U.S. patent application number 17/015905 was filed with the patent office on 2021-05-06 for vaccine composition.
This patent application is currently assigned to TURNSTONE LIMITED PARTNERSHIP. The applicant listed for this patent is TURNSTONE LIMITED PARTNERSHIP. Invention is credited to John Cameron BELL, Brian LICHTY, Jonathan POL, David F. STOJDL.
Application Number | 20210128706 17/015905 |
Document ID | / |
Family ID | 1000005345491 |
Filed Date | 2021-05-06 |
![](/patent/app/20210128706/US20210128706A1-20210506\US20210128706A1-2021050)
United States Patent
Application |
20210128706 |
Kind Code |
A1 |
STOJDL; David F. ; et
al. |
May 6, 2021 |
VACCINE COMPOSITION
Abstract
There is described a kit for use in inducing an immune response
in a mammal, the kit includes: a first virus that expresses MAGEA3,
Human Papilloma Virus E6/E7 fusion protein, human Six-Transmembrane
Epithelial Antigen of the Prostate protein, or Cancer Testis
Antigen 1, or a variant thereof as an antigenic protein and that is
formulated to generate an immunity to the protein or variant
thereof in the mammal. The kit also includes a Maraba MG1 virus
encoding the same antigen, or a variant of the same antigen. The
Maraba MG1 virus is formulated to induce the immune response in the
mammal. The first virus is immunologically distinct from the Maraba
MG1 virus.
Inventors: |
STOJDL; David F.; (Ottawa,
CA) ; BELL; John Cameron; (Ottawa, CA) ;
LICHTY; Brian; (Brantford, CA) ; POL; Jonathan;
(Paris, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TURNSTONE LIMITED PARTNERSHIP |
Toronto |
|
CA |
|
|
Assignee: |
TURNSTONE LIMITED
PARTNERSHIP
Toronto
CA
|
Family ID: |
1000005345491 |
Appl. No.: |
17/015905 |
Filed: |
September 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16735470 |
Jan 6, 2020 |
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17015905 |
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16249616 |
Jan 16, 2019 |
10646557 |
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16735470 |
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14769035 |
Aug 19, 2015 |
10363293 |
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PCT/CA2014/050118 |
Feb 20, 2014 |
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16249616 |
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61767776 |
Feb 21, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/001186 20180801;
C12N 2710/20034 20130101; C12N 2740/15043 20130101; C12N 15/86
20130101; C12N 2760/20243 20130101; A61K 2039/572 20130101; C12N
2760/20232 20130101; C12N 7/00 20130101; C12N 2710/16234 20130101;
A61K 2039/5256 20130101; C12N 2760/20242 20130101; A61K 39/0005
20130101; C12N 2710/10343 20130101; A61K 39/0011 20130101; A61K
39/12 20130101; C12N 2760/20223 20130101; A61K 2039/545 20130101;
C07K 2319/00 20130101; A61K 39/001184 20180801; A61K 2039/55561
20130101; A61K 2039/585 20130101; A61K 2039/5254 20130101; A61K
35/766 20130101; C07K 2319/50 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 15/86 20060101 C12N015/86; A61K 39/12 20060101
A61K039/12; A61K 35/766 20060101 A61K035/766; C12N 7/00 20060101
C12N007/00 |
Claims
1. A method of treating cancer in a mammal said cancer being a
tumour expressing an HPV protein, said method comprising: a)
administering a first virus comprising a nucleic acid capable of
expressing a protein comprising an HPV tumour associated antigen,
wherein said first virus is capable of generating immunity to said
HPV tumour associated antigen; b) administering a second virus,
said second virus being a Vesiculovirus, said second virus
comprising a nucleic acid capable of expressing a HPV tumour
associated antigen, wherein said second virus is capable of
providing a therapeutic oncolytic effect in said mammal; wherein
said first virus is immunologically distinct and physically; and
wherein either (i) the HPV tumour associated antigen comprises an
amino acid sequence that is at least 80% identical to SEQ ID NO. 7;
or (ii) the second virus is Maraba MG1.
2. The method of claim 1, wherein said second virus is Maraba
MG1.
3. The method of claim 2, wherein the HPV tumour associated antigen
expressed by said nucleic acid in at least the first or second
virus comprises an amino acid sequence that is at least 80%
identical to SEQ ID NO: 7.
4. The method of claim 3, wherein the amino acid sequence is at
least 90% identical to SEQ ID NO: 7.
5. The method of claim 4, wherein the amino acid sequence comprises
SEQ ID NO: 7.
6. The method of claim 4, wherein the amino acid sequence consists
of SEQ ID NO: 7.
7. The method of claim 5, wherein the amino acid sequence is
encoded by the nucleotide sequence of SEQ ID NO: 8.
8. The method according to claim 5, wherein the Maraba MG1 genome
comprises a reverse complement and RNA version of a nucleotide
sequence of SEQ ID NO: 9.
9. The method of claim 1, wherein the HPV tumour associated antigen
expressed by said nucleic acid in at least the first or second
virus that comprises an amino acid sequence that is at least 80%
identical to SEQ ID NO: 7.
10. The method of claim 8, wherein the amino acid sequence is at
least 90% identical to SEQ ID NO: 7.
11. The method of claim 9, wherein the amino acid sequence
comprises SEQ ID NO: 7.
12. The method of claim 10, wherein the amino acid sequence
consists of SEQ ID NO: 7.
13. The method of claim 11, wherein the amino acid sequence is
encoded by the nucleotide sequence of SEQ ID NO: 8.
14. The method according to claim 1, wherein said first virus is
administered intramuscularly and said second virus is administered
intravenously.
15. The method according to claim 1, wherein said first and second
virus both express the same HPV tumour associated antigen.
16. The method of claim 14, wherein the HPV tumour associated
antigen comprises SEQ ID NO: 7.
17. The method of claim 1, wherein the cancer expresses HPV E6 or
E7 proteins as antigenic proteins.
18. The method of claim 1, wherein the cancer is cervical cancer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/735,470, filed Jan. 6, 2020, now abandoned,
which is a continuation of U.S. patent application Ser. No.
16/249,616, filed Jan. 16, 2019, now U.S. Pat. No. 10,646,557,
which is a continuation of U.S. patent application Ser. No.
14/769,035, filed Aug. 19, 2015, now U.S. Pat. No. 10,363,293,
which is a U.S. National Stage of International Patent Application
No. PCT/CA2014/050118, filed Feb. 20, 2014, which claims the
benefit of U.S. Provisional Patent Application No. 61/767,776,
filed Feb. 21, 2013, the content of each of which is incorporated
by reference herein in its entirety.
SEQUENCE LISTING
[0002] This application incorporates by reference in its entirety
the Computer Readable Form of a Sequence Listing in ASCII text
format submitted via EFS-Web. The Sequence Listing text file
submitted via EFS-Web is entitled "14596-060-999_SEQ_LISTING.txt,"
was created on Dec. 9, 2020 and is 102,079 bytes in size.
FIELD
[0003] The present disclosure relates to oncolytic viruses for
inducing an immune response.
BACKGROUND
[0004] Oncolytic viruses (OVs) specifically infect, replicate in
and kill malignant cells, leaving normal tissues unaffected.
Several OVs have reached advanced stages of clinical evaluation for
the treatment of various neoplasms (Russell S J. et al., (2012) Nat
Biotechnol 30:658-670). Once approved, such viral agents could
substitute or combine with standard cancer therapies and allow for
reduced toxicity and improved therapeutic efficacy.
[0005] In addition to the vesicular stomatitis virus (VSV) (Stojdl
D F. et al., (2000) Nat Med 6:821-825; Stojdl D F. et al., (2003)
Cancer Cell 4:263-275), other rhabdoviruses displaying oncolytic
activity have been described recently (Brun J. et al., (2010) Mol
Ther 18: 1440-1449; Mahoney DJ. et al., (2011) Cancer Cell
20:443-456). Among them, the non-VSV Maraba virus showed the
broadest oncotropism in vitro (WO 2009/016433). A mutant Maraba
virus with improved tumor selectivity and reduced virulence in
normal cells was engineered. The attenuated strain is a double
mutant strain containing both G protein (Q242R) and M protein
(L123W) mutations. In vivo, this attenuated strain, called MG1 or
Maraba MG1, demonstrated potent anti-tumor activity in xenograft
and syngeneic tumor models in mice, with superior therapeutic
efficacy than the attenuated VSV, VSV.6M51 (WO 2011/070440).
[0006] Data accumulated over the past several years has revealed
that anti-tumor efficacy of oncolytic viruses not only depends on
their direct oncolysis but may also depend on their ability to
stimulate anti-tumor immunity (Bridle S W. et al., (2010) Mol Ther
184:4269-4275). This immune-mediated tumor control seems to play a
critical role in the overall efficacy of OV therapy. Indeed,
tumor-specific adaptive immune cells can patrol the tissues and
destroy tumor cells that have been missed by the OV. Moreover,
their memory compartment can prevent tumor recurrence.
[0007] Various strategies have been developed to improve OV-induced
anti-tumor immunity (Pol J. et al., (2012) Virus Adaptation and
Treatment 4:1-21). Some groups have genetically engineered OV
expressing immunomostimulatory cytokines. A herpes simplex and a
vaccinia virus expressing Granulocyte-Macrophage Colony-Stimulating
Factor (GM-CSF) have respectively reached phase III and IIB of the
clinical evaluation for cancer therapy while a VSV expressing
IFN-.beta. has just entered phase I.
[0008] Another strategy, defined as an oncolytic vaccine, consists
of expressing a tumor antigen from the OV (Russell S J. et al.,
(2012) Nat Biotechnol 30:658-670). Previously, it has been
demonstrated that VSV could also be used as a cancer vaccine vector
(Bridle BW. et al., (2010) Mol Ther 184:4269-4275). When applied in
a heterologous prime-boost setting to treat a murine melanoma
model, a VSV-human dopachrome tautomerase (hDCT) oncolytic vaccine
not only induced an increased tumor-specific immunity to DCT but
also a concomitant reduction in antiviral adaptive immunity. As a
result, the therapeutic efficacy was dramatically improved with an
increase of both median and tong term survivals (WO 2010/105347).
Although VSV was shown to be effective using hDCT as a tumor
associated antigen, there is no way to predict what tumor
associated antigens will be effective in a heterologous prime-boost
setting.
[0009] It is desirable to provide a vaccine vector that can be used
to activate the patient's immune system to kill tumor cells with
reduced toxicity to normal tissues, for example by activating
antibodies and/or lymphocytes against a tumor associated antigen on
the tumor. It is desirable if such a vaccine vector displays both
oncolytic activity and an ability to boost adaptive cell
immunity.
SUMMARY
[0010] The following summary is intended to introduce the reader to
one or more inventions described herein but not to define any one
of them.
[0011] It is an object of the present disclosure to obviate or
mitigate at least one disadvantage of previous anti-cancer
vaccines.
[0012] The authors of the present disclosure have surprisingly
determined that MAGEA3, Human Papilloma Virus E6/E7 fusion protein,
human Six-Transmembrane Epithelial Antigen of the Prostate protein,
and Cancer Testis Antigen 1, are all able to be used in a
heterologous prime-boost setting to induce an immune response in a
mammal. These results are unexpected and not predictable since not
all tumor associated antigens are able to induce an immune response
via a heterologous prime-boost. For example, the authors of the
present disclosure also determined that Placenta-specific protein 1
(PLAC-1) and Epstein-Barr Nuclear Antigen 1 were unable to
stimulate an immune response via a heterologous prime-boost.
[0013] In a first aspect, there is provided a kit for use in
inducing an immune response in a mammal. The kit includes: a first
virus that expresses a protein comprising an amino acid sequence of
SEQ ID NO: 1, or a variant thereof, as an antigenic protein and
that is formulated to generate an immunity to the protein or
variant thereof in the mammal. The kit also includes a Maraba MG1
virus encoding a protein comprising an amino acid sequence SEQ ID
NO: 1, or a variant thereof, as an antigenic protein, the Maraba
MG1 virus formulated to induce the immune response in the mammal;
the first virus being immunologically distinct from the Maraba MG1
virus. The antigenic protein expressed by the first virus and the
antigenic protein expressed by the Maraba MG1 virus may be
identical.
[0014] The first virus, the Maraba MG1 virus, or both, may be
formulated for administration as isolated viruses.
[0015] The Maraba MG1 virus may include a reverse complement and
RNA version of a transgene comprising a nucleotide sequence of SEQ
ID NO: 2. The Maraba MG1 virus may include a reverse complement and
RNA version of a codon optimized transgene comprising a nucleotide
sequence of SEQ ID NO: 3.
[0016] The first virus may include a transgene comprising a
nucleotide sequence of SEQ ID NO: 2 or 3, or may include a reverse
complement and RNA version of a transgene comprising a nucleotide
sequence of SEQ ID NO: 2 or 3, depending on whether the first virus
is a positive sense RNA virus, a DNA virus, or a negative sense RNA
virus.
[0017] The two viruses may be capable of expressing different
variants of the protein that comprises the sequence of SEQ ID NO:
1. The variant of the protein comprising an amino acid sequence of
SEQ ID NO: 1 that is expressed by the first virus, the Maraba MG1
virus, or both, may include at least one tumor associated epitope
selected from the group consisting of: FLWGPRALV (SEQ ID NO: 27),
KVAELVHFL (SEQ ID NO: 28), EGDCAPEEK (SEQ ID NO: 35),
KKLLTQHFVQENYLEY (SEQ ID NO: 36), and RKVAELVHFLLLKYR (SEQ ID NO:
37), and be at least 70% identical to SEQ ID NO: 1. Preferably, the
variant will be at least 80% identical to SEQ ID NO: 1. More
preferably, the variant will be at least 90% identical to SEQ ID
NO: 1. Even more preferably, the variant will be at least 95%
identical to SEQ ID NO: 1.
[0018] The variant of the protein comprising an amino acid sequence
of SEQ ID NO: 1 that is expressed by the first virus, the Maraba
MG1 virus, or both, may have an amino acid sequence of SEQ ID NO:
4. The nucleotide sequence that encodes the variant may include a
nucleotide sequence of SEQ ID NO: 5.
[0019] The Maraba MG1 virus may include a reverse complement and
RNA version of a nucleotide sequence of SEQ ID NO: 5. The first
virus may include a transgene comprising a nucleotide sequence of
SEQ ID NO: 5, or may include a reverse complement and RNA version
of a transgene comprising a nucleotide sequence of SEQ ID NO: 5,
depending on whether the first virus is a positive sense RNA virus,
a DNA virus, or a negative sense RNA virus.
[0020] If the first virus is a negative sense RNA virus, one of
either the Maraba MG1 virus or the first virus may include a
reverse complement and RNA version of a nucleotide sequence of SEQ
ID NO: 2 or 3, and the other of the Maraba MG1 virus and the first
virus may include a reverse complement and RNA version of SEQ ID
NO: 5.
[0021] If the first virus is a positive sense RNA virus or a DNA
virus, the Maraba MG1 virus may include a reverse complement and
RNA version of a nucleotide sequence of SEQ ID NO: 2 or 3, and the
first virus may include a nucleotide sequence of SEQ ID NO: 5.
Alternatively, the Maraba MG1 virus may include a reverse
complement and RNA version of a nucleotide sequence of SEQ ID NO:
5, and the first virus may include a nucleotide sequence of SEQ ID
NO: 2 or 3.
[0022] One of either the Maraba MG1 virus or the first virus may be
capable of expressing a protein that comprises the sequence of SEQ
ID NO: 1 or 4, and the other of the Maraba MG1 virus and the first
virus may be capable of expressing a protein that comprises the
other sequence.
[0023] The first virus may be an adenovirus.
[0024] According to another aspect, there is provided an isolated
Maraba MG1 viral particle having a genome that encodes a protein
comprising an amino acid sequence of SEQ ID NO: 1, or a variant
thereof.
[0025] The variant of the protein comprising an amino acid sequence
of SEQ ID NO: 1 may have an amino acid sequence of SEQ ID NO:
4.
[0026] The genome may include a reverse complement and RNA version
of a nucleotide sequence of SEQ ID NO: 2 or 3.
[0027] The genome may include a reverse complement and RNA version
of a nucleotide sequence of SEQ ID NO: 5.
[0028] The genome may include a reverse complement and RNA version
of a nucleotide sequence of SEQ ID NO: 6.
[0029] In another aspect, there is provided a kit for use in
inducing an immune response in a mammal. The kit includes: a first
virus that expresses a protein comprising an amino acid sequence of
SEQ ID NO: 7, or a variant thereof, as an antigenic protein and
that is formulated to generate an immunity to the protein or
variant thereof in the mammal. The kit also includes a Maraba MG1
virus encoding a protein comprising an amino acid sequence of SEQ
ID NO: 7, or a variant thereof, as an antigenic protein, the Maraba
MG1 virus formulated to induce the immune response in the mammal;
the first virus being immunologically distinct from the Maraba MG1
virus. The antigenic protein expressed by the first virus and the
antigenic protein expressed by the Maraba MG1 virus may be
identical.
[0030] The first virus, the Maraba MG1 virus, or both, may be
formulated for administration as isolated viruses.
[0031] If the first virus is a negative sense RNA virus, the Maraba
MG1 virus, the first virus, or both may include a reverse
complement and RNA version of a codon optimized transgene
comprising a nucleotide sequence of SEQ ID NO: 8. If the first
virus is a DNA virus or a positive sense RNA virus, the first virus
may include a codon optimized transgene comprising a nucleotide
sequence of SEQ ID NO: 8.
[0032] The variant of the protein comprising an amino acid sequence
of SEQ ID NO: 7 that is expressed by the first virus, the Maraba
MG1 virus, or both, may include at least one tumor associated
epitope and be at least 70% identical to SEQ ID NO: 7. Preferably,
the variant will be at least 80% identical to SEQ ID NO: 7. More
preferably, the variant will be at least 90% identical to SEQ ID
NO: 7. Even more preferably, the variant will be at least 95%
identical to SEQ ID NO: 7.
[0033] One of either the Maraba MG1 virus or the first virus may be
capable of expressing a protein that comprises the sequence of SEQ
ID NO: 7, and the other of the Maraba MG1 virus and the first virus
may be capable of expressing a variant of a protein that comprises
the sequence of SEQ ID NO: 7. The two viruses may be capable of
expressing different variants of the protein that comprises the
sequence of SEQ ID NO: 7.
[0034] The first virus may be a lentivirus.
[0035] According to another aspect, there is provided an isolated
Maraba MG1 viral particle having a genome that encodes a protein
comprising an amino acid sequence of SEQ ID NO: 7, or a variant
thereof.
[0036] The genome may include a reverse complement and RNA version
of a nucleotide sequence of SEQ ID NO: 8.
[0037] The genome may include a nucleotide sequence that is the
reverse complement and RNA version of SEQ ID NO: 9.
[0038] In another aspect, there is provided a kit for use in
inducing an immune response in a mammal. The kit includes: a first
virus that expresses a protein comprising an amino acid sequence of
SEQ ID NO: 10, or a variant thereof, as an antigenic protein and
that is formulated to generate an immunity to the protein or
variant thereof in the mammal. The kit also includes a Maraba MG1
virus encoding a protein comprising an amino acid sequence of SEQ
ID NO: 10, or a variant thereof, as an antigenic protein, the
Maraba MG1 virus formulated to induce the immune response in the
mammal; the first virus being immunologically distinct from the
Maraba MG1 virus. The antigenic protein expressed by the first
virus and the antigenic protein expressed by the Maraba MG 1 virus
may be identical.
[0039] The first virus, the Maraba MG1 virus, or both, may be
formulated for administration as isolated viruses.
[0040] If the first virus is a negative sense RNA virus, the Maraba
MG1 virus, the first virus, or both may include a reverse
complement and RNA version of a codon optimized transgene
comprising a nucleotide sequence of SEQ ID NO: 11. If the first
virus is a DNA virus or a positive sense RNA virus, the first virus
may include a codon optimized transgene comprising a nucleotide
sequence of SEQ ID NO: 11.
[0041] The variant of the protein comprising an amino acid sequence
of SEQ ID NO: 10 that is expressed by the first virus, the Maraba
MG1 virus, or both, may include at least one tumor associated
epitope and be at least 70% identical to SEQ ID NO: 10. Preferably,
the variant will be at least 80% identical to SEQ ID NO: 10. More
preferably, the variant will be at least 90% identical to SEQ ID
NO: 10. Even more preferably, the variant will be at least 30 95%
identical to SEQ ID NO: 10.
[0042] One of either the Maraba MG1 virus or the first virus may be
capable of expressing a protein that comprises the sequence of SEQ
ID NO: 10, and the other of the Maraba MG1 virus and the first
virus may be capable of expressing a variant of a protein that
comprises the sequence of SEQ ID NO: 10. The two viruses may be
capable of expressing different variants of the protein that
comprises the sequence of SEQ ID NO: 10.
[0043] The first virus may be a lentivirus.
[0044] According to another aspect, there is provided an isolated
Maraba MG1 viral particle having a genome that encodes a protein
comprising an amino acid sequence of SEQ ID NO: 10, or a variant
thereof.
[0045] The genome may include a reverse complement and RNA version
of a nucleotide sequence of SEQ ID NO: 11.
[0046] The genome may include a nucleotide sequence that is the
reverse complement and RNA version of SEQ ID NO: 12.
[0047] In another aspect, there is provided a kit for use in
inducing an immune response in a mammal. The kit includes: a first
virus that expresses a protein comprising an amino acid sequence of
SEQ ID NO: 13, or a variant thereof, as an antigenic protein and
that is formulated to generate an immunity to the protein or
variant thereof in the mammal. The kit also includes a Maraba MG1
virus encoding a protein comprising an amino acid sequence of SEQ
ID NO: 13, or a variant thereof, as an antigenic protein, the
Maraba MG1 virus formulated to induce the immune response in the
mammal; the first virus being immunologically distinct from the
Maraba MG1 virus. The antigenic protein expressed by the first
virus and the antigenic protein expressed by the Maraba MG1 virus
may be identical.
[0048] The first virus, the Maraba MG1 virus, or both, may be
formulated for administration as isolated viruses.
[0049] If the first virus is a negative sense RNA virus, the Maraba
MG1 virus, the first virus, or both may include a reverse
complement and RNA version of a codon optimized transgene
comprising a nucleotide sequence of SEQ ID NO: 14. If the first
virus is a DNA virus or a positive sense RNA virus, the first virus
may include a codon optimized transgene comprising a nucleotide
sequence of SEQ ID NO: 14.
[0050] The variant of the protein comprising an amino acid sequence
of SEQ ID NO: 13 that is expressed by the first virus, the Maraba
MG1 virus, or both, may include at least one tumor associated
epitope and be at least 70% identical to SEQ ID NO: 13. Preferably,
the variant will be at least 80% identical to SEQ ID NO: 13. More
preferably, the variant will be at least 90% identical to SEQ ID
NO: 13. Even more preferably, the variant will be at least 95%
identical to SEQ ID NO: 13.
[0051] One of either the Maraba MG1 virus or the first virus may be
capable of expressing a protein that comprises the sequence of SEQ
ID NO: 13, and the other of the Maraba MG1 virus and the first
virus may be capable of expressing a variant of a protein that
comprises the sequence of SEQ ID NO: 13. The two viruses may be
capable of expressing different variants of the protein that
comprises the sequence of SEQ ID NO: 13.
[0052] The first virus may be a lentivirus.
[0053] According to another aspect, there is provided an isolated
Maraba MG1 viral particle having a genome that encodes a protein
comprising an amino acid sequence of SEQ ID NO: 13, or a variant
thereof.
[0054] The genome may include a reverse complement and RNA version
of a nucleotide sequence of SEQ ID NO: 14.
[0055] The genome may include a nucleotide sequence that is the
reverse complement and RNA version of SEQ ID NO: 15.
[0056] Other aspects and features of the present disclosure will
become apparent to those ordinarily skilled in the art upon review
of the following description of specific embodiments in conjunction
with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] Embodiments of the present disclosure will now be described,
by way of example only, with reference to the attached Figures.
[0058] FIG. 1A shows the CD8.sup.+ or CD4.sup.+ T-cell responses in
tumor-bearing mice administered with MG1-hDCT.
[0059] FIG. 1B shows the therapeutic efficacy of MG1-hDCT
administered as a priming vector only in a metastatic lung cancer
mouse model.
[0060] FIG. 2 shows the comparison of the immune response of a
prime-boost vaccination in C57/BI6 mice with Ad-hDCT as the priming
vector and either Maraba MG1-hDCT or VSV-hDCT as the boosting
vector.
[0061] FIG. 3 shows the T-cell response in a metastatic lung cancer
mouse model following Ad-empty or Ad-hDCT, as the priming vector
only or following prime-boost vaccination with Ad-hDCT, as the
priming vector and either Maraba MG1 GFP or Maraba MG1-hDCT, as the
boosting vector.
[0062] FIG. 4 shows the survival plot in a metastatic lung cancer
mouse model following Ad-empty or Ad-hDCT, as the priming vector
only or following prime-boost vaccination with Ad-hDCT, as the
priming vector and either Maraba MG1 GFP or Maraba MG1-hDCT, as the
boosting vector.
[0063] FIG. 5 shows the survival plot in a metastatic brain cancer
mouse model following Ad-empty or Ad-hDCT, as the priming vector
only or following prime-boost vaccination with Ad-hDCT, as the
priming vector and Maraba MG1-hDCT, as the boosting vector.
[0064] FIG. 6 is a diagram of the priming vector Ad-MAGEA3, the
boosting vector Maraba MG1-MAGEA3 and the prime-boost strategy
utilized in a primate toxicity/immunogenicity study.
[0065] FIG. 7 shows the average T-cell response in primates given
Ad-MAGEA3 as the priming vector and a high or low dose of
MG1-MAGEA3 as the boosting vector. The T-cell responses were
determined after 5, 13 and 84 days following the boosting
vector.
[0066] FIG. 8 shows the T-cell responses in individual primates
given Ad-MAGEA3 as the priming vector and MG1-MAGEA3 as the
boosting vector after 5 days following the boosting vector. The
T-cell responses were stratified in relation to the MAGEA3 peptide
pool used to stimulate the response.
[0067] FIG. 9 shows the survival plot in a metastatic lung cancer
mouse model following Ad-hDCT versus Ad-hDCT plus Cyclophosphamide,
as the priming vector only or following prime-boost vaccination
with Ad-hDCT versus Ad-hDCT plus Cyclophosphamide, as the priming
vector and VSV-hDCT, as the boosting vector.
DETAILED DESCRIPTION
[0068] The present disclosure provides a kit for use in inducing an
immune response in a mammal. The kit includes a first virus that
expresses MAGEA3, a Human Papilloma Virus E6/E7 fusion protein,
human Six-Transmembrane Epithelial Antigen of the Prostate protein,
or Cancer Testis Antigen 1, or a variant thereof, as an antigen and
that is formulated to generate an immunity to the antigen in the
mammal. The kit also includes a Maraba MG1 virus encoding the same
antigen, or a variant of the same antigen, the Maraba MG1 virus
formulated to induce the immune response in the mammal. The first
virus is immunologically distinct from the Maraba MG1 virus so that
it may act as the "prime" in a heterologous prime-boost
vaccination.
[0069] Prime:boost immunizations can be given with unmatched
vaccine delivery methods while using the same antigen, in a
`heterologous` prime-boost format; or with matched vaccine delivery
methods, in a `homologous` prime-boost. Heterologous prime-boost
methods are preferable when using vectored vaccine platforms as
homologous vaccination would lead to boosting of responses to both
the vector and the transgene in the secondary response. In
contrast, a heterologous system focuses the secondary response
(that is, the boosted response) on the antigen as responses against
the first and the second vector are primary responses, and are
therefore much less robust.
[0070] In the present disclosure, the first virus and the Maraba
MG1 virus are used in a heterologous prime-boost format.
[0071] The antigenic proteins listed above are self-antigens
already tolerized by the immune system through a tightly controlled
process of negative selection in the thymus (Kruisbeek A M and
Amsen D, (1996) Curr Opin Immunol 8:233-244; Stockinger B (1999)
Adv Immunol 71:229-265) or peripheral tolerization. The major
challenge with developing vaccines to these antigenic proteins, and
any other self-antigens, is to induce a strong immune response
directed selectively against cancer cells. Although a number of
tumor associated antigenic peptides have been discovered, the
authors of the present disclosure have determined that is
impossible to predict which tumor associated antigenic peptides can
be successfully used to develop vaccines.
[0072] Melanoma antigen, family A,3 (MAGEA3) is a "cancer testis
antigen". The MAGE family of genes encoding tumor specific antigens
is discussed in De Plaen et al., Immunogenetics 40:360-369 (1994),
MAGEA3 is expressed in a wide variety of tumors including melanoma,
colorectal and lung. This protein was used by the authors of the
present disclosure as the antigenic protein in both the first virus
and the Maraba MG 1 virus. The authors also used a variant of the
MAGEA3 protein as the antigenic protein in both the first virus and
the Maraba MG 1 virus.
