compound bonded to at least one substrate
molecule bonded to a therapeutically active substance (e.g.,
polynucleotides described herein), where the therapeutically active
substance is released by the cleavage of the substrate molecule by
the catalytically active nucleic acid.
[0460] In one embodiment, the RNA vaccines may be formulated in a
nanoparticle comprising an inner core comprising a non-cellular
material and an outer surface comprising a cellular membrane. The
cellular membrane may be derived from a cell or a membrane derived
from a virus. As a non-limiting example, the nanoparticle may be
made by the methods described in International Patent Publication
No. WO2013052167, herein incorporated by reference in its entirety.
As another non-limiting example, the nanoparticle described in
International Patent Publication No. WO2013052167, herein
incorporated by reference in its entirety, may be used to deliver
the RNA vaccines described herein.
[0461] In one embodiment, the RNA vaccines may be formulated in
porous nanoparticle-supported lipid bilayers (protocells).
Protocells are described in International Patent Publication No.
WO2013056132, the contents of which are herein incorporated by
reference in its entirety.
[0462] In one embodiment, the RNA vaccines described herein may be
formulated in polymeric nanoparticles as described in or made by
the methods described in U.S. Pat. Nos. 8,420,123 and 8,518,963 and
European Patent No. EP2073848B1, the contents of each of which are
herein incorporated by reference in their entirety. As a
non-limiting example, the polymeric nanoparticle may have a high
glass transition temperature such as the nanoparticles described in
or nanoparticles made by the methods described in U.S. Pat. No.
8,518,963, the contents of which are herein incorporated by
reference in its entirety. As another non-limiting example, the
polymer nanoparticle for oral and parenteral formulations may be
made by the methods described in European Patent No. EP2073848B1,
the contents of which are herein incorporated by reference in its
entirety.
[0463] In another embodiment, the RNA vaccines described herein may
be formulated in nanoparticles used in imaging. The nanoparticles
may be liposome nanoparticles such as those described in US Patent
Publication No US20130129636, herein incorporated by reference in
its entirety. As a non-limiting example, the liposome may comprise
gadolinium(III)2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N'--
amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-acetic acid and a
neutral, fully saturated phospholipid component (see e.g., US
Patent Publication No US20130129636, the contents of which is
herein incorporated by reference in its entirety).
[0464] In one embodiment, the nanoparticles which may be used in
the present invention are formed by the methods described in U.S.
Patent Application No. US20130130348, the contents of which is
herein incorporated by reference in its entirety.
[0465] The nanoparticles of the present invention may further
include nutrients such as, but not limited to, those which
deficiencies can lead to health hazards from anemia to neural tube
defects (see e.g, the nanoparticles described in International
Patent Publication No WO2013072929, the contents of which is herein
incorporated by reference in its entirety). As a non-limiting
example, the nutrient may be iron in the form of ferrous, ferric
salts or elemental iron, iodine, folic acid, vitamins or
micronutrients.
[0466] In one embodiment, the RNA vaccines of the present invention
may be formulated in a swellable nanoparticle. The swellable
nanoparticle may be, but is not limited to, those described in U.S.
Pat. No. 8,440,231, the contents of which is herein incorporated by
reference in its entirety. As a non-limiting embodiment, the
swellable nanoparticle may be used for delivery of the RNA vaccines
of the present invention to the pulmonary system (see e.g., U.S.
Pat. No. 8,440,231, the contents of which is herein incorporated by
reference in its entirety).
[0467] The RNA vaccines of the present invention may be formulated
in polyanhydride nanoparticles such as, but not limited to, those
described in U.S. Pat. No. 8,449,916, the contents of which is
herein incorporated by reference in its entirety.
[0468] The nanoparticles and microparticles of the present
invention may be geometrically engineered to modulate macrophage
and/or the immune response. In one aspect, the geometrically
engineered particles may have varied shapes, sizes and/or surface
charges in order to incorporated the polynucleotides of the present
invention for targeted delivery such as, but not limited to,
pulmonary delivery (see e.g., International Publication No
WO2013082111, the contents of which is herein incorporated by
reference in its entirety). Other physical features the
geometrically engineering particles may have include, but are not
limited to, fenestrations, angled arms, asymmetry and surface
roughness, charge which can alter the interactions with cells and
tissues. As a non-limiting example, nanoparticles of the present
invention may be made by the methods described in International
Publication No WO2013082111, the contents of which is herein
incorporated by reference in its entirety.
[0469] In one embodiment, the nanoparticles of the present
invention may be water soluble nanoparticles such as, but not
limited to, those described in International Publication No.
WO2013090601, the contents of which is herein incorporated by
reference in its entirety. The nanoparticles may be inorganic
nanoparticles which have a compact and zwitterionic ligand in order
to exhibit good water solubility. The nanoparticles may also have
small hydrodynamic diameters (HD), stability with respect to time,
pH, and salinity and a low level of non-specific protein
binding.
[0470] In one embodiment the nanoparticles of the present invention
may be developed by the methods described in US Patent Publication
No. US20130172406, the contents of which are herein incorporated by
reference in its entirety.
[0471] In one embodiment, the nanoparticles of the present
invention are stealth nanoparticles or target-specific stealth
nanoparticles such as, but not limited to, those described in US
Patent Publication No. US20130172406; the contents of which is
herein incorporated by reference in its entirety. The nanoparticles
of the present invention may be made by the methods described in US
Patent Publication No. US20130172406, the contents of which are
herein incorporated by reference in its entirety.
[0472] In another embodiment, the stealth or target-specific
stealth nanoparticles may comprise a polymeric matrix. The
polymeric matrix may comprise two or more polymers such as, but not
limited to, polyethylenes, polycarbonates, polyanhydrides,
polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides, polyacetals, polyethers, polyesters, poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines,
polyesters, polyanhydrides, polyethers, polyurethanes,
polymethacrylates, polyacrylates, polycyanoacrylates or
combinations thereof.
[0473] In one embodiment, the nanoparticle may be a
nanoparticle-nucleic acid hybrid structure having a high density
nucleic acid layer. As a non-limiting example, the
nanoparticle-nucleic acid hybrid structure may made by the methods
described in US Patent Publication No. US20130171646, the contents
of which are herein incorporated by reference in its entirety. The
nanoparticle may comprise a nucleic acid such as, but not limited
to, polynucleotides described herein and/or known in the art.
[0474] At least one of the nanoparticles of the present invention
may be embedded in in the core a nanostructure or coated with a low
density porous 3-D structure or coating which is capable of
carrying or associating with at least one payload within or on the
surface of the nanostructure. Non-limiting examples of the
nanostructures comprising at least one nanoparticle are described
in International Patent Publication No. WO2013123523, the contents
of which are herein incorporated by reference in its entirety.
[0475] The mRNA cancer vaccines of the present invention may be
administered by any route which results in a therapeutically
effective outcome. These include, but are not limited, to
intradermal, intramuscular, and/or subcutaneous administration. The
present invention provides methods comprising administering mRNA
cancer vaccines and in accordance with the invention to a subject
in need thereof. The exact amount required will vary from subject
to subject, depending on the species, age, and general condition of
the subject, the severity of the disease, the particular
composition, its mode of administration, its mode of activity, and
the like. Compositions in accordance with the invention are
typically formulated in dosage unit form for ease of administration
and uniformity of dosage. It will be understood, however, that the
total daily usage of the compositions of the present invention may
be decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically effective,
prophylactically effective, or appropriate imaging dose level for
any particular patient will depend upon a variety of factors
including the disorder being treated and the severity of the
disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
compound employed; the duration of the treatment; drugs used in
combination or coincidental with the specific compound employed;
and like factors well known in the medical arts.
[0476] In certain embodiments, compositions in accordance with the
present invention may be administered at dosage levels sufficient
to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about
0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about
0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about
0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50
mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg
to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from
about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about
25 mg/kg, of subject body weight per day, one or more times a day,
to obtain the desired therapeutic, diagnostic, prophylactic, or
imaging effect (see e.g., the range of unit doses described in
International Publication No WO2013078199, herein incorporated by
reference in its entirety). The desired dosage may be delivered
three times a day, two times a day, once a day, every other day,
every third day, every week, every two weeks, every three weeks, or
every four weeks. In certain embodiments, the desired dosage may be
delivered using multiple administrations (e.g., two, three, four,
five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen, or more administrations). When multiple administrations
are employed, split dosing regimens such as those described herein
may be used.
[0477] A mRNA cancer vaccine pharmaceutical composition described
herein can be formulated into a dosage form described herein, such
as an intranasal, intratracheal, or injectable (e.g., intravenous,
intraocular, intravitreal, intramuscular, intradermal,
intracardiac, intraperitoneal, and subcutaneous).
[0478] In some embodiments, the RNA (e.g., mRNA) vaccine
compositions may be administered at dosage levels sufficient to
deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005
mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5
mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to
30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg
to 25 mg/kg, of subject body weight per day, one or more times a
day, per week, per month, etc. to obtain the desired therapeutic,
diagnostic, prophylactic, or imaging effect (see e.g., the range of
unit doses described in International Publication No WO2013078199,
herein incorporated by reference in its entirety). The desired
dosage may be delivered three times a day, two times a day, once a
day, every other day, every third day, every week, every two weeks,
every three weeks, every four weeks, every 2 months, every three
months, every 6 months, etc. In certain embodiments, the desired
dosage may be delivered using multiple administrations (e.g., two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve,
thirteen, fourteen, or more administrations). When multiple
administrations are employed, split dosing regimens such as those
described herein may be used. In some embodiments, the RNA vaccine
compositions may be administered at dosage levels sufficient to
deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to
about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg,
about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about
0.005 mg/kg. In some embodiments, the RNA vaccine compositions may
be administered once or twice (or more) at dosage levels sufficient
to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg,
0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.
[0479] In some embodiments, the RNA vaccine compositions may be
administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0
and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90,
Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and
3 months later, Day 0 and 6 months later, Day 0 and 9 months later,
Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2
years later, Day 0 and 5 years later, or Day 0 and 10 years later)
at a total dose of or at dosage levels sufficient to deliver a
total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg,
0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275
mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg,
0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600
mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg,
0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925
mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages and
frequency of administration are encompassed by the present
disclosure. For example, a the RNA vaccine composition may be
administered three or four times.
[0480] In some embodiments, the RNA vaccine compositions may be
administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0
and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90,
Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and
3 months later, Day 0 and 6 months later, Day 0 and 9 months later,
Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2
years later, Day 0 and 5 years later, or Day 0 and 10 years later)
at a total dose of or at dosage levels sufficient to deliver a
total dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg.
[0481] In some embodiments the RNA vaccine for use in a method of
vaccinating a subject is administered the subject a single dosage
of between 10 .mu.g/kg and 400 .mu.g/kg of the nucleic acid vaccine
in an effective amount to vaccinate the subject. In some
embodiments the RNA vaccine for use in a method of vaccinating a
subject is administered the subject a single dosage of between 10
.mu.g and 400 .mu.g of the nucleic acid vaccine in an effective
amount to vaccinate the subject.
[0482] This invention is not limited in its application to the
details of construction and the arrangement of components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments and of being practiced or
of being carried out in various ways. Also, the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of "including,"
"comprising," or "having," "containing," "involving," and
variations thereof herein, is meant to encompass the items listed
thereafter and equivalents thereof as well as additional items.
EXAMPLES
Example 1. Manufacture of Polynucleotides
[0483] According to the present invention, the manufacture of
polynucleotides and or parts or regions thereof may be accomplished
utilizing the methods taught in U.S. Ser. No. 61/800,049 filed Mar.
15, 2013 entitled "Manufacturing Methods for Production of RNA
Transcripts" (Attorney Docket number M500), the contents of which
is incorporated herein by reference in its entirety.
[0484] Purification methods may include those taught in U.S.
Provisional Patent Application No. 61/799,872, U.S. Provisional
Patent Application No. 61/794,842, U.S. Provisional Patent
Application 61/800,326, each of which is incorporated herein by
reference in its entirety.
[0485] Detection and characterization methods of the
polynucleotides may be performed as taught in U.S. Provisional
Patent Application No. 61/799,780 and U.S. Provisional Patent
Application No. 61/798,945, each of which is incorporated herein by
reference in its entirety.
[0486] Characterization of the polynucleotides of the invention may
be accomplished using a procedure selected from the group
consisting of polynucleotide mapping, reverse transcriptase
sequencing, charge distribution analysis, and detection of RNA
impurities, wherein characterizing comprises determining the RNA
transcript sequence, determining the purity of the RNA transcript,
or determining the charge heterogeneity of the RNA transcript. Such
methods are taught in, for example, U.S. Provisional Patent
Application No. 61/799,905 and U.S. Provisional Patent Application
No. 61/800,110, the contents of each of which is incorporated
herein by reference in its entirety.
Example 2. Chimeric Polynucleotide Synthesis
Introduction
[0487] According to the present invention, two regions or parts of
a chimeric polynucleotide may be joined or ligated using
triphosphate chemistry.
[0488] According to this method, a first region or part of 100
nucleotides or less is chemically synthesized with a 5'
monophosphate and terminal 3'desOH or blocked OH. If the region is
longer than 80 nucleotides, it may be synthesized as two strands
for ligation.
[0489] If the first region or part is synthesized as a
non-positionally modified region or part using in vitro
transcription (IVT), conversion the 5'monophosphate with subsequent
capping of the 3' terminus may follow.
[0490] Monophosphate protecting groups may be selected from any of
those known in the art.
[0491] The second region or part of the chimeric polynucleotide may
be synthesized using either chemical synthesis or IVT methods. IVT
methods may include an RNA polymerase that can utilize a primer
with a modified cap. Alternatively, a cap of up to 130 nucleotides
may be chemically synthesized and coupled to the IVT region or
part.
[0492] It is noted that for ligation methods, ligation with DNA T4
ligase, followed by treatment with DNAse should readily avoid
concatenation.
[0493] The entire chimeric polynucleotide need not be manufactured
with a phosphate-sugar backbone. If one of the regions or parts
encodes a polypeptide, then it is preferable that such region or
part comprise a phosphate-sugar backbone.
[0494] Ligation is then performed using any known click chemistry,
orthoclick chemistry, solulink, or other bioconjugate chemistries
known to those in the art.
Synthetic Route
[0495] The chimeric polynucleotide is made using a series of
starting segments. Such segments include:
[0496] (a) Capped and protected 5' segment comprising a normal 3'OH
(SEG. 1)
[0497] (b) 5' triphosphate segment which may include the coding
region of a polypeptide and comprising a normal 3'OH (SEG. 2)
[0498] (c) 5' monophosphate segment for the 3' end of the chimeric
polynucleotide (e.g., the tail) comprising cordycepin or no 3'OH
(SEG. 3)
[0499] After synthesis (chemical or IVT), segment 3 (SEG. 3) is
treated with cordycepin and then with pyrophosphatase to create the
5'monophosphate.
[0500] Segment 2 (SEG. 2) is then ligated to SEG. 3 using RNA
ligase. The ligated polynucleotide is then purified and treated
with pyrophosphatase to cleave the diphosphate. The treated
SEG.2-SEG. 3 construct is then purified and SEG. 1 is ligated to
the 5' terminus. A further purification step of the chimeric
polynucleotide may be performed.
[0501] Where the chimeric polynucleotide encodes a polypeptide, the
ligated or joined segments may be represented as: 5'UTR (SEG. 1),
open reading frame or ORF (SEG. 2) and 3'UTR+PolyA (SEG. 3).
[0502] The yields of each step may be as much as 90-95%.
Example 3: PCR for cDNA Production
[0503] PCR procedures for the preparation of cDNA are performed
using 2.times.KAPA HIFI.TM. HotStart ReadyMix by Kapa Biosystems
(Woburn, Mass.). This system includes 2.times.KAPA ReadyMix 12.5
.mu.l; Forward Primer (10 uM) 0.75 .mu.l; Reverse Primer (10 uM)
0.75 .mu.l; Template cDNA-100 ng; and dH.sub.20 diluted to 25.0
.mu.l. The reaction conditions are at 95.degree. C. for 5 min. and
25 cycles of 98.degree. C. for 20 sec, then 58.degree. C. for 15
sec, then 72.degree. C. for 45 sec, then 72.degree. C. for 5 min.
then 4.degree. C. to termination.
[0504] The reaction is cleaned up using Invitrogen's PURELINK.TM.
PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions
(up to 5 .mu.g). Larger reactions will require a cleanup using a
product with a larger capacity. Following the cleanup, the cDNA is
quantified using the NANODROP.TM. and analyzed by agarose gel
electrophoresis to confirm the cDNA is the expected size. The cDNA
is then submitted for sequencing analysis before proceeding to the
in vitro transcription reaction.
Example 4. In Vitro Transcription (IVT)
[0505] The in vitro transcription reaction generates
polynucleotides containing uniformly modified polynucleotides. Such
uniformly modified polynucleotides may comprise a region or part of
the polynucleotides of the invention. The input nucleotide
triphosphate (NTP) mix is made in-house using natural and
un-natural NTPs.
[0506] A typical in vitro transcription reaction includes the
following:
TABLE-US-00008 1 Template cDNA 1.0 .mu.g 2 10x transcription buffer
(400 mM Tris-HCl pH 8.0, 2.0 .mu.l 190 mM MgCl.sub.2, 50 mM DTT, 10
mM Spermidine) 3 Custom NTPs (25 mM each) 7.2 .mu.l 4 RNase
Inhibitor 20 U 5 T7 RNA polymerase 3000 U 6 dH.sub.20 Up to 20.0
.mu.l. and 7 Incubation at 37.degree. C for 3 hr-5 hrs.
[0507] The crude IVT mix may be stored at 4.degree. C. overnight
for cleanup the next day. 1 U of RNase-free DNase is then used to
digest the original template. After 15 minutes of incubation at
37.degree. C., the mRNA is purified using Ambion's MEGACLEAR.TM.
Kit (Austin, Tex.) following the manufacturer's instructions. This
kit can purify up to 500 .mu.g of RNA. Following the cleanup, the
RNA is quantified using the NanoDrop and analyzed by agarose gel
electrophoresis to confirm the RNA is the proper size and that no
degradation of the RNA has occurred.
Example 5. In Vivo Immunogeniticy Assay with mRNA Cancer
Vaccines
[0508] An MC38 immunogenicity study using mRNA vaccines in mice was
performed. mRNA antigens: three MC38 neoepitopes Adpgk, Dpagt1,
Reps1 having formats: 25mer, TMG, secreted CD40L-TMG fusion
protein) were generated. The positive control was a benchmark
comparison to 25-mer peptide immunization+anti-CD40+poly(I:C)
(Yadav et al, Nature 2015). Mice were immunized on days 0, 7, and
14. A readout was measured on Days 3, 10, and 17; followed by MC38
challenge on day 21 and sacrifice on day 35.
[0509] Characterization of the epitope-specific T cell population
was made by frequency of antigen-specific T cell population by
dextramers staining. A cytokine profile was generated:
Intracellular cytokine staining (IFN.gamma., TNF.alpha., IL-2) and
ELISPOT (upon MC38 mutant peptide stimulation). The following
memory and T cell differentiation markers: CD44, CD62L, IL7R,
KLRG1, CD122 and exhaustion markers: PD1, Lag3, Tim3, 2B4 were
used.
[0510] The results showing that mRNA vaccine induced an antigen
specific CD8 response are shown in FIG. 1. Results showing that
mRNA vaccines induced antigen specific effector/memory CD8 T cells
are shown in FIG. 2.
[0511] Some of the considerations for antigen designs include MHC
classes, Expression localization, Polypeptide format and
configuration, and Potency enhancing motifs. A multi-factorial
consideration of antigen design of mRNA-based neoepitopes is shown
in FIGS. 3 (schematic) and 4 (table).
Example 6. Method Development of FACS-Based MHC-Presentation
[0512] Objective: Validation of FACS-based assay of mRNA encoded
epitopes in MCF7 (HLA*201). The mRNA used was a combination a
concatemer of four different epitopes:
mut.gp100(T209M)+mut.tyrpsoinase(N271D)+mut.CDK4(R24C)+mut.MART1(A27L)
TMG.G25 (1/2){circumflex over ( )}3.nPEST seq: control mRNA of
tandem minigene of three repeats of mut.gp100(T209M). Protein
production was detected using an Anti-mut.MART1(A27L) TCRmer-PE and
Anti-HLA antibodies.
[0513] The method involved: MCF7 transfected with 250 ng mRNA using
LF2000; Peptide-pulsed control preparation: MCF7 were left
un-pulsed or pulsed with synthetic peptides in serum-free RPMI for
3 h at 37 C; and FACS analysis with anti-HLA and TCRmer (specific
for mutant MART1-HLA*201 complex) at .about.20 h.
[0514] The data is shown in FIG. 5. Specific MHC1/mut.MART1peptide
presentation by anti-mut.MART1TCRmer was detected on MCF7
cells.
Example 7. T Cell Response Elicited with mRNA Encoding Concatamers
of 20 Epitopes
[0515] mRNA concatamers induced both class I and class II T cell
responses. CA60 encodes 20 epitopes derived from the mutanome of a
patient. It includes 5 murine class II epitopes, 10 murine class I
epitopes, a murine positive control (SIINFEKL (SEQ ID NO: 6),
derived from ovalbumin), and 4 human (HLA-A2) epitopes (not shown).
Mice were immunized with 10 ug mRNA twice (prime+boost at day 14)
and spleen cells were analyzed at day 21 by flow cytometry.
[0516] The data are shown in FIG. 7. Four out of ten Class I
epitopes and five out of five class II epitopes were immunogenic.
The epitopes showed responses two-fold over the unstimulated
control. Some Class I predicted epitopes showed some level of cross
presentation.
[0517] No differences were observed between RP and no-RP for known
A2 concatamers. The same 5 out of 19 epitopes that showed responses
in ELISpot after restimulation have CD8+IFNg+ specific responses.
Data are shown in FIG. 8.
Example 8. Epitopes are Immunogenic Irrespective of Position within
mRNA Concatamer
[0518] The epitopes were immunogenic irrespective of their position
within the mRNA. CA80 and CA81 encode the same 20 epitopes known to
elicit T cell responses. They include 5 class II epitopes, 10
murine class I epitopes, a murine positive control (SIINFEKL (SEQ
ID NO: 6), derived from ovalbumin), and 4 human (HLA-A2) epitopes
(not shown). CA80 and CA81 differ only in the relative positions of
the different epitopes. Mice were immunized with 10 ug mRNA twice
(prime+boost at day 14) and spleen cells were analyzed at day 21 by
flow cytometry.
[0519] The data are shown in FIG. 9A. Eight out of 10 class I
epitopes and three out of five class II epitopes were immunogenic.
The epitopes showed responses eight-fold over the unstimulated
control. The same level of immunogenicity was observed irrespective
of the position within the mRNA. FIG. 9B shows that there is a
strong correlation (R squared=0.78) between percent frequency of
CD8+IFNg+ cells and interferon-gamma spot forming units (SFUs) in
ELISpot assays.
Example 9. Phase I, Open-Label Study to Assess Safety,
Tolerability, and Immunogenicity of mRNA Vaccine in Patients with
Solid Tumors
[0520] A phase I, open-label study to assess the safety,
tolerability, and immunogenicity of mRNA 4379 alone in patients
with resected solid tumors, and in combination with pembrolizumab
(a humanized anti-PD-1 antibody) in patients with unresectable
solid tumors is performed.
[0521] Objectives: Primary: safety & tolerability of mRNA-4379
in patients with resected solid tumors (Part A) &
mRNA-4379+pembrolizumab in patients with unresectable solid tumors
(Part B)
[0522] Secondary: Part A: RFS in patients with resected solid
tumors treated.
Part B: ORR, DOR, PFS & OS in patients with unresectable solid
tumors (pembro label)
[0523] Exploratory Study Objectives: Immunogenicity
[0524] Methodology: Two-part, open-label, 3+3 dose-escalation:
fixed dose of either 0.1 mg, 0.2 mg or 0.4 mg of mRNA-4379
administered via intramuscular (IM) injections once during 21-day
cycles for a maximum of 4 doses over 4 cycles.
[0525] A schematic of the mRNA component of mRNA-4379 is shown in
FIG. 10. mRNA-4379 contains a canonical dinucleotide mammalian cap
1 structure at the 5' end comprised of a 7-methyl guanosine linked
in a 5'-5' triphosphate configuration to the penultimate nucleotide
that is methylated at the 2' position of the ribose sugar (Kozak,
1991; Fechter and Brownlee, 2005). The cap structure is required
for initiation of translation. Following the cap structure is the
48-nt 5' untranslated region (5' UTR) that has been optimized to
facilitate initiation of translation. The 5' UTR ends at the AUG
methionine start codon encoding the first amino acid of the protein
coding region, or open reading frame (ORF), of mRNA-4379 which will
be uniquely defined for each patient. The ORF of mRNA-4379 ends
with the three mammalian stop codons linked in a row
(5'-UGA-UAA-UAG-3' (SEQ ID NO: 1132)) that start a common,
pre-specified 3' UTR nt sequence that has been optimized to promote
mRNA stabilization. mRNA-4379 ends with an approximately 100-nt
adenosine homopolymer, the polyA tail, which is required for mRNA
stabilization and protein translation. Both the cap structure at
the 5' end and the polyA tail at the 3' end are required for
mRNA-4379 to be translated by the cellular translational machinery.
RNA lacking either the 5' cap or the 3' polyA tail cannot be
translated and therefore will not produce protein. Any degradant of
mRNA-4379 lacking either the cap 1 structure on the 5' end or the
polyA tail on the 3' end would not produce any protein.
[0526] An example of the general molecular sequence of mRNA-4379 is
provided in FIG. 11, in which the patient specific coding region is
depicted by reference as (N). The nucleosides in mRNA-4379 are
chemically identical to naturally-occurring mammalian mRNA
nucleosides, with the exception that the uridine nucleoside
normally present in mammalian mRNA is fully replaced with
N1-methyl-pseudouridine, a naturally-occurring pyrimidine base
present in mammalian tRNAs (Rozenski, Crain et al. 1999; Kariko,
Buckstein et al. 2005). This nucleoside is included in mRNA-4379 in
place of the normal uridine base to minimize the indiscriminate
recognition of mRNA-4379 by pathogen-associated molecular pattern
(PAMP) receptors (e.g., Toll-like receptors (TLR), Desmet and
Ishii, 2012).
EQUIVALENTS
[0527] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0528] All references, including patent documents, disclosed herein
are incorporated by reference in their entirety.
