U.S. patent application number 14/138629 was filed with the patent office on 2014-05-15 for brachyury polypeptides and methods for use.
This patent application is currently assigned to The United States of America, as represented by he Secretary, Department of Health and Human Servi. 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 Andrei P. Kozlov, Claudia M. Palena, Jeffrey Schlom, Kwong-Yok Tsang.
Application Number | 20140135488 14/138629 |
Document ID | / |
Family ID | 39650527 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140135488 |
Kind Code |
A1 |
Schlom; Jeffrey ; et
al. |
May 15, 2014 |
BRACHYURY POLYPEPTIDES AND METHODS FOR USE
Abstract
It is disclosed herein that Brachyury is expressed in human
tumors, specifically in tumors of the small intestine, stomach,
kidney, bladder, uterus, ovary, and testes, as well as in lung,
colon and prostate carcinomas. Immunogenic Brachyury polypeptides
are disclosed herein. These polypeptides can be used in diagnostic
assays for Brachyury expression, as well as for inducing an immune
response to Brachyury. Polynucleotides encoding the immunogenic
Brachyury polypeptides, vectors including these polypeptides, host
cells transformed with these vectors, and methods of using these
polypeptides, polynucleotides, vectors, and host cells are
provided. Methods of diagnosing a Brachyury-expressing cancer are
also provided. Exemplary cancers include small lung, colon,
intestine, stomach, kidney, bladder, uterus, ovary, and testes and
prostate cancers. Methods of treating cancer are also
disclosed.
Inventors: |
Schlom; Jeffrey; (Potomac,
MD) ; Palena; Claudia M.; (Potomac, MD) ;
Kozlov; Andrei P.; (St. Petersburg, RU) ; Tsang;
Kwong-Yok; (Bethesda, 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 he Secretary, Department of Health and Human
Servi
Bethesda
MD
|
Family ID: |
39650527 |
Appl. No.: |
14/138629 |
Filed: |
December 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13460587 |
Apr 30, 2012 |
8613933 |
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14138629 |
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12528796 |
Aug 26, 2009 |
8188214 |
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PCT/US2008/055185 |
Feb 27, 2008 |
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13460587 |
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60904236 |
Feb 28, 2007 |
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Current U.S.
Class: |
536/24.5 |
Current CPC
Class: |
A61P 35/04 20180101;
C07K 14/4702 20130101; A61P 43/00 20180101; A61P 35/00 20180101;
G01N 33/56972 20130101; G01N 2333/70517 20130101; A61K 2039/53
20130101; C12N 15/113 20130101; A61P 37/04 20180101; Y02A 50/30
20180101; A61K 39/00 20130101; C07K 14/82 20130101; G01N 33/574
20130101; A61P 35/02 20180101; Y02A 50/466 20180101 |
Class at
Publication: |
536/24.5 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1.-21. (canceled)
22. An antisense molecule or an siRNA that specifically binds a
nucleotide sequence encoding SEQ ID NO: 1 for use in a method of
inhibiting growth or metastasis of a tumor in a subject, comprising
selecting a subject having the tumor; and administering a
therapeutically effective amount of the antisense molecule or siRNA
to the subject having the tumor, thereby inhibiting growth or
metastasis of the tumor in the subject, wherein the cancer cell is
from a cancer of the small intestine, stomach cancer, kidney
cancer, bladder cancer, uterine cancer, ovarian cancer, testicular
cancer, lung cancer, breast cancer, bronchial tube cancer, colon
cancer, prostate cancer, or a B cell tumor.
23. The antisense molecule of claim 22, wherein the cancer cell is
from a B cell tumour, wherein the B cell tumor is chronic
lymphocytic leukemia.
24. The antisense molecule or siRNA of claim 22, comprising
administering to the subject a therapeutically effective amount of
an siRNA.
25. The antisense molecule or siRNA of claim 22, further comprising
administering to the subject a therapeutically effective amount of
an isolated polypeptide comprising at most twelve consecutive amino
acids, wherein the isolated polypeptide comprises the amino acid
sequence set forth as one of: (a) WLLPGTSTX.sub.1 (SEQ ID NO: 3),
wherein X.sub.1 is a leucine (L) or a valine (V); (b)
SX.sub.2YX.sub.3SLX.sub.4SX.sub.5 (SEQ ID NO: 18), wherein X.sub.2
and X.sub.5 are either a valine or a leucine, wherein X.sub.3 is
proline (P), serine (S), threonine (T), leucine (L), or valine (V)
and wherein X.sub.4 is tryptophan (W), valine (V), leucine (L),
isoleucine (I), serine (S) or threorine (T); (c)
WLLX.sub.6GTSTX.sub.7 (SEQ ID NO: 19), wherein X.sub.6 is serine
(S), threonine (T), isoleucine (I), valine (V) and wherein X.sub.7
is leucine (L) or valine; (d) X.sub.8LIASTTPV (SEQ ID NO: 20,
wherein X.sub.8 is one of tyrosine (Y) or tryptophan (W); (e)
X.sub.9LIASX.sub.10TPV (SEQ ID NO: 21), wherein X.sub.9 is an
arginine (R), tyrosine (Y) or tryptophan (W) and X.sub.10 is a
valine (V), lysine (L), isoleucine (I), serine (S) or threonine
(T); or (f) ALYSFLLDFV (SEQ ID NO: 22).
Description
PRIORITY CLAIM
[0001] This is a continuation of U.S. patent application Ser. No.
12/528,796, filed Aug. 26, 2009, which is the U.S. national stage
of PCT Application No. PCT/US2008/055185, filed Feb. 27, 2008,
which was published in English under PCT Article 21(2), which in
turn claims the benefit of U.S. Provisional Application No.
60/904,236, filed Feb. 28, 2007. All of the prior applications are
incorporated herein by reference in their entirety.
FIELD
[0002] This application relates to the field of cancer
therapeutics, specifically to molecules such as immunogenic
peptides and inhibitory nucleic acids for the treatment of
cancer.
BACKGROUND
[0003] Brachyury (also known as "T") was identified in mice as a
dominant short tail mutant that is also a recessive lethal;
homozygous T/T embryos die in mid-gestation due to a failure of
posterior mesoderm formation (Chesley, J. Exp. Zool., 70: 429-459,
1935). The murine Brachyury gene has been cloned (Herrmann et al.,
Nature (Lond.), 343: 617-622, 1990), as well as the homologs in
other species, such as humans. The expression of the human
homologue of the mouse Brachyury was detected by RT-PCR in the
notochord remnant, the nucleus pulposus, of human abortuses at
14-15 weeks gestation (Edwards et al., Genome Res., 6: 226-233,
1996).