[0073] Human Papilloma Virus (HPV) oncoproteins E6/E7 are
constitutively expressed in cervical cancer (Zur Hausen, H (1996)
Biochem Biophys Acta 1288:F55-F78). Furthermore, IIPV types 16 and
18 are the cause of 75% of cervical cancer (Walboomers J M (1999) J
Pathol 189:12-19). The authors of the present disclosure used a
fusion protein of the E6/E7 oncoproteins of HPV types 16 and 18 as
the antigenic protein. The fusion protein was expressed using a
nucleotide sequence coexpressing HPV type 16/18 E6/E7 as a fusion
protein, which was mutated to remove oncogenic potential. The
fusion protein was used by the authors of the present disclosure as
the antigenic protein in both the first virus and the Maraba MG1
virus.
[0074] Six-Transmembrane Epithelial Antigen of the Prostate
(huSTEAP) is a recently identified protein shown to be
overexpressed in prostate cancer and up-regulated in multiple
cancer cell lines, including pancreas, colon, breast, testicular,
cervical, bladder, ovarian, acute lymphocytic leukemia and Ewing
sarcoma (Hubert R S et al., (1999) Proc Natl Acad Sci
96:14523-14528). The STEAP gene encodes a protein with six
potential membrane-spanning regions flanked by hydrophilic amino-
and carboxyl-terminal domains. This protein was used by the authors
of the present disclosure as the antigenic protein in both the
first virus and the Maraba MG1 virus.
[0075] Cancer Testis Antigen 1 (NYESO1) is a cancer/testis antigen
expressed in normal adult tissues, such as testis and ovary, and in
various cancers (Nicholaou T et al., (2006) Immunol Cell Biel
84:303-317). Cancer testis antigens are a unique family of
antigens, which have restricted expression to testicular germ cells
in a normal adult but are aberrantly expressed on a variety of
solid tumors, including soft tissue sarcomas, melanoma and
epithelial cancers. This protein was used by the authors of the
present disclosure as the antigenic protein in both the first virus
and the Maraba MG 1 virus.
[0076] In contrast to the successful use of the MAGEA3, HPV E6/E7
fusion, the huSTEAP, and the NYESO1 proteins as antigenic proteins
in a heterologous prime-boost vaccine, the authors of the present
disclosure determined that Epstein-Barr Nuclear Antigen 1 (EBDNA1,
SEQ ID NO: 16, encoded by SEQ ID NO: 17) was unable to generate a
similar immune response. EBDNA1 is a multifunctional viral protein
associated with Epstein-Barr virus (EBV) (Sibille H et al., (2003)
Proc Natl Acad Sci 100:10989-10994) and consistently expressed in
EBV-associated tumors (Young L S et al., (2004) Nature
Reviews--Cancer 4:757-768). EBNA1 has a glycine-alanine repeat
sequence that separates the protein into amino- and
carboxy-terminal domains (Young LS (2004) Nature Reviews--Cancer
4:757-768). This sequence also seems to stabilize the protein,
preventing proteasomal breakdown, as well as impairing antigen
processing and MHC class I-restricted antigen presentation. This
thereby inhibits the CDB-restricted cytotoxic T cell response
against virus-infected cells (Levitskaya J et al., (1995) Nature
375:685-688).
[0077] Placenta-specific protein 1 (PLAC-1) is another example of a
tumor associated antigenic protein that was unable to generate an
immune response in a heterologous prime-boost vaccine.
[0078] In the context of the present disclosure, a "variant" of a
tumor associated antigenic protein refers to a protein that (a)
includes at least one tumor associated antigenic epitope from the
tumor associated antigenic protein and (b) is at least 70%
identical to the tumor associated antigenic protein. Preferably,
the variant will be at least 80% identical to the tumor associated
antigenic protein. More preferably, the variant will be at least
90% identical to the tumor associated antigenic protein. Even more
preferably, the variant will be at least 95% identical to the tumor
associated antigenic protein. Variants with higher sequence
identities have increased likelihood that the epitopes are
presented in a similar 3-dimensional manner to the wild type
antigenic proteins.
[0079] Generally, a tumor associated antigenic epitope may be
identified by breaking up the whole antigenic protein into
overlapping series of peptides, or by generating libraries of
random peptides, and looking for T cell responses by stimulating
PBMCs or splenocytes from animals vaccinated with the protein
target using the peptide pools. Pools having a response identify
that peptide as a potential antigenic epitope. This approach is
discussed by Morris, GE in Encyclopedia of Life Sciences, 2007,
page 1-3 (doi:10.1002/9780470015902.a0002624.pub2).
[0080] A database summarizing well accepted antigenic epitopes is
provided by Van der Bruggen P, Stroobant V, Vigneron N, Van den
Eynde Bin "Database of T cell-defined human tumor antigens: the
2013 update." Cancer Immun 2013 13:15 and at
www.cancerimmunity.org/peptide/.
[0081] Tumor associated antigenic epitopes have been already
identified for MAGEA3. Accordingly, a variant of the MAGEA3 protein
may be, for example, an antigenic protein that includes at least
one tumor associated antigenic epitope selected from the group
consisting of: EVDPIGHLY (SEQ ID NO: 26), FLWGPRALV (SEQ ID NO:
27), KVAELVHFL (SEQ ID NO: 28), TFPDLESEF (SEQ ID NO: 29),
VAELVHFLL (SEQ ID NO: 30), MEVDPIGHLY (SEQ ID NO: 31), REPVTKAEML
(SEQ ID NO: 32), AELVHFLLL (SEQ ID NO: 33), WQYFFPVIF (SEQ ID NO:
34), EGDCAPEEK (SEQ ID NO: 35), KKLLTQHFVQENYLEY (SEQ ID NO: 36),
RKVAELVHFLLLKYR (SEQ ID NO: 37), ACYEFLWGPRALVETS (SEQ ID NO: 38),
VIFSKASSSLQL (SEQ ID NO: 39), VFGIELMEVDPIGHL (SEQ ID NO: 40),
GDNQIMPKAGLLIIV (SEQ ID NO: 41), TSYVKVLHHMVKISG (SEQ ID NO: 42),
RKVAELVHFLLLKYRA (SEQ ID NO: 43), and FLLLKYRAREPVTKAE (SEQ ID NO:
44); and that is at least 70% identical to the MAGEA3 protein.
[0082] It may be desirable for variants of a tumor associated
antigenic protein to include only antigenic epitopes that have high
allelic frequencies, such as frequencies greater than 40% of the
population. Accordingly, preferred examples of variants of MAGEA3
may include proteins that include at least one antigenic epitope
selected from the group consisting of: FLWGPRALV (SEQ ID NO: 27),
KVAELVHFL (SEQ ID NO: 28), EGDCAPEEK (SEQ ID NO: 35),
KKLLTQHFVQENYLEY (SEQ ID NO: 36), and RKVAELVHFLLLKYR (SEQ ID NO:
37) and that is at least 70% identical to the MAGEA3 protein.
[0083] The antigen expressed by the first virus does not need to
have exactly the same sequence as the antigen expressed by the
Maraba MG1 virus. The antigen expressed by Maraba MG1 must only
induce an overlapping immune response to the antigen expressed by
the first virus. For example, the first virus may express the
MAGEA3 and the Maraba MG virus may express a MAGEA3 variant, or
vice versa. Since both MAGEA3 and the variant of MAGEA3 induce
overlapping immune responses (as they both include at least one
identical tumor associated antigenic sequence), the first virus
acts as the prime and the Maraba MG1 virus acts as the boost. It is
sufficient that the immune response generated in the mammal to the
first antigen results in an immune response primarily to the MAGEA3
or MAGEA3 variant when the Maraba MG1 virus is administered.
[0084] In the context of the present disclosure, it should be
understood that all discussions of, and references to, a `protein
expressed by a virus` more exactly refer to a protein expressed by
a cell infected with the virus since viruses do not themselves have
the capability to express proteins. Similarly, all discussions of,
and references to, a `virus that expresses a protein` or `virus
capable of expressing a protein` more exactly refer to a virus that
includes the genetic information necessary for the protein to be
expressed by a cell infected with the virus.
[0085] The kit may additionally include an immune-potentiating
compound, such as cyclophosphamide (CPA), that increases the prime
immune response to the tumor associated antigenic protein generated
in the mammal by administrating the first virus. Cyclophosphamide
is a chemotherapeutic agent that may lead to enhanced immune
responses against the tumor associated antigenic protein. In a
synergistic murine melanoma 25 tumor model, CPA administered prior
to the priming vector significantly increased survival, while CPA
administered prior to the boosting vector did not.
[0086] The therapeutic approach disclosed herein combines: (1) a
viral vaccine, and (2) Maraba MG1 virus as an oncolytic viral
vaccine, both expressing MAGEA3, Human Papilloma Virus E6/E7 fusion
protein, human Six-Transmembrane Epithelial Antigen of the Prostate
protein, or Cancer Testis Antigen 1, or a variant thereof. Boosting
with the oncolytic vaccine may lead to both tumor debulking by the
oncolytic virus and a large increase in the number of
tumor-specific CTL (cytotoxic T-lymphocytes) in animals primed by
the viral vaccine. Paradoxically, this methodology actually
generates larger anti-tumor responses in tumor-bearing, as compared
to tumor-free, animals since the replication of oncolytic virus is
amplified in the tumor-bearing animals, which leads to an increase
in the number of antigen-specific Tumor Infiltrating Lymphocytes
(TILs), when compared to the replication of oncolytic virus in the
tumor-free animals and the associated number of antigen-specific
Tumor Infiltrating Lymphocytes (TILs).
[0087] The expression products of these genes are processed into
peptides, which, in turn, are expressed on cell surfaces. This can
lead to lysis of the tumor cells by specific CTLs. The T cell
response to foreign antigens includes both cytolytic T lymphocytes
and helper T lymphocytes. CD8.sup.+ cytotoxic or cytolytic T cells
(CTLs) are T cells which, when activated, lyse cells that present
the appropriate antigen presented by HLA class I molecules.
CD4.sup.+ T helper cells are T cells which secrete cytokines to
stimulate macrophages and antigen-producing B cells which present
the appropriate antigen by HLA class II molecules on their
surface.
[0088] The protein "MAGEA3" may be also referred to as "MAGE-A3"
and stands for melanoma-associated antigen 3. The antigenic MAGEA3
protein according to the present disclosure is a protein that
includes the amino acid sequence of SEQ ID NO: 1. This amino acid
sequence may be encoded by the nucleotide sequence of SEQ ID NO: 2.
Alternatively, the amino acid sequence may be encoded by a codon
optimized transgene that includes the nucleotide sequence of SEQ ID
NO: 3. A negative sense RNA virus that expresses the protein of SEQ
ID NO: 1 may include a reverse complement and RNA version of a
polynucleotide of SEQ ID NO: 2 or 3. A positive sense RNA virus or
a DNA virus that expresses the protein of SEQ ID NO: 1 may include
a sequence that is SEQ ID NO: 2 or 3.
[0089] An example of an antigenic MAGEA3 variant protein according
to the present disclosure is a protein that includes the amino acid
sequence of SEQ ID NO: 4. This amino acid sequence may be encoded
by the nucleotide sequence of SEQ ID NO: 5. A negative sense RNA
virus that expresses the protein of SEQ ID NO: 4 may include an RNA
polynucleotide which includes a sequence that is a reverse
complement and RNA version of SEQ ID NO: 5. A DNA virus or RNA
virus that expresses the protein of SEQ ID NO: 4 may include a
sequence that is SEQ ID NO: 5.
[0090] One example of such a negative sense RNA virus is a Maraba
virus that includes the reverse complement and RNA version of SEQ
ID NO: 6.
[0091] The antigenic protein "E6/E7 fusion protein" or "Human
Papilloma Virus E6/E7 fusion protein" according to the present
disclosure is a protein that includes the amino acid sequence of
SEQ ID NO: 7. This amino acid sequence may be encoded by the
nucleotide sequence of SEQ ID NO: 8. A negative sense RNA virus
that expresses the protein of SEQ ID NO: 7 may include a reverse
complement and RNA version of a polynucleotide of SEQ ID NO: 8. A
DNA virus or a positive sense RNA virus that expresses the protein
of SEQ ID NO: 7 may include a polynucleotide of SEQ ID NO: 8. One
example of such a negative sense RNA virus is a Maraba virus that
includes the reverse complement and RNA version of SEQ ID NO:
9.
[0092] The protein "huSTEAP" or "human Six-Transmembrane Epithelial
Antigen of the Prostate protein" according to the present
disclosure is a protein that includes the amino acid sequence of
SEQ ID NO: 10. This amino acid sequence may be encoded by the
nucleotide sequence of SEQ ID NO: 11. A negative sense RNA virus
that expresses the protein of SEQ ID NO: 10 may include a reverse
complement and RNA version of a polynucleotide of SEQ ID NO: 11. A
DNA virus or RNA virus that expresses the protein of SEQ ID NO: 10
may include a sequence that is SEQ ID NO: 11. One example of such a
negative sense RNA virus is a Maraba virus that includes the
reverse complement and RNA version of SEQ ID NO: 12.
[0093] The protein "NYESO1" or "human Cancer Testis Antigen 1"
according to the present disclosure is a protein that includes the
amino acid sequence of SEQ ID NO: 13. This amino acid sequence may
be encoded by the nucleotide sequence of SEQ ID NO: 14. A negative
sense RNA virus that expresses the protein of SEQ ID NO: 13 may
include a reverse complement and RNA version of a polynucleotide of
SEQ ID NO: 14. A DNA virus or RNA virus that expresses the protein
of SEQ ID NO: 13 may include a sequence that is SEQ ID NO: 14. One
example of such a negative sense RNA virus is a Maraba virus that
includes the reverse complement and RNA version of SEQ ID NO:
15.
[0094] The above noted sequences are shown in Appendix A.
[0095] The term "mammal" refers to humans as well as non-human
mammals. The term "cancer" is used herein to encompass any cancer
that expresses the tumor associated antigenic protein (that is:
MAGEA3, Human Papilloma Virus E6/E7 fusion protein, human
Six-Transmembrane Epithelial Antigen of the Prostate protein, or
Cancer Testis Antigen 1) used in the viruses of interest.
[0096] For example, when considering MAGEA3 as an antigenic
protein, the term "cancer" encompasses any cancer that expresses
MAGEA3 as an antigen. Examples of such a cancer include, but are
not limited to, melanoma, non-small cell lung cancer, head and neck
cancer, colorectal cancer, and bladder cancer.
[0097] When considering E6/E7 fusion protein as an antigenic
protein, the term "cancer" encompasses any cancer that expresses E6
and E7 proteins as antigenic proteins. Examples of such a cancer
include, but are not limited to, cervical cancer.
[0098] The first virus, the Maraba MG1 virus, or both may be
independently administered to the mammal intravenously,
intramuscularly, intraperitoneally, or intranasally. Following
administration of the viruses, an immune response is generated by
the mammal within an immune response interval, e.g. within about 4
days, and extending for months, years, or potentially life.
[0099] The first virus may be any virus that induces an immune
response to the tumor associated antigenic protein or variant
thereof after the first virus is administered to the patient.
Viruses that may be used according to the present disclosure
include, for example: adenovirus (Ad), poxvirus, retrovirus, and
alpha virus. An example of a poxvirus is vaccinia virus. An example
of a retrovirus is lentivirus. An example of an alpha virus is
semliki forest virus.
[0100] To establish an immune response to the tumor associated
antigenic protein or variant thereof, vaccination using the first
virus and the Maraba MG1 virus may be conducted using
well-established techniques. As one of skill in the art will
appreciate, the amount of virus required to generate an immune
response will vary with a number of factors, including, for
example, the selected antigen, the viral vector used to deliver the
antigen, and the mammal to be treated, e.g. species, age, size,
etc. In this regard, for example, intramuscular administration of
at least about 10.sup.7 PFU of Adenoviral vector to a mouse is
sufficient to generate an immune response. A corresponding amount
would be sufficient for administration to a human to generate an
immune response.
[0101] Once an immune response has been generated in the mammal by
administration of the first virus, Maraba MG1 virus encoding the
tumor associated antigenic protein or a variant thereof is
administered in an amount suitable for oncolytic viral therapy
within a suitable immune response interval. A suitable immune
response interval may be, for example, at least about 24 hours,
preferably at least about 2-4 days or longer, e.g. at least about 1
week, or at least about 2 weeks. The amount of Maraba MG1 virus
suitable for oncolytic viral therapy will vary with the mammal to
be treated, as will be appreciated by one of skill in the art. For
example, 10.sup.8 PFU of Maraba MG1 virus encoding MAGEA3
administered IV to a mouse is sufficient for oncolytic therapy. A
corresponding amount would be sufficient for use in a human.
[0102] Maraba MG1 virus encoding tumor associated antigenic protein
or a variant thereof may be prepared by incorporating a reverse
complement of a transgene encoding the tumor associated antigenic
protein or a variant thereof into the Maraba MG1 virus using
standard recombinant technology. For example, the reverse
complement of the transgene may be incorporated into the genome of
the Marama MG1 virus, or alternatively, may be incorporated into
the virus using a plasmid incorporating the transgene. The
transgene encoding the tumor may be a codon optimized
transgene.
EXAMPLES
[0103] The oncolytic Maraba MG1 is a potent oncolytic vaccine
platform. While unable to prime detectable responses against a
melanoma-associated antigen, Maraba MG1-vaccine displayed the
ability to boost preexisting tumor-specific CD4.sup.+ and CD8.sup.+
T-cell immunity. When applied to the treatment of syngeneic murine
melanoma tumor models, Maraba-MG1-mediated recall immunization
resulted in an extension of the median survival with complete
remission in more than 20% of the animals treated.
[0104] In a primate toxicity study heterologous prime-boost
vaccination with an Ad-MAGEA3 prime followed by a Maraba-MG1-MAGEA3
boost resulted in T-cell responses that were comparable to those
obtained in syngeneic murine tumor models demonstrating that in an
outbred primate population the prime-boost oncolytic vaccine
strategy gives immune responses comparable to animal models where
tumors can be engrafted and a dramatic extension of survival is
attained.
[0105] The authors of the present disclosure also determined that
proteins having the sequence SEQ ID NOs: 7, 10, or 13 could be used
to stimulate an immune response in a patient using a heterologous
prime boost with Maraba MG1. In contrast, the authors of the
present disclosure determined that administration of a first virus
expressing EBDNA-1 protein or Placenta-specific protein 1 (PLAC-1)
followed by administration of Maraba-MG1 expressing EBDNA-1 protein
or PLAC-1, respectively, was unable to stimulate an immune
response.
Example 1: MG1-hDCT is a Weak Priming Vector but a Potent Boosting
Vector
[0106] Ad-empty and Ad-hDCT are replication-deficient adenoviruses
(E1/E3-deletion) based on the human serotype 5 (Lane C. et al.,
(2004) Cancer Research 64:1509-1514; Ng P. et al., (2001) Mol Ther
3:809-815). The replication-deficient adenovirus vector was
engineered to express the hDCT transgene, which encodes the full
length human melanoma associated antigen DCT (dopachrome
tautomerase) while Ad-empty has no transgene. The resulting
adenovirus vector is termed "Ad-hDCT".
[0107] The MG1 variant of Maraba virus was engineered to express
the human form of the melanoma-associated antigen hDCT transgene.
The resulting MG1 virus vector is termed "MG1-hDCT" or "Maraba
MG1-hDCT". Other virus vectors are named using a similar
convention.
[0108] Recombinant Maraba and VSV were generated by transgene
insertion between the G and L viral genes. VSV-hDCT derives from
the wild-type Indiana strain of the VSV (Bridle BW. et al. (2009)
17:1814-1821; Lawson N D. et al., (1995) Proc Natl Acad Sci USA
92:4477-4481). MG1-GFP (Green Flourescent Protein used as a control
non-immunogenic transgene insertion) and MG1-hDCT derive from the
attenuated strain MG1 of Maraba virus. Prior to in vivo studies,
DCT (and GFP) expression from the virus was confirmed by western
blot of lysates from infected Vero cells cultured in alpha-MEM
containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 1 00
mg/ml streptomycin (all from Invitrogen, Grand Island, N.Y.).
[0109] The therapeutic efficacy of MG1-hDCT administered as a
monotherapy was evaluated initially. In order to generate lung
metastases, C57BI/6 mice (8-10 weeks old at study initiation) were
injected i.v. with 2.5.times.10.sup.5 B16-F10 cells (murine
melanoma cells expressing the murine DCT antigen) in 200 .mu.l
saline water. The oncolytic vaccine was injected systemically 5 or
14 days later and T-cell responses against the melanoma antigen DCT
were measured in the blood at day 19. The virus was administered
systemically at a high dose (10.sup.9 pfu i.v in 200 .mu.l PBS).
T-cell responses were measured by isolating PBMCs or splenocytes
and stimulating them with the SVYDFFVWL (SEQ ID NO: 45) (SVY) or
KFFHRTCKCTGNFA (SEQ ID NO: 46), (KFF) peptides corresponding to the
MHC-I or MHC-II restricted immunodominant epitopes of DCT,
respectively. Responding T-cells were detected after intracellular
cytokine staining (ICS) for IFN-.gamma. by flow cytometry.
[0110] As shown in FIGS. 1A and 1B, MG1-hDCT was unable to prime
DCT-specific CD8.sup.+ or CD4.sup.+ T-cell responses in
tumor-bearing mice (FIG. 1A). Administered alone, the MG1-hDCT
vaccine did not improve tumor outcome. Indeed, mice treated 14 days
post-tumor challenge reached endpoint in a similar timeframe as
untreated mice: after 20 days for the Ad-empty control group versus
21 days for the Ad-empty+MG1-hDCT group (FIG. 1B). Moreover,
survival was not extended even when mice were treated with MG1-hDCT
as early as 5 days after tumor engraftment (MG1-hDCT group, FIG.
1B). In conclusion, not only did MG1-hDCT fail to induce anti-DCT
immunity but its oncolytic activity offered no therapeutic benefit.
These results demonstrate that MG1-hDCT is unable to prime
significant T-cell responses against the tumor antigen DCT and is
thus a weak priming vector.
[0111] It was previously reported that an oncolytic VSV vector
serves as a potent booster of pre-existing immunity (Bridle BW. et
al., (2010) Mol Ther 184:4269-4275; WO 2010/105347). In the present
disclosure, the ability of Maraba MG1 virus to serve as a booster
vaccine was examined. Adenoviral vectors were used as priming
vectors and administered intramuscularly (i.m.) at a total dose of
2.times.10.sup.8 pfu (1.times.10.sup.8 pfu in 50 .mu.l PBS per
thigh). For adenovirus injection, mice were anesthetized in a
sealed chamber containing 5% inhalation isoflurane. Using Ad-hDCT
as a priming vector, MG1-hDCT was evaluated as a booster of
pre-existing DCT-specific responses. To evaluate Maraba virus as a
boosting vector, various routes of administration were evaluated.
An oncolytic dose of 1.times.10.sup.9 pfu of virus was administered
that is well tolerated in this mouse strain and an interval of 12
days post-Ad priming was selected as this was the longest interval
that would be feasible in the tumor model. When this dose of
MG1-Maraba-hDCT was administered by intravenous (i.v.), intranasal
(i.n.) and intramuscular (i.m.) routes, the i.v. route proved to be
far superior as measured by ICS for IFN-.gamma. in peripheral
CD8.sup.+ T-cells: 28.33%.+-.3.82 by i.v. versus 4.73%.+-.1.52 i.n.
versus 13.84%.+-.1.88 i.m. The responses were measured at day 5
post-Maraba administration coinciding with the peak of the
MG1-hDCT-mediated boost response. In the intravenously boosted
animals a significant proportion of DCT-specific CD8.sup.+ T-cells
was also measured in the spleen with a 3-fold increase in mice
administered with both vaccine vectors compared to animals primed
only: 3.45%.+-.0.45 in Ad-hDCT group versus 11.02%.+-.2.14 in the
Ad-hDCT+MG1-hDCT immunized animals (p=0.0085''). While Ad-hDCT was
unable to induce a detectable DCT-specific CD4.sup.+ T-cell
population in the blood and a barely detectable population in the
spleen, the MG1 Maraba-hDCT booster was able to generate clear
systemic CD4.sup.+ T-cell response but only when administered i.v.
(0.30%.+-.0.11). The response was also detectable in the spleen
with 0.14%.+-.0.03 of splenic CD4.sup.+ T-cells reacting to DCT KFF
peptide exposure. Similar to VSV, maximal immune boosting by MG1
Maraba virus is achieved by i.v. administration. In conclusion,
systemic delivery of a Maraba-vectored vaccine at a dose of
10.sup.9 pfu appeared to allow for efficient boosting of both
antigen-specific CD8.sup.+ and CD4.sup.+ T-cell populations. For
this reason, this route and dose were used for Maraba MG1
administration in subsequent in vivo experiments.
[0112] To show that Maraba MG1-hDCT is a more potent boosting
vector than VSV-hDCT, C57/B16 mice were primed with Ad-hDCT (Ad-BHG
was included as a control vector lacking a transgene) and then
boosted with an intravenous dose of either VSV-hDCT or Maraba-hDCT
14 days later. Immune analysis of CD8.sup.+ T cell responses were
measured in peripheral blood at day 5 post-boosting vector. At an
equivalent dose the response induced by Maraba vaccination was 3-8
fold as large as the VSV-induced responses (FIG. 2).
Example 2: MG1-hDCT Vaccine Strategy in Murine Models of Cancer
[0113] The therapeutic efficacy of MG 1-hDCT administered as a
boosting vector was subsequently investigated. Five days following
B16-F10 engraftment to generate lung metastases in animals, animals
received an Ad-hDCT priming vaccine and this was followed 9 days
later by a single i.v. dose of MG1 Maraba-hDCT as an oncolytic
booster vaccine. Ad-hDCT prime-MG1-hDCT boost vaccination generated
a very strong DCT-specific CD8.sup.+ T-cell response (mean %
IFN-.gamma..sup.+ CD8.sup.+ T-cells=27.54.+-.2.17, FIG. 3) that was
14 times higher than in non-boosted mice (1.95%.+-.0.29 in Ad-hDCT
group and 1.91%.+-.0.59 in Ad-hDCT+MG1-GFP group, FIG. 3).
Similarly, DCT-specific CD4.sup.+ T-cell responses were measured in
MG1-hDCT boosted animals while rarely detected in primed only mice
(mean % IFN-.gamma..sup.+CD4.sup.+ T-cells=0.25%.+-.0.06 in
Ad-hDCT+MG1-hDCT group versus <0.05% in Ad-hDCT and
Ad-hDCT+MG1-GFP groups, FIG. 3).
[0114] Looking at treatment outcome, Ad-hDCT immunization allowed a
10-day extension of the median survival compared to untreated mice:
31 days for Ad-hDCT treatment versus 20.5 days for Ad-empty group
(FIG. 4). Ad-hDCT treatment followed by MG1 Maraba-GFP oncolytic
treatment did not improve survival (27.5 days median survival for
Ad-hDCT+MG1-GFP group, FIG. 4). However, boosting anti-tumor
immunity with the Maraba MG1-DCT vaccine dramatically improved
tumor outcome with a 20-day extension of the median survival
compared to Ad-hDCT primed only animals (51 days for
Ad-hDCT+MG1-hDCT group, FIG. 4). More importantly, the oncolytic
MG1-hDCT booster treatment resulted in 23.3% long-term survival
(FIG. 4).
[0115] In order to characterize the respective contribution of
tumor-specific CD4.sup.+ and CD8.sup.+ T-cell responses in the
therapeutic efficacy, each T-cell compartment was selectively
depleted (data not shown). Depletion of the CD8.sup.+ T-cell
population at the time of the boost abrogated the therapeutic
benefit of MG1-hDCT administration. On the contrary, CD4.sup.+
T-cells depletion appeared not to affect significantly the
therapeutic efficacy indicating that Maraba immune boosting of
CD8.sup.+ T cells is CD4.sup.+-independent. While the critical role
of CD8.sup.+ T-cells in controlling tumor growth is admitted, these
results show that boosting tumor-specific CD8.sup.+ T-cells with
Maraba vaccine is a potent way of improving cancer therapy.
[0116] Finally, the efficacy of the prime-boost strategy involving
Maraba vaccine was also evaluated in a very challenging
intracranial B16-F10 model of metastatic melanoma brain cancer.
Ad-hDCT-mediated immunotherapy significantly improved survival of
melanoma brain met-bearing mice with a median extended from 15 days
for Ad-empty controls to 25.5 days for the Ad-hDCT group (FIG. 5).
As previously reported, such therapeutic efficacy demonstrates the
ability of the tumor-specific effector T-cells raised to cross the
blood-brain barrier and infiltrate the tumor bed (Bridle BW. et
al., (2010) Mol Ther 184:4269-4275). The additional administration
of a Maraba MG1-hDCT oncolytic booster further improved tumor
outcome with a median survival reaching 42 days together with cures
observed in 21.4% of treated animals (Ad-hDCT+MG1-hDCT group, FIG.
5).