Sequence CWU 1
1
113214PRTArtificial SequenceSynthetic Polypeptide 1Gly Phe Leu
Gly124PRTArtificial SequenceSynthetic Polypeptide 2Lys Val Ser
Arg135PRTArtificial SequenceSynthetic Polypeptide 3Thr Val Gly Leu
Arg1 545PRTArtificial SequenceSynthetic Polypeptide 4Pro Met Gly
Leu Pro1 555PRTArtificial SequenceSynthetic Polypeptide 5Pro Met
Gly Ala Pro1 568PRTArtificial SequenceSynthetic Polypeptide 6Ser
Ile Ile Asn Phe Glu Lys Leu1 5742DNAArtificial SequenceSynthetic
Polynucleotide 7aagcttagcg gccgcaccat gcgggtcacg gcgccccgaa cc
42830DNAArtificial SequenceSynthetic Polynucleotide 8ctgcagggag
ccggcccagg tctcggtcag 30933DNAArtificial SequenceSynthetic
Polynucleotide 9ggatccatcg tgggcattgt tgctggcctg gct
331042DNAArtificial SequenceSynthetic Polynucleotide 10gaattcagtc
tcgagtcaag ctgtgagaga cacatcagag cc 421176PRTHomo sapiens 11Met Gln
Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val
Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25
30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys
35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys
Glu 50 55 60Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly65 70
751241PRTArtificial SequenceSynthetic Polypeptide 12Glu Gly Ala Met
Val Ala Ala Thr Gln Gly Ala Ala Ala Ala Ala Gly1 5 10 15Ser Gly Ala
Gly Thr Gly Gly Gly Thr Ala Ser Gly Gly Thr Glu Gly 20 25 30Gly Ser
Ala Glu Ser Glu Gly Ala Lys 35 401339PRTArtificial
SequenceSynthetic Polypeptide 13Ala Met Val Ala Ala Thr Gln Gly Ala
Ala Ala Ala Ala Gly Ser Gly1 5 10 15Ala Gly Thr Gly Gly Gly Thr Ala
Ser Gly Gly Thr Glu Gly Gly Ser 20 25 30Ala Glu Ser Glu Gly Ala Lys
351426PRTArtificial SequenceSynthetic Polypeptide 14Gly Gly Gly Gly
Gly Gly Gly Gly Ala Gly Ala Ala Gly Gly Gly Gly1 5 10 15Gly Gly Glu
Asn Tyr Asp Asp Pro His Lys 20 251538PRTArtificial
SequenceSynthetic Polypeptide 15Met Val Ala Ala Thr Gln Gly Ala Ala
Ala Ala Ala Gly Ser Gly Ala1 5 10 15Gly Thr Gly Gly Gly Thr Ala Ser
Gly Gly Thr Glu Gly Gly Ser Ala 20 25 30Glu Ser Glu Gly Ala Lys
351617PRTArtificial SequenceSynthetic Polypeptide 16Gln Leu Leu Cys
Gly Ala Ala Ile Gly Thr His Glu Asp Asp Lys Tyr1 5 10
15Arg1722PRTArtificial SequenceSynthetic Polypeptide 17Phe Ser His
His Phe Glu Asp Ala Asp Asn Ile Tyr Ile Phe Leu Glu1 5 10 15Leu Cys
Ser Arg Lys Ser 201815PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 18Tyr Xaa Leu Val Gly Ala Gly Ala Ile Gly Cys Glu Leu
Leu Lys1 5 10 151926PRTArtificial SequenceSynthetic Polypeptide
19Ile Pro Glu Ser Cys Ser Phe Gly Tyr His Ala Gly Gly Trp Gly Lys1
5 10 15Pro Pro Val Asp Glu Thr Gly Lys Pro Leu 20
252036PRTArtificial SequenceSynthetic Polypeptide 20Val Ala Ala Thr
Gln Gly Ala Ala Ala Ala Ala Gly Ser Gly Ala Gly1 5 10 15Thr Gly Gly
Gly Thr Ala Ser Gly Gly Thr Glu Gly Gly Ser Ala Ser 20 25 30Glu Gly
Ala Lys 352122PRTArtificial SequenceSynthetic Polypeptide 21Ser Glu
Ala Asp Ile Glu Gly Pro Leu Pro Ala Lys Asp Ile His Leu1 5 10 15Asp
Leu Pro Ser Asn Asn 202216PRTArtificial SequenceSynthetic
Polypeptide 22His Phe Asn Ala Leu Gly Gly Trp Gly Glu Leu Gln Asn
Ser Val Lys1 5 10 152312PRTArtificial SequenceSynthetic Polypeptide
23Phe Ala Gln Ala Leu Gly Leu Thr Glu Ala Val Lys1 5
102417PRTArtificial SequenceSynthetic Polypeptide 24Thr Ser Val Leu
Ala Ala Ala Asn Pro Ile Glu Ser Gln Trp Asn Pro1 5 10
15Lys2516PRTArtificial SequenceSynthetic Polypeptide 25Gln Leu Leu
Gln Ala Asn Pro Ile Leu Glu Ser Phe Gly Asn Ala Lys1 5 10
152616PRTArtificial SequenceSynthetic Polypeptide 26Thr Ser Ile Leu
Ala Ala Ala Asn Pro Ile Ser Gly His Tyr Asp Arg1 5 10
152715PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(3)Xaa can be any naturally occurring
amino acid 27Ile Xaa Xaa Ala Asn Pro Leu Leu Glu Ala Phe Gly Asn
Ala Lys1 5 10 152817PRTArtificial SequenceSynthetic Polypeptide
28Leu Tyr Gly Ala Gln Phe His Pro Glu Val Gly Leu Thr Glu Asn Gly1
5 10 15Lys2922PRTArtificial SequenceSynthetic Polypeptide 29Pro Gln
Gly Gln Ala Pro Pro Leu Ser Gln Ala Gln Gly His Pro Gly1 5 10 15Ile
Gln Thr Pro Gln Arg 203027PRTArtificial SequenceSynthetic
Polypeptide 30Ala Ala Ala Ser Ala Ala Ala Ala Ser Ala Ala Ser Gly
Ser Pro Gly1 5 10 15Pro Gly Glu Gly Ser Ala Gly Gly Glu Lys Arg 20
253116PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(4)Xaa can be any naturally occurring
amino acid 31Ile Xaa Xaa Xaa Phe Leu Gly Ala Ser Leu Lys Asp Glu
Val Leu Lys1 5 10 153221PRTArtificial SequenceSynthetic Polypeptide
32Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly1
5 10 15Ile Ile Pro Pro His 203322PRTArtificial SequenceSynthetic
Polypeptide 33Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly
His Pro Gly1 5 10 15Ile Ile Pro Pro His Ala 203419PRTArtificial
SequenceSynthetic Polypeptide 34Ile Leu Ile Ser Leu Ala Thr Gly His
Arg Glu Glu Gly Gly Glu Asn1 5 10 15Leu Asp Gln3527PRTArtificial
SequenceSynthetic Polypeptide 35Leu Ser Glu Leu Thr Gln Gln Leu Ala
Gln Ala Thr Gly Lys Pro Pro1 5 10 15Gln Tyr Ile Ala Val His Val Val
Pro Asp Gln 20 253628PRTArtificial SequenceSynthetic Polypeptide
36Leu Ser Glu Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly Lys Pro Pro1
5 10 15Gln Tyr Ile Ala Val His Val Val Pro Asp Gln Leu 20
253719PRTArtificial SequenceSynthetic Polypeptide 37Asp Ala Thr Asn
Val Gly Asp Glu Gly Gly Phe Ala Pro Asn Ile Leu1 5 10 15Glu Asn
Lys3814PRTArtificial SequenceSynthetic Polypeptide 38Ile Leu Ala
Gln Ala Thr Ser Asp Leu Val Asn Ala Ile Lys1 5 103915PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(2)..(3)Xaa can be any
naturally occurring amino acidmisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 39Val Xaa Xaa Val Xaa Gln His Ala
Val Gly Ile Val Val Asn Lys1 5 10 154023PRTArtificial
SequenceSynthetic Polypeptide 40Gly Ser Leu Ala Glu Ala Val Gly Ser
Pro Pro Pro Ala Ala Thr Pro1 5 10 15Thr Pro Thr Pro Pro Thr Arg
204119PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 41Ser Xaa Gly Leu Pro Val Gly Ala Val Ile Asn Cys Ala
Asp Asn Thr1 5 10 15Gly Ala Lys4223PRTArtificial SequenceSynthetic
Polypeptide 42Tyr Cys Phe Ser Glu Met Ala Pro Val Cys Ala Val Val
Gly Gly Ile1 5 10 15Leu Ala Gln Glu Ile Val Lys 204320PRTArtificial
SequenceSynthetic Polypeptide 43His Val Tyr Gly Tyr Ser Met Ala Tyr
Gly Pro Ala Gln His Ala Ile1 5 10 15Ser Thr Glu Lys
204420PRTArtificial SequenceSynthetic Polypeptide 44Leu Trp Gln Leu
Ser Lys Pro Arg Pro Gly Cys Ser Val Leu Gly Pro1 5 10 15Leu Pro Leu
Leu 204518PRTArtificial SequenceSynthetic Polypeptide 45Met Ile Leu
Ile Gln Asp Gly Ser Gln Asn Thr Asn Val Asp Lys Pro1 5 10 15Leu
Arg4622PRTArtificial SequenceSynthetic Polypeptide 46Thr Tyr Ser
Met Val Val Val Pro Leu Tyr Asp Thr Leu Gly Pro Gly1 5 10 15Ala Ile
Arg Tyr Ile Ile 204722PRTArtificial SequenceSynthetic Polypeptide
47His Phe Ala Met Met His Gly Gly Thr Gly Phe Ala Gly Ile Asp Ser1
5 10 15Ser Ser Pro Glu Val Lys 204821PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(2)..(2)Xaa can be any
naturally occurring amino acid 48Gly Xaa Leu Lys Pro Gly Met Val
Val Thr Phe Ala Pro Val Asn Val1 5 10 15Thr Thr Glu Val Lys
204915PRTArtificial SequenceSynthetic Polypeptide 49Phe Asn Ala Leu
Phe Ala Gln Gly Asn Tyr Ser Glu Ala Ala Lys1 5 10
155024PRTArtificial SequenceSynthetic Polypeptide 50Gly Pro Ile His
Ile Gly Gly Pro Pro Gly Phe Ala Ser Ser Ser Gly1 5 10 15Lys Pro Gly
Pro Thr Val Ile Lys 205115PRTArtificial SequenceSynthetic
Polypeptide 51Gly Phe Gly Phe Val Thr Phe Asp Asp His Asp Pro Val
Asp Lys1 5 10 155220PRTArtificial SequenceSynthetic Polypeptide
52Asp Gln Gly Ser Cys Gly Ser Cys Trp Ala Phe Gly Ala Val Glu Ala1
5 10 15Ile Ser Asp Arg 205321PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 53Gly Xaa Asn Phe Gly Phe Gly Asp Ser Arg Gly Gly Gly
Gly Asn Phe1 5 10 15Gly Pro Gly Pro Gly 205419PRTArtificial
SequenceSynthetic Polypeptide 54His Asp Leu Phe Asp Ser Gly Phe Gly
Gly Gly Ala Gly Val Glu Thr1 5 10 15Gly Gly Lys5515PRTArtificial
SequenceSynthetic Polypeptide 55Cys Tyr Leu Phe Gly Gly Leu Ala Asn
Asp Ser Glu Asp Pro Lys1 5 10 155620PRTArtificial SequenceSynthetic
Polypeptide 56Thr Thr Glu Asp Ser Val Met Leu Asn Gly Phe Gly Thr
Val Val Asn1 5 10 15Ala Leu Gly Lys 205720PRTArtificial
SequenceSynthetic Polypeptide 57Leu Thr Glu Gly Leu His Gly Phe His
Val His Glu Phe Gly Asp Asn1 5 10 15Thr Ala Gly Cys
205811PRTArtificial SequenceSynthetic Polypeptide 58Gly Tyr Ala Phe
Ile Glu Tyr Glu His Glu Arg1 5 105913PRTArtificial
SequenceSynthetic Polypeptide 59Met Phe Ile Gly Gly Leu Ser Trp Asp
Thr Ser Lys Lys1 5 106013PRTArtificial SequenceSynthetic
Polypeptide 60Met Phe Ile Gly Gly Leu Ser Trp Asp Thr Thr Lys Lys1
5 106112PRTArtificial SequenceSynthetic Polypeptide 61Ser Met Gly
Phe Ile Gly His Tyr Leu Asp Gln Lys1 5 106212PRTArtificial
SequenceSynthetic Polypeptide 62Ser Met Gly Phe Ile Gly His Tyr Leu
Asp Gln Lys1 5 106321PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 63Ala Leu Xaa Gly Gly Ile Gly Phe Ile His His Asn Cys
Thr Pro Glu1 5 10 15Phe Gln Ala Asn Glu 206419PRTArtificial
SequenceSynthetic Polypeptide 64Asn Leu Gln Ser Thr Phe Ser Gly Phe
Gly Phe Ile Asn Ser Glu Asn1 5 10 15Val Phe Lys6515PRTArtificial
SequenceSynthetic Polypeptide 65Gly Phe Cys Phe Ile Thr Tyr Thr Asp
Glu Glu Pro Val Lys Lys1 5 10 156612PRTArtificial SequenceSynthetic
Polypeptide 66Met Pro Met Phe Ile Val Asn Thr Asn Val Pro Arg1 5
106719PRTArtificial SequenceSynthetic Polypeptide 67Val Ser Glu Ile
Phe Val Glu Leu Gln Gly Phe Leu Ala Ala Glu Gln1 5 10 15Asp Ile
Arg6811PRTArtificial SequenceSynthetic Polypeptide 68Gly Phe Cys
Phe Leu Glu Tyr Glu Asp His Lys1 5 106915PRTArtificial
SequenceSynthetic Polypeptide 69Gln Ala Val Ser Met Phe Leu Gly Ala
Val Glu Glu Ala Lys Lys1 5 10 157017PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 70Lys Pro Xaa Lys Pro Met Gln Phe Leu Gly Asp Glu Glu
Thr Val Arg1 5 10 15Lys7122PRTArtificial SequenceSynthetic
Polypeptide 71Gly Ala Ala Glu Pro His Thr Ile Ala Ala Phe Leu Gly
Gly Ala Ala1 5 10 15Ala Gln Glu Val Ile Lys 207217PRTArtificial
SequenceSynthetic Polypeptide 72Met Ile Pro Cys Asp Phe Leu Ile Pro
Val Gln Thr Gln His Pro Ile1 5 10 15Arg7319PRTArtificial
SequenceSynthetic Polypeptide 73Gln Gly Ala Pro Thr Ser Phe Leu Pro
Pro Glu Ala Ser Gln Leu Lys1 5 10 15Pro Asp Arg7414PRTArtificial
SequenceSynthetic Polypeptide 74Ser Thr Gly Gly Ala Pro Thr Phe Asn
Val Thr Val Thr Lys1 5 107513PRTArtificial SequenceSynthetic
Polypeptide 75Met Val Tyr Met Phe Gln Tyr Asp Ser Thr His Gly Lys1
5 107622PRTArtificial SequenceSynthetic Polypeptide 76His Phe Pro
Met Thr His Gly Asn Thr Gly Phe Ser Gly Ile Glu Ser1 5 10 15Ser Ser
Pro Glu Val Lys 207712PRTArtificial SequenceSynthetic Polypeptide
77Ala Val Ala Phe Ser Pro Val Thr Glu Leu Lys Lys1 5
107822PRTArtificial SequenceSynthetic Polypeptide 78Gly Phe Gly Phe
Val Thr Phe Ser Ser Met Ala Glu Val Asp Ala Ala1 5 10 15Met Ala Ala
Arg Pro His 207915PRTArtificial SequenceSynthetic Polypeptide 79Thr
Cys Gly Phe Asp Phe Thr Gly Ala Val Glu Asp Ile Ser Lys1 5 10
158018PRTArtificial SequenceSynthetic Polypeptide 80Glu Tyr Ser Gly
Leu Ser Asp Gly Tyr Gly Phe Thr Thr Asp Leu Phe1 5 10 15Gly
Arg8123PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 81Gly Gln His Val Xaa Gly Ser Pro Phe Gln Phe Thr Val
Gly Pro Leu1 5 10 15Gly Glu Gly Gly Ala His Lys 208214PRTArtificial
SequenceSynthetic Polypeptide 82Gly Phe Gly Phe Val Asp Phe Asn Ser
Glu Glu Asp Ala Lys1 5 108310PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 83Phe Xaa Phe Val Glu Phe Glu Asp Pro Arg1 5
108410PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 84Phe Xaa Phe Val Glu Phe Glu Asp Pro Arg1 5
108517PRTArtificial SequenceSynthetic Polypeptide 85Ile Glu Leu Phe
Val Gly Gly Glu Leu Ile Asp Pro Ala Asp Asp Arg1 5 10
15Lys8613PRTArtificial SequenceSynthetic Polypeptide 86Met Phe Val
Gly Gly Leu Ser Trp Asp Thr Ser Lys Lys1 5 108716PRTArtificial
SequenceSynthetic Polypeptide 87Ala Phe Ser Ala Phe Val Gly Gln Met
His Gln Gln Gly Ile Leu Lys1 5 10 158817PRTArtificial
SequenceSynthetic Polypeptide 88Gly Ile Leu Phe Val Gly Ser Gly Val
Ser Gly Gly Glu Glu Gly Ala1 5 10 15Arg8918PRTArtificial
SequenceSynthetic Polypeptide 89Ile Ile Ala Phe Val Gly Ser Pro Val
Glu Asp Asn Glu Lys Asp Leu1 5 10 15Val Lys9010PRTArtificial
SequenceSynthetic Polypeptide 90Asp Tyr Ala Phe Val His Phe Glu Asp
Arg1 5 109115PRTArtificial SequenceSynthetic Polypeptide 91Gly Tyr
Ala Phe Val His Phe Glu Thr Gln Glu Ala Ala Asp Lys1 5 10
159215PRTArtificial SequenceSynthetic Polypeptide 92Gly Tyr Gly Phe
Val His Phe Glu Thr Gln Glu Ala Ala Glu Arg1 5 10
159310PRTArtificial SequenceSynthetic Polypeptide 93Asn Tyr Gly Phe
Val His Ile Glu Asp Lys1 5 109418PRTArtificial SequenceSynthetic
Polypeptide 94Ile Thr Leu Pro Val Asp Phe Val Thr Ala Asp Lys Phe
Asp Glu Asn1 5 10 15Ala
Lys9515PRTArtificial SequenceSynthetic Polypeptide 95Gly Phe Gly
Phe Val Thr Phe Asp Asp His Asp Pro Val Asp Lys1 5 10
159617PRTArtificial SequenceSynthetic Polypeptide 96Leu Pro Asn Phe
Gly Phe Val Val Phe Asp Asp Ser Glu Pro Val Gln1 5 10
15Lys9715PRTArtificial SequenceSynthetic Polypeptide 97Gly Phe Gly
Phe Val Tyr Phe Gln Asn His Asp Ala Ala Asp Lys1 5 10
159811PRTArtificial SequenceSynthetic Polypeptide 98Tyr Gln Phe Trp
Asp Thr Gln Pro Val Pro Lys1 5 109917PRTArtificial
SequenceSynthetic Polypeptide 99Gln Leu Leu Cys Gly Ala Ala Ile Gly
Thr His Glu Asp Asp Lys Tyr1 5 10 15Arg10017PRTArtificial
SequenceSynthetic Polypeptide 100Gln Leu Leu Cys Gly Ala Ala Ile
Gly Thr His Glu Asp Asp Lys Tyr1 5 10 15Arg10126PRTArtificial
SequenceSynthetic Polypeptide 101Pro Pro Ala Gly Gly Gly Gly Gly
Ala Gly Gly Ala Gly Gly Gly Pro1 5 10 15Pro Pro Gly Pro Pro Gly Ala
Gly Asp Arg 20 2510221PRTArtificial SequenceSynthetic Polypeptide
102Phe Gly Gly Ser Phe Ala Gly Ser Phe Gly Gly Ala Gly Gly His Ala1
5 10 15Pro Gly Val Ala Arg 2010326PRTArtificial SequenceSynthetic
Polypeptide 103Cys Asn Pro Ile Ile Ser Gly Leu Tyr Gln Gly Ala Gly
Gly Pro Gly1 5 10 15Pro Gly Gly Phe Gly Ala Gln Gly Pro Lys 20
2510422PRTArtificial SequenceSynthetic Polypeptide 104Pro Gly Leu
Asn Leu Pro Pro Pro Ile Gly Gly Ala Gly Pro Pro Leu1 5 10 15Gly Leu
Pro Lys Pro Lys 2010525PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 105Gln Pro Xaa Val Asp Gly Phe Leu Val Gly Gly Ala Ser
Leu Lys Pro1 5 10 15Glu Phe Val Asp Ile Ile Asn Ala Lys 20
2510615PRTArtificial SequenceSynthetic Polypeptide 106Val Thr Gly
Asp His Ile Pro Thr Pro Gln Asp Leu Pro Gln Arg1 5 10
1510712PRTArtificial SequenceSynthetic Polypeptide 107Tyr Gly Gly
Glu Leu Val Pro His Phe Pro Ala Arg1 5 1010818PRTArtificial
SequenceSynthetic Polypeptide 108Tyr Gln Gly Ala Gly Gly Pro Gly
Pro Gly Gly Phe Gly Ala Gln Gly1 5 10 15Pro Lys10919PRTArtificial
SequenceSynthetic Polypeptide 109Glu Tyr Phe Gly Gly Phe Gly Glu
Val Glu Ser Ile Glu Leu Pro Met1 5 10 15Asp Asn
Lys11015PRTArtificial SequenceSynthetic Polypeptide 110Ala Leu Val
Leu Gly Gly Phe Ala His Met Asp Thr Glu Thr Lys1 5 10
1511118PRTArtificial SequenceSynthetic Polypeptide 111Val Ser His
Val Ser Thr Gly Gly Gly Ala Ser Leu Glu Leu Leu Glu1 5 10 15Gly
Lys11219PRTArtificial SequenceSynthetic Polypeptide 112Ala Glu Gly
Gly Gly Gly Gly Gly Arg Pro Gly Ala Pro Ala Ala Gly1 5 10 15Asp Gly
Lys11317PRTArtificial SequenceSynthetic Polypeptide 113Arg Gly Gly
Gly Gly Gly Gly Ser Gly Gly Ile Gly Tyr Pro Tyr Pro1 5 10
15Arg11423PRTArtificial SequenceSynthetic Polypeptide 114Asn Met
Gly Gly Pro Tyr Gly Gly Gly Asn Tyr Gly Pro Gly Gly Ser1 5 10 15Gly
Gly Ser Gly Gly Tyr Gly 2011521PRTArtificial SequenceSynthetic
Polypeptide 115Gly Thr Gly Gly Val Asp Thr Ala Ala Thr Gly Gly Val
Phe Asp Ile1 5 10 15Ser Asn Leu Asp Arg 2011616PRTArtificial
SequenceSynthetic Polypeptide 116His Phe Asn Ala Leu Gly Gly Trp
Gly Glu Leu Gln Asn Ser Val Lys1 5 10 1511725PRTArtificial
SequenceSynthetic Polypeptide 117Pro Glu Ser Cys Ser Phe Gly Tyr
His Ala Gly Gly Trp Gly Lys Pro1 5 10 15Pro Val Asp Glu Thr Gly Lys
Pro Leu 20 2511815PRTArtificial SequenceSynthetic Polypeptide
118Ser Ser Leu Pro Asn Phe Cys Gly Ile Phe Asn His Leu Glu Arg1 5
10 1511919PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 119Ala Met Ala Leu Xaa Gly Gly Ile Gly Phe Ile His His
Asn Cys Thr1 5 10 15Pro Glu Phe12023PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 120Ala Met Ala Leu Xaa Gly Gly Ile Gly Phe Ile His His
Asn Cys Thr1 5 10 15Pro Glu Phe Gln Ala Asn Glu
2012122PRTArtificial SequenceSynthetic Polypeptide 121Glu Trp Ile
Lys Pro Ile Met Phe Ser Gly Gly Ile Gly Ser Met Glu1 5 10 15Ala Asp
His Ile Ser Lys 2012214PRTArtificial SequenceSynthetic Polypeptide
122Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys1 5
1012319PRTArtificial SequenceSynthetic Polypeptide 123Glu Met Ala
Pro Val Cys Ala Val Val Gly Gly Ile Leu Ala Gln Glu1 5 10 15Ile Val
Lys12414PRTArtificial SequenceSynthetic Polypeptide 124Leu Ala Phe
His Gly Ile Leu Leu His Gly Leu Glu Asp Arg1 5 1012514PRTArtificial
SequenceSynthetic Polypeptide 125Met Gly Val Val Ala Gly Ile Leu
Val Gln Asn Val Leu Lys1 5 1012620PRTArtificial SequenceSynthetic
Polypeptide 126Phe Thr Ala Ser Ala Gly Ile Gln Val Val Gly Asp Asp
Leu Thr Val1 5 10 15Thr Asn Pro Lys 2012721PRTArtificial
SequenceSynthetic Polypeptide 127Thr Pro Tyr Gln Ile Ala Cys Gly
Ile Ser Gln Gly Leu Ala Asp Asn1 5 10 15Thr Val Ile Ala Lys
2012816PRTArtificial SequenceSynthetic Polypeptide 128Tyr Pro Ile
Glu His Gly Ile Val Thr Asn Trp Asp Asp Met Glu Lys1 5 10
1512921PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(10)..(10)Xaa can be any naturally occurring
amino acid 129Val Ala Ser Gly Ile Pro Ala Gly Trp Xaa Gly Leu Asp
Cys Gly Pro1 5 10 15Glu Ser Ser Lys Lys 2013016PRTArtificial
SequenceSynthetic Polypeptide 130Leu Phe Val Gly Gly Leu Asp Trp
Ser Thr Thr Gln Glu Thr Leu Arg1 5 10 1513116PRTArtificial
SequenceSynthetic Polypeptide 131His Gly Gly Ser Leu Gly Leu Gly
Leu Ala Ala Met Gly Thr Ala Arg1 5 10 1513216PRTArtificial
SequenceSynthetic Polypeptide 132Ile Phe Val Gly Gly Leu Ser Ala
Asn Thr Val Val Glu Asp Val Lys1 5 10 1513316PRTArtificial
SequenceSynthetic Polypeptide 133Leu Phe Ile Gly Gly Leu Ser Phe
Glu Thr Thr Asp Asp Ser Leu Arg1 5 10 1513416PRTArtificial
SequenceSynthetic Polypeptide 134Leu Phe Ile Gly Gly Leu Ser Phe
Glu Thr Thr Asp Glu Ser Leu Arg1 5 10 1513516PRTArtificial
SequenceSynthetic Polypeptide 135Leu Phe Ile Gly Gly Leu Ser Phe
Glu Thr Thr Glu Glu Ser Leu Arg1 5 10 1513613PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(3)..(3)Xaa can be any
naturally occurring amino acid 136Met Phe Xaa Gly Gly Leu Ser Trp
Asp Thr Ser Lys Lys1 5 1013713PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 137Met Phe Xaa Gly Gly Leu Ser Trp Asp Thr Ser Lys Lys1
5 1013813PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 138Met Phe Xaa Gly Gly Leu Ser Trp Asp Thr Ser Lys Lys1
5 1013915PRTArtificial SequenceSynthetic Polypeptide 139Asp Ala Val
Ser Gly Met Gly Val Ile Val His Ile Ile Glu Lys1 5 10
1514010PRTArtificial SequenceSynthetic Polypeptide 140Gly Gly Asn
Phe Gly Phe Gly Asp Ser Arg1 5 1014126PRTArtificial
SequenceSynthetic Polypeptide 141Gly Thr Thr Gly Ser Gly Ala Gly
Ser Gly Gly Pro Gly Gly Leu Thr1 5 10 15Ser Ala Ala Pro Ala Gly Gly
Asp Lys Lys 20 2514223PRTArtificial SequenceSynthetic Polypeptide
142Ile Ile Ser Gly Leu Tyr Gln Gly Ala Gly Gly Pro Gly Pro Gly Gly1
5 10 15Phe Gly Ala Gln Gly Pro Lys 2014323PRTArtificial
SequenceSynthetic Polypeptide 143Ile Ile Ser Gly Leu Tyr Gln Gly
Ala Gly Gly Pro Gly Pro Gly Gly1 5 10 15Phe Gly Ala Gln Gly Pro Lys
2014422PRTArtificial SequenceSynthetic Polypeptide 144Gly Gly Gly
Leu Leu Ile Gly Gly Gln Ala Trp Asp Trp Ala Asn Gln1 5 10 15Gly Glu
Asp Glu Arg Val 2014523PRTArtificial SequenceSynthetic Polypeptide
145Gly Asn Phe Gly Gly Ser Phe Ala Gly Ser Phe Gly Gly Ala Gly Gly1
5 10 15His Ala Pro Gly Val Ala Arg 2014622PRTArtificial
SequenceSynthetic Polypeptide 146Asn Phe Gly Gly Ser Phe Ala Gly
Ser Phe Gly Gly Ala Gly Gly His1 5 10 15Ala Pro Gly Val Ala Arg
2014722PRTArtificial SequenceSynthetic Polypeptide 147Asn Phe Gly
Gly Ser Phe Ala Gly Ser Phe Gly Gly Ala Gly Gly His1 5 10 15Ala Pro
Gly Val Ala Arg 2014822PRTArtificial SequenceSynthetic Polypeptide
148Asn Phe Gly Gly Ser Phe Ala Gly Ser Phe Gly Gly Ala Gly Gly His1
5 10 15Ala Pro Gly Val Ala Arg 2014933PRTArtificial
SequenceSynthetic Polypeptide 149Ser Ala Ala Asp Thr Lys Pro Gly
Thr Thr Gly Ser Gly Ala Gly Ser1 5 10 15Gly Gly Pro Gly Gly Leu Thr
Ser Ala Ala Pro Ala Gly Gly Asp Lys 20 25 30Lys15036PRTArtificial
SequenceSynthetic Polypeptide 150Ala Ala Thr Gln Gly Ala Ala Ala
Ala Ala Gly Ser Gly Ala Gly Thr1 5 10 15Gly Gly Gly Thr Ala Ser Gly
Gly Thr Glu Gly Gly Ser Ala Glu Ser 20 25 30Glu Gly Ala Lys
3515119PRTArtificial SequenceSynthetic Polypeptide 151Gly Ser Ser
Gly Gly Ser Gly Ala Lys Pro Ser Asp Ala Ala Ser Glu1 5 10 15Ala Ala
Arg15221PRTArtificial SequenceSynthetic Polypeptide 152Ile Gln Phe
His Phe His Trp Gly Ser Leu Asp Gly Gln Gly Ser Glu1 5 10 15His Thr
Val Asp Lys 2015318PRTArtificial SequenceSynthetic Polypeptide
153Met Ile Leu Ile Gln Asp Gly Ser Gln Asn Thr Asn Val Asp Lys Pro1
5 10 15Leu Arg15410PRTArtificial SequenceSynthetic Polypeptide
154Lys Gly Thr Phe Thr Asp Asp Leu His Lys1 5 1015525PRTArtificial
SequenceSynthetic Polypeptide 155Gln Gln Ser His Phe Ala Met Met
His Gly Gly Thr Gly Phe Ala Gly1 5 10 15Ile Asp Ser Ser Ser Pro Glu
Val Lys 20 2515620PRTArtificial SequenceSynthetic Polypeptide
156Val Ala Val Leu Ile Ser Gly Thr Gly Ser Asn Leu Gln Ala Leu Ile1
5 10 15Asp Ser Thr Arg 2015716PRTArtificial SequenceSynthetic
Polypeptide 157Phe Leu Ala Ala Gly Thr His Leu Gly Gly Thr Asn Leu
Asp Phe Gln1 5 10 1515814PRTArtificial SequenceSynthetic
Polypeptide 158Leu Val Leu Gly Thr His Thr Ser Asp Glu Gln Asn His
Leu1 5 1015920PRTArtificial SequenceSynthetic Polypeptide 159Thr
Gly Gly Val Asp Thr Ala Ala Thr Gly Gly Val Phe Asp Ile Ser1 5 10
15Asn Leu Asp Arg 2016020PRTArtificial SequenceSynthetic
Polypeptide 160Thr Gly Gly Val Asp Thr Ala Ala Val Gly Gly Val Phe
Asp Val Ser1 5 10 15Asn Ala Asp Arg 2016118PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(3)..(3)Xaa can be any
naturally occurring amino acid 161Ala Val Xaa Ile Val Ala Ala Gly
