U.S. patent application number 16/107559 was filed with the patent office on 2019-02-14 for brachyury protein, non-poxvirus non-yeast vectors encoding brachyury protein, and their use.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Claudia M. Palena, Jeffrey Schlom.
Application Number | 20190046619 16/107559 |
Document ID | / |
Family ID | 49304319 |
Filed Date | 2019-02-14 |
United States Patent
Application |
20190046619 |
Kind Code |
A1 |
Schlom; Jeffrey ; et
al. |
February 14, 2019 |
BRACHYURY PROTEIN, NON-POXVIRUS NON-YEAST VECTORS ENCODING
BRACHYURY PROTEIN, AND THEIR USE
Abstract
Brachyury protein can be used to induce Brachyury-specific CD4+
T cells in vivo and ex vivo. It is also disclosed that Brachyury
protein can be used to stimulate the production of both
Brachyury-specific CD4+ T cells and Brachyury-specific CD8+ T cells
in a subject, such as a subject with cancer. In some embodiments,
the methods include the administration of a Brachyury protein. In
additional embodiments, the methods include the administration of a
nucleic acid encoding the Brachyury protein, such as in a non-pox
non-yeast vector. In further embodiments, the method include the
administration of host cells expressing the Brachyury protein.
Inventors: |
Schlom; Jeffrey; (Potomac,
MD) ; Palena; Claudia M.; (Potomac, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
49304319 |
Appl. No.: |
16/107559 |
Filed: |
August 21, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14428308 |
Mar 13, 2015 |
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PCT/US2013/059737 |
Sep 13, 2013 |
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16107559 |
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61701525 |
Sep 14, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55583
20130101; A61K 39/0011 20130101; C07K 14/82 20130101; A61K 45/06
20130101; G01N 33/57415 20130101; C12N 2710/16234 20130101; C12N
15/86 20130101; Y02A 50/466 20180101; A61K 39/39558 20130101; A61N
5/10 20130101; C07K 14/4702 20130101; A61K 39/0005 20130101; A61K
39/39 20130101; Y02A 50/30 20180101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; G01N 33/574 20060101 G01N033/574; A61N 5/10 20060101
A61N005/10; A61K 39/39 20060101 A61K039/39; C12N 15/86 20060101
C12N015/86; C07K 14/82 20060101 C07K014/82; C07K 14/47 20060101
C07K014/47; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for inducing an immune response comprising a Brachyury
specific CD4+ T cell response comprising administering to a subject
an effective amount of an adenoviral vector encoding: (a) a protein
comprising an amino acid sequence at least 90% identical to the
amino acid sequence set forth as SEQ ID NO: 1; or (b) a polypeptide
comprising at least 15 consecutive amino acids of the amino acid
sequence set forth as SEQ ID NO: 1 that specifically binds a Major
Histocompatibility Class (MHC class II) molecule, or through
internalization and cross-presentation can bind to MHC Class I;
thereby inducing the immune response comprising a Brachyury
specific CD4+ T cell response.
2. The method of claim 1, wherein the immune response further
comprises a Brachyury specific CD8+ T cell response.
3. The method of claim 1, further comprising measuring the
Brachyury specific CD4+ T cell response.
4. (canceled)
5. The method of claim 1, wherein the subject is human.
6. The method of claim 1, wherein the subject has cancer.
7. The method of claim 6, wherein the cancer is a breast cancer,
small intestine cancer, stomach cancer, kidney cancer, bladder
cancer, uterus cancer, ovarian cancer, testes cancer, lung cancer,
colon cancer, prostate cancer, chronic lymphocytic leukemia (CLL),
a B cell lymphoma, a Burkitt's lymphoma or a Hodgkin's
lymphoma.
8.-11. (canceled)
12. The method of claim 1, comprising administering to the subject
an effective amount of the adenoviral vector encoding the
polypeptide or the protein sufficient to induce Brachyury specific
CD4+ T cells and/or CD8+ T cells.
13. (canceled)
14. The method of claim 1, wherein the polypeptide comprises 15 to
435 consecutive amino acids of the amino acid sequence set forth as
SEQ ID NO: 1.
15. The method of claim 1, wherein the polypeptide comprises at
least 20 consecutive amino acids of the amino acid sequence set
forth as SEQ ID NO: 1.
16.-17. (canceled)
18. The method of claim 1, wherein the adenoviral vector encodes a
costimulatory molecule.
19. The method of claim 18, wherein the costimulatory molecule is
one or more of B7-1, B7-2, LFA-3 or ICAM-1.
20. The method of claim 1, wherein the adenoviral vector comprises
a DNA sequence encoding an immunostimulatory molecule, wherein the
immunostimulatory molecule is selected from the group consisting of
IL-2, ICAM-1, LFA-3, CD72, GM-CSF, TNF-.alpha., IFN-.gamma., IL-12,
and IL-6.
21. (canceled)
22. The method of claim 1, further comprising administering to the
subject an effective amount of an adjuvant.
23. The method of claim 22, wherein the adjuvant is chitosan.
24. The method of claim 1, further comprising administering to the
subject a therapeutically effective amount of an agent selected
from the group consisting of a chemotherapeutic agent, radiation, a
small molecule targeted therapeutic, and monoclonal antibodies.
25. The method of claim 25, wherein the agent is an epithelial
growth factor receptor inhibitor, a transforming growth factor
(TGF)-.beta. inhibitor, or a tyrosine kinase inhibitor.
26. The method claim 1, wherein the subject has cancer, and wherein
the cancer is a chemotherapy resistant cancer or a radiation
resistant cancer.
27.-49. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional application of U.S.
patent application Ser. No. 14/428,308, filed Mar. 13, 2015, now
abandoned, which claims the benefit of U.S. national phase of
International Patent Application No. PCT/US2013/059737, filed Sep.
13, 2013, which claims the benefit of U.S. Provisional Patent
Application No. 61/701,525, filed Sep. 14, 2012, each of which is
incorporated by reference in its entirety herein.
SEQUENCE LISTING
[0002] Incorporated by reference in its entirety herein is a
nucleotide/amino acid sequence listing submitted concurrently
herewith and identified as follows: One 14,638 Byte ASCII (Text)
file named "740352_ST25.TXT," created on Aug. 21, 2018.
[0003] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand. In
the accompanying sequence listing:
[0004] SEQ ID NO: 1 is an amino acid sequence of a human Brachyury
protein.
[0005] SEQ ID NO: 2 is a nucleic acid sequence encoding a human
Brachyury protein.
[0006] SEQ ID NO: 3 is an amino acid sequence of a murine Brachyury
protein.
[0007] SEQ ID NO: 4 is a nucleic acid sequence encoding a murine
Brachyury protein.
[0008] SEQ ID NO: 5 is a Brachyury class IIA epitope.
[0009] SEQ ID NO: 6 is a Brachyury class IIB epitope.
FIELD
[0010] This application relates to the field of cancer
therapeutics, specifically to the use of a Brachyury protein and
non-poxvirus, non-yeast vectors encoding a Brachyury protein for
the treatment of cancer.
BACKGROUND
[0011] The Brachyury gene was initially cloned from mouse
developmental mutants characterized by an arrest in mesoderm
formation (Hermann et al, Nature 1990; 343:617-22) has been
recognized as gene that is important in mesoderm development during
gastrulation. Brachyury is a member of a family of transcription
factors, designated T-box transcription factors, these factors are
characterized by a conserved DNA-binding domain (Papaioannou et
al., Bioessays 1998; 20:9-19). These transcription factors play an
essential role in the formation and organization of mesoderm in
vertebrates (see, for example, Edwards et al., Genome Res 1996;
6:226-33). In addition to the important role of the T-box proteins
in the control of developmental processes, several members of this
family are deregulated in cancer. For example, the human Tbx2 gene
has been reported to be amplified in pancreatic cancer cell lines
(Mahlamaki et al., Genes Chromosomes Cancer 2002, 35:353-8) and is
overexpressed in BRCA-1- and BRCA-2-mutated breast tumors (Sinclair
et al., Cancer Res 2002; 62:3587-91). In addition, Tbx3 expression
has been shown to be augmented in certain human breast cancer cell
lines (Fan et al., Cancer Res 2004; 64:5132-9). Expression of
Brachyury has also been documented in human teratocarcinoma lines:
a subset of germ cell tumors, teratocarcinomas are embryonal
carcinoma cells with competence for mesoderm differentiation
(Gokhale et al., Cell Growth Differ 2000; 11:157-62) and in
chordomas (see, for example, Vojovic et al., J Pathol 2006;
209:157-65).
[0012] Immunotherapeutic interventions against cancer depend on the
identification of tumor antigens able to elicit a host immune
response against the tumor cells. Good targets are molecules that
are selectively expressed by malignant cells and that are also
essential for malignant transformation and/or tumor progression.
The epithelial-mesenchymal transition (EMT) has been recognized as
a key step during the progression of primary tumors into metastases
(Thiery et al., Nat Rev Cancer 2002; 2:442-54). Several molecules
have been identified that play a key role in EMT during tumor
progression (Huber et al., Curr Opin Cell Biol 2005; 17:548-58),
among them the transcription factors Twist, Snail, and Slug (Yang
et al., Cell 2004; 117:927-39; Cano et al., Nat Cell Biol 2000; 2:
76-83). Molecules that trigger EMT could function to prevent tumor
invasion and metastasis. However, a need remains for reagents that
induce an effective immune response to cancer, including a CD4 and
a CD8 T cell response.
SUMMARY
[0013] It is disclosed herein that Brachyury protein or a Brachyury
polypeptide can be used to induce Brachyury-specific CD4+ T cells
in vivo and ex vivo. It is also disclosed that Brachyury protein
and Brachyury polypeptides can be used to stimulate the production
of both Brachyury-specific CD4+ T cells and Brachyury-specific CD8+
T cells. Brachyury is expressed in numerous human cancers, such as
in cancer of the small intestine, stomach, kidney bladder, uterus,
ovary, testes, lung, colon, prostate, bronchial tube, chronic
lymphocytic leukemia (CLL), other B cell-based malignancies, and
breast cancer, such as infiltrating ductal carcinomas of the
breast. Thus, Brachyury protein, Brachyury polypeptides, and
nucleic acids encoding Brachyury protein and/or polypeptides, can
be used to produce Brachyury specific CD4+ T cells, and CD8+ T
cells, that can be used for the treatment or prevention of
cancer.
[0014] In some embodiments, methods are disclosed for inducing CD4+
Brachyury-specific T cells and/or CD8+ Brachyury specific T cells.
The methods include the use of a Brachyury protein, a Brachyury
polypeptide, nucleic acids encoding the Brachyury protein and/or
Brachyury polypeptides, or host cells expressing the Brachyury
protein or polypeptide, such as such as a Salmonella or Lisleria
host cells. These agents can be administered either alone or in
conjunction with another agent, such as a cytokine and/or another
cancer therapy. In some embodiments, methods are disclosed for
treating a subject with a cancer, such as a breast cancer, cancer
of the small intestine, stomach, kidney, bladder, uterus, ovaries,
testes lung, colon or prostate, or a tumor of B cell origin, or for
preventing these cancers in a subject. In some embodiments, the
methods include measuring Brachyury-specific CD4+ T cells. In
further embodiments, the methods also induce CD8+ Brachyury
specific T cells.
[0015] Non-pox non-yeast vectors encoding a Brachyury protein are
disclosed that can be used to induce CD4+ Brachyury-specific T
cells and/or CD8+ Brachyury-specific T cells. In some non-limiting
examples, the vector is an alphavirus, a lentiviurs, an adenovirus,
a measles virus or a poliovirus vector. In additional embodiments,
host cells transformed with these vectors, such as Salmonella and
Listeria host cells are provided.
[0016] In additional embodiments, methods are provided for
inhibiting the growth of a cancer cell in a subject. These methods
include contacting a dendritic cell with a protein comprising an
amino acid sequence at least 90% identical to the amino acid
sequence set forth as SEQ ID NO: 1, a polypeptide comprising at
least 15 consecutive amino acids of the amino acid sequence set
forth at SEQ ID NO: 1 that specifically binds a Major
Histocompatibility Class (MHC class II) molecule, or a Listeria or
Salmonella host cell expressing the protein or the polypeptide
thereby preparing a specific antigen presenting cell. These methods
also include administering the antigen presenting cell to the
subject, thereby inducing an immune response and inhibiting the
growth of the cancer cell.
[0017] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. Brachyury-specific CD4+ T cells can be expanded from
PBMCs of normal donors by culture in the presence of purified
recombinant Brachyury protein. Dendritic cells (DCs) from 2 normal
donors were prepared by culture in the presence of GM-CSF and IL-4.
On day 5, a purified recombinant Brachyury protein was added (10
g/ml) for 48 hours. For donor 2, an additional culture was set up
using purified HSA (human serum albumin) control protein (10
.mu.g/ml). On day 7, protein-pulsed DCs were harvested, irradiated
(20 Gy) and used as antigen-presenting cells (APCs) to stimulate
autologous PBMCs (ratio DC:PBMCs equal to 1:10). On days 3 and 5,
IL-2 (20 U/ml) was added to the cultures. T cells were harvested on
day 7 and CD4+ T cells were isolated by negative selection with
magnetic beads. CD4+ T cells were stimulated in similar manner for
an additional 7-day cycle. On day 7, CD4+ T cells were re-isolated
by using magnetic beads and evaluated for IFN-gamma production in
response to autologous, irradiated PBMCs (ratio PBMCs:T cells equal
to 3:1) alone or pulsed with control HSA protein vs. Brachyury
protein (10 .mu.g/ml). Culture supernatants were collected at 96
hours and evaluated for IFN-gamma by ELISA.
[0019] FIG. 2. A Brachyury-specific CD4 T cell line releases
cytokines and chemokines when stimulated with a class-II DRB1*0401
binding Brachyury peptide. Brachyury class IIA epitope (SEQ ID NO:
5) and Brachyury class IIB epitope (SEQ ID NO: 6).
[0020] FIG. 3A-3E. Brachyury induces an epithelial-to-mesenchymal
transition (EMT) in breast carcinoma cells. (A) MCF7-pcDNA and
MCF7-phBrachyury stable transfectants grown on plastic surface for
bright field images (top panels) and grown immunofluorescence
analysis of E-cadherin expression (green signal); blue signal
represents DAPI-stained nuclei (bottom panels). (B) Membrane images
from in vitro cell migration (top panels) and ECM invasion assays
(bottom panels) for MCF7-pcDNA and MCF7-pBrachyury cells. Results
are representative of three experiments. (C, D) Real-time PCR was
performed on indicated cell pairs for Brachyury, Fibronectin, and
Vimentin. Values (mean.+-.SEM) are expressed as a ratio to the
endogenous control GAPDH. (E) Immunofluoresent analysis of
Fibronectin expression in MDA-MB-436-con.shRNA and
MDA-MB-436-Br.shRNA stable transfectants (original magnification
20.times.). The green signal represents staining for Fibronectin;
the blue signal represents the DAPI-stained nuclei.
[0021] FIGS. 4A-4D. Effect of Brachyury expression on stem cell
marker expression and mammosphere growth of tumor cells. Real-time
PCR was performed for indicated genes on cDNA from (A) MCF7-pcDNA
and MCF7-phBrachyury cells and (C) MDA-MB-436-con.shRNA and
MDA-MB-436-Br.shRNA cells. Values (mean.+-.SEM) are expressed as a
ratio to the endogenous control GAPDH. Mammospheres were grown from
the MCF7 (B) or the MDA-MB-436 (D) tumor cell pairs on
ultra-low-attachment plates. Primary mammospheres were dissociated
and re-plated for secondary cultures. Bright field images of
mammospheres at 10.times. magnification and mean number of
mammospheres per 10.times. microscope field are shown for secondary
cultures in the left and right panels, respectively. Error bars
indicate SEM of 8-10 measurements.
[0022] FIGS. 5A-5D. Expression of Brachyury mRNA in breast
carcinoma tissues. (A) Real-time PCR was performed for Brachyury,
Twist, Snail, and Slug on human breast primary tumor tissue cDNA
from 41 breast cancer patients. As controls, 7 samples of normal
breast cDNA were also analyzed, each obtained from a histologically
normal section of breast from a patient with cancer or fibrocystic
disease. (B) Real-time PCR was performed for Brachyury on human
primary breast tumor tissue cDNA from 107 invasive ductal
adenocarcinomas, 6 invasive lobular adenocarcinomas, and 5 mixed
ductal/lobular adenocarcinomas. As controls, 7 samples of normal
breast cDNA were also analyzed, each obtained from a histologically
normal section of breast from a patient with cancer or fibrocystic
disease. All values and the means for each group are expressed as a
ratio to the endogenous control GAPDH. Brachyury expression is
shown for (B) breast primary tumor tissues from stages I-III
grouped together, (C) breast primary tumor tissues grouped by
histological tumor grade (Nottingham grading), (D) breast primary
tumor tissues grouped by ER and PR expression (ER+PR+ versus
ER-PR-).
[0023] FIGS. 6A-6F. Immunohistochemical detection of Brachyury in
primary breast carcinoma and metastatic tissues. Transmitted light
photomicrographs of tissue sections stained for Brachyury
expression in (A) a primary infiltrating ductal carcinoma, Grade 3
(patient 11); (B) a primary infiltrating ductal carcinoma, Grade 3
and (C) corresponding lymph node metastasis from the same patient
(patient 6); (D, E) bone metastatic lesions from two different
breast cancer patients (patients 22 and 23); (F) brain metastatic
lesion from a breast cancer patient (patient 24). The brown signal
represents staining for Brachyury. Magnification 20.times.
(A-F).
[0024] FIG. 7A-7C. Immunogenicity of Brachyury. (A) Detection of
IgG antibodies against Brachyury in the serum of normal donors and
metastatic breast cancer patients. Shown is the number of positive
cases in each group, stratified by titer of IgG as determined by
ELISA assay. Statistical analysis was performed, comparing breast
vs. normal donors. Brachyury-specific CTLs were generated from the
peripheral blood of a prostate cancer patient via stimulation with
a Brachyury-derived peptide. Cytotoxic activity was assessed in a
16-h assay against (B) HLA-A2.sup.+/Brachyury.sup.+ MCF7 cells or
HLA-A2.sup.-/Brachyury.sup.+ MDA-MB-436 cells, and (C)
HLA-A2.sup.+/Brachyury.sup.+ MDA-MB-231 cells. The
effector-to-target (E:T) ratios are indicated; major
histocompatibility complex (MHC)-restriction was analyzed by
pre-incubation of the targets with control IgG or a HLA-A specific
antibody.
DETAILED DESCRIPTION
[0025] It is disclosed herein that Brachyury protein and Brachyury
polypeptides of greater than 15 amino acids in length can be used
to induce Brachyury-specific CD4+ T cells in vivo and ex vivo. It
is also disclosed that Brachyury protein and Brachyury polypeptides
can be used to stimulate the production of both Brachyury-specific
CD4+ T cells and Brachyury-specific CD8+ T cells. Brachyury protein
is expressed in numerous human cancers, such as cancer of the small
intestine, stomach, kidney bladder, uterus, ovary, testes, lung,
colon, prostate, bronchial tube, chronic lymphocytic leukemia
(CLL), other B cell-based malignancies and breast cancer, such as
infiltrating ductal carcinomas of the breast and thus the method
disclosed herein can be used to treat or prevent these cancers. In
specific non-limiting examples, the breast cancer is an estrogen
receptor negative and progesterone receptor negative breast cancer.
In additional non-limiting examples, the cancer is any cancer that
is radiation resistant and/or chemotherapy resistant. The cancer
can express Brachyury or have the potential to express
Brachyury.
[0026] Non-pox non-yeast vectors encoding a Brachyury protein or a
Brachyury polypeptide, and host cells expressing Brachyury are
disclosed, these vectors and host cells can be used to induce CD4+
Brachyury-specific T cells and/or CD8+ T cells. In some
non-limiting examples, these vectors are adenovirus vectors,
alphavirus vectors, lentivirus vectors, poliovirus vectors,
Listeria vectors, Salmonella vectors or measles virus vectors. In
additional embodiments, host cells transformed with these vectors,
and methods of using these proteins, polynucleotides, vectors, and
host cells are provided. In some examples the host cells are
Salmonella or Listeria host cells.
[0027] Thus, methods are provided for inducing CD4+
Brachyury-specific T cells and/or CD8+ T cells. The methods include
the use of a Brachyury protein, Brachyury polypeptide, dendritic
cells expressing Brachyury epitopes, nucleic acids encoding
Brachyury protein and/or polyhpeptides, including non-pox non-yeast
vectors encoding the Brachyury protein and/or the Brachyury
polypeptide to induce the production of CD4+ Brachyury specific T
cells. In some embodiments, methods are disclosed for treating a
subject having cancer, such as, but not limited to, a cancer of the
small intestine, stomach, kidney bladder, uterus, ovary, testes,
lung, colon, prostate, bronchial tube, chronic lymphocytic leukemia
(CLL), other B cell-based malignancies, or breast cancer, such as
an infiltrating ductal carcinoma or estrogen receptor negative and
progesterone receptor negative breast cancers. Any of these cancers
can be chemotherapy resistant and/or radiation resistant. The
cancer can express Brachyury or have the potential to express
Brachyury. Methods are also disclosed for preventing these
cancers.
[0028] These methods include inducing CD4+ Brachyury-specific T
cells; the method can also include inducing CD8+ Brachyury-specific
T cells. The Brachyury protein, Brachyury polypeptide, dendritic
cells, nucleic acid, or non-pox non-yeast vector encoding the
Brachyury protein can be administered to the subject either alone
or in conjunction with a second agent, such as radiation therapy
and/or chemotherapy.
[0029] In some embodiments, the Brachyury protein comprises an
amino acid sequence at least 90% identical, or at least 95%
identical, to the amino acid sequence set forth as SEQ ID NO: 1. In
other embodiments, the Brachyury protein comprises, or consists of,
the amino acid sequence set forth as SEQ ID NO: 1, the amino acid
sequence set forth as SEQ ID NO: 1 without the N-terminal
methionine, or the amino acid sequence set forth as SEQ ID NO: 1,
with substitutions at position 177 (Asp vs. Gly, respectively),
position 368 (Thr vs. Ser, respectively) and position 409 (Asn vs.
Asp, respectively).
[0030] In further embodiments, a Brachyury polypeptide comprises at
least 15 amino acids of the amino acid sequence set forth as SEQ ID
NO: 1, such as at least 20, at least 30, at least 40, at least 50,
at least 60, at least 70, at least 80, at least 90, at least 100,
at least 200 amino acids of the amino acid sequence set forth as
SEQ ID NO: 1, wherein the entirety of SEQ ID NO: 1 is not included
in the polypeptide. In additional embodiments, a Brachyury
polpeptide is 15 to 100 amino acids of SEQ ID NO: 1, such as 15 to
200 amino acids, 15 to 300 amino acids, 15 to 400 amino acids, or
15 to 435 amino acids of SEQ ID NO: 1.
[0031] In additional embodiments, methods are provided for
inhibiting the growth of a cancer cell in a subject. These methods
include contacting a dendritic cell with a protein comprising an
amino acid sequence at least 90% identical to the amino acid
sequence set forth as SEQ ID NO: 1, a polypeptide comprising at
least 15 consecutive amino acids of the amino acid sequence set
forth at SEQ ID NO: 1 that specifically binds a Major
Histocompatibility Class (MHC class II) molecule, or a Listeria or
Salmonella host cell expressing the protein, thereby preparing a
specific antigen presenting cell. These methods also include
administering the antigen presenting cell to the subject, thereby
inducing an immune response and inhibiting the growth of the cancer
cell.
Terms
[0032] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0033] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of specific terms are
provided:
[0034] Adenovirus: A virus of the family Adenoviridae, which are
medium-sized (90-100 nm), nonenveloped icosahedral viruses composed
of a nucleocapsid and a double-stranded linear DNA genome. The
adenovirus genome is linear, non-segmented double-stranded (ds) DNA
that is between 26 and 45 kb. This allows the virus to
theoretically carry 22 to 40 genes. The linear dsDNA genome is able
to replicate in the nucleus of mammalian cells using the host's
replication machinery. However, adenoviral DNA does not integrate
into the genome and is not replicated during cell division.
[0035] Adeno-associated Virus: Adeno-associated virus (AAV) is a
small virus that infects humans and some other primate species. AAV
is not currently known to cause disease and consequently the virus
causes a very mild immune response. AAV can infect both dividing
and non-dividing cells and may incorporate its genome into that of
the host cell. The AAV genome is built of single-stranded
deoxyribonucleic acid (ssDNA), either positive- or negative-sensed,
which is about 4.7 kilobase long. The genome comprises inverted
terminal repeats (ITRs) at both ends of the DNA strand, and two
open reading frames (ORFs): rep and cap. Rep is composed of four
overlapping genes encoding Rep proteins required for the AAV life
cycle, and Cap contains overlapping nucleotide sequences of capsid
proteins: VP1, VP2 and VP3, which interact together to form a
capsid of an icosahedral symmetry. For gene therapy, ITRs seem to
be the only sequences required in cis next to the therapeutic gene:
structural (cap) and packaging (rep) genes can be delivered in
trans.
[0036] Adjuvant: A vehicle used to enhance antigenicity. Adjuvants
include a suspension of minerals (alum, aluminum hydroxide, or
phosphate) on which antigen is adsorbed; or water-in-oil emulsion
in which antigen solution is emulsified in mineral oil (Freund
incomplete adjuvant), sometimes with the inclusion of killed
mycobacteria (Freund's complete adjuvant) to further enhance
antigenicity (inhibits degradation of antigen and/or causes influx
of macrophages). Immunstimulatory oligonucleotides (such as those
including a CpG motif) can also be used as adjuvants (for example
see U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371;
6,239,116; 6,339,068; 6,406,705; and U.S. Pat. No. 6,429,199).
Adjuvants include biological molecules (a "biological adjuvant"),
such as costimulatory molecules. Exemplary adjuvants include IL-2,
RANTES, GM-CSF, TNF-.alpha., IFN-.gamma., G-CSF, LFA-3, CD72, B7-1,
B7-2, OX-40L and 4-1 BBL. Another exemplary adjuvant is chitosan.
Another adjuvant is Bacillus-Calmette-Guerin adjvant.
[0037] Alphavirus: A virus that belongs to the group IV Togaviridae
family of viruses. The alphaviruses are small, spherical, enveloped
viruses with a genome of a single positive sense strand RNA. The
total genome length ranges between 11,000 and 12,000 nucleotides,
and has a 5' cap, and 3' poly-A tail. The four non-structural
protein genes are encoded in the 5' two-thirds of the genome, while
the three structural proteins are translated from a subgenomic mRNA
colinear with the 3' one-third of the genome. The alphavirusus
include the Ross River virus, Sindbis virus, Semliki Forest virus,
and Venezuelan equine encephalitis virus.
[0038] Antigen: A compound, composition, or substance that can
stimulate the production of antibodies or a T cell response in an
animal, including compositions that are injected or absorbed into
an animal. An antigen reacts with the products of specific humoral
or cellular immunity, including those induced by heterologous
immunogens. The term "antigen" includes all related antigenic
epitopes. "Epitope" or "antigenic determinant" refers to a site on
an antigen to which B and/or T cells respond. In one embodiment, T
cells respond to the epitope, when the epitope is presented in
conjunction with an MHC molecule. Epitopes can be formed both from
contiguous amino acids or noncontiguous amino acids juxtaposed by
tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5, about 9, or
about 8-10 amino acids in a unique spatial conformation, but is
generally not more than 20 amino acids in length. Methods of
determining spatial conformation of epitopes include, for example,
x-ray crystallography and 2-dimensional nuclear magnetic
resonance.