[0004] Brachyury has generally proved a valuable marker for
recognition of mesodennal differentiation (Herrmann et al., Trends
Genet., 10: 280-286, 1994). For example, apart from expression in
embryos themselves, Brachyury has been reported to be activated
during the differentiation of certain murine EC and ES cell lines
differentiating along mesodennal lineages in vitro (see, for
example, Bain et al., Biochem. Biophys. Res. Commun., 223: 691-694,
1996). In humans, Brachyury has been shown to be expressed in
teratocarcinomas (Gokhele et al., Cell Growth and Differentiation
11:157-62, 2000), chordomas (Vujovic et al., J. Pathol. 2: 157-65,
2006) and hemagioblastomas (Glasker et al., Cancer Res. 66:
4167-4172, 2006).
[0005] Immunotherapy involves evoking an immune response against
cancer cells based on their production of target antigens.
Immunotherapy based on cell-mediated immune responses involves
generating a cell-mediated response to cells that produce
particular antigenic determinants, while immunotherapy based on
humoral immune responses involves generating specific antibodies to
cells that produce particular antigenic determinants.
[0006] Recent studies show that immunotherapy of cancer patients
may be dramatically improved by the finding that CD8.sup.+ CTLs
recognize and kill tumor cells that display peptides from
tumor-associated antigens within MHC Class I molecules. In clinical
studies it has been found that effector CD8.sup.+ T cells play a
major role in tumor regression. For example, several tumor antigens
in prostate cancer models have been identified and HLA
allele-specific peptides from those prostate cancer-associated
antigens have been identified as CD8.sup.+ T cell epitopes. For
example, HLA-A2.1 binding peptides were described that were derived
from prostate specific antigen (PSA) (Correale et al., J Immunol
161:3186, 1998), prostate-specific membrane antigen (PSMA) (Tjoa et
al., Prostate 28:65, 1996), prostate stem cell antigen (PSCA)
(Kiessling et al., Int J Cancer 102:390, 2002), and prostate acid
phosphatase (Peshwa et al., Prostate 36:129, 1998). For PSA,
clinical trials are in progress using different vaccine strategies.
However, there clearly is a need to identify additional antigens to
aid in the diagnosis of cancers of different organs, and to produce
peptides that can be used for immunotherapy of other types of
cancer.
SUMMARY
[0007] It is disclosed herein that Brachyury is expressed in human
tumors, specifically in tumors of the small intestine, stomach,
kidney, bladder, uterus, ovary, and testes, as well as in lung,
colon and prostate carcinomas. Immunogenic Brachyury polypeptides
are disclosed herein. These Brachyury polypeptides can be used for
inducing an immune response to Brachyury, as well as in diagnostic
assays for Brachyury expression. In one example, the polypeptide is
at most twelve consecutive amino acids in length, wherein the
isolated polypeptide comprises the amino acid sequence set forth as
WLLPGTSTX.sub.1 (SEQ ID NO: 3), wherein X.sub.1 is a leucine (L) or
a valine (V).
[0008] Polynucleotides encoding the immunogenic Brachyury
polypeptides, vectors including these polypeptides, host cells
transformed with these vectors, and methods of using these
polypeptides, polynucleotides, vectors, and host cells are provided
herein. In one embodiment, a composition is disclosed that includes
a first recombinant virus which has incorporated into a viral
genome or infectable portion thereof a nucleic acid encoding the
immunogenic Brachyury polypeptide and a second recombinant 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 co-expression of the polypeptide and the B7-1,
B7-2, or B7-1 and B7-2 encoding genes or DNA sequences.
[0009] Methods of diagnosing a Brachyury-expressing cancer are also
provided, that include the use of the disclosed immunogenic
Brachyury polypeptides, nucleic acids encoding these polypeptides,
or antibodies that specifically bind these polypeptides. Exemplary
cancers include lung, colon, small intestine, stomach, kidney,
bladder, uterus, ovary, and testes and prostate cancers.
[0010] Methods of inducing an immune response to Brachyury are also
disclosed. The methods include the use of the immunogenic Brachyury
polypeptides disclosed herein, nucleic acids encode these
polypeptides, and/or viral vectors encoding an immunogenic
Brachyury polypeptide, alone or in conjunction with other agents,
such as B7-1, B7-2, and/or a cytokine and/or with traditional
cancer therapies, such as surgery, radiation therapy and/or
chemotherapy. Methods are disclosed for treating a subject having a
tumor, such as, but not limited to, a small intestine, stomach,
kidney bladder, uterus, ovary, testes, lung, colon or prostate
tumor. Methods are also disclosed for treating a subject having a
breast tumor, bronchial tube tumor, chronic lymphocytic leukemia
(CLL) and other B cell-based malignancies. These methods include
inducing an immune response to Brachyury and/or using an inhibitory
nucleic acid, such as an siRNA or antisense molecule, to decrease
Brachyury expression in order to treat the tumor.
[0011] 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 FIGURES
[0012] FIGS. 1A-1C are a set of digital images of RT-PCR analysis
of Brachyury expression in human normal and tumor tissues. FIG. 1A
is a digital image of RT-PCR results from human multiple tissues
cDNA panels I and II. FIG. 1B is a digital image of RT-PCR results
from various human blood fraction cDNAs. FIG. 1C is a digital image
of results from RT-PCR of cDNA from tumor tissues (each tissue from
an individual cancer patient) that were amplified for expression of
Brachyury (upper panel) and GAPDH (bottom panel). Human DNA was
used as a positive control for the PCR reaction; water was added to
the tubes labeled as negative control.
[0013] FIGS. 2A-2B are graphs illustrating the binding of predicted
peptides to HLA-A0201 molecules. FIG. 2A is a bar graph of results
wherein peptides at 25 .mu.M were analyzed for binding to T2 cells;
a positive control (CAP1-6D) and an HLA-A3 binding peptide
(negative control) were used at the same concentration. MFI
indicates mean fluorescence intensity. FIG. 2B is a line graph of
results showing an analysis of half-life of peptide-MHC complexes
that was conducted as described in Materials and Methods. For each
peptide and the positive control CAP1-6D, half-life time is given
in minutes.
[0014] FIG. 3A-3C are graphs showing cytokine production and
cytotoxic activity of CTLs specific for three Brachyury derived
peptides. FIG. 3A is a bar graph showing results obtained when CD8+
T cells generated from PBMC of a healthy donor against peptides
T-p2, T-p3, and T-p4 were stimulated for 24 hours in the presence
of Brachyury (T)-specific peptides or irrelevant peptide-pulsed
autologous DCs. IFN-.gamma. was evaluated in the supernatants by
ELISA. FIG. 3B is a line graph showing cytotoxic activity (6-hour
assay) of CTLs generated with peptides T-p2 and T-p3 against
peptide-pulsed C1R-A2 targets. Two effector-to-target ratios (E:T)
were used as indicated. C1R-A2 cells were pulsed with 25 .mu.M of
T-p2 peptide (closed circles), T-p3 peptide (open circles),
irrelevant CAP1-6D peptide (close triangles), and without peptide
(open triangles). FIG. 3C is a line graph of results obtained when
T2 cells were pulsed with various concentrations of T-p2 peptide as
indicated and used as targets with T-p2 CTLs (at an
effector-to-targets ratio equal to 12.5:1).