Val Gly Glu Phe Glu Ala Gly Ile1 5 10 15Ser Lys16220PRTArtificial
SequenceSynthetic Polypeptide 162Glu Ile Leu Thr Leu Leu Gln Gly
Val His Gln Gly Ala Gly Phe Gln1 5 10 15Asp Ile Pro Lys
2016317PRTArtificial SequenceSynthetic Polypeptide 163Met Lys Pro
Leu Met Gly Val Ile Tyr Val Pro Leu Thr Asp Lys Glu1 5 10
15Lys16420PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 164Glu Cys Ile Ser Xaa His Val Gly Gln Ala Gly Val Gln
Ile Gly Asn1 5 10 15Ala Cys Trp Glu 2016516PRTArtificial
SequenceSynthetic Polypeptide 165His Phe Asn Ala Leu Gly Gly Trp
Gly Glu Leu Gln Asn Ser Val Lys1 5 10 1516624PRTArtificial
SequenceSynthetic Polypeptide 166Glu Ser Cys Ser Phe Gly Tyr His
Ala Gly Gly Trp Gly Lys Pro Pro1 5 10 15Val Asp Glu Thr Gly Lys Pro
Leu 201679PRTArtificial SequenceSynthetic Polypeptide 167Ala Gly
Tyr Val Thr His Leu Met Lys1 516826PRTArtificial SequenceSynthetic
Polypeptide 168Thr Met Phe Ser Ser Glu Val Gln Phe Gly His Ala Gly
Ala Cys Ala1 5 10 15Asn Gln Ala Ser Glu Thr Ala Val Ala Lys 20
2516917PRTArtificial SequenceSynthetic Polypeptide 169Met Pro Phe
Pro Val Asn His Gly Ala Ser Ser Glu Asp Thr Leu Leu1 5 10
15Lys17018PRTArtificial SequenceSynthetic Polypeptide 170Phe Phe
Leu His His Leu Ile Ala Glu Ile His Thr Ala Glu Ile Arg1 5 10 15Ala
Thr17117PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acidmisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 171Asn Xaa Ser Ala Xaa Gln Val Leu Ile Glu His Ile Gly
Asn Leu Asp1 5 10 15Arg17215PRTArtificial SequenceSynthetic
Polypeptide 172Gly Gly Tyr Val Leu His Ile Gly Thr Ile Tyr Gly Asp
Leu Lys1 5 10 1517312PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 173Asp Xaa His Leu Gly Gly Glu Asp Phe Asp Asn Arg1 5
1017420PRTArtificial SequenceSynthetic Polypeptide 174Gly Ile Leu
Gly Pro Pro Pro Pro Ser Phe His Leu Gly Gly Pro Ala1 5 10 15Val Gly
Pro Arg 2017519PRTArtificial SequenceSynthetic Polypeptide 175Pro
Thr Pro Pro Pro Thr Leu His Leu Val Pro Glu Pro Ala Ala Pro1 5 10
15Pro Pro Pro17625PRTArtificial SequenceSynthetic Polypeptide
176Tyr Gly Pro Gln Tyr Gly His Pro Pro Pro Pro Pro Pro Pro Pro Glu1
5 10 15Tyr Gly Pro His Ala Asp Ser Pro Val 20 2517717PRTArtificial
SequenceSynthetic Polypeptide 177Lys His Ser Gly Pro Asn Ser Ala
Asp Ser Ala Asn Asp Gly Phe Val1 5 10 15Arg17816PRTArtificial
SequenceSynthetic Polypeptide 178Arg Pro Glu Leu Leu Thr His Ser
Thr Thr Glu Val Thr Gln Pro Arg1 5 10 1517917PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(2)..(2)Xaa can be any
naturally occurring amino acid 179Leu Xaa Gly His Val Gly Phe Asp
Ser Leu Pro Asp Gln Leu Val Asn1 5 10 15Lys18019PRTArtificial
SequenceSynthetic Polypeptide 180Ala Ala Ser Ala Thr Gln Thr Ile
Ala Ala Ala Gln His Ala Ala Ser1 5 10 15Thr Pro
Lys18114PRTArtificial SequenceSynthetic Polypeptide 181Cys Leu Thr
Gln Ser Gly Ile Ala Gly Gly Tyr Lys Pro Phe1 5 1018218PRTArtificial
SequenceSynthetic Polypeptide 182Glu Leu Ala Gln Ile Ala Gly Arg
Pro Thr Glu Asp Glu Asp Glu Lys1 5 10 15Glu Lys18316PRTArtificial
SequenceSynthetic Polypeptide 183Ala Ile Thr Ile Ala Gly Val Pro
Gln Ser Val Thr Glu Cys Val Lys1 5 10 1518412PRTArtificial
SequenceSynthetic Polypeptide 184Gly Leu Cys Ala Ile Ala Gln Ala
Glu Ser Leu Arg1 5 1018521PRTArtificial SequenceSynthetic
Polypeptide 185Lys Pro Thr Ala Leu
Ile Gly Val Ala Ala Ile Gly Gly Ala Phe Ser1 5 10 15Glu Gln Ile Leu
Lys 2018621PRTArtificial SequenceSynthetic Polypeptide 186Asp Tyr
Met Asn Val Gln Cys His Ala Cys Ile Gly Gly Thr Asn Val1 5 10 15Gly
Glu Asp Ile Arg 2018723PRTArtificial SequenceSynthetic Polypeptide
187Asn Thr Gln Asn Phe Gln Ser Leu His Asn Ile Gly Ser Val Val Gln1
5 10 15His Ser Glu Gly Lys Pro Leu 2018822PRTArtificial
SequenceSynthetic Polypeptide 188Leu Lys Pro Pro Thr Leu Ile His
Gly Gln Ala Pro Ser Ala Gly Leu1 5 10 15Pro Ser Gln Lys Pro Lys
2018913PRTArtificial SequenceSynthetic Polypeptide 189Val Leu Ile
Ile Gly Gly Gly Asp Gly Gly Val Leu Arg1 5 1019017PRTArtificial
SequenceSynthetic Polypeptide 190Gly Cys Ile Thr Ile Ile Gly Gly
Gly Asp Thr Ala Thr Cys Cys Ala1 5 10 15Lys19117PRTArtificial
SequenceSynthetic Polypeptide 191Gly Arg Pro Ser Glu Thr Gly Ile
Ile Gly Ile Ile Asp Pro Glu Cys1 5 10 15Arg19220PRTArtificial
SequenceSynthetic Polypeptide 192Glu Ala Phe Gly Trp His Ala Ile
Ile Val Asp Gly His Ser Val Glu1 5 10 15Glu Leu Cys Lys
2019317PRTArtificial SequenceSynthetic Polypeptide 193Leu Ala Ala
Ala Ile Leu Gly Gly Val Asp Gln Ile His Ile Lys Pro1 5 10
15Gly19414PRTArtificial SequenceSynthetic Polypeptide 194Leu Tyr
Ser Ile Leu Gly Thr Thr Leu Lys Asp Glu Gly Lys1 5
1019518PRTArtificial SequenceSynthetic Polypeptide 195Met Ile Leu
Ile Gln Asp Gly Ser Gln Asn Thr Asn Val Asp Lys Pro1 5 10 15Leu
Arg19617PRTArtificial SequenceSynthetic Polypeptide 196Leu Ala Met
Gln Glu Phe Met Ile Leu Pro Val Gly Ala Ala Asn Phe1 5 10
15Arg19718PRTArtificial SequenceSynthetic Polypeptide 197Val Pro
Tyr Leu Ile Ala Gly Ile Gln His Ser Cys Gln Asp Ile Gly1 5 10 15Ala
Lys19818PRTArtificial SequenceSynthetic Polypeptide 198Thr Val Ala
Gly Gly Val His Ile Ser Gly Leu His Thr Glu Ser Ala1 5 10 15Pro
Arg19920PRTArtificial SequenceSynthetic Polypeptide 199Val Ala Val
Leu Ile Ser Gly Thr Gly Ser Asn Leu Gln Ala Leu Ile1 5 10 15Asp Ser
Thr Arg 2020025PRTArtificial SequenceSynthetic Polypeptide 200Gly
Ile Thr Ala Ile Gly Gly Thr Ser Thr Ile Ser Ser Glu Gly Thr1 5 10
15Gln His Ser Tyr Ser Glu Glu Glu Lys 20 2520118PRTArtificial
SequenceSynthetic Polypeptide 201Ala Gly Val Ser Ile Ser Val Val
His Gly Asn Leu Ser Glu Glu Ala1 5 10 15Ala Lys20226PRTArtificial
SequenceSynthetic Polypeptide 202His Val Thr Gln Ala His Val Gln
Thr Gly Ile Thr Ala Ala Pro Pro1 5 10 15Pro His Pro Gly Ala Pro His
Pro Pro Gln 20 2520321PRTArtificial SequenceSynthetic Polypeptide
203Ala Gly Leu Phe Leu Pro Gly Ser Val Gly Ile Thr Asp Pro Cys Glu1
5 10 15Ser Gly Asn Phe Arg 2020416PRTArtificial SequenceSynthetic
Polypeptide 204Ala Phe Ala His Ile Thr Gly Gly Gly Leu Leu Glu Asn
Ile Pro Arg1 5 10 1520512PRTArtificial SequenceSynthetic
Polypeptide 205Ile Leu Ala Gln Ile Thr Gly Thr Glu His Leu Lys1 5
1020616PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 206Thr Phe Xaa Asn Ile Thr Pro Ala Glu Val Gly Val Leu
Val Gly Lys1 5 10 1520715PRTArtificial SequenceSynthetic
Polypeptide 207His Ser Ser Gly Ile Val Ala Asp Leu Ser Glu Gln Ser
Leu Lys1 5 10 1520823PRTArtificial SequenceSynthetic Polypeptide
208Glu Asp Gly Asn Glu Glu Asp Lys Glu Asn Gln Gly Asp Glu Thr Gln1
5 10 15Gly Gln Gln Pro Pro Gln Arg 2020922PRTArtificial
SequenceSynthetic Polypeptide 209Pro Gly Pro Ser Gly Ile Thr Ile
Pro Gly Lys Pro Gly Ala Gln Gly1 5 10 15Val Pro Gly Pro Pro Gly
2021022PRTArtificial SequenceSynthetic Polypeptide 210Gly Leu Thr
Lys Pro Ala Ala Leu Ala Ala Ala Pro Ala Lys Pro Gly1 5 10 15Gly Ala
Gly Gly Ser Lys 2021113PRTArtificial SequenceSynthetic Polypeptide
211Leu Gly Ala Gln Leu Ala Asp Leu His Leu Asp Asn Lys1 5
1021218PRTArtificial SequenceSynthetic Polypeptide 212Ser Leu Val
Ala Ser Leu Ala Glu Pro Asp Phe Val Val Thr Asp Phe1 5 10 15Ala
Lys21319PRTArtificial SequenceSynthetic Polypeptide 213Met Ser Leu
Pro Leu Leu Ala Gly Gly Val Ala Asp Asp Ile Asn Thr1 5 10 15Asn Lys
Lys21422PRTArtificial SequenceSynthetic Polypeptide 214Gln Pro Tyr
Ala Val Ser Glu Leu Ala Gly His Gln Thr Ser Ala Glu1 5 10 15Ser Trp
Gly Thr Gly Arg 2021514PRTArtificial SequenceSynthetic Polypeptide
215Val Thr Val Ala Gly Leu Ala Gly Lys Asp Pro Val Gln Cys1 5
1021613PRTArtificial SequenceSynthetic Polypeptide 216Ile Ile Thr
Leu Ala Gly Pro Thr Asn Ala Ile Phe Lys1 5 1021714PRTArtificial
SequenceSynthetic Polypeptide 217Ser Thr His Gly Leu Ala Ile Leu
Gly Pro Glu Asn Pro Lys1 5 1021815PRTArtificial SequenceSynthetic
Polypeptide 218Ala Ser Ala Glu Leu Ala Leu Gly Glu Asn Ser Glu Val
Leu Lys1 5 10 1521919PRTArtificial SequenceSynthetic Polypeptide
219Ile Leu Ile Ser Leu Ala Thr Gly His Arg Glu Glu Gly Gly Glu Asn1
5 10 15Leu Asp Gln22018PRTArtificial SequenceSynthetic Polypeptide
220Ala Met Ser Arg Pro Phe Gly Val Ala Leu Leu Phe Gly Gly Val Asp1
5 10 15Glu Lys22121PRTArtificial SequenceSynthetic Polypeptide
221Leu Gln Ala Thr Ala His Ala Gln Ala Gln Leu Gly Cys Pro Val Ile1
5 10 15Ile His Pro Gly Arg 2022216PRTArtificial SequenceSynthetic
Polypeptide 222Ile Leu Ala Gly Leu Gly Phe Asp Pro Glu Met Gln Asn
Arg Pro Thr1 5 10 1522332PRTArtificial SequenceSynthetic
Polypeptide 223Pro Glu Arg Pro Gln Gln Leu Pro His Gly Leu Gly Gly
Ile Gly Met1 5 10 15Gly Leu Gly Pro Gly Gly Gln Pro Ile Asp Ala Asn
His Leu Asn Lys 20 25 3022424PRTArtificial SequenceSynthetic
Polypeptide 224Gln Leu Met Gln Leu Ile Gly Pro Ala Gly Leu Gly Gly
Leu Gly Gly1 5 10 15Leu Gly Ala Leu Thr Gly Pro Gly
2022516PRTArtificial SequenceSynthetic Polypeptide 225His Phe Asn
Ala Leu Gly Gly Trp Gly Glu Leu Gln Asn Ser Val Lys1 5 10
1522611PRTArtificial SequenceSynthetic Polypeptide 226Met Gly Ala
Gly Leu Gly His Gly Met Asp Arg1 5 1022722PRTArtificial
SequenceSynthetic Polypeptide 227Thr His Met Thr Ala Ile Val Gly
Met Ala Leu Gly His Arg Pro Ile1 5 10 15Pro Asn Gln Pro Pro Thr
2022825PRTArtificial SequenceSynthetic Polypeptide 228Pro His Gly
Leu Gly Gly Ile Gly Met Gly Leu Gly Pro Gly Gly Gln1 5 10 15Pro Ile
Asp Ala Asn His Leu Asn Lys 20 2522919PRTArtificial
SequenceSynthetic Polypeptide 229Ala Ser Gln Gly Asp Ser Ile Ser
Ser Gln Leu Gly Pro Ile His Pro1 5 10 15Pro Pro
Arg23018PRTArtificial SequenceSynthetic Polypeptide 230Val Trp Gln
Leu Gly Ser Ser Ser Pro Asn Phe Thr Leu Glu Gly His1 5 10 15Glu
Lys23112PRTArtificial SequenceSynthetic Polypeptide 231Tyr Val Ala
Thr Leu Gly Val Glu Val His Pro Leu1 5 1023213PRTArtificial
SequenceSynthetic Polypeptide 232Lys Leu Ile Ala Asp Tyr Ser Pro
Asp Asp Ile Phe Asn1 5 1023313PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 233Thr Xaa Gly Leu Ile Phe Val Val Asp Ser Asn Asp Arg1
5 1023417PRTArtificial SequenceSynthetic Polypeptide 234Val Pro Glu
Phe Gln Phe Leu Ile Gly Asp Glu Ala Ala Thr His Leu1 5 10
15Lys23518PRTArtificial SequenceSynthetic Polypeptide 235Cys Asn
Ile Asn Leu Leu Pro Leu Pro Asp Pro Ile Pro Ser Gly Leu1 5 10 15Met
Glu23622PRTArtificial SequenceSynthetic Polypeptide 236Leu Ile Thr
Glu Met Val Ala Leu Asn Pro Asp Phe Lys Pro Pro Ala1 5 10 15Asp Tyr
Lys Pro Pro Ala 2023715PRTArtificial SequenceSynthetic Polypeptide
237Asn Gln Val Ala Leu Asn Pro Gln Asn Thr Val Phe Asp Ala Lys1 5
10 1523815PRTArtificial SequenceSynthetic Polypeptide 238Gly Leu
Leu Lys Pro Gly Leu Asn Val Val Leu Glu Gly Pro Lys1 5 10
1523917PRTArtificial SequenceSynthetic Polypeptide 239Gly Val Asn
Leu Pro Gly Ala Ala Val Asp Leu Pro Ala Val Ser Glu1 5 10
15Lys24020PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 240Ile Ser Xaa Gly Leu Pro Val Gly Ala Val Ile Asn Cys
Ala Asp Asn1 5 10 15Thr Gly Ala Lys 2024114PRTArtificial
SequenceSynthetic Polypeptide 241Gly Gln Val Cys Leu Pro Val Ile
Ser Ala Glu Asn Trp Lys1 5 1024220PRTArtificial SequenceSynthetic
Polypeptide 242Glu Ile Leu Thr Leu Leu Gln Gly Val His Gln Gly Ala
Gly Phe Gln1 5 10 15Asp Ile Pro Lys 2024320PRTArtificial
SequenceSynthetic Polypeptide 243Asn Asn Gln Phe Gln Ala Leu Leu
Gln Tyr Ala Asp Pro Val Ser Ala1 5 10 15Gln His Ala Lys
2024416PRTArtificial SequenceSynthetic Polypeptide 244Leu Phe Ile
Gly Gly Leu Ser Phe Glu Thr Thr Asp Asp Ser Leu Arg1 5 10
1524514PRTArtificial SequenceSynthetic Polypeptide 245Ala Ile Gln
Leu Ser Gly Ala Glu Gln Leu Glu Ala Leu Lys1 5 1024620PRTArtificial
SequenceSynthetic Polypeptide 246Asp Val Ser Ile Glu Asp Ser Val
Ile Ser Leu Ser Gly Asp His Cys1 5 10 15Ile Ile Gly Arg
2024716PRTArtificial SequenceSynthetic Polypeptide 247Glu Tyr Leu
Leu Ser Gly Asp Ile Ser Glu Ala Glu His Cys Leu Lys1 5 10
1524820PRTArtificial SequenceSynthetic Polypeptide 248Val Val Ile
Ser Ser Asp Gly Gln Phe Ala Leu Ser Gly Ser Trp Asp1 5 10 15Gly Thr
Leu Arg 2024917PRTArtificial SequenceSynthetic Polypeptide 249Val
His Glu Gln Leu Ala Ala Leu Ser Gln Gly Pro Ile Ser Lys Pro1 5 10
15Lys25023PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acidmisc_feature(5)..(6)Xaa can be any naturally occurring
amino acid 250Leu Val Xaa Leu Xaa Xaa Glu Thr Ala Leu Leu Ser Ser
Gly Phe Ser1 5 10 15Leu Glu Asp Pro Gln Thr His
2025117PRTArtificial SequenceSynthetic Polypeptide 251Gly Pro Asp
Gly Leu Thr Ala Phe Glu Ala Thr Asp Asn Gln Ala Ile1 5 10
15Lys25218PRTArtificial SequenceSynthetic Polypeptide 252Ala Leu
Tyr Trp Leu Ser Gly Leu Thr Cys Thr Glu Gln Asn Phe Ile1 5 10 15Ser
Lys25313PRTArtificial SequenceSynthetic Polypeptide 253Ile Ile Thr
Leu Thr Gly Pro Thr Asn Ala Ile Phe Lys1 5 1025413PRTArtificial
SequenceSynthetic Polypeptide 254Leu Ala Thr Gln Leu Thr Gly Pro
Val Met Pro Val Arg1 5 1025514PRTArtificial SequenceSynthetic
Polypeptide 255Phe Pro Ser Leu Leu Thr His Asn Glu Asn Met Val Ala
Lys1 5 1025619PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acidmisc_feature(5)..(5)Xaa can be any naturally occurring
amino acidmisc_feature(9)..(9)Xaa can be any naturally occurring
amino acid 256Leu Glu Xaa Leu Xaa Thr Ile Asn Xaa Gly Leu Thr Ser
Ile Ala Asn1 5 10 15Leu Pro Lys25715PRTArtificial SequenceSynthetic
Polypeptide 257Ala Leu Leu Leu Leu Leu Val Gly Gly Val Asp Gln Ser
Pro Arg1 5 10 1525818PRTArtificial SequenceSynthetic Polypeptide
258Gly Lys Pro Val Gly Leu Val Gly Val Thr Glu Leu Ser Asp Ala Gln1
5 10 15Lys Lys25915PRTArtificial SequenceSynthetic Polypeptide
259Val Asn Val Ala Gly Leu Val Leu Ala Gly Ser Ala Asp Phe Lys1 5
10 1526019PRTArtificial SequenceSynthetic Polypeptide 260Gln Gly
Tyr Ile Gly Ala Ala Leu Val Leu Gly Gly Val Asp Val Thr1 5 10 15Gly
Pro His26114PRTArtificial SequenceSynthetic Polypeptide 261Leu Tyr
Thr Leu Val Leu Thr Asp Pro Asp Ala Pro Ser Arg1 5
1026214PRTArtificial SequenceSynthetic Polypeptide 262Ala Gln Ile
His Asp Leu Val Leu Val Gly Gly Ser Thr Arg1 5 1026320PRTArtificial
SequenceSynthetic Polypeptide 263Leu Asn His Val Ala Ala Gly Leu
Val Ser Pro Ser Leu Lys Ser Asp1 5 10 15Thr Ser Ser Lys
2026416PRTArtificial SequenceSynthetic Polypeptide 264Ile Glu Val
Gly Leu Val Val Gly Asn Ser Gln Val Ala Phe Glu Lys1 5 10
1526521PRTArtificial SequenceSynthetic Polypeptide 265Gly Tyr His
Gln Ser Ala Ser Glu His Gly Leu Val Val Ile Ala Pro1 5 10 15Asp Thr
Ser Pro Arg 2026621PRTArtificial SequenceSynthetic Polypeptide
266Gly Tyr His Gln Ser Ala Ser Glu His Gly Leu Val Val Ile Ala Pro1
5 10 15Asp Thr Ser Pro Arg 2026720PRTArtificial SequenceSynthetic
Polypeptide 267Gln Asp His Pro Trp Leu Leu Ser Gln Asn Leu Val Val
Lys Pro Asp1 5 10 15Gln Leu Ile Lys 2026815PRTArtificial
SequenceSynthetic Polypeptide 268Met Gly Leu Ala Met Gly Gly Gly
Gly Gly Ala Ser Phe Asp Arg1 5 10 1526927PRTArtificial
SequenceSynthetic Polypeptide 269Gln Leu Pro His Gly Leu Gly Gly
Ile Gly Met Gly Leu Gly Pro Gly1 5 10 15Gly Gln Pro Ile Asp Ala Asn
His Leu Asn Lys 20 2527016PRTArtificial SequenceSynthetic
Polypeptide 270Val Val Val Leu Met Gly Ser Thr Ser Asp Leu Gly His
Cys Glu Lys1 5 10 1527115PRTArtificial SequenceSynthetic
Polypeptide 271Met Ala Leu Ile Gln Met Gly Ser Val Glu Glu Ala Val
Gln Ala1 5 10 1527215PRTArtificial SequenceSynthetic Polypeptide
272Thr Thr Gly Phe Gly Met Ile Tyr Asp Ser Leu Asp Tyr Ala Lys1 5
10 1527316PRTArtificial SequenceSynthetic Polypeptide 273Trp Leu
Leu Ala Glu Met Leu Gly Asp Leu Ser Asp Ser Gln Leu Lys1 5 10
1527417PRTArtificial SequenceSynthetic Polypeptide 274Gln Ala Gln
Tyr Leu Gly Met Ser Cys Asp Gly Pro Phe Lys Pro Asp1 5 10
15His27513PRTArtificial SequenceSynthetic Polypeptide 275Ala His
Ser Ser Met Val Gly Val Asn Leu Pro Gln Lys1 5 1027616PRTArtificial
SequenceSynthetic Polypeptide 276Ser Gly Pro Val Val Ala Met Val
Trp Glu Gly Leu Asn Val Val Lys1 5 10 1527724PRTArtificial
SequenceSynthetic Polypeptide 277Val Asn Thr Gln Asn Phe Gln Ser
Leu His Asn Ile Gly Ser Val Val1 5 10 15Gln His Ser Glu Gly Lys Pro
Leu 2027812PRTArtificial SequenceSynthetic Polypeptide 278Leu Tyr
Val Ser Asn Leu Gly Ile Gly His Thr Arg1 5 1027917PRTArtificial
SequenceSynthetic Polypeptide 279Val Tyr Val Gly Asn Leu Gly Asn
Asn Gly Asn Lys Thr Glu Leu Glu1 5 10
15Arg28018PRTArtificial SequenceSynthetic Polypeptide 280Ile Val
Asp Leu Leu Gln Met Leu Glu Met Asn Met Ala Ile Ala Phe1 5 10 15Pro
Ala28116PRTArtificial SequenceSynthetic Polypeptide 281Val Leu Ala
Gln Asn Ser Gly Phe Asp Leu Gln Glu Thr Leu Val Lys1 5 10
1528225PRTArtificial SequenceSynthetic Polypeptide 282Gln Gln Ser
His Phe Pro Met Thr His Gly Asn Thr Gly Phe Ser Gly1 5 10 15Ile Glu
Ser Ser Ser Pro Glu Val Lys 20 2528314PRTArtificial
SequenceSynthetic Polypeptide 283Ile Leu Ile Ala Asn Thr Gly Met
Asp Thr Asp Lys Ile Lys1 5 1028412PRTArtificial SequenceSynthetic
Polypeptide 284Asn Asn Thr Val Thr Pro Gly Gly Lys Pro Asn Lys1 5
1028519PRTArtificial SequenceSynthetic Polypeptide 285Val Val Asn
Val Ala Asn Val Gly Ala Val Pro Ser Gly Gln Asp Asn1 5 10 15Ile His
Arg28617PRTArtificial SequenceSynthetic Polypeptide 286Met Pro Phe
Pro Val Asn His Gly Ala Ser Ser Glu Asp Thr Leu Leu1 5 10
15Lys2879PRTArtificial SequenceSynthetic Polypeptide 287Arg Pro Lys
Asp Pro Gly His Pro Tyr1 528815PRTArtificial SequenceSynthetic
Polypeptide 288Glu Leu Asp Ile Met Glu Pro Lys Val Pro Asp Asp Ile
Tyr Lys1 5 10 1528922PRTArtificial SequenceSynthetic Polypeptide
289Ala Glu Thr Ser Gln Gln Glu Ala Ser Glu Gly Gly Asp Pro Ala Ser1
5 10 15Pro Ala Leu Ser Leu Ser 2029016PRTArtificial
SequenceSynthetic Polypeptide 290Leu Leu Ala Ala Gln Asn Pro Leu
Ser Gln Ala Asp Arg Pro His Gln1 5 10 1529117PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 291Pro Asp Asn Phe Xaa Phe Gly Gln
Ser Gly Ala Gly Asn Asn Trp Ala1 5 10 15Lys29216PRTArtificial
SequenceSynthetic Polypeptide 292Met Ile Ala Gly Gln Val Leu Asp
Ile Asn Leu Ala Ala Glu Pro Lys1 5 10 1529322PRTArtificial
SequenceSynthetic Polypeptide 293Ile Ile Leu Asn Ser His Ser Pro
Ala Gly Ser Ala Ala Ile Ser Gln1 5 10 15Gln Asp Phe His Pro Lys
2029426PRTArtificial SequenceSynthetic Polypeptide 294Gly Ala Val
Ala Val Ser Ala Ala Pro Gly Ser Ala Ala Pro Ala Ala1 5 10 15Gly Ser
Ala Pro Ala Ala Ala Glu Glu Lys 20 2529547PRTArtificial
SequenceSynthetic Polypeptide 295Ser Ala Ala Gly Ala Ala Gly Ser
Ala Gly Gly Ser Ser Gly Ala Ala1 5 10 15Gly Ala Ala Gly Gly Gly Ala
Gly Ala Gly Thr Arg Pro Gly Asp Gly 20 25 30Gly Thr Ala Ser Ala Gly
Ala Ala Gly Pro Gly Ala Ala Thr Lys 35 40 4529620PRTArtificial
SequenceSynthetic Polypeptide 296Phe Thr Ala Ser Ala Gly Ile Gln
Val Val Gly Asp Asp Leu Thr Val1 5 10 15Thr Asn Pro Lys
2029720PRTArtificial SequenceSynthetic Polypeptide 297Phe Gly Ile
Val Thr Ser Ser Ala Gly Thr Gly Thr Thr Glu Asp Thr1 5 10 15Glu Ala
Lys Lys 2029824PRTArtificial SequenceSynthetic Polypeptide 298Ser
Leu Tyr Gln Ser Ala Gly Val Ala Pro Glu Ser Phe Glu Tyr Ile1 5 10
15Glu Ala His Gly Thr Gly Thr Lys 2029913PRTArtificial
SequenceSynthetic Polypeptide 299Val Ser Glu Ile Asp Glu Met Phe
Glu Ala Arg Lys Met1 5 1030018PRTArtificial SequenceSynthetic
Polypeptide 300Phe Gly Gly Ser Phe Ala Gly Ser Phe Gly Gly Ala Gly
Gly His Ala1 5 10 15Pro Gly30121PRTArtificial SequenceSynthetic
Polypeptide 301Phe Gly Gly Ser Phe Ala Gly Ser Phe Gly Gly Ala Gly
Gly His Ala1 5 10 15Pro Gly Val Ala Arg 2030221PRTArtificial
SequenceSynthetic Polypeptide 302Phe Gly Gly Ser Phe Ala Gly Ser
Phe Gly Gly Ala Gly Gly His Ala1 5 10 15Pro Gly Val Ala Arg
2030321PRTArtificial SequenceSynthetic Polypeptide 303Phe Gly Gly
Ser Phe Ala Gly Ser Phe Gly Gly Ala Gly Gly His Ala1 5 10 15Pro Gly
Val Ala Arg 2030432PRTArtificial SequenceSynthetic Polypeptide
304Ala Ala Asp Thr Lys Pro Gly Thr Thr Gly Ser Gly Ala Gly Ser Gly1
5 10 15Gly Pro Gly Gly Leu Thr Ser Ala Ala Pro Ala Gly Gly Asp Lys
Lys 20 25 3030535PRTArtificial SequenceSynthetic Polypeptide 305Ala
Thr Gln Gly Ala Ala Ala Ala Ala Gly Ser Gly Ala Gly Thr Gly1 5 10
15Gly Gly Thr Ala Ser Gly Gly Thr Glu Gly Gly Ser Ala Glu Ser Glu
20 25 30Gly Ala Lys 3530615PRTArtificial SequenceSynthetic
Polypeptide 306Glu Ile Glu Leu Ile Gly Ser Gly Gly Phe Gly Gln Val
Phe Lys1 5 10 1530728PRTArtificial SequenceSynthetic Polypeptide
307Lys Pro Gly Thr Thr Gly Ser Gly Ala Gly Ser Gly Gly Pro Gly Gly1
5 10 15Leu Thr Ser Ala Ala Pro Ala Gly Gly Asp Lys Lys 20
2530820PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acidmisc_feature(7)..(7)Xaa can be any naturally occurring
amino acid 308Leu Tyr Ala Asn Xaa Val Xaa Ser Gly Gly Thr Thr Met
Tyr Pro Gly1 5 10 15Ile Ala Asp Arg 2030910PRTArtificial
SequenceSynthetic Polypeptide 309Arg Ser Gly Lys Tyr Asp Leu Asp
Phe Lys1 5 1031016PRTArtificial SequenceSynthetic Polypeptide
310His Asp Gly Tyr Gly Ser His Gly Pro Leu Leu Pro Leu Pro Ser Arg1
5 10 1531113PRTArtificial SequenceSynthetic Polypeptide 311Ser Leu
Phe Ser Ser Ile Gly Glu Val Glu Ser Ala Lys1 5 1031213PRTArtificial
SequenceSynthetic Polypeptide 312Leu Gln Ser Ile Gly Thr Glu Asn
Thr Glu Glu Asn Arg1 5 1031318PRTArtificial SequenceSynthetic
Polypeptide 313Ser Leu Val Ala Ser Leu Ala Glu Pro Asp Phe Val Val
Thr Asp Phe1 5 10 15Ala Lys31421PRTArtificial SequenceSynthetic
Polypeptide 314Ala Glu Pro Met Gly Glu Lys Pro Val Gly Ser Leu Ala
Gly Ile Gly1 5 10 15Glu Val Leu Gly Lys 2031515PRTArtificial
SequenceSynthetic Polypeptide 315Val Gln Glu Ala Ile Asn Ser Leu
Gly Gly Ser Val Phe Pro Lys1 5 10 1531624PRTArtificial
SequenceSynthetic Polypeptide 316Ser Ala Ala Ala Ala Ser Ala Ala
Ser Gly Ser Pro Gly Pro Gly Glu1 5 10 15Gly Ser Ala Gly Gly Glu Lys
Arg 2031719PRTArtificial SequenceSynthetic Polypeptide 317Gln Thr
Ile Asp Asn Ser Gln Gly Ala Tyr Gln Glu Ala Phe Asp Ile1 5 10 15Ser
Lys Lys31819PRTArtificial SequenceSynthetic Polypeptide 318Phe Gly
Ile Val Thr Ser Ser Ala Gly Thr Gly Thr Thr Glu Asp Thr1 5 10 15Glu
Ala Lys31920PRTArtificial SequenceSynthetic Polypeptide 319Phe Gly
Ile Val Thr Ser Ser Ala Gly Thr Gly Thr Thr Glu Asp Thr1 5 10 15Glu
Ala Lys Lys 2032012PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 320Xaa Ser Ser Phe Asp Leu Asp Tyr Asp Phe Gln Arg1 5
1032121PRTArtificial SequenceSynthetic Polypeptide 321Phe Gln Ala
Gly Thr Ser Lys Pro Leu His Ser Ser Gly Ile Asn Val1 5 10 15Asn Ala
Ala Pro Phe 2032221PRTArtificial SequenceSynthetic Polypeptide