[0039] An antigen can be a tissue-specific antigen, or a
disease-specific antigen. These terms are not exclusive, as a
tissue-specific antigen can also be a disease specific antigen. A
tissue-specific antigen is expressed in a limited number of
tissues, such as a single tissue. Specific, non-limiting examples
of a tissue specific antigen are a prostate specific antigen, a
uterine specific antigen, and/or a testes specific antigen. A
tissue specific antigen may be expressed by more than one tissue,
such as, but not limited to, an antigen that is expressed in more
than one reproductive tissue, such as in both prostate and uterine
tissue. A disease-specific antigen is expressed coincidentally with
a disease process. Specific non-limiting examples of a
disease-specific antigen are an antigen whose expression correlates
with, or is predictive of, tumor formation, such as prostate cancer
and/or uterine cancer and/or testicular cancer. A disease-specific
antigen can be an antigen recognized by T cells or B cells.
[0040] Amplification: Of a nucleic acid molecule (e.g., a DNA or
RNA molecule) refers to use of a technique that increases the
number of copies of a nucleic acid molecule in a specimen. An
example of amplification is the polymerase chain reaction, in which
a biological sample collected from a subject is contacted with a
pair of oligonucleotide primers, under conditions that allow for
the hybridization of the primers to a nucleic acid template in the
sample. The primers are extended under suitable conditions,
dissociated from the template, and then re-annealed, extended, and
dissociated to amplify the number of copies of the nucleic acid.
The product of amplification can be characterized by
electrophoresis, restriction endonuclease cleavage patterns,
oligonucleotide hybridization or ligation, and/or nucleic acid
sequencing using standard techniques. Other examples of
amplification include strand displacement amplification, as
disclosed in U.S. Pat. No. 5,744,311; transcription-free isothermal
amplification, as disclosed in U.S. Pat. No. 6,033,881; repair
chain reaction amplification, as disclosed in WO 90/01069; ligase
chain reaction amplification, as disclosed in EP-A-320 308; gap
filling ligase chain reaction amplification, as disclosed in U.S.
Pat. No. 5,427,930; and NASBA.TM. RNA transcription-free
amplification, as disclosed in U.S. Pat. No. 6,025,134.
[0041] Antibody: Immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, i.e., molecules that
contain an antigen binding site that specifically binds
(immunoreacts with) an antigen, such as Brachyury protein.
[0042] A naturally occurring antibody (e.g., IgG, IgM, IgD)
includes four polypeptide chains, two heavy (H) chains and two
light (L) chains interconnected by disulfide bonds. However, it has
been shown that the antigen-binding function of an antibody can be
performed by fragments of a naturally occurring antibody. Thus,
these antigen-binding fragments are also intended to be designated
by the term "antibody." Specific, non-limiting examples of binding
fragments encompassed within the term antibody include (i) a Fab
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) an F.sub.d fragment consisting of the V.sub.H and
C.sub.H1 domains; (iii) an Fv fragment consisting of the VL and VH
domains of a single arm of an antibody, (iv) a dAb fragment (Ward
et al., Nature 341:544-546, 1989) which consists of a V.sub.H
domain; (v) an isolated complementarity determining region (CDR);
and (vi) a F(ab')2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region.
[0043] Immunoglobulins and certain variants thereof are known and
many have been prepared in recombinant cell culture (e.g., see U.S.
Pat. Nos. 4,745,055; 4,444,487; WO 88/03565; EP 256,654; EP
120,694; EP 125,023; Faoulkner et al., Nature 298:286, 1982;
Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann Rev.
Immunol 2:239, 1984). Humanized antibodies and fully human
antibodies are also known in the art.
[0044] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects.
[0045] Brachyury: The Brachyury gene is known to be important for
the development of mesoderm during gastrulation. Brachyury is the
founding member of a family of transcription factors, designated
T-box transcription factors, characterized by a conserved
DNA-binding domain (Papaioannou and Silver, Bioessays 20(1):9-19,
1998), that has an essential role in the formation and organization
of mesoderm in vertebrates (see, for example, Kispert and Herrmann,
Embo J 12(8):3211-20, 1993). For example, in Xenopus, Brachyury is
an early-immediate response gene of mesoderm inducers, such as
activin or TGF-.beta., and injection of Brachyury mRNA in embryos
is sufficient to induce ectopic mesoderm development (Smith et al.,
Cell 67(1):79-87, 1991). In addition to the fundamental role of the
T-box proteins in the control of developmental processes, several
members of this family appear to be deregulated in cancer. The
human Thx2 gene has been reported to be amplified in pancreatic
cancer cell lines (Mahlamaki et al., Genes Chromosomes Cancer
35(4):353-8, 2002) and over-expressed in BRCA-1- and BRCA-2-mutated
breast tumors (Sinclair et al., Cancer Res 62(13):3587-9, 2002).
Brachyury expression has been reported in human teratocarcinoma
lines and chordomas (Vujovic et al, J Pathol 209(2): 157-65, 2006).
Exemplary human brachyury amino acid and nucleic acid sequences are
set forth in GENBANK.RTM. Accession No NP_003172 and GENBANK.RTM.
Accession No. NM_003181, as available on Feb. 23, 2007,
incorporated herein by reference, and are provided below.
[0046] Breast cancer: A neoplastic condition of breast tissue that
can be benign or malignant. The most common type of breast cancer
is ductal carcinoma. Ductal carcinoma in situ is a non-invasive
neoplastic condition of the ducts. Lobular carcinoma is not an
invasive disease but is an indicator that a carcinoma may develop.
Infiltrating (malignant) carcinoma of the breast can be divided
into stages (I IIA, IIB, IIIA, IIIB, and IV). Tumor size staging
and node involvement staging can be combined into a single clinical
staging number, as exemplified below.
TABLE-US-00001 Tumor size staging Node involvement staging Clinical
stage T1 N0 I T1 N1 IIA T2 N0 IIA T2 N1 IIB T3 N0 IIB T1-T2 N2 IIIA
T3 N1 IIIA T3 N2 IIIA T4 N0-N2 IIIB
[0047] Breast carcinomas lose the typical histology and
architecture of normal breast glands. Generally, carcinoma cells
overgrow the normal cells and lose their ability to differentiate
into glandular like structures. The degree of loss of
differentiation in general is related to the aggressiveness of the
tumor. For example, "in situ" carcinoma by definition retains the
basement membrane intact, whereas as it progresses to "invasive",
the tumor shows breakout of basement membranes. Thus one would not
expect to see, within breast carcinomas, staining of a discrete
layer of basal cells as seen in normal breast tissue. For a
discussion of the physiology and histology of normal breast and
breast carcinoma, see Ronnov-Jessen, L., Petersen, O. W. &
Bissell, M. J. Cellular changes involved in conversion of normal to
malignant breast: importance of the stromal reaction. Physiol Rev
76, 69-125 (1996).
[0048] Breast cancers can be divided into groups based on their
expression profiles. Basal-type carcinomas usually are negative for
expression of estrogen receptor (ER) and negative for expression of
HER2 (erbB2) and progesterone receptor (PR), and thus are referred
to as "triple-negative breast cancers" or "TNBC." This type of
breast cancer is also denoted ER.sup.-/HER2.sup.-/PR.sup.- and
represents about 15-20% of all breast cancer, and generally cannot
be treated using Her2 targeted or estrogen targeted therapies. It
is believed that the aggressive nature of this cancer is correlated
with an enrichment for cancer stem cells (CSC) with a
CD44.sup.+CD24.sup.-/lo phenotype. In some embodiments, basal
carcinomas are negative for expression of progesterone receptor
(PR), positive for expression of epidermal growth factor receptor
(EGFR), and positive for expression of cytokeratin 5 (CK5). This
phenotype is denoted as follows:
ER.sup.-/PR.sup.-/HER2.sup.-/CK5.sup.+/EGFR.sup.+.
[0049] Cancer or Tumor: A malignant neoplasm that has undergone
characteristic anaplasia with loss of differentiation, increased
rate of growth, invasion of surrounding tissue, and is capable of
metastasis. For example, prostate cancer is a malignant neoplasm
that arises in or from prostate tissue, ovarian cancer is a
malignant neoplasm that arises in or from ovarian tissue, colon
cancer is a malignant neoplasm that arises in or from colon tissue,
and lung cancer is a malignant neoplasm that arises in the lungs.
Residual cancer is cancer that remains in a subject after any form
of treatment given to the subject to reduce or eradicate the
cancer. Metastatic cancer is a cancer at one or more sites in the
body other than the site of origin of the original (primary) cancer
from which the metastatic cancer is derived. Cancer includes, but
is not limited to, sarcomas and carcinomas. Prostate cancer is a
malignant tumor, generally of glandular origin, of the prostate.
Prostate cancers include adenocarcinomas and small cell
carcinomas.
[0050] cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and regulatory sequences that
determine transcription. cDNA is synthesized in the laboratory by
reverse transcription from messenger RNA extracted from cells.
[0051] Chemotherapeutic agents: Any chemical agent with therapeutic
usefulness in the treatment of diseases characterized by abnormal
cell growth. Such diseases include tumors, neoplasms, and cancer as
well as diseases characterized by hyperplastic growth such as
psoriasis. In one embodiment, a chemotherapeutic agent is an agent
of use in treating breast and/or prostate cancer. In one
embodiment, a chemotherapeutic agent is radioactive compound. One
of skill in the art can readily identify a chemotherapeutic agent
of use (e.g. see Slapak and Kufe, Principles of Cancer Therapy,
Chapter 86 in Harrison's Principles of Internal Medicine, 14th
edition, Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical
Oncology 2.sup.nd ed., .COPYRGT. 2000 Churchill Livingstone, Inc;
Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy,
2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F,
Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St.
Louis, Mosby-Year Book, 1993). Combination chemotherapy is the
administration of more than one agent to treat cancer, such as the
administration of a non-pox non-yeast vector encoding Brachyury in
combination with a radioactive or chemical compound to a
subject.
[0052] Conservative variants: "Conservative" amino acid
substitutions are those substitutions that do not substantially
affect or decrease an activity or antigenicity of an antigenic
epitope of Brachyury. Specific, non-limiting examples of a
conservative substitution include the following examples:
TABLE-US-00002 Original Residue Conservative Substitutions Al Ser
Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln
Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met;
Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu
[0053] The term conservative variant also includes the use of a
substituted amino acid in place of an unsubstituted parent amino
acid, provided that antibodies raised to the substituted
polypeptide also immunoreact with the unsubstituted polypeptide,
and/or that the substituted polypeptide retains the function of the
unstubstituted polypeptide. Non-conservative substitutions are
those that reduce an activity or antigenicity.
[0054] CD4: Cluster of differentiation factor 4, a T cell surface
protein that mediates interaction with the MHC Class II molecule.
CD4 also serves as the primary receptor site for HIV on T cells
during HIV infection. Cells that express CD4 are often helper T
cells.
[0055] CD8: Cluster of differentiation factor 8, a T cell surface
protein that mediates interaction with the MHC Class I molecule.
Cells that express CD8 are often cytotoxic T cells.
[0056] Consists Essentially Of/Consists Of: With regard to a
polypeptide or protein, a polypeptide (or protein) that consists
essentially of a specified amino acid sequence if it does not
include any additional amino acid residues. However, the
polypeptide (or protein) can include additional non-peptide
components, such as labels (for example, fluorescent, radioactive,
or solid particle labels), sugars or lipids. With regard to a
polypeptide or protein, a polypeptide or protein that consists of a
specified amino acid sequence does not include any additional amino
acid residues, nor does it include additional non-peptide
components, such as lipids, sugars or labels.
[0057] Costimulatory molecule: Although engagement of the TCR with
peptide-MHC delivers one signal to the T cell, this signal alone
can be insufficient to activate the T cell. Costimulatory molecules
are molecules that, when bound to their ligand, deliver a second
signal required for the T cell to become activated. The most
well-known costimulatory molecule on the T cell is CD28, which
binds to either B7-1 (also called CD80) or B7-2 (also known as
CD86). An additional costimulatory molecule is B7-3. Accessory
molecules that also provide a second signal for the activation of T
cells include intracellular adhesion molecule (ICAM-1 and ICAM-2),
leukocyte function associated antigen (LFA-1, LFA-2 and LFA-3).
Integrins and tumor necrosis factor (TNF) superfamily members can
also serve as co-stimulatory molecules.
[0058] Degenerate variant: A polynucleotide encoding an epitope of
Brachyury that includes a sequence that is degenerate as a result
of the genetic code. There are 20 natural amino acids, most of
which are specified by more than one codon. Therefore, all
degenerate nucleotide sequences are included in this disclosure as
long as the amino acid sequence of the Brachyury protein encoded by
the nucleotide sequence is unchanged.
[0059] Dendritic cell (DC): Dendritic cells are the principle
antigen presenting cells (APCs) involved in primary immune
responses. Dendritic cells include plasmacytoid dendritic cells and
myeloid dendritic cells. Their major function is to obtain antigen
in tissues, migrate to lymphoid organs and present the antigen in
order to activate T cells. Immature dendritic cells originate in
the bone marrow and reside in the periphery as immature cells.
[0060] Diagnostic: Identifying the presence or nature of a
pathologic condition, such as, but not limited to, a cancer, such
as small intestine, stomach, kidney, bladder, uterus, ovary,
testes, lung, colon or prostate cancer. Diagnostic methods differ
in their sensitivity and specificity. The "sensitivity" of a
diagnostic assay is the percentage of diseased individuals who test
positive (percent of true positives). The "specificity" of a
diagnostic assay is 1 minus the false positive rate, where the
false positive rate is defined as the proportion of those without
the disease who test positive. While a particular diagnostic method
may not provide a definitive diagnosis of a condition, it suffices
if the method provides a positive indication that aids in
diagnosis. "Prognostic" means predicting the probability of
development (for example, severity) of a pathologic condition, such
as prostate cancer, or metastasis.
[0061] Epithelial-to-Mesenchymal Transition: The epithelium is the
covering of internal and external surfaces of the body, including
the lining of vessels and other small cavities, that consists of
cells joined by biological cementing substances. Generally, fully
differentiated epithelial cells express proteins characteristic of
a differentiated phenotype, such as insulin, and have a limited
capacity to proliferate. The mesenchyme is the meshwork of loosely
organized embryonic connective tissue in the mesoderm from which
are formed the connective tissues of the body, along with the blood
vessels and lymphatic vessels. Vimentin is one marker of
mesenchymal cells. Mesenchymal cells generally have a greater
capacity to proliferate in vitro than epithelial cells and are not
fully differentiated. An "epithelial-to-mesenchymal" transition is
a biological process wherein a cell, or a population of cells, from
an epithelial phenotype convert to a less differentiated
mesenchymal phenotype. A "mesenchymal-to-epithelial" transition is
a biological process wherein a cell, or a population of cells,
convert from a less differentiated mesenchymal phenotype to a more
differentiated epithelial phenotype.
[0062] Epitope: An antigenic determinant. These are particular
chemical groups or peptide sequences on a molecule that are
antigenic (that elicit a specific immune response). An antibody
specifically binds a particular antigenic epitope on a polypeptide.
Epitopes can be formed both from contiguous amino acids or
noncontiguous amino acids juxtaposed by tertiary folding of a
protein. Epitopes formed from contiguous amino acids are typically
retained on exposure to denaturing solvents whereas epitopes formed
by tertiary folding are typically lost on treatment with denaturing
solvents. An epitope typically includes at least 3, and more
usually, at least 5, about 9, or 8 to 10 amino acids, and generally
not more than 20 amino acids, in a unique spatial conformation.
Methods of determining spatial conformation of epitopes include,
for example, x-ray crystallography and 2-dimensional nuclear
magnetic resonance. See, e.g., "Epitope Mapping Protocols" in
Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
In one embodiment, an epitope binds an MHC molecule, such as an HLA
molecule or a DR molecule. These molecules bind polypeptides having
the correct anchor amino acids separated by about eight to about
ten amino acids, such as nine amino acids.
[0063] Estrogen Receptor (ER): A receptor that is activated by the
hormone 17.beta.-estradiol (estrogen). The main function of the
estrogen receptor is as a DNA binding transcription factor that
regulates gene expression. Estrogen receptors are over-expressed in
around 70% of breast cancer cases, referred to as "ER positive" or
"ER.sup.+." Therapy for ER.sup.+ breast cancer involves selective
estrogen receptor modulators (SERMS) which behave as ER antagonists
in breast tissue or aromatase inhibitors. ER status is also used to
determine sensitivity of breast cancer lesions to tamoxifen and
aromatase inhibitors.
[0064] Expression Control Sequences: Nucleic acid sequences that
regulate the expression of a heterologous nucleic acid sequence to
which they are operatively linked. Expression control sequences are
operatively linked to a nucleic acid sequence when the expression
control sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus,
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signal for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of mRNA, and stop codons. The term "control
sequences" is intended to include, at a minimum, components whose
presence can influence expression, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences. Expression control
sequences can include a promoter.
[0065] A promoter is a minimal sequence sufficient to direct
transcription. Also included are those promoter elements which are
sufficient to render promoter-dependent gene expression
controllable for cell-type specific, tissue-specific, or inducible
by external signals or agents; such elements may be located in the
5' or 3' regions of the gene. Both constitutive and inducible
promoters are included (see e.g., Bitter et al., Methods in
Enzymology 153:516-544, 1987). For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like
can be used. In one embodiment, when cloning in mammalian cell
systems, promoters derived from the genome of mammalian cells (such
as the metallothionein promoter) or from mammalian viruses (such as
the retrovirus long terminal repeat; the adenovirus late promoter;
the vaccinia virus 7.5K promoter) can be used. Promoters are also
disclosed herein that are effective when included in a poxviral
vector. Promoters produced by recombinant DNA or synthetic
techniques can also be used to provide for transcription of the
nucleic acid sequences.
[0066] HER2: Human Epidermal growth factor Receptor 2 (Her2) is
also known as Her 2/neu (or ErbB-2, ERBB2). It is a member of the
ErbB protein family (also known as the epidermal growth factor
receptor family). HER2 has also been designated as CD340 (cluster
of differentiation 340) and p185. HER2 is notable for its role in
the pathogenesis of breast cancer and as a target of treatment. It
is a cell membrane surface-bound receptor tyrosine kinase and is
normally involved in the signal transduction pathways leading to
cell growth and differentiation.
[0067] Approximately 15-20 percent of breast cancers have an
amplification of the HER2 gene or overexpression of its protein
product. Overexpression of this receptor in breast cancer has been
associated with increased disease recurrence and worse prognosis.
Because of its prognostic role, breast tumors are routinely checked
for overexpression of HER2. Overexpression also occurs in other
cancer such as ovarian cancer, stomach cancer, and biologically
aggressive forms of uterine cancer, such as uterine serous
endometrial carcinoma.
[0068] Heterologous: Originating from separate genetic sources or
species. A polypeptide that is heterologous to Brachyury originates
from a nucleic acid that does not encode Brachyury. In specific,
non-limiting examples, with regard to a polypeptide comprising
Brachyury, a heterologous amino acid sequence includes a
3-galactosidase, a maltose binding protein, and albumin, hepatitis
B surface antigen, or an immunoglobulin amino acid sequence.
Generally, an antibody that specifically binds to a protein of
interest, such as Brachyury, will not specifically bind to a
heterologous protein.
[0069] Host cells: Cells in which a vector can be propagated and
its DNA expressed. The cell may be prokaryotic or eukaryotic. The
cell can be mammalian, such as a human cell. The term also includes
any progeny of the subject host cell. It is understood that all
progeny may not be identical to the parental cell since there may
be mutations that occur during replication. However, such progeny
are included when the term "host cell" is used.
[0070] Immune response: A response of a cell of the immune system,
such as a B cell, T cell, or monocyte, to a stimulus. In one
embodiment, the response is specific for a particular antigen (an
"antigen-specific response"). In one embodiment, an immune response
is a T cell response, such as a CD4+ response or a CD8+ response.
In another embodiment, the response is a B cell response, and
results in the production of specific antibodies.
[0071] Immunogenic polypeptide and Immnogenic Protein: A protein or
peptide which comprises an allele-specific motif or other sequence
such that the peptide will bind an MHC molecule and induce a T cell
response, or a B cell response (e.g. antibody production) against
the antigen.
[0072] Immunogenic peptides are generally 7 to 20 amino acids in
length, such as 9 to 12 amino acids in length. In one example, an
immunogenic polypeptide includes an allele-specific motif or other
sequence such that the peptide will bind an MHC molecule and induce
a T cell response against the antigen (protein) from which the
immunogenic polypeptide is derived. In one embodiment, immunogenic
peptides are identified using sequence motifs or other methods,
such as neural net or polynomial determinations, known in the art.
Typically, algorithms are used to determine the "binding threshold"
of peptides to select those with scores that give them a high
probability of binding at a certain affinity and will be
immunogenic. The algorithms are based either on the effects on MHC
binding of a particular amino acid at a particular position, the
effects on antibody binding of a particular amino acid at a
particular position, or the effects on binding of a particular
substitution in a motif-containing peptide. Within the context of
an immunogenic peptide, a "conserved residue" is one which appears
in a significantly higher frequency than would be expected by
random distribution at a particular position in a peptide. In one
embodiment, a conserved residue is one where the MHC structure may
provide a contact point with the immunogenic peptide. In one
example, an immunogenic "Brachyury polypeptide" is a series of
contiguous amino acid residues from the Brachyury protein generally
between 7 and 20 amino acids in length, such as about 8 to 11
residues in length. Specific immunogenic Brachyury polypeptides are
9 or 10 amino acid residues in length, or at most 12 amino acids in
length.
[0073] Immunogenic peptides and proteins can also be identified by
measuring their binding to a specific MHC protein (Class I or Class
II) and by their ability to stimulate CD4 and/or CD8 when presented
in the context of the MHC protein. The characteristics of
immunogenic polypeptides, are disclosed, for example, in PCT
Publication No. WO 00/12706, which is incorporated herein by
reference.
[0074] Generally, an immunogenic Brachyury protein includes a
number of immunogenic polypeptides, and can be used to induce an
immune response in a subject, such as a CD4+ T cell response. In
one example, an immunogenic Brachyury protein, when bound to a
Major Histocompatibility Complex Class II molecule, activates CD4+
T cells against cells expressing wild-type Brachyury protein,
and/or when bound to a Major Histocompatibility Complex Class I
molecule, activates cytotoxic T lymphocytes (CTLs) against cells
expressing wild-type Brachyury protein. Induction of CTLs using
synthetic peptides and CTL cytotoxicity assays are known in the
art, see U.S. Pat. No. 5,662,907, which is incorporated herein by
reference.
[0075] Immunogenic composition: A composition, such as a
composition comprising a Brachyury protein or a nucleic acid
encoding the Brachyury protein, that induces a measurable T cell
response against cells expressing Brachyury protein, or induces a
measurable B cell response (such as production of antibodies that
specifically bind Brachyury) against a Brachyury protein. For in
vitro use, the immunogenic composition can consist of the isolated
nucleic acid, vector including the nucleic acid/or immunogenic
protein. For in vivo use, the immunogenic composition will
typically comprise the nucleic acid, vector including the nucleic
acid, and or immunogenic protein, in pharmaceutically acceptable
carriers, and/or other agents. An immunogenic composition can
optionally include an adjuvant, a costimulatory molecule, or a
nucleic acid encoding a costimulatory molecule.
[0076] Immunostimulatory molecule: Molecules that stimulate the
cells of the immune system including costimulatory molecules,
cytokines and immunostimulatory nucleic acids, such as those that
include a CpG motif.
[0077] Inhibiting or treating a disease: Inhibiting a disease, such
as cancer growth, refers to inhibiting the full development of a
disease. In several examples, inhibiting a disease refers to
lessening symptoms of a cancer, such as preventing the development
of paraneoplastic syndrome in a person who is known to have a
cancer, or lessening a sign or symptom of the cancer or reducing
cancer volume. "Treatment" refers to a therapeutic intervention
that ameliorates a sign or symptom of a disease or pathological
condition related to the disease, such as the cancer.
[0078] Isolated: An "isolated" biological component (such as a
nucleic acid or protein or organelle) has been substantially
separated or purified away from other biological components in the
cell of the organism in which the component naturally occurs, i.e.,
other chromosomal and extra-chromosomal DNA and RNA, proteins and
organelles. Nucleic acids and proteins that have been "isolated"
include nucleic acids and proteins purified by standard
purification methods. The term also embraces nucleic acids and
proteins prepared by recombinant expression in a host cell as well
as chemically synthesized nucleic acids.
[0079] Label: A detectable compound or composition that is
conjugated directly or indirectly to another molecule to facilitate
detection of that molecule. Specific, non-limiting examples of
labels include fluorescent tags, enzymatic linkages, and
radioactive isotopes.
[0080] Lentiviral vector: Lentiviruses are a subclass of
Retroviruses. Lentiviral vectors can integrate into the genome of
non-dividing cells. This feature of Lentiviruses is unique, as
other Retroviruses can infect only dividing cells. The viral genome
in the form of RNA is reverse-transcribed when the virus enters the
cell to produce DNA, which is then inserted into the genome at a
random position by the viral integrase enzyme. The vector, now
called a provirus, remains in the genome and is passed on to the
progeny of the cell when it divides. Lentiviral vectors include
HIV-1, HIV-2, SIV (simian immunodeficiency virus), EIAV (equine
infectious anaemia virus), FIV (feline immunodeficiency virus),
CAEV (Caprine arthritis encephalitis virus), and VMV (Visna/maedi
virus) vectors. Lentiviral vectors also encompass chimeric
lentiviruses derived from at least two different lentiviruses.
[0081] Linker sequence: A linker sequence is an amino acid sequence
that covalently links two polypeptide domains. Linker sequences can
be included in the between the Brachyury proteins disclosed herein
to provide rotational freedom to the linked polypeptide domains. By
way of example, in a recombinant molecule comprising two Brachyury
proteins, linker sequences can be provided between them, so that
the proteins comprises Brachyury protein-linker-Brachyury protein.
Linker sequences, which are generally between 2 and 25 amino acids
in length, are well known in the art and include, but are not
limited to, four glycines and a serine spacer described by
Chaudhary et al., Nature 339:394-397, 1989.
[0082] Listeria: A Gram-positive bacilli. The genus Listeria
currently contains seven species: L. grayi. L. innocua, L.
ivanovii, L. monocytogenes, L. murrayi, L. seeligeri, and L.
welshimeri. L. monocytogenes is an intracellular bacterium that has
been used as a vector to deliver genes in vitro.
[0083] Lymphocytes: A type of white blood cell that is involved in
the immune defenses of the body. There are two main types of
lymphocytes: B cells and T cells.
[0084] Major Histocompatibility Complex (MHC): A generic
designation meant to encompass the histocompatability antigen
systems described in different species, including the human
leukocyte antigens ("HLA").
[0085] Mammal: This term includes both human and non-human mammals.
Similarly, the term "subject" includes both human and veterinary
subjects.
[0086] Neoplasm: An abnormal cellular proliferation, which includes
benign and malignant tumors, as well as other proliferative
disorders.