Example 3: Failure of Vaccine Strategy to Induce an Anti-mPLAC1 T
Cell Response
[0117] Although Maraba MG1 and VSV were able to act as boosting
vectors using hDCT as a tumor associated antigen, not all tumor
associated antigens can be used in a heterologous prime-boost
vaccine strategy. The authors of the present disclosure tested a
heterologous prime-boost vaccine strategy using huAd5-mPLAC1 as the
priming vector and VSV-mPLAC1 as the boosting vector.
[0118] PLAC1 is a recently described tumor associated antigen
expressed in the placenta but has also been reported in several
tumor cell lines and in tumors of patients breast, lung, liver,
gastric and colorectal cancers (Silva, W A et al., (2007) Cancer
Immun 7:18).
[0119] Ad-mPLAC1 is a replication-deficient adenoviruses
(E1/E3-deletion) based on the human serotype 5 (Lane C. et al.,
(2004) Cancer Research 64:1509-1514; Ng P. et al., (2001) Mol Ther
3:809-815). The replication-deficient adenovirus vector was
engineered to express the mPLAC1 transgene, which encodes the full
length murine antigen PLAC1 (placenta-specific 1), the resulting
adenovirus vector is termed "Ad-mPLAC1" or "huAd5-mPLAC1".
[0120] VSV virus was engineered to express the human form of the
melanoma-associated antigen mPLAC1 transgene. The resulting
VSV-virus vector is termed "VSVmPLAC1". Recombinant VSV was
generated by transgene insertion between the G and L viral genes.
VSV-mPLAC1 derives from the wild-type Indiana strain of the VSV
(Bridle B W. Et al. (2009) 17:1814-1821; Lawson N D. et al., (1995)
Proc Natl Acad Sci USA 92:4477-4481).
[0121] C57BI/6 mice were primed with Ad-mPLAC1 (2.times.10.sup.9
PFU IM injection) and then boosted with a single i.v.dose.of
VSV-mPLAC1 (2.times.10.sup.9 PFU) 14 days later. T-cell responses
were measured by isolating splenocytes and stimulating them with
individual 15 mmer peptides form an overlapping PLAC1 peptide
library for a total of 6 hours with golgi plug added 1 hour into
the stimulation. Following stimulation the splenocytes were stained
for CD4, CD8 and IFN.gamma. and analyzed on FACSCanto and FlowJo.
Responding T-cells were detected after intracellular cytokine
staining (ICS) for IFN-.gamma. by flow cytometry. None of the
mPLAC1 peptides were able to stimulate IFN-.gamma. production in
either CD8 or CD4 T cells.
Example 4: Construction of Oncolytic Vaccine Vectors with MAGEA3 or
a Variant Thereof
[0122] Ad-MAGEA3 is a replication-deficient adenovirus
(E1/E3-deletion) based on the human serotype 5 (Lane C. et al.,
(2004) Cancer Research 64:1509-1514; Ng P. et al., (2001) Mol Ther
3:809-815) containing the full-length human MAGEA3 gene. Maraba
MG1-hMAGEA3 has been developed and contains the codon-optimized
full length human MAGEA3 gene inserted between the G and L viral
genes of the MG1 double mutant of Maraba virus (Brun J. et al.,
(2010) Mol Ther 18:1440-1449). The MAGEA3 sequence (NCBI Gene ID:
4102 www.ncbi.nlm.nih.gov/gene/4102) was codon optimized for
expression in mammalian cells and then synthesized with a FLAG tag
on 3' end and with Mlul restriction sites on both 3' and 5' ends.
This sequence was ligated into the shuttle vector pMRB-MG1/pNF at
its Mlul site (between G and L genes) which contains part of the
Maraba-MG1 genome from the beginning of G to the end of L genes,
flanked by Kpnl and Nhel sites, respectively. The entire region
from Kpnl to Nhel, now containing MAGEA3 Flag between G and L was
then removed from pMRB-MG1/pNF and ligated back into the pMRB-MG1
genomic plasmid using Kpnl and Nhel sites. Maraba-MG1-MAGEA3 Flag
was then rescued and plaque purified. This is illustrated in FIG.
6.
[0123] A full length human MAGEA3 protein expressed by the
adenovirus may include the amino acid sequence of SEQ ID NO: 1. The
adenovirus may include a nucleotide sequence of SEQ ID NO: 2.
Alternatively, the amino acid sequence may be encoded by a codon
optimized transgene that includes the nucleotide sequence of SEQ ID
NO: 3. Accordingly, the adenovirus may include the codon-optimized
nucleotide sequence of SEQ ID NO: 3.
[0124] The Maraba MG1 virus may include a reverse complement and
RNA version of a nucleotide sequence of SEQ ID NO: 2.
Alternatively, the amino acid sequence may be encoded by a codon
optimized transgene that includes the nucleotide sequence of SEQ ID
NO: 3. Accordingly, the Maraba MG1 virus may include the reverse
complement and RNA version of the codon-optimized nucleotide
sequence of SEQ ID NO: 3.
[0125] One variant of MAGEA3 is a protein that includes the amino
acid sequence of SEQ ID NO: 4. This amino acid sequence may be
encoded by the nucleotide sequence of SEQ ID NO: 5. The adenovirus
may include a nucleotide sequence of SEQ ID NO: 5. The Maraba MG1
virus may include a reverse complement and RNA version of a
nucleotide sequence of SEQ ID NO: 5.
[0126] A negative sense RNA virus, such as a Maraba virus, that
expresses the protein of SEQ ID NO: 4 may include an RNA
polynucleotide which includes a sequence that is a reverse
complement and RNA version of SEQ ID NO: 6.
Example 5: MG1-MAGEA3 Vaccine Immune Response in Healthy
Primates
[0127] Healthy cynomolgous monkeys were used in a study designed to
collect toxicity and immunogenicity data for developing the
potential MG1-MAGEA3 oncolytic vaccine for human use. The use of
the cynomolgous monkeys maximizes the likelihood of identifying
responses that are quantitatively and qualitatively similar to
those expected in humans. Prior to study start primates were
acclimated for 4-6 weeks from the time of animal arrival until the
time of vascular access port implantation surgery. After a minimum
of 2-3 weeks following surgery, animals were vaccinated with a
non-replicating adenovirus Ad-MAGEA3 priming vector, injected in
each leg, 0.5 ml per dose totaling 1.times.10.sup.10 pfu by slow IM
injection. For the Ad-MAGEA3/MG1-MAGEA3 prime boost study,
Ad-MAGEA3 prime occurred at either 2 weeks (-14 days) or 4 weeks
(-28 days) prior to MG1-MAGEA3 boost. Therefore Ad-MAGEA3
administration occurred on Day -14 or on Day -28 and MG1-MAGEA3
boost on Days 0 and 3. The rationale for Ad-MAGEA3 dosage level
comes from the literature, and from previous experiments
demonstrating that a dose of 1.times.10.sup.10 pfu in Macaques (and
humans) is a safe dose with no observed toxicities (Bett et al.
Vaccine, 2010). For animals in the 2 week boosted group, MG1-MAGEA3
virus was injected i.v. at either a low dose 1.times.10.sup.10 or a
high dose 1.times.10.sup.11 at experiment days 0 and 3 (14 and 17
days after Ad-MAGEA3). For animals in the 4 week boosted group,
MG1-MAGEA3 virus was injected i.v. at either a low dose
1.times.10.sup.10 or a high dose 1.times.10.sup.11 at experiment
days 0 and 3, (28 and 31 days after the Ad-MAGEA3). Boosting Virus
was infused in 30 mL of sterile buffered saline (pH 7.5) over 30
minutes through the vascular access port. The rationale for MG
1-MAGEA3 low dosage level comes from pre-clinical studies that
demonstrate that the murine maximum tolerable dose is
1.times.10.sup.9. The relative body surface area scale-up to
Macaques equates this to 3.5.times.10.sup.10 total pfu. The
rationale for MG1-MAGEA3 high dosage level comes from a pilot
Non-Human Primate (NHP) toxicology study, where there was no
observed toxicity at a dose level of 2.times.10.sup.11 pfu. Animals
in the prime boost study were either sacrificed early (Day 14) or
late (Day 84). For the Ad-MAGEA3/MG1-MAGEA3 prime boost study,
blood samples were taken from all animals at 5 distinct time
points. For animals in the 2 week heterologous prime-boost cohort,
blood samples were collected prior to any vaccination and on a day
prior to Day -14 (Baseline) and on experiment Days 5, 13 and 84.
For animals in the 4 week heterologous prime-boost cohort, blood
samples were collected prior to any vaccination and on a day prior
to Day -28 (Baseline), and on experiment Days 5, 13, and 84.
[0128] To assess immune responses in the primates to the
heterologous prime-boost vaccination with Ad-MAGEA3/MG1-MAGEA3,
Peripheral Blood Mononuclear Cells (PBMCs) were incubated for 4
hours (last 3 hours in presence of Brefeldin A) with a pool of 10
hMAGE-A3 peptides for T-cell (re-) stimulation (or left
unstimulated for evaluation of the background). Peptides were from
an overlapping peptide library covering the whole hMAGE-A3 antigen
from N to C-termini in 87 peptides (15-mer each). After
stimulation, T-cells were stained with fluorescent anti-CD8 and
anti-CD4 antibodies for 25 minutes. After this surface staining,
cells were permeabilized and fixed with BD Cytofix/Cytoperm for 20
minutes. Then, hMAGE-A3-specific T-cells were detected by looking
at cytokine expression by intracellular staining with fluorescent
anti-IFN.gamma. and anti-TNF.alpha. antibodies for 25 minutes. Cell
analysis was performed on BD Canto flow cytometer.
[0129] FIG. 7 shows the average CD8.sup.+ T-cell immune responses
of monkeys given high and low dose MG1-MAGEA3 as a boosting vector
following an Ad-MAGEA3 prime. In the low dose MG1-MAGEA3 animals
there is a significant increase in CD8.sup.+ T-cell response 5 days
following the boost, which drops off over time while in the high
dose MG1-MAGEA3 animals there is a similar significant increase in
CD8.sup.+ T-cell response 5 days following the boost, which is
sustained at a higher level over time. FIG. 8 shows that all of the
animals in the study exhibited a significant increase in CD8.sup.+
T-cell response 5 days following the boost with MG1-MAGEA3
irrespective of high or low dose. These peak T-cell responses in
Primates demonstrate that in an outbred population the prime-boost
oncolytic vaccine strategy gives immune responses comparable to
animal models where tumors can be engrafted and a dramatic
extension of survival is attained.
Example 6: Construction and Immune Testing of Lentiviral Priming
Vectors and Oncolytic Vaccine Vectors Expressing Human Papilloma
Virus E6/E7 Fusion Protein
[0130] The HPV transgene is a fusion of HPV serotype 16 full-length
wild-type E6 (gi/4927720/gb/AAD33252.1/AF125673_1 E6 Human
papillomavirus type 16) and E7 (gi/4927721/gb/AAD33253.1/AF125673_2
E7 Human papillomavirus type 16) sequences and HPV serotype 18
full-length wild-type E6 (gi/137758/sp/P06463.1/VE6_HPV18 RecName:
Full=Protein E6) and E7 (gi/137792/sp/P06788.2/VE7 HPV18 RecName:
Full=Protein E7) sequences with deletions in all 4 nucleotide
sequences to remove zinc fingers required for Rb or p53 binding
(removing oncogenic potential of the proteins). The resulting
fusion protein has a flexible glycine linker plus AAY sequence
(which serves as a proteasomal cleavage site to ensure that each
antigen is proteolytically degraded to the peptides normally
generated for antigen presentation). This codon-optimized fusion
nucleotide sequence gives rise to a 527 amino acid HPV16/18 E6/E7
fusion protein (SEQ ID NO: 7).
[0131] Lentiviruses expressing Human Papilloma Virus E6/E7 fusion
transgene were made using the pDY.EG.WS lentivirus vector. The
modified HPV transgene was PCR amplified using primers containing
the EcoRI restriction site (forward primer
ACTGGAATTCATGCATCAGAAGCGAACTGC, SEQ ID NO: 18) and the BamHI
restriction site (reverse primer ACTGGGATCCTCACTGCTGGGAGGCACAC, SEQ
ID NO: 19). The HPV transgene PCR product was agarose gel purified.
The pDY.EG.WS lentivirus vector was cut at the EcoRI and BamHI
sites to remove eGFP, was agarose gel purified, and was subjected
to dephosphorylation using CIAP (Invitrogen Catalogue 18009-019).
The cut vector was then subjected to additional agarose gel
purification. The HPV transgene PCR product was then ligated into
the EcoRI/BamHI cut vector using T4 DNA ligase (Invitrogen). The
ligation reaction was subjected to a transformation using competent
cells, and plasmid DNA from positive colonies was subjected to
mini-prep amplification. The pDY.EG.WS lentivirus vector expressing
the modified HPV transgene was then subjected to maxi-prep
amplification. The lentivirus expressing Human Papilloma Virus
E6/E7 fusion transgene were rescued on 293T cells after
transfection of 6.4 .mu.g of each of three plasmids: the pDY.EG.WS
lentivirus vector expressing the modified HPV transgene, the
packaging pCMV-8.84 plasmid, and the envelope pMD2G plasmid. Virus
supernatants were pooled, and filtered through a 0.45 .mu.M filter
and centrifuged for 120 minutes at 50,000.times.g at 16.degree. C.
The lentivirus expressing Human Papilloma Virus E6/E7 fusion
transgene was resuspended in PBS, and stored at -80.degree. C.
[0132] Maraba MG1 was engineered to contain a Papilloma Virus E6/E7
fusion transgene inserted between the G and L viral genes of the
MG1 double mutant of Maraba virus (Brun J. et al., (2010) Mol Ther
18:1440-1449). The transgene sequence (SEQ ID NO: 8) was codon
optimized for expression in mammalian cells. The resulting Maraba
MG1 containing the HPV E6/E7 is designated, generally,
"Maraba-MG1-HPV E6/E7". A modified Maraba MG1 backbone was used to
facilitate cloning. A silent mutation was introduced into the L
gene of the Maraba MG1 genome backbone to remove one of the Mlul
sites. The second Mlul site was replaced with a BsiWI site at the
cloning region between G and L. These modifications to the Maraba
MG1 genome backbone allowed for a more direct cloning system than
that described in the Brun et al. paper as it avoids using the
shuttle plasmid pMRB-MG1/pNF. The HPV E6/E7 fused transgene
sequence was ligated into the modified Maraba MG1 genome backbone
at its Mlul site and BsiWI site (at cloning region between G and L)
The Maraba-MG1-HPV E6/E7 was then rescued (as previously described
in Brun et al., (2010) Mol Ther 18:1440-1449), plaque purified
once, and subjected to opti-prep purification.). The Maraba-MG1-HPV
E6/E7 has a genomic sequence that is the reverse complement and RNA
version of SEQ ID NO: 9.
[0133] Generally, animals were immunized by administration of the
priming vector (lentivirus-HPV E6/E7+poly I:C as an adjuvant) at
day 0 and by administration of 1e9 PFU of the boosting vector
(Maraba-MG1-HPV E6/E7) at day 14. Control animals were
prime-boosted with viral vectors encoding GFP instead of the HPV
E6/E7 transgene as a control non-immunogenic transgene insertion.
Analysis of the prime response was conducted at day 14 and of the
boost response at day 19. Each lentivirus-HPVE6/E7 preparation was
made with 250 ug poly I:C added as an adjuvant to the priming virus
and then split between 5 animals for each virus. Mice were
anesthetized with isoflurane and 30 uL of lentivirus-HPV E6/E7/poly
I:C was injected into each hind foot pad. The remaining virus was
injected subcutaneously near the left inguinal lymph node. 14 days
after prime, blood was collected and analyzed by flow cytometry.
Mice were then boosted with 1.times.10.sup.9 PFU MG1-HPV E6/E7
intravenously. 5 days following the boost, blood was drawn and
immune responses were assessed by flow cytometry.
[0134] Immune analysis was performed as follows: Blood was
collected via retro-orbital bleeding using heparinzied capillary
tube and blood was collected into heparin. Red blood cells were
then lysed using ACK lysis buffer and the resulting PBMCs were
analyzed for immune responses to the tumor antigens. PBMCs were
either incubated in the absence of peptide or stimulated with 2
ug/mL peptides (RAHYNIVTF) (SEQ ID NO: 47) for a total of 5 hours
with golgi plug added 1 hour into the stimulation. Following
stimulation the PBMCs were stained for CD4, CD8 and IFN.gamma. and
analyzed on FACSCanto and FlowJo. Responding T-cells were detected
after intracellular cytokine staining (ICS) for IFN-.gamma. by flow
cytometry. Values from unstimulated PBMCs were considered
background and subtracted from values obtained from stimulated
PBMCs. Data represents mean+/-SEM. In Table 1 it is demonstrated
that the HPV E6/E7 peptides were able to stimulate IFN-.gamma.
production in CD8 cells indicating the existence of an immune
response.
TABLE-US-00001 TABLE 1 IMMUNE RESPONSE to HPV E6/E7 PRIME-BOOST
Percentage of CD8 T Cells Secreting Interferon (IFN) .sup..gamma.
Immune Group Stimulatory Control Group Lentivirus-HPV E6/E7 Prime
Peptide Lentivirus-GFP Prime MG1-HPV E6/E7 Boost Epitope MG1-GFP
Boost (N = 5) RAHYNIVTF 0.0033 .+-. 0.0033 0.03 .+-. 0.025 0.036
.+-. 0.012 5.9 .+-. 2.7 (SEQ ID NO: 47) (after prime) (after boost)
(after prime) (after boost)
Example 7: Construction and Immune Testing of Lentiviral Priming
Vectors and Oncolytic Vaccine Vectors Expressing Cancer Testis
Antigen 1
[0135] The NYESO1 transgene is full-length wild-type sequence (SEQ
ID NO: 14) codon-optimized for expression in human and mouse to
give rise to a 180 amino acid protein (SEQ ID NO: 13).
[0136] Lentiviruses expressing Cancer Testis Antigen 1 transgene
were made using the pDY.EG.WS lentivirus vector. The NYESO1
transgene was PCR amplified using primers containing the BamHI
restriction site (forward primer ACTGGGATCCATGCAGGCCGAGGGCAGAG, SEQ
ID NO: 20) and the BamHI restriction site (reverse primer
ACTGGGATCCTCATCTTCTCTGGCCGCTGG, SEQ ID NO: 21). The NYESO1
transgene PCR product was agarose gel purified. The pDY.EG.WS
lentivirus vector was cut at the BamHI site to remove eGFP, was
agarose gel purified, and was subjected to dephosphorylation using
CIAP (Invitrogen Catalogue 18009-019). The cut vector was then
subjected to additional agarose gel purification. The NYESO1
transgene PCR product was then ligated into the BamHI cut vector
using T4 DNA ligase (Invitrogen). The ligation reaction was
subjected to a transformation using competent cells, and plasmid
DNA from positive colonies was subjected to mini-prep
amplification. The pDY.EG.WS lentivirus vector expressing the
modified HPV transgene was then subjected to maxi-prep
amplification. The lentivirus expressing NYESO1 transgene were
rescued on 293T cells after transfection of 6.4 .mu.g of each of
three plasmids: the pDY.EG.WS lentivirus vector expressing the
NYESO1 transgene, the packaging pCMV-8.84 plasmid, and the envelope
pMD2G plasmid. Virus supernatants were pooled, and filtered through
a 0.45 .mu.M filter and centrifuged for 120 minutes at
50,000.times.g at 16.degree. C. The lentivirus expressing NYESO1
transgene was resuspended in PBS, and stored at -80.degree. C.
[0137] Maraba MG1 was engineered to contain Cancer Testis Antigen 1
transgene inserted between the G and L viral genes of the MG1
double mutant of Maraba virus (Brun J. et al., (2010) Mol Ther
18:1440-1449). The transgene sequence was codon optimized for
expression in mammalian cells. The resulting Maraba MG1 containing
the NYESO1 protein is designated as "Maraba-MG1-NYESO1" or
"MG1-NYESO1".
[0138] The NYESO1 transgene was ligated into the shuttle vector
pMRB-MG1/pNF at its Mlul site (between G and L genes) which
contains part of the Maraba-MG1 genome from the beginning of G to
the end of L genes, flanked by Kpnl and Nhel sites, respectively.
The entire region from Kpnl to Nhel, now containing the NYESO1
transgene inserted between G and L was then removed from
pMRB-MG1/pNF and ligated back into the pMRB-MG1 genomic plasmid
using Kpnl and Nhel sites. The Maraba-MG1-NYESO1 was then rescued
(as previously described Brun J. et al., (2010) Mol Ther 18:
1440-1449). The Maraba-MG1-NYESO1 was plaque purified 3 times, and
purified via sucrose cushion purification. The Maraba-MG1-NYESO1
virus has a genomic sequence that is the reverse complement and RNA
version of SEQ ID NO: 15.
[0139] Generally, animals were immunized by administration of the
priming vector (lentivirus-NYESO1+poly I:C as an adjuvant) at day 0
and by administration of 1e9 PFU of the boosting vector
(Maraba-MG1-NYESO1) at day 14. Control animals were prime-boosted
with viral vectors encoding GFP instead of the NYESO1 transgene as
a control non-immunogenic transgene insertion. Analysis of the
prime response was conducted at day 14 and day 19. Each
lentivirus-NYESO1 preparation was made with 250 ug poly I:C added
as an adjuvant to the priming virus and then split between 5
animals for each virus. Mice were anesthetized with isoflurane and
30 uL of lentivirus-NYESO1/poly I:C was injected into each hind
foot pad. The remaining virus was injected subcutaneously near the
left inguinal lymph node. 14 days after prime, blood was collected
and analyzed by flow cytometry. Mice were then boosted with
1.times.10.sup.9 PFU MG1-NYESO1 intravenously. Five days following
the boost, blood was drawn and immune responses were assessed by
flow cytometry.
[0140] Immune analysis was performed as follows: Blood was
collected via retro-orbital bleeding using heparinzied capillary
tube and blood was collected into heparin. Red blood cells were
then lysed using ACK lysis buffer and the resulting PBMCs were
analyzed for immune responses to the tumor antigens. PBMCs were
either incubated in the absence of peptide or stimulated with 2
ug/mL peptides (RGPESRLL) (SEQ ID NO: 48) for a total of 5 hours
with golgi plug added 1 hour into the stimulation. Following
stimulation the PBMCs were stained for CD4, CD8 and IFN.gamma. and
analyzed on FACSCanto and FlowJo. Responding T-cells were detected
after intracellular cytokine staining (ICS) for IFN-.gamma. by flow
cytometry. Values from unstimulated PBMCs were considered
background and subtracted from values obtained from stimulated
PBMCs. Data represents mean+/-SEM. In Table 2 it is demonstrated
that the NYESO1 peptides were able to stimulate IFN-.gamma.
production in CD8 cells indicating the existence of an immune
response.
TABLE-US-00002 TABLE 2 IMMUNE RESPONSE to NYESO1 PRIME-BOOST
Percentage of CD8 T Cells Secreting Interferon (IFN) .sup..gamma.
Immune Group Stimulatory Control Group Lentivirus-NYESO1 Prime
Peptide Lentivirus-GFP Prime MG1-NYESO1 Boost Epitope MG1-GFP Boost
(N = 5) RGPESRLL 0 .+-. 0 0.013 .+-. 0.0088 0.027 .+-. 0.015 12.33
(SEQ ID NO: 48) (after prime) (after boost) (after prime) (after
boost)
Example 8: Construction and Immune Testing of Lentiviral Priming
Vectors and Oncolytic Vaccine Vectors Expressing Human
Six-Transmembrane Epithelial Antigen of the Prostate Protein
[0141] The huSTEAP transgene is full-length wild-type sequence (SEQ
ID NO: 11) codon-optimized for expression in human and mouse to
give rise to a 341 amino acid protein (SEQ ID NO: 10).
[0142] Lentiviruses expressing human Six-Transmembrane Epithelial
Antigen of the Prostate protein were made using the pDY.EG.WS
lentivirus vector. The huSTEAP transgene was PCR amplified using
primers containing the EcoRI restriction site (forward primer
ACTGGAATTCATGGAATCACGGAAGGACATC, SEQ ID NO: 22) and the BamHI
restriction site (reverse primer ACTGGGATCCTTAAAGCTTCAGCTGGCTACAG,
SEQ ID NO: 23). The huSTEAP transgene PCR product was agarose gel
purified. The pDY.EG.WS lentivirus vector was cut at the
EcoRI/BamHI site to remove eGFP, was agarose gel purified, and was
subjected to dephosphorylation using CIAP (Invitrogen Catalogue
18009-019). The cut vector was then subjected to additional agarose
gel purification. The huSTEAP transgene PCR product was then
ligated into the EcoRI/BamHI cut vector using T4 DNA ligase
(Invitrogen). The ligation reaction was subjected to a
transformation using competent cells, and plasmid DNA from positive
colonies was subjected to mini-prep amplification. The pDY.EG.WS
lentivirus vector expressing the modified huSTEAP transgene was
then subjected to maxi-prep amplification. The lentivirus
expressing huSTEAP transgene were rescued on 293T cells after
transfection of 6.4 .mu.g of each of three plasmids: the pDY.EG.WS
lentivirus vector expressing the huSTEAP transgene, the packaging
pCMV-8.84 plasmid, and the envelope pMD2G plasmid. Virus
supernatants were pooled, and filtered through a 0.45 .mu.M filter
and centrifuged for 120 minutes at 50,000.times.g at 16.degree. C.
The lentivirus expressing huSTEAP transgene was resuspended in PBS,
and stored at -80.degree. C.
[0143] Maraba MG1 was engineered to contain human Six-Transmembrane
Epithelial Antigen of the Prostate transgene inserted between the G
and L viral genes of the MG1 double mutant of Maraba virus (Brun J.
et al., (2010) Mol Ther 18:1440-1449). The transgene sequence was
codon optimized for expression in mammalian cells. The resulting
Maraba MG1 containing the huSTEAP protein is designated as
"Maraba-MG1-huSTEAP" or "MG1-huSTEAP". A modified Maraba MG1
backbone was used to facilitate cloning. A silent mutation was
introduced into the L gene of the Maraba MG1 genome backbone to
remove one of the Mlul sites. The second Mlul site was replaced
with a BsiWI site at the cloning region between G and L. These
modifications to the Maraba MG1 genome backbone allowed for a more
direct cloning system than that described in the Brun et al. paper
as it avoids using the shuttle plasmid pMRB-MG1/pNF. The huSTEAP
transgene sequence was ligated into the modified Maraba MG1 genome
backbone at its Mlul and BsiWI site (at cloning region between G
and L). The Maraba-MG1-huSTEAP was then rescued (as previously
described in Brun J. et al., (2010) Mol Ther 18:1440-1449), plaque
purified once, and subjected to opti-prep purification. The
Maraba-MG1-huSTEAP has a genomic sequence that is the reverse
complement and RNA version of SEQ ID NO: 12.
[0144] Generally, animals were immunized by administration of the
priming vector (lentivirus-huSTEAP+poly I:C as an adjuvant) at day
0 and by administration of 1e9 PFU of the boosting vector
(Maraba-MG1-huSTEAP) at day 14. Control animals were prime-boosted
with viral vectors encoding GFP instead of the huSTEAP transgene as
a control non-immunogenic transgene insertion. Analysis of the
prime response was conducted at day 14 and day 19. Each
lentivirus-huSTEAP preparation was made with 250 ug poly I:C added
as an adjuvant to the priming virus and then split between 5
animals for each virus. Mice were anesthetized with isoflurane and
30 uL of lentivirus-huSTEAP/poly I:C was injected into each hind
foot pad. The remaining virus was injected subcutaneously near the
left inguinal lymph node. 14 days after prime, blood was collected
and analyzed by flow cytometry. Mice were then boosted with
lx10.sup.9 PFU MG1-huSTEAP intravenously. Five days following the
boost, blood was drawn and immune responses were assessed by flow
cytometry.
[0145] Immune analysis was performed as follows: Blood was
collected via retro-orbital bleeding using heparinzied capillary
tube and blood was collected into heparin. Red blood cells were
then lysed using ACK lysis buffer and the resulting PBMCs were
analyzed for immune responses to the tumor antigens. PBMCs were
either incubated in the absence of peptide or stimulated with
peptides for a total of 5 hours with golgi plug added 1 hour into
the stimulation. PBMCs were either incubated in the absence of
peptide or stimulated with 2 ug/mL peptides (RSRYKLL) (SEQ ID NO:
49) for a total of 5 hours with golgi plug added 1 hour into the
stimulation. Following stimulation the PBMCs were stained for CD4,
CD8 and IFN.gamma. and analyzed on FACSCanto and FlowJo. Responding
T-cells were detected after intracellular cytokine staining (ICS)
for IFN-.gamma. by flow cytometry. Values from unstimulated PBMCs
were considered background and subtracted from values obtained from
stimulated PBMCs. Data 20 represents mean+/-SEM. In Table 3 it is
demonstrated that the huSTEAP peptides were able to stimulate
IFN-.gamma. production in CD8 cells indicating the existence of an
immune response.