[0015] FIGS. 4A-4F are graphs showing the Cytotoxic activity of
Brachyury-specific CTLs against tumor targets. FIG. 4A is a line
graph of results obtained when T-p2 CTLs from a normal donor were
used as effectors against various tumor targets in an .sup.111In
16-hour release assay, as indicated. FIG. 4B is a bar graph of
results obtained when .sup.111In-labeled H441 tumor cells were
incubated with 25 .mu.g/ml of a control IgG, anti-HLA-class I, or
anti-HLA-class II MAb for 1 hour before the addition of T-p2 T
cells. The E:T ratio was 20:1. FIG. 4C is a line graph of results
obtained when CTLs established from the blood of a colorectal
cancer patient (patient 1) and (FIG. 4D) an ovarian cancer patient
(patient 2) were used after three IVS for cytotoxic killing of H441
and AsPC-1 tumor cells. FIG. 4E is a bar graph showing cytotoxic
killing of LNCAP tumor cells by T-p2 T cells derived from patient
1. .sup.111In-labeled LNCAP tumor cells were incubated with 25
.mu.g/ml of a control IgG or an anti-HLA-A2,28 MAb for 1 hour
before the addition of T-p2 T cells. FIG. 4F is a bar graph and a
digital image of results obtained when T cells derived from patient
2 were used as effectors against various tumor targets, as
indicated. Shown in the digital image is the expression of
Brachyury and .beta.-actin mRNA by RT-PCR in each tumor cell
line.
[0016] FIGS. 5A-5B are digital images showing that stable knockdown
of Brachyury expression induces a mesenchymal-to-epithelial
transition in NCI-H460 lung carcinoma cancer cells. FIG. 5A is a
digital image of an RT-PCR analysis of Brachyury and .beta.-actin
mRNA expression in NCI-H460 lung carcinoma cells stably transfected
with a control shRNA or a Brachyury-specific shRNA construct
(Br.shRNA clones 1 and 2). FIG. 5B is a digital image of the
results obtained when the same cell lines were analyzed by western
blot for expression of human fibronectin, vimentin,
.gamma.-catenin, and .beta.-actin.
[0017] FIGS. 6A-6B are digital images and bar graphs showing that
the loss of Brachyury impairs the migratory and invasive properties
of NCI-H460 lung carcinoma cells in vitro. In vitro assay of (FIG.
6A) cell migration and (FIG. 6B) cell invasion in NCI-H460 lung
carcinoma cells stably transfected with a control shRNA or a
Brachyury-specific shRNA construct (Br.shRNA clone 2). Experiments
(n=3) were conducted in triplicate samples of each cell line as
described in the Examples section. The graph shows results from one
representative experiment. Each bar represents the results for an
individual replicate assay.+-.SEM. Representative images of the
bottom side of the filters for each cell line under .times.10
magnification are also shown. Statistical analysis of Student's
t-test was performed.
SEQUENCE LISTING
[0018] The Sequence Listing is submitted as an ASCII text file
[4239-77527-10_Sequence_Listing.txt, Apr. 25, 2012, 11.6 KB], which
is incorporated by reference herein.
[0019] 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:
[0020] SEQ ID NO: 1 is an exemplary amino acid sequence for a
Brachyury protein.
[0021] SEQ ID NO: 2 is an exemplary nucleic acid sequence encoding
a Brachyury polypeptide.
[0022] SEQ ID NO: 3 is the amino acid sequence of an immunogenic
Brachyury polypeptide.
[0023] SEQ ID NOS: 4-11 are the nucleic acid sequences of
primers.
[0024] SEQ ID NO: 12 is the amino acid sequence of a
carcinoembryonic antigen (CEA) polypeptide.
[0025] SEQ ID NO: 13 is the amino acid sequence of a human
immunodeficiency virus (HIV) polypeptide.
[0026] SEQ ID NOS: 14-22 are the amino acid sequence of exemplary
Brachyury polypeptides.
DETAILED DESCRIPTION
[0027] It is disclosed herein that Brachyury is expressed in human
tumors, specifically in tumors of the small intestine, stomach,
kidney, bladder, uterus, ovary, and testes, as well as in lung,
colon and prostate carcinomas. Brachyury is also expressed in
chronic lymphocytic leukemia and other B cell malignancies.
Immunogenic Brachyury polypeptides are disclosed herein. Nucleic
acids encoding these polypeptides, vectors including these nucleic
acids, and host cells transformed with the vectors are also
disclosed. Methods for inducing an immune response to a tumor cell
expressing Brachyury are also disclosed, as are methods for
detecting a tumor that expresses Brachyury. Methods for treatment
are also disclosed herein for the treatment of a tumor that
expresses Brachyury, wherein the method includes administering an
inhibitory nucleic acid.
TERMS
[0028] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common teens 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).
[0029] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of specific terms are
provided, along with particular examples:
[0030] 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. No. 6,194,388; U.S. Pat. No. 6,207,646; U.S. Pat. No.
6,214,806; U.S. Pat. No. 6,218,371; U.S. Pat. No. 6,239,116; U.S.
Pat. No. 6,339,068; U.S. Pat. No. 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 41 BBL.
[0031] 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. Methods of
determining spatial conformation of epitopes include, for example,
x-ray crystallography and 2-dimensional nuclear magnetic
resonance.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.HI
domains; (ii) an F.sub.d fragment consisting of the V.sub.H and
C.sub.HI 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').sub.2 fragment, a bivalent fragment comprising
two Fab fragments linked by a disulfide bridge at the hinge
region.
[0036] Immunoglobulins and certain variants thereof are known and
many have been prepared in recombinant cell culture (e.g., see U.S.
Pat. No. 4,745,055; U.S. Pat. No. 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.
[0037] 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.
[0038] Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA)
has two strands, a 5'->3' strand, referred to as the plus
strand, and a 3'->5' strand (the reverse complement), referred
to as the minus strand. Because RNA polymerase adds nucleic acids
in a 5'->3' direction, the minus strand of the DNA serves as the
template for the RNA during transcription. Thus, the RNA formed
will have a sequence complementary to the minus strand and
identical to the plus strand (except that U is substituted for
T).
[0039] Antisense molecules are molecules that are specifically
hybridizable or specifically complementary to either RNA or the
plus strand of DNA. Sense molecules are molecules that are
specifically hybridizable or specifically complementary to the
minus strand of DNA. Antigene molecules are either antisense or
sense molecules directed to a dsDNA target.