[0087] Oligonucleotide: A linear polynucleotide sequence of up to
about 100 nucleotide bases in length.
[0088] Open reading frame (ORF): A series of nucleotide triplets
(codons) coding for amino acids without any internal termination
codons. These sequences are usually translatable into a
peptide.
[0089] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence, such as a
sequence that encodes a Brachyury protein. Generally, operably
linked DNA sequences are contiguous and, where necessary to join
two protein-coding regions, in the same reading frame.
[0090] Peptide Modifications: Brachyury proteins include synthetic
embodiments of peptides described herein. In addition, analogs
(non-peptide organic molecules), derivatives (chemically
functionalized peptide molecules obtained starting with the
disclosed peptide sequences) and variants (homologs) of these
proteins can be utilized in the methods described herein. Each
protein or polypeptide of this disclosure is comprised of a
sequence of amino acids, which may be either L- and/or D-amino
acids, naturally occurring and otherwise.
[0091] Protein and polypeptides can be modified by a variety of
chemical techniques to produce derivatives having essentially the
same activity as the unmodified peptides, and optionally having
other desirable properties. For example, carboxylic acid groups of
the protein, whether carboxyl-terminal or side chain, can be
provided in the form of a salt of a pharmaceutically-acceptable
cation or esterified to form a C.sub.1-C.sub.16 ester, or converted
to an amide of formula NR.sub.1R.sub.2 wherein R.sub.1 and R.sub.2
are each independently H or C.sub.1-C.sub.16 alkyl, or combined to
form a heterocyclic ring, such as a 5- or 6-membered ring. Amino
groups of the peptide, whether amino-terminal or side chain, can be
in the form of a pharmaceutically-acceptable acid addition salt,
such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic,
tartaric and other organic salts, or can be modified to
C.sub.1-C.sub.16 alkyl or dialkyl amino or further converted to an
amide.
[0092] Hydroxyl groups of the peptide side chains may be converted
to C.sub.1-C.sub.16 alkoxy or to a C.sub.1-C.sub.16 ester using
well-recognized techniques. Phenyl and phenolic rings of the
peptide side chains may be substituted with one or more halogen
atoms, such as fluorine, chlorine, bromine or iodine, or with
C.sub.1-C.sub.16 alkyl, C.sub.1-C.sub.16 alkoxy, carboxylic acids
and esters thereof, or amides of such carboxylic acids. Methylene
groups of the peptide side chains can be extended to homologous
C.sub.2-C.sub.4 alkylenes. Thiols can be protected with any one of
a number of well-recognized protecting groups, such as acetamide
groups. Those skilled in the art will also recognize methods for
introducing cyclic structures into proteins and polypeptides to
select and provide conformational constraints to the structure that
result in enhanced stability.
[0093] Peptidomimetic and organomimetic embodiments are envisioned,
whereby the three-dimensional arrangement of the chemical
constituents of such peptido- and organomimetics mimic the
three-dimensional arrangement of the peptide backbone and component
amino acid side chains, resulting in such peptido- and
organomimetics of a Brachyury protein having measurable or enhanced
ability to generate an immune response. For computer modeling
applications, a pharmacophore is an idealized three-dimensional
definition of the structural requirements for biological activity.
Peptido- and organomimetics can be designed to fit each
pharmacophore with current computer modeling software (using
computer assisted drug design or CADD). See Walters,
"Computer-Assisted Modeling of Drugs," in Klegerman & Groves,
eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo
Grove, Ill., pp. 165-174 and Principles of Pharmacology, Munson
(ed.) 1995, Ch. 102, for descriptions of techniques used in CADD.
Also included are mimetics prepared using such techniques.
[0094] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers of use are conventional. Remington's
Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co.,
Easton, Pa., 15th Edition (1975), describes compositions and
formulations suitable for pharmaceutical delivery of the fusion
proteins herein disclosed.
[0095] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(such as powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0096] A "therapeutically effective amount" is a quantity of a
composition or a cell to achieve a desired effect in a subject
being treated. For instance, this can be the amount of Brachyury
protein or a vector encoding a Brachyury protein necessary to
induce an immune response, inhibit cancer growth, reduce cancer
volume, prevent cancer, or to measurably alter outward symptoms of
the cancer. When administered to a subject, a dosage will generally
be used that will achieve target tissue concentrations (for
example, in lymphocytes) that has been shown to achieve an in vitro
effect.
[0097] Plasmid: A DNA molecule that is separate from, and can
replicate independently of, the chromosomal DNA. They are
double-stranded and, in many cases, circular. Generally, a gene to
be replicated is inserted into copies of a plasmid containing genes
that make cells resistant to particular antibiotics and a multiple
cloning site (MCS, or polylinker), which is a short region
containing several commonly used restriction sites allowing the
easy insertion of DNA fragments at this location.
[0098] Poliovirus: A human enterovirus and member of the family of
Picornaviridae; the wild-type poliovirus causes poliomyelitis.
Poliovirus is composed of an RNA genome and a protein capsid. The
wild-type genome is a single-stranded positive-sense RNA genome
that is about 7500 nucleotides long. The viral particle is about 30
nanometres in diameter with icosahedral symmetry.
[0099] Polynucleotide: The term polynucleotide or nucleic acid
sequence refers to a polymeric form of nucleotide at least 10 bases
in length. A recombinant polynucleotide includes a polynucleotide
that is not immediately contiguous with both of the coding
sequences with which it is immediately contiguous (one on the 5'
end and one on the 3' end) in the naturally occurring genome of the
organism from which it is derived. The term therefore includes, for
example, a recombinant DNA which is incorporated into a vector;
into an autonomously replicating plasmid or virus; or into the
genomic DNA of a prokaryote or eukaryote, or which exists as a
separate molecule (e.g., a cDNA) independent of other sequences.
The nucleotides can be ribonucleotides, deoxyribonucleotides, or
modified forms of either nucleotide. The term includes single- and
double-stranded forms of DNA.
[0100] Polypeptide: A chain of amino acids, generally greater than
eight amino acids in length, such as greater than fifteen amino
acids in length, which can be post-translationally modified (e.g.,
glycosylation or phosphorylation) that is not the complete
wild-type protein. A polypeptide can be at least 15, at least 20,
at least 30, at least 40, at least 50, at least 60, at least 70, at
least 80, at least 90, at least 100, at least 200 amino acids in
length. Thus, a polypeptide can be, for example, 20-300, 30-300,
40-300, 50-300, 60-300, 70-300, 80-300, 90-300, 100-300, or 200-300
amino acids in length. In additional embodiments, a polypeptide is
15 to 10-, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100,
55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 85-100, 90-100 or
95-100 amino acids in length. In further embodiments a polypeptide
is up to 433, 434 or 435 amino acids in length.
[0101] Protein: A chain of amino acids, generally greater than 100
amino acids in length, that has a specific function in a cell and
is a complete wild-type protein or the complete wild type protein
without the N-terminal methionine. A protein can be
post-translationally modified. In one embodiment, the protein is a
Brachyury protein.
[0102] Measles virus (Mobrillivirus): A negative strand RNA virus
belonging the Parmyoviridiae family that causes measles.
Heterologous genes can be inserted into the viral genome. The non
segmented genome of measles virus has an anti-message polarity
which results in a genomic RNA which, when purified, is not
translated either in vivo or in vitro and is not infectious.
[0103] Poxvirus: Four genera of poxviruses infect humans: orthopox,
parapox, yatapox, molluscipox. Orthopox includes smallpox virus
(variola), vaccinia virus, cowpox virus, and monkeypox virus.
Parapox includes the orf virus, pseudocowpox, and bovine papular
stomatitis virus. Yatapox inlcudes tanapox virus, and yaba monkey
tumor virus. Molluscipox includes molluscum contagiosum virus
(MCV). Poxviridae viral particles (virions) are generally enveloped
(external enveloped virion--EEV), though the intracellular mature
virion (IMV) form of the virus, which contains different envelope,
is also infectious.
[0104] Vaccinia virus is used as an effective tool for heterologous
protein expression. Vaccinia virus enters cells mainly by cell
fusion. This virus contains three classes of genes, early,
intermediate and late, that are transcribed by viral RNA polymerase
and associated transcription factors. Vaccinia virus replicates its
genome in cytoplasm of the infected cells and after late gene
expression virion morphogenesis produces intracellular mature
virion (IMV) that contains envelope, although the origin of the
envelope membrane is still unknown. IMV is transported to Golgi,
wherein the intracellular enveloped virus (IEV) is formed. IEV
transports along microtubules to reach cell periphery and fuse with
plasma membrane to become cell-associated enveloped virus (CEV)
that triggers actin tails on cell surfaces or forms the
extracellular enveloped virion (EEV), which is believed to be
important for long range dissemination within the host
organism.
[0105] A "non-poxviral vector" is a vector that is not included in
the four genera of poxviruses.
[0106] Progesterone receptor (PR): A receptor, also known as NR3C3
(nuclear receptor subfamily 3, group C, member 3), that is a
steroid receptor that specifically binds progesterone. The
progesterone receptor is not expressed on triple negative basal
breast cancer cells.
[0107] Probes and primers: A probe comprises an isolated nucleic
acid attached to a detectable label or reporter molecule. Primers
are short nucleic acids, preferably DNA oligonucleotides, of about
15 nucleotides or more in length. Primers may be annealed to a
complementary target DNA strand by nucleic acid hybridization to
form a hybrid between the primer and the target DNA strand, and
then extended along the target DNA strand by a DNA polymerase
enzyme. Primer pairs can be used for amplification of a nucleic
acid sequence, for example by polymerase chain reaction (PCR) or
other nucleic-acid amplification methods known in the art. One of
skill in the art will appreciate that the specificity of a
particular probe or primer increases with its length. Thus, for
example, a primer comprising 20 consecutive nucleotides will anneal
to a target with a higher specificity than a corresponding primer
of only 15 nucleotides. Thus, in order to obtain greater
specificity, probes and primers can be selected that comprise about
20, 25, 30, 35, 40, 50 or more consecutive nucleotides.
[0108] Purified: Brachyury proteins and nucleic acids as disclosed
herein can be purified (and/or synthesized) by any of the means
known in the art (see, e.g., Guide to Protein Purification, ed.
Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and
Scopes, Protein Purification: Principles and Practice, Springer
Verlag, New York, 1982). Substantial purification denotes
purification from other proteins, nucleic acids, or cellular
components. The term purified does not require absolute purity;
rather, it is intended as a relative term. A substantially purified
protein is at least about 60%, 70%, 80%, 90%, 95%, 98% or 99% pure.
Thus, in one specific, non-limiting example, a substantially
purified protein is at least 90% free of other proteins or cellular
components. In additional embodiments, a nucleic acid or cell
preparation is purified such that the nucleic acid or cell
represents at least about 60% (such as, but not limited to, 70%,
80%, 90%, 95%, 98% or 99%) of the total nucleic acid or cell
content of the preparation, respectively. Thus, in one specific,
non-limiting example, a substantially purified nucleic acid is at
least 90% free of other nucleic acids or cellular components.
[0109] Recombinant: A recombinant nucleic acid is one that has a
sequence that is not naturally occurring or has a sequence that is
made by an artificial combination of two otherwise separated
segments of sequence. This artificial combination is often
accomplished by chemical synthesis or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids,
e.g., by genetic engineering techniques.
[0110] Replication defective: A viral vector that cannot further
replicate and package its genomes. In one non-limiting example,
when the cells of a subject are infected with a vector, a
heterologous in the vector is expressed in the subject's cells,
however, due to the fact that the patient's cells lack essential
genes. Examples are the rev and cap genes for AAV, or gag, pol and
env for a lentivirus. Generally, the genes necessary to replicate
and package are not present, such that and wild-type virus cannot
be formed in the subject's cells.
[0111] Salmonella: A genus of rod-shaped, Gram-negative,
non-spore-forming, predominantly motile enterobacteria with
diameters around 0.7 to 1.5 .mu.m, lengths from 2 to 5 .mu.m, and
flagella which grade in all directions (i.e. peritrichous). They
are chemoorganotrophs, obtaining their energy from oxidation and
reduction reactions using organic sources, and are facultative
anaerobes. Salmonella can be used as delivery vector for
therapeutic proteins, by including plasmids, such as those with
truncated tetA genes in the host cell. Attenuated S. typhimirium
can be transformed with DNA plasmids, such as, but not limited to,
pIRES (Invitrogen) and used as a carrier for delivery of
polypeptides and proteins.
[0112] Selectively hybridize: Hybridization under moderately or
highly stringent conditions that excludes non-related nucleotide
sequences.
[0113] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(for example, GC v. AT content), and nucleic acid type (for
example, RNA versus DNA) of the hybridizing regions of the nucleic
acids can be considered in selecting hybridization conditions. An
additional consideration is whether one of the nucleic acids is
immobilized, for example, on a filter.
[0114] A specific example of progressively higher stringency
conditions is as follows: 2.times.SSC/0.1% SDS at about room
temperature (hybridization conditions); 0.2.times.SSC/0.1%/SDS at
about room temperature (low stringency conditions);
0.2.times.SSC/0.1% SDS at about 42.degree. C. (moderate stringency
conditions); and 0.1.times.SSC at about 68.degree. C. (high
stringency conditions). One of skill in the art can readily
determine variations on these conditions (e.g., Molecular Cloning:
A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined
empirically.
[0115] Sequence identity: The similarity between amino acid
sequences is expressed in terms of the similarity between the
sequences, otherwise referred to as sequence identity. Sequence
identity is frequently measured in terms of percentage identity (or
similarity or homology); the higher the percentage, the more
similar the two sequences are. Homologs or variants of a Brachyury
protein will possess a relatively high degree of sequence identity
when aligned using standard methods.
[0116] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981;
Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Higgins and
Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989;
Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson
and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et
al., Nature Genet. 6:119, 1994, presents a detailed consideration
of sequence alignment methods and homology calculations.
[0117] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol. Biol. 215:403, 1990) is available from several
sources, including the National Center for Biotechnology
Information (NCBI, Bethesda, Md.) and on the internet, for use in
connection with the sequence analysis programs blastp, blastn,
blastx, tblastn and tblastx. A description of how to determine
sequence identity using this program is available on the NCBI
website on the internet.
[0118] Homologs and variants of a Brachyury protein are typically
characterized by possession of at least 75%, for example at least
80%, sequence identity, or at least 90% i sequence identity,
counted over the full length alignment with the amino acid sequence
of Brachyury using the NCBI Blast 2.0, gapped blastp set to default
parameters. For comparisons of amino acid sequences of greater than
about 30 amino acids, the Blast 2 sequences function is employed
using the default BLOSUM62 matrix set to default parameters, (gap
existence cost of 11, and a per residue gap cost of 1). When
aligning short peptides (fewer than around 30 amino acids), the
alignment should be performed using the Blast 2 sequences function,
employing the PAM30 matrix set to default parameters (open gap 9,
extension gap 1 penalties). Proteins with even greater similarity
to the reference sequences will show increasing percentage
identities when assessed by this method, such as at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, or at least
99% sequence identity. When less than the entire sequence is being
compared for sequence identity, homologs and variants will
typically possess at least 80% sequence identity over short windows
of 10-20 amino acids, and can possess sequence identities of at
least 85% or at least 90% or 95% depending on their similarity to
the reference sequence. Methods for determining sequence identity
over such short windows are available at the NCBI website on the
internet. One of skill in the art will appreciate that these
sequence identity ranges are provided for guidance only; it is
entirely possible that strongly significant homologs could be
obtained that fall outside of the ranges provided.
[0119] Specific binding agent: An agent that binds substantially
only to a defined target. Thus a Brachyury specific binding agent
is an agent that binds substantially to a Brachyury protein. In one
embodiment, the specific binding agent is a monoclonal or
polyclonal antibody that specifically binds Brachyury protein.
[0120] T Cell: A white blood cell critical to the immune response.
T cells include, but are not limited to, CD4+ T cells and CD8.sup.+
T cells. A CD4+ T lymphocyte is an immune cell that carries a
marker on its surface known as "cluster of differentiation 4" (CD4)
and is MHC Class II restricted. These cells, often called helper T
cells, help orchestrate the immune response, including antibody
responses as well as killer T cell responses. CD8.sup.+ T cells
carry the "cluster of differentiation 8" (CD8) marker and are MHC
Class I restricted. In one embodiment, a CD8 T cell is a cytotoxic
T lymphocyte. In another embodiment, a CD8 cell is a suppressor T
cell.
[0121] Therapeutically active protein: An agent composed of amino
acids, such as a Brachyury protein, that causes induction of an
immune response, as measured by clinical response (for example
increase in a population of immune cells, increased cytolytic
activity against cells that express Brachyury, or measurable
reduction of tumor burden). Therapeutically active molecules can
also be made from nucleic acids. Examples of a nucleic acid based
therapeutically active molecule is a nucleic acid sequence that
encodes a Brachyury protein, wherein the nucleic acid sequence is
operably linked to a control element such as a promoter.
[0122] In one embodiment, a therapeutically effective amount of a
composition, such as a Brachyury protein or a vector encoding the
Brachyury protein, is an amount used to generate an immune
response, or to treat or prevent cancer in a subject. In several
examples, "treatment" refers to a therapeutic intervention that
ameliorates a sign or symptom of a cancer, or a reduction in tumor
burden.
[0123] Transduced: A transduced cell is a cell into which has been
introduced a nucleic acid molecule by molecular biology techniques.
As used herein, the term transduction encompasses all techniques by
which a nucleic acid molecule might be introduced into such a cell,
including transfection with viral vectors, transformation with
plasmid vectors, and introduction of naked DNA by electroporation,
lipofection, and particle gun acceleration.
[0124] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. A vector may also
include one or more selectable marker gene and other genetic
elements known in the art. Vectors include plasmid vectors,
including plasmids for expression in gram negative and gram
positive bacterial cell. Exemplary vectors include those for
expression in E. coli and Salmonella. Vectors also include poxviral
vectors, such as, but are not limited to, retrovirus, orthopox,
avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha
virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus
vectors. Vectors also include vectors for expression in yeast
cells.
[0125] Yeast: Unicellular microorganisms that belong to one of
three classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. A
yeast can be a non-pathogenic strain such as Saccharomyces
cerevisiae. Yeawst strains include Saccharomyces, Candida (which
can be pathogenic), Cryptococcus, Hansemdla, Kluyveromyces, Pichia,
Rhodotorula, Schizosaccharomyces and Yarrowia. Yeast genera include
Saccharomyces, Candida, Hansenula. Pichia or Schizosaccharomyces.
Species of yeast strains include Saccharomyces cerevisiae,
Saccharomyces carlsbergensis, Candida albicans, Candida kefvr,
Candida tropicalis, Cryptococcus laurentii. Cryptococcus
neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces
fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var.
lactis, Pichia pastoris. Rhodotorula rubra, Schizosaccharomyces
pombe, and Yarrowia lipolytica.
[0126] Yeast vehicles" include, but are not limited to, a live
intact (whole) yeast microorganism (i.e., a yeast cell having all
its components including a cell wall), a killed (dead) or
inactivated intact yeast microorganism, or derivatives of intact
yeast including: a yeast spheroplast (i.e., a yeast cell lacking a
cell wall), a yeast cytoplast (i.e., a yeast cell lacking a cell
wall and nucleus), a yeast ghost (i.e., a yeast cell lacking a cell
wall, nucleus and cytoplasm), a subcellular yeast membrane extract
or fraction thereof (also referred to as a yeast membrane particle
or a subcellular yeast particle), any other yeast particle, or a
yeast cell wall preparation. A "non-yeast vector" is a composition
that does not include yeast vehicles.
[0127] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. The term "comprises"
means "includes." Similarly, comprising "A or B" includes "A," "B,"
and both "A and B." It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including explanations of terms, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
[0128] Immunogenic Brachyury Protein and Brachyury
Polyepeptides
[0129] Brachyury (also known as "T-protein") is a protein which is
transcribed in the mesoderm. In one embodiment, the Brachyury
protein has a sequence set forth as:
TABLE-US-00003 (SEQ ID NO: 1)
MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLE
ESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDF
VAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPV
SFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFP
ETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGD
SQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTL
RSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHP
SMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFF
RGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRL IASWTPVSPPSM (see
also GENBANK .RTM. Accession No NP_003172 and GENBANK .RTM.
Accession No. NM_003181, as available on Feb. 23, 2007,
incorporated herein by reference).
[0130] Using the genetic code, one of skill in the art can readily
produce a nucleic acid sequence encoding Brachyury. In one example,
Brachyury protein is encoded by a nucleic acid having a sequence
set forth as:
TABLE-US-00004 (SEQ ID NO: 2) tttgcttttg cttatttccg tccatttccc
tctctgcgcg cggaccttcc ttttccagat ggtgagagcc gcggggacac ccgacgccgg
ggcaggctga tccacgatcc tgggtgtgcg taacgccgcc tggggctccg tgggcgaggg
acgtgtgggg acaggtgcac cggaaactgc cagactggag agttgaggca tcggaggcgc
gagaacagca ctactactgc ggcgagacga gcgcggcgca tcccaaagcc cggccaaatg
cgctcgtccc tgggagggga gggaggcgcg cctggagcgg ggacagtctt ggtccgcgcc
ctcctcccgg gtctgtgccg ggacccggga cccgggagcc gtcgcaggtc tcggtccaag
gggccccttt tctcggaagg gcggcggcca agagcaggga aggtggatct caggtagcga
gtctgggctt cggggacggc ggggagggga gccggacggg aggatgagct cccctggcac
cgagagcgcg ggaaagagcc tgcagtaccg agtggaccac ctgctgagcg ccgtggagaa
tgagctgcag gcgggcagcg agaagggcga ccccacagag cgcgaactgc gcgtgggcct
ggaggagagc gagctgtggc tgcgcttcaa ggagctcacc aatgagatga tcgtgaccaa
gaacggcagg aggatgtttc cggtgctgaa ggtgaacgtg tctggcctgg accccaacgc
catgtactcc ttcctgctgg acttcgtggc ggcggacaac caccgctgga agtacgtgaa
cggggaatgg gtgccggggg gcaagccgga gccgcaggcg cccagctgcg tctacatcca
ccccgactcg cccaacttcg gggcccactg gatgaaggct cccgtctcct tcagcaaagt
caagctcacc aacaagctca acggaggggg ccagatcatg ctgaactcct tgcataagta
tgagcctcga atccacatag tgagagttgg gggtccacag cgcatgatca ccagccactg
cttccctgag acccagttca tagcggtgac tgcttatcag aacgaggaga tcacagctct
taaaattaag tacaatccat ttgcaaaagc tttccttgat gcaaaggaaa gaagtgatca
caaagagatg atggaggaac ccggagacag ccagcaacct gggtactccc aatgggggtg
gcttcttcct ggaaccagca ccctgtgtcc acctgcaaat cctcatcctc agtttggagg
tgccctctcc ctcccctcca cgcacagctg tgacaggtac ccaaccctga ggagccaccg
gtcctcaccc taccccagcc cctatgctca tcggaacaat tctccaacct attctgacaa
ctcacctgca tgtttatcca tgctgcaatc ccatgacaat tggtccagcc ttggaatgcc
tgcccatccc agcatgctcc ccgtgagcca caatgccagc ccacctacca gctccagtca
gtaccccagc ctgtggtctg tgagcaacgg cgccgtcacc ccgggctccc aggcagcagc
cgtgtccaac gggctggggg cccagttctt ccggggctcc cccgcgcact acacacccct
cacccatccg gtctcggcgc cctcttcctc gggatcccca ctgtacgaag gggcggccgc
ggccacagac atcgtggaca gccagtacga cgccgcagcc caaggccgcc tcatagcctc
atggacacct gtgtcgccac cttccatgtg aagcagcaag gcccaggtcc cgaaagatgc
agtgactttt tgtcgtggca gccagtggtg actggattga cctactaggt acccagtggc
agtctcaggt taagaaggaa atgcagcctc agtaacttcc ttttcaaagc agtggaggag
cacacggcac ctttccccag agccccagca tcccttgctc acacctgcag tagcggtgct
gtcccaggtg gcttacagat gaacccaact gtggagatga tgcagttggc ccaacctcac
tgacggtgaa aaaatgtttg ccagggtcca gaaacttttt ttggtttatt tctcatacag
tgtattggca actttggcac accagaattt gtaaactcca ccagtcctac tttagtgaga
taaaaagcac actcttaatc ttcttccttg ttgctttcaa gtagttagag ttgagctgtt
aaggacagaa taaaatcata gttgaggaca gcaggtttta gttgaattga aaatttgact
gctctgcccc ctagaatgtg tgtattttaa gcatatgtag ctaatctctt gtgttgttaa
actataactg tttcatattt ttcttttgac aaagtagcca aagacaatca gcagaaagca
ttttctgcaa aataaacgca atatgcaaaa tgtgattcgt ccagttatta gtgaagcccc
tccttttgtg agtatttact gtttattg.
[0131] In other embodiments, Brachyury protein has an amino acid
sequence at least 90% identical to SEQ ID NO: 1, for example a
polypeptide that is at least or about 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%0 or 99% identical to SEQ ID NO: 1. Brachyury proteins are
disclosed herein that can be used to induce an immune response (are
immunogenic), wherein the Brachyury protein can produce a
Brachyury-specific CD4+ T cell response. In some embodiments, the
Brachyury protein produces a Brachyury specific CD4+ T cell
response and a Brachyury Specific CD8+ T cell response.
[0132] SEQ ID NO: 1 provides an exemplary sequence for the
full-length Brachyury; another full length Brachyury is this amino
acid sequence with the N-terminal methionine removed. In some
examples, the Brachyury protein includes the amino acid sequence
set forth as SEQ ID NO: 1, with substitutions at position 177 (Asp
vs. Gly, respectively), position 368 (Thr vs. Ser, respectively)
and position 409 (Asn vs. Asp, respectively). Thus, these sequences
can be used to induce a Brachyury specific CD4+ T cell
response.
[0133] Positions 41 to 223 of the amino acid sequence set forth as
SEQ ID NO: 1 represents the T-box DNA binding domain of human
Brachyury, and the T-box domain in other Brachyury sequences,
including Brachyury sequences from other species, can be readily
identified by comparison to these sequences. As used herein,
reference to a T-box domain of a Brachyury protein can include an
additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39 or 40 consecutive amino acids of the
Brachyury sequence on the N-terminal and/or the C-terminal end of
the defined T-box domain (e.g., on either side of positions 41-223
of SEQ ID NO: 1). In some embodiments, the Brachyury protein
comprises the T-box domain of SEQ ID NO: 1 and induces a Brachyury
specific CD4+ T cell response. In some embodiment, the polypeptide
includes the T-box DNA binding domain or a portion thereof.
[0134] Human Brachyury has very high homology with Brachyury from
other animal species and therefore, the sequences of Brachyury from
other organisms can be utilized, particularly where these sequences
are identical, substantially homologous, and elicit an effective
immune response against the target antigen (e.g., native Brachyury
expressed by a tumor cell). For example, murine Brachyury, which
was cloned by Hermann and colleagues in 1990 (Hermann et al.,
supra), and is approximately 85% identical to human Brachyury at
the nucleotide level. Murine Brachyury is approximately 91%
identical to human Brachyury at the amino acid level. With respect
to Brachyury from other animals, at the amino acid level, human
Brachyury is 99.5% identical to Brachyury from Pan troglodytes,
90.1% identical to Brachyury from Canis lupus familiaris, 88.5%
identical to Brachyury from Bos Taurus, 92.2% identical to
Brachyury from Rattus norvegicus, and 80.9% identical to Brachyury
from Gallus gallus. Nucleic acids encoding these Brachyury proteins
can be used in the poxviral vectors and methods disclosed herein.