322His Ile Gly Gly Pro Pro Gly Phe Ala Ser Ser Ser Gly Lys Pro Gly1
5 10 15Pro Thr Val Ile Lys 2032321PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 323Xaa Ser Ser Gly Pro Gly Ala Ser Ser Gly Thr Ser Gly
Asp His Gly1 5 10 15Glu Leu Val Val Arg 2032419PRTArtificial
SequenceSynthetic Polypeptide 324Glu Leu Val Ser Ser Ser Ser Ser
Gly Ser Asp Ser Asp Ser Glu Val1 5 10 15Asp Lys
Lys32512PRTArtificial SequenceSynthetic Polypeptide 325Met Asp Ser
Thr Glu Pro Pro Tyr Ser Gln Lys Arg1 5 1032616PRTArtificial
SequenceSynthetic Polypeptide 326Val Val Val Leu Met Gly Ser Thr
Ser Asp Leu Gly His Cys Glu Lys1 5 10 1532716PRTArtificial
SequenceSynthetic Polypeptide 327Ile Leu Asp Ser Val Gly Ile Glu
Ala Asp Asp Asp Arg Leu Asn Lys1 5 10 1532816PRTArtificial
SequenceSynthetic Polypeptide 328Ser Thr Gln Pro Ile Ser Ser Val
Gly Lys Pro Ala Ser Val Ile Lys1 5 10 1532914PRTArtificial
SequenceSynthetic Polypeptide 329Ala Leu Gln Ser Val Gly Gln Ile
Val Gly Glu Val Leu Lys1 5 1033022PRTArtificial SequenceSynthetic
Polypeptide 330Val Ser Ser Leu Ala Glu Gly Ser Val Thr Ser Val Gly
Ser Val Asn1 5 10 15Pro Ala Glu Asn Phe Arg 2033116PRTArtificial
SequenceSynthetic Polypeptide 331Thr Gly Ser Ile Ser Ser Ser Val
Ser Val Pro Ala Lys Pro Glu Arg1 5 10 1533219PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(2)..(7)Xaa can be any
naturally occurring amino acid 332Tyr Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Ser Gln Ser Tyr Gly Gly Tyr Glu1 5 10 15Asn Gln
Lys33313PRTArtificial SequenceSynthetic Polypeptide 333Ile Tyr Trp
Gly Thr Ala Thr Thr Gly Lys Pro His Val1 5 1033411PRTArtificial
SequenceSynthetic Polypeptide 334Met Val Gln Thr Ala Val Val Pro
Val Lys Lys1 5 1033514PRTArtificial SequenceSynthetic Polypeptide
335Met Met Leu Gly Thr Glu Gly Gly Glu Gly Phe Val Val Lys1 5
1033613PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 336Xaa Thr Phe Ile Ala Ile Lys Pro Asp Gly Val Gln Arg1
5 1033718PRTArtificial SequenceSynthetic Polypeptide 337Val Ser His
Val Ser Thr Gly Gly Gly Ala Ser Leu Glu Leu Leu Glu1 5 10 15Gly
Lys33831PRTArtificial SequenceSynthetic Polypeptide 338Ala Ala Ala
Ala Ala Gly Ser Gly Ala Gly Thr Gly Gly Gly Thr Ala1 5 10 15Ser Gly
Gly Thr Glu Gly Gly Ser Ala Glu Ser Glu Gly Ala Lys 20 25
3033921PRTArtificial SequenceSynthetic Polypeptide 339Thr Ile Gly
Asn Ser Cys Gly Thr Ile Gly Leu Ile His Ala Val Ala1 5 10 15Asn Asn
Gln Asp Lys 2034016PRTArtificial SequenceSynthetic Polypeptide
340Thr Gly Glu Glu Ile Phe Gly Thr Ile Gly Met Arg Pro Asn Ala Lys1
5 10 1534120PRTArtificial SequenceSynthetic Polypeptide 341Thr Thr
Gln Phe Ser Cys Thr Leu Gly Glu Lys Phe Glu Glu Thr Thr1 5 10 15Ala
Asp Gly Arg 2034211PRTArtificial SequenceSynthetic Polypeptide
342Gly Cys Thr Ala Thr Leu Gly Asn Phe Ala Lys1 5
1034312PRTArtificial SequenceSynthetic Polypeptide 343Tyr Val Ala
Thr Leu Gly Val Glu Val His Pro Leu1 5 1034415PRTArtificial
SequenceSynthetic Polypeptide 344Leu Ala Ala Thr Asn Ala Leu Leu
Asn Ser Leu Glu Phe Thr Lys1 5 10 1534518PRTArtificial
SequenceSynthetic Polypeptide 345Gly Pro Gly Ala Ser Ser Gly Thr
Ser Gly Asp His Gly Glu Leu Val1 5 10 15Val Arg34610PRTArtificial
SequenceSynthetic Polypeptide 346Ser Thr Thr Thr Gly His Leu Ile
Tyr Lys1 5 1034735PRTArtificial SequenceSynthetic Polypeptide
347Ala Leu Ser Ala Ala Asp Thr Lys Pro Gly Thr Thr Gly Ser Gly Ala1
5 10 15Gly Ser Gly Gly Pro Gly Gly Leu Thr Ser Ala Ala Pro Ala Gly
Gly 20 25 30Asp Lys Lys 3534810PRTArtificial SequenceSynthetic
Polypeptide 348Ser Thr Thr Thr Gly His Leu Ile Tyr Lys1 5
1034918PRTArtificial SequenceSynthetic Polypeptide 349Val Thr Ile
Ile Gly Pro Ala Thr Val Gly Gly Ile Lys Pro Gly Cys1 5 10 15Phe
Lys35022PRTArtificial SequenceSynthetic Polypeptide 350Thr Val Val
Phe Ser His Pro Pro Ile Gly Thr Val Gly Leu Thr Glu1 5 10 15Asp Glu
Ala Ile His Lys 2035119PRTArtificial SequenceSynthetic Polypeptide
351Gly Ser Pro Thr Ser Leu Gly Thr Trp Gly Ser Trp Ile Gly Pro Asp1
5 10 15His Asp Lys35220PRTArtificial SequenceSynthetic Polypeptide
352Gly Ser Pro Thr Ser Leu Gly Thr Trp Gly Ser Trp Ile Gly Pro Asp1
5 10 15His Asp Lys Phe 2035316PRTArtificial SequenceSynthetic
Polypeptide 353Ile His Phe Pro Leu Ala Thr Tyr Ala Pro Val Ile Ser
Ala Glu Lys1 5 10 1535413PRTArtificial SequenceSynthetic
Polypeptide 354Ala Asn Pro Gln Val Gly Val Ala Phe Pro His Ile Lys1
5 1035515PRTArtificial SequenceSynthetic Polypeptide 355Thr Cys Thr
Thr Val Ala Phe Thr Gln Val Asn Ser Glu Asp Lys1 5 10
1535616PRTArtificial SequenceSynthetic Polypeptide 356Val Leu Thr
Gly Val Ala Gly Glu Asp Ala Glu Cys His Ala Ala Lys1 5 10
1535720PRTArtificial SequenceSynthetic Polypeptide 357Asn Ile Pro
Pro Tyr Phe Val Ala Leu Val Pro Gln Glu Glu Glu Leu1 5 10 15Asp Asp
Gln Lys 2035819PRTArtificial SequenceSynthetic Polypeptide 358Gly
Gln Glu Thr Ala Val Ala Pro Ser Leu Val Ala Pro Ala Leu Asn1 5 10
15Lys Pro Lys35920PRTArtificial SequenceSynthetic Polypeptide
359Gln Gly Gln Glu Thr Ala Val Ala Pro Ser Leu Val Ala Pro Ala Leu1
5 10 15Asn Lys Pro Lys 2036020PRTArtificial SequenceSynthetic
Polypeptide 360Gly Phe Val Thr Phe Ser Ser Met Ala Glu Val Asp Ala
Ala Met Ala1 5 10 15Ala Arg Pro His 2036117PRTArtificial
SequenceSynthetic Polypeptide 361Val Asp Tyr Tyr Thr Thr Thr Pro
Ala Leu Val Phe Gly Lys Pro Val1 5 10 15Arg36217PRTArtificial
SequenceSynthetic Polypeptide 362Val Asp Tyr Tyr Thr Thr Thr Pro
Ala Leu Val Phe Gly Lys Pro Val1 5 10 15Arg36327PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 363Ala Ser Gln Pro Xaa Val Asp Gly
Phe Leu Val Gly Gly Ala Ser Leu1 5 10 15Lys Pro Glu Phe Val Asp Ile
Ile Asn Ala Lys 20 2536417PRTArtificial SequenceSynthetic
Polypeptide 364Thr Ile Ile Gly Pro Ala Thr Val Gly Gly Ile Lys Pro
Gly Cys Phe1 5 10 15Lys36519PRTArtificial SequenceSynthetic
Polypeptide 365Gly Gly Val Asp Thr Ala Ala Val Gly Gly Val Phe Asp
Val Ser Asn1 5 10 15Ala Asp Arg36611PRTArtificial SequenceSynthetic
Polypeptide 366Thr Thr Val His Ala Ile Thr Ala Thr Gln Lys1 5
1036720PRTArtificial SequenceSynthetic Polypeptide 367Glu Glu Val
Arg Pro Gln Asp Thr Val Ser Val Ile Gly Gly Val Ala1 5 10 15Gly Gly
Ser Lys 2036811PRTArtificial SequenceSynthetic Polypeptide 368Gln
Val Ile Gly Thr Gly Ser Phe Phe Pro Lys1 5 1036916PRTArtificial
SequenceSynthetic Polypeptide 369Ala Ser Gly Asn Tyr Ala Thr Val
Ile Ser His Asn Pro Glu Thr Lys1 5 10 1537017PRTArtificial
SequenceSynthetic Polypeptide 370Met Lys Pro Leu Met Gly Val Ile
Tyr Val Pro Leu Thr Asp Lys Glu1 5 10 15Lys37116PRTArtificial
SequenceSynthetic Polypeptide 371Phe Ser Val Cys Val Leu Gly Asp
Gln Gln His Cys Asp Glu Ala Lys1 5 10 1537221PRTArtificial
SequenceSynthetic Polypeptide 372Glu Asn Ala Phe Cys Asn Leu Ala
Ala Ile Val Pro Asp Ser
Val Gly1 5 10 15Arg His Ser Pro Ala 2037320PRTArtificial
SequenceSynthetic Polypeptide 373Ala Tyr Val Gly Asn Leu Pro Phe
Asn Thr Val Gln Gly Asp Ile Asp1 5 10 15Ala Ile Phe Lys
2037415PRTArtificial SequenceSynthetic Polypeptide 374Thr Leu Thr
Thr Val Gln Gly Ile Ala Asp Asp Tyr Asp Lys Lys1 5 10
1537519PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(4)..(4)Xaa can be any naturally occurring
amino acid 375Cys Ile Ser Xaa His Val Gly Gln Ala Gly Val Gln Ile
Gly Asn Ala1 5 10 15Cys Trp Glu37623PRTArtificial SequenceSynthetic
Polypeptide 376Thr His Ala Leu Gln Trp Pro Ser Leu Thr Val Gln Trp
Leu Pro Glu1 5 10 15Val Thr Lys Pro Glu Gly Lys
2037732PRTArtificial SequenceSynthetic Polypeptide 377Ala Ser Val
Pro Ala Gly Gly Ala Val Ala Val Ser Ala Ala Pro Gly1 5 10 15Ser Ala
Ala Pro Ala Ala Gly Ser Ala Pro Ala Ala Ala Glu Glu Lys 20 25
3037814PRTArtificial SequenceSynthetic Polypeptide 378Tyr Glu Glu
Val Ser Val Ser Gly Phe Glu Glu Phe His Arg1 5 1037917PRTArtificial
SequenceSynthetic Polypeptide 379Cys Met Thr Thr Val Ser Trp Asp
Gly Asp Lys Leu Gln Cys Val Gln1 5 10 15Lys38016PRTArtificial
SequenceSynthetic Polypeptide 380Met His Gly Gly Gly Pro Thr Val
Thr Ala Gly Leu Pro Leu Pro Lys1 5 10 1538120PRTArtificial
SequenceSynthetic Polypeptide 381Leu Ala Leu Val Thr Gly Gly Glu
Ile Ala Ser Thr Phe Asp His Pro1 5 10 15Glu Leu Val Lys
2038220PRTArtificial SequenceSynthetic Polypeptide 382Leu Glu Gly
Thr Leu Leu Lys Pro Asn Met Val Thr Pro Gly His Ala1 5 10 15Cys Thr
Gln Lys 2038311PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 383Xaa Val Val Glu Ser Ala Tyr Glu Val Ile Lys1 5
1038415PRTArtificial SequenceSynthetic Polypeptide 384Ile Leu Ala
Gln Val Val Gly Asp Val Asp Thr Ser Leu Pro Arg1 5 10
1538522PRTArtificial SequenceSynthetic Polypeptide 385Cys Phe Ser
Glu Met Ala Pro Val Cys Ala Val Val Gly Gly Ile Leu1 5 10 15Ala Gln
Glu Ile Val Lys 2038620PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(7)..(7)Xaa can be any naturally occurring
amino acid 386Glu Thr Glu Asp Thr Phe Xaa Ala Asp Leu Val Val Gly
Leu Cys Thr1 5 10 15Gly Gln Ile Lys 2038714PRTArtificial
SequenceSynthetic Polypeptide 387Glu Gly Pro Ala Val Val Gly Gln
Phe Ile Gln Asp Val Lys1 5 1038819PRTArtificial SequenceSynthetic
Polypeptide 388Met Leu Ile Ser Gly Tyr Ala Leu Asn Cys Val Val Gly
Ser Gln Gly1 5 10 15Met Pro Lys38914PRTArtificial SequenceSynthetic
Polypeptide 389His Trp Pro Phe Met Val Val Asn Asp Ala Gly Arg Pro
Lys1 5 1039016PRTArtificial SequenceSynthetic Polypeptide 390Ser
Gly Pro Val Val Ala Met Val Trp Glu Gly Leu Asn Val Val Lys1 5 10
1539118PRTArtificial SequenceSynthetic Polypeptide 391Ala Leu Gln
Asp Glu Trp Asp Ala Val Met Leu His Ser Phe Thr Leu1 5 10 15Arg
Gln39214PRTArtificial SequenceSynthetic Polypeptide 392Glu Tyr Phe
Ser Trp Glu Gly Ala Phe Gln His Val Gly Lys1 5 1039322PRTArtificial
SequenceSynthetic Polypeptide 393Ala Thr Val Ala Ser Gly Ile Pro
Ala Gly Trp Met Gly Leu Asp Cys1 5 10 15Gly Pro Glu Ser Ser Lys
2039423PRTArtificial SequenceSynthetic Polypeptide 394Ala Thr Val
Ala Ser Gly Ile Pro Ala Gly Trp Met Gly Leu Asp Cys1 5 10 15Gly Pro
Glu Ser Ser Lys Lys 2039520PRTArtificial SequenceSynthetic
Polypeptide 395Asp Cys Ala Phe Tyr Asp Pro Thr His Ala Trp Ser Gly
Gly Leu Asp1 5 10 15His Gln Leu Lys 2039618PRTArtificial
SequenceSynthetic Polypeptide 396Gln Phe Gln Ala Leu Leu Gln Tyr
Ala Asp Pro Val Ser Ala Gln His1 5 10 15Ala Lys39721PRTArtificial
SequenceSynthetic Polypeptide 397Glu Gln Pro Gln His Pro Leu His
Val Thr Tyr Ala Gly Ala Ala Val1 5 10 15Asp Glu Leu Gly Lys
2039812PRTArtificial SequenceSynthetic Polypeptide 398Thr Phe Ser
Tyr Ala Gly Phe Glu Met Gln Pro Lys1 5 1039921PRTArtificial
SequenceSynthetic Polypeptide 399Gly Tyr Ile Trp Asn Tyr Gly Ala
Ile Pro Gln Thr Trp Glu Asp Pro1 5 10 15Gly His Asn Asp Lys
2040023PRTArtificial SequenceSynthetic Polypeptide 400Asp Tyr Thr
Gly Tyr Asn Asn Tyr Tyr Gly Tyr Gly Asp Tyr Ser Asn1 5 10 15Gln Gln
Ser Gly Tyr Gly Lys 2040113PRTArtificial SequenceSynthetic
Polypeptide 401Gln Ser Gly Tyr Gly Gly Gln Thr Lys Pro Ile Phe Arg1
5 1040220PRTArtificial SequenceSynthetic Polypeptide 402Val Pro Leu
Ile Glu Ser Gly Thr Ala Gly Tyr Leu Gly Gln Val Thr1 5 10 15Thr Ile
Lys Lys 2040320PRTArtificial SequenceSynthetic Polypeptide 403Gly
Ile Leu Gly Tyr Thr Glu His Gln Val Val Ser Ser Asp Phe Asn1 5 10
15Ser Asp Thr His 2040422PRTArtificial SequenceSynthetic
Polypeptide 404Gly Ile Leu Gly Tyr Thr Glu His Gln Val Val Ser Ser
Asp Phe Asn1 5 10 15Ser Asp Thr His Ser Ser 2040516PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 405Gln Thr Cys Val Xaa His Tyr Thr
Gly Met Leu Glu Asp Gly Lys Lys1 5 10 1540619PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 406Gln Thr Cys Val Xaa His Tyr Thr
Gly Met Leu Glu Asp Gly Lys Lys1 5 10 15Phe Asp
Ser4079PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 407Ala Xaa Tyr Val Thr His Leu Met Lys1
54085PRTArtificial SequenceSynthetic Polypeptide 408Lys Val Ser Val
Arg1 540919PRTArtificial SequenceSynthetic Polypeptide 409Met Asp
Leu Ala Ala Ala Ala Glu Pro Gly Ala Gly Ser Gln His Leu1 5 10 15Glu
Val Arg41041PRTArtificial SequenceSynthetic Polypeptide 410Glu Gly
Ala Met Val Ala Ala Thr Gln Gly Ala Ala Ala Ala Ala Gly1 5 10 15Ser
Gly Ala Gly Thr Gly Gly Gly Thr Ala Ser Gly Gly Thr Glu Gly 20 25
30Gly Ser Ala Glu Ser Glu Gly Ala Lys 35 4041118PRTArtificial
SequenceSynthetic Polypeptide 411Gly Thr Ser Phe Asp Ala Ala Ala
Thr Ser Gly Gly Ser Ala Ser Ser1 5 10 15Glu Lys41239PRTArtificial
SequenceSynthetic Polypeptide 412Ala Met Val Ala Ala Thr Gln Gly
Ala Ala Ala Ala Ala Gly Ser Gly1 5 10 15Ala Gly Thr Gly Gly Gly Thr
Ala Ser Gly Gly Thr Glu Gly Gly Ser 20 25 30Ala Glu Ser Glu Gly Ala
Lys 3541314PRTArtificial SequenceSynthetic Polypeptide 413Gly Ile
Leu Ala Ala Asp Glu Ser Thr Gly Ser Ile Ala Lys1 5
1041429PRTArtificial SequenceSynthetic Polypeptide 414Pro Ala Ala
Pro Ala Leu Ser Ala Ala Asp Thr Lys Pro Gly Thr Thr1 5 10 15Gly Ser
Gly Ala Gly Ser Gly Gly Pro Gly Gly Leu Thr 20 2541538PRTArtificial
SequenceSynthetic Polypeptide 415Met Val Ala Ala Thr Gln Gly Ala
Ala Ala Ala Ala Gly Ser Gly Ala1 5 10 15Gly Thr Gly Gly Gly Thr Ala
Ser Gly Gly Thr Glu Gly Gly Ser Ala 20 25 30Glu Ser Glu Gly Ala Lys
3541618PRTArtificial SequenceSynthetic Polypeptide 416Ser Ser Ile
Gln Ala Thr Thr Ala Ala Gly Ser Gly His Pro Thr Ser1 5 10 15Cys
Cys41718PRTArtificial SequenceSynthetic Polypeptide 417Asn Glu Ala
Ile Gln Ala Ala His Asp Ala Val Ala Gln Glu Gly Gln1 5 10 15Cys
Arg41822PRTArtificial SequenceSynthetic Polypeptide 418Gln Phe Gly
Leu Pro Ala Glu Ala Val Glu Ala Ala Asn Lys Gly Asp1 5 10 15Val Glu
Ala Phe Ala Lys 2041921PRTArtificial SequenceSynthetic Polypeptide
419Leu Val Ile Pro Asn Thr Leu Ala Val Asn Ala Ala Gln Asp Ser Thr1
5 10 15Asp Leu Val Ala Lys 2042016PRTArtificial SequenceSynthetic
Polypeptide 420Glu Ala Leu Ala Ala Met Asn Ala Ala Gln Val Lys Pro
Leu Gly Lys1 5 10 1542121PRTArtificial SequenceSynthetic
Polypeptide 421Ala Pro Arg Pro Pro Val Ser Ala Ala Ser Gly Arg Pro
Gln Asp Asp1 5 10 15Thr Asp Ser Ser Arg 2042225PRTArtificial
SequenceSynthetic Polypeptide 422Gly Asp Pro Gln Glu Ala Lys Pro
Gln Glu Ala Ala Val Ala Pro Glu1 5 10 15Lys Pro Pro Ala Ser Asp Glu
Thr Lys 20 2542316PRTArtificial SequenceSynthetic Polypeptide
423Glu Gly Asp Met Ile Val Cys Ala Ala Tyr Ala His Glu Leu Pro Lys1
5 10 1542414PRTArtificial SequenceSynthetic Polypeptide 424Gly Ile
Leu Ala Ala Asp Glu Ser Thr Gly Ser Ile Ala Lys1 5
1042519PRTArtificial SequenceSynthetic Polypeptide 425Pro Glu Glu
Ala Cys Ser Phe Ile Leu Ser Ala Asp Phe Pro Ala Leu1 5 10 15Val Val
Lys42624PRTArtificial SequenceSynthetic Polypeptide 426Gly Trp Asn
Ala Tyr Ile Asp Asn Leu Met Ala Asp Gly Thr Cys Gln1 5 10 15Asp Ala
Ala Ile Val Gly Tyr Lys 2042714PRTArtificial SequenceSynthetic
Polypeptide 427Tyr Leu Ala Ala Asp Lys Asp Gly Asn Val Thr Cys Glu
Arg1 5 1042816PRTArtificial SequenceSynthetic Polypeptide 428Leu
Pro Val Asp Phe Val Thr Ala Asp Lys Phe Asp Glu Asn Ala Lys1 5 10
1542913PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 429Thr Xaa Glu Ala Glu Ala Ala His Gly Thr Val Thr Arg1
5 1043012PRTArtificial SequenceSynthetic Polypeptide 430Thr Val Phe
Ala Glu His Ile Ser Asp Glu Cys Lys1 5 1043113PRTArtificial
SequenceSynthetic Polypeptide 431Thr Val Phe Ala Glu His Ile Ser
Asp Glu Cys Lys Arg1 5 1043216PRTArtificial SequenceSynthetic
Polypeptide 432Asp Leu Glu Ala Glu His Val Glu Val Glu Asp Thr Thr
Leu Asn Arg1 5 10 1543311PRTArtificial SequenceSynthetic
Polypeptide 433Cys Ala Glu Ile Ala His Asn Val Ser Ser Lys1 5
1043428PRTArtificial SequenceSynthetic Polypeptide 434Glu Ala Ala
Ala Ala Gly Gly Gly Val Gly Ala Gly Ala Gly Gly Gly1 5 10 15Cys Gly
Pro Gly Gly Ala Asp Ser Ser Lys Pro Arg 20 2543515PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(2)..(2)Xaa can be any
naturally occurring amino acid 435Tyr Xaa Leu Val Gly Ala Gly Ala
Ile Gly Cys Glu Leu Leu Lys1 5 10 1543615PRTArtificial
SequenceSynthetic Polypeptide 436Leu Ile Tyr Ala Gly Lys Ile Leu
Asn Asp Asp Thr Ala Leu Lys1 5 10 1543720PRTArtificial
SequenceSynthetic Polypeptide 437Phe Gly Asp Asn Thr Ala Gly Cys
Thr Ser Ala Gly Pro His Phe Asn1 5 10 15Pro Leu Ser Arg
2043813PRTArtificial SequenceSynthetic Polypeptide 438Ile Ile Thr
Leu Ala Gly Pro Thr Asn Ala Ile Phe Lys1 5 1043917PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 439Glu Ile Val His Xaa Gln Ala Gly
Gln Cys Gly Asn Gln Ile Gly Ala1 5 10 15Lys44019PRTArtificial
SequenceSynthetic Polypeptide 440Ala Ile Cys Ala Gly Pro Thr Ala
Leu Leu Ala His Glu Ile Gly Phe1 5 10 15Gly Ser
Lys44118PRTArtificial SequenceSynthetic Polypeptide 441Ser His Glu
His Ser Pro Ser Asp Leu Glu Ala His Phe Val Pro Leu1 5 10 15Val
Lys44212PRTArtificial SequenceSynthetic Polypeptide 442Glu Leu Gln
Ala His Gly Ala Asp Glu Leu Leu Lys1 5 1044318PRTArtificial
SequenceSynthetic Polypeptide 443Ser Trp Ala Asp Leu Val Asn Ala
His Val Val Pro Gly Ser Gly Val1 5 10 15Val Lys44412PRTArtificial
SequenceSynthetic Polypeptide 444Thr Phe Ile Ala Ile Lys Pro Asp
Gly Val Gln Arg1 5 1044521PRTArtificial SequenceSynthetic
Polypeptide 445Gly Tyr Ile Trp Asn Tyr Gly Ala Ile Pro Gln Thr Trp
Glu Asp Pro1 5 10 15Gly His Asn Asp Lys 2044615PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(5)..(6)Xaa can be any
naturally occurring amino acid 446Ala Thr Ala Thr Xaa Xaa Ala Lys
Pro Gln Ile Thr Asn Pro Lys1 5 10 1544719PRTArtificial
SequenceSynthetic Polypeptide 447Thr Thr Glu Thr Ala Gln His Ala
Gln Gly Ala Lys Pro Gln Val Gln1 5 10 15Pro Gln
Lys44818PRTArtificial SequenceSynthetic Polypeptide 448Val Ala Ser
Tyr Leu Leu Ala Ala Leu Gly Gly Asn Ser Ser Pro Ser1 5 10 15Ala
Lys44914PRTArtificial SequenceSynthetic Polypeptide 449Ile Ala Leu
Pro Ala Pro Arg Gly Ser Gly Thr Ala Ser Asp1 5 1045014PRTArtificial
SequenceSynthetic Polypeptide 450Gln Ile Gly Asn Val Ala Ala Leu
Pro Gly Ile Val His Arg1 5 1045121PRTArtificial SequenceSynthetic
Polypeptide 451Asp Gly Thr Val Leu Cys Glu Leu Ile Asn Ala Leu Tyr
Pro Glu Gly1 5 10 15Gln Ala Pro Val Lys 2045222PRTArtificial
SequenceSynthetic Polypeptide 452Asp Gly Thr Val Leu Cys Glu Leu
Ile Asn Ala Leu Tyr Pro Glu Gly1 5 10 15Gln Ala Pro Val Lys Lys
2045314PRTArtificial SequenceSynthetic Polypeptide 453Asp Phe Thr
Val Ser Ala Met His Gly Asp Met Asp Gln Lys1 5 1045416PRTArtificial
SequenceSynthetic Polypeptide 454Leu Val Thr Asp Cys Val Ala Ala
Met Asn Pro Asp Ala Val Leu Arg1 5 10 1545514PRTArtificial
SequenceSynthetic Polypeptide 455His Glu Leu Gln Ala Asn Cys Tyr
Glu Glu Val Lys Asp Arg1 5 1045625PRTArtificial SequenceSynthetic
Polypeptide 456Cys Ser Leu Gln Ala Ala Ala Ile Leu Asp Ala Asn Asp
Ala His Gln1 5 10 15Thr Glu Thr Ser Ser Ser Gln Val Lys 20
2545720PRTArtificial SequenceSynthetic Polypeptide 457Ser Gly Leu
Gly Arg Pro Gln Leu Gln Gly Ala Pro Ala Ala Glu Pro1 5 10 15Met Ala
Val Pro 2045818PRTArtificial SequenceSynthetic Polypeptide 458Gln
Glu Thr Ala Val Ala Pro Ser Leu Val Ala Pro Ala Leu Asn Lys1 5 10
15Pro Lys45915PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 459Ala Gln Xaa Ala Ala Pro Ala Ser Val Pro Ala Gln Ala
Pro Lys1 5 10 1546021PRTArtificial SequenceSynthetic Polypeptide
460Gly Glu Thr Ile Phe Val Thr Ala Pro His Glu Ala Thr Ala Gly Ile1
5 10 15Ile Gly Val Asn Arg 2046118PRTArtificial SequenceSynthetic
Polypeptide 461Glu Tyr Ser Ser Glu Leu Asn Ala Pro Ser Gln Glu Ser
Asp Ser His1 5 10 15Pro Arg46220PRTArtificial SequenceSynthetic
Polypeptide 462Asp Gln Val Thr Ala Gln Glu Ile Phe Gln Asp Asn His
Glu Asp Gly1 5 10 15Pro Thr Ala Lys 2046319PRTArtificial
SequenceSynthetic Polypeptide 463Leu His Glu Glu Glu Ile Gln Glu
Leu Gln Ala Gln Ile Gln Glu Gln1 5 10 15His Val
Gln46418PRTArtificial SequenceSynthetic Polypeptide 464Gln Gln Gln
Arg Pro Leu Glu Ala Gln Pro Ser Ala Pro Gly His Ser1 5 10
15Val Lys46522PRTArtificial SequenceSynthetic Polypeptide 465Ala
Ala His Thr Ala Asn Phe Leu Leu Asn Ala Ser Gly Ser Thr Ser1 5 10
15Thr Pro Ala Pro Ser Arg 2046616PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(4)Xaa can be any naturally occurring
amino acid 466Ile Xaa Xaa Xaa Phe Leu Gly Ala Ser Leu Lys Asp Glu
Val Leu Lys1 5 10 1546724PRTArtificial SequenceSynthetic
Polypeptide 467Asn Val Glu Glu Ala Asp Ala Ala Met Ala Ala Ser Pro
His Ala Val1 5 10 15Asp Gly Asn Thr Val Glu Leu Lys
2046816PRTArtificial SequenceSynthetic Polypeptide 468Ala Leu Leu
Val Thr Ala Ser Gln Cys Gln Gln Pro Ala Glu Asn Lys1 5 10
1546931PRTArtificial SequenceSynthetic Polypeptide 469Gln Ser Ser
Trp Gly Met Met Gly Met Leu Ala Ser Gln Gln Asn Gln1 5 10 15Ser Gly
Pro Ser Gly Asn Asn Gln Asn Gln Gly Asn Met Gln Arg 20 25
3047016PRTArtificial SequenceSynthetic Polypeptide 470Leu Pro Pro
Gly Phe Ser Ala Ser Ser Thr Val Glu Lys Pro Ser Lys1 5 10
1547116PRTArtificial SequenceSynthetic Polypeptide 471Ala Ala Val
Pro Ser Gly Ala Ser Thr Gly Ile Tyr Glu Ala Leu Glu1 5 10
1547216PRTArtificial SequenceSynthetic Polypeptide 472Leu Asn Cys
Gln Val Ile Gly Ala Ser Val Asp Ser His Phe Cys His1 5 10
1547320PRTArtificial SequenceSynthetic Polypeptide 473Gly Ala Asn
Gln Tyr Thr Phe His Leu Glu Ala Thr Glu Asn Pro Gly1 5 10 15Ala Leu
Ile Lys 2047422PRTArtificial SequenceSynthetic Polypeptide 474Leu
Ser Glu Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly Lys Pro Pro1 5 10
15Gln Tyr Ile Ala Val His 2047545PRTArtificial SequenceSynthetic
Polypeptide 475Ala Gly Glu Gln Glu Gly Ala Met Val Ala Ala Thr Gln
Gly Ala Ala1 5 10 15Ala Ala Ala Gly Ser Gly Ala Gly Thr Gly Gly Gly
Thr Ala Ser Gly 20 25 30Gly Thr Glu Gly Gly Ser Ala Glu Ser Glu Gly
Ala Lys 35 40 4547619PRTArtificial SequenceSynthetic Polypeptide
476Ile Ser Ser Ile Gln Ala Thr Thr Ala Ala Gly Ser Gly His Pro Thr1
5 10 15Ser Cys Cys47713PRTArtificial SequenceSynthetic Polypeptide
477Gly Leu Gly Ala Thr Thr His Pro Thr Ala Ala Val Lys1 5
1047817PRTArtificial SequenceSynthetic Polypeptide 478Ile Glu Pro
Pro Pro Leu Asp Ala Val Ile Glu Ala Glu His Thr Leu1 5 10
15Arg47919PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 479Ser Xaa Gly Leu Pro Val Gly Ala Val Ile Asn Cys Ala
Asp Asn Thr1 5 10 15Gly Ala Lys48018PRTArtificial SequenceSynthetic
Polypeptide 480Val Asn Val Ala Asn Val Gly Ala Val Pro Ser Gly Gln
Asp Asn Ile1 5 10 15His Arg48113PRTArtificial SequenceSynthetic
Polypeptide 481Gln Phe Leu Glu Cys Ala Gln Asn Gln Gly Asp Ile Lys1
5 1048220PRTArtificial SequenceSynthetic Polypeptide 482Gly Gly Leu