[0040] 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 mesodeim in vertebrates (see, for example, Kispert and Hellmann,
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 Tbx2 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 previously reported in human
teratocarcinoma lines: a subset of germ cell tumors,
teratocarcinomas are embryonal carcinoma cells with competence for
mesoderm differentiation (Fan et al., Cancer Res 64(15):5132-9,
2004), as well as in 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.sub.--003172 and GENBANK.RTM. Accession No. NM.sub.--003181, as
available on Feb. 23, 2007, incorporated herein by reference
[0041] Cancer or Tumor: A malignant neoplasm that has undergone
characteristic anaplasia with loss of differentiation, increase
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.
[0042] 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.
[0043] 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-00001 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
The term conservative variation 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.
Non-conservative substitutions are those that reduce an activity or
antigenicity.
[0044] 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.
[0045] 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.
[0046] Chemotherapy; 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 neoplasms
such as solid tumors. In one embodiment, a chemotherapeutic agent
is a radioactive molecule. 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).
The immunogenic Brachyury polypeptides disclosed herein can be used
in conjunction with additional chemotherapeutic agents.
[0047] Consists Essentially Of/Consists Of: With regard to a
polypeptide, a polypeptide that consists essentially of a specified
amino acid sequence if it does not include any additional amino
acid residues. However, the polypeptide 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, a polypeptide 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.
[0048] 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.
[0049] 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 polypeptide
encoded by the nucleotide sequence is unchanged.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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 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 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.
[0054] Expression Control Sequences: Nucleic acid sequences that
regulate the expression of a heterologous nucleic acid sequence to
which it is 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.
[0055] 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 produced
by recombinant DNA or synthetic techniques can also be used to
provide for transcription of the nucleic acid sequences.
[0056] 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 one
specific, non-limiting example, a polypeptide comprising nine
consecutive amino acids from Brachyury, or at most 12 consecutive
amino acids from Brachyury, and 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 will not specifically bind to a heterologous
protein.
[0057] 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.
[0058] 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.
[0059] Immunogenic peptide: A peptide which comprises an
allele-specific motif or other sequence such that the peptide will
bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL")
response, or a B cell response (e.g. antibody production) against
the antigen from which the immunogenic peptide is derived.
[0060] 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.
[0061] Immunogenic peptides can also be identified by measuring
their binding to a specific MHC protein (e.g. HLA-A02.01) 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.
[0062] In one example, an immunogenic "Brachyury peptide" 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 disclosed herein that are 9 or 10 amino acid
residues in length, or at most 12 amino acids in length. Generally,
immunogenic Brachyury polypeptide can be used to induce an immune
response in a subject, such as a B cell response or a T cell
response. In one example, an immunogenic Brachyury polypeptide,
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 known in the art, see U.S.
Pat. No. 5,662,907, which is incorporated herein by reference. In
one example, an immunogenic peptide includes an allele-specific
motif or other sequence such that the peptide will bind an MHC
molecule and induce a cytotoxic T lymphocyte ("CTL") response
against the antigen from which the immunogenic peptide is
derived.
[0063] Immunogenic composition: A composition comprising an
immunogenic Brachyury polypeptide or a nucleic acid encoding the
immunogenic Brachyury polypeptide that induces a measurable CTL
response against cells expressing Brachyury polypeptide, or induces
a measurable B cell response (such as production of antibodies that
specifically bind Brachyury) against a Brachyury polypeptide. For
in vitro use, the immunogenic composition can consist of the
isolated nucleic acid, vector including the nucleic acid/or
immunogenic peptide. For in vivo use, the immunogenic composition
will typically comprise the nucleic acid, vector including the
nucleic acid, and or immunogenic polypeptide, 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. A
Brachyury polypeptide, or nucleic acid encoding the polypeptide,
can be readily tested for its ability to induce a CTL by
art-recognized assays.
[0064] Inhibiting or treating a disease: Inhibiting a disease, such
as tumor growth, refers to inhibiting the full development of a
disease. In several examples, inhibiting a disease refers to
lessening symptoms of a tumor, 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 tumor or reducing
tumor 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 tumor.
[0065] 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.
[0066] 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.
[0067] 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 epitopes disclosed herein
to provide rotational freedom to the linked polypeptide domains and
thereby to promote proper domain folding and presentation to the
MHC. By way of example, in a recombinant polypeptide comprising two
Brachyury domains, linker sequences can be provided between them,
such as a polypeptide comprising Brachyury
polypeptide-linker-Brachyury polypeptide. 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, the
glycine(4)-serine spacer (GGGGS.times.3) described by Chaudhary et
al., Nature 339:394-397, 1989.
[0068] 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.
[0069] Major Histocompatibility Complex (MHC): A generic
designation meant to encompass the histocompatability antigen
systems described in different species, including the human
leukocyte antigens ("HLA").
[0070] Mammal: This term includes both human and non-human mammals.
Similarly, the term "subject" includes both human and veterinary
subjects.
[0071] Neoplasm: An abnormal cellular proliferation, which includes
benign and malignant tumors, as well as other proliferative
disorders.
[0072] Oligonucleotide: A linear polynucleotide sequence of up to
about 100 nucleotide bases in length.
[0073] 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.
[0074] 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 polypeptide. Generally, operably
linked DNA sequences are contiguous and, where necessary to join
two protein-coding regions, in the same reading frame.
[0075] Peptide Modifications: Brachyury epitopes 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
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.
[0076] Peptides 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.
[0077] 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 the peptides of this invention
to select and provide conformational constraints to the structure
that result in enhanced stability.
[0078] 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 an immunogenic Brachyury polypeptide 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 Klegennan & 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.
[0079] 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.
[0080] 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.
[0081] 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 necessary to
induce an immune response, inhibit tumor growth, reduce tumor
volume or to measurably alter outward symptoms of the tumor. 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.
[0082] 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.
[0083] Polypeptide: Any chain of amino acids, regardless of length
or post-translational modification (e.g., glycosylation or
phosphorylation). In one embodiment, the polypeptide is a Brachyury
polypeptide. A polypeptide can be between 3 and 30 amino acids in
length. In one embodiment, a polypeptide is from about 7 to about
25 amino acids in length. In yet another embodiment, a polypeptide
is from about 8 to about 12 amino acids in length. In yet another
embodiment, a peptide is about 9 amino acids in length. With regard
to polypeptides, the word "about" indicates integer amounts. Thus,
in one example, a polypeptide "about" 9 amino acids in length is
from 8 to 10 amino acids in length.
[0084] 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.
[0085] Purified: The epitopes of Brachyury 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 or cellular components. 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 90% free of other proteins or cellular components.
[0086] Thus, the term purified does not require absolute purity;
rather, it is intended as a relative term. For example, a purified
nucleic acid is one in which the nucleic acid is more enriched than
the nucleic acid in its natural environment within a cell. 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.
[0087] 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.
[0088] Selectively hybridize: Hybridization under moderately or
highly stringent conditions that excludes non-related nucleotide
sequences.