Generally, the T-box domain of these Brachyury proteins is included
in the region of amino acids 1-223. These polypeptides can be used
to induce a Brachyury specific CD4+ T cell response.
[0135] Mouse and human Brachyury differ by two amino acids (at
positions 26 and 96) in the T-box region. The murine Brachyury has
the amino acid sequence set forth as:
TABLE-US-00005 (SEQ ID NO: 3, 436 amino acids)
MSSPGTESAGKSLQYRVDHLLSAVESELQAGSEKGDPTERELRVGLE
ESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDF
VTADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPV
SFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFP
ETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERNDHKDVMEEPGD
CQQPGYSQWGWLVPGAGTLCPPASSHPQFGGSLSLPSTHGCERYPAL
RNHRSSPYPSPYAHRNSSPTYADNSSACLSMLQSHDNWSSLGVPGHT
SMLPVSHNASPPTGSSQYPSLWSVSNGTITPGSQTAGVSNGLGAQFF
RGSPAHYTPLTHTVSAATSSSSGSPMYEGAATVTDISDSQYDTAQSL LIASWTPVSPPSM
A nucleotide sequence encoding murine Brachyury is:
TABLE-US-00006 (SEQ ID NO: 4) ggctccgcag agtgaccctt tttcttggaa
aagcggtggc gagagaagtg aaggtggctg ttgggtaggg agtcaagact cctggaaggt
ggagagggtg gcgggaggat gagctcgccgggcacagaga gcgcagggaa gagcctgcag
taccgagtgg accacctgct cagcgccgtggagagcgagc tgcaggcggg cagcgagaag
ggagacccca ccgaacgcga actgcgagtg ggcctggagg agagcgagct gtggctgcgc
ttcaaggagc taactaacga gatgattgtg accaagaacg gcaggaggat gttcccggtg
ctgaaggtaa atgtgtcagg cctggacccc aatgccatgt actctttctt gctggacttc
gtgacggctg acaaccaccg ctggaaatat gtgaacgggg agtgggtacc tgggggcaaa
ccagagcctc aggcgcccag ctgcgtctac atccacccag actcgcccaa ttttggggcc
cactggatga aggcgcctgt gtctttcagc aaagtcaaac tcaccaacaa gctcaatgga
gggggacaga tcatgttaaa ctccttgcat aagtatgaac ctcggattca catcgtgaga
gttgggggcc cgcaacgcat gatcaccagc cactgctttc ccgagaccca gttcatagct
gtgactgcct accagaatga ggagattaca gcccttaaaa ttaaatacaa cccatttgct
aaagccttcc ttgatgccaa agaaagaaac gaccacaaag atgtaatgga ggaaccgggg
gactgccagc agccggggta ttcccaatgg gggtggcttg ttcctggtgc tggcaccctc
tgcccgcctg ccagctccca ccctcagttt ggaggctcgc tctctctccc ctccacacac
ggctgtgaga ggtacccagc tctaaggaac caccggtcat cgccctaccc cagcccctat
gctcatcgga acagctctcc aacctatgcg gacaattcat ctgcttgtct gtccatgctg
cagtcccatg ataactggtc tagcctcgga gtgcctggcc acaccagcat gctgcctgtg
agtcataacg ccagcccacc tactggctct agccagtatc ccagtctctg gtctgtgagc
aatggtacca tcaccccagg ctcccagaca gctggggtgt ccaacgggct gggagctcag
ttctttcgag gctcccctgc acattacaca ccactgacgc acacggtctc agctgccacg
tcctcgtctt ctggttctcc gatgtatgaa ggggctgcta cagtcacaga catttctgac
agccagtatg acacggccca aagcctcctc atagcctcgt ggacacctgt gtcaccccca
tctatgtgaa ttgaactttc ctccatgtgc tgagacttgt aacaaccggt gtcaactgga
tcttctaggc tcaaagtggc aggctcttgg gacaagggaa aaataaataa ataaaagcta
gatactaaca actccatttt caaataagag caataataca tgtcctataa tcatgttcta
cagcctcttg tttgatacct acagtagtga tatgtgtcct acattatgaa gccaaggaca
gagagacggc tgtggtccag ttttttgtga ctggcagtta atcagagtcc tttgctaggt
agggtcctat atcttgtgtt tctctacaac atatatgtga ctttgaaatc ctggaattcg
tccaccccct gtcctacttt agtgagacac aaggtacacc tctaatgtcc tcccttgttg
ccttagagta gttaactttg aggacagaaa aaagcatagc cagaagattg taactgaacc
gtcaactgtt ctgcccttgg aacatgccta ctttaagcac acgtagcttt ttgtgttggg
aagtcaactg tatggatact tttctgttga caaagtagcc aaagacaatc tgcagaaagt
gttttctgca caataaaggc aatatatagc acctgg, See also the amino acid
and nucleic acid sequences set forth in GENBANK .RTM. Accession No.
NM_009309 (GI: 118130357), Oct. 29, 2011, incorporated herein by
reference.
Positions 41 to 223 of SEQ ID NO:4 represent the T-box DNA binding
domain of murine Brachyury. These Brachyury proteins can also be
used to induce a Brachyury specific T cell response.
[0136] In one embodiment, the Brachyury protein includes, consists
essentially of, or consists of, an amino acid sequence at least at
least 90% identical to SEQ ID NO: 1, for example a polypeptide that
is about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to
SEQ ID NO: 1. In another embodiment, the Brachyury proteins do not
include the first amino acid of SEQ ID NO: 1 (methionine). IN
further embodiments, the Brachyury protein includes, or consists
of, amino acids 2-435 of SEQ ID NO: 1. In yet another embodiment,
the Brachyury protein includes, or consists of, the amino acid
sequence set forth as SEQ ID NO:1, with substitutions at position
177 (Asp vs. Gly, respectively), position 368 (Thr vs. Ser,
respectively) and position 409 (Asn vs. Asp, respectively).
[0137] In some examples, the Brachyury protein includes amino acids
1-15 of SEQ ID NO: 1. In yet another embodiment, the Brachyury
protein includes, consists essentially of, or consists of, the
amino acid sequence set forth as SEQ ID NO: 3.
[0138] Brachyury polypeptides are also of use in the methods
disclosed herein. The These Brachyury polypeptides include at least
15, at least 20, at least 30, at least 40, at least 50, at least
60, at least 70, at least 80, at least 90, at least 100, at least
200, or at least 300, or at least 400 amino acids of a Brachyury
protein, such as 435 amino acids of a Brachyury protein. The
Brachyury protein can include 15, 20, 30, 40, 50, 60, 70, 80, 90,
100, 200, 300 or 400 amino acids of a Brachyury protein. In some
embodiments, a Brachyury polypeptide is 15-400, 20-400, 30-400,
40-400, 50-400, 60-400, 70-400, 80-400, 90-400, 100-400, or 200-400
amino acids of a Brachyury protein. In other embodiments, a
Brachyury polypeptide is 15-300, 20-300, 30-300, 40-300, 50-300,
60-300, 70-300, 80-300, 90-300, 100-300, or 200-300 amino acids of
a Brachyury protein. In additional embodiments, the Brachyury
polypeptide is 15 to 10-, 20-100, 25-100, 30-100, 35-100, 40-100,
45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100,
85-100, 90-100 or 95-100 amino acids of any of the Brachyury
proteins disclosed herein. The Brachyury polypeptide can be 15, 50,
100, 150, 250, 300, 350, 400, 430, 431, 432, 433 or 434 amino acids
in length. The Brachyury polypeptide can be 15-430, 15-431, 15-432,
15-433, 15-434 or 15-435 amino acids in length. An exemplary
polypeptide is shown in FIG. 2.
[0139] In further embodiments, a Brachyury polypeptide is 15-20
amino acids in length, such as 15-17 amino acids in length, such as
15, 16, 17, 17, 19 or 20 amino acids in length and binds MHC Class
II. The MHC Class II antigen can be encoded by a HLA-DP, HLA-DR,
HLA-B, HLA-DQA1 or HLA-DQB1 allele.
[0140] It is disclosed herein that Brachyury protein, Brachyury
polypeptides, nucleic acids encoding Brachyury proteins and
polypeptides, and non-pox non-yeast viral vectors including a
polynucleotide encoding a Brachyury protein can be used to induce
Brachyury specific CD4+ T cells in a subject. In additional
embodiments the Brachyury protein, Brachyury polypeptide,
polynucleotide encoding a Brachyury protein or polypeptide, or
non-pox non-yeast vector include the polynucleotide induce a
Brachyury specific CD8+ T cell response, or both a Brachyury
specific CD4+ T cell response and a CD8+ T cell response.
[0141] In several embodiments, the isolated Brachyury protein or
polypeptide is included in a fusion protein. Thus, the fusion
protein can include the Brachyury protein or Brachyury polypeptide
(see above) and a second heterologous moiety, such as a myc
protein, an enzyme or a carrier (such as a hepatitis carrier
protein or bovine serum albumin) covalently linked to the Brachyury
protein or polypeptide. Thus, in several specific non-limiting
examples, the fusion protein includes a Brachyury protein (or
Brachyury polypeptide) and six sequential histidine residues, a
.beta.-galactosidase amino acid sequence, and/or an immunoglobulin
amino acid sequence.
[0142] Brachyury proteins or polypeptides that are linked to a
carrier are also of use in the disclosed methods. Generally, a
carrier is an immunogenic macromolecule to which an antigenic
molecule can be bound. When bound to a carrier, the bound Brachyury
protein or Brachyury polypeptide becomes more immunogenic. Carriers
are chosen to increase the immunogenicity of the bound molecule
and/or to elicit higher titers of antibodies against the carrier
which are diagnostically, analytically, and/or therapeutically
beneficial. Covalent linking of a molecule to a carrier can confer
enhanced immunogenicity and T cell dependence (see Pozsgay et al.,
PNAS 96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976;
Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers include
polymeric carriers, which can be natural (for example,
polysaccharides, polypeptides or proteins from bacteria or
viruses), semi-synthetic or synthetic materials containing one or
more functional groups to which a reactant moiety can be attached.
Bacterial products and viral proteins (such as hepatitis B surface
antigen and core antigen) can also be used as carriers, as well as
proteins from higher organisms such as keyhole limpet hemocyanin,
horseshoe crab hemocyanin, edestin, mammalian serum albumins, and
mammalian immunoglobulins. Suitable carriers include, but are not
limited to, a hepatitis B small envelope protein HBsAg. This
protein has the capacity to self-assemble into aggregates and can
form viral-like particles. The preparation of HBsAg is well
documented, see for example European Patent Application Publication
No. EP-A-0 226 846, European Patent Application Publication No.
EP-A-0 299 108 and PCT Publication No. WO 01/117554, and the amino
acid sequence disclosed, for example, in Tiollais et al., Nature,
317: 489, 1985, and European Patent Publication No. EP-A-0 278 940,
and PCT Publication No. WO 91/14703, all of which are incorporated
herein by reference.
[0143] In other embodiments, only the Brachyury protein or
polypeptide is utilized. Thus, a second heterologous moiety is
non-covalently linked to the Brachyury protein or polypeptide.
[0144] Nucleic Acids Encoding Brachyury Protein and
Polypeptides
[0145] Nucleic acids that encode a Brachyury protein and/or
polypeptide can readily be produced. These nucleic acids include
DNA, cDNA and RNA sequences which encode the Brachyury polypeptide
of interest. Silent mutations in the coding sequence result from
the degeneracy (i.e., redundancy) of the genetic code, whereby more
than one codon can encode the same amino acid residue. Thus, for
example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG;
serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC;
asparagine can be encoded by AAT or AAC; aspartic acid can be
encoded by GAT or GAC; cysteine can be encoded by TGT or TGC;
alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be
encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and
isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the
standard genetic code can be found in various sources (e.g., L.
Stryer, 1988, Biochemistry, 3.sup.rd Edition, W.H. 5 Freeman and
Co., NY).
[0146] A nucleic acid encoding a Brachyury protein can be cloned or
amplified by in vitro methods, such as the polymerase chain
reaction (PCR), the ligase chain reaction (LCR), the
transcription-based amplification system (TAS), the self-sustained
sequence replication system (3SR) and the Q.beta. replicase
amplification system (QB). For example, a polynucleotide encoding
the Brachyury protein can be isolated by polymerase chain reaction
of cDNA using primers based on the DNA sequence of the molecule. A
wide variety of cloning and in vitro amplification methodologies
are well known to persons skilled in the art. PCR methods are
described in, for example, U.S. Pat. No. 4,683,195; Mullis et al.,
Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich,
ed., PCR Technology, (Stockton Press, N Y, 1989). Polynucleotides
also can be isolated by screening genomic or cDNA libraries with
probes selected from the sequences of the desired polynucleotide
under stringent hybridization conditions.
[0147] A polynucleotide sequence encoding a Brachyury protein or
polypeptide can be operatively linked to expression control
sequences. An expression control sequence operatively linked to a
coding sequence is ligated such that expression of the coding
sequence is achieved under conditions compatible with the
expression control sequences. The expression control sequences
include, but are not limited to, appropriate promoters, enhancers,
transcription terminators, a start codon (i.e., ATG) in front of a
protein-encoding gene, splicing signal for introns, maintenance of
the correct reading frame of that gene to permit proper translation
of mRNA, and stop codons. Suitable promoters include, but are not
limited to, an SV40 early promoter, RSV promoter, adenovirus major
late promoter, human CMV immediate early I promoter, poxvirus
promoter, 30K promoter, I3 promoter, sE/L promoter, 7.5K promoter,
40K promoter, and C1 promoter. T DNA vaccines are described in U.S.
Pat. Nos. 5,589,466; 5,973,972, which are each incorporated herein
by reference. In addition to the delivery protocols described in
those applications, alternative methods of delivering DNA are
described in U.S. Pat. Nos. 4,945,050 and 5,036,006, which are both
incorporated herein by reference.
[0148] Plasmids have been designed with a number of goals in mind,
such as achieving regulated high copy number and avoiding potential
causes of plasmid instability in bacteria, and providing means for
plasmid selection that are compatible with human therapeutic use.
Particular attention has been paid to the dual requirements of gene
therapy plasmids. First, they are suitable for maintenance and
fermentation in E. coli, so that large amounts of DNA can be
produced and purified. Second, they are safe and suitable for use
in human patients and animals. The first requirement calls for high
copy number plasmids that can be selected for and stably maintained
relatively easily during bacterial fermentation. The second
requirement calls for attention to elements such as selectable
markers and other coding sequences. In some embodiments plasmids
that encode a Brachyury protein or polypeptide are composed of: (1)
a high copy number replication origin, (2) a selectable marker,
such as, but not limited to, the neo gene for antibiotic selection
with kanamycin, (3) transcription termination sequences, and (4) a
multicloning site for incorporation of various nucleic acid
cassettes; and (5) a nucleic acid sequence encoding a Brachyury
protein and/or a Brachyury polypeptide.
[0149] There are numerous plasmid vectors that are known in the art
for inducing a nucleic acid encoding a protein. These include, but
are not limited to, the vectors disclosed in U.S. Pat. Nos.
6,103,470; 7,598,364; 7,989,425; and U.S. Pat. No. 6,416,998, which
are incorporated herein by reference.
[0150] Non-Pox Non-Yeast Vectors
[0151] Non-poxviral non-yeast vectors can be used to express the
Brachyury proteins and/or polypeptides disclosed herein. These
vectors are not poxvirus vectors, and thus are not an orthopox,
suipox, avipox, or capripox virus vector. Orthopox include
vaccinia, ectromelia, and raccoon pox. One example of an orthopox
is vaccinia. Avipox includes fowlpox, canary pox and pigeon pox.
Capripox include goatpox and sheeppox. An example of a suipox is
swinepox vector. Exemplary pox viral vectors for expression as
described for example, in U.S. Pat. No. 6,165,460, which is
incorporated herein by reference. The vaccinia virus genome is
known in the art. It is composed of a HIND F13L region, TK region,
and an HA region. Recombinant vaccinia virus has been used to
incorporate an exogenous gene for expression of the exogenous gene
product (see, for example, Perkus et al. Science 229:981-984, 1985;
Kaufman et al. Inti J. Cancer 48:900-907, 1991; Moss Science
252:1662, 1991). Baxby and Paoletti (Vaccine 10:8-9, 1992) disclose
the construction and use as a vector, of the non-replicating
poxvirus, including canarypox virus, fowlpox virus and other avian
species. Sutter and Moss (Proc. Nat'l. Acad. Sci U.S.A.
89:10847-10851, 1992) and Sutter et al. (Virology 1994) disclose
the construction and use as a vector, the non-replicating
recombinant Ankara virus (MVA, modified vaccinia Ankara) in the
construction and use of a vector. These vectors are not used in the
present methods.
[0152] The vectors disclosed herein are also non-yeast vectors.
Thus, the disclosed vectors are not used for expression in yeast
such as S. cerevisiae or Kluyveromyces lactis. Thus, the disclosed
vectors generally do not include all the required elements for
expression in yeast. As examples, promoters are known to be of use
in yeast expression systems such as the constitutive promoters
plasma membrane H.sup.+-ATPase (PMA1), glyceraldehyde-3-phosphate
dehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcohol
dehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5).
The promoters are not utilized in the presently disclosed vectors.
In addition, many inducible promoters, such as GAL1-10 (induced by
galactose), PHO5 (induced by low extracellular inorganic
phosphate), and tandem heat shock HSE elements (induced by
temperature elevation to 37.degree. C.) are not used in the present
vectors. Promoters that direct variable expression in response to a
titratable inducer include the methionine-responsive MET3 and MET25
promoters and copper-dependent CUP1 promoters; these promoters are
not utilized. In additional examples, the vectors do not include
yeast nutritional markers (such as URA3, ADE3, HIS1, and others)
for selection in yeast.
[0153] A number of non-pox non-yeast viral vectors can be utilized,
including polyoma, SV40 (Madzak et al., 1992, J. Gen. Virol.,
73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol.
Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques,
6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin
et al., 1992, Proc. Nad. Acad. Sci. USA, 89:2581-2584; Rosenfeld et
al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids
Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene
Ther., 1:241-256), vaccinia virus (Mackett et al., 1992,
Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992,
Curr. Top. Microbiol. Immunol., 158:91-123; On et al., 1990, Gene,
89:279-282), herpes viruses including HSV and EBV (Margolskee,
1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al.,
1992, J. Virol., 66:29522965; Fink et al., 1992. Hum. Gene Ther.
3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371;
Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199), Sindbis
viruses (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167;
U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.
Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al.,
1996, Proc. Natl. Acad. Sci. USA 93:11371-11377), human herpesvirus
vectors (HHV) such as HHV-6 and HHV-7, and retroviruses of avian
(Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754;
Petropouplos et al., 1992, J. Virol., 66:3391-3397), murine
(Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24; Miller et
al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al., 1984, Mol.
Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401-407),
and human origin (Page et al., 1990, J. Virol., 64:5370-5276;
Buchschalcher et al., 1992, J. Virol., 66:2731-2739). Baculovirus
(Autographa californica multinuclear polyhedrosis virus; AcMNPV)
vectors can be used. Vectors can be obtained from commercial
sources (such as PharMingen, San Diego, Calif.; Protein Sciences
Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.). Suitable
vectors are disclosed, for example, in U.S. Published Patent
Application No. 2010/0247486, which is incorporated herein by
reference. In specific non-limiting examples, the vectors are
retrovirus vectors (for example, lentivirus vectors), measles virus
vectors, alphavirus vectors, baculovirus vectors, Sindbis virus
vectors, adenovirus and poliovirus vectors. These vectors include a
polynucleotide that encodes a Brachyury protein or Brachyury
polypeptide.
[0154] Non-pox non-yeast vectors that encode a Brachyury protein or
Brachyury polypeptide include at least one expression control
element operationally linked to the nucleic acid sequence encoding
the Brachyury protein or polypeptide. The expression control
elements are inserted in the vector to control and regulate the
expression of the nucleic acid sequence. Examples of expression
control elements of use in these vectors includes, but is not
limited to, lac system, operator and promoter regions of phage
lambda, promoters derived from polyoma, adenovirus, retrovirus or
SV40. Additional operational elements include, but are not limited
to, leader sequence, termination codons, polyadenylation signals
and any other sequences necessary for the appropriate transcription
and subsequent translation of the nucleic acid sequence encoding
the Brachyury protein or the Brachyury polypeptide in the host
system. The expression vector can contain additional elements
necessary for the transfer and subsequent replication of the
expression vector containing the nucleic acid sequence in the host
system. Examples of such elements include, but are not limited to,
origins of replication and selectable markers. It will further be
understood by one skilled in the art that such vectors can be
constructed using conventional methods (Ausubel et al., (1987) in
"Current Protocols in Molecular Biology," John Wiley and Sons, New
York, N.Y.) and are commercially available.
[0155] Optionally, the vector can encode one or more
immunostimulatory molecules, such as IL-2, IL-6, IL-12, LFA (for
example, LFA-1, LFA-2 and/or LFA-3), CD72, RANTES, G-CSF, GM-CSF,
TNF-.alpha., IFN-.gamma., ICAM-1, B7-1, B7-2, other B7 related
molecules, OX-40L or 41 BBL, or combinations of these molecules.
These immuostimulatory molecules can be used as biological
adjuvants (see, for example, Salgaller et al., 1998, J. Surg.
Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl
1):S61-6, Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper
et al., 2000, Adv. Exp. Med. Biol. 465:381-90). In several
examples, the vector can encode IL-2, RANTES, GM-CSF, TNF-.alpha.,
IFN-.gamma., G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1, B7-2, OX-40L, 41
BBL and/or ICAM-1.
[0156] Basic techniques for preparing recombinant DNA viruses
containing a heterologous DNA sequence encoding a Brachyury protein
or Brachyury polypeptide are known in the art. Such techniques
involve, for example, homologous recombination between the DNA
sequences flanking the DNA sequence in a donor plasmid and
homologous sequences present in a parental virus (Mackett et al.,
1982, Proc. Natl. Acad. Sci. USA 79:7415-7419). In particular,
recombinant viral vectors can be used in delivering the gene. The
vector can be constructed, for example, by steps known in the art,
including using a unique restriction endonuclease site that is
naturally present or artificially inserted in the parental viral
vector to insert the heterologous DNA encoding the Brachyury
protein or Brachyury polypeptide.
[0157] Generally, a DNA donor vector contains the following
elements: (i) a prokaryotic origin of replication, so that the
vector may be amplified in a prokaryotic host; (ii) a gene encoding
a marker which allows selection of prokaryotic host cells that
contain the vector (e.g., a gene encoding antibiotic resistance);
(iii) at least one DNA sequence encoding the Brachyury protein or
Brachyury polypeptide located adjacent to a transcriptional
promoter capable of directing the expression of the sequence; and
(iv) DNA sequences homologous to the region of the parent virus
genome where the foreign gene(s) will be inserted, flanking the
construct of element (iii). Methods for constructing donor plasmids
for the introduction of multiple foreign genes into viral virus are
described in PCT Publication no WO 91/19803, incorporated herein by
reference.
[0158] Generally, DNA fragments for construction of the donor
vector, including fragments containing transcriptional promoters
and fragments containing sequences homologous to the region of the
parent virus genome into which foreign DNA sequences are to be
inserted, can be obtained from genomic DNA or cloned DNA fragments.
The donor plasmids can be mono, di-, or multivalent (i.e., can
contain one or more inserted foreign DNA sequences). The donor
vector can contain an additional gene that encodes a marker that
will allow identification of recombinant viruses containing
inserted foreign DNA. Several types of marker genes can be used to
permit the identification and isolation of recombinant viruses.
These include genes that encode antibiotic or chemical resistance
(e.g., see Spyropoulos et al., 1988, J. Virol. 62:1046; Falkner and
Moss, 1988, J. Virol. 62:1849; Franke et al., 1985, Mol. Cell.
Biol. 5:1918), as well as genes such as the E. coli lacZ gene, that
permit identification of recombinant viral plaques by colorimetric
assay (Panicali et al., 1986, Gene 47:193-199).
[0159] The DNA gene sequence to be inserted into the virus can be
placed into a donor plasmid into which DNA homologous to a section
of DNA such as that of the insertion site of the virus where the
DNA is to be inserted has been inserted. Separately the DNA gene
sequence to be inserted is ligated to a promoter. The promoter-gene
linkage is positioned in the plasmid construct so that the
promoter-gene linkage is flanked on both ends by DNA homologous to
a DNA sequence flanking a region of viral DNA that is the desired
insertion region. With a parental adenoviral vector, an adenoviral
promoter is used. Similarly, with a parental lentiviral vector, a
lentivirus promoter is used. The resulting plasmid construct is
then amplified by growth within E. coli bacteria and isolated.
Next, the isolated plasmid containing the DNA gene sequence to be
inserted is transfected into a cell culture, along with the
parental virus. Recombination between homologous viral DNA in the
plasmid and the viral genome respectively results in a recombinant
virus modified by the presence of the promoter-gene construct in
its genome, at a site that does not affect virus viability.
[0160] As noted above, the DNA sequence is inserted into a region
(insertion region) in the virus that does not affect virus
viability of the resultant recombinant virus. One of skill in the
art can readily identify such regions in a virus by, for example,
randomly testing segments of virus DNA for regions that allow
recombinant formation without seriously affecting virus viability
of the recombinant.
[0161] Homologous recombination between donor plasmid DNA and viral
DNA in an infected cell can result in the formation of recombinant
viruses that incorporate the desired elements. Appropriate host
cells for in vivo recombination are generally eukaryotic cells that
can be infected by the virus and transfected by the plasmid vector.
Examples of such cells suitable for use with viral vectors are
fibroblasts, HuTK143 (human) cells, and CV-1 and BSC-40 (both
monkey kidney) cells. Infection of cells with a virus and
transfection of these cells with plasmid vectors is accomplished by
techniques standard in the art (see U.S. Pat. No. 4,603,112 and PCT
Publication No. WO 89/03429). Following in vivo recombination,
recombinant viral progeny can be identified. For example,
co-integration of a gene encoding a marker or indicator gene with
the foreign gene(s) of interest, as described above, can be used to
identify recombinant progeny. One specific non-limiting example of
an indicator gene is the E. coli lacZ gene. Recombinant viruses
expressing beta-galactosidase can be selected using a chromogenic
substrate for the enzyme (Panicali et al., 1986, Gene 47:193). Once
a recombinant virus has been identified, a variety of well-known
methods can be used to assay the expression of the Brachyury
protein or Brachyury polypeptide encoded by the inserted DNA
fragment. These methods include black plaque assay (an in situ
enzyme immunoassay performed on viral plaques), Western blot
analysis, radioimmunoprecipitation (RIPA), and enzyme immunoassay
(EIA).
[0162] This disclosure encompasses a recombinant vector comprising
more than one antigen of interest for the purpose of having a
multivalent vaccine. For example, the recombinant vectors, such as
a viral vector, can comprise the virus genome or portions thereof,
the nucleic acid sequence encoding the Brachyury protein or
Brachyury polypeptide and a nucleic acid sequence encoding a
carrier, such as, but not limited to, hepatitis B surface
antigen.