Thr Asp Glu Ala Ala Leu Ser Cys Cys Ser Asp Ala Asp1 5 10 15Pro Ser
Thr Lys 2048311PRTArtificial SequenceSynthetic Polypeptide 483Ile
Leu Ser Cys Gly Glu Val Ile His Val Lys1 5 1048414PRTArtificial
SequenceSynthetic Polypeptide 484Ala Ile Val Asp Cys Gly Phe Glu
His Pro Ser Glu Val Gln1 5 1048514PRTArtificial SequenceSynthetic
Polypeptide 485His Tyr Tyr Glu Val Ser Cys His Asp Gln Gly Leu Cys
Arg1 5 1048615PRTArtificial SequenceSynthetic Polypeptide 486Met
Leu Val Gln Cys Met Gln Asp Gln Glu His Pro Ser Ile Arg1 5 10
1548715PRTArtificial SequenceSynthetic Polypeptide 487Asp Val Val
Ile Cys Pro Asp Ala Ser Leu Glu Asp Ala Lys Lys1 5 10
1548816PRTArtificial SequenceSynthetic Polypeptide 488Ser Gly Tyr
Ala Phe Val Asp Cys Pro Asp Glu His Trp Ala Met Lys1 5 10
1548919PRTArtificial SequenceSynthetic Polypeptide 489Phe Val Leu
Cys Pro Glu Cys Glu Asn Pro Glu Thr Asp Leu His Val1 5 10 15Asn Pro
Lys49014PRTArtificial SequenceSynthetic Polypeptide 490Ile Ala Ile
Leu Thr Cys Pro Phe Glu Pro Pro Lys Pro Lys1 5 1049114PRTArtificial
SequenceSynthetic Polypeptide 491Ile Ala Ile Leu Thr Cys Pro Phe
Glu Pro Pro Lys Pro Lys1 5 1049225PRTArtificial SequenceSynthetic
Polypeptide 492Ala Ala Thr Glu Gln Tyr His Gln Val Leu Cys Pro Gly
Pro Ser Gln1 5 10 15Asp Asp Pro Leu His Pro Leu Asn Lys 20
2549314PRTArtificial SequenceSynthetic Polypeptide 493Ala Ile Val
Ile Cys Pro Thr Asp Glu Asp Leu Lys Asp Arg1 5 1049419PRTArtificial
SequenceSynthetic Polypeptide 494Ala Thr Ala His Ala Gln Ala Gln
Leu Gly Cys Pro Val Ile Ile His1 5 10 15Pro Gly
Arg49514PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 495Ile Ile Pro Gly Xaa Met Cys Gln Gly Gly Asp Phe Thr
Arg1 5 1049614PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 496Ile Ile Pro Gly Xaa Met Cys Gln Gly Gly Asp Phe Thr
Arg1 5 1049719PRTArtificial SequenceSynthetic Polypeptide 497Pro
Ala Leu Tyr Trp Leu Ser Gly Leu Thr Cys Thr Glu Gln Asn Phe1 5 10
15Ile Ser Lys49817PRTArtificial SequenceSynthetic Polypeptide
498Met Thr Val Gly Cys Val Ala Gly Asp Glu Glu Ser Tyr Glu Val Phe1
5 10 15Lys49917PRTArtificial SequenceSynthetic Polypeptide 499Ala
Thr Val Ala Phe Cys Asp Ala Gln Ser Thr Gln Glu Ile His Glu1 5 10
15Lys50016PRTArtificial SequenceSynthetic Polypeptide 500Asn Tyr
Gly Ile Leu Ala Asp Ala Thr Glu Gln Val Gly Gln His Lys1 5 10
1550110PRTArtificial SequenceSynthetic Polypeptide 501Leu Gln Asp
Cys Glu Gly Leu Ile Val Arg1 5 1050220PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(7)..(7)Xaa can be any
naturally occurring amino acidmisc_feature(10)..(10)Xaa can be any
naturally occurring amino acid 502Gln Ile Ser Ala Gly Tyr Xaa Pro
Val Xaa Asp Cys His Thr Ala His1 5 10 15Ile Ala Cys Lys
2050320PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(7)..(7)Xaa can be any naturally occurring
amino acidmisc_feature(10)..(10)Xaa can be any naturally occurring
amino acid 503Gln Ile Ser Ala Gly Tyr Xaa Pro Val Xaa Asp Cys His
Thr Ala His1 5 10 15Ile Ala Cys Lys 2050419PRTArtificial
SequenceSynthetic Polypeptide 504Thr Gly Glu Pro Cys Cys Asp Trp
Val Gly Asp Glu Gly Ala Gly His1 5 10 15Phe Val
Lys50519PRTArtificial SequenceSynthetic Polypeptide 505Asp Ala Thr
Asn Val Gly Asp Glu Gly Gly Phe Ala Pro Asn Ile Leu1 5 10 15Glu Asn
Lys50613PRTArtificial SequenceSynthetic Polypeptide 506Asn Ile Leu
Asp Phe Pro Gln His Val Ser Pro Ser Lys1 5 1050714PRTArtificial
SequenceSynthetic Polypeptide 507Asp His Val Val Ser Asp Phe Ser
Glu His Gly Ser Leu Lys1 5 1050818PRTArtificial SequenceSynthetic
Polypeptide 508Ala Asp Asn Glu Leu Ser Pro Glu Cys Leu Asp Gly Ala
Gln His Phe1 5 10 15Leu Lys50918PRTArtificial SequenceSynthetic
Polypeptide 509Ile Gln Thr Leu Gly Tyr Phe Pro Val Gly Asp Gly Asp
Phe Pro His1 5 10 15Gln Lys51019PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acidmisc_feature(5)..(7)Xaa can be any naturally occurring
amino acid 510Asp Xaa Gln Glu Xaa Xaa Xaa Phe Leu Leu Asp Gly Leu
His Glu Asp1 5 10 15Leu Asn Arg51118PRTArtificial SequenceSynthetic
Polypeptide 511Met Ile Leu Ile Gln Asp Gly Ser Gln Asn Thr Asn Val
Asp Lys Pro1 5 10 15Leu Arg51215PRTArtificial SequenceSynthetic
Polypeptide 512Phe Thr Ile Ser Asp His Pro Gln Pro Ile Asp Pro Leu
Leu Lys1 5 10 1551318PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 513Val Asn Pro Thr Xaa Phe Phe Asp Ile Ala Val Asp Gly
Glu Pro Leu1 5 10 15Gly Arg51417PRTArtificial SequenceSynthetic
Polypeptide 514Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp Val
Pro Asn Ile1 5 10 15Lys51520PRTArtificial SequenceSynthetic
Polypeptide 515Asn Gly Ser Ile Tyr Asn Pro Glu Val Leu Asp Ile Thr
Glu Glu Thr1 5 10 15Leu His Ser Arg 2051613PRTArtificial
SequenceSynthetic Polypeptide 516Gln His Ile Val Asn Asp Met Asn
Pro Gly Asn Leu His1 5 1051713PRTArtificial SequenceSynthetic
Polypeptide 517Met Leu Leu Asp Ser Glu Gln His Pro Cys Gln Leu Lys1
5 1051813PRTArtificial SequenceSynthetic Polypeptide 518Glu Gly Leu
Met Leu Asp Ser His Glu Glu Leu Tyr Lys1 5 1051915PRTArtificial
SequenceSynthetic Polypeptide 519Thr Ala Gly Asp Thr His Leu Gly
Gly Glu Asp Phe Asp Asn Arg1 5 10 1552021PRTArtificial
SequenceSynthetic Polypeptide 520Pro Gly Gly Leu Leu Leu Gly Asp
Val Ala Pro Asn Phe Glu Ala Asn1 5 10 15Thr Thr Val Gly Arg
2052118PRTArtificial SequenceSynthetic Polypeptide 521Asn Asp Gly
Ala Thr Ile Leu Ser Met Met Asp Val Asp His Gln Ile1 5 10 15Ala
Lys52219PRTArtificial SequenceSynthetic Polypeptide 522Gly Glu Glu
Gly Leu Thr Leu Asn Leu Glu Asp Val Gln Pro His Asp1 5 10 15Leu Gly
Lys52318PRTArtificial SequenceSynthetic Polypeptide 523Thr Ile Asp
Asn Ser Gln Gly Ala Tyr Gln Glu Ala Phe Asp Ile Ser1 5 10 15Lys
Lys52418PRTArtificial SequenceSynthetic Polypeptide 524Ile Val Gly
Phe Phe Asp Asp Ser Phe Ser Glu Ala His Ser Glu Phe1 5 10 15Leu
Lys52521PRTArtificial SequenceSynthetic Polypeptide 525Gln Glu Glu
Ala Ser Gly Val Ala Leu Gly Glu Ala Pro Asp His Ser1 5 10 15Tyr Glu
Ser Leu Arg 2052614PRTArtificial SequenceSynthetic Polypeptide
526Asp Pro Val Gln Glu Ala Trp Ala Glu Asp Val Asp Leu Arg1 5
1052716PRTArtificial SequenceSynthetic Polypeptide 527Pro Met Ile
Tyr Ile Cys Gly Glu Cys His Thr Glu Asn Glu Ile Lys1 5 10
1552815PRTArtificial SequenceSynthetic Polypeptide 528His Met Ser
Glu Phe Met Glu Cys Asn Leu Asn Glu Leu Val Lys1 5 10
1552915PRTArtificial SequenceSynthetic Polypeptide 529Met Gly Tyr
Ala Glu Glu Ala Pro Tyr Asp Ala Ile His Val Gly1 5 10
1553014PRTArtificial SequenceSynthetic Polypeptide 530Met Ala Asp
Gln Leu Thr Glu Glu Gln Ile Ala Glu Phe Lys1 5 1053112PRTArtificial
SequenceSynthetic Polypeptide 531Tyr Leu Ala Glu Phe Ala Thr Gly
Asn Asp Arg Lys1 5 1053217PRTArtificial SequenceSynthetic
Polypeptide 532Leu Ala Glu Leu Glu Glu Phe Ile Asn Gly Pro Asn Asn
Ala His Ile1 5 10 15Gln5339PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 533Xaa Glu Phe Thr Asp His Leu Val Lys1
553423PRTArtificial SequenceSynthetic Polypeptide 534Phe Cys Val
Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp1 5 10 15Ser Gly
Gly Pro Val Val Cys 2053515PRTArtificial SequenceSynthetic
Polypeptide 535Leu Leu Ala Glu Gly His Pro Asp Pro Asp Ala Glu Leu
Gln Arg1 5 10 1553610PRTArtificial SequenceSynthetic Polypeptide
536Gly Leu Thr Glu Gly Leu His Gly Phe His1 5 1053710PRTArtificial
SequenceSynthetic Polypeptide 537Gly Leu Thr Glu Gly Leu His Gly
Phe His1 5 1053819PRTArtificial SequenceSynthetic Polypeptide
538Phe Val His Trp Tyr Val Gly Glu Gly Met Glu Glu Gly Glu Phe Ser1
5 10 15Glu Ala Arg53913PRTArtificial SequenceSynthetic Polypeptide
539Met Leu Leu His Glu Gly Gln His Pro Ala Gln Leu Arg1 5
1054018PRTArtificial SequenceSynthetic Polypeptide 540Tyr His Gly
Tyr Thr Phe Ala Asn Leu Gly Glu His Glu Phe Val Glu1 5 10 15Glu
Lys54112PRTArtificial SequenceSynthetic Polypeptide 541Thr Val Phe
Ala Glu His Ile Ser Asp Glu Cys Lys1 5 1054215PRTArtificial
SequenceSynthetic Polypeptide 542Val Ile Leu Glu Glu His Ser Thr
Cys Glu Asn Glu Val Ser Lys1 5 10 1554315PRTArtificial
SequenceSynthetic Polypeptide 543Leu Leu Thr Glu Ile His Gly Gly
Ala Gly Gly Pro Ser Gly Arg1 5 10 1554416PRTArtificial
SequenceSynthetic Polypeptide 544Ala His Leu Met Glu Ile Gln Val
Asn Gly Gly Thr Val Ala Glu Lys1 5 10 1554518PRTArtificial
SequenceSynthetic Polypeptide 545Ile Ser Trp Leu Asp Ala Asn Thr
Leu Ala Glu Lys Asp Glu Phe Glu1 5 10 15His Lys54618PRTArtificial
SequenceSynthetic Polypeptide 546Ile Ser Trp Leu Asp Ala Asn Thr
Leu Ala Glu Lys Asp Glu Phe Glu1 5 10 15His Lys54712PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(1)..(1)Xaa can be any
naturally occurring amino acid 547Xaa Glu Lys Phe Glu Asp Glu Asn
Phe Ile Leu Lys1 5 1054822PRTArtificial SequenceSynthetic
Polypeptide 548Ser Ala Val Glu Ala Gly Ser Glu Val Ser Glu Lys Pro
Gly Gln Glu1 5 10 15Ala Pro Val Leu Pro Lys 2054922PRTArtificial
SequenceSynthetic Polypeptide 549Ser Ala Val Glu Ala Gly Ser Glu
Val Ser Glu Lys Pro Gly Gln Glu1 5 10 15Ala Pro Val Leu Pro Lys
2055013PRTArtificial SequenceSynthetic Polypeptide 550Ile Leu Asn
Glu Lys Pro Thr Thr Asp Glu Pro Glu Lys1 5 1055114PRTArtificial
SequenceSynthetic Polypeptide 551Tyr Leu Ala Glu Lys Tyr Glu Trp
Asp Val Ala Glu Ala Arg1 5 1055215PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(6)..(6)Xaa can be any naturally occurring
amino acid 552Cys Leu Glu Leu Phe Xaa Glu Leu Ala Glu Asp Lys Glu
Asn Tyr1 5 10 1555316PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(6)..(6)Xaa can be any naturally occurring
amino acid 553Cys Leu Glu Leu Phe Xaa Glu Leu Ala Glu Asp Lys Glu
Asn Tyr Lys1 5 10 1555411PRTArtificial SequenceSynthetic
Polypeptide 554Met Glu Glu Leu His Asn Gln Glu Val Gln Lys1 5
1055526PRTArtificial SequenceSynthetic Polypeptide 555Gly Val Asn
Val Ala Gly Val Ser Leu Gln Glu Leu Asn Pro Glu Met1 5 10 15Gly Thr
Asp Asn Asp Ser Glu Asn Trp Lys 20 2555616PRTArtificial
SequenceSynthetic Polypeptide 556Ala Ser Asp Ile Ala Met Thr Glu
Leu Pro Pro Thr His Pro Ile Arg1 5 10 1555716PRTArtificial
SequenceSynthetic Polypeptide 557Val Val Val Ala Glu Asn Phe Asp
Glu Ile Val Asn Asn Glu Asn Lys1 5 10 1555816PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(2)..(2)Xaa can be any
naturally occurring amino acid 558Ile Xaa Glu Gly Cys Glu Glu Pro
Ala Thr His Asn Ala Leu Ala Lys1 5 10 1555913PRTArtificial
SequenceSynthetic Polypeptide 559Val Thr Glu Gln Gly Ala Glu Leu
Ser Asn Glu Glu Arg1 5 1056014PRTArtificial SequenceSynthetic
Polypeptide 560Ala Val Thr Glu Gln Gly His Glu Leu Ser
Asn Glu Glu Arg1 5 1056121PRTArtificial SequenceSynthetic
Polypeptide 561Gln Val Asp Gln Glu Glu Pro His Val Glu Glu Gln Gln
Gln Gln Thr1 5 10 15Pro Ala Glu Asn Lys 2056221PRTArtificial
SequenceSynthetic Polypeptide 562Gln Val Asp Gln Glu Glu Pro His
Val Glu Glu Gln Gln Gln Gln Thr1 5 10 15Pro Ala Glu Asn Lys
2056314PRTArtificial SequenceSynthetic Polypeptide 563Val Val Phe
Glu Gln Thr Lys Val Ile Ala Asp Asn Val Lys1 5 1056423PRTArtificial
SequenceSynthetic Polypeptide 564Asn Ile Phe Val Gly Glu Asn Ile
Leu Glu Glu Ser Glu Asn Leu His1 5 10 15Asn Ala Asp Gln Pro Leu Arg
2056514PRTArtificial SequenceSynthetic Polypeptide 565Leu Phe Ile
His Glu Ser Ile His Asp Glu Val Val Asn Arg1 5 1056625PRTArtificial
SequenceSynthetic Polypeptide 566Val Thr Asn Gly Ile Glu Glu Pro
Leu Glu Glu Ser Ser His Glu Pro1 5 10 15Glu Pro Glu Pro Glu Ser Glu
Thr Lys 20 2556712PRTArtificial SequenceSynthetic Polypeptide
567Gly Ile Val Glu Glu Ser Val Thr Gly Val His Arg1 5
1056818PRTArtificial SequenceSynthetic Polypeptide 568Gln Cys Pro
Ser Val Val Ser Leu Leu Ser Glu Ser Tyr Asn Pro His1 5 10 15Val
Arg56914PRTArtificial SequenceSynthetic Polypeptide 569Ala Ser Leu
Gln Glu Thr His Phe Asp Ser Thr Gln Thr Lys1 5 1057010PRTArtificial
SequenceSynthetic Polypeptide 570Thr Phe Gly Glu Thr His Pro Phe
Thr Lys1 5 1057117PRTArtificial SequenceSynthetic Polypeptide
571Val Met Leu Gly Glu Thr Asn Pro Ala Asp Ser Lys Pro Gly Thr Ile1
5 10 15Arg57217PRTArtificial SequenceSynthetic Polypeptide 572Val
Met Leu Gly Glu Thr Asn Pro Ala Asp Ser Lys Pro Gly Thr Ile1 5 10
15Arg57317PRTArtificial SequenceSynthetic Polypeptide 573Val Met
Leu Gly Glu Thr Asn Pro Ala Asp Ser Lys Pro Gly Thr Ile1 5 10
15Arg57418PRTArtificial SequenceSynthetic Polypeptide 574Gly Ala
Asp Phe Leu Val Thr Glu Val Glu Asn Gly Gly Ser Leu Gly1 5 10 15Ser
Lys57521PRTArtificial SequenceSynthetic Polypeptide 575Leu Pro Thr
Glu Ala Tyr Ile Ser Val Glu Glu Val His Asp Asp Gly1 5 10 15Thr Pro
Thr Ser Lys 2057621PRTArtificial SequenceSynthetic Polypeptide
576Leu Pro Thr Glu Ala Tyr Ile Ser Val Glu Glu Val His Asp Asp Gly1
5 10 15Thr Pro Thr Ser Lys 2057718PRTArtificial SequenceSynthetic
Polypeptide 577Met Glu Glu Val Pro His Asp Cys Pro Gly Ala Asp Ser
Ala Gln Ala1 5 10 15Gly Arg57815PRTArtificial SequenceSynthetic
Polypeptide 578Val Asp Glu Asn Cys Val Gly Phe Asp His Thr Val Lys
Pro Val1 5 10 1557921PRTArtificial SequenceSynthetic Polypeptide
579Val His Val Val Pro Asp Gln Leu Met Ala Phe Gly Gly Ser Ser Glu1
5 10 15Pro Cys Ala Leu Cys 2058015PRTArtificial SequenceSynthetic
Polypeptide 580Ile Trp Cys Phe Gly Pro Asp Gly Thr Gly Pro Asn Ile
Leu Thr1 5 10 1558110PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 581Tyr Val Xaa Phe Gly Pro His Ala Gly Lys1 5
1058218PRTArtificial SequenceSynthetic Polypeptide 582Glu Phe Ala
Gly Phe Gln Cys Gln Ile Gln Phe Gly Pro His Asn Glu1 5 10 15Gln
Lys58317PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 583Lys Pro Xaa Lys Pro Met Gln Phe Leu Gly Asp Glu Glu
Thr Val Arg1 5 10 15Lys58413PRTArtificial SequenceSynthetic
Polypeptide 584Met Val Tyr Met Phe Gln Tyr Asp Ser Thr His Gly Lys1
5 1058515PRTArtificial SequenceSynthetic Polypeptide 585Glu Glu Leu
Gly Phe Arg Pro Glu Tyr Ser Ala Ser Gln Leu Lys1 5 10
1558610PRTArtificial SequenceSynthetic Polypeptide 586His Leu Glu
Phe Ser His Asp Gln Tyr Arg1 5 1058715PRTArtificial
SequenceSynthetic Polypeptide 587Thr Cys Gly Phe Asp Phe Thr Gly
Ala Val Glu Asp Ile Ser Lys1 5 10 1558814PRTArtificial
SequenceSynthetic Polypeptide 588Gly Phe Gly Phe Val Asp Phe Asn
Ser Glu Glu Asp Ala Lys1 5 1058914PRTArtificial SequenceSynthetic
Polypeptide 589Gly Phe Gly Phe Val Asp Phe Asn Ser Glu Glu Asp Ala
Lys1 5 1059010PRTArtificial SequenceSynthetic Polypeptide 590Asn
Tyr Gly Phe Val His Ile Glu Asp Lys1 5 1059115PRTArtificial
SequenceSynthetic Polypeptide 591Gly Phe Gly Phe Val Thr Phe Asp
Asp His Asp Pro Val Asp Lys1 5 10 1559217PRTArtificial
SequenceSynthetic Polypeptide 592Leu Pro Asn Phe Gly Phe Val Val
Phe Asp Asp Ser Glu Pro Val Gln1 5 10 15Lys59315PRTArtificial
SequenceSynthetic Polypeptide 593Gln Leu Leu Cys Gly Ala Ala Ile
Gly Thr His Glu Asp Asp Lys1 5 10 1559417PRTArtificial
SequenceSynthetic Polypeptide 594Gln Leu Leu Cys Gly Ala Ala Ile
Gly Thr His Glu Asp Asp Lys Tyr1 5 10 15Arg59516PRTArtificial
SequenceSynthetic Polypeptide 595Met Thr Asn Gly Phe Ser Gly Ala
Asp Leu Thr Glu Ile Cys Gln Arg1 5 10 1559622PRTArtificial
SequenceSynthetic Polypeptide 596Val Gln Gly Glu Val Met Glu Gly
Ala Asp Asn Gln Gly Ala Gly Glu1 5 10 15Gln Gly Arg Pro Val Arg
2059721PRTArtificial SequenceSynthetic Polypeptide 597Met Gly Gly
His Gly Tyr Gly Gly Ala Gly Asp Ala Ser Ser Gly Phe1 5 10 15His Gly
Gly His Phe 2059836PRTArtificial SequenceSynthetic Polypeptide
598Leu Gly Asn Val Leu Gly Gly Leu Ile Ser Gly Ala Gly Gly Gly Gly1
5 10 15Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 20 25 30Thr Ala Met Arg 3559921PRTArtificial SequenceSynthetic
Polypeptide 599Phe Gly Gly Ser Phe Ala Gly Ser Phe Gly Gly Ala Gly
Gly His Ala1 5 10 15Pro Gly Val Ala Arg 2060021PRTArtificial
SequenceSynthetic Polypeptide 600Phe Gly Gly Ser Phe Ala Gly Ser
Phe Gly Gly Ala Gly Gly His Ala1 5 10 15Pro Gly Val Ala Arg
2060115PRTArtificial SequenceSynthetic Polypeptide 601Val Leu Val
Val Gly Ala Gly Gly Ile Gly Cys Glu Leu Leu Lys1 5 10
1560220PRTArtificial SequenceSynthetic Polypeptide 602Val Thr Ala
Asp His Gly Pro Ala Val Ser Gly Ala His Asn Thr Ile1 5 10 15Ile Cys
Ala Arg 2060314PRTArtificial SequenceSynthetic Polypeptide 603Cys
Glu Ala Leu Ala Gly Ala Pro Leu Asp Asn Ala Pro Lys1 5
1060414PRTArtificial SequenceSynthetic Polypeptide 604Cys Glu Ala
Leu Ala Gly Ala Pro Leu Asp Asn Ala Pro Lys1 5 1060514PRTArtificial
SequenceSynthetic Polypeptide 605Ser Thr Gly Gly Ala Pro Thr Phe
Asn Val Thr Val Thr Lys1 5 1060614PRTArtificial SequenceSynthetic
Polypeptide 606Lys Gly Cys Asp Val Val Val Ile Pro Ala Gly Val Pro
Arg1 5 1060715PRTArtificial SequenceSynthetic Polypeptide 607Phe
Ser Pro Ala Gly Val Glu Gly Cys Pro Ala Leu Pro His Lys1 5 10
1560813PRTArtificial SequenceSynthetic Polypeptide 608His Ser Ser
Leu Ala Gly Cys Gln Ile Ile Asn Tyr Arg1 5 1060919PRTArtificial
SequenceSynthetic Polypeptide 609Ser Ser Glu Val Gly Tyr Asp Ala
Met Ala Gly Asp Phe Val Asn Met1 5 10 15Val Glu
Lys61018PRTArtificial SequenceSynthetic Polypeptide 610Ser Ile Glu
Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile1 5 10 15Gly
Arg61118PRTArtificial SequenceSynthetic Polypeptide 611Ser Ile Glu
Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile1 5 10 15Gly
Arg61215PRTArtificial SequenceSynthetic Polypeptide 612Val Thr Gly
Asp His Ile Pro Thr Pro Gln Asp Leu Pro Gln Arg1 5 10
1561315PRTArtificial SequenceSynthetic Polypeptide 613Val Thr Gly
Asp His Ile Pro Thr Pro Gln Asp Leu Pro Gln Arg1 5 10
1561417PRTArtificial SequenceSynthetic Polypeptide 614Asn Gly Asp
Thr Phe Leu Gly Gly Glu Asp Phe Asp Gln Ala Leu Leu1 5 10
15Arg61516PRTArtificial SequenceSynthetic Polypeptide 615Ile Val
Tyr Ile Cys Cys Gly Glu Asp His Thr Ala Ala Leu Thr Lys1 5 10
1561617PRTArtificial SequenceSynthetic Polypeptide 616Met Val Asp
Gly Asn Val Ser Gly Glu Phe Thr Asp Leu Val Pro Glu1 5 10
15Lys61712PRTArtificial SequenceSynthetic Polypeptide 617Met Ala
Ala Gln Gly Glu Pro Gln Val Gln Phe Lys1 5 1061820PRTArtificial
SequenceSynthetic Polypeptide 618Gln Ala Leu Ala Val His Leu Ala
Leu Gln Gly Glu Ser Ser Ser Glu1 5 10 15His Phe Leu Lys
2061916PRTArtificial SequenceSynthetic Polypeptide 619Ala Phe Tyr
Asn Asn Val Leu Gly Glu Tyr Glu Glu Tyr Ile Thr Lys1 5 10
1562013PRTArtificial SequenceSynthetic Polypeptide 620Leu Leu Asn
Gln Met Asp Gly Phe Asp Thr Leu His Arg1 5 1062115PRTArtificial
SequenceSynthetic Polypeptide 621Gly Leu Thr Glu Gly Leu His Gly
Phe His Val His Glu Phe Gly1 5 10 1562222PRTArtificial
SequenceSynthetic Polypeptide 622Gly Leu Thr Glu Gly Leu His Gly
Phe His Val His Glu Phe Gly Asp1 5 10 15Asn Thr Ala Gly Cys Thr
2062322PRTArtificial SequenceSynthetic Polypeptide 623Gly Leu Thr
Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp1 5 10 15Asn Thr
Ala Gly Cys Thr 2062422PRTArtificial SequenceSynthetic Polypeptide
624Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp1
5 10 15Asn Thr Ala Gly Cys Thr 2062522PRTArtificial
SequenceSynthetic Polypeptide 625Gly Leu Thr Glu Gly Leu His Gly
Phe His Val His Glu Phe Gly Asp1 5 10 15Asn Thr Ala Gly Cys Thr
2062622PRTArtificial SequenceSynthetic Polypeptide 626Gly Leu Thr
Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp1 5 10 15Asn Thr
Ala Gly Cys Thr 2062724PRTArtificial SequenceSynthetic Polypeptide
627Ala Ala Asp Ser Tyr Phe Ser Leu Leu Gln Gly Phe Ile Asn Ser Leu1
5 10 15Asp Glu Ser Thr Gln Glu Ser Lys 2062821PRTArtificial
SequenceSynthetic Polypeptide 628Ile Asn Pro Tyr Leu Leu Gly Thr
Met Ala Gly Gly Ala Ala Asp Cys1 5 10 15Ser Phe Trp Glu Arg
2062920PRTArtificial SequenceSynthetic Polypeptide 629Gln His Asp
Leu Phe Asp Ser Gly Phe Gly Gly Gly Ala Gly Val Glu1 5 10 15Thr Gly
Gly Lys 2063019PRTArtificial SequenceSynthetic Polypeptide 630Thr
Thr His Phe Val Glu Gly Gly Asp Ala Gly Asn Arg Glu Asp Gln1 5 10
15Ile Asn Arg63119PRTArtificial SequenceSynthetic Polypeptide
631Thr Thr His Phe Val Glu Gly Gly Asp Ala Gly Asn Arg Glu Asp Gln1
5 10 15Ile Asn Arg63221PRTArtificial SequenceSynthetic Polypeptide
632Ser Gln Pro Ile Ala Gln Gln Pro Leu Gln Gly Gly Asp His Ser Gly1
5 10 15Asn Tyr Gly Tyr Lys 2063322PRTArtificial SequenceSynthetic
Polypeptide 633Gly Thr Asp Gly Thr Asp Asn Pro Leu Ser Gly Gly Asp
Gln Tyr Gln1 5 10 15Asn Ile Thr Val His Arg 2063417PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 634Gly Cys Ile Thr Xaa Ile Gly Gly
Gly Asp Thr Ala Thr Cys Cys Ala1 5 10 15Lys63538PRTArtificial
SequenceSynthetic Polypeptide 635Trp Gly Ser Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Arg Lys 20 25 30Ser Ser Ser Ala Ala Ala
3563621PRTArtificial SequenceSynthetic Polypeptide 636Leu Ala Ala
Gly Ser Leu Ala Ala Pro Gly Gly Gly Gly Gly Ser Ala1 5 10 15Gly Gly
Ala Arg Pro 2063716PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(5)Xaa can be any naturally occurring
amino acid 637Gly Ser Xaa Xaa Xaa Gly Gly Gly Ser Tyr Asn Asp Phe
Gly Asn Tyr1 5 10 1563820PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(6)..(6)Xaa can be any naturally occurring
amino acid 638Val Asn Ala Ala Asn Xaa Ser Leu Leu Gly Gly Gly Gly
Val Asp Gly1 5 10 15Cys Ile His Arg 2063923PRTArtificial
SequenceSynthetic Polypeptide 639Phe Cys Val Gly Phe Leu Glu Gly
Gly Lys Asp Ser Cys Gln Gly Asp1 5 10 15Ser Gly Gly Pro Val Val Cys
2064024PRTArtificial SequenceSynthetic Polypeptide 640Leu Val Asp
Gly Gln Ile Phe Cys Leu His Gly Gly Leu Ser Pro Ser1 5 10 15Ile Asp
Thr Leu Asp His Ile Arg 2064113PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 641Met Phe Xaa Gly Gly Leu Ser Trp Asp Thr Ser Lys Lys1
5 1064221PRTArtificial SequenceSynthetic Polypeptide 642Asp Pro Gln
Glu Leu Leu Glu Gly Gly Asn Gln Gly Glu Gly Asp Pro1 5 10 15Gln Ala
Glu Gly Arg 2064325PRTArtificial SequenceSynthetic Polypeptide
643Asn Met Gly Gly Pro Tyr Gly Gly Gly Asn Tyr Gly Pro Gly Gly Ser1
5 10 15Gly Gly Ser Gly Gly Tyr Gly Gly Arg 20 2564416PRTArtificial
SequenceSynthetic Polypeptide 644Arg Gly Gly Pro Gly Gly Pro Gly
Gly Pro Gly Gly Pro Met Gly Arg1 5 10 1564519PRTArtificial
SequenceSynthetic Polypeptide 645Ser Val Leu Asp Asp Trp Phe Pro
Leu Gln Gly Gly Gln Gly Gln Val1 5 10 15His Leu
Arg64616PRTArtificial SequenceSynthetic Polypeptide 646Ile Ile Met
Glu Tyr Leu Gly Gly Gly Ser Ala Leu Asp Leu Leu Arg1 5 10
1564723PRTArtificial SequenceSynthetic Polypeptide 647Ser His Phe
Ala Met Met His Gly Gly Thr Gly Phe Ala Gly Ile Asp1 5 10 15Ser Ser