TABLE-US-00003 TABLE 3 IMMUNE RESPONSE to huSTEAP PRIME-BOOST
Percentage of CD8 T Cells Secreting Interferon (IFN) .sup..gamma.
Immune Group Stimulatory Control Group Lentivirus-huSTEAP Prime
Peptide Lentivirus-GFP Prime MG1- huSTEAP Boost Epitope MG1-GFP
Boost (N = 5) RSYRYKLL 0.0033 .+-. 0.0033 0.0033 .+-. 0.0033 0.008
.+-. 0.0508 0.0406 .+-. 0.11 (SEQ ID NO: (after prime) (after
boost) (after prime) (after boost) 49)
Example 9: Construction and Immune Testing of Lentiviral Priming
Vectors and Oncolytic Vaccine Vectors Expressing Epstein-Barr
Nuclear Antigen 1
[0146] The EBDNA1 transgene is a partial nucleotide sequence of
full-length wild-type EBDNA1
(www.ncbi.nlm.nih.gov/protein/Q1HVF7.1) with the Glycine-Alanine
generating repetitive sequence deleted (which separates the protein
into amino- and carboxy-terminal domains). This sequence seems to
stabilize the protein, preventing proteasomal breakdown, as well as
impairing antigen processing and MHC class I-restricted antigen
presentation (Levitskaya Jet al., (1995) Nature 375:685-688). The
truncated EBDNA1 nucleotide sequence (SEQ ID NO: 17) was
codon-optimized for expression in human and mouse to give rise to a
238 amino acid protein (SEQ ID NO: 16).
[0147] Lentiviruses expressing Epstein-Barr Nuclear Antigen 1
protein were made using the pDY.EG.WS lentivirus vector. The
modified EBDNA1 transgene was PCR amplified using primers
containing the EcoRI restriction site (forward primer
ACTGGAATTCATGCCAGTCGGCCAGGCTG, SEQ ID NO: 24) and the BamHI
restriction site (reverse primer ACTGGGATCCTTATTCCTGCCCCTCTTCTCC,
SEQ ID NO: 25). The EBDNA1 transgene PCR product was agarose gel
purified. The pDY.EG.WS lentivirus vector was cut at the EcoRI and
BamHI sites to remove eGFP, was agarose gel purified, and was
subjected to dephosphorylation using CIAP (Invitrogen Catalogue
18009-019). The cut vector was then subjected to additional agarose
gel purification. The EBDNA1 transgene PCR product was then ligated
into the EcoRI/BamHI cut vector using T4 DNA ligase (Invitrogen).
The ligation reaction was subjected to a transformation using
competent cells, and plasmid DNA from positive colonies was
subjected to mini-prep amplification. The pDY.EG.WS lentivirus
vector expressing the EBDNA1 transgene was then subjected to
maxi-prep amplification. The lentivirus expressing EBDNA1 transgene
was rescued on 293T cells after transfection of 6.4 .mu.g of each
of three plasmids: the pDY.EG.WS lentivirus vector expressing the
EBDNA1 transgene, the packaging pCMV-8.84 plasmid, and the envelope
pMD2G plasmid. Virus supernatants were pooled, and filtered through
a 0.45 .mu.M filter and centrifuged for 120 minutes at
50,000.times.g at 16.degree. C. The lentivirus expressing EBDNA1
transgene was resuspended in PBS, and stored at -80.degree. C.
[0148] Maraba MG1 was engineered to contain Epstein-Barr Nuclear
Antigen 1 transgene inserted between the G and L viral genes of the
MG1 double mutant of Maraba virus (Brun J. et al., (2010) Mol Ther
18: 1440-1449). The transgene sequence was codon optimized for
expression in mammalian cells. The resulting Maraba MG1 containing
the EBVDNA1 protein is designated as "Maraba-MG1-EBVDNA1" or
"MG1-EDVDNA1". A modified Maraba MG1 backbone was used to
facilitate cloning. A silent mutation was introduced into the L
gene of the Maraba MG1 genome backbone to remove one of the Mlul
sites. The second Mlul site was replaced with a BsiWI site at the
cloning region between G and L. These modifications to the Maraba
MG1 genome backbone allowed for a more direct cloning system than
that described in the Brun et al. paper as it avoids using the
shuttle plasmid pMRB-MG1/pNF. The EBDNA1 transgene sequence was
ligated into the modified Maraba MG1 genome backbone at its Mlul
and BsiWI site (at cloning region between G and L). The
Maraba-MG1-EBDNA1 transgene was then rescued (as previously
described in Brun J. et al., (2010) Mol Ther 18:1440-1449), plaque
purified once, and subjected to opti-prep purification.
[0149] Generally, animals were immunized by administration of the
priming vector (lentivirus-EBDNA1+poly I:C as an adjuvant) at day 0
and by administration of 1e9 PFU of the boosting vector
(Maraba-MG1-EBDNA1) at day 14. Control animals were prime-boosted
with viral vectors encoding GFP instead of the TM transgene as a
control non-immunogenic transgene insertion. Analysis of the prime
response was conducted at day 14 and day 19. Each lentivirus-EBDNA1
preparation was made with 250 ug poly I:C added as an adjuvant to
the priming virus and then split between 5 animals for each virus.
Mice were anesthetized with isoflurane and 30 uL of
lentivirus-EBDNA1/poly I:C was injected into each hind foot pad.
The remaining virus was injected subcutaneously near the left
inguinal lymph node. 14 days after prime, blood was collected and
analyzed by flow cytometry. Mice were then boosted with
1.times.10.sup.9 PFU MG1-EBVDNA1 intravenously. Five days following
the boost, blood was drawn and immune responses were assessed by
flow cytometry.
[0150] Immune analysis was performed as follows: Blood was
collected via retro-orbital bleeding using heparinzied capillary
tube and blood was collected into heparin. Red blood cells were
then lysed using ACK lysis buffer and the resulting PBMCs were
analyzed for immune responses to the tumor antigens. PBMCs were
either incubated in the absence of peptide or stimulated with 2
ug/mL peptides (VYGGSKTSL) (SEQ ID NO: 50) for a total of 5 hours
with golgi 30 plug added 1 hour into the stimulation. Following
stimulation the PBMCs were stained for CD4, CD8 and IFN.gamma. and
analyzed on FACSCanto and FlowJo. Responding T-cells were detected
after intracellular cytokine staining (ICS) for IFN-.gamma. by flow
cytometry. Values from unstimulated PBMCs were considered
background and subtracted from values obtained from stimulated
PBMCs. Data represents mean+/-SEM. The EBVDNA1 peptides were unable
to stimulate IFN-.gamma. production in either CD8 T cells
indicating a lack of an immune response, as shown in Table 4.
TABLE-US-00004 TABLE 4 IMMUNE RESPONSE to EBVDNA1 PRIME-BOOST
Percentage of CD8 T Cells Secreting Interferon (IFN) .sup..gamma.
Immune Group Stimulatory Control Group Lentivirus- EBVDNA1Prime
Peptide Lentivirus-GFP Prime MG1- EBVDNA1 Boost Epitope MG1-GFP
Boost (N = 5) VYGGSKTSL 0.055 .+-. 0.015 0.01 .+-. 0.0058 0.008
.+-. 0.0049 0.09 .+-. 0.05 (SEQ ID NO: 50) (after prime) (after
boost) (after prime) (after boost)
Example 10: Effect of Cyclophosphamide on Adenovirus-OV Vaccine
Prime-Boost Strategy
[0151] Cyclophosphamide (CPA) is a chemotherapeutic agent used to
treat various types of cancer. High doses of this drug are required
for effective chemotherapy. High doses of CPA are thought to lead
to immunosuppression while low doses of the drug can lead to
enhanced immune responses against a variety of antigens.
Surprisingly, in the heterologous prime-boost strategy of the
current disclosure, CPA only results in an increase in immune
response when administered prior to the priming of the immune
system by the first virus.
[0152] In order to generate lung metastases, C57BI/6 mice (8-10
weeks old at study initiation) were injected with
2.5.times.10.sup.5 B16-F10 cells (murine melanoma cells expressing
the murine DCT antigen) in 200 .mu.l saline water i.v. at day 0.
Five days following B16-F10 engraftment, mice received an Ad-hDCT
priming vaccine (2.times.10.sup.8 pfu in 200 .mu.l PBS i.v.) and
this was followed 14 days later by a single i.v. dose of VSV-hDCT
(2.times.10.sup.9 pfu in 200 .mu.l PBS i.v.) as an oncolytic
booster vaccine. Additionally, mice either received vehicle or CPA
(1 mg/20 g mouse, i.p.) at day (-1) prior to the prime and/or day
13 prior to the boost. In FIG. 9 it can be seen that CPA given
prior to the priming vector significantly increases survival while
CPA administered prior to the boosting vector does not extend
survival (data not shown).
[0153] In the preceding description, for purposes of explanation,
numerous details are set forth in order to provide a thorough
understanding of the examples. The above-described examples are
intended to be exemplary only. Alterations, modifications and
variations can be effected to the particular examples by those of
skill in the art without departing from the scope, which is defined
solely by the claims appended hereto.
TABLE-US-00005 APPENDIX A Protein and Nucleotide Sequences Protein
sequence of full length, wild type, human MAGEA3 (SEQ ID NO: 1):
MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESP
DPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHF
LLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFA
TCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILG
DPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPH
ISYPPLHEWVLREGEE* DNA sequence encoding full length, wild type,
human MAGEA3 (SEQ ID NO: 2):
ATGCCTCTTGAGCAGAGGAGTCAGCACTGCAAGCCTGAAGAAGGCCTTGAGGCCCG
AGGAGAGGCCCTGGGCCTGGTGGGTGCGCAGGCTCCTGCTACTGAGGAGCAGGAGG
CTGCCTCCTCCTCTTCTACTCTAGTTGAAGTCACCCTGGGGGAGGTGCCTGCTGCCG
AGTCACCAGATCCTCCCCAGAGTCCTCAGGGAGCCTCCAGCCTCCCCACTACCATGA
ACTACCCTCTCTGGAGCCAATCCTATGAGGACTCCAGCAACCAAGAAGAGGAGGGG
CCAAGCACCTTCCCTGACCTGGAGTCCGAGTTCCAAGCAGCACTCAGTAGGAAGGT
GGCCGAGTTGGTTCATTTTCTGCTCCTCAAGTATCGAGCCAGGGAGCCGGTCACAAA
GGCAGAAATGCTGGGGAGTGTCGTCGGAAATTGGCAGTATTTCTTTCCTGTGATCTT
CAGCAAAGCTTCCAGTTCCTTGCAGCTGGTCTTTGGCATCGAGCTGATGGAAGTGGA
CCCCATCGGCCACTTGTACATCTTTGCCACCTGCCTGGGCCTCTCCTACGATGGCCTG
CTGGGTGACAATCAGATCATGCCCAAGGCAGGCCTCCTGATAATCGTCCTGGCCATA
ATCGCAAGAGAGGGCGACTGTGCCCCTGAGGAGAAAATCTGGGAGGAGCTGAGTGT
GTTAGAGGTGTTTGAGGGGAGGGAAGACAGTATCTTGGGGGATCCCAAGAAGCTGC
TCACCCAACATTTCGTGCAGGAAAACTACCTGGAGTACCGGCAGGTCCCCGGCAGT
GATCCTGCATGTTATGAATTCCTGTGGGGTCCAAGGGCCCTCGTTGAAACCAGCTAT
GTGAAAGTCCTGCACCATATGGTAAAGATCAGTGGAGGACCTCACATTTCCTACCCA
CCCCTGCATGAGTGGGTTTTGAGAGAGGGGGAAGAGTGA Codon optimized DNA
sequence encoding full length, wild type, human MAGEA3 protein (SEQ
ID NO: 3): ATGCCCCTGGAGCAGCGGTCTCAGCATTGCAAGCCAGAGGAGGGCCTCGAGGCGAG
GGGCGAGGCCCTCGGCTTGGTGGGGGCGCAGGCTCCTGCAACCGAGGAGCAAGAGG
CCGCATCCAGTTCCTCTACCCTGGTTGAGGTGACCTTGGGTGAGGTGCCCGCCGCGG
AGAGCCCCGACCCGCCTCAAAGCCCCCAGGGTGCCAGCTCCCTGCCCACAACAATG
AACTACCCACTCTGGAGTCAGTCTTACGAGGACAGTAGTAACCAAGAGGAGGAGGG
ACCCTCCACATTCCCAGACCTGGAGTCTGAATTCCAGGCAGCATTGTCTAGAAAAGT
GGCCGAATTGGTGCACTTCCTGCTGCTGAAGTATCGCGCCCGCGAGCCAGTCACAAA
AGCTGAAATGCTGGGTTCTGTCGTGGGAAATTGGCAGTACTTCTTCCCCGTGATCTT
CAGTAAAGCGTCCAGCTCCTTGCAGCTGGTCTTTGGTATCGAGCTGATGGAGGTGGA
TCCCATCGGCCATCTGTATATCTTTGCCACATGCCTGGGCCTGAGCTACGATGGCCT
GCTGGGCGACAACCAGATCATGCCAAAAGCTGGCCTGCTGATCATCGTTCTGGCTAT
CATCGCTAGAGAAGGAGATTGCGCCCCTGAAGAAAAGATCTGGGAGGAACTGAGCG
TCCTGGAAGTCTTTGAGGGTCGTGAAGACAGCATTCTCGGGGATCCCAAGAAGCTGC
TGACCCAGCACTTCGTGCAGGAGAACTATCTGGAGTACCGCCAGGTTCCCGGCAGC
GACCCCGCTTGCTACGAGTTCCTGTGGGGCCCCAGGGCCCTGGTCGAGACATCCTAC
GTGAAGGTCCTGCACCATATGGTTAAAATCAGCGGCGGCCCCCATATCTCTTATCCG
CCGCTCCACGAGTGGGTGCTCCGGGAGGGAGAGGAG Protein sequence of a variant
of full length, wild type, human MAGEA3 (SEQ ID NO: 4):
MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESP
DPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHF
LLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIFA
TCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILG
DPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPH
ISYPPLHEWVLREGEEDYKDDDDK* DNA sequence encoding a variant of full
length, wild type, human MAGEA3 (SEQ ID NO: 5):
ATGCCCCTGGAACAGCGGAGCCAGCACTGCAAGCCCGAGGAAGGCCTGGAAGCCA
GAGGCGAAGCCCTGGGACTGGTGGGAGCCCAGGCCCCTGCCACAGAAGAACAGGA
AGCCGCCAGCAGCAGCTCCACCCTGGTGGAAGTGACCCTGGGCGAAGTGCCTGCCG
CCGAGAGCCCTGATCCCCCTCAGTCTCCTCAGGGCGCCAGCAGCCTGCCCACCACCA
TGAACTACCCCCTGTGGTCCCAGAGCTACGAGGACAGCAGCAACCAGGAAGAGGAA
GGCCCCAGCACCTTCCCCGACCTGGAAAGCGAGTTCCAGGCCGCCCTGAGCCGGAA
GGTGGCAGAGCTGGTGCACTTCCTGCTGCTGAAGTACAGAGCCCGCGAGCCCGTGA
CCAAGGCCGAGATGCTGGGCAGCGTGGTGGGAAACTGGCAGTACTTCTTCCCCGTG
ATCTTCTCCAAGGCCAGCAGCTCCCTGCAGCTGGTGTTCGGCATCGAGCTGATGGAA
GTGGACCCCATCGGCCACCTGTACATCTTCGCCACCTGTCTGGGCCTGAGCTACGAC
GGCCTGCTGGGCGACAACCAGATCATGCCCAAGGCCGGCCTGCTGATCATCGTGCT
GGCCATCATTGCCCGCGAGGGCGACTGCGCCCCTGAGGAAAAGATCTGGGAGGAAC
TGAGCGTGCTGGAAGTGTTCGAGGGCAGAGAGGACAGCATCCTGGGCGACCCCAAG
AAGCTGCTGACCCAGCACTTCGTGCAGGAAAACTACCTGGAATACCGCCAGGTGCC
CGGCAGCGACCCCGCCTGTTACGAGTTCCTGTGGGGCCCCAGGGCTCTGGTGGAAA
CCAGCTACGTGAAGGTGCTGCACCACATGGTGAAAATCAGCGGCGGACCCCACATC
AGCTACCCCCCACTGCACGAGTGGGTGCTGAGAGAGGGCGAAGAGGACTACAAGG
ACGACGACGACAAATGA Protein sequence of HPV E6/E7 fusion protein (SEQ
ID NO: 7):
MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIV
YRDGNPYAVDKLKFYSKISEYRHYCYSVYGTTLEQQYNKPLCDLLIRINQKPLCPEEKQ
RHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLGGGGGAAYMARFEDPTRRPYKLP
DLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAAHKIDFYSRIRELR
HYSDSVYGDTLEKLTNTGLYNLLIRLRQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHS
CCNRARQERLQRRRETQVGGGGGAAYMHGDTPTLHEYMLDLQPETTDLYQLNDSSEE
EDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVPICSQ
KPGGGGGAAYMHGPKATLQDIVLHLEPQNEIPVDLLQLSDSEEENDEIDGVNHQHLPAR
RAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVPWCASQQ* DNA sequence
of HPV E6/E7 fusion protein (SEQ ID NO: 8):
ATGCATCAGAAGCGAACTGCTATGTTTCAGGACCCTCAGGAGCGGCCACGCAAACT
GCCTCAGCTGTGCACCGAACTGCAGACAACTATCCACGACATCATTCTGGAATGCGT
GTACTGTAAGCAGCAGCTGCTGAGGAGAGAGGTCTATGACTTCGCTTTTCGCGATCT
GTGCATCGTGTACCGAGACGGAAACCCATATGCAGTCGATAAGCTGAAGTTCTACA
GCAAGATCTCCGAATACAGGCATTACTGTTACAGCGTGTACGGGACCACACTGGAG
CAGCAGTATAACAAGCCCCTGTGCGACCTGCTGATCAGAATTAATCAGAAGCCCCT
GTGCCCTGAGGAAAAACAGAGGCACCTGGATAAGAAACAGAGATTTCATAACATCC
GAGGACGATGGACCGGGCGGTGCATGTCCTGCTGTAGAAGCTCCCGGACTCGACGA
GAGACCCAGCTGGGCGGAGGAGGAGGAGCAGCTTACATGGCACGATTCGAGGACC
CTACCCGAAGGCCATATAAGCTGCCCGACCTGTGCACAGAACTGAATACTTCTCTGC
AGGACATCGAGATTACATGCGTGTACTGTAAAACCGTCCTGGAGCTGACAGAAGTG
TTCGAGTTTGCTTTCAAGGACCTGTTTGTGGTCTACCGGGATTCAATCCCTCACGCAG
CCCATAAAATCGACTTCTACAGCAGGATCAGGGAACTGCGCCACTACTCCGACAGC
GTGTACGGGGATACACTGGAGAAGCTGACAAACACTGGCCTGTACAATCTGCTGAT
CCGACTGCGACAGAAGCCACTGAACCCAGCCGAAAAACTGAGACACCTGAACGAG
AAGAGACGGTTTCACAATATTGCAGGCCATTATAGGGGACAGTGCCATAGTTGCTGT
AATCGAGCCAGGCAGGAAAGACTGCAGCGCCGAAGGGAGACTCAAGTCGGCGGAG
GAGGAGGAGCTGCATACATGCACGGCGACACCCCCACACTGCATGAATATATGCTG
GATCTGCAGCCTGAGACTACCGACCTGTACCAGCTGAACGATTCTAGTGAGGAAGA
GGACGAAATCGACGGACCAGCAGGACAGGCAGAGCCTGACCGGGCCCACTATAAT
ATTGTGACATTCTGCTGTAAGTGCGATTCTACTCTGCGGCTGTGCGTGCAGAGTACT
CATGTCGACATCCGCACCCTGGAGGATCTGCTGATGGGGACTCTGGGCATCGTCCCA
ATTTGTAGCCAGAAACCAGGCGGCGGCGGCGGAGCAGCTTACATGCACGGACCCAA
GGCTACCCTGCAGGACATCGTGCTGCATCTGGAACCTCAGAATGAGATTCCAGTCGA
CCTGCTGCAGCTGAGTGATTCAGAAGAGGAAAACGACGAGATCGACGGCGTGAATC
ACCAGCATCTGCCTGCTAGACGGGCAGAGCCACAGCGACACACAATGCTGTGCATG
TGCTGTAAGTGTGAAGCCAGGATCAAGCTGGTGGTCGAGTCAAGCGCCGACGATCT
GCGCGCCTTCCAGCAGCTGTTCCTGAATACTCTGTCATTTGTCCCTTGGTGTGCCTCC
CAGCAGTGA Protein sequence of huSTEAP protein (SEQ ID NO: 10):
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCP
SELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMV
SITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLS
YPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIP
SVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIV
VLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQLKL* DNA sequence of huSTEAP
protein (SEQ ID NO: 11):
ATGGAATCACGGAAGGACATCACTAATCAGGAGGAACTGTGGAAAATGAAGCCAA
GAAGGAATCTGGAAGAGGACGACTATCTGCACAAGGACACCGGCGAAACAAGTAT
GCTGAAACGACCAGTGCTGCTGCACCTGCATCAGACTGCTCACGCAGACGAGTTTG
ATTGCCCCTCTGAACTGCAGCACACCCAGGAGCTGTTCCCACAGTGGCATCTGCCCA
TCAAGATTGCCGCTATCATTGCTTCACTGACATTTCTGTACACTCTGCTGAGAGAAGT
GATCCACCCCCTGGCCACCAGCCATCAGCAGTACTTCTATAAGATCCCTATCCTGGT
CATCAACAAGGTCCTGCCAATGGTGAGCATCACACTGCTGGCCCTGGTCTACCTGCC
TGGAGTGATCGCAGCCATTGTCCAGCTGCACAATGGGACAAAGTATAAGAAATTTC
CACATTGGCTGGATAAGTGGATGCTGACTAGGAAACAGTTCGGACTGCTGTCCTTCT
TTTTCGCCGTGCTGCACGCTATCTACAGCCTGTCCTATCCCATGAGGAGGAGCTACC
GGTATAAGCTGCTGAACTGGGCTTACCAGCAGGTGCAGCAGAACAAGGAGGACGCA
TGGATTGAACATGACGTGTGGCGCATGGAAATCTACGTGAGCCTGGGCATTGTCGG
ACTGGCCATCCTGGCTCTGCTGGCAGTGACCAGTATCCCTTCTGTCAGTGACTCACT
GACATGGAGAGAGTTTCACTACATTCAGAGCAAGCTGGGGATCGTGTCCCTGCTGCT
GGGCACCATCCATGCACTGATTTTTGCCTGGAACAAGTGGATCGATATCAAGCAGTT
CGTGTGGTATACTCCCCCTACCTTTATGATTGCCGTCTTCCTGCCCATCGTGGTCCTG
ATCTTCAAGTCCATCCTGTTCCTGCCTTGTCTGCGGAAGAAAATCCTGAAAATTCGG
CACGGATGGGAGGATGTCACCAAAATCAATAAGACTGAAATCTGTAGCCAGCTGAA GCTTTAA
Protein sequence of NYESO1 MAR protein (SEQ ID NO: 13):
MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPG
GGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPP
LPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPS GQRR*
DNA sequence of NYESO1 MAR (SEQ ID NO: 14):
ATGCAGGCCGAGGGCAGAGGCACAGGCGGATCTACAGGCGACGCCGATGGCCCTG
GCGGCCCTGGAATTCCTGACGGACCTGGCGGCAATGCCGGCGGACCCGGAGAAGCT
GGCGCCACAGGCGGAAGAGGACCTAGAGGCGCTGGCGCCGCTAGAGCTTCTGGACC
AGGCGGAGGCGCCCCTAGAGGACCTCATGGCGGAGCCGCCTCCGGCCTGAACGGCT
GTTGCAGATGTGGAGCCAGAGGCCCCGAGAGCCGGCTGCTGGAATTCTACCTGGCC
ATGCCCTTCGCCACCCCCATGGAAGCCGAGCTGGCCAGACGGTCCCTGGCCCAGGA
TGCTCCTCCTCTGCCTGTGCCCGGCGTGCTGCTGAAAGAATTCACCGTGTCCGGCAA
CATCCTGACCATCCGGCTGACTGCCGCCGACCACAGACAGCTCCAGCTGTCTATCAG
CTCCTGCCTGCAGCAGCTGAGCCTGCTGATGTGGATCACCCAGTGCTTTCTGCCCGT
GTTCCTGGCTCAGCCCCCCAGCGGCCAGAGAAGATGA Protein sequence of EBDNA1
(SEQ ID NO: 16):
MPVGQADYFEYHQEGGPDGEPDMPPGAIEQGPADDPGEGPSTGPRGQGDGGRRKKGG
WFGKHRGQGGSNQKFENIADGLRTLLARCHVERTTDEGTWVAGVFVYGGSKTSLYNL
RRGISLAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFIVFLQTHIFAEGLKDAIKDL
VMPKPAPTCNIKATVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDDGDEGGDGDEG EEGQE*
DNA sequence of EBDNA1 (SEQ ID NO: 17):
ATGCCAGTCGGCCAGGCTGATTACTTTGAATACCACCAGGAGGGGGGACCAGACGG
AGAACCAGACATGCCACCAGGAGCCATTGAACAGGGACCAGCAGACGATCCTGGA
GAGGGACCATCAACTGGACCCCGAGGACAGGGGGACGGCGGAAGGAGAAAGAAAG
GGGGATGGTTCGGAAAGCACCGAGGACAGGGAGGGAGCAACCAGAAATTTGAAAA
TATCGCTGACGGCCTGCGAACACTGCTGGCAAGGTGCCATGTGGAGAGAACCACAG
ATGAAGGCACATGGGTCGCCGGAGTGTTCGTCTACGGCGGAAGCAAGACTTCCCTG
TATAACCTGCGGCGCGGCATCTCTCTGGCCATTCCACAGTGCCGGCTGACCCCTCTG
AGTCGCCTGCCATTCGGGATGGCTCCTGGACCAGGACCACAGCCTGGACCACTGAG
GGAGTCCATCGTGTGCTACTTCATTGTCTTTCTGCAGACACACATCTTTGCCGAAGGC
CTGAAGGACGCCATCAAGGACCTGGTCATGCCCAAGCCTGCACCAACTTGCAATAT
CAAGGCCACCGTGTGCAGTTTCGACGATGGCGTGGACCTGCCCCCTTGGTTTCCACC
TATGGTGGAGGGAGCCGCTGCAGAAGGGGACGATGGCGATGACGGGGACGATGGG
GATGAAGGCGGGGACGGCGATGAGGGAGAAGAGGGGCAGGAATAA
Sequence CWU 1
1
501314PRTHomo sapiens 1Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys
Pro Glu Glu Gly Leu1 5 10 15Glu Ala Arg Gly Glu Ala Leu Gly Leu Val
Gly Ala Gln Ala Pro Ala 20 25 30Thr Glu Glu Gln Glu Ala Ala Ser Ser
Ser Ser Thr Leu Val Glu Val 35 40 45Thr Leu Gly Glu Val Pro Ala Ala
Glu Ser Pro Asp Pro Pro Gln Ser 50 55 60Pro Gln Gly Ala Ser Ser Leu
Pro Thr Thr Met Asn Tyr Pro Leu Trp65 70 75 80Ser Gln Ser Tyr Glu
Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser 85 90 95Thr Phe Pro Asp
Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys 100 105 110Val Ala
Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu 115 120
125Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln
130 135 140Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu
Gln Leu145 150 155 160Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro
Ile Gly His Leu Tyr 165 170 175Ile Phe Ala Thr Cys Leu Gly Leu Ser
Tyr Asp Gly Leu Leu Gly Asp 180 185 190Asn Gln Ile Met Pro Lys Ala
Gly Leu Leu Ile Ile Val Leu Ala Ile 195 200 205Ile Ala Arg Glu Gly
Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu 210 215 220Leu Ser Val
Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly225 230 235
240Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu
245 250 255Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu
Phe Leu 260 265 270Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val
Lys Val Leu His 275 280 285His Met Val Lys Ile Ser Gly Gly Pro His
Ile Ser Tyr Pro Pro Leu 290 295 300His Glu Trp Val Leu Arg Glu Gly
Glu Glu305 3102945DNAHomo sapiens 2atgcctcttg agcagaggag tcagcactgc
aagcctgaag aaggccttga ggcccgagga 60gaggccctgg gcctggtggg tgcgcaggct
cctgctactg aggagcagga ggctgcctcc 120tcctcttcta ctctagttga
agtcaccctg ggggaggtgc ctgctgccga gtcaccagat 180cctccccaga