[0089] 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.
[0090] A specific example of progressively higher stringency
conditions is as follows: 2.times.SC/0.1% SDS at about room
temperature (hybridization conditions); 0.2.times.SC/0.1% SDS at
about room temperature (low stringency conditions);
0.2.times.SC/0.1% SDS at about 42.degree. C. (moderate stringency
conditions); and 0.1.times.SC 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.
[0091] 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
polypeptide will possess a relatively high degree of sequence
identity when aligned using standard methods.
[0092] 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.
[0093] 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.
[0094] Homologs and variants of a Brachyury polypeptide are
typically characterized by possession of at least 75%, for example
at least 80%, 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.
[0095] Small interfering RNAs: Synthetic or naturally-produced
small double stranded RNAs (dsRNAs) that can induce gene-specific
inhibition of expression in invertebrate and vertebrate species are
provided. These RNAs are suitable for interference or inhibition of
expression of a target gene and comprise double stranded RNAs of
about 15 to about 40 nucleotides containing a 3' and/or 5' overhang
on each strand having a length of 0- to about 5-nucleotides,
wherein the sequence of the double stranded RNAs is essentially
identical to a portion of a coding region of the target gene for
which interference or inhibition of expression is desired. The
double stranded RNAs can be formed from complementary ssRNAs or
from a single stranded RNA that forms a hairpin or from expression
from a DNA vector.
[0096] 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 polypeptide. In
one embodiment, the specific binding agent is a monoclonal or
polyclonal antibody that specifically binds Brachyury.
[0097] T Cell: A white blood cell critical to the immune response.
T cells include, but are not limited to, CD4.sup.+ T cells and
CD8.sup.+ T cells. A CD4.sup.+ T lymphocyte is an immune cell that
carries a marker on its surface known as "cluster of
differentiation 4" (CD4). These cells, also known as 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. In one
embodiment, a CD8 T cell is a cytotoxic T lymphocyte. In another
embodiment, a CD8 cell is a suppressor T cell.
[0098] Therapeutically active polypeptide: An agent, such as an
epitope of Brachyury 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
epitope, wherein the nucleic acid sequence is operably linked to a
control element such as a promoter. Another example of a
therapeutically active molecule is an antisense molecule or a siRNA
for Brachyury.
[0099] In one embodiment, a therapeutically effective amount of a
composition, such as a Brachyury polypeptide, is an amount used to
generate an immune response, or to treat 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.
[0100] 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.
[0101] 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 viral
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.
[0102] 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. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. 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. The term "comprises"
means "includes." 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.
Immunogenic Brachyury Peptides
[0103] Brachyury (also known as "T-protein") is a polypeptide which
is transcribed in the mesoderm. In one embodiment, the polypeptide
has a sequence set forth as:
TABLE-US-00002 (SEQ ID NO: 1)
MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEES
ELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAAD
NHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVK
LTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVT
AYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWG
WLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPY
AHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTS
SSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSA
PSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSM
(see also GENBANK.RTM. Accession No NP.sub.--003172 and
GENBANK.RTM. Accession No. NM.sub.--003181, as available on Feb.
23, 2007, incorporated herein by reference).
[0104] In other embodiments, Brachyury has an amino acid sequence
at least 90% identical to SEQ ID NO: 1, for example a polypeptide
that has at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% sequence identity to SEQ ID NO: 1.
[0105] Using the genetic code, one of skill in the art can readily
produce a nucleic acid sequence encoding Brachyury. In one example,
Brachyury is encoded by a nucleic acid having a sequence set forth
as:
TABLE-US-00003 (SEQ ID NO: 1) 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 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
[0106] Immunogenic fragments of Brachyury (and Brachyury itself),
can be chemically synthesized by standard methods. If desired,
polypeptides can also be chemically synthesized by emerging
technologies. One such process is described in W. Lu et al.,
Federation of European Biochemical Societies Letters. 429:31-35,
1998. Polypeptides can also be produced using molecular genetic
techniques, such as by inserting a nucleic acid encoding Brachyury
or an epitope thereof into an expression vector, introducing the
expression vector into a host cell, and isolating the polypeptide
(see below).
[0107] Brachyury polypeptides are disclosed herein that can be used
to induce an immune response (are immunogenic). These peptides
comprise at most twelve amino acids, such as eleven, ten amino
acids, or nine consecutive amino acids of a Brachyury
polypeptide.
[0108] An isolated polypeptide is disclosed that includes at most
twelve consecutive amino acids from Brachyury, wherein the isolated
polypeptide comprises the amino acid sequence set forth as
WLLPGTSTX.sub.1 (SEQ ID NO: 3), wherein X.sub.1 is a leucine (L) or
a valine (V). In some embodiments, amino acid 1 (X.sub.1) is a
leucine. In additional embodiments, amino acid 1 (X.sub.1) is a
valine. In one example the polypeptide consists essentially of the
amino acid sequence set forth as SEQ ID NO: 3. Thus, in one
example, the polypeptide consists essentially of SEQ ID NO: 3,
wherein amino acid X.sub.1 is a valine (V), and in another example
the polypeptide consists essentially of SEQ ID NO: 3, wherein amino
acid X.sub.1 is a leucine. In additional examples, the polypeptide
is eleven amino acids in length or ten amino acids in length. In
further examples, the isolated polypeptide consists of the amino
acid sequence set forth as SEQ ID NO: 3.
[0109] In additional embodiments the isolated Brachyury polypeptide
is nine to twelve amino acids in length and comprises the amino
acid sequence set forth as SQYPSLWSV (SEQ ID NO: 14), WLLPGTSTL
(SEQ ID NO: 15), RLIASWTPV (SEQ ID NO: 16), or AMYSFLLDFV (SEQ ID
NO: 17). In several examples, the isolated Brachyury polypeptide is
nine or ten amino acids in length, and comprises one of the amino
acid sequences set forth as SEQ ID NOs: 14-17. In additional
examples, the isolated Brachyury polypeptide consists of the amino
acid sequence set forth as one of SEQ ID NO: 14-17.