Ser Pro Glu Val Lys 2064822PRTArtificial SequenceSynthetic
Polypeptide 648Gln Gly Phe Gln Leu Thr His Ser Leu Gly Gly Gly Thr
Gly Ser Gly1 5 10 15Met Gly Thr Leu Leu Ile 2064919PRTArtificial
SequenceSynthetic Polypeptide 649Met Ala Asp Tyr Leu Ile Ser Gly
Gly Thr Ser Tyr Val Pro Asp Asp1 5 10 15Gly Leu
Thr65013PRTArtificial SequenceSynthetic Polypeptide 650Val Thr Val
Ala Gly Gly Val His Ile Ser Gly Leu His1 5 1065114PRTArtificial
SequenceSynthetic Polypeptide 651Val Thr Val Ala Gly Gly Val His
Ile Ser Gly Leu His Thr1 5 1065215PRTArtificial SequenceSynthetic
Polypeptide 652Val Thr Val Ala Gly Gly Val His Ile Ser Gly Leu His
Thr Glu1 5 10 1565320PRTArtificial SequenceSynthetic Polypeptide
653Tyr Ala Val Ser Glu Leu Ala Gly His Gln Thr Ser Ala Glu Ser Trp1
5 10 15Gly Thr Gly Arg 2065413PRTArtificial SequenceSynthetic
Polypeptide 654Thr Phe Gln Gly His Thr Asn Glu Val Asn Ala Ile Lys1
5 1065514PRTArtificial SequenceSynthetic Polypeptide 655Gly Asp Gly
Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys1 5 1065618PRTArtificial
SequenceSynthetic Polypeptide 656Val Thr Ile Ile Gly Pro Ala Thr
Val Gly Gly Ile Lys Pro Gly Cys1 5
10 15Phe Lys65720PRTArtificial SequenceSynthetic Polypeptide 657Phe
Ser Leu Pro Gly Met Glu His Val Tyr Gly Ile Pro Glu His Ala1 5 10
15Asp Asn Leu Arg 2065816PRTArtificial SequenceSynthetic
Polypeptide 658Ile Phe Val Gly Gly Ile Pro His Asn Cys Gly Glu Thr
Glu Leu Arg1 5 10 1565918PRTArtificial SequenceSynthetic
Polypeptide 659Leu Pro Pro Ser Gly Ala Val Pro Val Thr Gly Ile Pro
Pro His Val1 5 10 15Val Lys66013PRTArtificial SequenceSynthetic
Polypeptide 660Met Asp Gly Ile Val Pro Asp Ile Ala Val Gly Thr Lys1
5 106619PRTArtificial SequenceSynthetic Polypeptide 661Arg Gly Ile
Trp His Asn Asp Asn Lys1 566220PRTArtificial SequenceSynthetic
Polypeptide 662Gly Lys Pro Glu Ile Glu Gly Lys Pro Glu Ser Glu Gly
Glu Pro Gly1 5 10 15Ser Glu Thr Arg 2066322PRTArtificial
SequenceSynthetic Polypeptide 663Tyr Asp Ile Asn Ala His Ala Cys
Val Thr Gly Lys Pro Ile Ser Gln1 5 10 15Gly Gly Ile His Gly Arg
2066420PRTArtificial SequenceSynthetic Polypeptide 664Glu Leu Thr
Gln Gln Leu Ala Gln Ala Thr Gly Lys Pro Pro Gln Tyr1 5 10 15Ile Ala
Val His 2066517PRTArtificial SequenceSynthetic Polypeptide 665Asn
Pro Lys Pro Phe Leu Asn Gly Leu Thr Gly Lys Pro Val Met Val1 5 10
15Lys66616PRTArtificial SequenceSynthetic Polypeptide 666Cys Pro
Ser Ile Leu Gly Gly Leu Ala Pro Glu Lys Asp Gln Pro Lys1 5 10
1566721PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(10)..(10)Xaa can be any naturally occurring
amino acid 667Val Ala Ser Gly Ile Pro Ala Gly Trp Xaa Gly Leu Asp
Cys Gly Pro1 5 10 15Glu Ser Ser Lys Lys 2066812PRTArtificial
SequenceSynthetic Polypeptide 668Gln Val Leu Gln Gly Leu Asp Tyr
Leu His Ser Lys1 5 1066914PRTArtificial SequenceSynthetic
Polypeptide 669Gly Ala Leu Glu Gly Leu Pro Arg Pro Pro Pro Pro Val
Lys1 5 1067016PRTArtificial SequenceSynthetic Polypeptide 670Leu
Phe Ile Gly Gly Leu Ser Phe Glu Thr Thr Asp Glu Ser Leu Arg1 5 10
1567116PRTArtificial SequenceSynthetic Polypeptide 671Val Phe Val
Gly Gly Leu Ser Pro Asp Thr Ser Glu Glu Gln Ile Lys1 5 10
1567213PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 672Met Phe Xaa Gly Gly Leu Ser Trp Asp Thr Ser Lys Lys1
5 1067320PRTArtificial SequenceSynthetic Polypeptide 673Asn Val Ile
Ile Trp Gly Asn His Ser Ser Thr Gln Tyr Pro Asp Val1 5 10 15Asn His
Ala Lys 2067419PRTArtificial SequenceSynthetic Polypeptide 674Leu
Leu Ser Gly Leu Ala Glu Gly Leu Gly Gly Asn Ile Glu Gln Leu1 5 10
15Val Ala Arg67514PRTArtificial SequenceSynthetic Polypeptide
675Leu Val Ile Asn Gly Asn Pro Ile Thr Ile Phe Gln Glu Arg1 5
1067616PRTArtificial SequenceSynthetic Polypeptide 676Ser Ala Ala
Met Leu Gly Asn Ser Glu Asp His Thr Ala Leu Ser Arg1 5 10
1567711PRTArtificial SequenceSynthetic Polypeptide 677Ile Phe Gln
Gly Asn Val His Asn Phe Glu Lys1 5 1067815PRTArtificial
SequenceSynthetic Polypeptide 678Asn Asn Pro Pro Thr Leu Glu Gly
Asn Tyr Ser Lys Pro Leu Lys1 5 10 1567911PRTArtificial
SequenceSynthetic Polypeptide 679Met Val Gly Pro Ala Val Ile Val
Asp Lys Lys1 5 1068014PRTArtificial SequenceSynthetic Polypeptide
680Met Met Leu Gly Pro Glu Gly Gly Glu Gly Phe Val Val Lys1 5
1068116PRTArtificial SequenceSynthetic Polypeptide 681Ser Ile Tyr
Glu Ala Leu Gly Gly Pro His Asp Pro Asn Val Ala Lys1 5 10
1568213PRTArtificial SequenceSynthetic Polypeptide 682Thr Phe Gln
Gly Pro Asn Cys Pro Ala Thr Cys Gly Arg1 5 1068311PRTArtificial
SequenceSynthetic Polypeptide 683Ile Met Gly Pro Asn Tyr Thr Pro
Gly Lys Lys1 5 1068413PRTArtificial SequenceSynthetic Polypeptide
684Met Val Ile Ile Thr Gly Pro Pro Glu Ala Gln Phe Lys1 5
1068520PRTArtificial SequenceSynthetic Polypeptide 685Ala Phe Gly
Leu Thr Asp Asp Gln Val Ser Gly Pro Pro Ser Ala Pro1 5 10 15Ala Glu
Asp Arg 2068614PRTArtificial SequenceSynthetic Polypeptide 686Thr
Val Gln Gly Pro Pro Thr Ser Asp Asp Ile Phe Glu Arg1 5
1068715PRTArtificial SequenceSynthetic Polypeptide 687Phe Val Ile
Gly Gly Pro Gln Gly Asp Ala Gly Leu Thr Gly Arg1 5 10
1568813PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 688Ile Ile Thr Leu Xaa Gly Pro Thr Asn Ala Ile Phe Lys1
5 1068921PRTArtificial SequenceSynthetic Polypeptide 689Lys Pro Pro
Thr Leu Ile His Gly Gln Ala Pro Ser Ala Gly Leu Pro1 5 10 15Ser Gln
Lys Pro Lys 2069011PRTArtificial SequenceSynthetic Polypeptide
690Arg Gly Gln Gly Gly Tyr Pro Gly Lys Pro Arg1 5
1069118PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(6)..(6)Xaa can be any naturally occurring
amino acid 691Arg Pro Asp Asn Phe Xaa Phe Gly Gln Ser Gly Ala Gly
Asn Asn Trp1 5 10 15Ala Lys69218PRTArtificial SequenceSynthetic
Polypeptide 692Gly Leu Leu Ala Leu Ser Ser Ala Leu Ser Gly Gln Ser
His Leu Ala1 5 10 15Ile Lys69322PRTArtificial SequenceSynthetic
Polypeptide 693Ala Leu Pro Pro Val Leu Thr Thr Val Asn Gly Gln Ser
Pro Pro Glu1 5 10 15His Ser Ala Pro Ala Lys 2069413PRTArtificial
SequenceSynthetic Polypeptide 694Gln Ser Gly Tyr Gly Gly Gln Thr
Lys Pro Ile Phe Arg1 5 1069511PRTArtificial SequenceSynthetic
Polypeptide 695Leu Ser Gly Gln Thr Asn Ile His Leu Ser Lys1 5
1069618PRTArtificial SequenceSynthetic Polypeptide 696Val Val Leu
Met Ser His Leu Gly Arg Pro Asp Gly Val Pro Met Pro1 5 10 15Asp
Lys69719PRTArtificial SequenceSynthetic Polypeptide 697Val Val Leu
Met Ser His Leu Gly Arg Pro Asp Gly Val Pro Met Pro1 5 10 15Asp Lys
Tyr69817PRTArtificial SequenceSynthetic Polypeptide 698Gln Gln Ser
Ile Ala Gly Ser Ala Asp Ser Lys Pro Ile Asp Val Ser1 5 10
15Arg69920PRTArtificial SequenceSynthetic Polypeptide 699Val Thr
Leu Gly Pro Val Pro Glu Ile Gly Gly Ser Glu Ala Pro Ala1 5 10 15Pro
Gln Asn Lys 2070022PRTArtificial SequenceSynthetic Polypeptide
700Asn Phe Gly Gly Ser Phe Ala Gly Ser Phe Gly Gly Ala Gly Gly His1
5 10 15Ala Pro Gly Val Ala Arg 2070117PRTArtificial
SequenceSynthetic Polypeptide 701Met Met Asp Tyr Leu Gln Gly Ser
Gly Glu Thr Pro Gln Thr Asp Val1 5 10 15Arg70218PRTArtificial
SequenceSynthetic Polypeptide 702Asp Ser Val Trp Gly Ser Gly Gly
Gly Gln Gln Ser Val Asn His Leu1 5 10 15Val Lys70317PRTArtificial
SequenceSynthetic Polypeptide 703Pro Gln Val Ala Ile Ile Cys Gly
Ser Gly Leu Gly Gly Leu Thr Asp1 5 10 15Lys70428PRTArtificial
SequenceSynthetic Polypeptide 704Pro Thr Ser Ser Glu Gln Gly Gly
Leu Glu Gly Ser Gly Ser Ala Ala1 5 10 15Gly Glu Gly Lys Pro Ala Leu
Ser Glu Glu Glu Arg 20 2570522PRTArtificial SequenceSynthetic
Polypeptide 705Thr Val Glu Gln Leu Leu Thr Gly Ser Pro Thr Ser Pro
Thr Val Glu1 5 10 15Pro Glu Lys Pro Thr Arg 2070614PRTArtificial
SequenceSynthetic Polypeptide 706Gly Cys Leu Glu Gly Ser Gln Gly
Thr Gln Ala Leu His Lys1 5 1070722PRTArtificial SequenceSynthetic
Polypeptide 707Leu Leu Ala Val Ser Ala Pro Ala Leu Gln Gly Ser Arg
Pro Gly Glu1 5 10 15Thr Glu Glu Asn Val Arg 2070820PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(2)..(3)Xaa can be any
naturally occurring amino acid 708Ile Xaa Xaa Gly Ser Ser Gly Ala
Gln Gly Ser Gly Gly Gly Ser Thr1 5 10 15Ser Ala His Tyr
2070915PRTArtificial SequenceSynthetic Polypeptide 709Val Ala Phe
Thr Gly Ser Thr Glu Val Gly His Leu Ile Gln Lys1 5 10
1571016PRTArtificial SequenceSynthetic Polypeptide 710Val Val Val
Leu Met Gly Ser Thr Ser Asp Leu Gly His Cys Glu Lys1 5 10
1571117PRTArtificial SequenceSynthetic Polypeptide 711Met Val Glu
Leu Leu Gly Ser Tyr Thr Glu Asp Asn Ala Ser Gln Ala1 5 10
15Arg71214PRTArtificial SequenceSynthetic Polypeptide 712Ile Tyr
Trp Gly Thr Ala Thr Thr Gly Lys Pro His Val Ala1 5
1071315PRTArtificial SequenceSynthetic Polypeptide 713Ile Val Gly
Phe Cys Trp Gly Gly Thr Ala Val His His Leu Met1 5 10
1571423PRTArtificial SequenceSynthetic Polypeptide 714Gly Val Val
Pro Leu Ala Gly Thr Asp Gly Glu Thr Thr Thr Gln Gly1 5 10 15Leu Asp
Gly Leu Ser Glu Arg 2071516PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acidmisc_feature(4)..(4)Xaa can be any naturally occurring
amino acidmisc_feature(6)..(6)Xaa can be any naturally occurring
amino acid 715Gly Xaa Val Xaa Phe Xaa Gly Thr Asp His Ile Asp Gln
Trp Asn Lys1 5 10 1571612PRTArtificial SequenceSynthetic
Polypeptide 716Ser Val Ser Gly Thr Asp Val Gln Glu Glu Cys Arg1 5
1071714PRTArtificial SequenceSynthetic Polypeptide 717Met Met Leu
Gly Thr Glu Gly Gly Glu Gly Phe Val Val Lys1 5 1071820PRTArtificial
SequenceSynthetic Polypeptide 718Ile Ala Phe His Gln Asp Gly Ser
Leu Ala Gly Thr Gly Gly Leu Asp1 5 10 15Ala Phe Gly Arg
2071916PRTArtificial SequenceSynthetic Polypeptide 719Leu Asn Phe
Ser His Gly Thr His Glu Tyr His Ala Glu Thr Ile Lys1 5 10
1572015PRTArtificial SequenceSynthetic Polypeptide 720Leu Val Leu
Gly Thr His Thr Ser Asp Glu Gln Asn His Leu Val1 5 10
1572122PRTArtificial SequenceSynthetic Polypeptide 721Ala Leu His
Trp Leu Val Leu Gly Thr His Thr Ser Asp Glu Gln Asn1 5 10 15His Leu
Val Val Ala Arg 2072213PRTArtificial SequenceSynthetic Polypeptide
722Val Leu Ser Gly Thr Ile His Ala Gly Gln Pro Val Lys1 5
1072316PRTArtificial SequenceSynthetic Polypeptide 723Ile Ile Thr
Ile Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln Tyr1 5 10
1572411PRTArtificial SequenceSynthetic Polypeptide 724Gly Gly Thr
Ser Asp Val Glu Val Asn Glu Lys1 5 1072516PRTArtificial
SequenceSynthetic Polypeptide 725Val Leu Thr Gly Val Ala Gly Glu
Asp Ala Glu Cys His Ala Ala Lys1 5 10 1572620PRTArtificial
SequenceSynthetic Polypeptide 726Thr Gly Gly Val Asp Thr Ala Ala
Val Gly Gly Val Phe Asp Val Ser1 5 10 15Asn Ala Asp Arg
2072716PRTArtificial SequenceSynthetic Polypeptide 727Phe Ile Val
Asp Gly Trp His Glu Met Asp Ala Glu Asn Pro Leu His1 5 10
1572826PRTArtificial SequenceSynthetic Polypeptide 728Thr Met Phe
Ser Ser Glu Val Gln Phe Gly His Ala Gly Ala Cys Ala1 5 10 15Asn Gln
Ala Ser Glu Thr Ala Val Ala Lys 20 2572922PRTArtificial
SequenceSynthetic Polypeptide 729Pro Ile Tyr Asp Val Leu Gln Met
Val Gly His Ala Asn Arg Pro Leu1 5 10 15Gln Asp Asp Glu Gly Arg
2073010PRTArtificial SequenceSynthetic Polypeptide 730Glu Trp Ala
His Ala Thr Ile Ile Pro Lys1 5 1073110PRTArtificial
SequenceSynthetic Polypeptide 731Lys His Glu Ala Asn Asn Pro Gln
Leu Lys1 5 1073210PRTArtificial SequenceSynthetic Polypeptide
732Met Val Asn His Phe Ile Ala Glu Phe Lys1 5 1073310PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(3)..(3)Xaa can be any
naturally occurring amino acid 733Leu Val Xaa His Phe Val Glu Glu
Phe Lys1 5 1073417PRTArtificial SequenceSynthetic Polypeptide
734Met Pro Phe Pro Val Asn His Gly Ala Ser Ser Glu Asp Thr Leu Leu1
5 10 15Lys73523PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 735Asn Xaa Cys Trp Glu Leu Tyr Cys Leu Glu His Gly Ile
Gln Pro Asp1 5 10 15Gly Gln Met Pro Ser Asp Lys
2073623PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 736Asn Xaa Cys Trp Glu Leu Tyr Cys Leu Glu His Gly Ile
Gln Pro Asp1 5 10 15Gly Gln Met Pro Ser Asp Lys
2073720PRTArtificial SequenceSynthetic Polypeptide 737Val His Ala
Gly Pro Phe Ala Asn Ile Ala His Gly Asn Ser Ser Ile1 5 10 15Ile Ala
Asp Arg 2073818PRTArtificial SequenceSynthetic Polypeptide 738Ile
Asn Gln Val Phe His Gly Ser Cys Ile Thr Glu Gly Asn Glu Leu1 5 10
15Thr Lys73915PRTArtificial SequenceSynthetic Polypeptide 739Phe
Glu Leu Gln His Gly Thr Glu Glu Gln Gln Glu Glu Val Arg1 5 10
1574018PRTArtificial SequenceSynthetic Polypeptide 740Glu Gln Gln
Glu Ala Ile Glu His Ile Asp Glu Val Gln Asn Glu Ile1 5 10 15Asp
Arg74121PRTArtificial SequenceSynthetic Polypeptide 741Ala Val Glu
Ala Leu Ala Ala Ala Leu Ala His Ile Ser Gly Ala Thr1 5 10 15Ser Val
Asp Gln Arg 2074213PRTArtificial SequenceSynthetic Polypeptide
742Arg His Leu Ala Pro Thr Gly Asn Ala Pro Ala Ser Arg1 5
1074318PRTArtificial SequenceSynthetic Polypeptide 743Leu Leu Thr
Asp Phe Cys Thr His Leu Pro Asn Leu Pro Asp Ser Thr1 5 10 15Ala
Lys74416PRTArtificial SequenceSynthetic Polypeptide 744Val Asp Glu
Phe Val Thr His Asn Leu Ser Phe Asp Glu Ile Asn Lys1 5 10
1574519PRTArtificial SequenceSynthetic Polypeptide 745Ala Thr Leu
Glu Leu Thr His Asn Trp Gly Thr Glu Asp Asp Glu Thr1 5 10 15Gln Ser
Tyr74616PRTArtificial SequenceSynthetic Polypeptide 746Glu Glu Phe
Thr Ala Phe Leu His Pro Glu Glu Tyr Asp Tyr Met Lys1 5 10
1574712PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 747Gln Xaa Phe His Pro Glu Gln Leu Ile Thr Gly Lys1 5
1074823PRTArtificial SequenceSynthetic Polypeptide 748Pro Val Thr
His Asn Leu Pro Thr Val Ala His Pro Ser Gln Ala Pro1 5 10 15Ser Pro
Asn Gln Pro Thr Lys 2074917PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(6)Xaa can be any naturally occurring
amino acid 749Ala Xaa Xaa Xaa Xaa Xaa Gln His Gln Ala Gly Gln Ala
Pro His Leu1 5 10 15Gly75017PRTArtificial SequenceSynthetic
Polypeptide 750Cys Asn Phe Thr Asp Gly Ala Leu Val Gln His Gln Glu
Trp Asp Gly1 5 10 15Lys75113PRTArtificial SequenceSynthetic
Polypeptide 751Gly Val Leu His Gln Phe Ser Gly Thr Glu Thr Asn Lys1
5 1075216PRTArtificial SequenceSynthetic Polypeptide 752Gln Ile Gly
Ala Val Val Ser His Gln Ser Ser Val Ile Pro Asp Arg1 5 10
1575319PRTArtificial SequenceSynthetic Polypeptide 753Ile Glu Pro
Asn Glu Val Thr His Ser Gly Asp Thr Gly Val Glu Thr1 5 10 15Asp Gly
Arg75415PRTArtificial SequenceSynthetic Polypeptide 754His Tyr Ala
His Thr Asp Cys Pro Gly His Ala Asp Tyr Val Lys1 5 10
1575511PRTArtificial SequenceSynthetic Polypeptide 755Thr Ile Cys
Ser His Val Gln Asn Met Ile Lys1 5 1075613PRTArtificial
SequenceSynthetic Polypeptide 756Leu Leu Gly His Trp Glu Glu Ala
Ala His Asp Leu Ala1 5 1075712PRTArtificial SequenceSynthetic
Polypeptide 757Thr Tyr Thr Ile Ala Asn Gln Phe Pro Leu Asn Lys1 5
1075817PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(4)..(4)Xaa can be any naturally occurring
amino acid 758Asn Pro Thr Xaa Phe Phe Asp Ile Ala Val Asp Gly Glu
Pro Leu Gly1 5 10 15Arg75915PRTArtificial SequenceSynthetic
Polypeptide 759Leu Val Ser Ile Gly Ala Glu Glu Ile Val Asp Gly Asn
Ala Lys1 5 10 1576021PRTArtificial SequenceSynthetic Polypeptide
760Thr Thr Asp Gly Val Tyr Glu Gly Val Ala Ile Gly Gly Asp Arg Tyr1
5 10 15Pro Gly Ser Thr Phe 2076115PRTArtificial SequenceSynthetic
Polypeptide 761Thr His Ile Asn Ile Val Val Ile Gly His Val Asp Ser
Gly Lys1 5 10 1576219PRTArtificial SequenceSynthetic Polypeptide
762Asp Asn Asp Phe Cys Gly Thr Asp Met Thr Ile Gly Thr Asp Ser Ala1
5 10 15Leu His Arg76318PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 763Val Leu Xaa Asn Met Glu Ile Gly Thr Ser Leu Phe Asp
Glu Glu Gly1 5 10 15Ala Lys76416PRTArtificial SequenceSynthetic
Polypeptide 764Val Cys Thr Leu Ala Ile Ile Asp Pro Gly Asp Ser Asp
Ile Ile Arg1 5 10 1576517PRTArtificial SequenceSynthetic
Polypeptide 765Gly Cys Ile Thr Ile Ile Gly Gly Gly Asp Thr Ala Thr
Cys Cys Ala1 5 10 15Lys76614PRTArtificial SequenceSynthetic
Polypeptide 766Thr Phe Asn Gln Val Glu Ile Lys Pro Glu Met Ile Gly
His1 5 1076713PRTArtificial SequenceSynthetic Polypeptide 767Cys
Gln Leu Glu Ile Asn Phe Asn Thr Leu Gln Thr Lys1 5
1076812PRTArtificial SequenceSynthetic Polypeptide 768His Leu Glu
Ile Asn Pro Asp His Pro Ile Val Glu1 5 1076915PRTArtificial
SequenceSynthetic Polypeptide 769His Leu Glu Ile Asn Pro Asp His
Ser Ile Ile Glu Thr Leu Arg1 5 10 1577018PRTArtificial
SequenceSynthetic Polypeptide 770Val Pro Tyr Leu Ile Ala Gly Ile
Gln His Ser Cys Gln Asp Ile Gly1 5 10 15Ala Lys77110PRTArtificial
SequenceSynthetic Polypeptide 771Val Leu Ser Ile Gln Ser His Val
Ile Arg1 5 1077210PRTArtificial SequenceSynthetic Polypeptide
772Glu Leu Gly Ile Thr Ala Leu His Ile Lys1 5 1077310PRTArtificial
SequenceSynthetic Polypeptide 773Leu Val Ala Ile Val Asp Pro His
Ile Lys1 5 1077413PRTArtificial SequenceSynthetic Polypeptide
774Thr Leu Thr Ile Val Asp Thr Gly Ile Gly Met Thr Lys1 5
1077512PRTArtificial SequenceSynthetic Polypeptide 775Leu Val Ala
Ile Val Asp Val Ile Asp Gln Asn Arg1 5 1077614PRTArtificial
SequenceSynthetic Polypeptide 776Gln Ile Ile Leu Glu Lys Glu Glu
Thr Glu Glu Leu Lys Arg1 5 1077710PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 777Xaa Lys His Pro Asp Ala Asp Ser Leu Tyr1 5
1077815PRTArtificial SequenceSynthetic Polypeptide 778Cys Ile Gly
Lys Pro Gly Gly Ser Leu Asp Asn Ser Glu Gln Lys1 5 10
1577924PRTArtificial SequenceSynthetic Polypeptide 779His His Ile
Tyr Leu Glu Gly Thr Leu Leu Lys Pro Asn Met Val Thr1 5 10 15Pro Gly
His Ala Cys Thr Gln Lys 2078019PRTArtificial SequenceSynthetic
Polypeptide 780Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly Lys Pro Pro
Gln Tyr Ile1 5 10 15Ala Val His78125PRTArtificial SequenceSynthetic
Polypeptide 781Ser Ser Pro Pro Glu Leu Pro Asp Val Met Lys Pro Gln
Asp Ser Gly1 5 10 15Ser Ser Ala Asn Glu Gln Ala Val Gln 20
2578213PRTArtificial SequenceSynthetic Polypeptide 782Leu Gln Glu
Leu Glu Lys Tyr Pro Gly Ile Gln Thr Arg1 5 1078312PRTArtificial
SequenceSynthetic Polypeptide 783Trp Ile Gly Leu Asp Leu Ser Asn
Gly Lys Pro Arg1 5 1078414PRTArtificial SequenceSynthetic
Polypeptide 784Met Pro Phe Leu Glu Leu Asp Thr Asn Leu Pro Ala Asn
Arg1 5 1078520PRTArtificial SequenceSynthetic Polypeptide 785Glu
Thr Ala Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp Pro Gln Thr1 5 10
15His Ala Asn Arg 2078617PRTArtificial SequenceSynthetic
Polypeptide 786Glu Ala Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly Thr
Ile Thr Thr1 5 10 15Lys78711PRTArtificial SequenceSynthetic
Polypeptide 787Tyr Glu Leu Gly Arg Pro Ala Ala Asn Thr Lys1 5
1078820PRTArtificial SequenceSynthetic Polypeptide 788Gly Asn Pro
Ile Cys Ser Leu His Asp Gln Gly Ala Gly Gly Asn Gly1 5 10 15Asn Val
Leu Lys 2078916PRTArtificial SequenceSynthetic Polypeptide 789Val
Ile Leu His Leu Lys Glu Asp Gln Thr Glu Tyr Leu Glu Glu Arg1 5 10
1579020PRTArtificial SequenceSynthetic Polypeptide 790Ile Gln Gln
Leu Cys Glu Asp Ile Ile Gln Leu Lys Pro Asp Val Val1 5 10 15Ile Thr
Glu Lys 2079120PRTArtificial SequenceSynthetic Polypeptide 791Ile
Gln Gln Leu Cys Glu Asp Ile Ile Gln Leu Lys Pro Asp Val Val1 5 10
15Ile Thr Glu Lys 2079210PRTArtificial SequenceSynthetic
Polypeptide 792Thr Leu Asn Asn Asp Ile Met Leu Ile Lys1 5
1079315PRTArtificial SequenceSynthetic Polypeptide 793Asn Gln Val
Ala Leu Asn Pro Gln Asn Thr Val Phe Asp Ala Lys1 5 10
1579415PRTArtificial SequenceSynthetic Polypeptide 794Asn Gln Val
Ala Leu Asn Pro Gln Asn Thr Val Phe Asp Ala Lys1 5 10
1579519PRTArtificial SequenceSynthetic Polypeptide 795Ser Thr Ala
Thr Leu Ala Trp Gly Val Asn Leu Pro Ala His Thr Val1 5 10 15Ile Ile
Lys79613PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 796Glu Xaa Leu Glu Leu Pro Glu Asp Glu Glu Glu Lys Lys1
5 1079717PRTArtificial SequenceSynthetic Polypeptide 797Gly Val Asn
Leu Pro Gly Ala Ala Val Asp Leu Pro Ala Val Ser Glu1 5 10
15Lys79810PRTArtificial SequenceSynthetic Polypeptide 798Arg Leu
Pro Pro Ala Ala Gly Asp Glu Pro1 5 107999PRTArtificial
SequenceSynthetic Polypeptide 799Leu Asp Leu Pro Pro Tyr Glu Thr
Phe1 580018PRTArtificial SequenceSynthetic Polypeptide 800Asp Gly
Asp Ser Val Met Val Leu Pro Thr Ile Pro Glu Glu Glu Ala1 5 10 15Lys
Lys80117PRTArtificial SequenceSynthetic Polypeptide 801Glu Ile Val
His Leu Gln Ala Gly Gln Cys Gly Asn Gln Ile Gly Ala1 5 10
15Lys80220PRTArtificial SequenceSynthetic Polypeptide 802Asp Val
Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys1 5 10 15Ile
Ile Gly Arg 2080320PRTArtificial SequenceSynthetic Polypeptide
803Ser Ser Ala Pro Gly Pro Leu Glu Leu Asp Leu Thr Gly Asp Leu Glu1
5 10 15Ser Phe Lys Lys 2080411PRTArtificial SequenceSynthetic
Polypeptide 804Phe Leu Glu Met Cys Asn Asp Leu Leu Ala Arg1 5
1080515PRTArtificial SequenceSynthetic Polypeptide 805Thr Thr Gly
Phe Gly Met Ile Tyr Asp Ser Leu Asp Tyr Ala Lys1 5 10
1580612PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 806Xaa Met Asn Pro Thr Asn Thr Val Phe Asp Ala Lys1 5
1080712PRTArtificial SequenceSynthetic Polypeptide 807Glu Asp Ala
Met Ala Met Val Asp His Cys Leu Lys1 5 1080818PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(3)..(3)Xaa can be any
naturally occurring amino acidmisc_feature(5)..(5)Xaa can be any
naturally occurring amino acidmisc_feature(9)..(9)Xaa can be any
naturally occurring amino acid 808Ala Asn Xaa Val Xaa Ser Gly Gly
Xaa Thr Met Tyr Pro Gly Ile Ala1 5 10 15Asp Arg80918PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(3)..(3)Xaa can be any
naturally occurring amino acidmisc_feature(5)..(5)Xaa can be any
naturally occurring amino acidmisc_feature(9)..(9)Xaa can be any
naturally occurring amino acid 809Ala Asn Xaa Val Xaa Ser Gly Gly
Xaa Thr Met Tyr Pro Gly Ile Ala1 5 10 15Asp Arg81018PRTArtificial
SequenceSynthetic Polypeptide 810Ala Leu Gln Asp Leu Glu Asn Ala
Ala Ser Gly Asp Ala Ala Val His1 5 10 15Gln Arg81115PRTArtificial
SequenceSynthetic Polypeptide 811Asp Pro Val Thr Asn Leu Asn Asn
Ala Phe Glu Val Ala Glu Lys1 5 10 1581213PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(1)..(1)Xaa can be any
naturally occurring amino acid 812Xaa Asn Ala Gly Pro Asn Thr Asn
Gly Ser Gln Phe Phe1 5 1081318PRTArtificial SequenceSynthetic
Polypeptide 813Asn Tyr Ser Val Phe Tyr Tyr Glu Ile Gln Asn Ala Pro
Glu Gln Ala1 5 10 15Cys His81414PRTArtificial SequenceSynthetic
Polypeptide 814Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu Asp Lys Ile
Arg1 5 1081524PRTArtificial SequenceSynthetic Polypeptide 815Tyr