gtcctcaggg agcctccagc ctccccacta ccatgaacta ccctctctgg
240agccaatcct atgaggactc cagcaaccaa gaagaggagg ggccaagcac
cttccctgac 300ctggagtccg agttccaagc agcactcagt aggaaggtgg
ccgagttggt tcattttctg 360ctcctcaagt atcgagccag ggagccggtc
acaaaggcag aaatgctggg gagtgtcgtc 420ggaaattggc agtatttctt
tcctgtgatc ttcagcaaag cttccagttc cttgcagctg 480gtctttggca
tcgagctgat ggaagtggac cccatcggcc acttgtacat ctttgccacc
540tgcctgggcc tctcctacga tggcctgctg ggtgacaatc agatcatgcc
caaggcaggc 600ctcctgataa tcgtcctggc cataatcgca agagagggcg
actgtgcccc tgaggagaaa 660atctgggagg agctgagtgt gttagaggtg
tttgagggga gggaagacag tatcttgggg 720gatcccaaga agctgctcac
ccaacatttc gtgcaggaaa actacctgga gtaccggcag 780gtccccggca
gtgatcctgc atgttatgaa ttcctgtggg gtccaagggc cctcgttgaa
840accagctatg tgaaagtcct gcaccatatg gtaaagatca gtggaggacc
tcacatttcc 900tacccacccc tgcatgagtg ggttttgaga gagggggaag agtga
9453942DNAArtificial Sequencecodon optimized nucleotide sequence
encoding full length, wild type, human MAGEA3 protein 3atgcccctgg
agcagcggtc tcagcattgc aagccagagg agggcctcga ggcgaggggc 60gaggccctcg
gcttggtggg ggcgcaggct cctgcaaccg aggagcaaga ggccgcatcc
120agttcctcta ccctggttga ggtgaccttg ggtgaggtgc ccgccgcgga
gagccccgac 180ccgcctcaaa gcccccaggg tgccagctcc ctgcccacaa
caatgaacta cccactctgg 240agtcagtctt acgaggacag tagtaaccaa
gaggaggagg gaccctccac attcccagac 300ctggagtctg aattccaggc
agcattgtct agaaaagtgg ccgaattggt gcacttcctg 360ctgctgaagt
atcgcgcccg cgagccagtc acaaaagctg aaatgctggg ttctgtcgtg
420ggaaattggc agtacttctt ccccgtgatc ttcagtaaag cgtccagctc
cttgcagctg 480gtctttggta tcgagctgat ggaggtggat cccatcggcc
atctgtatat ctttgccaca 540tgcctgggcc tgagctacga tggcctgctg
ggcgacaacc agatcatgcc aaaagctggc 600ctgctgatca tcgttctggc
tatcatcgct agagaaggag attgcgcccc tgaagaaaag 660atctgggagg
aactgagcgt cctggaagtc tttgagggtc gtgaagacag cattctcggg
720gatcccaaga agctgctgac ccagcacttc gtgcaggaga actatctgga
gtaccgccag 780gttcccggca gcgaccccgc ttgctacgag ttcctgtggg
gccccagggc cctggtcgag 840acatcctacg tgaaggtcct gcaccatatg
gttaaaatca gcggcggccc ccatatctct 900tatccgccgc tccacgagtg
ggtgctccgg gagggagagg ag 9424322PRTArtificial Sequencevariant of
MAGEA3 protein derived from homo sapiens 4Met Pro Leu Glu Gln Arg
Ser Gln His Cys Lys Pro Glu Glu Gly Leu1 5 10 15Glu Ala Arg Gly Glu
Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala 20 25 30Thr Glu Glu Gln
Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val 35 40 45Thr Leu Gly
Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser 50 55 60Pro Gln
Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp65 70 75
80Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser
85 90 95Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg
Lys 100 105 110Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg
Ala Arg Glu 115 120 125Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val
Val Gly Asn Trp Gln 130 135 140Tyr Phe Phe Pro Val Ile Phe Ser Lys
Ala Ser Ser Ser Leu Gln Leu145 150 155 160Val Phe Gly Ile Glu Leu
Met Glu Val Asp Pro Ile Gly His Leu Tyr 165 170 175Ile Phe Ala Thr
Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp 180 185 190Asn Gln
Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile 195 200
205Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu
210 215 220Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile
Leu Gly225 230 235 240Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val
Gln Glu Asn Tyr Leu 245 250 255Glu Tyr Arg Gln Val Pro Gly Ser Asp
Pro Ala Cys Tyr Glu Phe Leu 260 265 270Trp Gly Pro Arg Ala Leu Val
Glu Thr Ser Tyr Val Lys Val Leu His 275 280 285His Met Val Lys Ile
Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu 290 295 300His Glu Trp
Val Leu Arg Glu Gly Glu Glu Asp Tyr Lys Asp Asp Asp305 310 315
320Asp Lys5969DNAArtificial Sequencenucleotide sequence encoding
variant of MAGEA3 protein derived from homo sapiens 5atgcccctgg
aacagcggag ccagcactgc aagcccgagg aaggcctgga agccagaggc 60gaagccctgg
gactggtggg agcccaggcc cctgccacag aagaacagga agccgccagc
120agcagctcca ccctggtgga agtgaccctg ggcgaagtgc ctgccgccga
gagccctgat 180ccccctcagt ctcctcaggg cgccagcagc ctgcccacca
ccatgaacta ccccctgtgg 240tcccagagct acgaggacag cagcaaccag
gaagaggaag gccccagcac cttccccgac 300ctggaaagcg agttccaggc
cgccctgagc cggaaggtgg cagagctggt gcacttcctg 360ctgctgaagt
acagagcccg cgagcccgtg accaaggccg agatgctggg cagcgtggtg
420ggaaactggc agtacttctt ccccgtgatc ttctccaagg ccagcagctc
cctgcagctg 480gtgttcggca tcgagctgat ggaagtggac cccatcggcc
acctgtacat cttcgccacc 540tgtctgggcc tgagctacga cggcctgctg
ggcgacaacc agatcatgcc caaggccggc 600ctgctgatca tcgtgctggc
catcattgcc cgcgagggcg actgcgcccc tgaggaaaag 660atctgggagg
aactgagcgt gctggaagtg ttcgagggca gagaggacag catcctgggc
720gaccccaaga agctgctgac ccagcacttc gtgcaggaaa actacctgga
ataccgccag 780gtgcccggca gcgaccccgc ctgttacgag ttcctgtggg
gccccagggc tctggtggaa 840accagctacg tgaaggtgct gcaccacatg
gtgaaaatca gcggcggacc ccacatcagc 900taccccccac tgcacgagtg
ggtgctgaga gagggcgaag aggactacaa ggacgacgac 960gacaaatga
969614297DNAArtificial Sequencenucleotide encoding an artificial
Maraba virus that expresses a MAGEA3 protein 6aagcttgata tcattcagga
cgagcctcag actccagcgt aactggactg caatcaactc 60actggctcac cttcacgggt
gggcctttct tcggtagaaa atcaaaggat cttcttgaga 120tccttttttt
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
180ggtttgtttg ccggatcaag agctaccaac tctttttccg aggtaactgg
cttcagcaga 240gcgcagatac caaatactgt tcttctagtg tagccgtagt
taggccacca cttcaagaac 300tctgtagcac cgcctacata cctcgctctg
ctaatcctgt taccagtggc tgctgccagt 360ggcgataagt cgtgtcttac
cgggttggac tcaagacgat agttaccgga taaggcgcag 420cggtcgggct
gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
480gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
agggagaaag 540gcggacaggt atccggtaag cggcagggtc ggaacaggag
agcgcacgag ggagcttcca 600gggggaaacg cctggtatct ttatagtcct
gtcgggtttc gccacctctg acttgagcat 660cgatttttgt gatgctcgtc
aggggggcgg agcctatgga aaaacgccag caacgcagaa 720aggcccaccc
gaaggtgagc caggtgatta catttgggcc ctcatcagag gttttcaccg
780tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
gtgaagcgat 840tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
gtttctccag aagcgttaat 900gtctggcttc tgataaagcg ggccatgtta
agggcggttt tttcctgttt ggtcatttag 960aaaaactcat cgagcatcaa
gtgaaactgc aatttattca tatcaggatt atcaatacca 1020tatttttgaa
aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
1080atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
acaacctatt 1140aatttcccct cgtcaaaaat aaggttatca agtgagaaat
caccatgagt gacgactgaa 1200tccggtgaga atggcaaaag cttatgcatt
tctttccaga cttgttcaac aggccagcca 1260ttacgctcgt catcaaaatc
actcgcacca accaaaccgt tattcattcg tgattgcgcc 1320tgagcgagac
gaaatacgcg atcgccgtta aaaggacaat tacaaacagg aatcgaatgc
1380aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
aggatattct 1440tctaatacct ggaatgctgt tttccctggg atcgcagtgg
tgagtaacca tgcatcatca 1500ggagtacgga taaaatgctt gatggtcgga
agaggcataa attccgtcag ccagtttagc 1560ctgaccatct catctgtaac
atcattggca acgctacctt tgccatgttt cagaaacaac 1620tctggcgcat
cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
1680tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
tcgcggcctc 1740gagcaagacg tttcccgttg aatatggctc attttagctt
ccttagctcc tgaaaatctc 1800gataactcaa aaaatacgcc cggtagtgat
cttatttcat tatggtgaaa gttggaacct 1860cttacgtgcc gatcaagtca
aaagcctccg gtcggaggct tttgactttc tgctatggag 1920gtcaggtatg
atttaaatgg tcagtattga gcgatatcta gagaattcgt ctaatacgac
1980tcactatagg gacgaagaca aacaaaccat tgatagaatt aagaggctca
tgaaaatcct 2040taacagcgtt caaaatgtct gttacagtca agagagtcat
tgatgattca ctcatcaccc 2100ccaaattgcc tgcgaatgag gaccctgtgg
agtaccctgc tgattatttc aaaaagtccc 2160gtgatattcc ggtgtacata
aacacgacca aaagtttgtc tgatttgcgg ggctatgttt 2220atcaaggcct
aaagtcaggc aacatctcta taattcatgt caacagttat ctgtatgcag
2280cattaaaaga gatcagagga aaattggaca gagattggat cacctttggt
atccaaatcg 2340gaaaaacagg agatagcgtg gggatattcg atttactgac
cctaaaacct ctagatggtg 2400ttttaccaga tggggtgtct gatgctactc
gaactagctc agacgatgca tggcttccac 2460tgtatctatt ggggttatac
agagttggtc gaacacagat gccagaatac aggaagaagc 2520tgatggatgg
tctgattaat caatgtaaga tgatcaatga gcagtttgaa ccactgttgc
2580cagaaggaag agatgtcttt gatgtctggg gaaatgacag caattacaca
aagattgtgg 2640ccgctgtaga tatgttcttc catatgttca aaaagcatga
gaaggcctct ttcaggtatg 2700gcacaatagt gtcaagattt aaggattgtg
cagcattggc tacatttggt catctgtgta 2760agatcactgg tatgtccact
gaagatgtga caacttggat tctaaacagg gaggtggctg 2820atgagatggt
tcaaatgatg tacccaggac aggagataga taaggctgat tcttacatgc
2880cttatctaat cgacttaggt ctgtcctcaa aatctccata ttcatcagtt
aaaaatccag 2940ctttccattt ttggggtcaa ttgaccgcat tgttactgag
atcaaccaga gccagaaatg 3000cacgtcagcc ggatgacatc gagtatacat
ccctgaccac tgctgggctg ttgtatgcat 3060atgccgttgg ttcgtctgca
gacctggctc aacaattcta cgttggggac aacaagtatg 3120tgccagaaac
tggagatgga ggattaacca ccaatgcacc gccacaaggg cgagatgtgg
3180tcgagtggct tagttggttt gaagatcaaa acagaaaacc taccccagac
atgctcatgt 3240atgctaagag agctgtcagt gctttacaag gattgaggga
gaagacgatt ggcaagtacg 3300ccaagtcaga gtttgacaaa tgacaactca
ctcaccatat gtattactac ctttgcttca 3360tatgaaaaaa actaacagcg
atcatggatc agctatcaaa ggtcaaggaa ttccttaaga 3420cttacgcgca
gttggatcaa gcagtacaag agatggatga cattgagtct cagagagagg
3480aaaagactaa ttttgatttg tttcaggaag aaggattgga gattaaggag
aagccttcct 3540attatcgggc agatgaagaa gagattgatt cagatgaaga
cagcgtggat gatgcacaag 3600acttagggat acgtacatca acaagtccca
tcgaggggta tgtggatgag gagcaggatg 3660attatgagga tgaggaagtg
aacgtggtgt ttacatcgga ctggaaacag cctgagctgg 3720aatccgacgg
ggatgggaaa actctccgat tgacgatacc agatggattg actggggagc
3780agaagtcgca atggcttgcc acgattaagg cagttgttca gagtgctaaa
tattggaaca 3840tctcagaatg ttcatttgag agttatgagc aaggggtttt
gattagagag agacaaatga 3900ctcctgatgt ctacaaagtc actcctgttt
taaatgctcc accggttcaa atgacagcta 3960atcaagatgt ttggtctctc
agcagcactc catttacatt tttgcccaag aaacaaggtg 4020tgactccatt
gaccatgtcc ttagaagaac tcttcaacac ccgaggtgaa ttcatatctc
4080tgggaggaaa cgggaaaatg agtcaccggg aggccatcat tctagggttg
agacacaaga 4140agctctataa tcaagccaga ctaaagtata acttagcttg
aatatgaaaa aaactaacag 4200atatcaaaag atatctctaa ctcagtccat
tgtgttcagt tcaatcatga gctctctcaa 4260gaaaattttg ggtattaaag
ggaaagggaa gaaatctaag aaattaggta tggctccccc 4320accctatgaa
gaagagactc caatggaata ttctccaagt gcaccttatg ataagtcatt
4380gtttggagtc gaagatatgg atttccatga tcaacgtcaa ctccgatatg
agaaatttca 4440cttctcattg aagatgactg tgagatcaaa caaaccattt
cgaaattatg atgacgttgc 4500agcagcggtg tccaattggg atcatatgta
catcggcatg gcaggaaaac gtccttttta 4560taagatatta gcattcatgg
gttctactct attgaaggct acaccagccg tctgggctga 4620ccaaggacag
ccagaatatc atgctcactg tgagggacga gcttacttgc cgcatcggtt
4680agggccgacc cctccgatgt tgaatgtccc tgaacatttt cgccgtccat
ttaacatcgg 4740attattcaga gggacaatcg acataaccct ggtacttttc
gatgatgaat ctgtagattc 4800tgccccggtc atatgggatc attttaatgc
atccagattg agcagcttca gagaaaaggc 4860tttgttgttt ggtttgattc
tagaaaagaa agccactggg aattgggtat tggactctat 4920tagtcatttc
aagtaattat cacaagtgtt gaggtgatgg gcagactatg aaaaaaacta
4980acagggttca aacactcttg atcgaggtac ccagttatat ttgttacaac
aatgttgaga 5040ctttttctct tttgtttctt ggccttagga gcccactcca
aatttactat agtattccct 5100catcatcaaa aagggaattg gaagaatgtg
ccttccacat atcattattg cccttctagt 5160tctgaccaga attggcataa
tgatttgact ggagttagtc ttcatgtgaa aattcccaaa 5220agtcacaaag
ctatacaagc agatggctgg atgtgccacg ctgctaaatg ggtgactact
5280tgtgacttca gatggtacgg acccaaatac atcacgcatt ccatacactc
tatgtcaccc 5340accctagaac agtgcaagac cagtattgag cagacaaagc
aaggagtttg gattaatcca 5400ggctttcccc ctcaaagctg cggatatgct
acagtgacgg atgcagaggt ggttgttgta 5460caagcaacac ctcatcatgt
gttggttgat gagtacacag gagaatggat tgactcacaa 5520ttggtggggg
gcaaatgttc caaggaggtt tgtcaaacgg ttcacaactc gaccgtgtgg
5580catgctgatt acaagattac agggctgtgc gagtcaaatc tggcatcagt
ggatatcacc 5640ttcttctctg aggatggtca aaagacgtct ttgggaaaac
cgaacactgg attcaggagt 5700aatcactttg cttacgaaag tggagagaag
gcatgccgta tgcagtactg cacacgatgg 5760ggaatccgac taccttctgg
agtatggttt gaattagtgg acaaagatct cttccaggcg 5820gcaaaattgc
ctgaatgtcc tagaggatcc agtatctcag ctccttctca gacttctgtg
5880gatgttagtt tgatacaaga cgtagagagg atcttagatt actctctatg
ccaggagacg 5940tggagtaaga tacgagccaa gcttcctgta tctccagtag
atctgagtta tctcgcccca 6000aaaaatccag ggagcggacc ggccttcact
atcattaatg gcactttgaa atatttcgaa 6060acaagataca tcagagttga
cataagtaat cccatcatcc ctcacatggt gggaacaatg 6120agtggaacca
cgactgagcg tgaattgtgg aatgattggt atccatatga agacgtagag
6180attggtccaa atggggtgtt gaaaactccc actggtttca agtttccgct
gtacatgatt 6240gggcacggaa tgttggattc cgatctccac aaatcctccc
aggctcaagt cttcgaacat 6300ccacacgcaa aggacgctgc atcacagctt
cctgatgatg agactttatt ttttggtgac 6360acaggactat caaaaaaccc
agtagagtta gtagaaggct ggttcagtag ctggaagagc 6420acattggcat
cgttctttct gattataggc ttgggggttg cattaatctt catcattcga
6480attattgttg cgattcgcta taaatacaag gggaggaaga cccaaaaaat
ttacaatgat 6540gtcgagatga gtcgattggg aaataaataa cagatgacgc
atgagggtca gatcagattt 6600acagcgtaag tgtgatattt aggattataa
aggttcctta attttaattt gttacgcgtt 6660gtatgaaaaa aactcatcaa
cagccatcgc caccatgccc ctggaacagc ggagccagca 6720ctgcaagccc
gaggaaggcc tggaagccag aggcgaagcc ctgggactgg tgggagccca
6780ggcccctgcc acagaagaac aggaagccgc cagcagcagc tccaccctgg
tggaagtgac 6840cctgggcgaa gtgcctgccg ccgagagccc tgatccccct
cagtctcctc agggcgccag 6900cagcctgccc accaccatga actaccccct
gtggtcccag agctacgagg acagcagcaa 6960ccaggaagag gaaggcccca
gcaccttccc cgacctggaa agcgagttcc aggccgccct 7020gagccggaag
gtggcagagc tggtgcactt cctgctgctg aagtacagag cccgcgagcc
7080cgtgaccaag gccgagatgc tgggcagcgt ggtgggaaac tggcagtact
tcttccccgt 7140gatcttctcc aaggccagca gctccctgca gctggtgttc
ggcatcgagc tgatggaagt 7200ggaccccatc ggccacctgt acatcttcgc
cacctgtctg ggcctgagct acgacggcct 7260gctgggcgac aaccagatca
tgcccaaggc cggcctgctg atcatcgtgc tggccatcat 7320tgcccgcgag
ggcgactgcg cccctgagga aaagatctgg gaggaactga gcgtgctgga
7380agtgttcgag ggcagagagg acagcatcct gggcgacccc aagaagctgc
tgacccagca 7440cttcgtgcag gaaaactacc tggaataccg ccaggtgccc
ggcagcgacc ccgcctgtta 7500cgagttcctg tggggcccca gggctctggt
ggaaaccagc tacgtgaagg tgctgcacca 7560catggtgaaa atcagcggcg
gaccccacat cagctacccc ccactgcacg agtgggtgct 7620gagagagggc
gaagaggact acaaggacga cgacgacaaa tgagtctgag cacgagttgc
7680agacgcgttg tatgaaaaaa actcatcaac agccatcatg gatgttaacg
attttgagtt 7740gcatgaggac tttgcattgt ctgaagatga ctttgtcact
tcagaatttc tcaatccgga 7800agaccaaatg acatacctga atcatgccga
ttataatttg aattctccct taatcagcga 7860tgatattgat ttcctgatca
agaaatataa tcatgagcaa attccgaaaa tgtgggatgt 7920caagaattgg
gagggagtgt tagagatgtt gacagcctgt caagccagtc caattttatc
7980tagcactatg cataagtggg tgggaaagtg gctcatgtct gatgatcatg
acgcaagcca 8040aggcttcagt tttcttcatg aagtggacaa agaagctgat
ctgacgtttg aggtggtgga 8100gacattcatt agaggatggg gaggtcgaga
attgcagtac aagaggaaag acacatttcc 8160ggactccttt agagttgcag
cctcattgtg tcaaaaattc cttgatttgc acaaactcac 8220tctgataatg
aattcagtct ctgaagtcga acttaccaac ctagcaaaga attttaaagg
8280aaaaaacagg aaagcaaaaa gcggaaatct gataaccaga ttgagggttc
ccagtttagg 8340tcctgctttt gtgactcagg gatgggtgta catgaagaag
ttggaaatga ttatggatcg 8400gaattttttg ttgatgttga aagacgttat
catcgggagg atgcagacga tcctgtccat 8460gatctcaaga gatgataatc
tcttctccga gtctgatatc tttactgtat taaagatata 8520ccggataggg
gataagatat tagaaaggca agggacaaag ggttacgact tgatcaaaat
8580gattgagcct atttgtaact taaagatgat gaatctggca cgtaaatatc
gtcctctcat 8640ccctacattt cctcattttg aaaaacatat tgctgactct
gttaaggaag gatcgaaaat 8700agacaaaggg attgagttta tatatgatca
cattatgtca atccctggtg tggacttgac 8760cttagttatt tacggatcat
ttcggcactg gggtcatcct tttatcaact actatgaggg 8820cttagagaag
ctacacaagc aggttacaat gcccaagact attgacagag aatatgcaga
8880atgtcttgct agtgatctgg caagaatcgt tcttcagcaa caattcaatg
aacataagaa 8940atggtttgtt gatgtagata aagtcccaca atcccatcct
ttcaaaagcc atatgaaaga 9000gaatacttgg cctactgcag cccaagttca
ggattacggc gatcgctggc atcagctccc 9060actcatcaaa tgcttcgaaa
tcccagattt gttagatcca tcgatcatct actcagacaa 9120aagtcattcc
atgaaccggt ctgaagtact acgacatgta agacttacac ctcatgtgcc
9180cattccaagc aggaaagtat tgcagacaat gttggagact aaggcaacag
actggaaaga 9240gtttttaaag aaaattgacg aagaggggtt agaggatgat
gatcttgtca taggactcaa 9300agggaaagag agagaattaa aaattgcggg
aagattcttt tctttgatgt cctggaagct 9360cagagagtat tttgtcatca
ctgagtattt gattaagacg cactttgtcc cgatgtttaa 9420agggttgacc
atggcggatg acttgacagc ggtgataaag aagatgatgg acacatcttc
9480aggacaaggc ttagataatt atgaatccat ttgtatagcc aaccatattg
actatgagaa 9540gtggaacaat catcaaagaa aagagtcgaa cgggcccgtg
ttcaaggtga tgggtcaatt 9600cttgggatat ccacgtctga ttgagagaac
tcatgaattt tttgagaaga gtctgatata 9660ttacaatgga cgaccagatc
tgatgcgggt tcgaggaaat tctctagtca acgcctcatc 9720tttaaatgtc
tgctgggagg gtcaagctgg gggattagaa ggactgcgac agaagggatg
9780gagtattcta aatttgcttg tcattcagag agaagcaaaa ataaggaaca
ccgccgtgaa 9840agtgctagct caaggtgaca atcaggtgat atgtactcag
tataaaacga agaaatcccg 9900gaatgatatt gagcttaagg cagctctaac
acagatggta tctaataatg agatgattat 9960gtctgcgatt aaatcaggca
ccgagaaact gggtcttttg attaatgatg atgagacaat 10020gcaatctgct
gattacctca attacgggaa ggttcccatt ttcagaggag taatcagagg
10080ccttgagaca aaaagatggt cacgcgtgac ctgtgtgaca aatgatcaga
ttccaacgtg 10140tgcgaacatt atgagctctg tgtcaactaa tgcattaact
gtagcccatt ttgccgagaa 10200tccagtcaat gccatcattc agtataacta
ctttggaaca tttgcaaggc tactgctgat 10260gatgcatgac cccgctctga
ggatctctct gtatgaagtc caatcaaaaa ttccaggact 10320tcacagtttg
acatttaaat attctatgtt gtatctggat ccttcgatag gaggagtctc
10380cggaatgtca ctctcgagat tcctcataag atcatttcca gatccagtga
cagaaagttt 10440ggcgttctgg aaatttatcc actctcatgc aagaagcgat
tcattaaagg agatatgtgc 10500agtttttgga aatcctgaaa ttgcaagatt
tcggctaact catgtcgata aattggtgga 10560agacccaacc tcattgaaca
tagctatggg aatgagtcct gctaatctat taaagacaga 10620ggtaaaaaaa
tgtctactgg aatcaaggca gagcatcaag aaccagattg taagagatgc
10680tactatttac ctacaccatg aggaagacaa acttcgtagt ttcttatggt
ccataacacc 10740actgttccct cggttcttga gtgaattcaa atctgggaca
ttcatcggag tagcagatgg 10800cctgatcagc ttatttcaga actctaggac
tattcgaaat tcttttaaaa agcgttatca 10860cagggaactt gatgatttaa
taatcaagag cgaagtttcc tcacttatgc atttgggtaa 10920gctacatttg
aggcgaggct cagttcgtat gtggacttgc tcttctactc aggctgatct
10980tctccgattc cggtcatggg gaagatctgt tataggaacc acagtccctc
atcccttaga 11040gatgttagga caacatttta aaaaggagac tccttgcagt
gcttgcaaca tatccggatt 11100agactatgta tctgtccact gtccgaatgg
gattcatgac gtttttgaat cacgtggtcc 11160actccctgca tatttgggtt
ctaaaacatc cgaatcaact tcgatcttgc agccgtggga 11220gagagagagt
aaagtaccgt tgattaagcg tgccacaagg cttcgtgatg caatttcatg
11280gtttgtgtct cccgactcta acttggcctc aactatcctt aagaacataa
atgcattaac 11340aggagaagaa tggtcaaaga agcagcatgg atttaaaagg
acgggatcgg cgttacacag 11400gttctccaca tccaggatga gtcatggtgg
ttttgcttct cagagtacgg ctgccttgac 11460tagattgatg gcaactactg
acactatgag agatctggga gaacagaact atgatttcct 11520gtttcaggcg
acattattgt atgctcaaat aaccacaact gtagtcagga atggatcatt
11580tcatagctgc acggaccatt accatataac ctgcaaatct tgtctgaggg
ccattgatga 11640gattaccttg gattcagcga tggaatatag ccctccagat
gtatcatcag ttttacaatc 11700ttggaggaat ggagaaggct cttggggaca
tgaagtgaaa caaatatacc cagttgaagg 11760tgactggagg ggactatctc
ctgttgaaca atcttatcaa gtcggacgct gtatcgggtt 11820tctgttcggt
gatctggcgt atagaaaatc atcccatgca gatgatagct ccatgtttcc
11880gttatctata caaaacaaag tcagaggaag aggcttttta aaagggctta
tggatgggtt 11940aatgagagcc agttgttgcc aggtgatcca tcgtcgaagc
ttagcccatc tgaagagacc 12000ggctaatgca gtctatggag ggctgattta
tttgatagac aaattgagtg catctgcccc 12060ttttctttca ctgacgagac
atggaccttt aagggaagaa ttagaaactg ttccacataa 12120gataccgact
tcttatccta cgagcaaccg agatatgggg gtgatagttc gtaattattt
12180taaatatcag tgcagactgg tagaaaaagg tcggtacaag acacattatc
ctcaattgtg 12240gcttttctca gatgtgctgt ccattgattt cttaggaccc
ctgtctatat cttcaactct 12300attgggtatt ctgtataaac agacgttatc
ttctcgagac aaaaatgagt tgagagaact 12360cgctaacttg tcttcattgt
tgagatcagg agaaggatgg gaagatatcc atgtcaaatt 12420cttctctaag
gacactttac tctgccctga agagatccga catgcgtgca aatttgggat
12480tgctaaggaa tccgctgttt taagctatta tcctccttgg tctcaagagt
cttatggagg 12540catcacctcg atccccgtat atttttcgac caggaagtat
cccaaaattt tagatgtccc 12600tcctcgggtt caaaacccat tggtctcggg
tctacgattg gggcaactcc ctactggagc 12660acattataag attaggagca
ttgtaaagaa caagaacctt cgttatagag atttccttag 12720ttgtggggat