[0110] In additional embodiments, the Brachyury polypeptide is nine
to twelve amino acids in length, and comprises the amino acid
sequence: SX.sub.2YX.sub.3SLX.sub.4SX.sub.5 (SEQ ID NO: 18),
wherein X.sub.2 and X.sub.5 are either a valine or a leucine,
wherein X.sub.3 is proline (P), serine (S), threonine (T), leucine
(L), or valine (V) and wherein X.sub.4 is tryptophan (W), valine
(V), leucine (L), isoleucine (I), serine (S) or threorine (T). In
further examples, the Brachyury polypeptide is nine, ten or eleven
amino acids in length, and comprises the amino acid sequence set
forth as SEQ ID NO: 18, wherein X.sub.5 is one of valine or a
leucine. In additional examples, the Brachyury polypeptide consists
of the amino acid sequence set forth as SEQ ID NO: 18 wherein
X.sub.5 is a valine or a leucine. The following exemplary Brachyury
polypeptides are encompassed by the present disclosure:
TABLE-US-00004 T-p1a: SLYPSLWSV (SEQ ID NO: 18, wherein X.sub.2 is
L, X.sub.3 is P, X.sub.4 is W and X.sub.5 is V) T-p1b: SLYPSLWSL
(SEQ ID NO: 18, wherein X.sub.2 is L, X.sub.3 is P, X.sub.4 is W
and X.sub.5 is L) T-p1c: SX.sub.2YSSLWSV (SEQ ID NO: 18, wherein
X.sub.2 is Q or L, X.sub.3 is Y, X.sub.4 is W and X.sub.5 is V)
T-p1d: SX.sub.2YTSLWSV (SEQ ID NO: 18, wherein X.sub.2 is Q or L,
X.sub.3 is T, X.sub.4 is W, and X.sub.5 is V) T-p1e:
SX.sub.2YLSLWSV (SEQ ID NO: 18, wherein X.sub.2 is Q or L, X.sub.3
is L, X.sub.4 is W and X.sub.5 is V) T-p1f: SX.sub.2YVSLWSV (SEQ ID
NO: 18, wherein X.sub.2 is Q or L, X.sub.3 is V, X.sub.4 is W and
X.sub.5 is V) T-p1g: SX.sub.2YPSLVSV (SEQ ID NO: 18, wherein
X.sub.2 is Q or L, X.sub.3 is P, X.sub.4 is V and X.sub.5 is V)
T-p1h: SX.sub.2YPSLLSV (SEQ ID NO: 18, wherein X.sub.2 is Q or L,
X.sub.3 is P, X.sub.4 is L and X.sub.5 is V) T-p1i: SX.sub.2YPSLISV
(SEQ ID NO: 18, wherein X.sub.2 is Q or L, X.sub.3 is P, X.sub.4 is
I and X.sub.5 is V) T-p1j: SX.sub.2YPSLSSV (SEQ ID NO: 18, wherein
X.sub.2 is Q or L, X.sub.3 is P, X.sub.4 is S and X.sub.5 is V)
T-p1k: SX.sub.2YPSLTSV (SEQ ID NO: 18, wherein X.sub.2 is Q or L,
X.sub.3 is P, X.sub.4 is T and X.sub.5 is V)
[0111] In further embodiments, the Brachyury polypeptide is nine to
twelve amino acids in length, and comprises the amino acid
sequence: WLLX.sub.6GTSTX.sub.7 (SEQ ID NO: 19), wherein X.sub.6 is
serine (S), threonine (T), isoleucine (I), valine (V) and wherein
X.sub.7 is leucine (L) or valine. In further examples, the
Brachyury polypeptide is nine or ten amino acids in length, and
comprises the amino acid sequence set forth as SEQ ID NO: 19,
wherein X.sub.7 is one of valine or a leucine. In additional
examples, the Brachyury polypeptide consists of the amino acid
sequence set forth as SEQ ID NO: 19 wherein X.sub.5-7 is a valine
or a leucine. The following exemplary polypeptides are encompassed
by this disclosure:
TABLE-US-00005 Tp2b: WLLSGTSTX.sub.7 (SEQ ID NO: 19, wherein
X.sub.6 is S, and X.sub.7 is L or V) Tp2c: WLLTGTSTX.sub.7 (SEQ ID
NO: 19, wherein X.sub.6 is T, and X.sub.7 is L or V) Tp2d:
WLLIGTSTX.sub.7 (SEQ ID NO: 19, wherein X.sub.6 is I, and X.sub.7
is L or V) Tp2e: WLLVGTSTX.sub.7 (SEQ ID NO: 19, wherein X.sub.6 is
V, and X.sub.7 is L or V)
[0112] In additional embodiments, the Brachyury polypeptide is nine
to twelve amino acids in length, and comprises the amino acid
sequence X.sub.8LIASTTPV (SEQ ID NO: 20), wherein X.sub.8 is
tyrosine (Y) or tryptophan (W). The Brachyury polypeptide can be
nine or ten amino acids in length, and comprises the amino acid
sequence set forth as X.sub.8LIASTTPV (SEQ ID NO: 20, wherein
X.sub.8 is one of tyrosine (Y) or tryptophan (W). In additional
examples, the Brachyury polypeptide consists of the amino acid
sequence set forth as SEQ ID NO: 20 wherein X.sub.8 is a tryptophan
or a tyrosine. Thus, the following polypeptides are encompassed by
the present disclosure:
TABLE-US-00006 T-p3a: YLIASWTPV (SEQ ID NO: 20, wherein X.sub.8 is
Y) T-p3b: WLIASWTPV (SEQ ID NO: 20, wherein X.sub.8 is W)
[0113] In another set of embodiments, the isolated Brachyury
polypeptide is nine to twelve amino acids in length, and comprises
the amino acid sequence: X.sub.9LIASX.sub.10TPV (SEQ ID NO: 21),
wherein X.sub.9 is an arginine (R), tyrosine (Y) or tryptophan (W)
and X.sub.10 is a valine (V), lysine (L), isoleucine (I), serine
(S) or threonine (T). In some examples, the isolated Brachyury
polypeptide is nine or ten amino acids in length, and comprises the
amino acid sequence set forth as X.sub.9LIASTTPV (SEQ ID NO: 21,
X.sub.9 is an arginine, tyrosine or tryptophan and X.sub.10 is a
valine, lysine, isoleucine, serine or threonine). In additional
examples, the Brachyury polypeptide consists of the amino acid
sequence set forth as SEQ ID NO: 21 wherein X.sub.9 is an arginine,
tyrosine or tryptophan and X.sub.10 is a valine, serine,
isoleucine, or threonine. Thus, the following polypeptides are
encompassed by the present disclosure:
TABLE-US-00007 Tp3c: X.sub.9LIASVTPV (SEQ ID NO: 21, wherein
X.sub.9 = R, Y, or W And X.sub.10 is V) Tp3d: X.sub.9LIASLTPV (SEQ
ID NO: 21, wherein X.sub.9 = R, Y, or W And X.sub.10 is V) Tp3e:
X.sub.9LIASITPV (SEQ ID NO: 21, wherein X.sub.9 = R, Y, or W And
X.sub.10 is V) Tp3f: X.sub.9LIASSTPV (SEQ ID NO: 21, wherein
X.sub.9 = R, Y, or W And X.sub.10 is V) Tp3g: X.sub.9LIASTTPV (SEQ
ID NO: 21, wherein X.sub.9 = R, Y, or W And X.sub.10 is V)
[0114] In an additional embodiment, the isolated Brachyury
polypeptide is ten to twelve amino acids in length, and comprises
the amino acid sequence ALYSFLLDFV (SEQ ID NO: 22, T-p4a). In some
examples, the isolated Brachyury polypeptide is ten or eleven amino
acids in length and comprises ALYSFLLDFV (SEQ ID NO: 22). In an
additional example, the isolated Brachyury polypeptide consists of
ALYSFLLDFV (SEQ ID NO: 22).