Tyr Phe Asn His Ile Thr Asn Ala Ser Gln Trp Glu Arg Pro Ser1 5 10
15Gly Asn Ser Ser Ser Gly Gly Lys 2081611PRTArtificial
SequenceSynthetic Polypeptide 816Thr Asn Asp Trp Glu Asp His Leu
Ala Val Lys1 5 1081713PRTArtificial SequenceSynthetic Polypeptide
817Ala Phe His Asn Glu Ala Gln Val Asn Pro Glu Arg Lys1 5
1081813PRTArtificial SequenceSynthetic Polypeptide 818Asn Cys Leu
Thr Asn Phe His Gly Met Asp Leu Thr Arg1 5 1081914PRTArtificial
SequenceSynthetic Polypeptide 819Thr Asn Val Ala Asn Phe Pro Gly
His Ser Gly Pro Ile Thr1 5 1082018PRTArtificial SequenceSynthetic
Polypeptide 820Ile Leu Asn Asn Gly His Ala Phe Asn Val Glu Phe Asp
Asp Ser Gln1 5 10 15Asp Lys82114PRTArtificial SequenceSynthetic
Polypeptide 821Ile Glu Gln Leu Gln Asn His Glu Asn Glu Asp Ile Tyr
Lys1 5 1082223PRTArtificial SequenceSynthetic Polypeptide 822Pro
Val Phe Val His Ala Gly Pro Phe Ala Asn Ile Ala His Gly Asn1 5 10
15Ser Ser Ile Ile Ala Asp Arg 2082314PRTArtificial
SequenceSynthetic Polypeptide 823Val Trp Tyr Val Ser Asn Ile Asp
Gly Thr His Ile Ala Lys1 5 1082419PRTArtificial SequenceSynthetic
Polypeptide 824Cys Asp Glu Val Met Gln Leu Leu Leu Glu Asn Leu Gly
Asn Glu Asn1 5 10 15Val His Arg82519PRTArtificial SequenceSynthetic
Polypeptide 825Gln Asp Gln Arg Pro Leu His Pro Val Ala Asn Pro His
Ala Glu Ile1 5 10 15Ser Thr Lys82612PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 826Xaa Asn Pro Leu Asp Ala Gly Ala Ala Glu Pro Ile1 5
1082719PRTArtificial SequenceSynthetic Polypeptide 827Leu Ile Pro
Gln Leu Val Ala Asn Val Thr Asn Pro Asn Ser Thr Glu1 5 10 15His Met
Lys82816PRTArtificial SequenceSynthetic Polypeptide 828Ser Ala Ala
Met Leu Gly Asn Ser Glu Asp His Thr Ala Leu Ser Arg1 5 10
1582929PRTArtificial SequenceSynthetic Polypeptide 829Asn Tyr Gln
Gln Asn Tyr Gln Asn Ser Glu Ser Gly Glu Lys Asn Glu1 5 10 15Gly Ser
Glu Ser Ala Pro Glu Gly Gln Ala Gln Gln Arg 20 2583018PRTArtificial
SequenceSynthetic Polypeptide 830Leu Gly Glu Met Trp Asn Asn Thr
Ala Ala Asp Asp Lys Gln Pro Tyr1 5 10 15Glu Lys83117PRTArtificial
SequenceSynthetic Polypeptide 831Ile Met Gln Asn Thr Asp Pro His
Ser Gln Glu Tyr Val Glu His Leu1 5 10 15Lys83214PRTArtificial
SequenceSynthetic Polypeptide 832Ile Leu Ile Ala Asn Thr Gly Met
Asp Thr Asp Lys Ile Lys1 5 1083320PRTArtificial SequenceSynthetic
Polypeptide 833Ala Trp Val Trp Asn Thr His Ala Asp Phe Ala Asp Glu
Cys Pro Lys1 5 10 15Pro Glu Leu Leu 2083418PRTArtificial
SequenceSynthetic Polypeptide 834Asp His Ala Ser Ile Gln Met Asn
Val Ala Glu Val Asp Lys Val Thr1 5 10 15Gly Arg83512PRTArtificial
SequenceSynthetic Polypeptide 835Ala Leu Ala Asn Val Asn Ile Gly
Ser Leu Ile Cys1 5 1083613PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(4)..(5)Xaa can be any naturally occurring
amino acid 836Glu His Gly Xaa Xaa Thr Asn Trp Asp Asp Met Glu Lys1
5 1083716PRTArtificial SequenceSynthetic Polypeptide 837Ser Ala Ala
Gln Ala Ala Ala Gln Thr Asn Ser Asn Ala Ala Gly Lys1 5 10
1583819PRTArtificial SequenceSynthetic Polypeptide 838Glu Glu Thr
Phe Glu Ala Ala Met Leu Gly Gln Ala Glu Glu Val Val1 5 10 15Gln Glu
Arg83918PRTArtificial SequenceSynthetic Polypeptide 839Pro Pro Tyr
Asp Glu Gln Thr Gln Ala Phe Ile Asp Ala Ala Gln Glu1 5 10 15Ala
Arg84015PRTArtificial SequenceSynthetic Polypeptide 840Leu Glu Gln
Gly Gln Ala Ile Asp Asp Leu Met Pro Ala Gln Lys1 5 10
1584116PRTArtificial SequenceSynthetic Polypeptide 841Ser Leu His
Gln Ala Ile Glu Gly Asp Thr Ser Gly Asp Phe Leu Lys1 5 10
1584216PRTArtificial SequenceSynthetic Polypeptide 842Gln Leu Gln
Gln Ala Gln Ala Ala Gly Ala Glu Gln Glu Val Glu Lys1 5 10
1584319PRTArtificial SequenceSynthetic Polypeptide 843Tyr Leu Glu
Val Val Leu Asn Thr Leu Gln Gln Ala Ser Gln Ala Gln1 5 10 15Val Asp
Lys84419PRTArtificial SequenceSynthetic Polypeptide 844Tyr Leu Glu
Val Val Leu Asn Thr Leu Gln Gln Ala Ser Gln Ala Gln1 5 10 15Val Asp
Lys84520PRTArtificial SequenceSynthetic Polypeptide 845Phe Leu Ser
Glu Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly Lys Pro1 5 10 15Pro Gln
Tyr Ile 2084621PRTArtificial SequenceSynthetic Polypeptide 846Phe
Leu Ser Glu Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly Lys Pro1 5 10
15Pro Gln Tyr Ile Ala 2084723PRTArtificial SequenceSynthetic
Polypeptide 847Phe Leu Ser Glu Leu Thr Gln Gln Leu Ala Gln Ala Thr
Gly Lys Pro1 5 10 15Pro Gln Tyr Ile Ala Val His
2084814PRTArtificial SequenceSynthetic Polypeptide 848Met Thr Ser
Met Gly Gln Ala Thr Trp Ser Asp Pro His Lys1 5 1084919PRTArtificial
SequenceSynthetic Polypeptide 849Glu Glu Leu Gly Leu Ile Glu Gln
Ala Tyr Asp Asn Pro His Glu Ala1 5 10 15Leu Ser
Arg85017PRTArtificial SequenceSynthetic Polypeptide 850Ser Leu Gly
Thr Ile Gln Gln Cys Cys Asp Ala Ile Asp His Leu Cys1 5 10
15Arg85125PRTArtificial SequenceSynthetic Polypeptide 851Ala Ala
Ala Ala Ala Ala Gln Gln Gln Gln Gln Cys Gly Gly Gly Gly1 5 10 15Ala
Thr Lys Pro Ala Val Ser Gly Lys 20 2585215PRTArtificial
SequenceSynthetic Polypeptide 852Asn Ser Cys Asn Gln Cys Asn Glu
Pro Arg Pro Glu Asp Ser Arg1 5 10 1585319PRTArtificial
SequenceSynthetic Polypeptide 853Val Leu Ile Ala Phe Ala Gln Tyr
Leu Gln Gln Cys Pro Phe Glu Asp1 5 10 15His Val
Lys85414PRTArtificial
SequenceSynthetic Polypeptide 854Asp Ser Leu Leu Gln Asp Gly Glu
Phe Ser Met Asp Leu Arg1 5 1085518PRTArtificial SequenceSynthetic
Polypeptide 855Tyr Phe Leu Gly Ser Ile Val Asn Phe Ser Gln Asp Pro
Asp Val His1 5 10 15Phe Lys85618PRTArtificial SequenceSynthetic
Polypeptide 856Val Phe Ser Trp Leu Gln Gln Glu Gly His Leu Ser Glu
Glu Glu Met1 5 10 15Ala Arg85712PRTArtificial SequenceSynthetic
Polypeptide 857Val Met Ser Gln Glu Ile Gln Glu Gln Leu His Lys1 5
1085811PRTArtificial SequenceSynthetic Polypeptide 858Lys Gln Glu
Pro Val Lys Pro Glu Glu Gly Arg1 5 1085918PRTArtificial
SequenceSynthetic Polypeptide 859Leu Trp Tyr Cys Asp Leu Gln Gln
Glu Ser Ser Gly Ile Ala Gly Ile1 5 10 15Leu Lys86014PRTArtificial
SequenceSynthetic Polypeptide 860Lys Gln Glu Tyr Asp Glu Ser Gly
Pro Ser Ile Val His Arg1 5 1086119PRTArtificial SequenceSynthetic
Polypeptide 861Glu Thr Glu Ala Ile Cys Phe Phe Val Gln Gln Phe Thr
Asp Met Glu1 5 10 15His Asn Arg86213PRTArtificial SequenceSynthetic
Polypeptide 862Val Thr Glu Gln Gly Ala Glu Leu Ser Asn Glu Glu Arg1
5 1086317PRTArtificial SequenceSynthetic Polypeptide 863Ala Tyr Met
Gly Asn Val Leu Gln Gly Gly Glu Gly Gln Ala Pro Thr1 5 10
15Arg86414PRTArtificial SequenceSynthetic Polypeptide 864Ala Val
Thr Glu Gln Gly His Glu Leu Ser Asn Glu Glu Arg1 5
1086518PRTArtificial SequenceSynthetic Polypeptide 865Val Ala His
Thr Phe Val Val Asp Val Ala Gln Gly Thr Gln Val Thr1 5 10 15Gly
Arg86611PRTArtificial SequenceSynthetic Polypeptide 866Val Gly Gln
Gly Tyr Pro His Asp Pro Pro Lys1 5 1086718PRTArtificial
SequenceSynthetic Polypeptide 867Ile Tyr Ala Val Glu Ala Ser Thr
Met Ala Gln His Ala Glu Val Leu1 5 10 15Val Lys86821PRTArtificial
SequenceSynthetic Polypeptide 868Thr Leu Ala Ile Tyr Phe Glu Val
Val Asn Gln His Asn Ala Pro Ile1 5 10 15Pro Gln Gly Gly Arg
2086918PRTArtificial SequenceSynthetic Polypeptide 869Glu Leu Ala
Gln Ile Ala Gly Arg Pro Thr Glu Asp Glu Asp Glu Lys1 5 10 15Glu
Lys87018PRTArtificial SequenceSynthetic Polypeptide 870Met Asp Glu
Met Ala Thr Thr Gln Ile Ser Lys Asp Glu Leu Asp Glu1 5 10 15Leu
Lys87115PRTArtificial SequenceSynthetic Polypeptide 871Tyr Pro His
Leu Gly Gln Lys Pro Gly Gly Ser Asp Phe Leu Arg1 5 10
1587219PRTArtificial SequenceSynthetic Polypeptide 872Thr Met Leu
Glu Leu Leu Asn Gln Leu Asp Gly Phe Gln Pro Asn Thr1 5 10 15Gln Val
Lys87319PRTArtificial SequenceSynthetic Polypeptide 873Ile Leu Leu
Glu Leu Leu Asn Gln Met Asp Gly Phe Asp Gln Asn Val1 5 10 15Asn Val
Lys87413PRTArtificial SequenceSynthetic Polypeptide 874Leu Leu Asn
Gln Met Asp Gly Phe Asp Thr Leu His Arg1 5 1087520PRTArtificial
SequenceSynthetic Polypeptide 875Phe Gln Glu Ser Ala Glu Ala Ile
Leu Gly Gln Asn Ala Ala Tyr Leu1 5 10 15Gly Glu Leu Lys
2087621PRTArtificial SequenceSynthetic Polypeptide 876His Pro Cys
Phe Ile Ile Ala Glu Ile Gly Gln Asn His Gln Gly Asp1 5 10 15Leu Asp
Val Ala Lys 2087722PRTArtificial SequenceSynthetic Polypeptide
877Leu Leu Gln Asp His Pro Trp Leu Leu Ser Gln Asn Leu Val Val Lys1
5 10 15Pro Asp Gln Leu Ile Lys 2087817PRTArtificial
SequenceSynthetic Polypeptide 878Ala Leu Pro Ala Val Gln Gln Asn
Asn Leu Asp Glu Asp Leu Ile Arg1 5 10 15Lys87913PRTArtificial
SequenceSynthetic Polypeptide 879Ala Leu Gly Gln Asn Pro Thr Asn
Ala Glu Val Leu Lys1 5 1088014PRTArtificial SequenceSynthetic
Polypeptide 880Asn Tyr Gln Gln Asn Tyr Gln Asn Ser Glu Ser Gly Glu
Lys1 5 1088129PRTArtificial SequenceSynthetic Polypeptide 881Asn
Tyr Gln Gln Asn Tyr Gln Asn Ser Glu Ser Gly Glu Lys Asn Glu1 5 10
15Gly Ser Glu Ser Ala Pro Glu Gly Gln Ala Gln Gln Arg 20
2588222PRTArtificial SequenceSynthetic Polypeptide 882Cys Gly Ala
Pro Ser Ala Thr Gln Pro Ala Thr Ala Glu Thr Gln His1 5 10 15Ile Ala
Asp Gln Val Arg 2088326PRTArtificial SequenceSynthetic Polypeptide
883Gln Ala Ala Ala Ala Ala Ala Gln Gln Gln Gln Gln Cys Gly Gly Gly1
5 10 15Gly Ala Thr Lys Pro Ala Val Ser Gly Lys 20
2588419PRTArtificial SequenceSynthetic Polypeptide 884Ile Asp Val
Thr Asp Phe Leu Ser Met Thr Gln Gln Asp Ser His Ala1 5 10 15Pro Leu
Arg88515PRTArtificial SequenceSynthetic Polypeptide 885Ile Gly Ser
Cys Thr Gln Gln Asp Val Glu Leu His Val Gln Lys1 5 10
1588614PRTArtificial SequenceSynthetic Polypeptide 886Leu Phe Pro
Leu Asn Gln Gln Asp Val Pro Asp Lys Phe Lys1 5 1088718PRTArtificial
SequenceSynthetic Polypeptide 887Ile Gly Gln Gln Pro Gln Gln Pro
Gly Ala Pro Pro Gln Gln Asp Tyr1 5 10 15Thr Lys88827PRTArtificial
SequenceSynthetic Polypeptide 888His Gln Ala Ala Ala Ala Ala Ala
Gln Gln Gln Gln Gln Cys Gly Gly1 5 10 15Gly Gly Ala Thr Lys Pro Ala
Val Ser Gly Lys 20 2588912PRTArtificial SequenceSynthetic
Polypeptide 889Met Phe Thr Gln Gln Gln Pro Gln Glu Leu Ala Arg1 5
1089019PRTArtificial SequenceSynthetic Polypeptide 890Leu Gln Gln
Gln Gln Arg Pro Glu Asp Ala Glu Asp Gly Ala Glu Gly1 5 10 15Gly Gly
Lys89120PRTArtificial SequenceSynthetic Polypeptide 891Leu Gln Gln
Gln Gln Arg Pro Glu Asp Ala Glu Asp Gly Ala Glu Gly1 5 10 15Gly Gly
Lys Arg 2089221PRTArtificial SequenceSynthetic Polypeptide 892Ser
Ser Glu Ala Asp Met Glu Cys Leu Asn Gln Arg Pro Pro Glu Asn1 5 10
15Pro Asp Thr Asp Lys 2089324PRTArtificial SequenceSynthetic
Polypeptide 893Ser Ser Glu Ala Asp Met Glu Cys Leu Asn Gln Arg Pro
Pro Glu Asn1 5 10 15Pro Asp Thr Asp Lys Asn Val Gln
2089424PRTArtificial SequenceSynthetic Polypeptide 894Asn Val Asn
Pro Glu Ser Gln Leu Ile Gln Gln Ser Glu Gln Ser Glu1 5 10 15Ser Glu
Thr Ala Gly Ser Thr Lys 2089517PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 895Pro Asp Asn Phe Xaa Phe Gly Gln Ser Gly Ala Gly Asn
Asn Trp Ala1 5 10 15Lys89619PRTArtificial SequenceSynthetic
Polypeptide 896Ser Gln Thr Cys Glu Phe Asn Met Ile Glu Gln Ser Gly
Pro Pro His1 5 10 15Glu Pro Arg89723PRTArtificial SequenceSynthetic
Polypeptide 897Ser Ala Val Leu Pro Pro Glu Asp Met Ser Gln Ser Gly
Pro Ser Gly1 5 10 15Ser His Pro Gln Gly Pro Arg
2089815PRTArtificial SequenceSynthetic Polypeptide 898Ile Glu Phe
Leu Gln Ser His Glu Asn Gln Glu Ile Tyr Gln Lys1 5 10
1589916PRTArtificial SequenceSynthetic Polypeptide 899Asn Thr Val
Ser Gln Ser Ile Ser Gly Asp Pro Glu Ile Asp Lys Lys1 5 10
1590015PRTArtificial SequenceSynthetic Polypeptide 900Leu Leu Ile
His Gln Ser Leu Ala Gly Gly Ile Ile Gly Val Lys1 5 10
1590118PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 901Met Val Xaa Tyr Leu Ala Asn Leu Thr Gln Ser Gln Ile
Ala Leu Asn1 5 10 15Glu Lys90222PRTArtificial SequenceSynthetic
Polypeptide 902Pro Pro Lys Pro Glu Pro Phe Gln Phe Gly Gln Ser Ser
Gln Lys Pro1 5 10 15Pro Val Ala Gly Gly Lys 2090321PRTArtificial
SequenceSynthetic Polypeptide 903Asn Gly Asn Tyr Cys Val Leu Gln
Met Asp Gln Ser Tyr Lys Pro Asp1 5 10 15Glu Asn Glu Val Arg
2090419PRTArtificial SequenceSynthetic Polypeptide 904Ile Leu Val
Gly Asp Val Gly Gln Thr Val Asp Asp Pro Tyr Ala Thr1 5 10 15Phe Val
Lys90518PRTArtificial SequenceSynthetic Polypeptide 905Ala Asp Asp
Val Asp Leu Glu Gln Val Ala Asn Glu Thr His Gly His1 5 10 15Val
Gly90619PRTArtificial SequenceSynthetic Polypeptide 906Ala Asp Asp
Val Asp Leu Glu Gln Val Ala Asn Glu Thr His Gly His1 5 10 15Val Gly
Ala90717PRTArtificial SequenceSynthetic Polypeptide 907Ser Ile Asn
Phe Leu His Gln Val Cys His Asp Gln Thr Pro Thr Thr1 5 10
15Lys90819PRTArtificial SequenceSynthetic Polypeptide 908Cys Thr
Thr Val Ala Phe Thr Gln Val Asn Ser Glu Asp Lys Gly Ala1 5 10 15Leu
Ala Lys90912PRTArtificial SequenceSynthetic Polypeptide 909Gln Gln
Leu Gln Gln Val Pro Gly Leu Leu His Arg1 5 1091012PRTArtificial
SequenceSynthetic Polypeptide 910Ser Gln Gln Tyr Pro Ala Ala Arg
Pro Ala Glu Pro1 5 1091118PRTArtificial SequenceSynthetic
Polypeptide 911Asp Phe Cys Ile Gln Val Gly Arg Asn Ile Ile His Gly
Ser Asp Ser1 5 10 15Val Lys91218PRTArtificial SequenceSynthetic
Polypeptide 912Val Leu Met Ser His Leu Gly Arg Pro Asp Gly Val Pro
Met Pro Asp1 5 10 15Lys Tyr91319PRTArtificial SequenceSynthetic
Polypeptide 913Val Leu Met Ser His Leu Gly Arg Pro Asp Gly Val Pro
Met Pro Asp1 5 10 15Lys Tyr Ser91414PRTArtificial SequenceSynthetic
Polypeptide 914Ala Gln Val Ala Arg Pro Gly Gly Asp Thr Ile Phe Gly
Lys1 5 1091514PRTArtificial SequenceSynthetic Polypeptide 915Ala
Gln Val Ala Arg Pro Gly Gly Asp Thr Ile Phe Gly Lys1 5
1091617PRTArtificial SequenceSynthetic Polypeptide 916Phe Met Ser
Val Gln Arg Pro Gly Pro Tyr Asp Arg Pro Gly Thr Ala1 5 10
15Arg91713PRTArtificial SequenceSynthetic Polypeptide 917Val Leu
Val Glu Arg Ser Ala Ala Glu Thr Val Thr Lys1 5 1091814PRTArtificial
SequenceSynthetic Polypeptide 918Phe Leu Pro Ser Ala Arg Ser Ser
Pro Ala Ser Ser Pro Glu1 5 1091921PRTArtificial SequenceSynthetic
Polypeptide 919Arg Pro Glu Leu Gly Ser Glu Gly Leu Gly Ser Ala Ala
His Gly Ser1 5 10 15Gln Pro Asp Leu Arg 2092017PRTArtificial
SequenceSynthetic Polypeptide 920Met Pro Asp Gln Gly Met Thr Ser
Ala Asp Asp Phe Phe Gln Gly Thr1 5 10 15Lys92122PRTArtificial
SequenceSynthetic Polypeptide 921Asp Val Pro Ala Pro Ser Thr Ser
Ala Asp Lys Val Glu Ser Leu Asp1 5 10 15Val Asp Ser Glu Ala Lys
2092217PRTArtificial SequenceSynthetic Polypeptide 922Gln Val Cys
Leu Pro Val Ile Ser Ala Glu Asn Trp Lys Pro Ala Thr1 5 10
15Lys92320PRTArtificial SequenceSynthetic Polypeptide 923Gly Phe
Gly Ser Gly Asp Asp Pro Tyr Ser Ser Ala Glu Pro His Val1 5 10 15Ser
Gly Val Lys 2092421PRTArtificial SequenceSynthetic Polypeptide
924Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe1
5 10 15Asn Pro Leu Ser Arg 2092521PRTArtificial SequenceSynthetic
Polypeptide 925Thr Tyr Phe Ser Cys Thr Ser Ala His Thr Ser Thr Gly
Asp Gly Thr1 5 10 15Ala Met Ile Thr Arg 2092614PRTArtificial
SequenceSynthetic Polypeptide 926Thr Tyr Ser Leu Gly Ser Ala Leu
Arg Pro Ser Thr Ser Arg1 5 1092717PRTArtificial SequenceSynthetic
Polypeptide 927Val Ser Asp Gln Glu Leu Gln Ser Ala Asn Ala Ser Val
Asp Asp Ser1 5 10 15Arg92819PRTArtificial SequenceSynthetic
Polypeptide 928Ala Pro Gly Ser Ala Ala Pro Ala Ala Gly Ser Ala Pro
Ala Ala Ala1 5 10 15Glu Glu Lys92919PRTArtificial SequenceSynthetic
Polypeptide 929Ala Pro Gly Ser Ala Ala Pro Ala Ala Gly Ser Ala Pro
Ala Ala Ala1 5 10 15Glu Glu Lys93019PRTArtificial SequenceSynthetic
Polypeptide 930Ala Pro Gly Ser Ala Ala Pro Ala Ala Gly Ser Ala Pro
Ala Ala Ala1 5 10 15Glu Glu Lys93120PRTArtificial SequenceSynthetic
Polypeptide 931Ala Pro Gly Ser Ala Ala Pro Ala Ala Gly Ser Ala Pro
Ala Ala Ala1 5 10 15Glu Glu Lys Lys 2093215PRTArtificial
SequenceSynthetic Polypeptide 932Asn Glu Gly Ser Glu Ser Ala Pro
Glu Gly Gln Ala Gln Gln Arg1 5 10 1593320PRTArtificial
SequenceSynthetic Polypeptide 933Gln Val Glu Pro Leu Asp Pro Pro
Ala Gly Ser Ala Pro Gly Glu His1 5 10 15Val Phe Val Lys
2093418PRTArtificial SequenceSynthetic Polypeptide 934Pro Thr Gly
Glu Ala Gly Pro Ser Cys Ser Ser Ala Ser Asp Lys Leu1 5 10 15Pro
Arg93514PRTArtificial SequenceSynthetic Polypeptide 935Tyr Tyr Thr
Ser Ala Ser Gly Asp Glu Met Val Ser Leu Lys1 5 1093620PRTArtificial
SequenceSynthetic Polypeptide 936Asn Gln Gln Gly Ala His Ser Ala
Leu Ser Ser Ala Ser Thr Ser Ser1 5 10 15His Asn Leu Gln
2093716PRTArtificial SequenceSynthetic Polypeptide 937Glu Ala Leu
Leu Ser Ser Ala Val Asp His Gly Ser Asp Glu Val Lys1 5 10
1593819PRTArtificial SequenceSynthetic Polypeptide 938Asp Tyr Met
Val Glu Ile Asp Ile Leu Ala Ser Cys Asp His Pro Asn1 5 10 15Ile Val
Lys93911PRTArtificial SequenceSynthetic Polypeptide 939Met Glu Ser
Cys Gly Ile His Glu Thr Thr Phe1 5 1094012PRTArtificial
SequenceSynthetic Polypeptide 940Gln Leu Ser Ser Cys Leu Pro Asn
Ile Val Pro Lys1 5 1094112PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 941Leu Ile Xaa Ser Asp Gly His Glu Phe Ile Val Lys1 5
1094219PRTArtificial SequenceSynthetic Polypeptide 942Glu Ile Val
Asp Gly Gly Val Ile Leu Glu Ser Asp Pro Gln Gln Val1 5 10 15Val His
Arg94315PRTArtificial SequenceSynthetic Polypeptide 943Ser Leu Glu
Asp Ala Leu Ser Ser Asp Thr Ser Gly His Phe Arg1 5 10
1594420PRTArtificial SequenceSynthetic Polypeptide 944Val Gly Val
Glu Ala His Val Asp Ile His Ser Asp Val Pro Lys Gly1 5 10 15Ala Asn
Ser Phe 2094516PRTArtificial SequenceSynthetic Polypeptide 945Val
Ile Leu Gly Ser Glu Ala Ala Gln Gln His Pro Glu Glu Val Arg1 5 10
1594610PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(1)Xaa can be any naturally occurring
amino acid 946Xaa Ser Glu Asp Lys Gly Ala Leu Ala Lys1 5
1094720PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(5)..(6)Xaa can be any naturally occurring
amino acid 947Gly Gly Thr Ser Xaa Xaa Ser Ser Glu Gly Thr Gln His
Ser Tyr Ser1 5 10 15Glu Glu Glu Lys 2094824PRTArtificial
SequenceSynthetic Polypeptide 948Cys Ala Leu Gly Gly Thr Ser Glu
Leu Ser Ser Glu Gly Thr Gln His1 5 10 15Ser Tyr Ser Glu Glu Glu Lys
Tyr 2094915PRTArtificial SequenceSynthetic Polypeptide 949Met Asp
Pro Asn Ile Val Gly Ser Glu His Tyr Asp Val Ala Arg1 5 10
1595021PRTArtificial SequenceSynthetic Polypeptide 950Ser Pro Ala
Pro Ser Ser Val Pro Leu Gly Ser Glu Lys Pro Ser Asn1 5 10 15Val Ser
Gln Asp Arg 2095120PRTArtificial SequenceSynthetic Polypeptide
951Met Thr Gln Ala Gly Val Glu Glu Leu Glu Ser Glu Asn Lys Ile Pro1
5 10 15Ala Thr Gln Lys 2095213PRTArtificial SequenceSynthetic
Polypeptide 952Met Leu Leu Asp
Ser Glu Gln His Pro Cys Gln Leu Lys1 5 1095337PRTArtificial
SequenceSynthetic Polypeptide 953Gly Leu Gly Asn Val Leu Gly Gly
Leu Ile Ser Gly Ala Gly Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30Gly Thr Ala Met Arg
3595437PRTArtificial SequenceSynthetic Polypeptide 954Ile Met Asp
Asp Leu Thr Glu Val Leu Cys Ser Gly Ala Gly Gly Val1 5 10 15His Ser
Gly Gly Ser Gly Asp Gly Ala Gly Ser Gly Gly Pro Gly Ala 20 25 30Gln
Asn His Val Lys 3595535PRTArtificial SequenceSynthetic Polypeptide
955Ala Thr Gln Gly Ala Ala Ala Ala Ala Gly Ser Gly Ala Gly Thr Gly1
5 10 15Gly Gly Thr Ala Ser Gly Gly Thr Glu Gly Gly Ser Ala Glu Ser
Glu 20 25 30Gly Ala Lys 3595622PRTArtificial SequenceSynthetic
Polypeptide 956Leu Glu Pro Ala Pro Leu Asp Ser Leu Cys Ser Gly Ala
Ser Ala Glu1 5 10 15Glu Pro Thr Ser His Arg 2095712PRTArtificial
SequenceSynthetic Polypeptide 957Val Ile Gly Ser Gly Cys Asn Leu
Asp Ser Ala Arg1 5 1095814PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 958Trp Xaa Leu Asn Ser Gly Asp Gly Ala Phe Tyr Gly Pro
Lys1 5 1095922PRTArtificial SequenceSynthetic Polypeptide 959Phe
Phe Asp Met Ala Tyr Gln Gly Phe Ala Ser Gly Asp Gly Asp Lys1 5 10
15Asp Ala Trp Ala Val Arg 2096019PRTArtificial SequenceSynthetic
Polypeptide 960Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp
His Cys Ile1 5 10 15Ile Gly Arg96116PRTArtificial SequenceSynthetic
Polypeptide 961Glu Tyr Leu Leu Ser Gly Asp Ile Ser Glu Ala Glu His
Cys Leu Lys1 5 10 1596214PRTArtificial SequenceSynthetic
Polypeptide 962Asp Asp Gly Leu Phe Ser Gly Asp Pro Asn Trp Phe Pro
Lys1 5 1096321PRTArtificial SequenceSynthetic Polypeptide 963Trp
Gln His Asp Leu Phe Asp Ser Gly Phe Gly Gly Gly Ala Gly Val1 5 10
15Glu Thr Gly Gly Lys 2096418PRTArtificial SequenceSynthetic
Polypeptide 964Asp Ser Val Trp Gly Ser Gly Gly Gly Gln Gln Ser Val
Asn His Leu1 5 10 15Val Lys96525PRTArtificial SequenceSynthetic
Polypeptide 965Pro Glu Gly Pro Asn Glu Ala Glu Val Thr Ser Gly Lys
Pro Glu Gln1 5 10 15Glu Val Pro Asp Ala Glu Glu Glu Lys 20
2596610PRTArtificial SequenceSynthetic Polypeptide 966Val Gln Ser
Gly Asn Ile Asn Ala Ala Lys1 5 1096716PRTArtificial
SequenceSynthetic Polypeptide 967Tyr Gln Tyr Gly Gly Leu Asn Ser
Gly Arg Pro Val Thr Pro Pro Arg1 5 10 1596826PRTArtificial
SequenceSynthetic Polypeptide 968Val Leu Gln Ala Thr Val Val Ala
Val Gly Ser Gly Ser Lys Gly Lys1 5 10 15Gly Gly Glu Ile Gln Pro Val
Ser Val Lys 20 2596917PRTArtificial SequenceSynthetic Polypeptide
969Gly Ile Leu Phe Val Gly Ser Gly Val Ser Gly Gly Glu Glu Gly Ala1
5 10 15Arg97015PRTArtificial SequenceSynthetic Polypeptide 970Ile
Glu Phe Leu Gln Ser His Glu Asn Gln Glu Ile Tyr Gln Lys1 5 10
1597119PRTArtificial SequenceSynthetic Polypeptide 971Leu Asp Glu
Val Ile Thr Ser His Gly Ala Ile Glu Pro Asp Lys Asp1 5 10 15Asn Val
Arg97217PRTArtificial SequenceSynthetic Polypeptide 972Glu His Pro
Val Ile Glu Ser His Pro Asp Asn Ala Leu Glu Asp Leu1 5 10
15Arg97315PRTArtificial SequenceSynthetic Polypeptide 973Leu Ile
Gln Ser His Pro Glu Ser Ala Glu Asp Leu Gln Glu Lys1 5 10
1597413PRTArtificial SequenceSynthetic Polypeptide 974Thr Ile Val
Ile Thr Ser His Pro Gly Gln Ile Val Lys1 5 1097516PRTArtificial
SequenceSynthetic Polypeptide 975Ile Glu Trp Leu Glu Ser His Gln
Asp Ala Asp Ile Glu Asp Phe Lys1 5 10 1597617PRTArtificial
SequenceSynthetic Polypeptide 976Gly Tyr Pro His Leu Cys Ser Ile
Cys Asp Leu Pro Val His Ser Asn1 5 10 15Lys97721PRTArtificial
SequenceSynthetic Polypeptide 977Ser Glu Pro Cys Ala Leu Cys Ser
Leu His Ser Ile Gly Lys Ile Gly1 5 10 15Gly Ala Gln Asn Arg
2097813PRTArtificial SequenceSynthetic Polypeptide 978Leu Gln Ser
Ile Gly Thr Glu Asn Thr Glu Glu Asn Arg1 5 1097914PRTArtificial
SequenceSynthetic Polypeptide 979Leu Phe Ile His Glu Ser Ile His
Asp Glu Val Val Asn Arg1 5 1098024PRTArtificial SequenceSynthetic
Polypeptide 980Val Thr Phe Asn Ile Asn Asn Ser Ile Pro Pro Thr Phe