ggatctgggg ggatgaccgc ggcactattg agagaaaaca gacaaagtag
12780gggaatcttc aacagcctgt tagagttagc cggatctctt atgagaggag
catctccaga 12840gcctccaagt gcactggaga cgctcgggca agaacgatct
aggtgtgtga atggaagcac 12900atgttgggag tactcatctg acctaagcca
aaaagagaca tgggattact tcttaagatt 12960gaagagaggc ctgggtttga
ccgtggactt aatcaccatg gacatggagg tcagagaccc 13020taatacaagt
ttgatgatag aaaagaacct caaagtttat ctgcatcaga tattagaacc
13080aactggtgtc ttaatatata aaacatacgg gacccatatt gcgacacaaa
cagataatat 13140cctgacgata atcggtcctt tctttgagac ggttgaccta
gtccagtccg aatacagcag 13200ctcacaaacg tccgaggtct attttgtagg
acgaggcttg cgctctcatg ttgacgaacc 13260ctgggtggac tggccatcct
taatggacaa ttggagatcc atttatgctt ttcatgatcc 13320tactacagaa
tttatcagag caaaaaaagt ctgtgaaatt gacagtctta taggcattcc
13380ggctcaattc attccagacc catttgtaaa tctcgagacc atgctacaga
tagttggtgt 13440tccaacagga gtttcgcatg ccgcagctct attatcatca
caatatccaa atcaattggt 13500cacaacgtca atattttata tgacactcgt
gtcttattat aatgtaaacc atattcgaag 13560aagccccaag cctttctctc
ctccgtctga tggagtctca cagaacattg gttcagccat 13620agtcggacta
agtttttggg tgagtttgat ggagaatgat ctcggattat acaaacaggc
13680tctaggtgca ataaagacgt cattccctat tagatggtcc tctgtccaga
ccaaggatgg 13740gtttacacaa gaatggagaa ctaaaggaaa cggaattcct
aaagattgtc gtctctcaga 13800ctctttggct cagataggaa actggatcag
agcgatggaa ttggttagga acaaaacgag 13860gcaatcagga ttttctgaaa
ccctatttga tcaattctgc ggacttgcag accatcacct 13920caaatggcgg
aagttgggaa acagaacagg aattattgat tggctaaata atagaatttc
13980atccattgac aaatccatct tggtgaccaa aagtgatctg catgacgaga
actcatggag 14040ggagtgaaga tgtattcttc cacctctcat tgggtgatac
ccatatatga aaaaaactat 14100aagtacttta aactctcttt gttttttaat
gtatatctgg ttttgttgtt tccgtgccgg 14160ccatggtccc agcctcctcg
ctggcggccg gtgggcaaca ttccgagggg accgtcccct 14220cggtaatgac
gaatgggaca accccttggg gcctctaaac gggtcttgag gggttttttg
14280gtttaaacaa cgaattc 142977527PRTArtificial Sequencefusion
protein derived from E6 and E7 proteins of human papilloma virus
7Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro1 5
10 15Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His
Asp 20 25 30Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg
Arg Glu 35 40 45Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr
Arg Asp Gly 50 55 60Asn Pro Tyr Ala Val Asp Lys Leu Lys Phe Tyr Ser
Lys Ile Ser Glu65 70 75 80Tyr Arg His Tyr Cys Tyr Ser Val Tyr Gly
Thr Thr Leu Glu Gln Gln 85 90 95Tyr Asn Lys Pro Leu Cys Asp Leu Leu
Ile Arg Ile Asn Gln Lys Pro 100 105 110Leu Cys Pro Glu Glu Lys Gln
Arg His Leu Asp Lys Lys Gln Arg Phe 115 120 125His Asn Ile Arg Gly
Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg 130 135 140Ser Ser Arg
Thr Arg Arg Glu Thr Gln Leu Gly Gly Gly Gly Gly Ala145 150 155
160Ala Tyr Met Ala Arg Phe Glu Asp Pro Thr Arg Arg Pro Tyr Lys Leu
165 170 175Pro Asp Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile
Glu Ile 180 185 190Thr Cys Val Tyr Cys Lys Thr Val Leu Glu Leu Thr
Glu Val Phe Glu 195 200 205Phe Ala Phe Lys Asp Leu Phe Val Val Tyr
Arg Asp Ser Ile Pro His 210 215 220Ala Ala His Lys Ile Asp Phe Tyr
Ser Arg Ile Arg Glu Leu Arg His225 230 235 240Tyr Ser Asp Ser Val
Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr 245 250 255Gly Leu Tyr
Asn Leu Leu Ile Arg Leu Arg Gln Lys Pro Leu Asn Pro 260 265 270Ala
Glu Lys Leu Arg His Leu Asn Glu Lys Arg Arg Phe His Asn Ile 275 280
285Ala Gly His Tyr Arg Gly Gln Cys His Ser Cys Cys Asn Arg Ala Arg
290 295 300Gln Glu Arg Leu Gln Arg Arg Arg Glu Thr Gln Val Gly Gly
Gly Gly305 310 315 320Gly Ala Ala Tyr Met His Gly Asp Thr Pro Thr
Leu His Glu Tyr Met 325 330 335Leu Asp Leu Gln Pro Glu Thr Thr Asp
Leu Tyr Gln Leu Asn Asp Ser 340 345 350Ser Glu Glu Glu Asp Glu Ile
Asp Gly Pro Ala Gly Gln Ala Glu Pro 355 360 365Asp Arg Ala His Tyr
Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser 370 375 380Thr Leu Arg
Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu385 390 395
400Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Pro Ile Cys Ser Gln
405 410 415Lys Pro Gly Gly Gly Gly Gly Ala Ala Tyr Met His Gly Pro
Lys Ala 420 425 430Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro Gln
Asn Glu Ile Pro 435 440 445Val Asp Leu Leu Gln Leu Ser Asp Ser Glu
Glu Glu Asn Asp Glu Ile 450 455 460Asp Gly Val Asn His Gln His Leu
Pro Ala Arg Arg Ala Glu Pro Gln465 470 475 480Arg His Thr Met Leu
Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Lys 485 490 495Leu Val Val
Glu Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu 500 505 510Phe
Leu Asn Thr Leu Ser Phe Val Pro Trp Cys Ala Ser Gln Gln 515 520
52581584DNAArtificial Sequencenucleotide sequence encoding fusion
protein derived from E6 and E7 proteins of human papilloma virus
8atgcatcaga agcgaactgc tatgtttcag gaccctcagg agcggccacg caaactgcct
60cagctgtgca ccgaactgca gacaactatc cacgacatca ttctggaatg cgtgtactgt
120aagcagcagc tgctgaggag agaggtctat gacttcgctt ttcgcgatct
gtgcatcgtg 180taccgagacg gaaacccata tgcagtcgat aagctgaagt
tctacagcaa gatctccgaa 240tacaggcatt actgttacag cgtgtacggg
accacactgg agcagcagta taacaagccc 300ctgtgcgacc tgctgatcag
aattaatcag aagcccctgt gccctgagga aaaacagagg 360cacctggata
agaaacagag atttcataac atccgaggac gatggaccgg gcggtgcatg
420tcctgctgta gaagctcccg gactcgacga gagacccagc tgggcggagg
aggaggagca 480gcttacatgg cacgattcga ggaccctacc cgaaggccat
ataagctgcc cgacctgtgc 540acagaactga atacttctct gcaggacatc
gagattacat gcgtgtactg taaaaccgtc 600ctggagctga cagaagtgtt
cgagtttgct ttcaaggacc tgtttgtggt ctaccgggat 660tcaatccctc
acgcagccca taaaatcgac ttctacagca ggatcaggga actgcgccac
720tactccgaca gcgtgtacgg ggatacactg gagaagctga caaacactgg
cctgtacaat 780ctgctgatcc gactgcgaca gaagccactg aacccagccg
aaaaactgag acacctgaac 840gagaagagac ggtttcacaa tattgcaggc
cattataggg gacagtgcca tagttgctgt 900aatcgagcca ggcaggaaag
actgcagcgc cgaagggaga ctcaagtcgg cggaggagga 960ggagctgcat
acatgcacgg cgacaccccc acactgcatg aatatatgct ggatctgcag
1020cctgagacta ccgacctgta ccagctgaac gattctagtg aggaagagga
cgaaatcgac 1080ggaccagcag gacaggcaga gcctgaccgg gcccactata
atattgtgac attctgctgt 1140aagtgcgatt ctactctgcg gctgtgcgtg
cagagtactc atgtcgacat ccgcaccctg 1200gaggatctgc tgatggggac
tctgggcatc gtcccaattt gtagccagaa accaggcggc 1260ggcggcggag
cagcttacat gcacggaccc aaggctaccc tgcaggacat cgtgctgcat
1320ctggaacctc agaatgagat tccagtcgac ctgctgcagc tgagtgattc
agaagaggaa 1380aacgacgaga tcgacggcgt gaatcaccag catctgcctg
ctagacgggc agagccacag 1440cgacacacaa tgctgtgcat gtgctgtaag
tgtgaagcca ggatcaagct ggtggtcgag 1500tcaagcgccg acgatctgcg
cgccttccag cagctgttcc tgaatactct gtcatttgtc 1560ccttggtgtg
cctcccagca gtga 1584912754DNAArtificial Sequencenucleotide encoding
an artificial Maraba virus that expresses a human papilloma virus
E6/E7 fusion protein 9acgaagacaa acaaaccatt gatagaatta agaggctcat
gaaaatcctt aacagcgttc 60aaaatgtctg ttacagtcaa gagagtcatt gatgattcac
tcatcacccc caaattgcct 120gcgaatgagg accctgtgga gtaccctgct
gattatttca aaaagtcccg tgatattccg 180gtgtacataa acacgaccaa
aagtttgtct gatttgcggg gctatgttta tcaaggccta 240aagtcaggca
acatctctat aattcatgtc aacagttatc tgtatgcagc attaaaagag
300atcagaggaa aattggacag agattggatc acctttggta tccaaatcgg
aaaaacagga 360gatagcgtgg ggatattcga tttactgacc ctaaaacctc
tagatggtgt tttaccagat 420ggggtgtctg atgctactcg aactagctca
gacgatgcat ggcttccact gtatctattg 480gggttataca gagttggtcg
aacacagatg ccagaataca ggaagaagct gatggatggt 540ctgattaatc
aatgtaagat gatcaatgag cagtttgaac cactgttgcc agaaggaaga
600gatgtctttg atgtctgggg aaatgacagc aattacacaa agattgtggc
cgctgtagat 660atgttcttcc atatgttcaa aaagcatgag aaggcctctt
tcaggtatgg cacaatagtg 720tcaagattta aggattgtgc agcattggct
acatttggtc atctgtgtaa gatcactggt 780atgtccactg aagatgtgac
aacttggatt ctaaacaggg aggtggctga tgagatggtt 840caaatgatgt
acccaggaca ggagatagat aaggctgatt cttacatgcc ttatctaatc
900gacttaggtc tgtcctcaaa atctccatat tcatcagtta aaaatccagc
tttccatttt 960tggggtcaat tgaccgcatt gttactgaga tcaaccagag
ccagaaatgc acgtcagccg 1020gatgacatcg agtatacatc cctgaccact
gctgggctgt tgtatgcata tgccgttggt 1080tcgtctgcag acctggctca
acaattctac gttggggaca acaagtatgt gccagaaact 1140ggagatggag
gattaaccac caatgcaccg ccacaagggc gagatgtggt cgagtggctt
1200agttggtttg aagatcaaaa cagaaaacct accccagaca tgctcatgta
tgctaagaga 1260gctgtcagtg ctttacaagg attgagggag aagacgattg
gcaagtacgc caagtcagag 1320tttgacaaat gacaactcac tcaccatatg
tattactacc tttgcttcat atgaaaaaaa 1380ctaacagcga tcatggatca
gctatcaaag gtcaaggaat tccttaagac ttacgcgcag 1440ttggatcaag
cagtacaaga gatggatgac attgagtctc agagagagga aaagactaat
1500tttgatttgt ttcaggaaga aggattggag attaaggaga agccttccta
ttatcgggca 1560gatgaagaag agattgattc agatgaagac agcgtggatg
atgcacaaga cttagggata 1620cgtacatcaa caagtcccat cgaggggtat
gtggatgagg agcaggatga ttatgaggat 1680gaggaagtga acgtggtgtt
tacatcggac tggaaacagc ctgagctgga atccgacggg 1740gatgggaaaa
ctctccgatt gacgatacca gatggattga ctggggagca gaagtcgcaa
1800tggcttgcca cgattaaggc agttgttcag agtgctaaat attggaacat
ctcagaatgt 1860tcatttgaga gttatgagca aggggttttg attagagaga
gacaaatgac tcctgatgtc 1920tacaaagtca ctcctgtttt aaatgctcca
ccggttcaaa tgacagctaa tcaagatgtt 1980tggtctctca gcagcactcc
atttacattt ttgcccaaga aacaaggtgt gactccattg 2040accatgtcct
tagaagaact cttcaacacc cgaggtgaat tcatatctct gggaggaaac
2100gggaaaatga gtcaccggga ggccatcatt ctagggttga gacacaagaa
gctctataat 2160caagccagac taaagtataa cttagcttga atatgaaaaa
aactaacaga tatcaaaaga 2220tatctctaac tcagtccatt gtgttcagtt
caatcatgag ctctctcaag aaaattttgg 2280gtattaaagg gaaagggaag
aaatctaaga aattaggtat ggctccccca ccctatgaag 2340aagagactcc
aatggaatat tctccaagtg caccttatga taagtcattg tttggagtcg
2400aagatatgga tttccatgat caacgtcaac tccgatatga gaaatttcac
ttctcattga 2460agatgactgt gagatcaaac aaaccatttc gaaattatga
tgacgttgca gcagcggtgt 2520ccaattggga tcatatgtac atcggcatgg
caggaaaacg tcctttttat aagatattag 2580cattcatggg ttctactcta
ttgaaggcta caccagccgt ctgggctgac caaggacagc 2640cagaatatca
tgctcactgt gagggacgag cttacttgcc gcatcggtta gggccgaccc
2700ctccgatgtt gaatgtccct gaacattttc gccgtccatt taacatcgga
ttattcagag 2760ggacaatcga cataaccctg gtacttttcg atgatgaatc
tgtagattct gccccggtca 2820tatgggatca ttttaatgca tccagattga
gcagcttcag agaaaaggct ttgttgtttg 2880gtttgattct agaaaagaaa
gccactggga attgggtatt ggactctatt agtcatttca 2940agtaattatc
acaagtgttg aggtgatggg cagactatga aaaaaactaa cagggttcaa
3000acactcttga tcgaggtacc cagttatatt tgttacaaca atgttgagac
tttttctctt 3060ttgtttcttg gccttaggag cccactccaa atttactata
gtattccctc atcatcaaaa 3120agggaattgg aagaatgtgc cttccacata
tcattattgc ccttctagtt ctgaccagaa 3180ttggcataat gatttgactg
gagttagtct tcatgtgaaa attcccaaaa gtcacaaagc 3240tatacaagca
gatggctgga tgtgccacgc tgctaaatgg gtgactactt gtgacttcag
3300atggtacgga cccaaataca tcacgcattc catacactct atgtcaccca
ccctagaaca 3360gtgcaagacc agtattgagc agacaaagca aggagtttgg
attaatccag gctttccccc 3420tcaaagctgc
ggatatgcta cagtgacgga tgcagaggtg gttgttgtac aagcaacacc
3480tcatcatgtg ttggttgatg agtacacagg agaatggatt gactcacaat
tggtgggggg 3540caaatgttcc aaggaggttt gtcaaacggt tcacaactcg
accgtgtggc atgctgatta 3600caagattaca gggctgtgcg agtcaaatct
ggcatcagtg gatatcacct tcttctctga 3660ggatggtcaa aagacgtctt
tgggaaaacc gaacactgga ttcaggagta atcactttgc 3720ttacgaaagt
ggagagaagg catgccgtat gcagtactgc acacgatggg gaatccgact
3780accttctgga gtatggtttg aattagtgga caaagatctc ttccaggcgg
caaaattgcc 3840tgaatgtcct agaggatcca gtatctcagc tccttctcag
acttctgtgg atgttagttt 3900gatacaagac gtagagagga tcttagatta
ctctctatgc caggagacgt ggagtaagat 3960acgagccaag cttcctgtat
ctccagtaga tctgagttat ctcgccccaa aaaatccagg 4020gagcggaccg
gccttcacta tcattaatgg cactttgaaa tatttcgaaa caagatacat
4080cagagttgac ataagtaatc ccatcatccc tcacatggtg ggaacaatga
gtggaaccac 4140gactgagcgt gaattgtgga atgattggta tccatatgaa
gacgtagaga ttggtccaaa 4200tggggtgttg aaaactccca ctggtttcaa
gtttccgctg tacatgattg ggcacggaat 4260gttggattcc gatctccaca
aatcctccca ggctcaagtc ttcgaacatc cacacgcaaa 4320ggacgctgca
tcacagcttc ctgatgatga gactttattt tttggtgaca caggactatc
4380aaaaaaccca gtagagttag tagaaggctg gttcagtagc tggaagagca
cattggcatc 4440gttctttctg attataggct tgggggttgc attaatcttc
atcattcgaa ttattgttgc 4500gattcgctat aaatacaagg ggaggaagac
ccaaaaaatt tacaatgatg tcgagatgag 4560tcgattggga aataaataac
agatgacgca tgagggtcag atcagattta cagcgtaagt 4620gtgatattta
ggattataaa ggttccttaa ttttaatttg ttacgcgttg tatgaaaaaa
4680actcatcaac agccatcatg catcagaagc gaactgctat gtttcaggac
cctcaggagc 4740ggccacgcaa actgcctcag ctgtgcaccg aactgcagac
aactatccac gacatcattc 4800tggaatgcgt gtactgtaag cagcagctgc
tgaggagaga ggtctatgac ttcgcttttc 4860gcgatctgtg catcgtgtac
cgagacggaa acccatatgc agtcgataag ctgaagttct 4920acagcaagat
ctccgaatac aggcattact gttacagcgt gtacgggacc acactggagc
4980agcagtataa caagcccctg tgcgacctgc tgatcagaat taatcagaag
cccctgtgcc 5040ctgaggaaaa acagaggcac ctggataaga aacagagatt
tcataacatc cgaggacgat 5100ggaccgggcg gtgcatgtcc tgctgtagaa
gctcccggac tcgacgagag acccagctgg 5160gcggaggagg aggagcagct
tacatggcac gattcgagga ccctacccga aggccatata 5220agctgcccga
cctgtgcaca gaactgaata cttctctgca ggacatcgag attacatgcg
5280tgtactgtaa aaccgtcctg gagctgacag aagtgttcga gtttgctttc
aaggacctgt 5340ttgtggtcta ccgggattca atccctcacg cagcccataa
aatcgacttc tacagcagga 5400tcagggaact gcgccactac tccgacagcg
tgtacgggga tacactggag aagctgacaa 5460acactggcct gtacaatctg
ctgatccgac tgcgacagaa gccactgaac ccagccgaaa 5520aactgagaca
cctgaacgag aagagacggt ttcacaatat tgcaggccat tataggggac
5580agtgccatag ttgctgtaat cgagccaggc aggaaagact gcagcgccga
agggagactc 5640aagtcggcgg aggaggagga gctgcataca tgcacggcga
cacccccaca ctgcatgaat 5700atatgctgga tctgcagcct gagactaccg
acctgtacca gctgaacgat tctagtgagg 5760aagaggacga aatcgacgga
ccagcaggac aggcagagcc tgaccgggcc cactataata 5820ttgtgacatt
ctgctgtaag tgcgattcta ctctgcggct gtgcgtgcag agtactcatg
5880tcgacatccg caccctggag gatctgctga tggggactct gggcatcgtc
ccaatttgta 5940gccagaaacc aggcggcggc ggcggagcag cttacatgca
cggacccaag gctaccctgc 6000aggacatcgt gctgcatctg gaacctcaga
atgagattcc agtcgacctg ctgcagctga 6060gtgattcaga agaggaaaac
gacgagatcg acggcgtgaa tcaccagcat ctgcctgcta 6120gacgggcaga
gccacagcga cacacaatgc tgtgcatgtg ctgtaagtgt gaagccagga
6180tcaagctggt ggtcgagtca agcgccgacg atctgcgcgc cttccagcag
ctgttcctga 6240atactctgtc atttgtccct tggtgtgcct cccagcagtg
acgtacgtgt atgaaaaaaa 6300ctcatcaaca gccatcatgg atgttaacga
ttttgagttg catgaggact ttgcattgtc 6360tgaagatgac tttgtcactt
cagaatttct caatccggaa gaccaaatga catacctgaa 6420tcatgccgat
tataatttga attctccctt aatcagcgat gatattgatt tcctgatcaa
6480gaaatataat catgagcaaa ttccgaaaat gtgggatgtc aagaattggg
agggagtgtt 6540agagatgttg acagcctgtc aagccagtcc aattttatct
agcactatgc ataagtgggt 6600gggaaagtgg ctcatgtctg atgatcatga
cgcaagccaa ggcttcagtt ttcttcatga 6660agtggacaaa gaagctgatc
tgacgtttga ggtggtggag acattcatta gaggatgggg 6720aggtcgagaa
ttgcagtaca agaggaaaga cacatttccg gactccttta gagttgcagc
6780ctcattgtgt caaaaattcc ttgatttgca caaactcact ctgataatga
attcagtctc 6840tgaagtcgaa cttaccaacc tagcaaagaa ttttaaagga
aaaaacagga aagcaaaaag 6900cggaaatctg ataaccagat tgagggttcc
cagtttaggt cctgcttttg tgactcaggg 6960atgggtgtac atgaagaagt
tggaaatgat tatggatcgg aattttttgt tgatgttgaa 7020agacgttatc
atcgggagga tgcagacgat cctgtccatg atctcaagag atgataatct
7080cttctccgag tctgatatct ttactgtatt aaagatatac cggatagggg
ataagatatt 7140agaaaggcaa gggacaaagg gttacgactt gatcaaaatg
attgagccta tttgtaactt 7200aaagatgatg aatctggcac gtaaatatcg
tcctctcatc cctacatttc ctcattttga 7260aaaacatatt gctgactctg
ttaaggaagg atcgaaaata gacaaaggga ttgagtttat 7320atatgatcac
attatgtcaa tccctggtgt ggacttgacc ttagttattt acggatcatt
7380tcggcactgg ggtcatcctt ttatcaacta ctatgagggc ttagagaagc
tacacaagca 7440ggttacaatg cccaagacta ttgacagaga atatgcagaa
tgtcttgcta gtgatctggc 7500aagaatcgtt cttcagcaac aattcaatga
acataagaaa tggtttgttg atgtagataa 7560agtcccacaa tcccatcctt
tcaaaagcca tatgaaagag aatacttggc ctactgcagc 7620ccaagttcag
gattacggcg atcgctggca tcagctccca ctcatcaaat gcttcgaaat
7680cccagatttg ttagatccat cgatcatcta ctcagacaaa agtcattcca
tgaaccggtc 7740tgaagtacta cgacatgtaa gacttacacc tcatgtgccc
attccaagca ggaaagtatt 7800gcagacaatg ttggagacta aggcaacaga
ctggaaagag tttttaaaga aaattgacga 7860agaggggtta gaggatgatg
atcttgtcat aggactcaaa gggaaagaga gagaattaaa 7920aattgcggga
agattctttt ctttgatgtc ctggaagctc agagagtatt ttgtcatcac
7980tgagtatttg attaagacgc actttgtccc gatgtttaaa gggttgacca
tggcggatga 8040cttgacagcg gtgataaaga agatgatgga cacatcttca
ggacaaggct tagataatta 8100tgaatccatt tgtatagcca accatattga
ctatgagaag tggaacaatc atcaaagaaa 8160agagtcgaac gggcccgtgt
tcaaggtgat gggtcaattc ttgggatatc cacgtctgat 8220tgagagaact
catgaatttt ttgagaagag tctgatatat tacaatggac gaccagatct
8280gatgcgggtt cgaggaaatt ctctagtcaa cgcctcatct ttaaatgtct
gctgggaggg 8340tcaagctggg ggattagaag gactgcgaca gaagggatgg
agtattctaa atttgcttgt 8400cattcagaga gaagcaaaaa taaggaacac
cgccgtgaaa gtgctagctc aaggtgacaa 8460tcaggtgata tgtactcagt
ataaaacgaa gaaatcccgg aatgatattg agcttaaggc 8520agctctaaca
cagatggtat ctaataatga gatgattatg tctgcgatta aatcaggcac
8580cgagaaactg ggtcttttga ttaatgatga tgagacaatg caatctgctg
attacctcaa 8640ttacgggaag gttcccattt tcagaggagt aatcagaggc
cttgagacaa aaagatggtc 8700tcgagtgacc tgtgtgacaa atgatcagat
tccaacgtgt gcgaacatta tgagctctgt 8760gtcaactaat gcattaactg
tagcccattt tgccgagaat ccagtcaatg ccatcattca 8820gtataactac
tttggaacat ttgcaaggct actgctgatg atgcatgacc ccgctctgag
8880gatctctctg tatgaagtcc aatcaaaaat tccaggactt cacagtttga
catttaaata 8940ttctatgttg tatctggatc cttcgatagg aggagtctcc
ggaatgtcac tctcgagatt 9000cctcataaga tcatttccag atccagtgac
agaaagtttg gcgttctgga aatttatcca 9060ctctcatgca agaagcgatt
cattaaagga gatatgtgca gtttttggaa atcctgaaat 9120tgcaagattt
cggctaactc atgtcgataa attggtggaa gacccaacct cattgaacat
9180agctatggga atgagtcctg ctaatctatt aaagacagag gtaaaaaaat
gtctactgga 9240atcaaggcag agcatcaaga accagattgt aagagatgct
actatttacc tacaccatga 9300ggaagacaaa cttcgtagtt tcttatggtc
cataacacca ctgttccctc ggttcttgag 9360tgaattcaaa tctgggacat
tcatcggagt agcagatggc ctgatcagct tatttcagaa 9420ctctaggact
attcgaaatt cttttaaaaa gcgttatcac agggaacttg atgatttaat
9480aatcaagagc gaagtttcct cacttatgca tttgggtaag ctacatttga
ggcgaggctc 9540agttcgtatg tggacttgct cttctactca ggctgatctt
ctccgattcc ggtcatgggg 9600aagatctgtt ataggaacca cagtccctca
tcccttagag atgttaggac aacattttaa 9660aaaggagact ccttgcagtg
cttgcaacat atccggatta gactatgtat ctgtccactg 9720tccgaatggg
attcatgacg tttttgaatc acgtggtcca ctccctgcat atttgggttc
9780taaaacatcc gaatcaactt cgatcttgca gccgtgggag agagagagta
aagtaccgtt 9840gattaagcgt gccacaaggc ttcgtgatgc aatttcatgg
tttgtgtctc ccgactctaa 9900cttggcctca actatcctta agaacataaa
tgcattaaca ggagaagaat ggtcaaagaa 9960gcagcatgga tttaaaagga
cgggatcggc gttacacagg ttctccacat ccaggatgag 10020tcatggtggt
tttgcttctc agagtacggc tgccttgact agattgatgg caactactga
10080cactatgaga gatctgggag aacagaacta tgatttcctg tttcaggcga
cattattgta 10140tgctcaaata accacaactg tagtcaggaa tggatcattt
catagctgca cggaccatta 10200ccatataacc tgcaaatctt gtctgagggc
cattgatgag attaccttgg attcagcgat 10260ggaatatagc cctccagatg
tatcatcagt tttacaatct tggaggaatg gagaaggctc 10320ttggggacat
gaagtgaaac aaatataccc agttgaaggt gactggaggg gactatctcc
10380tgttgaacaa tcttatcaag tcggacgctg tatcgggttt ctgttcggtg
atctggcgta 10440tagaaaatca tcccatgcag atgatagctc catgtttccg
ttatctatac aaaacaaagt 10500cagaggaaga ggctttttaa aagggcttat
ggatgggtta atgagagcca gttgttgcca 10560ggtgatccat cgtcgaagct
tagcccatct gaagagaccg gctaatgcag tctatggagg 10620gctgatttat
ttgatagaca aattgagtgc atctgcccct tttctttcac tgacgagaca
10680tggaccttta agggaagaat tagaaactgt tccacataag ataccgactt
cttatcctac 10740gagcaaccga gatatggggg tgatagttcg taattatttt
aaatatcagt gcagactggt 10800agaaaaaggt cggtacaaga cacattatcc
tcaattgtgg cttttctcag atgtgctgtc 10860cattgatttc ttaggacccc
tgtctatatc ttcaactcta ttgggtattc tgtataaaca 10920gacgttatct
tctcgagaca aaaatgagtt gagagaactc gctaacttgt cttcattgtt
10980gagatcagga gaaggatggg aagatatcca tgtcaaattc ttctctaagg
acactttact 11040ctgccctgaa gagatccgac atgcgtgcaa atttgggatt
gctaaggaat ccgctgtttt 11100aagctattat cctccttggt ctcaagagtc
ttatggaggc atcacctcga tccccgtata 11160tttttcgacc aggaagtatc
ccaaaatttt agatgtccct cctcgggttc aaaacccatt 11220ggtctcgggt
ctacgattgg ggcaactccc tactggagca cattataaga ttaggagcat
11280tgtaaagaac aagaaccttc gttatagaga tttccttagt tgtggggatg
gatctggggg 11340gatgaccgcg gcactattga gagaaaacag acaaagtagg
ggaatcttca acagcctgtt 11400agagttagcc ggatctctta tgagaggagc