[0115] In several embodiments, the isolated Brachyury polypeptide
is include in a fusion protein. Thus, the fusion protein can
include the 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 polypeptide. In additional
embodiments, the protein consists of the Brachyury polypeptide.
Thus, a second heterologous moiety is non-covalently linked to the
Brachyury polypeptide. For example, the polypeptide can be nine or
ten acid amino acids in length, and consists of the sequence set
forth as one of SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,
SEQ ID NO: 21, SEQ ID NO: 22.
[0116] These above-described brachyury polypeptides are
immunogenic, and thus can be used to induce an immune response in a
subject. The Brachyury polypeptides disclosed herein do not include
all the additional consecutive amino acids of SEQ ID NO: 1. In one
embodiment, the polypeptide does not include amino acids 1-15 of
SEQ ID NO: 1.
[0117] Without being bound by theory, it is believed that the
presentation of peptides by MHC Class I molecules involves binding
to the cleft in an MHC Class I molecule through the anchor residues
of the peptide and ultimate presentation on the cell surface.
Depending upon the particular anchor residues, among other things,
certain peptides can bind more tightly to particular HLA molecules
than others. Peptides that bind well are usually "dominant"
epitopes, while those that bind less well are often "subdominant"
or "cryptic" epitopes. Dominant epitopes of either self proteins or
foreign proteins evoke strong tolerance or immune responses.
Subdominant or cryptic epitopes generate weak responses or no
responses at all. Without being bound by theory, tighter binding by
dominant epitopes to HLA molecules results in their denser
presentation on the cell surface, greater opportunity to react with
immune cells and greater likelihood of eliciting an immune response
or tolerance. MHC Class I molecules present epitopes from
endogenous proteins for presentation to CTL cells. HLA A, HLA B and
HLA C molecules bind peptides of about eight to ten amino acids in
length (such as nine amino acids in length) that have particular
anchoring residues. The anchoring residues recognized by an HLA
Class I molecule depend upon the particular allelic form of the HLA
molecule. A CD8+ T cell bears T cell receptors that recognize a
specific epitope when presented by a particular HLA molecule on a
cell. When a CTL precursor that has been stimulated by an antigen
presenting cell to become a cytotoxic T lymphocyte contacts a cell
that bears such an HLA-peptide complex, the CTL forms a conjugate
with the cell and destroys it. In several examples presented
herein, the polypeptides that are disclosed bind and are presented
by HLA-A2.1.
[0118] In several examples, the Brachyury polypeptide can be
repeated in series, such that the polypeptide includes several
copies of the immunogenic Brachyury polypeptide. However, only one
copy of the Brachyury polypeptide can be included in an immunogenic
molecule. In several examples, two, three, four, five copies of the
Brachyury polypeptide are included in an immunogenic molecule. The
copies of the Brachyury polypeptide can be separated by peptide
linkers.
[0119] In additional examples, the polypeptide can be a fusion
protein and can also include heterologous sequences to Brachyury
(such as amino acid sequences of at least nine amino acids in
length that are not included in SEQ ID NO: 1). Thus, in several
specific non-limiting examples, the immunogenic peptide is a fusion
polypeptide, for example the polypeptide includes six sequential
histidine residues, a .beta.-galactosidase amino acid sequence, or
an immunoglobulin amino acid sequence. The polypeptide can also be
covalently linked to a carrier. 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.
[0120] As noted above, the fusion polypeptide can optionally
include repetitions of one or more of the Brachyury polypeptides
disclosed herein. In one specific, non-limiting example, the
polypeptide includes two, three, four, five, or up to ten
repetitions of one of a Brachyury polypeptide. A linker sequence
can optionally be included between the Brachyury polypeptides. In
all of these examples, the polypeptide does not include the
full-length Brachyury amino acid sequence, such as the amino acid
sequence set forth as SEQ ID NO: 1.
[0121] The Brachyury polypeptides disclosed herein can be
chemically synthesized by standard methods, or can be produced
recombinantly. An exemplary process for polypeptide production is
described in Lu et al., Federation of European Biochemical
Societies Letters. 429:31-35, 1998. They can also be isolated by
methods including preparative chromatography and immunological
separations.
[0122] A Brachyury polypeptide can be covalently linked to a
carrier, which is an immunogenic macromolecule to which an
antigenic molecule can be bound. When bound to a carrier, the bound
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. Additional bacterial products for use as
carriers include bacterial wall proteins and other products (for
example, streptococcal or staphylococcal cell walls and
lipopolysaccharide (LPS)).
[0123] Polynucleotides encoding the Brachyury polypeptides
disclosed herein are also provided. These polynucleotides include
DNA, cDNA and RNA sequences which encode the 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).
[0124] A nucleic acid encoding a Brachyury polypeptide 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 Q13 replicase
amplification system (QB). For example, a polynucleotide encoding
the 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, NY, 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.
[0125] The polynucleotides encoding a Brachyury polypeptide include
a recombinant DNA which is incorporated into a vector in an
autonomously replicating plasmid or virus or into the genomic DNA
of a prokaryote or eukaryote, or which exists as a separate
molecule (such as a cDNA) independent of other sequences. The
nucleotides of the invention can be ribonucleotides,
deoxyribonucleotides, or modified focus of either nucleotide. The
term includes single and double forms of DNA.
[0126] In one embodiment, vectors are used for expression in yeast
such as S. cerevisiae or Kluyveromyces lactis. Several 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 (ADM), and pleiotropic
drug-resistant pump (PDR5). In addition, may inducible promoters
are of use, 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.).
Promoters that direct variable expression in response to a
titratable inducer include the methionine-responsive MET3 and MET25
promoters and copper-dependent CUP1 promoters. Any of these
promoters may be cloned into multicopy (2.mu.) or single copy (CEN)
plasmids to give an additional level of control in expression
level. The plasmids can include nutritional markers (such as URA3,
ADE3, HIS1, and others) for selection in yeast and antibiotic
resistance (AMP) for propagation in bacteria. Plasmids for
expression on K. lactis are known, such as pKLAC 1. Thus, in one
example, after amplification in bacteria, plasmids can be
introduced into the corresponding yeast auxotrophs by methods
similar to bacterial transformation.