[0163] The vectors of use in the methods disclosed herein are
non-yeast, non-poxviral vectors. Vectors that are useful include
adenovirus, alphavirus, lentivirus, measles virus and poliovirus
vectors. However, this disclosure is not limited to these types of
non-yeast non-poxviral vectors. Additional vectors herpes simplex
viruses, human papilloma virus, Simian immunodeficiency viruses,
human T cell lymphoma virus (HTLV), human foamy virus,
spumaviruses, mammalian type B retroviruses, mammalian type C
retroviruses, avian type C retroviruses, mammalian type D
retroviruses. Vectors also include Epstein-Barr virus vectors,
Moloney murine leukemia virus vectors, Harvey murine sarcoma virus
vectors, murine mammary tumor virus vectors, Rous sarcoma virus
vectors and nonviral plasmid vectors. Several types of vectors of
use are disclosed below. Compositions including these vectors are
of use in inducing a CD4+ T cell response to Brachyury and for the
treatment of cancer. These compositions also can be used to induce
a CD8+ T cell response to Brachyury.
[0164] Adenovirus Vectors
[0165] Adenovirus vectors (Ad) vectors can be produced that encode
a Brachyury protein or a Brachyury polypeptide and are of use in
the methods disclosed herein. These vectors are of use in the
methods disclosed herein, including replication competent,
replication deficient, gutless forms thereof, and adeno-associated
virus (AAV) vectors. Without being bound by theory, adenovirus
vectors are known to exhibit strong expression in vitro, excellent
titer, and the ability to transduce dividing and non-dividing cells
in vivo (Hitt et al., Adv in Virus Res 55:479-505, 2000). When used
in vivo these vectors lead to strong but transient gene expression
due to immune responses elicited to the vector backbone.
[0166] Adenoviral vectors are often constructed by insertion of a
nucleic acid encoding a Brachyury protein in place of, or in the
middle of, essential viral sequences such as those found at the E1
region of adenovirus (Berkner, BioTechniques, 6:616-629, 1988;
Graham et al., Methods in Molecular Biology, 7:109-128, Ed: Murcy,
The Human Press Inc., 1991). Inactivation of essential viral genes
by, for example, deletion or insertion, disables the adenovirus'
ability to replicate. To propagate such vectors in cell culture,
the deleted genes must be provided in trans (for example, the E1A
and E1B proteins in the case of an E1 delete vector). These
replication-defective adenoviruses are produced in packaging cells
engineered to complement the replication-incompetent virus by
expressing the subset of genetic elements deleted from their viral
genome. Potential sites for the insertion of a nucleic acid of
interest, such as a nucleic acid encoding a Brachyury protein, in
recombinant adenoviral vectors include, without limitation, the E1,
E2, E3 and the E4 region. In some embodiments, a recombinant
adenoviral vector is produced from a human adenovirus that has the
E1 region deleted and replaced with a nucleic acid encoding a
Brachyury protein or Brachyury polypeptide. The resulting viral
vector, with one or more of its essential genes inactivated, is
replication defective (Statford-Perricaudet et al., Human Gene
Therapy, 1:241-256, 1990).
[0167] The recombinant adenovirus vectors can include: (1) a
packaging site enabling the vector to be incorporated into
replication-defective Ad virions; and (2) the nucleic acid encoding
the Brachyury protein or Brachyury polypeptide. Other elements of
use for incorporation into infectious virions, include the 5' and
3' Ad ITRs; the E2 and E3 genes can be included in the vector. In
some embodiments, a nucleic acid encoding a Brachyury protein or
Brachyury polypeptide is inserted into adenovirus in the deleted
E1A, E1B or E3 region of the virus genome. In some embodiments, the
adenovirus vectors do not express one or more wild-type adenovirus
gene products, such as E1a, E1b, E2, E3, E4. In some non-limiting
examples, virions are typically used together with packaging cell
lines that complement the functions of E1, E2A, E4 and optionally
the E3 gene regions (see, for example, U.S. Pat. Nos. 5,872,005,
5,994,106, 6,133,028 and 6,127,175, incorporated by reference
herein in their entirety). Adenovirus vectors can be purified and
formulated using techniques known in the art.
[0168] In some embodiments, packaging cell lines such as the human
embryonic kidney 293 ("HEK-293" or "293") cell line (Graham et al.,
J. Gen. Virol., 36:59-72, 1977) or human embryonic retinoblast
("HER-911" or "911") cell line (Fallaux et al., Hum. Gene Ther.,
7:215-222, 1996), provide in trans the missing region, such as the
E1 region, so that the deleted or modified adenoviral vector can
replicate in such cells. Suitable adenoviral vectors are disclosed,
for example, in U.S. Patent Publication No. 20080193484, which is
incorporated herein by reference. Replication-defective adenovirus
virions encapsulating the recombinant adenovirus vectors can be
made by standard techniques known in the art using packaging cells
and packaging technology. Examples of these methods can be found,
for example, in U.S. Pat. No. 5,872,005, incorporated herein by
reference in its entirety.
[0169] Recombinant AAV vectors are characterized in that they are
capable of directing the expression and the production of the
selected transgenic products in targeted cells. Thus, the
recombinant vectors comprise at least all of the sequences of AAV
essential for encapsidation and the physical structures for
infection of target cells.
[0170] Recombinant AAV (rAAV) virions can be constructed such that
they include, as operatively linked components in the direction of
transcription, control sequences including transcriptional
initiation and termination sequences, and the nucleic acid encoding
the Brachyury protein or Brachyury polypeptide. These components
are bounded on the 5' and 3' end by functional AAV inverted
terminal repeat (ITR) sequences. By "functional AAV ITR sequences"
is meant that the ITR sequences function as intended for the
rescue, replication and packaging of the AAV virion. Hence, AAV
ITRs for use in the vectors need not have a wild-type nucleotide
sequence, and can be altered by the insertion, deletion or
substitution of nucleotides, or the AAV ITRs can be derived from
any of several AAV serotypes, provided they are functional. An AAV
vector is a vector derived from an adeno-associated virus serotype,
including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5,
AAV-6, AAV-7, AAV-8, etc. In some embodiments, the AAV vectors have
the wild type REP and CAP genes deleted in whole or part, but
retain functional flanking ITR sequences. These vectors can all be
used, without limitation, for the expression of a Brachyury
protein.
[0171] Alphavirus
[0172] Alphaviruses encoding a Brachyury protein or Brachyury
polypeptide are provided and are of use in the methods disclosed
herein. Alphaviruses are a set of serologically related
arthropod-borne viruses of the Togavirus family. Twenty-six known
viruses and virus subtypes have been classified within the
alphavirus genus utilizing the hemagglutination inhibition (HI)
assay. Briefly, the HI test segregates the 26 alphaviruses into
three major complexes: the Venezuelan encephalitis (VE) complex,
the Semliki Forest (SF) complex, and the western encephalitis (WE)
complex. In addition, four additional viruses, eastern encephalitis
(EE), Barmah Forest, Middelburg, and Ndumu, receive individual
classification based on the HI serological assay. Representative
examples of suitable alphaviruses include Aura (American Type
Culture Collection (ATCC) VR-368), Bebaru virus (ATCC VR-600, ATCC
VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64,
ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65,
ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369,
ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66),
Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo
virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna
virus (ATCC VR-372, ATCC VR-1245), Ross River virus (ATCC VR-373,
ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis
virus (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti
(ATCC VR-469), Una (ATCC VR-374), Venezuelan equine
encephalomyelitis (ATCC VR-69), Venezuelan equine encephalomyelitis
virus (ATCC VR-923, ATCC VR-1250 ATCC VR-1249, ATCC VR-532),
Western equine encephalomyelitis (ATCC VR-70, ATCC VR-1251, ATCC
VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC
VR-375), see U.S. Pat. No. 5,843,723, which is incorporated herein
by reference.
[0173] In some embodiments, and alphavirus vector is a Sinbis
virus. In some embodiments, recombinant alphavirus vector
constructs are utilized that include a 5' sequence which is capable
of initiating transcription of an alphavirus, a nucleotide sequence
encoding alphavirus nonstructural proteins, a viral junction region
which has been inactivated such that viral transcription of the
subgenomic fragment is prevented, an alphavirus RNA polymerase
recognition sequence, and a nucleic acid sequence encoding a
Brachyury protein. Alphavirus vector constructs which have
inactivated viral junction regions do not transcribe the subgenomic
fragment, making them suitable for a wide variety of
applications.
[0174] In some embodiments, the alphavirus such as Sinbis virus,
constructs are provided which contain a 5' promoter which is
capable of initiating the synthesis of viral RNA in vitro from
cDNA. The 5' promoters include both eukaryotic and prokaryotic
promoters, such as, for example, the .beta.-galactosidase promoter,
trpE promoter, lacZ promoter, T7 promoter, T3 promoter, SP6
promoter, SV40 promoter, CMV promoter, and MoMLV LTR.
Representative examples of such sequences include nucleotides 1-60,
and to a lesser extent nucleotides 150-210, of the wild-type
Sindbis virus, nucleotides 10-75 for tRNA Asparagine (Schlesinger
et al., U.S. Pat. No. 5,091,309, incorporated herein by refernece),
and 5' sequences from other Togaviruses which initiate
transcription.
[0175] Alphavirus vectors can contain sequences which encode
alphavirus nonstructural proteins (NSPs). As an example, for
Sindbis virus there are four nonstructural proteins, NSP1, NSP2,
NSP3 and NSP4, which encode proteins that enable the virus to
self-replicate. Nonstructural proteins 1 through 3 (NSP1-NSP3) are,
encoded by nucleotides 60 to 5750 of the wild-type Sindbis virus.
These proteins are produced as a polyprotein and later cleaved into
nonstructural proteins NSP1, NSP2, and NSP3. NSP4. The alphavirus
vector constructs can also include a viral junction region which
has been inactivated, such that viral transcription of the
subgenomic fragment is prevented. Briefly, the alphavirus viral
junction region normally controls transcription initiation of the
subgenomic mRNA. In the case of the Sindbis virus, the normal viral
junction region typically begins at approximately nucleotide number
7579 and continues through at least nucleotide number 7612 (and
possibly beyond), see U.S. Pat. No. 5,843,723 for the complete
sequence, incorporated herein by reference.
[0176] Several members of the alphavirus genus can be used as
"replicon" expression vectors. Replicon vectors may be utilized in
any of several formats, including DNA vector constructs, RNA
replicon vectors, and recombinant replicon particles (see below).
These include, for example, SIN (Xiong et al., Science
243:1188-1191, 1989; Dubensky et al., J. Virol. 70:508-519, 1996;
Hariharan et al., J. Virol. 72:950-958, 1998; Polo et al., PNAS
96:4598-4603, 1999), Semliki Forest virus (Liljestrom,
Bio/Technology 9:1356-1361, 1991; Berglund et al., Nat. Biotech.
16:562-565, 1998), VEE (Pushko et al. Virology 239:389-401, 1997),
and chimeras of multiple alphaviruses (U.S. Pat. No. 6,376,236; PCT
Publication No. WO2002099035; Perri et al., J. Virol.
77:10394-10403, 2003).
[0177] Alphavirus vector constructs are also disclosed in U.S. Pat.
Nos. 5,789,245; 5,843,723; 5,814,482, and U.S. Pat. No. 6,015,694;
PCT Publication No. WO 00/61772; and PCT Publication No. WO
02/99035. Generally, these vectors include a 5' sequence which
initiates transcription of alphavirus RNA, a nucleotide sequence
encoding alphavirus nonstructural proteins, a viral subgenomic
junction region promoter which directs the expression of an
adjacent heterologous nucleic acid sequence, an RNA polymerase
recognition sequence and a polyadenylate tract.
[0178] An alphavirus can be used as a replicon (a recombinant
alphavirus particle) that is a virus-like particle containing a
self-replicating alphavirus vector or "replicon" nucleic acid. The
replicon particle itself is generally considered to be replication
incompetent or "defective," that is no progeny replicon particles
will result when a host cell is infected with a replicon particle,
because genes encoding one or more structural proteins necessary
for packaging are deleted.
[0179] Although alphavirus vectors can be used directly for
administration in vivo as RNA, or delivered as a plasmid-based cDNA
(e.g., Eukaryotic Layered Vector Initiation System), often, for in
vivo vaccine and therapeutic applications, the alphavirus RNA
replicon vector or replicon RNA is first packaged into a virus-like
particle, comprising alphavirus structural proteins (e.g., capsid
protein and envelope glycoproteins). Alphavirus and replicons of
use are disclosed, for example, in Published U.S. Patent
Application No. 20110002958, which is incorporated herein by
reference. Because of their configuration, vector replicons do not
express these alphavirus structural proteins necessary for
packaging into recombinant alphavirus replicon particles. Thus, to
generate replicon particles, the structural proteins must be
provided in trans.
[0180] Packaging can be accomplished by a variety of methods,
including transient approaches such as co-transfection of in vitro
transcribed replicon and defective helper RNA(s) (Liljestrom,
Bio/Technology 9:1356-1361, 1991; Bredenbeek et al., I Virol.
67:6439-6446, 1993; Frolov et al., J. Virol. 71:2819-2829, 1997;
Pushko et al., Virology 239:389-401, 1997; U.S. Pat. No. 5,789,245
and U.S. Pat. No. 5,842,723) or plasmid DNA-based replicon and
defective helper constructs (Dubensky et al., J. Virol. 70:508-519,
1996), as well as introduction of alphavirus replicons into stable
packaging cell lines (PCL) (Polo et al., PNAS 96:4598-4603, 1999;
U.S. Pat. Nos. 5,789,245; 5,842,723; 6,015,694).
[0181] The trans packaging methodologies permit the modification of
one or more structural protein genes (for example, to incorporate
sequences of alphavirus variants such as the attenuated mutants,
see U.S. Pat. Nos. 5,789,245; 5,842,723; 6,015,694), followed by
the subsequent incorporation of the modified structural protein
into the final replicon particles. In addition, such packaging
permits the overall modification of alphavirus replicon particles
by packaging of a vector construct or RNA replicon derived from a
first alphavirus using structural proteins derived from a second
alphavirus different from that of the vector construct.
[0182] Measles Virus
[0183] Measles viruses encoding a Brachyury protein or Brachyury
polypeptide are provided and are of use in the methods disclosed
herein. The nucleic acid sequences of Measles Viruses are disclosed
in PCT Publication No. WO 98/13501, which provides the sequence of
a DNA copy of the positive strand (antigenomic) message sense RNA
of various wild-type of vaccine measles strains, including
Edmonston Wild-type strain, Moraten strain and Schwarz strain. PCT
Publication No. WO 97/06270, incorporated herein by reference,
discloses the production of recombinant measles vectors.
[0184] An attenuated strain of measles virus can also be used to
deliver a Brachyury protein or Brachyury polypeptide. The Moraten
attenuated form of the virus has been used world-wide as a vaccine
and has an excellent safety record (Hilleman, et al., J. Am. Med.
Assoc. 206: 587-590, 1968). Accordingly, in one embodiment, the
Moraten strain is used. The Moraten vaccine is commercially
available from MERCK.RTM. and is provided lyophilized in a vial
which when reconstituted to 0.5 ml comprises 10.sup.3 pfu/ml.
[0185] In a further embodiment, the Edmonston-B vaccine strain of
measles virus is used (MV-Edm) (Enders and Peebles, Proc. Soc. Exp.
Biol. Med. 86: 277-286, 1954). MV-Edm grows efficiently in tumor
cells but its growth is severely restricted in primary cultures of
human peripheral blood mononuclear cells, normal dermal
fibroblasts, and vascular smooth muscle cells. A form of the Enders
attenuated Edmonston strain is available commercially from Merck
(ATTENUVAX.RTM.). Other attenuated measles virus strains can also
be utilized, such as Leningrad-16, and Moscow-5 strains (Sinitsyna,
et al., Res. Virol. 141(5): 517-31, 1990), Schwarz strain
(Fourrier, et al., Pediatrie 24(1): 97-8, 1969), 9301B strain
(Takeda, et al. J. VIROL. 72/11: 8690-8696), the AIK-C strain
(Takehara, et al., Virus Res 26 (2): 167-75, 1992), and those
described in Schneider-Shaulies, et al., PNAS 92(2): 3943-7,
1995).
[0186] In some embodiments, the recombinant measles virus
nucleotide sequence comprises a replicon having a total number of
nucleotides which is a multiple of six. The "rule of six" is
expressed in the fact that the total number of nucleotides present
in the recombinant cDNA finally amount to a total number of
nucleotides which is a multiple of six, a rule which allows
efficient replication of genome RNA of the measles virus.
[0187] In additional embodiments, heterologous DNA, such as a
nucleic acid encoding Brachyury protein, is cloned in the measles
virus within an Additional Transcription Unit (ATU) inserted in the
cDNA corresponding to the antigenomic RNA of measles virus. The
location of the ATU can vary along the cDNA: it is however located
in such a site that it will benefit from the expression gradient of
the measles virus. Therefore, the ATU can be spread along the cDNA.
In one embodiment, the ATU is inserted in the N-terminal portion of
the sequence and especially within the region upstream from the
L-gene of the measles virus and upstream from the M gene of the
virus. In other embodiments, the ATU is inserted upstream from the
N gene of the virus, see U.S. Published Patent Application No.
2011/0129493, incorporated herein by reference. Particular cistrons
in the measles virus genome can targeted to modify genes whose
expression is associated with attenuation (Schneider-Shaulies et
at. PNAS 92(2): 3943-7, 1995, Takeda, et al. J. Virol. 72/11:
8690-8696, 1998). Thus, in one embodiment, a recombinant measles
virus strain is generated encoding a Brachyury protein or Brachyury
polypeptide in any of an H protein, a V protein, a C protein, and
combinations thereof.
[0188] Recombinant measles virus vectors include the plasmid
pTM-MVSchw which contains the cDNA resulting from reverse
transcription of the antigenomic RNA of measles virus and an
adapted expression control sequence including a promoter and
terminator for the T7 polymerase. Vectors are also disclosed, for
example, in U.S. Published Patent Application No. 2006/0013826,
which is incorporated herein by reference. These vectors are of use
in the methods disclosed herein.
[0189] Additional attenuated strains of measles virus can be
produced that express a Brachyury protein or Brachyury polypeptide.
Attenuated strains of viruses are obtained by serial passage of the
virus in cell culture (e.g., in non-human cells), until a virus is
identified which is immunogenic but not pathogenic. While wild type
virus will cause fatal infection in marmosets, vaccine strains do
not. Individuals receiving an attenuated measles virus vaccine do
not display classical measles symptoms. Attenuation is associated
with decreased viral replication (as measured in vivo by inability
to cause measles in monkeys), diminished viremia, and failure to
induce cytopathological effects in tissues (e.g., cell-cell fusion,
multinucleated cells). See U.S. Pat. No. 7,393,527, which is
incorporated herein by reference.
[0190] In one embodiment, an effective dose of an attenuated
measles virus encoding a Brachyury protein or Brachyury polypeptide
is produced by infecting a primary cell or a continuous cell line
with a starting innoculum of a stock comprising an attenuated
Moraten strain of measles virus (or an innoculum of an MMR stock)
or the MV-Edm strain or any of the other strains described above
and expanding the virus after serial passage. Cells or cell lines
include, but are not limited to, monkey kidney or testes cells or
monkey cell lines (e.g., Vero, KB, CV-1, BSC-1, and the like).
Viral replication in cells is observed as cell-cell fusion and
syncytia formation.
[0191] The attenuated measles virus is expanded until a desired
dose concentration is obtained in standard cell culture media. In
one embodiment, the therapeutically effective dose concentration is
about 10.sup.3 to 10.sup.12 pfu. In another embodiment of the
invention, the concentration is about 10.sup.5 to 10.sup.8 pfu.
Viral titer can be assayed by inoculating cells (e.g., Vero cells)
in culture dishes (e.g., such as 35 mm dishes). After 2-3 hours of
viral adsorption, the inoculum is removed and cells are overlaid
with a mixture of cell culture medium and agarose or
methylcellulose (e.g., 2 ml DMEM containing 5% FCS and 1% SeaPlaque
agarose). After about 3 to about 5 days, cultures are fixed with 1
ml of 10% trifluoroacetic acid for about 1 hour, then UV
cross-linked for 30 minutes. After removal of the agarose overlay,
cell monolayers are stained with crystal violet and plaques are
counted to determine viral titer. Virus is harvested from cell
syncytia by scraping cells from the dishes, subjecting them to
freeze/thawing (e.g., approximately two rounds), and centrifuging.
The cleared supernatants represent "plaque purified" virus.
[0192] Viral stocks are produced by infection of cell monolayers
(e.g., adsorption for about 1.5 hours at 37.degree. C.), followed
by scraping of infected cells into a suitable medium (e.g.,
Opti-MEM, Gibco-BRL) and freeze/thaw lysis (for example, 2 rounds).
Viral stocks are aliquoted, frozen and stored at 70.degree.
C.-80.degree. C. and can be stored at concentrations higher than
the therapeutically effective dose. In one embodiment, viral stock
is stored in a stabilizing solution. Stabilizing solutions are
known in the art, see for example, U.S. Pat. No. 4,985,244, and
U.S. Pat. No. 4,500,512.
[0193] Poliovirus
[0194] Polioviruses encoding a Brachyury protein or a Brachyury
polypeptide are provided and are of use in the methods disclosed
herein. The entire poliovirus genome has been cloned and sequenced
and the viral proteins identified. An infectious poliovirus cDNA is
also available which has allowed further genetic manipulation of
the virus (Racaniello V R et al., Science 214(4542) 916-919, 1981).
The wild-type genomic RNA molecule is 7433 nucleotides long,
polyadenylated at the 3' end and has a small covalently attached
viral protein (VPg) at the 5' terminus (Kitamura N et al., Nature
291:547-553; 1981 Racaniello V R et al., Proc. Natl. Acad. Sci. USA
78:4887-4891, 1981). Expression of the poliovirus genome occurs via
the translation of a single protein (polyprotein) which is
subsequently processed by virus encoded proteases (2A and 3C) to
give the mature structural (capsid) and nonstructural proteins
(Kitamura N et al., Nature 291:547-553, 1981; Koch F et al., The
Molecular Biology of Poliovirus, Springer-Verlag, Vienna, 1985).
Poliovirus replication is catalyzed by the virus-encoded
RNA-dependent RNA polymerase, which copies the genomic RNA to give
a complementary RNA molecule, which then serves as a template for
further RNA production (Koch F et al., supra; Kuhn R J et al., in D
J Rowlands et al. (ed.) Molecular Biology of Positive Strand RNA
viruses, Academic Press Ltd., London, 1987). The translation and
proteolytic processing of the poliovirus polyprotein is described
in Nicklin M J H et al., Bio/Technology 4:33-42, 1986.
[0195] The viral RNA genome encodes the necessary proteins required
for generation of new progeny RNA, as well as encapsidation of the
new RNA genomes. In vitro, poliovirus is lytic, resulting in the
complete destruction of permissive cells. Since the viral
replication cycle does not include any DNA intermediates, there is
no possibility of integration of viral DNA into the host
chromosomal DNA.
[0196] Early studies identified three poliovirus types based on
reactivity to antibodies (Koch F et al., supra, 1985). These three
serological types, designated as type I, type II, and type III,
have been further distinguished as having numerous nucleotide
differences in both the non-coding regions and the protein coding
regions. All three strains are suitable for use in delivering
heterologous proteins. In addition, there are also available
attenuated versions of all three strains of poliovirus.
[0197] Replicons can comprise deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA). Replicons are poliovirus-based
polynucleotides that lack a wild type poliovirus nucleic acid
necessary for encapsidation of the virus. Consequently, newly
encapsidated replicons cannot be produced following initial cell
entry in the absence of the missing nucleic acid. Replicons can
lack this nucleic acid as a result of any modification of the
wildtype poliovirus nucleic acid including, but not limited to,
deletions, insertions, and substitutions, including an insertion of
a nucleic acid encoding a Brachyury protein. In some embodiments,
poliovirus replicons lack a wild type poliovirus nucleic acid that
encodes at least a portion of a protein that is required for
encapsidation. Proteins necessary for replicon encapsidation
include proteins that are part of the capsid structure. Examples of
such proteins are those encoded by the VP1, VP2, VP3, and VP4 genes
of the poliovirus P1 capsid precursor region, the Vpg protein, and
those proteins that are necessary for proper processing of
structural proteins of the capsid structure, such as the proteases
responsible for cleaving the viral polyprotein. Thus, in some
embodiments, the poliovirus vector lacks nucleic acid sequences
encoding one or more of VP1, VP2, VP3, and VP4, genes of the
poliovirus P1 capsid precursor region, the Vpg protein, and encodes
a Brachyury protein or Brachyury polypeptide.
[0198] Replicons are typically introduced into a cell in an RNA
form. Encapsidated replicons are able to enter cells via
interaction of the capsid proteins with poliovirus receptor.
Replicons are fully capable of RNA replication (amplification) upon
introduction into cells and translation, in the correct reading
frame, of the single polyprotein through which expression of the
entire replicon genome occurs. Translation of replicon sequences
may be transient, usually lasting only about 24-48 hours. High
levels of replicon-encoded proteins can accumulate during the
translation period. Encapsidated replicons are able to enter cells
via interaction of the capsid proteins with the hPVR protein.
[0199] In some embodiments, replicons comprise RNA, including
sequences encoding Brachyury protein, and are encapsidated. In some
examples, the replicons have a deletion of the capsid (P1) gene and
are derived from the RNA genome of poliovirus type 1, type 2, type
3 or combinations thereof. Further, a nucleic acid encoding a
Brachyury protein or Brachyury polypeptide can be substituted for
part or all of the capsid (P1) gene such that the portion of the
capsid (P1) gene which remains, if any, is insufficient to support
encapsidation in vivo. Generally, the term "P1 replicons" refers to
replicons in which the entire nucleic acid encoding the P1 capsid
precursor protein has been deleted or altered such that the
proteins which are normally encoded by this nucleic acid are not
expressed or are expressed in a non-functional form. The proteins
that are normally encoded by the P capsid precursor region of the
poliovirus genome include the proteins encoded by the VP1, VP2,
VP3, and VP4 genes. P1 replicons, therefore, lack the VP1, VP2,
VP3, and VP4 genes or comprise unexpressible or non-functional
forms of the VP1, VP2, VP3, and VP4 genes. P1 replicons can include
a nucleic acid encoding a Brachyury protein or Brachyury
polypeptide substituted for the VP1, VP2, VP3, and VP4 genes.
[0200] In some embodiments, encapsidated replicons may be produced
by introducing both a replicon and a complementing expression
vector that provides the missing nucleic acid necessary for
encapsidation in trans to a host cell. A "replicon encapsidation
vector" refers to a non-poliovirus-based vector that comprises a
nucleic acid required for replicon encapsidation and provides the
required nucleic acid (or encoded protein) in trans. Replicon
encapsidation vectors can be introduced into a host cell prior to,
concurrently with, or subsequent to replicon introduction. Suitable
methods for encapsidation are disclosed in U.S. Pat. No. 6,680,169,
which is incorporated by reference herein. Methods which can be
used to prepare encapsidated replicons have been described Porter D
C et al., J. Virol. 67:3712-3719, 1993; Porter D C et al., 1995, J.
Virol. 69:1548-1555, 1995; PCT Publication No. WO 96/25173; U.S.
Pat. Nos. 5,614,413, 5,817,512; 6,063,384; and U.S. Pat. No.