atctccagag cctccaagtg cactggagac 11460gctcgggcaa gaacgatcta
ggtgtgtgaa tggaagcaca tgttgggagt actcatctga 11520cctaagccaa
aaagagacat gggattactt cttaagattg aagagaggcc tgggtttgac
11580cgtggactta atcaccatgg acatggaggt cagagaccct aatacaagtt
tgatgataga 11640aaagaacctc aaagtttatc tgcatcagat attagaacca
actggtgtct taatatataa 11700aacatacggg acccatattg cgacacaaac
agataatatc ctgacgataa tcggtccttt 11760ctttgagacg gttgacctag
tccagtccga atacagcagc tcacaaacgt ccgaggtcta 11820ttttgtagga
cgaggcttgc gctctcatgt tgacgaaccc tgggtggact ggccatcctt
11880aatggacaat tggagatcca tttatgcttt tcatgatcct actacagaat
ttatcagagc 11940aaaaaaagtc tgtgaaattg acagtcttat aggcattccg
gctcaattca ttccagaccc 12000atttgtaaat ctcgagacca tgctacagat
agttggtgtt ccaacaggag tttcgcatgc 12060cgcagctcta ttatcatcac
aatatccaaa tcaattggtc acaacgtcaa tattttatat 12120gacactcgtg
tcttattata atgtaaacca tattcgaaga agccccaagc ctttctctcc
12180tccgtctgat ggagtctcac agaacattgg ttcagccata gtcggactaa
gtttttgggt 12240gagtttgatg gagaatgatc tcggattata caaacaggct
ctaggtgcaa taaagacgtc 12300attccctatt agatggtcct ctgtccagac
caaggatggg tttacacaag aatggagaac 12360taaaggaaac ggaattccta
aagattgtcg tctctcagac tctttggctc agataggaaa 12420ctggatcaga
gcgatggaat tggttaggaa caaaacgagg caatcaggat tttctgaaac
12480cctatttgat caattctgcg gacttgcaga ccatcacctc aaatggcgga
agttgggaaa 12540cagaacagga attattgatt ggctaaataa tagaatttca
tccattgaca aatccatctt 12600ggtgaccaaa agtgatctgc atgacgagaa
ctcatggagg gagtgaagat gtattcttcc 12660acctctcatt gggtgatacc
catatatgaa aaaaactata agtactttaa actctctttg 12720ttttttaatg
tatatctggt tttgttgttt ccgt 1275410341PRTHomo sapiens 10Met Glu Ser
Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met1 5 10 15Lys Pro
Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr 20 25 30Gly
Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln 35 40
45Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr
50 55 60Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala
Ile65 70 75 80Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu
Val Ile His 85 90 95Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys
Ile Pro Ile Leu 100 105 110Val Ile Asn Lys Val Leu Pro Met Val Ser
Ile Thr Leu Leu Ala Leu 115 120 125Val Tyr Leu Pro Gly Val Ile Ala
Ala Ile Val Gln Leu His Asn Gly 130 135 140Thr Lys Tyr Lys Lys Phe
Pro His Trp Leu Asp Lys Trp Met Leu Thr145 150 155 160Arg Lys Gln
Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala 165 170 175Ile
Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu 180 185
190Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp
195 200 205Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu
Gly Ile 210 215 220Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr
Ser Ile Pro Ser225 230 235 240Val Ser Asp Ser Leu Thr Trp Arg Glu
Phe His Tyr Ile Gln Ser Lys 245 250 255Leu Gly Ile Val Ser Leu Leu
Leu Gly Thr Ile His Ala Leu Ile Phe 260 265 270Ala Trp Asn Lys Trp
Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr Pro 275 280 285Pro Thr Phe
Met Ile Ala Val Phe Leu Pro Ile Val Val Leu Ile Phe 290 295 300Lys
Ser Ile Leu Phe Leu Pro Cys Leu Arg Lys Lys Ile Leu Lys Ile305 310
315 320Arg His Gly Trp Glu Asp Val Thr Lys Ile Asn Lys Thr Glu Ile
Cys 325 330 335Ser Gln Leu Lys Leu 340111026DNAHomo sapiens
11atggaatcac ggaaggacat cactaatcag gaggaactgt ggaaaatgaa gccaagaagg
60aatctggaag aggacgacta tctgcacaag gacaccggcg aaacaagtat gctgaaacga
120ccagtgctgc tgcacctgca tcagactgct cacgcagacg agtttgattg
cccctctgaa 180ctgcagcaca cccaggagct gttcccacag tggcatctgc
ccatcaagat tgccgctatc 240attgcttcac tgacatttct gtacactctg
ctgagagaag tgatccaccc cctggccacc 300agccatcagc agtacttcta
taagatccct atcctggtca tcaacaaggt cctgccaatg 360gtgagcatca
cactgctggc cctggtctac ctgcctggag tgatcgcagc cattgtccag
420ctgcacaatg ggacaaagta taagaaattt ccacattggc tggataagtg
gatgctgact 480aggaaacagt tcggactgct gtccttcttt ttcgccgtgc
tgcacgctat ctacagcctg 540tcctatccca tgaggaggag ctaccggtat
aagctgctga actgggctta ccagcaggtg 600cagcagaaca aggaggacgc
atggattgaa catgacgtgt ggcgcatgga aatctacgtg 660agcctgggca
ttgtcggact ggccatcctg gctctgctgg cagtgaccag tatcccttct
720gtcagtgact cactgacatg gagagagttt cactacattc agagcaagct
ggggatcgtg 780tccctgctgc tgggcaccat ccatgcactg atttttgcct
ggaacaagtg gatcgatatc 840aagcagttcg tgtggtatac tccccctacc
tttatgattg ccgtcttcct gcccatcgtg 900gtcctgatct tcaagtccat
cctgttcctg ccttgtctgc ggaagaaaat cctgaaaatt 960cggcacggat
gggaggatgt caccaaaatc aataagactg aaatctgtag ccagctgaag 1020ctttaa
10261212196DNAArtificial Sequencenucleotide encoding an artificial
Maraba virus that expresses a human Six-Transmembrane Epithelial
Antigen of the Prostate protein 12acgaagacaa acaaaccatt gatagaatta
agaggctcat gaaaatcctt aacagcgttc 60aaaatgtctg ttacagtcaa gagagtcatt
gatgattcac tcatcacccc caaattgcct 120gcgaatgagg accctgtgga
gtaccctgct gattatttca aaaagtcccg tgatattccg 180gtgtacataa
acacgaccaa aagtttgtct gatttgcggg gctatgttta tcaaggccta
240aagtcaggca acatctctat aattcatgtc aacagttatc tgtatgcagc
attaaaagag 300atcagaggaa aattggacag agattggatc acctttggta
tccaaatcgg aaaaacagga 360gatagcgtgg ggatattcga tttactgacc
ctaaaacctc tagatggtgt tttaccagat 420ggggtgtctg atgctactcg
aactagctca gacgatgcat ggcttccact gtatctattg 480gggttataca
gagttggtcg aacacagatg ccagaataca ggaagaagct gatggatggt
540ctgattaatc aatgtaagat gatcaatgag cagtttgaac cactgttgcc
agaaggaaga 600gatgtctttg atgtctgggg aaatgacagc aattacacaa
agattgtggc cgctgtagat 660atgttcttcc atatgttcaa aaagcatgag
aaggcctctt tcaggtatgg cacaatagtg 720tcaagattta aggattgtgc
agcattggct acatttggtc atctgtgtaa gatcactggt 780atgtccactg
aagatgtgac aacttggatt ctaaacaggg aggtggctga tgagatggtt
840caaatgatgt acccaggaca ggagatagat aaggctgatt cttacatgcc
ttatctaatc 900gacttaggtc tgtcctcaaa atctccatat tcatcagtta
aaaatccagc tttccatttt 960tggggtcaat tgaccgcatt gttactgaga
tcaaccagag ccagaaatgc acgtcagccg 1020gatgacatcg agtatacatc
cctgaccact gctgggctgt tgtatgcata tgccgttggt 1080tcgtctgcag
acctggctca acaattctac gttggggaca acaagtatgt gccagaaact
1140ggagatggag gattaaccac caatgcaccg ccacaagggc gagatgtggt
cgagtggctt 1200agttggtttg aagatcaaaa cagaaaacct accccagaca
tgctcatgta tgctaagaga 1260gctgtcagtg ctttacaagg attgagggag
aagacgattg gcaagtacgc caagtcagag 1320tttgacaaat gacaactcac
tcaccatatg tattactacc tttgcttcat atgaaaaaaa 1380ctaacagcga
tcatggatca gctatcaaag gtcaaggaat tccttaagac ttacgcgcag
1440ttggatcaag cagtacaaga gatggatgac attgagtctc agagagagga
aaagactaat 1500tttgatttgt ttcaggaaga aggattggag attaaggaga
agccttccta ttatcgggca 1560gatgaagaag agattgattc agatgaagac
agcgtggatg atgcacaaga cttagggata 1620cgtacatcaa caagtcccat
cgaggggtat gtggatgagg agcaggatga ttatgaggat 1680gaggaagtga
acgtggtgtt tacatcggac tggaaacagc ctgagctgga atccgacggg
1740gatgggaaaa ctctccgatt gacgatacca gatggattga ctggggagca
gaagtcgcaa 1800tggcttgcca cgattaaggc agttgttcag agtgctaaat
attggaacat ctcagaatgt 1860tcatttgaga gttatgagca aggggttttg
attagagaga gacaaatgac tcctgatgtc 1920tacaaagtca ctcctgtttt
aaatgctcca ccggttcaaa tgacagctaa tcaagatgtt 1980tggtctctca
gcagcactcc atttacattt ttgcccaaga aacaaggtgt gactccattg
2040accatgtcct tagaagaact cttcaacacc cgaggtgaat tcatatctct
gggaggaaac 2100gggaaaatga gtcaccggga ggccatcatt ctagggttga
gacacaagaa gctctataat 2160caagccagac taaagtataa cttagcttga
atatgaaaaa aactaacaga tatcaaaaga 2220tatctctaac tcagtccatt
gtgttcagtt caatcatgag ctctctcaag aaaattttgg 2280gtattaaagg
gaaagggaag aaatctaaga aattaggtat ggctccccca ccctatgaag
2340aagagactcc aatggaatat
tctccaagtg caccttatga taagtcattg tttggagtcg 2400aagatatgga
tttccatgat caacgtcaac tccgatatga gaaatttcac ttctcattga
2460agatgactgt gagatcaaac aaaccatttc gaaattatga tgacgttgca
gcagcggtgt 2520ccaattggga tcatatgtac atcggcatgg caggaaaacg
tcctttttat aagatattag 2580cattcatggg ttctactcta ttgaaggcta
caccagccgt ctgggctgac caaggacagc 2640cagaatatca tgctcactgt
gagggacgag cttacttgcc gcatcggtta gggccgaccc 2700ctccgatgtt
gaatgtccct gaacattttc gccgtccatt taacatcgga ttattcagag
2760ggacaatcga cataaccctg gtacttttcg atgatgaatc tgtagattct
gccccggtca 2820tatgggatca ttttaatgca tccagattga gcagcttcag
agaaaaggct ttgttgtttg 2880gtttgattct agaaaagaaa gccactggga
attgggtatt ggactctatt agtcatttca 2940agtaattatc acaagtgttg
aggtgatggg cagactatga aaaaaactaa cagggttcaa 3000acactcttga
tcgaggtacc cagttatatt tgttacaaca atgttgagac tttttctctt
3060ttgtttcttg gccttaggag cccactccaa atttactata gtattccctc
atcatcaaaa 3120agggaattgg aagaatgtgc cttccacata tcattattgc
ccttctagtt ctgaccagaa 3180ttggcataat gatttgactg gagttagtct
tcatgtgaaa attcccaaaa gtcacaaagc 3240tatacaagca gatggctgga
tgtgccacgc tgctaaatgg gtgactactt gtgacttcag 3300atggtacgga
cccaaataca tcacgcattc catacactct atgtcaccca ccctagaaca
3360gtgcaagacc agtattgagc agacaaagca aggagtttgg attaatccag
gctttccccc 3420tcaaagctgc ggatatgcta cagtgacgga tgcagaggtg
gttgttgtac aagcaacacc 3480tcatcatgtg ttggttgatg agtacacagg
agaatggatt gactcacaat tggtgggggg 3540caaatgttcc aaggaggttt
gtcaaacggt tcacaactcg accgtgtggc atgctgatta 3600caagattaca
gggctgtgcg agtcaaatct ggcatcagtg gatatcacct tcttctctga
3660ggatggtcaa aagacgtctt tgggaaaacc gaacactgga ttcaggagta
atcactttgc 3720ttacgaaagt ggagagaagg catgccgtat gcagtactgc
acacgatggg gaatccgact 3780accttctgga gtatggtttg aattagtgga
caaagatctc ttccaggcgg caaaattgcc 3840tgaatgtcct agaggatcca
gtatctcagc tccttctcag acttctgtgg atgttagttt 3900gatacaagac
gtagagagga tcttagatta ctctctatgc caggagacgt ggagtaagat
3960acgagccaag cttcctgtat ctccagtaga tctgagttat ctcgccccaa
aaaatccagg 4020gagcggaccg gccttcacta tcattaatgg cactttgaaa
tatttcgaaa caagatacat 4080cagagttgac ataagtaatc ccatcatccc
tcacatggtg ggaacaatga gtggaaccac 4140gactgagcgt gaattgtgga
atgattggta tccatatgaa gacgtagaga ttggtccaaa 4200tggggtgttg
aaaactccca ctggtttcaa gtttccgctg tacatgattg ggcacggaat
4260gttggattcc gatctccaca aatcctccca ggctcaagtc ttcgaacatc
cacacgcaaa 4320ggacgctgca tcacagcttc ctgatgatga gactttattt
tttggtgaca caggactatc 4380aaaaaaccca gtagagttag tagaaggctg
gttcagtagc tggaagagca cattggcatc 4440gttctttctg attataggct
tgggggttgc attaatcttc atcattcgaa ttattgttgc 4500gattcgctat
aaatacaagg ggaggaagac ccaaaaaatt tacaatgatg tcgagatgag
4560tcgattggga aataaataac agatgacgca tgagggtcag atcagattta
cagcgtaagt 4620gtgatattta ggattataaa ggttccttaa ttttaatttg
ttacgcgttg tatgaaaaaa 4680actcatcaac agccatcatg gaatcacgga
aggacatcac taatcaggag gaactgtgga 4740aaatgaagcc aagaaggaat
ctggaagagg acgactatct gcacaaggac accggcgaaa 4800caagtatgct
gaaacgacca gtgctgctgc acctgcatca gactgctcac gcagacgagt
4860ttgattgccc ctctgaactg cagcacaccc aggagctgtt cccacagtgg
catctgccca 4920tcaagattgc cgctatcatt gcttcactga catttctgta
cactctgctg agagaagtga 4980tccaccccct ggccaccagc catcagcagt
acttctataa gatccctatc ctggtcatca 5040acaaggtcct gccaatggtg
agcatcacac tgctggccct ggtctacctg cctggagtga 5100tcgcagccat
tgtccagctg cacaatggga caaagtataa gaaatttcca cattggctgg
5160ataagtggat gctgactagg aaacagttcg gactgctgtc cttctttttc
gccgtgctgc 5220acgctatcta cagcctgtcc tatcccatga ggaggagcta
ccggtataag ctgctgaact 5280gggcttacca gcaggtgcag cagaacaagg
aggacgcatg gattgaacat gacgtgtggc 5340gcatggaaat ctacgtgagc
ctgggcattg tcggactggc catcctggct ctgctggcag 5400tgaccagtat
cccttctgtc agtgactcac tgacatggag agagtttcac tacattcaga
5460gcaagctggg gatcgtgtcc ctgctgctgg gcaccatcca tgcactgatt
tttgcctgga 5520acaagtggat cgatatcaag cagttcgtgt ggtatactcc
ccctaccttt atgattgccg 5580tcttcctgcc catcgtggtc ctgatcttca
agtccatcct gttcctgcct tgtctgcgga 5640agaaaatcct gaaaattcgg
cacggatggg aggatgtcac caaaatcaat aagactgaaa 5700tctgtagcca
gctgaagctt taacgtacgt gtatgaaaaa aactcatcaa cagccatcat
5760ggatgttaac gattttgagt tgcatgagga ctttgcattg tctgaagatg
actttgtcac 5820ttcagaattt ctcaatccgg aagaccaaat gacatacctg
aatcatgccg attataattt 5880gaattctccc ttaatcagcg atgatattga
tttcctgatc aagaaatata atcatgagca 5940aattccgaaa atgtgggatg
tcaagaattg ggagggagtg ttagagatgt tgacagcctg 6000tcaagccagt
ccaattttat ctagcactat gcataagtgg gtgggaaagt ggctcatgtc
6060tgatgatcat gacgcaagcc aaggcttcag ttttcttcat gaagtggaca
aagaagctga 6120tctgacgttt gaggtggtgg agacattcat tagaggatgg
ggaggtcgag aattgcagta 6180caagaggaaa gacacatttc cggactcctt
tagagttgca gcctcattgt gtcaaaaatt 6240ccttgatttg cacaaactca
ctctgataat gaattcagtc tctgaagtcg aacttaccaa 6300cctagcaaag
aattttaaag gaaaaaacag gaaagcaaaa agcggaaatc tgataaccag
6360attgagggtt cccagtttag gtcctgcttt tgtgactcag ggatgggtgt
acatgaagaa 6420gttggaaatg attatggatc ggaatttttt gttgatgttg
aaagacgtta tcatcgggag 6480gatgcagacg atcctgtcca tgatctcaag
agatgataat ctcttctccg agtctgatat 6540ctttactgta ttaaagatat
accggatagg ggataagata ttagaaaggc aagggacaaa 6600gggttacgac
ttgatcaaaa tgattgagcc tatttgtaac ttaaagatga tgaatctggc
6660acgtaaatat cgtcctctca tccctacatt tcctcatttt gaaaaacata
ttgctgactc 6720tgttaaggaa ggatcgaaaa tagacaaagg gattgagttt
atatatgatc acattatgtc 6780aatccctggt gtggacttga ccttagttat
ttacggatca tttcggcact ggggtcatcc 6840ttttatcaac tactatgagg
gcttagagaa gctacacaag caggttacaa tgcccaagac 6900tattgacaga
gaatatgcag aatgtcttgc tagtgatctg gcaagaatcg ttcttcagca
6960acaattcaat gaacataaga aatggtttgt tgatgtagat aaagtcccac
aatcccatcc 7020tttcaaaagc catatgaaag agaatacttg gcctactgca
gcccaagttc aggattacgg 7080cgatcgctgg catcagctcc cactcatcaa
atgcttcgaa atcccagatt tgttagatcc 7140atcgatcatc tactcagaca
aaagtcattc catgaaccgg tctgaagtac tacgacatgt 7200aagacttaca
cctcatgtgc ccattccaag caggaaagta ttgcagacaa tgttggagac
7260taaggcaaca gactggaaag agtttttaaa gaaaattgac gaagaggggt
tagaggatga 7320tgatcttgtc ataggactca aagggaaaga gagagaatta
aaaattgcgg gaagattctt 7380ttctttgatg tcctggaagc tcagagagta
ttttgtcatc actgagtatt tgattaagac 7440gcactttgtc ccgatgttta
aagggttgac catggcggat gacttgacag cggtgataaa 7500gaagatgatg
gacacatctt caggacaagg cttagataat tatgaatcca tttgtatagc
7560caaccatatt gactatgaga agtggaacaa tcatcaaaga aaagagtcga
acgggcccgt 7620gttcaaggtg atgggtcaat tcttgggata tccacgtctg
attgagagaa ctcatgaatt 7680ttttgagaag agtctgatat attacaatgg
acgaccagat ctgatgcggg ttcgaggaaa 7740ttctctagtc aacgcctcat
ctttaaatgt ctgctgggag ggtcaagctg ggggattaga 7800aggactgcga
cagaagggat ggagtattct aaatttgctt gtcattcaga gagaagcaaa
7860aataaggaac accgccgtga aagtgctagc tcaaggtgac aatcaggtga
tatgtactca 7920gtataaaacg aagaaatccc ggaatgatat tgagcttaag
gcagctctaa cacagatggt 7980atctaataat gagatgatta tgtctgcgat
taaatcaggc accgagaaac tgggtctttt 8040gattaatgat gatgagacaa
tgcaatctgc tgattacctc aattacggga aggttcccat 8100tttcagagga
gtaatcagag gccttgagac aaaaagatgg tctcgagtga cctgtgtgac
8160aaatgatcag attccaacgt gtgcgaacat tatgagctct gtgtcaacta
atgcattaac 8220tgtagcccat tttgccgaga atccagtcaa tgccatcatt
cagtataact actttggaac 8280atttgcaagg ctactgctga tgatgcatga
ccccgctctg aggatctctc tgtatgaagt 8340ccaatcaaaa attccaggac
ttcacagttt gacatttaaa tattctatgt tgtatctgga 8400tccttcgata
ggaggagtct ccggaatgtc actctcgaga ttcctcataa gatcatttcc
8460agatccagtg acagaaagtt tggcgttctg gaaatttatc cactctcatg
caagaagcga 8520ttcattaaag gagatatgtg cagtttttgg aaatcctgaa
attgcaagat ttcggctaac 8580tcatgtcgat aaattggtgg aagacccaac
ctcattgaac atagctatgg gaatgagtcc 8640tgctaatcta ttaaagacag
aggtaaaaaa atgtctactg gaatcaaggc agagcatcaa 8700gaaccagatt
gtaagagatg ctactattta cctacaccat gaggaagaca aacttcgtag
8760tttcttatgg tccataacac cactgttccc tcggttcttg agtgaattca
aatctgggac 8820attcatcgga gtagcagatg gcctgatcag cttatttcag
aactctagga ctattcgaaa 8880ttcttttaaa aagcgttatc acagggaact
tgatgattta ataatcaaga gcgaagtttc 8940ctcacttatg catttgggta
agctacattt gaggcgaggc tcagttcgta tgtggacttg 9000ctcttctact
caggctgatc ttctccgatt ccggtcatgg ggaagatctg ttataggaac
9060cacagtccct catcccttag agatgttagg acaacatttt aaaaaggaga
ctccttgcag 9120tgcttgcaac atatccggat tagactatgt atctgtccac
tgtccgaatg ggattcatga 9180cgtttttgaa tcacgtggtc cactccctgc
atatttgggt tctaaaacat ccgaatcaac 9240ttcgatcttg cagccgtggg
agagagagag taaagtaccg ttgattaagc gtgccacaag 9300gcttcgtgat
gcaatttcat ggtttgtgtc tcccgactct aacttggcct caactatcct
9360taagaacata aatgcattaa caggagaaga atggtcaaag aagcagcatg
gatttaaaag 9420gacgggatcg gcgttacaca ggttctccac atccaggatg
agtcatggtg gttttgcttc 9480tcagagtacg gctgccttga ctagattgat
ggcaactact gacactatga gagatctggg 9540agaacagaac tatgatttcc
tgtttcaggc gacattattg tatgctcaaa taaccacaac 9600tgtagtcagg
aatggatcat ttcatagctg cacggaccat taccatataa cctgcaaatc
9660ttgtctgagg gccattgatg agattacctt ggattcagcg atggaatata
gccctccaga 9720tgtatcatca gttttacaat cttggaggaa tggagaaggc
tcttggggac atgaagtgaa 9780acaaatatac ccagttgaag gtgactggag
gggactatct cctgttgaac aatcttatca 9840agtcggacgc tgtatcgggt
ttctgttcgg tgatctggcg tatagaaaat catcccatgc 9900agatgatagc
tccatgtttc cgttatctat acaaaacaaa gtcagaggaa gaggcttttt
9960aaaagggctt atggatgggt taatgagagc cagttgttgc caggtgatcc
atcgtcgaag 10020cttagcccat ctgaagagac cggctaatgc agtctatgga
gggctgattt atttgataga 10080caaattgagt gcatctgccc cttttctttc
actgacgaga catggacctt taagggaaga 10140attagaaact gttccacata
agataccgac ttcttatcct acgagcaacc gagatatggg 10200ggtgatagtt
cgtaattatt ttaaatatca gtgcagactg gtagaaaaag gtcggtacaa
10260gacacattat cctcaattgt ggcttttctc agatgtgctg tccattgatt
tcttaggacc 10320cctgtctata tcttcaactc tattgggtat tctgtataaa
cagacgttat cttctcgaga 10380caaaaatgag ttgagagaac tcgctaactt
gtcttcattg ttgagatcag gagaaggatg 10440ggaagatatc catgtcaaat
tcttctctaa ggacacttta ctctgccctg aagagatccg 10500acatgcgtgc
aaatttggga ttgctaagga atccgctgtt ttaagctatt atcctccttg
10560gtctcaagag tcttatggag gcatcacctc gatccccgta tatttttcga
ccaggaagta 10620tcccaaaatt ttagatgtcc ctcctcgggt tcaaaaccca
ttggtctcgg gtctacgatt 10680ggggcaactc cctactggag cacattataa
gattaggagc attgtaaaga acaagaacct 10740tcgttataga gatttcctta
gttgtgggga tggatctggg gggatgaccg cggcactatt 10800gagagaaaac
agacaaagta ggggaatctt caacagcctg ttagagttag ccggatctct
10860tatgagagga gcatctccag agcctccaag tgcactggag acgctcgggc
aagaacgatc 10920taggtgtgtg aatggaagca catgttggga gtactcatct
gacctaagcc aaaaagagac 10980atgggattac ttcttaagat tgaagagagg
cctgggtttg accgtggact taatcaccat 11040ggacatggag gtcagagacc
ctaatacaag tttgatgata gaaaagaacc tcaaagttta 11100tctgcatcag
atattagaac caactggtgt cttaatatat aaaacatacg ggacccatat
11160tgcgacacaa acagataata tcctgacgat aatcggtcct ttctttgaga
cggttgacct 11220agtccagtcc gaatacagca gctcacaaac gtccgaggtc
tattttgtag gacgaggctt 11280gcgctctcat gttgacgaac cctgggtgga
ctggccatcc ttaatggaca attggagatc 11340catttatgct tttcatgatc
ctactacaga atttatcaga gcaaaaaaag tctgtgaaat 11400tgacagtctt
ataggcattc cggctcaatt cattccagac ccatttgtaa atctcgagac
11460catgctacag atagttggtg ttccaacagg agtttcgcat gccgcagctc
tattatcatc 11520acaatatcca aatcaattgg tcacaacgtc aatattttat
atgacactcg tgtcttatta 11580taatgtaaac catattcgaa gaagccccaa
gcctttctct cctccgtctg atggagtctc 11640acagaacatt ggttcagcca
tagtcggact aagtttttgg gtgagtttga tggagaatga 11700tctcggatta
tacaaacagg ctctaggtgc aataaagacg tcattcccta ttagatggtc
11760ctctgtccag accaaggatg ggtttacaca agaatggaga actaaaggaa
acggaattcc 11820taaagattgt cgtctctcag actctttggc tcagatagga
aactggatca gagcgatgga 11880attggttagg aacaaaacga ggcaatcagg
attttctgaa accctatttg atcaattctg 11940cggacttgca gaccatcacc
tcaaatggcg gaagttggga aacagaacag gaattattga 12000ttggctaaat
aatagaattt catccattga caaatccatc ttggtgacca aaagtgatct
12060gcatgacgag aactcatgga gggagtgaag atgtattctt ccacctctca
ttgggtgata 12120cccatatatg aaaaaaacta taagtacttt aaactctctt
tgttttttaa tgtatatctg 12180gttttgttgt ttccgt 1219613180PRTHomo
sapiens 13Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp
Ala Asp1 5 10 15Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly
Asn Ala Gly 20 25 30Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly
Pro Arg Gly Ala 35 40 45Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly
Ala Pro Arg Gly Pro 50 55 60His Gly Gly Ala Ala Ser Gly Leu Asn Gly
Cys Cys Arg Cys Gly Ala65 70 75 80Arg Gly Pro Glu Ser Arg Leu Leu
Glu Phe Tyr Leu Ala Met Pro Phe 85 90 95Ala Thr Pro Met Glu Ala Glu
Leu Ala Arg Arg Ser Leu Ala Gln Asp 100 105 110Ala Pro Pro Leu Pro
Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val 115 120 125Ser Gly Asn
Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln 130 135 140Leu
Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met145 150
155 160Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro
Ser 165 170 175Gly Gln Arg Arg 18014543DNAHomo sapiens 14atgcaggccg
agggcagagg cacaggcgga tctacaggcg acgccgatgg ccctggcggc 60cctggaattc
ctgacggacc tggcggcaat gccggcggac ccggagaagc tggcgccaca
120ggcggaagag gacctagagg cgctggcgcc gctagagctt ctggaccagg