Asp Gly Glu1 5 10 15Glu Glu Pro Ser Gln Gly Gln Lys
2098123PRTArtificial SequenceSynthetic Polypeptide 981Asn Leu Asn
Thr Leu Cys Trp Ala Ile Gly Ser Ile Ser Gly Ala Met1 5 10 15His Glu
Glu Asp Glu Lys Arg 2098221PRTArtificial SequenceSynthetic
Polypeptide 982Glu Ala Ser Ala Thr Asn Ser Pro Cys Thr Ser Lys Pro
Ala Thr Pro1 5 10 15Ala Pro Ser Glu Lys 2098318PRTArtificial
SequenceSynthetic Polypeptide 983Pro Pro Asn Pro Asn Cys Tyr Val
Cys Ala Ser Lys Pro Glu Val Thr1 5 10 15Val Arg98410PRTArtificial
SequenceSynthetic Polypeptide 984Ile Cys Ser Lys Pro Val Val Leu
Pro Lys1 5 1098520PRTArtificial SequenceSynthetic Polypeptide
985Gln Phe His Phe His Trp Gly Ser Leu Asp Gly Gln Gly Ser Glu His1
5 10 15Thr Val Asp Lys 2098621PRTArtificial SequenceSynthetic
Polypeptide 986Gln Phe His Phe His Trp Gly Ser Leu Asp Gly Gln Gly
Ser Glu His1 5 10 15Thr Val Asp Lys Lys 2098720PRTArtificial
SequenceSynthetic Polypeptide 987Gly Asn Pro Ile Cys Ser Leu His
Asp Gln Gly Ala Gly Gly Asn Gly1 5 10 15Asn Val Leu Lys
2098816PRTArtificial SequenceSynthetic Polypeptide 988Glu Ala Asn
Phe Thr Val Ser Ser Met His Gly Asp Met Pro Gln Lys1 5 10
1598915PRTArtificial SequenceSynthetic Polypeptide 989Asn Gln Leu
Thr Ser Asn Pro Glu Asn Thr Val Phe Asp Ala Lys1 5 10
1599020PRTArtificial SequenceSynthetic Polypeptide 990Gln Val Leu
Val Gly Ser Tyr Cys Val Phe Ser Asn Gln Gly Gly Leu1 5 10 15Val His
Pro Lys 2099112PRTArtificial SequenceSynthetic Polypeptide 991Asp
Leu Gln Ser Asn Val Glu His Leu Thr Glu Lys1 5 1099214PRTArtificial
SequenceSynthetic Polypeptide 992Glu Glu Met Gln Ser Asn Val Glu
Val Val His Thr Tyr Arg1 5 1099323PRTArtificial SequenceSynthetic
Polypeptide 993Ala Pro Val Gln Pro Gln Gln Ser Pro Ala Ala Ala Pro
Gly Gly Thr1 5 10 15Asp Glu Lys Pro Ser Gly Lys
2099423PRTArtificial SequenceSynthetic Polypeptide 994Ala Pro Val
Gln Pro Gln Gln Ser Pro Ala Ala Ala Pro Gly Gly Thr1 5 10 15Asp Glu
Lys Pro Ser Gly Lys 2099522PRTArtificial SequenceSynthetic
Polypeptide 995Asn Asp Gly Pro Val Thr Ile Glu Leu Glu Ser Pro Ala
Pro Gly Thr1 5 10 15Ala Thr Ser Asp Pro Lys 2099620PRTArtificial
SequenceSynthetic Polypeptide 996Ile Asn Ser Leu Phe Leu Thr Asp
Leu Tyr Ser Pro Glu Tyr Pro Gly1 5 10 15Pro Ser His Arg
2099723PRTArtificial SequenceSynthetic Polypeptide 997Asn Gly Ser
Leu Asp Ser Pro Gly Lys Gln Asp Thr Glu Glu Asp Glu1 5 10 15Glu Glu
Asp Glu Lys Asp Lys 2099824PRTArtificial SequenceSynthetic
Polypeptide 998Ser Ala Ala Ala Ala Ser Ala Ala Ser Gly Ser Pro Gly
Pro Gly Glu1 5 10 15Gly Ser Ala Gly Gly Glu Lys Arg
2099924PRTArtificial SequenceSynthetic Polypeptide 999Ser Ala Ala
Ala Ala Ser Ala Ala Ser Gly Ser Pro Gly Pro Gly Glu1 5 10 15Gly Ser
Ala Gly Gly Glu Lys Arg 20100021PRTArtificial SequenceSynthetic
Polypeptide 1000Asn Ala Asp Thr Asp Leu Val Ser Trp Leu Ser Pro His
Asp Pro Asn1 5 10 15Ser Val Val Thr Lys 20100117PRTArtificial
SequenceSynthetic Polypeptide 1001Leu Ser Pro Pro Tyr Ser Ser Pro
Gln Glu Phe Ala Gln Asp Val Gly1 5 10 15Arg100218PRTArtificial
SequenceSynthetic Polypeptide 1002Ile Ile Ala Phe Val Gly Ser Pro
Val Glu Asp Asn Glu Lys Asp Leu1 5 10 15Val Lys100314PRTArtificial
SequenceSynthetic Polypeptide 1003Met Glu Ser Gln Glu Pro Thr Glu
Ser Ser Gln Asn Gly Lys1 5 10100412PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 1004Ala Xaa Ala Ser Gln Leu Asp Cys Asn Phe Leu Lys1 5
10100518PRTArtificial SequenceSynthetic Polypeptide 1005Ser Gln Gly
Asp Ser Ile Ser Ser Gln Leu Gly Pro Ile His Pro Pro1 5 10 15Pro
Arg100620PRTArtificial SequenceSynthetic Polypeptide 1006Leu Gly
Gly Leu Leu Lys Pro Thr Val Ala Ser Gln Asn Gln Asn Leu1 5 10 15Pro
Val Ala Lys 20100730PRTArtificial SequenceSynthetic Polypeptide
1007Ser Ser Trp Gly Met Met Gly Met Leu Ala Ser Gln Gln Asn Gln
Ser1 5 10 15Gly Pro Ser Gly Asn Asn Gln Asn Gln Gly Asn Met Gln Arg
20 25 30100815PRTArtificial SequenceSynthetic Polypeptide 1008Asp
Glu Tyr Leu Ile Asn Ser Gln Thr Thr Glu His Ile Val Lys1 5 10
15100922PRTArtificial SequenceSynthetic Polypeptide 1009Tyr Gln Leu
Gly Leu Ala Tyr Gly Tyr Asn Ser Gln Tyr Asp Glu Ala1 5 10 15Val Ala
Gln Phe Ser Lys 20101019PRTArtificial SequenceSynthetic Polypeptide
1010Gly Leu Leu Leu Leu Ser Val Val Val Thr Ser Arg Pro Glu Ala
Phe1 5 10 15Gln Pro His101116PRTArtificial SequenceSynthetic
Polypeptide 1011Arg Pro Ala Ser Val Ser Ser Ser Ala Ala Val Glu His
Glu Gln Arg1 5 10 15101219PRTArtificial SequenceSynthetic
Polypeptide 1012Phe Gly Ile Val Thr Ser Ser Ala Gly Thr Gly Thr Thr
Glu Asp Thr1 5 10 15Glu Ala Lys101320PRTArtificial
SequenceSynthetic Polypeptide 1013Phe Gly Ile Val Thr Ser Ser Ala
Gly Thr Gly Thr Thr Glu Asp Thr1 5 10 15Glu Ala Lys Lys
20101423PRTArtificial SequenceSynthetic Polypeptide 1014Ser Thr Ala
Ser Ala Pro Ala Ala Val Asn Ser Ser Ala Ser Ala Asp1 5 10 15Lys Pro
Leu Ser Asn Met Lys 20101516PRTArtificial SequenceSynthetic
Polypeptide 1015Glu Ala Leu Leu Ser Ser Ala Val Asp His Gly Ser Asp
Glu Val Lys1 5 10 15101617PRTArtificial SequenceSynthetic
Polypeptide 1016Val Ser Trp Leu Glu Tyr Glu Ser Ser Phe Ser Asn Glu
Glu Ala Gln1 5 10 15Lys101720PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(3)Xaa can be any naturally occurring
amino acid 1017Ile Xaa Xaa Gly Ser Ser Gly Ala Gln Gly Ser Gly Gly
Gly Ser Thr1 5 10 15Ser Ala His Tyr 20101821PRTArtificial
SequenceSynthetic Polypeptide 1018His Ile Gly Gly Pro Pro Gly Phe
Ala Ser Ser Ser Gly Lys Pro Gly1 5 10 15Pro Thr Val Ile Lys
20101922PRTArtificial SequenceSynthetic Polypeptide 1019Phe Glu Met
Tyr Glu Pro Ser Glu Leu Glu Ser Ser His Leu Thr Asp1 5 10 15Gln Asp
Asn Glu Ile Arg 20102021PRTArtificial SequenceSynthetic Polypeptide
1020Ser Pro Asp Asp Asp Leu Gly Ser Ser Asn Trp Glu Ala Ala Asp
Leu1 5 10 15Gly Asn Glu Glu Arg 20102128PRTArtificial
SequenceSynthetic Polypeptide 1021Gly Asp Ser Gln Val Ser Ser Asn
Pro Thr Ser Ser Pro Pro Gly Glu1 5 10 15Ala Pro Ala Pro Val Ser Val
Asp Ser Glu Pro Ser 20 25102218PRTArtificial SequenceSynthetic
Polypeptide 1022Phe Val Asn Gly Gln Pro Arg Pro Leu Glu Ser Ser Gln
Val Lys Tyr1 5 10 15Leu Arg102318PRTArtificial SequenceSynthetic
Polypeptide 1023Lys Pro Leu Thr Ser Ser Ser Ala Ala Pro Gln Arg Pro
Ile Ser Thr1 5 10 15Gln Arg102422PRTArtificial SequenceSynthetic
Polypeptide 1024Ile His Ile Gly Gly Pro Pro Gly Phe Ala Ser Ser Ser
Gly Lys Pro1 5 10 15Gly Pro Thr Val Ile Lys 20102519PRTArtificial
SequenceSynthetic Polypeptide 1025Glu Leu Val Ser Ser Ser Ser Ser
Gly Ser Asp Ser Asp Ser Glu Val1 5 10 15Asp Lys
Lys102612PRTArtificial SequenceSynthetic Polypeptide 1026Leu Leu
Asp Ser Ser Thr Val Thr His Leu Phe Lys1 5 10102723PRTArtificial
SequenceSynthetic Polypeptide 1027Pro Pro Pro Ala Ala Pro Pro Pro
Ser Ser Ser Ser Val Pro Glu Ala1 5 10 15Gly Gly Pro Pro Ile Lys Lys
20102820PRTArtificial SequenceSynthetic Polypeptide 1028Tyr Val Glu
Leu Phe Leu Asn Ser Thr Ala Gly Ala Ser Gly Gly Ala1 5 10 15Tyr Glu
His Arg 20102922PRTArtificial SequenceSynthetic Polypeptide 1029Ser
His Glu Leu Ser Asp Phe Gly Leu Glu Ser Thr Ala Gly Glu Ile1 5 10
15Pro Val Val Ala Ile Arg 20103022PRTArtificial SequenceSynthetic
Polypeptide 1030Glu Cys Glu Glu Glu Ala Ile Asn Ile Gln Ser Thr Ala
Pro Glu Glu1 5 10 15Glu His Glu Ser Pro Arg 20103119PRTArtificial
SequenceSynthetic Polypeptide 1031Glu Gly Thr Gly Ser Thr Ala Thr
Ser Ser Ser Ser Thr Ala Gly Ala1 5 10 15Ala Gly
Lys103218PRTArtificial SequenceSynthetic Polypeptide 1032Pro Leu
His Ser Ile Ile Ser Ser Thr Glu Ser Val Gln Gly Ser Thr1 5 10 15Ser
Lys103315PRTArtificial SequenceSynthetic Polypeptide 1033Val Ala
Phe Thr Gly Ser Thr Glu Val Gly His Leu Ile Gln Lys1 5 10
15103420PRTArtificial SequenceSynthetic Polypeptide 1034Leu Ala Leu
Val Thr Gly Gly Glu Ile Ala Ser Thr Phe Asp His Pro1 5 10 15Glu Leu
Val Lys 20103517PRTArtificial SequenceSynthetic Polypeptide 1035Ala
Thr Ile Glu Leu Cys Ser Thr His Ala Asn Asp Ala Ser Ala Leu1 5 10
15Arg103616PRTArtificial SequenceSynthetic Polypeptide 1036Val His
Ile Thr Leu Ser Thr His Glu Cys Ala Gly Leu Ser Glu Arg1 5 10
15103718PRTArtificial SequenceSynthetic Polypeptide 1037Glu Glu Glu
Glu Pro Gln Ala Pro Gln Glu Ser Thr Pro Ala Pro Pro1 5 10 15Lys
Lys103816PRTArtificial SequenceSynthetic Polypeptide 1038Ser Ile
Thr Ile Leu Ser Thr Pro Glu Gly Thr Ser Ala Ala Cys Lys1 5 10
15103919PRTArtificial SequenceSynthetic Polypeptide 1039Glu Thr Leu
Ala Ser Ser Asp Ser Phe Ala Ser Thr Gln Pro Thr His1 5 10 15Ser Trp
Lys104016PRTArtificial SequenceSynthetic Polypeptide 1040Val Val
Val Leu Met Gly Ser Thr Ser Asp Leu Gly His Cys Glu Lys1 5 10
15104124PRTArtificial SequenceSynthetic Polypeptide 1041Val Leu Leu
Ser Asn Leu Ser Ser Thr Ser His Val Pro Glu Val Asp1 5 10 15Pro Gly
Ser Ala Glu Leu Gln Lys 20104211PRTArtificial SequenceSynthetic
Polypeptide 1042Leu Phe Asp Ser Thr Thr Leu Glu His Gln Lys1 5
10104316PRTArtificial SequenceSynthetic Polypeptide 1043Thr Gln Leu
Glu Gly Leu Gln Ser Thr Val Thr Gly His Val Glu Arg1 5 10
15104421PRTArtificial SequenceSynthetic Polypeptide 1044Gly Ser Glu
Ser Gly Gly Ser Ala Val Asp Ser Val Ala Gly Glu His1 5 10 15Ser Val
Ser Gly Arg 20104515PRTArtificial SequenceSynthetic Polypeptide
1045Tyr Glu Ile Leu Gln Ser Val Asp Asp Ala Ala Ile Val Ile Lys1 5
10 15104619PRTArtificial SequenceSynthetic Polypeptide 1046Asn Asp
Leu Ser Ile Cys Gly Thr Leu His Ser Val Asp Gln Tyr Leu1 5 10 15Asn
Ile Lys104713PRTArtificial SequenceSynthetic Polypeptide 1047Ile
Leu Asp Ser Val Gly Ile Glu Ala Asp Asp Asp Arg1 5
10104816PRTArtificial SequenceSynthetic Polypeptide 1048Ile Leu Asp
Ser Val Gly Ile Glu Ala Asp Asp Asp Arg Leu Asn Lys1 5 10
15104916PRTArtificial SequenceSynthetic Polypeptide 1049Ile Tyr Val
Ala Ser Val His Gln Asp Leu Ser Asp Asp Asp Ile Lys1 5 10
15105012PRTArtificial SequenceSynthetic Polypeptide 1050Glu Leu Gln
Ser Val Lys Pro Gln Glu Ala Pro Lys1 5 10105118PRTArtificial
SequenceSynthetic Polypeptide 1051His Tyr Thr Glu Gly Ala Glu Leu
Val Asp Ser Val Leu Asp Val Val1 5 10 15Arg Lys105219PRTArtificial
SequenceSynthetic Polypeptide 1052Leu Ala Glu Gly Ser Val Thr Ser
Val Gly Ser Val Asn Pro Ala Glu1 5 10 15Asn Phe
Arg105319PRTArtificial SequenceSynthetic Polypeptide 1053Gly Ser
Pro Thr Ser Leu Gly Thr Trp Gly Ser Trp Ile Gly Pro Asp1 5 10 15His
Asp Lys105414PRTArtificial SequenceSynthetic Polypeptide 1054Val
Leu Asn Ser Tyr Trp Val Gly Glu Asp Ser Thr Tyr Lys1 5
10105511PRTArtificial SequenceSynthetic Polypeptide 1055Ser Leu Gly
Thr Ala Asp Val His Phe Glu Arg1 5 10105612PRTArtificial
SequenceSynthetic Polypeptide 1056Met Ala Gly Thr Ala Phe Asp Phe
Glu Asn Met Lys1 5 10105713PRTArtificial SequenceSynthetic
Polypeptide 1057Val Leu Ala Thr Ala Phe Asp Thr Thr Leu Gly Gly
Arg1 5 10105819PRTArtificial SequenceSynthetic Polypeptide 1058Val
Glu Leu Phe Leu Asn Ser Thr Ala Gly Ala Ser Gly Gly Ala Tyr1 5 10
15Glu His Arg105920PRTArtificial SequenceSynthetic Polypeptide
1059Ala Pro Pro Pro Ser Gly Ser Ala Val Ser Thr Ala Pro Gln Gln
Lys1 5 10 15Pro Ile Gly Lys 20106017PRTArtificial SequenceSynthetic
Polypeptide 1060Ser Gln Ile Phe Ser Thr Ala Ser Asp Asn Gln Pro Thr
Val Thr Ile1 5 10 15Lys106114PRTArtificial SequenceSynthetic
Polypeptide 1061Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro His Val
Ala1 5 10106214PRTArtificial SequenceSynthetic Polypeptide 1062Met
Met Leu Gly Thr Glu Gly Gly Glu Gly Phe Val Val Lys1 5
10106317PRTArtificial SequenceSynthetic Polypeptide 1063Phe Gly Ala
Val Trp Thr Gly Asp Asn Thr Ala Glu Trp Asp His Leu1 5 10
15Lys106415PRTArtificial SequenceSynthetic Polypeptide 1064Val Ser
His Val Ser Thr Gly Gly Gly Ala Ser Leu Glu Leu Leu1 5 10
15106516PRTArtificial SequenceSynthetic Polypeptide 1065Val Ser His
Val Ser Thr Gly Gly Gly Ala Ser Leu Glu Leu Leu Glu1 5 10
15106618PRTArtificial SequenceSynthetic Polypeptide 1066Val Ser His
Val Ser Thr Gly Gly Gly Ala Ser Leu Glu Leu Leu Glu1 5 10 15Gly
Lys106721PRTArtificial SequenceSynthetic Polypeptide 1067Ile Leu
Ile Ser Leu Ala Thr Gly His Arg Glu Glu Gly Gly Glu Asn1 5 10 15Leu
Asp Gln Ala Arg 20106819PRTArtificial SequenceSynthetic Polypeptide
1068Thr Leu Asp Gln Cys Ile Gln Thr Gly Val Asp Asn Pro Gly His
Pro1 5 10 15Phe Ile Lys106920PRTArtificial SequenceSynthetic
Polypeptide 1069Ser Gly Phe Thr Leu Asp Asp Val Ile Gln Thr Gly Val
Asp Asn Pro1 5 10 15Gly His Pro Tyr 20107014PRTArtificial
SequenceSynthetic Polypeptide 1070Asp Leu Thr Thr Gly Tyr Asp Asp
Ser Gln Pro Asp Lys Lys1 5 10107114PRTArtificial SequenceSynthetic
Polypeptide 1071Phe Phe Phe Gly Thr His Glu Thr Ala Phe Leu Gly Pro
Lys1 5 10107214PRTArtificial SequenceSynthetic Polypeptide 1072Phe
Pro Ser Leu Leu Thr His Asn Glu Asn Met Val Ala Lys1 5
10107317PRTArtificial SequenceSynthetic Polypeptide 1073Tyr Glu Asp
Ile Cys Pro Ser Thr His Asn Met Asp Val Pro Asn Ile1 5 10
15Lys107424PRTArtificial SequenceSynthetic Polypeptide 1074Asp Tyr
Ala Leu His Trp Leu Val Leu Gly Thr His Thr Ser Asp Glu1 5 10 15Gln
Asn His Leu Val Val Ala Arg 20107510PRTArtificial SequenceSynthetic
Polypeptide 1075Phe Gly Thr Ile Asn Ile Val His Pro Lys1 5
10107618PRTArtificial SequenceSynthetic Polypeptide 1076Ser Met Val
Asn Thr Lys Pro Glu Lys Thr Glu Glu Asp Ser Glu Glu1 5 10 15Val
Arg107729PRTArtificial SequenceSynthetic Polypeptide 1077Val Thr
Leu Leu Thr Pro Ala Gly Ala Thr Gly Ser Gly Gly Gly Thr1 5 10 15Ser
Gly Asp Ser Ser Lys Gly Glu Asp Lys Gln Asp Arg 20
25107823PRTArtificial SequenceSynthetic Polypeptide 1078Pro Gly Glu
Thr Leu Thr Glu Ile Leu Glu Thr Pro Ala Thr Ser Glu1 5 10 15Gln Glu
Ala Glu His Gln Arg 20107918PRTArtificial SequenceSynthetic
Polypeptide 1079Asn Ser Val Gln Thr Pro Val Glu Asn Ser Thr Asn Ser
Gln His Gln1 5 10 15Val Lys108016PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(3)Xaa can be any naturally occurring
amino acid 1080Ala Xaa Xaa Ile Thr Pro Val Pro Gly Gly Val Gly Pro
Met Thr Val1 5 10 15108121PRTArtificial SequenceSynthetic
Polypeptide 1081Ser Thr Val Leu Thr Pro Met Phe Val Glu Thr Gln Ala
Ser Gln Gly1 5 10 15Thr Leu Gln Thr Arg 20108215PRTArtificial
SequenceSynthetic Polypeptide 1082Thr Phe Thr Thr Gln Glu Thr Ile
Thr Asn Ala Glu Thr Ala Lys1 5 10 15108314PRTArtificial
SequenceSynthetic Polypeptide 1083Ser Pro Val Ser Thr Arg Pro Leu
Pro Ser Ala Ser Gln Lys1 5 10108419PRTArtificial SequenceSynthetic
Polypeptide 1084Thr Asn Glu Gln Trp Gln Met Ser Leu Gly Thr Ser Glu
Asp His Gln1 5 10 15His Phe Thr108518PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 1085Gln Glu Ile Ile Xaa Gln Leu Asp
Val Thr Thr Ser Glu Tyr Glu Lys1 5 10 15Glu Lys108624PRTArtificial
SequenceSynthetic Polypeptide 1086Leu Leu Ala Phe Leu Leu Ala Glu
Leu Gly Thr Ser Gly Ser Ile Asp1 5 10 15Gly Asn Asn Gln Leu Val Ile
Lys 20108717PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(2)Xaa can be any naturally occurring
amino acid 1087Leu Xaa Asn Met Glu Ile Gly Thr Ser Leu Phe Asp Glu
Glu Gly Ala1 5 10 15Lys108825PRTArtificial SequenceSynthetic
Polypeptide 1088Ala Glu Lys Pro Ala Glu Thr Pro Val Ala Thr Ser Pro
Thr Ala Thr1 5 10 15Asp Ser Thr Ser Gly Asp Ser Ser Arg 20
25108916PRTArtificial SequenceSynthetic Polypeptide 1089Leu Leu Glu
Thr Thr Asp Arg Pro Asp Gly His Gln Asn Asn Leu Arg1 5 10
15109019PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(8)..(9)Xaa can be any naturally occurring
amino acid 1090Ala Gln Thr Ile Thr Ser Glu Xaa Xaa Ser Thr Thr Thr
Thr Thr His1 5 10 15Ile Thr Lys109116PRTArtificial
SequenceSynthetic Polypeptide 1091Ala Asp Ala Val Gly Met Ser Thr
Val Pro Glu Val Ile Val Ala Arg1 5 10 15109216PRTArtificial
SequenceSynthetic Polypeptide 1092Ile His Phe Pro Leu Ala Thr Tyr
Ala Pro Val Ile Ser Ala Glu Lys1 5 10 15109316PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(3)..(3)Xaa can be any
naturally occurring amino acidmisc_feature(5)..(6)Xaa can be any
naturally occurring amino acid 1093Asp Thr Xaa Val Xaa Xaa Asp Thr
Tyr Asn Cys Asp Leu His Phe Lys1 5 10 15109414PRTArtificial
SequenceSynthetic Polypeptide 1094Val Val Ile Gly Met Asp Val Ala
Ala Ser Glu Phe Phe Arg1 5 10109520PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(2)..(6)Xaa can be any naturally occurring
amino acidmisc_feature(10)..(10)Xaa can be any naturally occurring
amino acid 1095Gly Xaa Xaa Xaa Xaa Xaa Ile Gly Leu Xaa Val Ala Asp
Leu Ala Glu1 5 10 15Ser Ile Met Lys 20109613PRTArtificial
SequenceSynthetic Polypeptide 1096Ala Asn Pro Gln Val Gly Val Ala
Phe Pro His Ile Lys1 5 10109723PRTArtificial SequenceSynthetic
Polypeptide 1097Pro Gln Glu Ala Lys Pro Gln Glu Ala Ala Val Ala Pro
Glu Lys Pro1 5 10 15Pro Ala Ser Asp Glu Thr Lys
20109811PRTArtificial SequenceSynthetic Polypeptide 1098His Phe Ser
Val Glu Gly Gln Leu Glu Phe Arg1 5 10109914PRTArtificial
SequenceSynthetic Polypeptide 1099Val Ala Thr Leu Gly Val Glu Val
His Pro Leu Val Phe His1 5 10110014PRTArtificial SequenceSynthetic
Polypeptide 1100His Trp Pro Phe Gln Val Ile Asn Asp Gly Asp Lys Pro
Lys1 5 10110118PRTArtificial SequenceSynthetic Polypeptide 1101Leu
Pro Val Pro Ala Phe Asn Val Ile Asn Gly Gly Ser His Ala Gly1 5 10
15Asn Lys110221PRTArtificial SequenceSynthetic Polypeptide 1102Glu
Val Ala Asn Gly Ile Glu Ser Leu Gly Val Lys Pro Asp Leu Pro1 5 10
15Pro Pro Pro Ser Lys 20110319PRTArtificial SequenceSynthetic
Polypeptide 1103Thr Tyr Tyr Asp Val Leu Gly Val Lys Pro Asn Ala Thr
Gln Glu Glu1 5 10 15Leu Lys Lys110418PRTArtificial
SequenceSynthetic Polypeptide 1104Glu Thr Val Ala Val Lys Pro Thr
Glu Asn Asn Glu Glu Glu Phe Thr1 5 10 15Ser Lys110513PRTArtificial
SequenceSynthetic Polypeptide 1105Ser Leu Leu Val Asn Pro Glu Gly
Pro Thr Leu Met Arg1 5 10110616PRTArtificial SequenceSynthetic
Polypeptide 1106Asn Trp Met Asn Ser Leu Gly Val Asn Pro His Val Asn
His Leu Tyr1 5 10 15110718PRTArtificial SequenceSynthetic
Polypeptide 1107His Gly Leu Leu Val Pro Asn Asn Thr Thr Asp Gln Glu
Leu Gln His1 5 10 15Ile Arg110818PRTArtificial SequenceSynthetic
Polypeptide 1108Gln Glu Leu Glu Phe Leu Glu Val Gln Glu Glu Tyr Ile
Lys Asp Glu1 5 10 15Gln Lys110920PRTArtificial SequenceSynthetic
Polypeptide 1109Leu Glu Gly Thr Leu Leu Lys Pro Asn Met Val Thr Pro
Gly His Ala1 5 10 15Cys Thr Gln Lys 20111011PRTArtificial
SequenceSynthetic Polypeptide 1110Phe Val Asn Val Val Pro Thr Phe
Gly Lys Lys1 5 10111116PRTArtificial SequenceSynthetic Polypeptide
1111Glu Asp Leu Val Phe Ile Phe Trp Ala Pro Glu Ser Ala Pro Leu
Lys1 5 10 15111219PRTArtificial SequenceSynthetic Polypeptide
1112Ala Ile Tyr Ile Asp Ala Ser Cys Leu Thr Trp Glu Gly Gln Gln
Phe1 5 10 15Gln Gly Lys111321PRTArtificial SequenceSynthetic
Polypeptide 1113Glu Gln Pro Gln His Pro Leu His Val Thr Tyr Ala Gly
Ala Ala Val1 5 10 15Asp Glu Leu Gly Lys 20111418PRTArtificial
SequenceSynthetic Polypeptide 1114Ser Pro Asp Gly His Leu Phe Gln
Val Glu Tyr Ala Gln Glu Ala Val1 5 10 15Lys Lys11158PRTArtificial
SequenceSynthetic Polypeptide 1115Asn Tyr Lys Pro Pro Ala Gln Lys1
5111611PRTArtificial SequenceSynthetic Polypeptide 1116Val Tyr Asn
Tyr Asn His Leu Met Pro Thr Arg1 5 10111715PRTArtificial
SequenceSynthetic Polypeptide 1117Leu Ala Glu Ala Glu Leu Glu Tyr
Asn Pro Glu His Val Ser Arg1 5 10 15111813PRTArtificial
SequenceSynthetic Polypeptide 1118Met Pro Tyr Gln Tyr Pro Ala Leu
Thr Pro Glu Gln Lys1 5 10111918PRTArtificial SequenceSynthetic
Polypeptide 1119Thr Ser Ser Ala Asn Asn Pro Asn Leu Met Tyr Gln Asp
Glu Cys Asp1 5 10 15Arg Arg112018PRTArtificial SequenceSynthetic
Polypeptide 1120Val Gly Ile Asn Tyr Gln Pro Pro Thr Val Val Pro Gly
Gly Asp Leu1 5 10 15Ala Lys112115PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(6)..(6)Xaa can be any naturally occurring
amino acid 1121Tyr Met Ala Cys Cys Xaa Leu Tyr Arg Gly Asp Val Val
Pro Lys1 5 10 15112211PRTArtificial SequenceSynthetic Polypeptide
1122Ser Tyr Cys Tyr Val Ser Lys Glu Glu Leu Lys1 5
10112310PRTArtificial SequenceSynthetic Polypeptide 1123Glu Ala Ala
Gly Ile Gly Ile Leu Thr Val1 5 10112410PRTArtificial
SequenceSynthetic Polypeptide 1124Glu Leu Ala Gly Ile Gly Ile Leu
Thr Val1 5 1011259PRTArtificial SequenceSynthetic Polypeptide
1125Tyr Met Asp Gly Thr Met Ser Gln Val1 51126140PRTArtificial
SequenceSynthetic Polypeptide 1126Met His His His His His His His
His His His Lys Glu Asn Gln Pro1 5 10 15Glu Asn Ser Gln Thr Pro Gly
Gly Gly Ser Val Pro Leu Ala His Ser 20 25 30Ser Ser Ala Phe Thr Ile
Met Asp Gln Val Pro Phe Ser Val Ser Val 35 40 45Ser Gln Leu Gln Ser
Ser Met His Asn Ala Leu His Ile Tyr Met Asp 50 55 60Gly Thr Met Ser
Gln Val Gln Gly Ser Ala Asn Asp Gly Val Gly Ala65 70 75 80Tyr Gly
Thr Val Tyr Lys Ala Cys Asp Pro His Ser Gly His Phe Val 85 90 95Ala
Leu Lys Ser Val Gly His Gly His Ser Tyr Thr Thr Ala Glu Glu 100 105
110Leu Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Val Leu Gly Lys
115 120 125Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 130 135
1401127115PRTArtificial SequenceSynthetic Polypeptide 1127Met His
His His His His His His His His His Lys Glu Asn Gln Pro1 5 10 15Glu
Asn Ser Gln Thr Pro Gly Gly Gly Ser Val Pro Leu Ala His Ser 20 25
30Ser Ser Ala Phe Thr Ile Met Asp Gln Val Pro Phe Ser Val Ser Val
35 40 45Ser Gln Leu Val Pro Leu Ala His Ser Ser Ser Ala Phe Thr Ile
Met 50 55 60Asp Gln Val Pro Phe Ser Val Ser Val Ser Gln Leu Val Pro
Leu Ala65 70 75 80His Ser Ser Ser Ala Phe Thr Ile Met Asp Gln Val
Pro Phe Ser Val 85 90 95Ser Val Ser Gln Leu Gly Lys Pro Ile Pro Asn
Pro Leu Leu Gly Leu 100 105 110Asp Ser Thr 115112851RNAArtificial
SequenceSynthetic Polynucleotidemisc_feature(1)..(1)Modified with
7-methylmisc_feature(1)..(1)Modified with inorganic
triphosphatemisc_feature(2)..(2)Modified with 2'-O-methyl
1128ggggaaauaa gagagaaaag aagaguaaga agaaauauaa gagccaccau g
511129226RNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(226)..(226)Modified with OH
1129ugauaauagg cuggagccuc gguggccaug cuucuugccc cuugggccuc
cccccagccc 60cuccuccccu uccugcaccc guacccccgu ggucuuugaa uaaagucuga
gugggcggca 120aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 180aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aucuag 226113012PRTArtificial SequenceSynthetic 1130His
Glu Tyr Gly Ala Glu Ala Leu Glu Arg Ala Gly1 5
1011315PRTArtificial SequenceSynthetic 1131Lys Phe Glu Arg Gln1
511329RNAArtificial SequenceSynthetic 1132ugauaauag 9
* * * * *