6,680,169.
[0201] Nonencapsidated replicons can be delivered directly to
target cells, for example by direct injection into, for example,
muscle cells (see, for example, Acsadi G et al., Nature
352(6338):815-818, 1991; Wolff J A et al., Science 247:1465-1468,
1990), or by electroporation, transfection mediated by calcium
phosphate, transfection mediated by DEAE-dextran, liposome-mediated
transfection or receptor-mediated nucleic acid uptake (see for
example Wu G et al., J. Biol. Chem. 263:14621-14624, 1988; Wilson J
M et al., J. Biol. Chem. 267:963-967, 1992; and U.S. Pat. No.
5,166,320), or other methods of delivering naked nucleic acids to
target cells.
[0202] Retroviral Vectors
[0203] Retroviral vectors, including lentiviral vectors encoding a
Brachyury protein and/or Brachyury polypeptide are provided and are
of use in the methods disclosed herein. Retroviral vectors have
been tested and found to be suitable delivery vehicles for the
stable introduction of a variety of genes of interest into the
genomic DNA of a broad range of target cells. Without being bound
by theory, the ability of retroviral vectors to deliver
unrearranged, single copy transgenes into cells makes retroviral
vectors well suited for transferring genes into cells. Further,
retroviruses enter host cells by the binding of retroviral envelope
glycoproteins to specific cell surface receptors on the host cells.
Consequently, pseudotyped retroviral vectors in which the encoded
native envelope protein is replaced by a heterologous envelope
protein that has a different cellular specificity than the native
envelope protein (e.g., binds to a different cell-surface receptor
as compared to the native envelope protein) can also be used.
[0204] Generally, retroviruses contain three major coding domains,
gag, pol, env, which code for essential virion proteins. Retroviral
vectors are of use wherein gag, pol and/or env are absent or not
functional. Retroviral vectors are disclosed, for example, in U.S.
Published Patent Application No. 20060286634, which is incorporated
herein by reference herein.
[0205] Thus retroviral vectors are provided which include, for
example, retroviral transfer vectors comprising a nucleic acid
encoding a Brachyury protein and retroviral packaging vectors
comprising one or more packaging elements. In some embodiments,
pseudotyped retroviral vectors are provided encoding a heterologous
or functionally modified envelope protein for producing pseudotyped
retrovirus.
[0206] There are many retroviruses and examples include: murine
leukemia virus (MLV), lentivirus such as human immunodeficiency
virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary
tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma
virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine
osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus
(Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian
myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus
(AEV). Other retroviruses suitable for use include, but are not
limited to, Avian Leukosis Virus, Bovine Leukemia Virus, Mink-Cell
Focus-Inducing Virus. The core sequence of the retroviral vectors
can be derived from a wide variety of retroviruses, including for
example, B, C, and D type retroviruses as well as spumaviruses and
lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring
Harbor Laboratory, 1985). An example of a retrovirus suitable for
use in the compositions and methods disclosed herein, includes, but
is not limited to, lentivirus.
[0207] One lentivirus is a human immunodeficiency virus (HIV), for
example, type 1 or 2 (i.e., HIV-1 or HIV-2). Other lentivirus
vectors include sheep Visna/maedi virus, feline immunodeficiency
virus (FIV), bovine lentivirus, simian immunodeficiency virus
(SIV), an equine infectious anemia virus (EIAV), and a caprine
arthritis-encephalitis virus (CAEV).
[0208] Lentiviruses share several structural virion proteins in
common, including the envelope glycoproteins SU (gp120) and TM
(gp41), which are encoded by the env gene; CA (p24), MA (p117) and
NC (p7-11), which are encoded by the gag gene; and RT, PR and IN
encoded by the pol gene. HIV-1 and HIV-2 contain accessory and
other proteins involved in regulation of synthesis and processing
virus RNA and other replicative functions. The accessory proteins,
encoded by the vif, vpr, vpu/vpx, and nef genes, can be omitted (or
inactivated) from the recombinant system. In addition, tat and rev
can be omitted or inactivated, such as by mutation or deletion.
[0209] Without being bound by theory, the use of lentivirus-based
gene transfer techniques generally relies on the in vitro
production of recombinant lentiviral particles carrying a highly
deleted viral genome in which a gene of interest, such as a nucleic
acid encoding a Brachyury protein or Brachyury polypeptide, is
accommodated. In particular, the recombinant lentivirus are
recovered through the in trans co-expression in a permissive cell
line of (1) the packaging constructs, i.e., a vector expressing the
Gag-Pol precursors together with Rev (alternatively expressed in
trans); (2) a vector expressing an envelope receptor, generally of
an heterologous nature, and (3) the transfer vector, consisting in
the viral cDNA deprived of all open reading frames, but maintaining
the sequences required for replication, incapsidation, and
expression, in which the sequences to be expressed are inserted. In
one embodiment the lentigen lentiviral vector described in Lu, X.
et al. Journal of gene medicine 6:963-973, 2004 is used to express
the Brachyury protein or Brachyury polypeptide. Suitable lentiviral
vectors are also disclosed, for example, in U.S. Published Patent
Application No. 20100062524, which is incorporated herein by
reference.
[0210] Retroviral packaging systems for generating producer cells
and producer cell lines that produce retroviruses, and methods of
making such packaging systems are known in the art. Generally, the
retroviral packaging systems include at least two packaging
vectors: a first packaging vector which includes a first nucleotide
sequence comprising a gag, a pol, or gag and pol genes; and a
second packaging vector which includes a second nucleotide sequence
comprising a heterologous or functionally modified envelope gene.
In some embodiments, the retroviral elements are derived from a
lentivirus, such as HIV. These vectors can lack a functional tat
gene and/or functional accessory genes (vif, vpr, vpu, vpx, nef).
In other embodiments, the system further comprises a third
packaging vector that comprises a nucleotide sequence comprising a
rev gene. The packaging system can be provided in the form of a
packaging cell that contains the first, second, and, optionally,
third nucleotide sequences.
[0211] First generation lentiviral vector packaging systems provide
separate packaging constructs for gag/pol and env, and typically
employ a heterologous or functionally modified envelope protein for
safety reasons. In second generation lentiviral vector systems, the
accessory genes, vif, vpr, vpu and nef, are deleted or inactivated.
Third generation lentiviral vector systems are those from which the
tat gene has been deleted or otherwise inactivated (e.g., via
mutation). Compensation for the regulation of transcription
normally provided by tat can be provided by the use of a strong
constitutive promoter, such as the human cytomegalovirus immediate
early (HCMV-IE) enhancer/promoter. Other promoters/enhancers can be
selected based on strength of constitutive promoter activity,
specificity for target tissue (e.g., liver-specific promoter), or
other factors relating to desired control over expression, as is
understood in the art. For example, in some embodiments, an
inducible promoter such as tet can be used to achieve controlled
expression. The gene encoding rev can be provided on a separate
expression construct, such that a typical third generation
lentiviral vector system will involve four plasmids: one each for
gagpol, rev, envelope and the transfer vector. Regardless of the
generation of packaging system employed, gag and pol can be
provided on a single construct or on separate constructs.
[0212] Typically, the packaging vectors are included in a packaging
cell, and are introduced into the cell via transfection,
transduction or infection. Methods for transfection, transduction
or infection are well known by those of skill in the art. A
retroviral vector of the present invention can be introduced into a
packaging cell line, via transfection, transduction or infection,
to generate a producer cell or cell line. The packaging vectors can
be introduced into human cells or cell lines by standard methods
including, for example, calcium phosphate transfection, lipofection
or electroporation. In some embodiments, the packaging vectors are
introduced into the cells together with a dominant selectable
marker, such as neo, DHFR, Gin synthetase or ADA, followed by
selection in the presence of the appropriate drug and isolation of
clones. A selectable marker gene can be linked physically to genes
encoding by the packaging vector.
[0213] Stable cell lines, wherein the packaging functions are
configured to be expressed by a suitable packaging cell, are known.
For example, see U.S. Pat. No. 5,686,279; and Ory et al., Proc.
Natl. Acad. Sci. 93:11400-11406, 1996, which describe packaging
cells. Zufferey et al., Nature Biotechnology 15:871-875, 1997
disclose a lentiviral packaging plasmid wherein sequences 3' of pol
including the HIV-1 envelope gene are deleted. The construct
contains tat and rev sequences and the 3' LTR is replaced with poly
A sequences. The 5' LTR and psi sequences are replaced by another
promoter, such as one which is inducible. For example, a CMV
promoter can be used.
[0214] The packaging vectors can include additional changes to the
packaging functions to enhance lentiviral protein expression and to
enhance safety. For example, all of the HIV sequences upstream of
gag can be removed. Also, sequences downstream of envelope can be
removed. Moreover, steps can be taken to modify the vector to
enhance the splicing and translation of the RNA.
[0215] A self-inactivating vector (SIN) can be used, which improves
the biosafety of the vector by deletion of the HIV-1 long terminal
repeat (LTR) as described, for example, by Zufferey et al., J.
Virology 72(12):9873-9880, 1998. Inducible vectors can also be
used, such as through a tet-inducible LTR.
[0216] Host Cells
[0217] DNA sequences encoding a Brachyury protein and/or Brachyury
polypeptide can be expressed from a vector in vitro by DNA transfer
into a suitable host cell. The term "host cell" also includes any
progeny of the subject host cell. It is understood that all progeny
may not be identical to the parental cell since there may be
mutations that occur during replication. Methods of stable
transfer, meaning that the foreign DNA is continuously maintained
in the host, are known in the art.
[0218] Hosts cells can include microbial, insect and mammalian host
cells. Methods of expressing DNA sequences having eukaryotic or
viral sequences in prokaryotes are well known in the art.
Non-limiting examples of suitable host cells include animal cells
(for example, mammalian cells, such as human). Techniques for the
propagation of mammalian cells in culture are well-known (see,
Jakoby and Pastan (eds), 1979, Cell Culture. Methods in Enzymology,
volume 58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.).
Examples of commonly used mammalian host cell lines are VERO and
HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although
cell lines may be used, such as cells designed to provide higher
expression desirable glycosylation patterns, or other features.
[0219] Transformation of a host cell with recombinant DNA can be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as, but not
limited to, E coli, competent cells which are capable of DNA uptake
can be prepared from cells harvested after exponential growth phase
and subsequently treated by the CaCl.sub.2) method using procedures
well known in the art. Alternatively, MgCl.sub.2 or RbCl can be
used. Transformation can also be performed after forming a
protoplast of the host cell if desired, or by electroporation.
[0220] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate coprecipitates, conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or infection with the poxvirus
vectors can be used. Eukaryotic cells can also be co-transformed
with polynucleotide sequences encoding a Brachyury protein or
Brachyury polypeptide, and a second foreign DNA molecule encoding a
selectable phenotype, such as the herpes simplex thymidine kinase
gene. Methods for using viral vectors to transform eukaryotic cells
are known, (see for example, Eukaryotic Viral Vectors, Cold Spring
Harbor Laboratory, Gluzman ed., 1982).
[0221] A nucleic acid encoding a Brachyury protein is expressed in
the host cell, such as a Salmonella host cell, for example S
typhimurium host cell, or a Listeria, such as a L. monocytogenes
host cell. A host cell-compatible promoter is any promoter or
promoter/enhancer that is able to initiate sufficient transcription
of the Brachyury protein or Brachyury polypeptide, such as in
amounts sufficient to induce a Brachyury specific CD4+ T cell
response when the host cell is introduced into the subject. Some
examples of expression control sequences are a promoter/enhancer
from the cytomegalovirus (CMV) immediate early gene 1 or the Rous
sarcoma virus (RSV) long terminal repeat or the simian virus 40
promoter or the adenovirus 2 major late promoter or the mouse
mammary tumor virus promoter (MMTV). These host cells can be used
in attenuated and/or heat-killed forms. These host cells can be
administered multiple times without eliciting host neutralizing
activity.
[0222] In some embodiments the host cell are utilized with an
adjuvant. Specific non-limiting examples of adjuvants of use are
GM-CSF, Bacillus-Calmette-Guerin adjvant or CD40 ligand
(CD40L).
[0223] Attenuated Bacteria: Listeria and Salmonella
[0224] Prokaryotic host cells include Listeria and Salmonella,
which can be used directly to provide a Brachyury protein and/or
Brachyury polypeptide, such as for use in the methods disclosed
herein. Thus, in some embodiments, an attenuated invasive
intracellular bacterium capable of infecting a mammalian host or
cell thereof, but having a decreased ability in intra- and
intercellular movement in the host as compared to a wild type
bacterium is utilized. In some embodiments, the bactium is
transformed with (a) a promoter activated when said bacterium is
present in the cytosol of a host cell, operably linked to a
structural gene or fragment thereof, encoding a polypeptide which
is lethal to the bacterium, (b) a host cell-compatible promoter,
operably linked to a structural gene or fragment thereof, encoding
a Brachyury protein, wherein (a) and (b) can be on the same plasmid
or different plasmids or (a) can be integrated into the bacterial
chromosome. The attenuated bacteria include a number of
intracellular bacteria, such as Salmonella, Yersinia, Renibacterium
and Listeria capable of intracellular growth (Coynault et al.,
Molecular Microbiology 22, 149-160, 1996; Hohmann et al., Vaccine
14, 19-14, 1996; Karem et al., Infection and Immuity 63, 4557-4563,
1995; O'Callaghan et al., Infection and Immunity 56, 419-423, 1988;
Sigwart et al., Infection and Immunity 57, 1858-61, 1989; and Sinha
et al., Infection and Immunity 65, 1566-1569, 1997).
[0225] The disclosed attenuated invasive bacteria can still invade
a host, or cells thereof, but are not pathogenic. In some
embodiments, attenuation can be achieved by mutation (see T.
Maniatis, et al. Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., 1989). In specific non-limiting
examples, attenuation can be caused by mutating the bacterial genes
which encode for pathogenic and/or toxic polypeptides. Such a
mutation can be achieved randomly, such as by chemical modification
and selected later, for example, for loss of function, or it can be
site directed. Thus in some embodiments, the bacterium is
attenuated by deletion, insertion or point mutations, to eliminate
the function of certain genes that encode polypeptides which lead
to pathogenesis. In some embodiments, the bacterium is attenuated
by deletion of an entire operon by chromosomal deletion, such as
the attenuated L. monocytogenes mutant strain DELTA2 that is a
derivative of the fully virulent wild-type strain EGD, and lacks
the entire lecithinase operon consisting of the genes mpl, actA and
plcB due to a chromosomal deletion. Due to the deletion of this
operon the inflammatory response caused by L. monocytogenes during
infection of a mammalian host is significantly reduced. Suitable
bacteria are disclosed for example, in U.S. Pat. No. 6,143,551,
which is incorporated by reference herein.
[0226] A variety of potential live Salmonella strains with
different attenuation levels, which subsequently serve as platforms
for the development of recombinant live Salmonella carrier strains
that express heterologous antigens. Such recombinant live
Salmonella vaccine carriers are equipped with modules comprising
variable gene cassettes that regulate the expression of
heterologous antigens in Salmonella and determine presentation of
the heterologous antigens to the host immune system. By
combinations of both systems, differently attenuated live
Salmonella vaccine strains and variable gene cassettes, a variety
of recombinant live carrier strains can be generated that have
broad application.
[0227] S. typhimurium contains two type III secretion systems for
virulence determinants. The first controls bacterial invasion of
epithelial cells, and is encoded by genes within a 40 kb
pathogenicity island (SPI1). The other is encoded by genes within a
second 40 kb pathogenicity island (SPI2) and is required for
systemic growth of this pathogen within its host. The genes located
on pathogenicity island SPI1 are mainly responsible for early steps
of the infection process, the invasion of non-phagocytic host cells
by the bacterium. For most of the SPI1 genes, mutations result in a
reduced invasiveness in vitro. However, mutants that are defective
in invasion are not necessarily avirulent. In comparison, virulence
studies of SPI2 mutants have shown them to be attenuated by at
least five orders of magnitude compared with the wild-type strain
after both oral and intraperitoneal inoculation of mice.
[0228] Many of the genes encoding components of the SPI2 secretion
system are located in a 25 kb segment of SPI2. SPI2 contains genes
for a type III secretion apparatus (ssa) and a two component
regulatory system (ssr), as well as candidate genes for a set of
secreted effectors (sse) and their specific chaperones (ssc). On
the basis of similarities with genes present in other bacterial
pathogens, the first 13 genes within the ssaK/U operon and ssaJ
encode components of the secretion system apparatus. A number of
additional genes, including ssaC, which encode a secretion system
apparatus protein and a two component regulatory protein,
respectively, are found in a region approximately 8 kb from
ssaJ.
[0229] Thus, an attenuated gram-negative cell can have inactivated
at least one gene selected from effector (sse) gene secretion
apparatus (ssa) genes, chaperon (ssc) genes and regulation (ssr)
genes. With regard to the sse genes are affected by the
inactivation, the inactivated gene is preferably sseC, sseD, sseE
or a combination thereof. As far as the ssr genes are affected by
the inactivation, preferably at least ssrB is inactivated. As far
as the ssc genes are affected by the inactivation, preferably at
least sscB is inactivated.
[0230] Attenuation can be the result of original mutations in SPI2
gene locus. Combination of the individual mutations in the SPI2
gene locus with each other, and with other known attenuating gene
mutations, such as aroA, results in a broad repertoire of
attenuation and immunogenicity. Different expression cassettes can
be introduced on these platforms, allowing further modulation of
the immune response directed against the heterologous antigens.
[0231] Pathogenic Salmonella or Listeria serve as a basis for the
construction of a panel of different live Salmonella vaccine
prototypes generated by gradual attenuations accomplished through
the introduction of defined SPI2 gene locus mutations. Each
resulting individual live Salmonella vaccine prototype is further
transformed into a multivalent recombinant vaccine by the
introduction of exchangeable DNA modules carrying (1) a nucleic
acid encoding a Brachyury protein or polypeptide and (2) adequate
expression systems executing efficacious antigen presentation to
the host immune system. In concert, these features elicit a
specific immune response, such as a Brachyury specific T cell
response.
[0232] The inactivation of the gene of the SPI2 locus (or
functional homologue thereof in cells other than Salmonella) is
effected by a mutation which may comprise deletion. In some
embodiments, the deletion is cause by insertion of a heterologous
nucleic acid, such as a nucleic acid encoding Brachyury into the
gene to be inactivated. With regard to Salmonella, pathogenic
Salmonella species are gradually attenuated by mutations in
individual virulence genes that are part of the SPI2 gene locus,
for example an sse gene coding for an effector protein, such as
sseC, ssed or sseE, or an ssc gene, such as sscB, coding for a
chaperone, or an ssr gene, such as ssrB, coding for a regulator.
Individual mutation of each of these genes leads to a unique
individual grade of attenuation, which, in turn, effects a
characteristic immune response at the mucosal, humoral and cellular
levels. The individual grade of attenuation can be moderately
increased by combinations of at least two gene mutations within the
SPI2 gene locus or by combination with a mutation in another
Salmonella gene known to attenuate virulence, such as an aro gene,
for eample aroA. A stronger grade of attenuation is achieved by
mutation of a virulence gene that is part of a polycistronic gene
cluster encoding several virulence factors, such as the
transcriptional unit comprising the sseC, sseD, sseE and sscB
genes, such that the mutation exerts a polar effect, disrupting
expression of the following genes. The grade of attenuation may
directly depend on the number of virulence genes that are affected
by the polar mutation as well as their individual characteristics.
Finally, the strongest attenuation is achieved when regulatory
genes, such as ssrB, are mutated. Again, each mode of attenuation
of Salmonella leads to the generation of a live Salmonella strain
that evokes an immune response, see U.S. Pat. No. 7,700,104, which
is incorporated herein by reference.
[0233] With regard to Listeria, L. monocytogenes is a
Gram-positive, facultative intracellular bacterium that lacks
lipopolysaccharide (LPS) and is also able to invade a wider range
of mammalian cells where it replicates in the cytosol as well
(Portnoy et al., Infect. Immun. 60, 1263, 1992). Since it invades
its host through the intestinal mucosal surface, L. monocytogenes
is also a candidate for oral vaccination. Shortly after infection,
bacteria are found in the spleen where professional APC are
abundant. Delivery of DNA to those cells is therefore significantly
enhanced by the use of suitably constructed L. monocytogenes.
Attenuated L. monocytogenes cells are lysed in the cytosol of the
host cell by the production of a Pac-dependent phage lysin
releasing plasmid DNA which carries a heterologous gene, such as a
nucleic acid encoding Brachyury protein, under the control of a
promoter, such as, but not limited to, the human cytomegalovirus
major immediate-early promoter/enhancer region. Beside the
advantages of avoiding the use of antibiotics, lysin-mediated
plasmid release is an efficient method comparable to eliminating
the bacteria by antibiotic treatment.
[0234] The attenuated bacterium can be a mutant of wild-type
Listeria which invades host cells and is released into the cytosol
of the infected cells with similar efficiencies as the wild-type
strain, but is impaired in intra- and intercellular movement. For
example, the mutant L. monocytogenes strain DELTA2 is unable to
polymerise host cell actin in the cytosol which L. monocytogenes
wild type strain uses for its movement inside the host cell.
Furthermore, due to the deletion of plcB, the bacterium is unable
to lyse the host cell membranes which the wild type strain lyses
upon entering neighboring cells. Mutant bacteria are therefore
unable to move from one infected cell into a neighboring cell
(cell-to-cell spread). This illustrates a decreased ability as
compared to wild type strains in intra- and inter-cellular
movement. In some embodiments the attenuated bacterium is a mutant
of L. monocytogenes which invades the host and is released into the
cytosol of the infected cells with similar efficiencies as the
wild-type strain, but it is not pathogenic, i.e., it doesn't cause
a disease. In specific non-limiting examples, the bacterium is L.
monocytogenes that lacks the entire lecithinase operon containing
the genes mpl, actA and plcB, and encodes a Brachyury protein or a
Brachyury polypeptide.
[0235] In some embodiments, a structural gene or fragment thereof
is also included, such as encoding a polypeptide which is lethal to
the bacterium is any polypeptide which when expressed in the
bacterium will result in the release of plasmid DNA and death of
the bacterium, for example by lysis of the bacterium. In some
embodiments, the polypeptide can be a bacteriophage lysin,
preferable the gene product of ply 118 or other
Listeria-phage-encoded lysins, for example the mureinhydrolase
encoded by the iap gene of L. monocytogenes or other iap-related
genes especially iap of L. grayi. The lysis protein PLY 118 is a
late gene product of the Listeria bacteriophage A118 necessary for
the release of progeny phages. PLY 118 is a highly active, cell
wall-hydrolyzing enzyme specific for Listeria (Loessner et al.,
Mol. Microbiol. 16, 1231, 1995). By a promoter activated when it is
present in an invasive bacterium which is in the cytosol of a host
cell, it is meant any promoter which, when the bacteria is inside
the infected cell, is (under the control of a transcription
activator which is) preferentially turned on, driving its
transcription. For example, the L. monocytogenes promoter PactA can
be used. The PactA promoter is controlled by the transcription
activator PrfA which regulates most of the known virulence genes of
L. monocytogenes and is specifically activated in the cytosol of
the infected host cells to interact with the actA promoter. Other
promoters which can be used are other promoters of L.
monocytogenes, such as those controlling the expression of inlC and
other genes for small internalins (Engelbrecht et al., Mol.
Microbiol. 21:823-837, 1996).
[0236] Therapeutic Methods and Pharmaceutical Compositions
[0237] The Brachyury proteins and Brachyury polypeptides disclosed
herein, nucleic acids encoding the Brachyury proteins or Brachyury
polypeptides, or host cells including these nucleic acids can be
used to generate an immune response in a subject, such as, but not
limited to, a Brachyury specific CD4+ T cell response and/or a
Brachyury CD8+ T cells response. In several examples, the subject
has a cancer that expresses Brachyury. In other embodiments, the
method is a method for preventing cancer in the subject. The
subject can be at risk of developing cancer. In specific
non-limiting examples, the subject has high grade prostatic
intraepithelial neoplasia, familial adenomatous polyposis, or
atypia of the breast. The the methods include administering to a
subject a therapeutically effective amount of one or more of the
Brachyury proteins and/or polypeptides disclosed herein, nucleic
acids encoding these Brachyury proteins or Brachyury polypeptides,
host cells, such as Listeria or Salmonella host cells, dendritic
cells presenting epitopes of the protein or polypeptide, and/or
vectors including these nucleic acids, in order to generate an
immune response.
[0238] The methods can include selecting a subject in need of
treatment, such as a subject with a cancer that expresses Brachyury
or a cancer with the potential to express Brachyury. In several
examples, the methods include selecting a subject with a cancer of
the small intestine, stomach, kidney, bladder, uterus, ovaries,
testes lung, colon, prostate, tumor of B cell origin (such as
chronic lymphocytic leukemia (CLL), a B cell lymphoma, Burkitt's
lymphoma or a Hodgkin's lymphoma) or breast cancer wherein the
cancer expresses Brachyury or has the potential to express
Brachyury. In some non-limiting examples, examples, the cancer is
radiation resistant and/or chemotherapy resistant. In additional
non-limiting examples, the subject has breast cancer, such as a
ductal carcinoma, for example an infiltrating ductal carcinoma or
an estrogen receptor negative and progesterone receptor negative
breast cancer. In further examples, the subject has high-grade
prostatic intraepithelial neoplasia, familial adenomatous
polyposis, or atypia of the breast.
[0239] In exemplary applications, compositions are administered to
a subject in an amount sufficient to raise an immune response to
Brachyury-expressing cells, such as a CD4+ T cell response. A
Brachyury specific CD8+ T cell response can also be induced using
the methods disclosed herein. Administration induces a sufficient
immune response to slow the proliferation of Brachyury-expressing
cells, or to inhibit their growth, or to reduce a sign or a symptom
of the cancer, or to prevent a cancer. Amounts effective for this
use will depend upon the severity of the disease, the general state
of the patient's health, and the robustness of the patient's immune
system. In one example, a therapeutically effective amount of the
composition is that which provides either subjective relief of a
symptom(s) or an objectively identifiable improvement as noted by
the clinician or other qualified observer. The composition can
include a Brachyury protein and/or a Brachyury polypeptide, a
nucleic acid encoding a Brachyury protein and/or a Brachyury
polypeptide, a vector including the nucleic acid, or a host cell
expressing the Brachyury protein and/or Brachyury polypeptide. It
should be noted that these compositions can be used in
combination.
[0240] The composition can be administered by any means known to
one of skill in the art (see Banga, A., "Parenteral Controlled
Delivery of Therapeutic Peptides and Proteins," in Therapeutic
Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster,
Pa., 1995). Thus, the composition can be administered either
locally or systemically, such as by intramuscular, subcutaneous,
intraperitoneal or intravenous injection, but even oral, nasal,
transdermal or anal administration is contemplated. In one
embodiment, administration is by subcutaneous or intramuscular
injection.
[0241] Use of Proteins, Polypeptides, Nucleic Acids and Host
Cells
[0242] When the Brachyury protein and/or the Brachyury polypeptide
is administered, to extend the time during which protein is
available to stimulate a response, the protein and/or polypeptide
can be provided as an implant, an oily injection, in a liposome, or
as a particulate system. The particulate system can be a
microparticle, a microcapsule, a microsphere, a nanocapsule, or
similar particle. (see, e.g., Banga, supra). A particulate carrier
based on a synthetic polymer has been shown to act as an adjuvant
to enhance the immune response, in addition to providing a
controlled release. Adjuvants can also be used in combination with
the protein, including, for example, chitosan, aluminum salts, an
immunostimulatory oligodeoxynucletoide, liposomes and/or one or
more cytokines. The Brachyury protein or polypeptide can be
administered in a liposome.