cggaggcgcc 180cctagaggac ctcatggcgg agccgcctcc ggcctgaacg
gctgttgcag atgtggagcc 240agaggccccg agagccggct gctggaattc
tacctggcca tgcccttcgc cacccccatg 300gaagccgagc tggccagacg
gtccctggcc caggatgctc ctcctctgcc tgtgcccggc 360gtgctgctga
aagaattcac cgtgtccggc aacatcctga ccatccggct gactgccgcc
420gaccacagac agctccagct gtctatcagc tcctgcctgc agcagctgag
cctgctgatg 480tggatcaccc agtgctttct gcccgtgttc ctggctcagc
cccccagcgg ccagagaaga 540tga 5431511725DNAArtificial
Sequencenucleotide encoding an artificial Maraba virus that
expresses a human Cancer Testis Antigen 1 protein 15acgaagacaa
acaaaccatt gatagaatta agaggctcat gaaaatcctt aacagcgttc 60aaaatgtctg
ttacagtcaa gagagtcatt gatgattcac tcatcacccc caaattgcct
120gcgaatgagg accctgtgga gtaccctgct gattatttca aaaagtcccg
tgatattccg 180gtgtacataa acacgaccaa aagtttgtct gatttgcggg
gctatgttta tcaaggccta 240aagtcaggca acatctctat aattcatgtc
aacagttatc tgtatgcagc attaaaagag 300atcagaggaa aattggacag
agattggatc acctttggta tccaaatcgg aaaaacagga 360gatagcgtgg
ggatattcga tttactgacc ctaaaacctc tagatggtgt tttaccagat
420ggggtgtctg atgctactcg aactagctca gacgatgcat ggcttccact
gtatctattg 480gggttataca gagttggtcg aacacagatg ccagaataca
ggaagaagct gatggatggt 540ctgattaatc aatgtaagat gatcaatgag
cagtttgaac cactgttgcc agaaggaaga 600gatgtctttg atgtctgggg
aaatgacagc aattacacaa agattgtggc cgctgtagat 660atgttcttcc
atatgttcaa aaagcatgag aaggcctctt tcaggtatgg cacaatagtg
720tcaagattta aggattgtgc agcattggct acatttggtc atctgtgtaa
gatcactggt 780atgtccactg aagatgtgac aacttggatt ctaaacaggg
aggtggctga tgagatggtt 840caaatgatgt acccaggaca ggagatagat
aaggctgatt cttacatgcc ttatctaatc 900gacttaggtc tgtcctcaaa
atctccatat tcatcagtta aaaatccagc tttccatttt 960tggggtcaat
tgaccgcatt gttactgaga tcaaccagag ccagaaatgc acgtcagccg
1020gatgacatcg agtatacatc cctgaccact gctgggctgt tgtatgcata
tgccgttggt 1080tcgtctgcag acctggctca acaattctac gttggggaca
acaagtatgt gccagaaact 1140ggagatggag gattaaccac caatgcaccg
ccacaagggc gagatgtggt cgagtggctt 1200agttggtttg aagatcaaaa
cagaaaacct accccagaca tgctcatgta tgctaagaga 1260gctgtcagtg
ctttacaagg attgagggag aagacgattg gcaagtacgc caagtcagag
1320tttgacaaat gacaactcac tcaccatatg tattactacc tttgcttcat
atgaaaaaaa 1380ctaacagcga tcatggatca gctatcaaag gtcaaggaat
tccttaagac ttacgcgcag 1440ttggatcaag cagtacaaga gatggatgac
attgagtctc agagagagga aaagactaat 1500tttgatttgt ttcaggaaga
aggattggag attaaggaga agccttccta ttatcgggca 1560gatgaagaag
agattgattc agatgaagac agcgtggatg atgcacaaga cttagggata
1620cgtacatcaa caagtcccat cgaggggtat gtggatgagg agcaggatga
ttatgaggat 1680gaggaagtga acgtggtgtt tacatcggac tggaaacagc
ctgagctgga atccgacggg 1740gatgggaaaa ctctccgatt gacgatacca
gatggattga ctggggagca gaagtcgcaa 1800tggcttgcca cgattaaggc
agttgttcag agtgctaaat attggaacat ctcagaatgt 1860tcatttgaga
gttatgagca aggggttttg attagagaga gacaaatgac tcctgatgtc
1920tacaaagtca ctcctgtttt aaatgctcca ccggttcaaa tgacagctaa
tcaagatgtt 1980tggtctctca gcagcactcc atttacattt ttgcccaaga
aacaaggtgt gactccattg 2040accatgtcct tagaagaact cttcaacacc
cgaggtgaat tcatatctct gggaggaaac 2100gggaaaatga gtcaccggga
ggccatcatt ctagggttga gacacaagaa gctctataat 2160caagccagac
taaagtataa cttagcttga atatgaaaaa aactaacaga tatcaaaaga
2220tatctctaac tcagtccatt gtgttcagtt caatcatgag ctctctcaag
aaaattttgg 2280gtattaaagg gaaagggaag aaatctaaga aattaggtat
ggctccccca ccctatgaag 2340aagagactcc aatggaatat tctccaagtg
caccttatga taagtcattg tttggagtcg 2400aagatatgga tttccatgat
caacgtcaac tccgatatga gaaatttcac ttctcattga 2460agatgactgt
gagatcaaac aaaccatttc gaaattatga tgacgttgca gcagcggtgt
2520ccaattggga tcatatgtac atcggcatgg caggaaaacg tcctttttat
aagatattag 2580cattcatggg ttctactcta ttgaaggcta caccagccgt
ctgggctgac caaggacagc 2640cagaatatca tgctcactgt gagggacgag
cttacttgcc gcatcggtta gggccgaccc 2700ctccgatgtt gaatgtccct
gaacattttc gccgtccatt taacatcgga ttattcagag 2760ggacaatcga
cataaccctg gtacttttcg atgatgaatc tgtagattct gccccggtca
2820tatgggatca ttttaatgca tccagattga gcagcttcag agaaaaggct
ttgttgtttg 2880gtttgattct agaaaagaaa gccactggga attgggtatt
ggactctatt agtcatttca 2940agtaattatc acaagtgttg aggtgatggg
cagactatga aaaaaactaa cagggttcaa 3000acactcttga tcgaggtacc
cagttatatt tgttacaaca atgttgagac tttttctctt 3060ttgtttcttg
gccttaggag cccactccaa atttactata gtattccctc atcatcaaaa
3120agggaattgg aagaatgtgc cttccacata tcattattgc ccttctagtt
ctgaccagaa 3180ttggcataat gatttgactg gagttagtct tcatgtgaaa
attcccaaaa gtcacaaagc 3240tatacaagca gatggctgga tgtgccacgc
tgctaaatgg gtgactactt gtgacttcag 3300atggtacgga cccaaataca
tcacgcattc catacactct atgtcaccca ccctagaaca 3360gtgcaagacc
agtattgagc agacaaagca aggagtttgg attaatccag gctttccccc
3420tcaaagctgc ggatatgcta cagtgacgga tgcagaggtg gttgttgtac
aagcaacacc 3480tcatcatgtg ttggttgatg agtacacagg agaatggatt
gactcacaat tggtgggggg 3540caaatgttcc aaggaggttt gtcaaacggt
tcacaactcg accgtgtggc atgctgatta 3600caagattaca gggctgtgcg
agtcaaatct ggcatcagtg gatatcacct tcttctctga 3660ggatggtcaa
aagacgtctt tgggaaaacc gaacactgga ttcaggagta atcactttgc
3720ttacgaaagt ggagagaagg catgccgtat gcagtactgc acacgatggg
gaatccgact 3780accttctgga gtatggtttg aattagtgga caaagatctc
ttccaggcgg caaaattgcc 3840tgaatgtcct agaggatcca gtatctcagc
tccttctcag acttctgtgg atgttagttt 3900gatacaagac gtagagagga
tcttagatta ctctctatgc caggagacgt ggagtaagat 3960acgagccaag
cttcctgtat ctccagtaga tctgagttat ctcgccccaa aaaatccagg
4020gagcggaccg gccttcacta tcattaatgg cactttgaaa tatttcgaaa
caagatacat 4080cagagttgac ataagtaatc ccatcatccc tcacatggtg
ggaacaatga gtggaaccac 4140gactgagcgt gaattgtgga atgattggta
tccatatgaa gacgtagaga ttggtccaaa 4200tggggtgttg aaaactccca
ctggtttcaa gtttccgctg tacatgattg ggcacggaat 4260gttggattcc
gatctccaca aatcctccca ggctcaagtc ttcgaacatc cacacgcaaa
4320ggacgctgca tcacagcttc ctgatgatga gactttattt tttggtgaca
caggactatc 4380aaaaaaccca gtagagttag tagaaggctg gttcagtagc
tggaagagca cattggcatc 4440gttctttctg attataggct tgggggttgc
attaatcttc atcattcgaa ttattgttgc 4500gattcgctat aaatacaagg
ggaggaagac ccaaaaaatt tacaatgatg tcgagatgag 4560tcgattggga
aataaataac agatgacgca tgagggtcag atcagattta cagcgtaagt
4620gtgatattta ggattataaa ggttccttaa ttttaatttg ttacgcgttg
tatgaaaaaa 4680actcatcaac agccatcgcc accatgcagg ccgagggcag
aggcacaggc ggatctacag 4740gcgacgccga tggccctggc ggccctggaa
ttcctgacgg acctggcggc aatgccggcg 4800gacccggaga agctggcgcc
acaggcggaa gaggacctag aggcgctggc gccgctagag 4860cttctggacc
aggcggaggc gcccctagag gacctcatgg cggagccgcc tccggcctga
4920acggctgttg cagatgtgga gccagaggcc ccgagagccg gctgctggaa
ttctacctgg 4980ccatgccctt cgccaccccc atggaagccg agctggccag
acggtccctg gcccaggatg 5040ctcctcctct gcctgtgccc ggcgtgctgc
tgaaagaatt caccgtgtcc ggcaacatcc 5100tgaccatccg gctgactgcc
gccgaccaca gacagctcca gctgtctatc agctcctgcc 5160tgcagcagct
gagcctgctg atgtggatca cccagtgctt tctgcccgtg ttcctggctc
5220agccccccag cggccagaga agatgagtcg acacgcgttg tatgaaaaaa
actcatcaac 5280agccatcatg gatgttaacg attttgagtt gcatgaggac
tttgcattgt ctgaagatga 5340ctttgtcact tcagaatttc tcaatccgga
agaccaaatg acatacctga atcatgccga 5400ttataatttg aattctccct
taatcagcga tgatattgat ttcctgatca agaaatataa 5460tcatgagcaa
attccgaaaa tgtgggatgt caagaattgg gagggagtgt tagagatgtt
5520gacagcctgt caagccagtc caattttatc tagcactatg cataagtggg
tgggaaagtg 5580gctcatgtct gatgatcatg acgcaagcca aggcttcagt
tttcttcatg aagtggacaa 5640agaagctgat ctgacgtttg aggtggtgga
gacattcatt agaggatggg gaggtcgaga 5700attgcagtac aagaggaaag
acacatttcc ggactccttt agagttgcag cctcattgtg 5760tcaaaaattc
cttgatttgc acaaactcac tctgataatg aattcagtct ctgaagtcga
5820acttaccaac ctagcaaaga attttaaagg aaaaaacagg aaagcaaaaa
gcggaaatct 5880gataaccaga ttgagggttc ccagtttagg tcctgctttt
gtgactcagg gatgggtgta 5940catgaagaag ttggaaatga ttatggatcg
gaattttttg ttgatgttga aagacgttat 6000catcgggagg atgcagacga
tcctgtccat gatctcaaga gatgataatc tcttctccga 6060gtctgatatc
tttactgtat taaagatata ccggataggg gataagatat tagaaaggca
6120agggacaaag ggttacgact tgatcaaaat gattgagcct atttgtaact
taaagatgat 6180gaatctggca cgtaaatatc gtcctctcat ccctacattt
cctcattttg aaaaacatat 6240tgctgactct gttaaggaag gatcgaaaat
agacaaaggg attgagttta tatatgatca 6300cattatgtca atccctggtg
tggacttgac cttagttatt tacggatcat ttcggcactg 6360gggtcatcct
tttatcaact actatgaggg cttagagaag ctacacaagc aggttacaat
6420gcccaagact attgacagag aatatgcaga atgtcttgct agtgatctgg
caagaatcgt 6480tcttcagcaa caattcaatg aacataagaa atggtttgtt
gatgtagata aagtcccaca 6540atcccatcct ttcaaaagcc atatgaaaga
gaatacttgg cctactgcag cccaagttca 6600ggattacggc gatcgctggc
atcagctccc actcatcaaa tgcttcgaaa tcccagattt 6660gttagatcca
tcgatcatct actcagacaa aagtcattcc atgaaccggt ctgaagtact
6720acgacatgta agacttacac ctcatgtgcc cattccaagc aggaaagtat
tgcagacaat 6780gttggagact aaggcaacag actggaaaga gtttttaaag
aaaattgacg aagaggggtt 6840agaggatgat gatcttgtca taggactcaa
agggaaagag agagaattaa aaattgcggg 6900aagattcttt tctttgatgt
cctggaagct cagagagtat tttgtcatca ctgagtattt 6960gattaagacg
cactttgtcc cgatgtttaa agggttgacc atggcggatg acttgacagc
7020ggtgataaag aagatgatgg acacatcttc aggacaaggc ttagataatt
atgaatccat 7080ttgtatagcc aaccatattg actatgagaa gtggaacaat
catcaaagaa aagagtcgaa 7140cgggcccgtg ttcaaggtga tgggtcaatt
cttgggatat ccacgtctga ttgagagaac 7200tcatgaattt tttgagaaga
gtctgatata ttacaatgga cgaccagatc tgatgcgggt 7260tcgaggaaat
tctctagtca acgcctcatc tttaaatgtc tgctgggagg gtcaagctgg
7320gggattagaa ggactgcgac agaagggatg gagtattcta aatttgcttg
tcattcagag 7380agaagcaaaa ataaggaaca ccgccgtgaa agtgctagct
caaggtgaca atcaggtgat 7440atgtactcag tataaaacga agaaatcccg
gaatgatatt gagcttaagg cagctctaac 7500acagatggta tctaataatg
agatgattat gtctgcgatt aaatcaggca ccgagaaact 7560gggtcttttg
attaatgatg atgagacaat gcaatctgct gattacctca attacgggaa
7620ggttcccatt ttcagaggag taatcagagg ccttgagaca aaaagatggt
cacgcgtgac 7680ctgtgtgaca aatgatcaga ttccaacgtg tgcgaacatt
atgagctctg tgtcaactaa 7740tgcattaact gtagcccatt ttgccgagaa
tccagtcaat gccatcattc agtataacta 7800ctttggaaca tttgcaaggc
tactgctgat gatgcatgac cccgctctga ggatctctct 7860gtatgaagtc
caatcaaaaa ttccaggact tcacagtttg acatttaaat attctatgtt
7920gtatctggat ccttcgatag gaggagtctc cggaatgtca ctctcgagat
tcctcataag 7980atcatttcca gatccagtga cagaaagttt ggcgttctgg
aaatttatcc actctcatgc 8040aagaagcgat tcattaaagg agatatgtgc
agtttttgga aatcctgaaa ttgcaagatt 8100tcggctaact catgtcgata
aattggtgga agacccaacc tcattgaaca tagctatggg 8160aatgagtcct
gctaatctat taaagacaga ggtaaaaaaa tgtctactgg aatcaaggca
8220gagcatcaag aaccagattg taagagatgc tactatttac ctacaccatg
aggaagacaa 8280acttcgtagt ttcttatggt ccataacacc actgttccct
cggttcttga gtgaattcaa 8340atctgggaca ttcatcggag tagcagatgg
cctgatcagc ttatttcaga actctaggac 8400tattcgaaat tcttttaaaa
agcgttatca cagggaactt gatgatttaa taatcaagag 8460cgaagtttcc
tcacttatgc atttgggtaa gctacatttg aggcgaggct cagttcgtat
8520gtggacttgc tcttctactc aggctgatct tctccgattc cggtcatggg
gaagatctgt 8580tataggaacc acagtccctc atcccttaga gatgttagga
caacatttta aaaaggagac 8640tccttgcagt gcttgcaaca tatccggatt
agactatgta tctgtccact gtccgaatgg 8700gattcatgac gtttttgaat
cacgtggtcc actccctgca tatttgggtt ctaaaacatc 8760cgaatcaact
tcgatcttgc agccgtggga gagagagagt aaagtaccgt tgattaagcg
8820tgccacaagg cttcgtgatg caatttcatg gtttgtgtct cccgactcta
acttggcctc 8880aactatcctt aagaacataa atgcattaac aggagaagaa
tggtcaaaga agcagcatgg 8940atttaaaagg acgggatcgg cgttacacag
gttctccaca tccaggatga gtcatggtgg 9000ttttgcttct cagagtacgg
ctgccttgac tagattgatg gcaactactg acactatgag 9060agatctggga
gaacagaact atgatttcct gtttcaggcg acattattgt atgctcaaat
9120aaccacaact gtagtcagga atggatcatt tcatagctgc acggaccatt
accatataac 9180ctgcaaatct tgtctgaggg ccattgatga gattaccttg
gattcagcga tggaatatag 9240ccctccagat gtatcatcag ttttacaatc
ttggaggaat ggagaaggct cttggggaca 9300tgaagtgaaa caaatatacc
cagttgaagg tgactggagg ggactatctc ctgttgaaca 9360atcttatcaa
gtcggacgct gtatcgggtt tctgttcggt gatctggcgt atagaaaatc
9420atcccatgca gatgatagct ccatgtttcc gttatctata caaaacaaag
tcagaggaag 9480aggcttttta aaagggctta tggatgggtt aatgagagcc
agttgttgcc aggtgatcca 9540tcgtcgaagc ttagcccatc tgaagagacc
ggctaatgca gtctatggag ggctgattta 9600tttgatagac aaattgagtg
catctgcccc ttttctttca ctgacgagac atggaccttt 9660aagggaagaa
ttagaaactg ttccacataa gataccgact tcttatccta cgagcaaccg
9720agatatgggg gtgatagttc gtaattattt taaatatcag tgcagactgg
tagaaaaagg 9780tcggtacaag acacattatc ctcaattgtg gcttttctca
gatgtgctgt ccattgattt 9840cttaggaccc ctgtctatat cttcaactct
attgggtatt ctgtataaac agacgttatc 9900ttctcgagac aaaaatgagt
tgagagaact cgctaacttg tcttcattgt tgagatcagg 9960agaaggatgg
gaagatatcc atgtcaaatt cttctctaag gacactttac tctgccctga
10020agagatccga catgcgtgca aatttgggat tgctaaggaa tccgctgttt
taagctatta 10080tcctccttgg tctcaagagt cttatggagg catcacctcg
atccccgtat atttttcgac 10140caggaagtat cccaaaattt tagatgtccc
tcctcgggtt caaaacccat tggtctcggg 10200tctacgattg gggcaactcc
ctactggagc acattataag attaggagca ttgtaaagaa 10260caagaacctt
cgttatagag atttccttag ttgtggggat ggatctgggg ggatgaccgc
10320ggcactattg agagaaaaca gacaaagtag gggaatcttc aacagcctgt
tagagttagc 10380cggatctctt atgagaggag catctccaga gcctccaagt
gcactggaga cgctcgggca 10440agaacgatct aggtgtgtga atggaagcac
atgttgggag tactcatctg acctaagcca 10500aaaagagaca tgggattact
tcttaagatt gaagagaggc ctgggtttga ccgtggactt 10560aatcaccatg
gacatggagg tcagagaccc taatacaagt ttgatgatag aaaagaacct
10620caaagtttat ctgcatcaga tattagaacc aactggtgtc ttaatatata
aaacatacgg 10680gacccatatt gcgacacaaa cagataatat cctgacgata
atcggtcctt tctttgagac 10740ggttgaccta gtccagtccg aatacagcag
ctcacaaacg tccgaggtct attttgtagg 10800acgaggcttg cgctctcatg
ttgacgaacc ctgggtggac tggccatcct taatggacaa 10860ttggagatcc
atttatgctt ttcatgatcc tactacagaa tttatcagag caaaaaaagt
10920ctgtgaaatt gacagtctta taggcattcc ggctcaattc attccagacc
catttgtaaa 10980tctcgagacc atgctacaga tagttggtgt tccaacagga
gtttcgcatg ccgcagctct 11040attatcatca caatatccaa atcaattggt
cacaacgtca atattttata tgacactcgt 11100gtcttattat aatgtaaacc
atattcgaag aagccccaag cctttctctc ctccgtctga 11160tggagtctca
cagaacattg gttcagccat agtcggacta agtttttggg tgagtttgat
11220ggagaatgat ctcggattat acaaacaggc tctaggtgca ataaagacgt
cattccctat 11280tagatggtcc tctgtccaga ccaaggatgg gtttacacaa
gaatggagaa ctaaaggaaa 11340cggaattcct aaagattgtc gtctctcaga
ctctttggct cagataggaa actggatcag 11400agcgatggaa ttggttagga
acaaaacgag gcaatcagga ttttctgaaa ccctatttga 11460tcaattctgc
ggacttgcag accatcacct caaatggcgg aagttgggaa acagaacagg
11520aattattgat tggctaaata atagaatttc atccattgac aaatccatct
tggtgaccaa 11580aagtgatctg catgacgaga actcatggag ggagtgaaga
tgtattcttc cacctctcat 11640tgggtgatac ccatatatga aaaaaactat
aagtacttta aactctcttt gttttttaat 11700gtatatctgg ttttgttgtt tccgt
1172516238PRTHomo sapiens 16Met Pro Val Gly Gln Ala Asp Tyr Phe Glu
Tyr His Gln Glu Gly Gly1 5 10 15Pro Asp Gly Glu Pro Asp Met Pro Pro
Gly Ala Ile Glu Gln Gly Pro 20 25 30Ala Asp Asp Pro Gly Glu Gly Pro
Ser Thr Gly Pro Arg Gly Gln Gly 35 40 45Asp Gly Gly Arg Arg Lys Lys
Gly Gly Trp Phe Gly Lys His Arg Gly 50 55 60Gln Gly Gly Ser Asn Gln
Lys Phe Glu Asn Ile Ala Asp Gly Leu Arg65 70 75 80Thr Leu Leu Ala
Arg Cys His Val Glu Arg Thr Thr Asp Glu Gly Thr 85 90 95Trp Val Ala
Gly Val Phe Val Tyr Gly Gly Ser Lys Thr Ser Leu Tyr 100 105 110Asn
Leu Arg Arg Gly Ile Ser Leu Ala Ile Pro Gln Cys Arg Leu Thr 115 120
125Pro Leu Ser Arg Leu Pro Phe Gly Met Ala Pro Gly Pro Gly Pro Gln
130 135 140Pro Gly Pro Leu Arg Glu Ser Ile Val Cys Tyr Phe Ile Val
Phe Leu145 150 155 160Gln Thr His Ile Phe Ala Glu Gly Leu Lys Asp
Ala Ile Lys Asp Leu 165 170 175Val Met Pro Lys Pro Ala Pro Thr Cys
Asn Ile Lys Ala Thr Val Cys 180 185 190Ser Phe Asp Asp Gly Val Asp
Leu Pro Pro Trp Phe Pro Pro Met Val 195 200 205Glu Gly Ala Ala Ala
Glu Gly Asp Asp Gly Asp Asp Gly Asp Asp Gly 210 215 220Asp Glu Gly
Gly Asp Gly Asp Glu Gly Glu Glu Gly Gln Glu225 230 23517717DNAHomo
sapiens 17atgccagtcg gccaggctga ttactttgaa taccaccagg aggggggacc
agacggagaa 60ccagacatgc caccaggagc cattgaacag ggaccagcag acgatcctgg
agagggacca 120tcaactggac cccgaggaca gggggacggc ggaaggagaa
agaaaggggg atggttcgga 180aagcaccgag gacagggagg gagcaaccag
aaatttgaaa atatcgctga cggcctgcga 240acactgctgg caaggtgcca
tgtggagaga accacagatg aaggcacatg ggtcgccgga 300gtgttcgtct
acggcggaag caagacttcc ctgtataacc tgcggcgcgg catctctctg
360gccattccac agtgccggct gacccctctg agtcgcctgc cattcgggat
ggctcctgga 420ccaggaccac agcctggacc actgagggag tccatcgtgt
gctacttcat tgtctttctg 480cagacacaca tctttgccga aggcctgaag
gacgccatca aggacctggt catgcccaag 540cctgcaccaa cttgcaatat
caaggccacc gtgtgcagtt tcgacgatgg cgtggacctg 600cccccttggt
ttccacctat ggtggaggga gccgctgcag aaggggacga tggcgatgac
660ggggacgatg gggatgaagg cggggacggc gatgagggag aagaggggca ggaataa
7171830DNAArtificial Sequencesynthesized primer 18actggaattc
atgcatcaga agcgaactgc 301929DNAArtificial Sequencesynthesized
primer 19actgggatcc tcactgctgg gaggcacac 292029DNAArtificial
Sequencesynthesized primer 20actgggatcc atgcaggccg agggcagag
292130DNAArtificial Sequencesynthesized primer 21actgggatcc
tcatcttctc tggccgctgg 302231DNAArtificial Sequencesynthesized
primer 22actggaattc atggaatcac ggaaggacat c 312332DNAArtificial
Sequencesynthesized primer 23actgggatcc ttaaagcttc agctggctac ag
322429DNAArtificial Sequencesynthesized primer 24actggaattc
atgccagtcg gccaggctg 292531DNAArtificial Sequencesynthesized primer
25actgggatcc ttattcctgc ccctcttctc c 31269PRTArtificial
Sequenceantigenic epitope from human MAGEA3 protein 26Glu Val Asp
Pro Ile Gly His Leu Tyr1 5279PRTArtificial Sequenceantigenic
epitope from human MAGEA3 protein 27Phe Leu Trp Gly Pro Arg Ala Leu
Val1 5289PRTArtificial Sequenceantigenic epitope from human MAGEA3
protein 28Lys Val Ala Glu Leu Val His Phe Leu1 5299PRTArtificial
Sequenceantigenic epitope from human MAGEA3 protein 29Thr Phe Pro
Asp Leu Glu Ser Glu Phe1 5309PRTArtificial Sequenceantigenic
epitope from human MAGEA3 protein 30Val Ala Glu Leu Val His Phe Leu
Leu1 53110PRTArtificial Sequenceantigenic epitope from human MAGEA3
protein 31Met Glu Val Asp Pro Ile Gly His Leu Tyr1 5
103210PRTArtificial Sequenceantigenic epitope from human MAGEA3
protein 32Arg Glu Pro Val Thr Lys Ala Glu Met Leu1 5
10339PRTArtificial Sequenceantigenic epitope from human MAGEA3
protein 33Ala Glu Leu Val His Phe Leu Leu Leu1 5349PRTArtificial
Sequenceantigenic epitope from human MAGEA3 protein 34Trp Gln Tyr
Phe Phe Pro Val Ile Phe1 5359PRTArtificial Sequenceantigenic
epitope from human MAGEA3 protein 35Glu Gly Asp Cys Ala Pro Glu Glu
Lys1 53616PRTArtificial Sequenceantigenic epitope from human MAGEA3
protein 36Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu
Glu Tyr1 5 10 153715PRTArtificial Sequenceantigenic epitope from
human MAGEA3 protein 37Arg Lys Val Ala Glu Leu Val His Phe Leu Leu
Leu Lys Tyr Arg1 5 10 153816PRTArtificial Sequenceantigenic epitope
from human MAGEA3 protein 38Ala Cys Tyr Glu Phe Leu Trp Gly Pro Arg
Ala Leu Val Glu Thr Ser1 5 10 153912PRTArtificial Sequenceantigenic
epitope from human MAGEA3 protein 39Val Ile Phe Ser Lys Ala Ser Ser
Ser Leu Gln Leu1 5 104015PRTArtificial Sequenceantigenic epitope
from human MAGEA3 protein 40Val Phe Gly Ile Glu Leu Met Glu Val Asp
Pro Ile Gly His Leu1 5 10 154115PRTArtificial Sequenceantigenic
epitope from human MAGEA3 protein 41Gly Asp Asn Gln Ile Met Pro Lys
Ala Gly Leu Leu Ile Ile Val1 5 10 154215PRTArtificial
Sequenceantigenic epitope from human MAGEA3 protein 42Thr Ser Tyr
Val Lys Val Leu His His Met Val Lys Ile Ser Gly1 5 10
154316PRTArtificial Sequenceantigenic epitope from human MAGEA3
protein 43Arg Lys Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr
Arg Ala1 5 10 154416PRTArtificial Sequenceantigenic epitope from
human MAGEA3 protein 44Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro
Val Thr Lys Ala Glu1 5 10 15459PRTArtificial SequenceMHC-I
restricted immunodominant epitope from melanoma associated antigen
dopachrome tautomerase 45Ser Val Tyr Asp Phe Phe Val Trp Leu1
54614PRTArtificial SequenceMHC-II restricted immunodominant epitope
from melanoma associated antigen dopachrome tautomerase 46Lys Phe
Phe His Arg Thr Cys Lys Cys Thr Gly Asn Phe Ala1 5
10479PRTArtificial Sequenceantigenic epitope 47Arg Ala His Tyr Asn
Ile Val Thr Phe1 5488PRTArtificial Sequenceantigenic epitope 48Arg
Gly Pro Glu Ser Arg Leu Leu1 5497PRTArtificial Sequenceantigenic
epitope 49Arg Ser Arg Tyr Lys Leu Leu1 5509PRTArtificial
Sequenceantigenic epitope 50Val Tyr Gly Gly Ser Lys Thr Ser Leu1
5
* * * * *
References