[0243] In one specific, non-limiting example, the Brachyury protein
is administered in a manner to direct the immune response to a
cellular response (that is, a Brachyury specific CD4+ response
and/or CD8+ response), rather than a humoral (antibody) response.
The Brachyury polypeptide can induce both a Brachyury specific CD4+
T cell response and a Brachyury specific CD8+ T cell response.
Methods for measuring a CD4+ and CD8+ T cell response are known in
the art, and include biological assays, ELISPOT assays, and
fluorescence activated cell sorting. An exemplary assay for
measuring Brachyury specific CD4+ T cells is disclosed in the
examples below.
[0244] In one specific, non-limiting example, a pharmaceutical
composition for intravenous administration would include about 0.1
.mu.g to 10 mg of immunogenic Brachyury protein and/or Brachyury
polypeptide per patient per day. Dosages from 0.1 up to about 100
mg per patient per day can be used, particularly if the agent is
administered to a secluded site and not into the circulatory or
lymph system, such as into a body cavity or into a lumen of an
organ. Actual methods for preparing administrable compositions will
be known or apparent to those skilled in the art and are described
in more detail in such publications as Remingtons Pharmaceutical
Sciences, 19.sup.th Ed., Mack Publishing Company, Easton, Pa.,
1995.
[0245] Optionally, one or more immunostimulatory molecules, such as
IL-2, IL-6, IL-12, LFA (for example, LFA-1, LFA-2 and/or LFA-3),
CD72, RANTES, G-CSF, GM-CSF, TNF-.alpha., IFN-.gamma., ICAM-1,
B7-1, B7-2, other B7 related molecules, OX-40L and/or or 41 BBL, or
combinations of these molecules, can be used as biological
adjuvants (see, for example, Salgaller et al., 1998, J. Surg.
Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl
1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper
et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can
be administered systemically (or locally) to the host. In several
examples, IL-2, RANTES, GM-CSF, TNF-.alpha., IFN-.gamma., G-CSF,
LFA-3, CD72, B7-1, B7-2, B7-1, B7-2, OX-40L, 41 BBL and/or ICAM-1
are administered. IL-15 or an IL-15/IL-15 receptor complex can be
administered.
[0246] A number of means for inducing cellular responses, both in
vitro and in vivo, are known. Lipids have been identified as agents
capable of assisting in priming T cells in vivo against various
antigens. For example, as described in U.S. Pat. No. 5,662,907,
palmitic acid residues can be attached to the alpha and epsilon
amino groups of a lysine residue and then linked (for example, via
one or more linking residues, such as glycine, glycine-glycine,
serine, serine-serine, or the like) to an immunogenic peptide or
protein. The lipidated peptide can then be injected directly in a
micellar form, incorporated in a liposome, or emulsified in an
adjuvant. As another example, E. coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime
tumor specific T cells when covalently attached to an appropriate
peptide or protein (see, Deres et al., Nature 342:561, 1989).
Further, as the induction of neutralizing antibodies can also be
primed with the same molecule conjugated to a protein which
displays an appropriate epitope, two compositions can be combined
to elicit both humoral and cell-mediated responses where that is
deemed desirable.
[0247] A pharmaceutical composition including a Brachyury protein
and/or Brachyury polypeptide is thus provided. These compositions
are used to generate an immune response, such as for
immunotherapy.
[0248] In one embodiment, the Brachyury protein and/or the
Brachyury polypeptide is mixed with an adjuvant containing two or
more of a stabilizing detergent, a micelle-forming agent, and an
oil. Suitable stabilizing detergents, micelle-forming agents, and
oils are detailed in U.S. Pat. No. 5,585,103; 5,709,860; 5,270,202;
and U.S. Pat. No. 5,695,770, all of which are incorporated by
reference. A stabilizing detergent is any detergent that allows the
components of the emulsion to remain as a stable emulsion. Such
detergents include polysorbate, 80 (TWEEN)
(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl;
manufactured by ICI Americas, Wilmington, Del.), TWEEN 40.TM.,
TWEEN 20.TM., TWEEN 60.TM., Zwittergent.TM. 3-12, TEEPOL HB7.TM.,
and SPAN 85.TM.. These detergents are usually provided in an amount
of approximately 0.05 to 0.5%, such as at about 0.2%. A micelle
forming agent is an agent which is able to stabilize the emulsion
formed with the other components such that a micelle-like structure
is formed. Such agents generally cause some irritation at the site
of injection in order to recruit macrophages to enhance the
cellular response. Examples of such agents include polymer
surfactants described by BASF Wyandotte publications, e.g.,
Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al.,
J. Immunol 129:1244, 1981, PLURONIC.TM. L62LF, L101, and L64,
PEG1000, and TETRONIC.TM. 1501, 150R1, 701, 901, 1301, and 130R1.
The chemical structures of such agents are well known in the art.
In one embodiment, the agent is chosen to have a
hydrophile-lipophile balance (HLB) of between 0 and 2, as defined
by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent can be
provided in an effective amount, for example between 0.5 and 10%,
or in an amount between 1.25 and 5%.
[0249] The oil included in the composition is chosen to promote the
retention of the antigen in oil-in-water emulsion, such as to
provide a vehicle for the desired antigen, and preferably has a
melting temperature of less than 65.degree. C. such that emulsion
is formed either at room temperature (about 20.degree. C. to
25.degree. C.), or once the temperature of the emulsion is brought
down to room temperature. Examples of such oils include squalene,
Squalane, EICOSANE.TM., tetratetracontane, glycerol, and peanut oil
or other vegetable oils. In one specific, non-limiting example, the
oil is provided in an amount between 1 and 10%, or between 2.5 and
5%. The oil should be both biodegradable and biocompatible so that
the body can break down the oil over time, and so that no adverse
affects, such as granulomas, are evident upon use of the oil.
[0250] In one embodiment, the adjuvant is a mixture of stabilizing
detergents, micelle-forming agent, and oil available under the name
PROVAX.RTM. (IDEC Pharmaceuticals, San Diego, Calif.). In other
embodiments, the Brachyury protein and/or Brachyury polypeptide are
included in a liposome.
[0251] Adjuvants can also be administered with the Brachyury
protein and/or Brachyury polypeptide. An adjuvant can be any
immunostimulatory molecule, such as a cytokine, immunostimulatory
nucleic acid, or a biological adjuvant (see above). The adjuvant
can be chitosan. Chitosan is a linear polysaccharide formed from
repeating beta (1-4 linked) N-acetyl-D-glucosamine and
D-glucosamine units, and is derived from the partial deacetylation
of chitin obtained from the shells of crustaceans. Chitosan can be
made commercially by a heterogeneous alkaline hydrolysis of chitin
to give a product which possesses a random distribution of
remaining acetyl moieties. The properties of chitosans depend upon
inter alia the degree of deacetylation, and the molecular weight.
Most commercially available chitosans contain a population of
chitosan molecules of varying molecular weights and varying
concentrations of the component N-acetyl-D-glucosamine and
D-glucosamine groups. The immunological properties of chitosans are
known to be linked to the ratio between the N-acetyl-D-glucosamine
and D-glucosamine groups. The efficacy of chitosans as adjuvants
depends to a considerable extent on the extent of the level of
deacetylation. Thus, in some embodiments, the chitosan is at least
80% deacetylated, see U.S. Pat. No. 6,534,065, which is
incorporated herein by reference.
[0252] Controlled release parenteral formulations can be made as
implants, oily injections, or as particulate systems. For a broad
overview of protein delivery systems, see Banga, Therapeutic
Peptides and Proteins: Formulation, Processing, and Delivery
Systems, Technomic Publishing Company, Inc., Lancaster, Pa., 1995.
Particulate systems include microspheres, microparticles,
microcapsules, nanocapsules, nanospheres, and nanoparticles.
Microcapsules contain the therapeutic protein as a central core. In
microspheres, the therapeutic agent is dispersed throughout the
particle. Particles, microspheres, and microcapsules smaller than
about 1 .mu.m are generally referred to as nanoparticles,
nanospheres, and nanocapsules, respectively. Capillaries have a
diameter of approximately 5 .mu.m so that only nanoparticles are
administered intravenously. Microparticles are typically around 100
.mu.m in diameter and are administered subcutaneously or
intramuscularly (see Kreuter, Colloidal Drug Delivery Systems, J.
Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342,
1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A.
Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339,
1992).
[0253] Polymers can be used for ion-controlled release. Various
degradable and nondegradable polymeric matrices for use in
controlled drug delivery are known in the art (Langer, Accounts
Chem. Res. 26:537, 1993). For example, the block copolymer,
polaxamer 407 exists as a viscous yet mobile liquid at low
temperatures but forms a semisolid gel at body temperature. It has
shown to be an effective vehicle for formulation and sustained
delivery of recombinant interleukin-2 and urease (Johnston et al.,
Pharm. Res. 9:425, 1992; and Pec. J. Parent. Sci. Tech. 44(2):58,
1990). Alternatively, hydroxyapatite has been used as a
microcarrier for controlled release of proteins (Ijntema et al.,
Int. J. Pharm. 112:215, 1994). In yet another aspect, liposomes are
used for controlled release as well as drug targeting of the
lipid-capsulated drug (Betageri et al., Liposome Drug Delivery
Systems, Technomic Publishing Co., Inc., Lancaster, Pa., 1993).
Numerous additional systems for controlled delivery of therapeutic
proteins are known (e.g., U.S. Pat. Nos. 5,055,303; 5,188,837;
4,235,871; 4,501,728; 4,837,028; 4,957,735; and U.S. Pat. Nos.
5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;
4,902,505; 5,506,206; 5,271,961; 5,254,342; and U.S. Pat. No.
5,534,496).
[0254] In another embodiment, the composition includes a nucleic
acid encoding a Brachyury protein and/or a Brachyury polypeptide. A
therapeutically effective amount of the polynucleotide encoding the
Brachyury protein and/or the Brachyury polypeptide can be
administered to a subject in order to generate an immune response.
In one specific, non-limiting example, a therapeutically effective
amount of the polynucleotide encoding the Brachyury protein and/or
Brachyury polypeptide is administered to a subject to treat or
prevent cancer.
[0255] Optionally, one or more immunostimulatory molecules and/or
costimulatory molecules, such as IL-2, IL-6, IL-12, LFA (for
example, LFA-1, LFA-2 and/or LFA-3), CD72, RANTES, G-CSF, GM-CSF,
TNF-.alpha., IFN-.gamma. ICAM-1, B7-1, B7-2, other B7 related
molecules, OX-40L or 41 BBL, or combinations of these molecules,
can be used as biological adjuvants (see, for example, Salgaller et
al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000. Cancer
J Sci. Am. 6(Suppl 1):561-6; Cao et al., 1998, Stem Cells 16(Suppl
1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90).
These molecules can be administered systemically (or locally) to
the host. In several examples, IL-2, RANTES, GM-CSF, TNF-.alpha.,
IFN-.gamma., G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1, B7-2, OX-40L, 41
BBL and/or ICAM-1 are administered. L-15 or an IL-15/IL-15 receptor
complex can be administered.
[0256] Optionally, a non-pox non-yeast vector is administered that
encodes one or more immunostimulatory or costimulatory molecules,
such as IL-2, IL-6, IL-12, IL-15, LFA (for example, LFA-1, LFA-2
and/or LFA-3), CD72, RANTES, G-CSF, GM-CSF, TNF-.alpha.,
IFN-.gamma., ICAM-1, B7-1, B7-2, other B7 related molecules, OX-40L
or 41 BBL, or combinations of these molecules (see, for example,
Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al.,
2000, Cancer J Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem
Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.
465:381-90). In several examples, the vector can encode IL-2,
RANTES, GM-CSF, TNF-.alpha., IFN-.gamma., G-CSF, LFA-3, CD72, B7-1,
B7-2, B7-1, B7-2, OX-40L, 41 BBL and/or ICAM-1. In various
embodiments, the nucleic acid encoding the biological adjuvant can
be cloned into same vector as the Brachyury protein or Brachyury
polypeptide coding sequence, or the nucleic acid can be cloned into
one or more separate vectors for co-administration. In addition,
nonspecific immunomodulating factors such as Bacillus
Cahnette-Guerin (BCG) and levamisole can be co-administered.
[0257] One approach to administration of nucleic acids is direct
immunization with plasmid DNA, such as with a mammalian expression
plasmid. As described above, the nucleotide sequence encoding a
Brachyury protein or polypeptide can be placed under the control of
a promoter to increase expression of the molecule.
[0258] Immunization by nucleic acid constructs is well known in the
art and taught, for example, in U.S. Pat. No. 5,643,578 (which
describes methods of immunizing vertebrates by introducing DNA
encoding a desired antigen to elicit a cell-mediated or a humoral
response), and U.S. Pat. No. 5,593,972 and U.S. Pat. No. 5,817,637
(which describe operably linking a nucleic acid sequence encoding
an antigen to regulatory sequences enabling expression). U.S. Pat.
No. 5,880,103 describes several methods of delivery of nucleic
acids encoding immunogenic peptides or other antigens to an
organism. The methods include liposomal delivery of the nucleic
acids (or of the synthetic peptides themselves), and
immune-stimulating constructs, or ISCOMS.TM., negatively charged
cage-like structures of 30-40 nm in size formed spontaneously on
mixing cholesterol and Quil A.TM. (saponin). Protective immunity
has been generated in a variety of experimental models of
infection, including toxoplasmosis and Epstein-Barr virus-induced
tumors, using ISCOMS.TM. as the delivery vehicle for antigens
(Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of antigen
as low as 1 .mu.g encapsulated in ISCOMS.TM. have been found to
produce Class I mediated CTL responses (Takahashi et al., Nature
344:873, 1990). In other embodiments, the nucleic acid can be
loaded onto gold microspheres by standard methods and introduced
into the skin by a device such as Bio-Rad's HELIOS.TM. Gene Gun.
The nucleic acids can be "naked," consisting of plasmids under
control of a strong promoter. Typically, the DNA is injected into
muscle, although it can also be injected directly into other sites,
including tissues in proximity to metastases. Dosages for injection
are usually around 0.5 .mu.g/kg to about 50 mg/kg, and typically
are about 0.005 mg/kg to about 5 mg/kg (see, for example, U.S. Pat.
No. 5,589,466).
[0259] In another approach to using nucleic acids for immunization,
a Brachyury protein or Brachyury polypeptide can also be expressed
by attenuated host cells or non-pox non-yeast viral vectors. These
vectors and host cells are disclosed above. Suitable non-pox
non-yeast viral vector, include an adenovirus, an alphvirus, a
lentivirus, a measles virus or a poliovirus vector. Suitable host
cell include an attenuated bacterium, such as Listeria or
Salmonella host cells.
[0260] A first recombinant non-pox virus encoding a Brachyury
protein can be used in conjunction with a second recombinant
non-pox virus which has incorporated into a viral genome or
infectable portion thereof one or more genes or DNA sequences
encoding B7-1, B7-2, or B7-1 and B7-2, wherein the composition is
able to coinfect a host cell resulting in coexpression of the
polypeptide and the B7-1, B7-2, or B7-1 and B7-2 encoding genes or
DNA sequences (see U.S. Pat. No. 6,893,869, and U.S. Pat. No.
6,045,908, which are incorporated by reference herein). The
expression of the B7 gene family has been shown to be an important
mechanism of anti-tumor responses in both mice and humans.
[0261] When a non-pox viral vector is utilized, it is desirable to
provide the recipient with a dosage of each recombinant virus in
the composition in the range of from about 10' to about 10.sup.10
plaque forming units/mg mammal, although a lower or higher dose can
be administered. The composition of recombinant viral vectors can
be introduced into a mammal either prior to any evidence of a
cancer, or to mediate regression of the disease in a mammal
afflicted with the cancer. Examples of methods for administering
the composition into mammals include, but are not limited to,
exposure of cells to the recombinant virus ex vivo, or injection of
the composition into the affected tissue or intravenous,
subcutaneous, intradermal or intramuscular administration of the
virus. Alternatively the recombinant viral vector or combination of
recombinant viral vectors may be administered locally by direct
injection into the cancerous lesion in a pharmaceutically
acceptable carrier. Generally, the quantity of recombinant non-pox
viral vector, carrying the nucleic acid sequence of a Brachyury
protein or Brachyury polypeptide to be administered is based on the
titer of virus particles. An exemplary range of the immunogen to be
administered is 10.sup.5 to 10.sup.10 virus particles per mammal,
such as a human.
[0262] In the embodiment where a combination of a first recombinant
viral vector carrying a nucleic acid sequence of a Brachyury
protein or Brachyury polypeptide and a second recombinant viral
vector carrying the nucleic acid sequence of one or more
costimulatory or immunostimulatory molecules is used, the mammal
can be immunized with different ratios of the first and second
recombinant viral vector. In one embodiment the ratio of the first
vector to the second vector is about 1:1, or about 1:3, or about
1:5. Optimal ratios of the first vector to the second vector may
easily be titered using the methods known in the art (see, for
example, U.S. Pat. No. 6,893,869, incorporated herein by
reference). Simultaneous production of an immunostimulatory
molecule and the Brachyury protein or Brachyury polypeptide
enhances the generation of specific effectors. Without being bound
by theory, dependent upon the specific immunostimulatory molecules,
different mechanisms might be responsible for the enhanced
immunogenicity: augmentation of help signal (IL-2), recruitment of
professional APC (GM-CSF), increase in CTL frequency (IL-2), effect
on antigen processing pathway and MHC expression (IFN.gamma. and
TNF.alpha.) and the like. For example, IL-2, IL-6, IL-15,
interferon, tumor necrosis factor, or a nucleic acid encoding these
molecules, can be administered in conjunction with a Brachyury
protein, or a nucleic acid encoding a Brachyury protein or
Brachyury polypeptide. The co-expression of a Brachyury protein or
Brachyury polypeptide together with at least one immunostimulatory
molecule can be effective in an animal model to show anti-tumor
effects.
[0263] Single or multiple administrations of the compositions are
administered depending on the dosage and frequency as required and
tolerated by the subject. In one embodiment, the dosage is
administered once as a bolus, but in another embodiment can be
applied periodically until a therapeutic result is achieved.
Generally, the dose is sufficient to induce an immune response to
Brachyury, to treat or ameliorate symptoms or signs of disease,
without producing unacceptable toxicity to the subject. Systemic or
local administration can be utilized.
[0264] Use of Antigen Presenting Cells
[0265] In another method, antigen presenting cells (APCs), such as
dendritic cells, are pulsed or co-incubated with a Brachyury
protein and/or Brachyury polypeptide in vitro. In one specific,
non-limiting example, the antigen presenting cells can be
autologous cells. A therapeutically effective amount of the antigen
presenting cells, such as dendritic cells presenting epitopes of
the protein or polypeptide can then be administered to a subject.
In some embodiments, the method is a method of treating and/or
preventing cancer in a subject.
[0266] The Brachyury protein and/or Brachyury polypeptide can be
delivered to the dendritic cells or to dendritic cell precursors
via any method known in the art, including, but not limited to,
pulsing dendritic cells directly with antigen, or utilizing a broad
variety of antigen delivery vehicles, such as, for example,
liposomes, or other vectors known to deliver antigen to cells. In
one specific, non-limiting example an antigenic formulation
includes about 0.1 .mu.g to about 1,000 .mu.g, or about 1 to about
100 .mu.g of a selected Brachyury protein. The Brachyury protein
can also be administered with agents that promote dendritic cell
maturation. Specific, non-limiting examples of agents of use are
interleukin-4 (IL-4) and granulocyte/macrophage colony stimulating
factor (GM-CSF), or flt-3 ligand (flt-3L). The preparation can also
contain buffers, excipients, and preservatives, amongst other
ingredients.
[0267] In one embodiment, mature antigen presenting cells are
generated to present the Brachyury protein and/or Brachyury
polypeptide epitopes. These dendritic cells are then administered
alone (or in combination with another agent) to a subject with a
cancer that expresses Brachyury, or has the potential to express
Brachyury, such as a small intestine, stomach, kidney, bladder,
uterus, ovary, testis, lung colon, prostate cancer, a tumor of B
cell origin (such as chronic lymphocytic leukemia (CLL), a B cell
lymphoma, Burkitt's lymphoma or a Hodgkin's lymphoma) or breast
cancer, such as an infiltrating ductal carcinoma or an estrogen
receptor negative and progesterone receptor negative breast cancer.
In some examples, the cancer is radiation resistant and/or
chemotherapy resistant. The antigen presenting cells can also be
administered to prevent these cancers. The subject can have high
grade prostatic intraepithelial neoplasia, familial adenomatous
polyposis, or atypia of the breast.
[0268] The cells can be administered to a subject to inhibit the
growth of cells of Brachyury expressing cancer or a cancer that has
the potential to express Brachyury. In these applications, a
therapeutically effective amount of activated antigen presenting
cells are administered to a subject suffering from a disease, in an
amount sufficient to raise an immune response to
Brachyury-expressing cells. The resulting immune response is
sufficient to slow the proliferation of such cells or to inhibit
their growth, or to reduce a sign or a symptom of the tumor.
[0269] In a supplemental method, any of these immunotherapies is
augmented by administering a cytokine, such as interleukin (IL)-2,
IL-3, IL-6, IL-10, IL-12, IL-15, GM-CSF, or interferons. In another
embodiment, the mature dendritic cells are administered in
conjunction with a chemotherapeutic agent.
[0270] Combination Therapy
[0271] In some embodiments, the subject is administered a Brachury
protein, a Brachyury polypeptide, a nucleic acid encoding a
Brachyury protein, a host cell expressing the Brachyury protein,
and/or dendritic cells, and is administered an additional agent. In
one example, this administration is sequential. However, the
administration can be simultaneous.
[0272] In some embodiments the subject has cancer. The cancer can
express Brachyury or have the potential to express Brachyury. Thus,
the additional agent can be a chemotherapeutic agent. Additional
agents include radiation, small molecule targeted therapies,
monoclonal antibodies, and/or checkpoint inhibitors, such as
anti-PD-1, anti-PD-L1, anti-CTLA-4, and others. In some
embodiments, the subject is administered an epithelial growth
factor receptor inhibitor, a transforming growth factor (TGF)-3
inhibitor, or a tyrosine kinase inhibitor.
[0273] In some embodiments, the additional chemotherapeutic agent
is an epithelial growth factor receptor (EGFR) inhibitor. Numerous
compounds are known that inhibit EGFR, see for example, U.S. Pat.
Nos. 5,196,446; 5,217,999; 5,459,061; 7,049,410; 6,355,678, which
are incorporated herein by reference. A number of EGFR inhibitors
are in clinical development, such as for the treatment of lung
cancer. These include tustuzumab, lapatinib, pertuzumab,
panitumumab, genfitinib (IRESSA.RTM.), erlotinib (TARCEVA.RTM.),
cetuximab (ERTIBUX.RTM.), afatinib, nectiumumab, nimotuzumab,
PF299804 (Pfizer), R05083945 (Roche), ABT-806 (Abbott) and AP2113
(Ariad). In other embodiments, the additional chemotherapeutic
agent is a tyrosine kinase inhibitor. These include, but are not
limited to avastin and termsirolimus.
[0274] Examples of additional agents of use are alkylating agents,
antimetabolites, natural products, or hormones and their
antagonists. Examples of alkylating agents include nitrogen
mustards (such as mechlorethamine, cyclophosphamide, melphalan,
uracil mustard or chlorambucil), alkyl sulfonates (such as
busulfan), nitrosoureas (such as carmustine, lomustine, semustine,
streptozocin, or dacarbazine). Examples of antimetabolites include
folic acid analogs (such as methotrexate), pyrimidine analogs (such
as 5-FU or cytarabine), and purine analogs, such as mercaptopurine
or thioguanine. Examples of natural products include vinca
alkaloids (such as vinblastine, vincristine, or vindesine),
epipodophyllotoxins (such as etoposide or teniposide), antibiotics
(such as dactinomycin, daunorubicin, doxorubicin, bleomycin,
plicamycin, or mitocycin C), and enzymes (such as L-asparaginase).
Examples of miscellaneous agents include platinum coordination
complexes (such as cis-diamine-dichloroplatinum II also known as
cisplatin), substituted ureas (such as hydroxyurea), methyl
hydrazine derivatives (such as procarbazine), and adrenocrotical
suppressants (such as mitotane and aminoglutethimide). Examples of
hormones and antagonists include adrenocorticosteroids (such as
prednisone), progestins (such as hydroxyprogesterone caproate,
medroxyprogesterone acetate, and magestrol acetate), estrogens
(such as diethylstilbestrol and ethinyl estradiol), antiestrogens
(such as tamoxifen), and androgens (such as testosterone
proprionate and fluoxymesterone). Examples of the most commonly
used chemotherapy drugs that can be concurrently administered with
the disclosed immunotherapy include Adriamycin, Alkeran, Ara-C,
BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan,
Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin,
Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone,
Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel),
Velban, Vincristine, VP-16, while some more newer drugs include
Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),
Leustatin, Navelbine, Rituxan STI-571, Taxotere. Topotecan
(Hycamtin). Xeloda (Capecitabine), Zevelin and calcitriol.
Non-limiting examples of immunomodulators that can be used include
AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon
(Genentech), GM-CSF (granulocyte macrophage colony stimulating
factor, Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human
immune globulin (Cutter Biological), IMREG (from Imreg of New
Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor;
Genentech).
[0275] The additional agent can be a small molecule, a vaccine, or
a biologic. For example, when the additional agent is a vaccine,
the vaccine can be a yeast-based or viral-based (e.g.,
poxviral-based) vaccine. Examples of viral vectors include
poxvirus, retrovirus, adenovirus, adeno-associated virus, herpes
virus, polio virus, alphavirus, baculorvirus, and Sindbis virus. In
one embodiment, the viral vector is a poxvirus selected from the
group consisting of orthopox, avipox, fowlpox, raccoon pox, rabbit
pox, capripox (e.g., sheep pox), leporipox, and suipox (e.g.,
swinepox). Examples of avipox viruses include fowlpox, pigeonpox,
and canarypox, such as ALVAC. Examples of orthopox viruses include
vaccinia, modified vaccinia Ankara (MVA), Wyeth, NYVAC, TROYVAC,
Dry-Vax, POXVAC-TC (Schering-Plough Corporation), and derivatives
thereof. For example, derivatives of the Wyeth strain include, but
are not limited to, derivatives which lack a functional K1L
gene.
[0276] The vaccine can encode any suitable antigen, such as the
Brachyury protein or Brachyury polypeptide described herein,
5-.alpha.-reductase, .alpha.-fetoprotein ("AFP"), AM-1, APC, April,
B melanoma antigen gene ("BAGE"), .beta.-catenin, Bc112, bcr-abl,
Brachyury, CA-125, caspase-8 ("CASP-8", also known as "FLICE"),
Cathepsins, CD19, CD20, CD21/complement receptor 2 ("CR2"),
CD22/BL-CAM, CD23/F.sub.c.epsilon.RII, CD33, CD35/complement
receptor 1 ("CR1"), CD44/PGP-1, CD45/leucocyte common antigen
("LCA"), CD46/membrane cofactor protein ("MCP"), CD52/CAMPATH-1,
CD55/decay accelerating factor ("DAF"), CD59/protectin, CDC27,
CDK4, carcinoembryonic antigen ("CEA"), c-myc, cyclooxygenase-2
("cox-2"), deleted in colorectal cancer gene ("DCC"), DcR3, E6/E7,
CGFR, EMBP, Dna78, farnesyl transferase, fibroblast growth
factor-8a ("FGF8a"), fibroblast growth factor-8b ("FGF8b"),
FLK-1/KDR, folic acid receptor, G250, G melanoma antigen gene
family ("GAGE-family"), gastrin 17, gastrin-releasing hormone,
ganglioside 2 ("GD2")/ganglioside 3 ("GD3")/ganglioside-monosialic
acid-2 ("GM2"), gonadotropin releasing hormone ("GnRH"),
UDP-GlcNAc:R.sub.1Man(.alpha.1-6)R.sub.2 [GlcNAc to
Man(.alpha.1-6)] .beta.1,6-N-acetylglucosaminyltransferase V ("GnT
V"), GPI, gp100/Pme 17, gp-100-in4, gp15, gp75/tyrosine-related
protein-1 ("gp75/TRP-1"), human chorionic gonadotropin ("hCG"),
heparanase, Her2/neu, human mammary tumor virus ("HMTV"), 70
kiloDalton heat-shock protein ("HSP70"), human telomerase reverse
transcriptase ("hTERT"), insulin-like growth factor receptor-1
("IGFR-1"), interleukin-13 receptor ("IL-13R"), inducible nitric
oxide synthase ("iNOS"), Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA,
LKLR-FUT, melanoma antigen-encoding family ("MAGE-family",
including at least MAGE-1, MAGE-2, MAGE-3, and MAGE-4),
mammaglobin, MAP17, Melan-A/melanoma antigen recognized by
T-cells-1 ("MART-1"), mesothelin, MIC A/B, MT-MMPs, mucin,
testes-specific antigen NY-ESO-1, osteonectin, p15, P170/MDR1, p53,
p97/melanotransferrin, PAI-1, platelet-derived growth factor
("PDGF"), .mu.PA, PRAME, probasin, progenipoietin,
prostate-specific antigen ("PSA"), prostate-specific membrane
antigen ("PSMA"), RAGE-1, Rb, RCAS1, SART-1, SSX-family, STAT3,
STn, TAG-72, transforming growth factor-alpha ("TGF-.alpha."),
transforming growth factor-beta ("TGF-.beta."), Thymosin-beta-15,
tumor necrosis factor-alpha ("TNF-.alpha."), TP1, TRP-2,
tyrosinase, vascular endothelial growth factor ("VEGF"), ZAG,
p161NK4, and/or glutathione-S-transferase ("GST").
[0277] In one embodiment, the vaccine is PROSTVAC.TM., which is a
sequentially dosed combination of two different poxviruses each
encoding prostate specific antigen (PSA) plus three immune
enhancing co-stimulatory molecules, B7.1, ICAM-1, and LFA-3
(TRICOM). The first poxvirus is Vaccinia-PSA-TRICOM, and the second
poxvirus is Fowlpox-PSA-TRICOM.
[0278] Thus, the invention provides a method for treating or
preventing cancer in a subject comprising administering to the
subject a combination therapy comprising:
[0279] (1) (a) a protein comprising an amino acid sequence at least
90% identical to the amino acid sequence set forth as SEQ ID NO: 1;
(b) a polypeptide comprising at least 15 consecutive amino acids of
the amino acid sequence set forth at SEQ ID NO: 1; (c) a nucleic
acid encoding the protein or the polypeptide; (d) a host cell
expressing the protein or the polypeptide; or (e) a non-pox
non-yeast vector encoding the protein or the polypeptide, and
[0280] (2) a small molecule, a vaccine (e.g., a pox-viral or yeast
vaccine as described above), or a biologic,
[0281] thereby treating or preventing cancer in the subject.
EXAMPLES
Example 1: Induction of CD4+ Cells Using Brachyury Protein or
Brachyury Polypeptide
[0282] FIGS. 1 and 2 show the induction of CD4+ cells using
Brachyury protein and a Brachyury polypeptide.
[0283] I. FIG. 1
[0284] Methods:
[0285] Dendritic cells (DCs) from 2 normal donors were prepared
from the adherent cell fraction of peripheral blood mononuclear
cells (PBMCs) by culture in the presence of GM-CSF and IL-4. On day
5, a purified, recombinant full length Brachyury protein was added
to the cultures (10 .mu.g/ml) for 48 hours. For donor 2, an
additional culture was set up using purified HSA (human serum
albumin) control protein (10 .mu.g/ml). On day 7, protein-pulsed
DCs were harvested, irradiated (20 Gy) and used as
antigen-presenting cells (APCs) to stimulate autologous PBMCs
(ratio DC:PBMCs equal to 1:10). On days 3 and 5, IL-2 (20 U/ml) was
added to the cultures. Cells were harvested on day 7 and CD4+ T
cells were isolated by negative selection utilizing CD4
purification magnetic beads (Miltenyi Biotec). CD4+ T cells were
subsequently stimulated in a similar manner for an additional 7-day
cycle. On day 7, CD4+ T cells were re-purified by using CD4
purification magnetic beads, and evaluated for IFN-gamma production
in response to autologous, irradiated PBMCs (ratio PBMCs:CD4+ T
cells equal to 3:1) alone or pulsed with control HSA protein vs.
Brachyury protein (10 .mu.g/ml). Culture supernatants were
collected at 96 hours and evaluated for IFN-gamma by ELISA.
[0286] Summary of Findings:
[0287] As shown in FIG. 1, Brachyury-specific CD4+ T cells can be
expanded from the blood of normal donors by culture of PBMCs in the
presence of autologous DCs pulsed with a purified, full length
Brachyury protein. After 2 rounds of in vitro stimulation,
Brachyury-specific CD4+ T cells specifically released IFN-gamma in
response to stimulation with autologous PBMCs pulsed with purified
Brachyury protein (full length, recombinant) but not with a
control, irrelevant protein (HAS).
[0288] II. FIG. 2
[0289] Methods: A CD4+ Brachyury-specific T-cell line (T-BRA) was
generated from a prostate patient vaccinated with a PSA-based
vaccine. CD40L-matured autologous DCs were used as
antigen-presenting cells (APCs). PBMCs obtained on day 90
post-vaccination were added to the APCs and pulsed with 10 .mu.g/mL
of Brachyury 9-mer agonist peptide (WLLPGTSTV) at an effector:APC
ratio of 10:1. The culture was then incubated for 3 days at
37.degree. C. in a humidified atmosphere containing 5% CO.sub.2.
The culture was then supplemented with recombinant human IL-7 and
IL-15 at a concentration of 10 ng/ml for 5 days. The 3-day
incubation with peptide and 5-day IL-7 and IL-15 supplement
constituted one in vitro stimulation (IVS) cycle. Autologous DCs
were used as APCs for 3 in vitro stimulation (IVS) cycles.
Irradiated (23,000 rads) autologous EBV-transformed B cells were
used as APCs after the third IVS cycle. For re-stimulation with
EBV-transformed B cells, peptides at a concentration of 10 .mu.g/mL
were used to pulse the autologous EBV-transformed B cells at an
effector:APC ratio of 1:3. CD4+ T cells were then isolated from the
cell culture at the end of the 4th IVS and stimulated with a
Brachyury class II 15-mer peptide (Brachyury class
IIB)(QWGWLLPGTSTL). The cultures were then supplemented with
recombinant human IL-7 and IL-15 at a concentration of 10 ng/mL for
5 days. The CD4+ T cell line was then assayed for specificity to
the Brachyury class IIB epitope by stimulation with APCs pulsed
with the Brachyury class IIB or a control Brachyury class IIA
epitope.
Example 2: Brachyury in Carcinoma
[0290] FIGS. 3-7 show the results achieved.
TABLE-US-00007 TABLE 1 Expression of Brachyury in primary breast
carcinoma tissues by immunohistochemistry utilizing a murine
monoclonal anti-brachyury Ab Tumor Adjacent Distal Pt # %
Positivity Intensity tissue tissue 1 80 ++ pos neg 2 80 ++ pos neg
3 90 ++ pos neg 4 90 + neg neg 5 90 + pos neg 6 30 + neg neg 7 15
+++ neg neg 8 60 + neg neg 9 focal + neg neg 10 70 + neg neg 11 40
+ neg neg 12 30 + neg neg 13 focal + neg neg 14 90 +++ neg neg 15
90 -/+ pos neg 16 30 + neg neg 17 neg neg neg neg 18 50 ++ neg neg
19 neg neg neg neg 20 neg neg neg neg 21 40 ++ pos neg 22 30 ++ pos
neg 23 85 ++ pos neg 24 80 +++ pos neg 25 30 + pos neg 26 50 ++ pos
neg 27 65 ++ pos NA 28 40 +++ pos neg 29 25 ++ pos NA 30 15 + pos
NA Pos = positive; neg = negative; NA = not available
TABLE-US-00008 TABLE 2 Brachyury expression in primary breast
carcinoma tissues by immunohistochemistry by lymph node status,
tumor grade, and hormone receptor expression Number of Tumor tissue
tumors positive sample for Brachyury Lymph node status:
Node-negative 13/15 (86.7%)* Node-positive 10/11 (93.3%) Grade: G1
2/3 (66.7%) G2 -- G3 25/27 (92.6%) ER/PR expression: ER+ PR+ 4/6
(66.7%) ER- PR- 21/22 (95.5%) ER/PR/HER2 expression: ER+ PR+ HER2+
1/2 (50.0%) ER+ PR+ HER2- 3/4 (75.0%) ER- PR- HER2+ 12/12 (100.0%)
ER- PR- HER2- 9/10 (90.0%) Statistical analysis was performed,
comparing node-positive vs. node-negative, grade 3 vs. grade 1, and
ER- PR- vs. ER+ PR+ samples. *Numbers in parentheses indicate
percentage.
TABLE-US-00009 TABLE 3 Expression of Brachyury in metastatic breast
carcinoma lesions by immunohistochemistry using a murine monoclonal
anti-brachyury Ab Brachyury Pt # Site % Positivity Intensity 6
Breast primary tumor 30 + 6 Met.sup.+ lymph node (a) 90 + 6
Met.sup.+ lymph node (b) 90 + 6 Non-met lymph node neg neg 9 Breast
primary tumor focal + 9 Met.sup.+ lymph node (a) 60 ++ 9 Met.sup.+
lymph node (b) 60 ++ 9 Non-met lymph node neg neg 31 Pleura 90 + 32
Bone 90 ++ 33 Bone 90 + 34 Brain 70 ++
[0291] Human matched breast primary tumor tissues and metastatic
lymph nodes from two patients were analyzed for Brachyury
expression by immunohistochemistry. The breast primary tumor tissue
samples were from infiltrating ductal adenocarcinomas. Two lymph
nodes positive for metastasis from each patient (a, b) and 1 lymph
node negative for metastasis from same patient (c) were assayed.
Metastatic lesions from additional patients were also analyzed for
Brachyury expression by immunohistochemistry. Adjacent and distal
breast tissues in the samples were negative for Brachyury
expression.
[0292] The breast primary tumor tissue samples were from 30
infiltrating ductal adenocarcinomas. Brachyury expression for each
sample is reported as staining in tumor cells (% positivity,
intensity), staining in breast adjacent tissue, and staining in
breast distal tissue.
[0293] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
Sequence CWU 1
1
61435PRTHomo sampiens 1Met Ser Ser Pro Gly Thr Glu Ser Ala Gly Lys
Ser Leu Gln Tyr Arg 1 5 10 15 Val Asp His Leu Leu Ser Ala Val Glu
Asn Glu Leu Gln Ala Gly Ser 20 25 30 Glu Lys Gly Asp Pro Thr Glu
Arg Glu Leu Arg Val Gly Leu Glu Glu 35 40 45 Ser Glu Leu Trp Leu
Arg Phe Lys Glu Leu Thr Asn Glu Met Ile Val 50 55 60 Thr Lys Asn
Gly Arg Arg Met Phe Pro Val Leu Lys Val Asn Val Ser 65 70 75 80 Gly
Leu Asp Pro Asn Ala Met Tyr Ser Phe Leu Leu Asp Phe Val Ala 85 90
95 Ala Asp Asn His Arg Trp Lys Tyr Val Asn Gly Glu Trp Val Pro Gly
100 105 110 Gly Lys Pro Glu Pro Gln Ala Pro Ser Cys Val Tyr Ile His
Pro Asp 115 120 125 Ser Pro Asn Phe Gly Ala His Trp Met Lys Ala Pro
Val Ser Phe Ser 130 135 140 Lys Val Lys Leu Thr Asn Lys Leu Asn Gly
Gly Gly Gln Ile Met Leu 145 150 155 160 Asn Ser Leu His Lys Tyr Glu
Pro Arg Ile His Ile Val Arg Val Gly 165 170 175 Gly Pro Gln Arg Met
Ile Thr Ser His Cys Phe Pro Glu Thr Gln Phe 180 185 190 Ile Ala Val
Thr Ala Tyr Gln Asn Glu Glu Ile Thr Ala Leu Lys Ile 195 200 205 Lys
Tyr Asn Pro Phe Ala Lys Ala Phe Leu Asp Ala Lys Glu Arg Ser 210 215
220 Asp His Lys Glu Met Met Glu Glu Pro Gly Asp Ser Gln Gln Pro Gly
225 230 235 240 Tyr Ser Gln Trp Gly Trp Leu Leu Pro Gly Thr Ser Thr
Leu Cys Pro 245 250 255 Pro Ala Asn Pro His Pro Gln Phe Gly Gly Ala
Leu Ser Leu Pro Ser 260 265 270 Thr His Ser Cys Asp Arg Tyr Pro Thr
Leu Arg Ser His Arg Ser Ser 275 280 285 Pro Tyr Pro Ser Pro Tyr Ala
His Arg Asn Asn Ser Pro Thr Tyr Ser 290 295 300 Asp Asn Ser Pro Ala
Cys Leu Ser Met Leu Gln Ser His Asp Asn Trp 305 310 315 320 Ser Ser
Leu Gly Met Pro Ala His Pro Ser Met Leu Pro Val Ser His 325 330 335
Asn Ala Ser Pro Pro Thr Ser Ser Ser Gln Tyr Pro Ser Leu Trp Ser 340
345 350 Val Ser Asn Gly Ala Val Thr Pro Gly Ser Gln Ala Ala Ala Val
Ser 355 360 365 Asn Gly Leu Gly Ala Gln Phe Phe Arg Gly Ser Pro Ala
His Tyr Thr 370 375 380 Pro Leu Thr His Pro Val Ser Ala Pro Ser Ser
Ser Gly Ser Pro Leu 385 390 395 400 Tyr Glu Gly Ala Ala Ala Ala Thr
Asp Ile Val Asp Ser Gln Tyr Asp 405 410 415 Ala Ala Ala Gln Gly Arg
Leu Ile Ala Ser Trp Thr Pro Val Ser Pro 420 425 430 Pro Ser Met 435
22518DNAHomo sapiens 2tttgcttttg cttatttccg tccatttccc tctctgcgcg
cggaccttcc ttttccagat 60ggtgagagcc gcggggacac ccgacgccgg ggcaggctga
tccacgatcc tgggtgtgcg 120taacgccgcc tggggctccg tgggcgaggg
acgtgtgggg acaggtgcac cggaaactgc 180cagactggag agttgaggca
tcggaggcgc gagaacagca ctactactgc ggcgagacga 240gcgcggcgca
tcccaaagcc cggccaaatg cgctcgtccc tgggagggga gggaggcgcg
300cctggagcgg ggacagtctt ggtccgcgcc ctcctcccgg gtctgtgccg
ggacccggga 360cccgggagcc gtcgcaggtc tcggtccaag gggccccttt
tctcggaagg gcggcggcca 420agagcaggga aggtggatct caggtagcga
gtctgggctt cggggacggc ggggagggga 480gccggacggg aggatgagct
cccctggcac cgagagcgcg ggaaagagcc tgcagtaccg 540agtggaccac
ctgctgagcg ccgtggagaa tgagctgcag gcgggcagcg agaagggcga
600ccccacagag cgcgaactgc gcgtgggcct ggaggagagc gagctgtggc
tgcgcttcaa 660ggagctcacc aatgagatga tcgtgaccaa gaacggcagg
aggatgtttc cggtgctgaa 720ggtgaacgtg tctggcctgg accccaacgc
catgtactcc ttcctgctgg acttcgtggc 780ggcggacaac caccgctgga
agtacgtgaa cggggaatgg gtgccggggg gcaagccgga 840gccgcaggcg
cccagctgcg tctacatcca ccccgactcg cccaacttcg gggcccactg
900gatgaaggct cccgtctcct tcagcaaagt caagctcacc aacaagctca
acggaggggg 960ccagatcatg ctgaactcct tgcataagta tgagcctcga
atccacatag tgagagttgg 1020gggtccacag cgcatgatca ccagccactg
cttccctgag acccagttca tagcggtgac 1080tgcttatcag aacgaggaga
tcacagctct taaaattaag tacaatccat ttgcaaaagc 1140tttccttgat
gcaaaggaaa gaagtgatca caaagagatg atggaggaac ccggagacag
1200ccagcaacct gggtactccc aatgggggtg gcttcttcct ggaaccagca
ccctgtgtcc 1260acctgcaaat cctcatcctc agtttggagg tgccctctcc
ctcccctcca cgcacagctg 1320tgacaggtac ccaaccctga ggagccaccg
gtcctcaccc taccccagcc cctatgctca 1380tcggaacaat tctccaacct
attctgacaa ctcacctgca tgtttatcca tgctgcaatc 1440ccatgacaat
tggtccagcc ttggaatgcc tgcccatccc agcatgctcc ccgtgagcca
1500caatgccagc ccacctacca gctccagtca gtaccccagc ctgtggtctg
tgagcaacgg 1560cgccgtcacc ccgggctccc aggcagcagc cgtgtccaac
gggctggggg cccagttctt 1620ccggggctcc cccgcgcact acacacccct
cacccatccg gtctcggcgc cctcttcctc 1680gggatcccca ctgtacgaag
gggcggccgc ggccacagac atcgtggaca gccagtacga 1740cgccgcagcc
caaggccgcc tcatagcctc atggacacct gtgtcgccac cttccatgtg
1800aagcagcaag gcccaggtcc cgaaagatgc agtgactttt tgtcgtggca
gccagtggtg 1860actggattga cctactaggt acccagtggc agtctcaggt
taagaaggaa atgcagcctc 1920agtaacttcc ttttcaaagc agtggaggag
cacacggcac ctttccccag agccccagca 1980tcccttgctc acacctgcag
tagcggtgct gtcccaggtg gcttacagat gaacccaact 2040gtggagatga
tgcagttggc ccaacctcac tgacggtgaa aaaatgtttg ccagggtcca
2100gaaacttttt ttggtttatt tctcatacag tgtattggca actttggcac
accagaattt 2160gtaaactcca ccagtcctac tttagtgaga taaaaagcac
actcttaatc ttcttccttg 2220ttgctttcaa gtagttagag ttgagctgtt
aaggacagaa taaaatcata gttgaggaca 2280gcaggtttta gttgaattga
aaatttgact gctctgcccc ctagaatgtg tgtattttaa 2340gcatatgtag
ctaatctctt gtgttgttaa actataactg tttcatattt ttcttttgac
2400aaagtagcca aagacaatca gcagaaagca ttttctgcaa aataaacgca
atatgcaaaa 2460tgtgattcgt ccagttatta gtgaagcccc tccttttgtg
agtatttact gtttattg 25183436PRTMus musculus 3Met Ser Ser Pro Gly
Thr Glu Ser Ala Gly Lys Ser Leu Gln Tyr Arg 1 5 10 15 Val Asp His
Leu Leu Ser Ala Val Glu Ser Glu Leu Gln Ala Gly Ser 20 25 30 Glu
Lys Gly Asp Pro Thr Glu Arg Glu Leu Arg Val Gly Leu Glu Glu 35 40
45 Ser Glu Leu Trp Leu Arg Phe Lys Glu Leu Thr Asn Glu Met Ile Val
50 55 60 Thr Lys Asn Gly Arg Arg Met Phe Pro Val Leu Lys Val Asn
Val Ser 65 70 75 80 Gly Leu Asp Pro Asn Ala Met Tyr Ser Phe Leu Leu
Asp Phe Val Thr 85 90 95 Ala Asp Asn His Arg Trp Lys Tyr Val Asn
Gly Glu Trp Val Pro Gly 100 105 110 Gly Lys Pro Glu Pro Gln Ala Pro
Ser Cys Val Tyr Ile His Pro Asp 115 120 125 Ser Pro Asn Phe Gly Ala
His Trp Met Lys Ala Pro Val Ser Phe Ser 130 135 140 Lys Val Lys Leu
Thr Asn Lys Leu Asn Gly Gly Gly Gln Ile Met Leu 145 150 155 160 Asn
Ser Leu His Lys Tyr Glu Pro Arg Ile His Ile Val Arg Val Gly 165 170
175 Gly Pro Gln Arg Met Ile Thr Ser His Cys Phe Pro Glu Thr Gln Phe
180 185 190 Ile Ala Val Thr Ala Tyr Gln Asn Glu Glu Ile Thr Ala Leu
Lys Ile 195 200 205 Lys Tyr Asn Pro Phe Ala Lys Ala Phe Leu Asp Ala
Lys Glu Arg Asn 210 215 220 Asp His Lys Asp Val Met Glu Glu Pro Gly
Asp Cys Gln Gln Pro Gly 225 230 235 240 Tyr Ser Gln Trp Gly Trp Leu
Val Pro Gly Ala Gly Thr Leu Cys Pro 245 250 255 Pro Ala Ser Ser His
Pro Gln Phe Gly Gly Ser Leu Ser Leu Pro Ser 260 265 270 Thr His Gly
Cys Glu Arg Tyr Pro Ala Leu Arg Asn His Arg Ser Ser 275 280 285 Pro
Tyr Pro Ser Pro Tyr Ala His Arg Asn Ser Ser Pro Thr Tyr Ala 290 295
300 Asp Asn Ser Ser Ala Cys Leu Ser Met Leu Gln Ser His Asp Asn Trp
305 310 315 320 Ser Ser Leu Gly Val Pro Gly His Thr Ser Met Leu Pro
Val Ser His 325 330 335 Asn Ala Ser Pro Pro Thr Gly Ser Ser Gln Tyr
Pro Ser Leu Trp Ser 340 345 350 Val Ser Asn Gly Thr Ile Thr Pro Gly
Ser Gln Thr Ala Gly Val Ser 355 360 365 Asn Gly Leu Gly Ala Gln Phe
Phe Arg Gly Ser Pro Ala His Tyr Thr 370 375 380 Pro Leu Thr His Thr
Val Ser Ala Ala Thr Ser Ser Ser Ser Gly Ser 385 390 395 400 Pro Met
Tyr Glu Gly Ala Ala Thr Val Thr Asp Ile Ser Asp Ser Gln 405 410 415
Tyr Asp Thr Ala Gln Ser Leu Leu Ile Ala Ser Trp Thr Pro Val Ser 420
425 430 Pro Pro Ser Met 435 42046DNAMus musculus 4ggctccgcag
agtgaccctt tttcttggaa aagcggtggc gagagaagtg aaggtggctg 60ttgggtaggg
agtcaagact cctggaaggt ggagagggtg gcgggaggat gagctcgccg
120ggcacagaga gcgcagggaa gagcctgcag taccgagtgg accacctgct
cagcgccgtg 180gagagcgagc tgcaggcggg cagcgagaag ggagacccca
ccgaacgcga actgcgagtg 240ggcctggagg agagcgagct gtggctgcgc
ttcaaggagc taactaacga gatgattgtg 300accaagaacg gcaggaggat
gttcccggtg ctgaaggtaa atgtgtcagg cctggacccc 360aatgccatgt
actctttctt gctggacttc gtgacggctg acaaccaccg ctggaaatat
420gtgaacgggg agtgggtacc tgggggcaaa ccagagcctc aggcgcccag
ctgcgtctac 480atccacccag actcgcccaa ttttggggcc cactggatga
aggcgcctgt gtctttcagc 540aaagtcaaac tcaccaacaa gctcaatgga
gggggacaga tcatgttaaa ctccttgcat 600aagtatgaac ctcggattca
catcgtgaga gttgggggcc cgcaacgcat gatcaccagc 660cactgctttc
ccgagaccca gttcatagct gtgactgcct accagaatga ggagattaca
720gcccttaaaa ttaaatacaa cccatttgct aaagccttcc ttgatgccaa
agaaagaaac 780gaccacaaag atgtaatgga ggaaccgggg gactgccagc
agccggggta ttcccaatgg 840gggtggcttg ttcctggtgc tggcaccctc
tgcccgcctg ccagctccca ccctcagttt 900ggaggctcgc tctctctccc
ctccacacac ggctgtgaga ggtacccagc tctaaggaac 960caccggtcat
cgccctaccc cagcccctat gctcatcgga acagctctcc aacctatgcg
1020gacaattcat ctgcttgtct gtccatgctg cagtcccatg ataactggtc
tagcctcgga 1080gtgcctggcc acaccagcat gctgcctgtg agtcataacg
ccagcccacc tactggctct 1140agccagtatc ccagtctctg gtctgtgagc
aatggtacca tcaccccagg ctcccagaca 1200gctggggtgt ccaacgggct
gggagctcag ttctttcgag gctcccctgc acattacaca 1260ccactgacgc
acacggtctc agctgccacg tcctcgtctt ctggttctcc gatgtatgaa
1320ggggctgcta cagtcacaga catttctgac agccagtatg acacggccca
aagcctcctc 1380atagcctcgt ggacacctgt gtcaccccca tctatgtgaa
ttgaactttc ctccatgtgc 1440tgagacttgt aacaaccggt gtcaactgga
tcttctaggc tcaaagtggc aggctcttgg 1500gacaagggaa aaataaataa
ataaaagcta gatactaaca actccatttt caaataagag 1560caataataca
tgtcctataa tcatgttcta cagcctcttg tttgatacct acagtagtga
1620tatgtgtcct acattatgaa gccaaggaca gagagacggc tgtggtccag
ttttttgtga 1680ctggcagtta atcagagtcc tttgctaggt agggtcctat
atcttgtgtt tctctacaac 1740atatatgtga ctttgaaatc ctggaattcg
tccaccccct gtcctacttt agtgagacac 1800aaggtacacc tctaatgtcc
tcccttgttg ccttagagta gttaactttg aggacagaaa 1860aaagcatagc
cagaagattg taactgaacc gtcaactgtt ctgcccttgg aacatgccta
1920ctttaagcac acgtagcttt ttgtgttggg aagtcaactg tatggatact
tttctgttga 1980caaagtagcc aaagacaatc tgcagaaagt gttttctgca
caataaaggc aatatatagc 2040acctgg 2046515PRTArtificial
SequenceSynthetic 5Arg Pro Met Phe Pro Val Leu Lys Val Asn Val Ser
Gly Leu Asp 1 5 10 15 615PRTArtificial SequenceSynthetic 6Gln Trp
Gly Trp Leu Leu Pro Gly Thr Ser Thr Leu Cys Pro Pro 1 5 10 15
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