[0127] The brachyury peptides can be expressed in a variety of
yeast strains. For example, seven pleiotropic drug-resistant
transporters, YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11, and PDR15,
together with their activating transcription factors, PDR1 and
PDR3, have been simultaneously deleted in yeast host cells,
rendering the resultant strain sensitive to drugs. Yeast strains
with altered lipid composition of the plasma membrane, such as the
erg6 mutant defective in ergosterol biosynthesis, can also be
utilized. Proteins that are highly sensitive to proteolysis can be
expressed in a yeast lacking the master vacuolar endopeptidase
Pep4, which controls the activation of other vacuolar hydrolases.
Heterologous expression in strains carrying temperature-sensitive
(ts) alleles of genes can be employed if the corresponding null
mutant is inviable.
[0128] Viral vectors can also be prepared encoding the brachyury
polypeptides disclosed herein. A number of viral vectors have been
constructed, 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) 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 are also known in
the art, and may be obtained from commercial sources (such as
PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden,
Conn.; Stratagene, La Jolla, Calif.).
[0129] Thus, in one embodiment, the polynucleotide encoding a
Brachyury polypeptide is included in a viral vector. Suitable
vectors include retrovirus vectors, orthopox vectors, avipox
vectors, fowlpox vectors, capripox vectors, suipox vectors,
adenoviral vectors, herpes virus vectors, alpha virus vectors,
baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors
and poliovirus vectors. Specific exemplary vectors are poxvirus
vectors such as vaccinia virus, fowlpox virus and a highly
attenuated vaccinia virus (MVA), adenovirus, baculovirus and the
like.
[0130] Pox viruses useful in practicing the present invention
include orthopox, suipox, avipox, and capripox virus. Orthopox
include vaccinia, ectromelia, and raccoon pox. One example of an
orthopox of use is vaccinia. Avipox includes fowlpox, canary pox
and pigeon pox. Capripox include goatpox and sheeppox. In one
example, the suipox is swinepox. Examples of pox viral vectors for
expression as described for example, in U.S. Pat. No. 6,165,460,
which is incorporated herein by reference. Other viral vectors that
can be used include other DNA viruses such as herpes virus and
adenoviruses, and RNA viruses such as retroviruses and polio.
[0131] In some cases, vaccinia viral vectors may elicit a strong
antibody response. Thus, while numerous boosts with vaccinia
vectors are possible, its repeated use may not be useful in certain
instances. However, this sensitivity problem can be minimized by
using pox from different genera for boosts. In one example, when
the first or initial pox virus vector is vaccinia, the second and
subsequent pox virus vectors are selected from the pox viruses from
a different genus such as suipox, avipox, capripox or an orthopox
immunogenically distinct from vaccinia.
[0132] 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. Int. J. Cancer 48:900-907, 1991; Moss Science 252:1662, 1991).
A gene encoding an antigen of interest, such as an immunogenic
Brachyury polypeptide, can be incorporated into the HIND F13L
region or alternatively incorporated into the TK region of
recombinant vaccinia virus vector (or other nonessential regions of
the vaccinia virus genome). 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.
[0133] Suitable vectors are disclosed, for example, in U.S. Pat.
No. 6,998,252, which is incorporated herein by reference. In one
example, a recombinant poxvirus, such as a recombinant vaccinia
virus is synthetically modified by insertion of a chimeric gene
containing vaccinia regulatory sequences or DNA sequences
functionally equivalent thereto flanking DNA sequences which in
nature are not contiguous with the flanking vaccinia regulatory DNA
sequences that encode a Brachyury polypeptide. The recombinant
virus containing such a chimeric gene is effective at expressing
the Brachyury polypeptide. In one example, the vaccine viral vector
comprises (A) a segment comprised of (i) a first DNA sequence
encoding a Brachyury polypeptide and (ii) a poxvirus promoter,
wherein the poxvirus promoter is adjacent to and exerts
transcriptional control over the DNA sequence encoding a Brachyury
polypeptide; and, flanking said segment, (B) DNA from a
nonessential region of a poxvirus genome. The viral vector can
encode a selectable marker. In one example, the poxvirus includes,
for example, a thymidine kinase gene (see U.S. Pat. No. 6,998,252,
which is incorporated herein by reference).
[0134] Poxviral vectors that encode a Brachyury polypeptide include
at least one expression control element operationally linked to the
nucleic acid sequence encoding the Brachyury polypeptide. The
expression control elements are inserted in the poxviral 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, yeast promoters and 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
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 are easily 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.
[0135] Basic techniques for preparing recombinant DNA viruses
containing a heterologous DNA sequence encoding the Brachyury
polypeptide, are known in the art. Such techniques involve, for
example, homologous recombination between the viral DNA sequences
flanking the DNA sequence in a donor plasmid and homologous
sequences present in the parental virus (Mackett et al., 1982,
Proc. Natl. Acad. Sci. USA 79:7415-7419). In particular,
recombinant viral vectors such as a poxyviral vector can be used in
delivering the gene. The vector can be constructed for example by
steps known in the art, such as steps analogous to the methods for
creating synthetic recombinants of the fowlpox virus described in
U.S. Pat. No. 5,093,258, incorporated herein by reference. Other
techniques include using a unique restriction endonuclease site
that is naturally present or artificially inserted in the parental
viral vector to insert the heterologous DNA.
[0136] 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 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 pox virus are described in WO91/19803,
incorporated herein by reference.
[0137] 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).
[0138] The DNA gene sequence to be inserted into the virus can be
placed into a donor plasmid, such as an E. coli or a Salmonella
plasmid construct, into which DNA homologous to a section of DNA
such as that of the insertion site of the poxvirus 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 pox DNA that is the desired insertion region.
With a parental pox viral vector, a pox 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, for example chick embryo fibroblasts, along with the
parental virus, for example poxvirus. Recombination between
homologous pox DNA in the plasmid and the viral genome respectively
results in a recombinant poxvirus modified by the presence of the
promoter-gene construct in its genome, at a site that does not
affect virus viability.
[0139] 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. One region that can readily be used and is
present in many viruses is the thymidine kinase (TK) gene. The TK
gene has been found in all pox virus genomes examined, including
leporipoxvirus (Upton et al., 1986, J. Virology 60:920); shope
fibromavirus; capripoxvirus (Gershon et al., 1989, J. Gen. Virol.
70:525) Kenya sheep-1; orthopoxvirus (Weir et al., 1983, J. Virol.
46:530) vaccinia (Esposito et al., 1984, Virology 135:561);
monkeypox and variola virus (Hruby et al., 1983, PNAS 80:3411)
vaccinia (Kilpatrick et al., 1985, Virology 143:399); Yaba monkey
tumor virus; avipoxvirus (Binns et al., 1988, J. Gen. Virol.
69:1275); fowipox; (Boyle et al., 1987, Virology 156:355); fowlpox
(Schnitzlein et al., 1988, J. Virological Methods 20:341); fowlpox,
quailpox; entomopox (Lytvyn et al., 1992, J. Gen. Virol.
73:3235-3240). In vaccinia, in addition to the TK region, other
insertion regions include, for example, the HindIII fragment. In
fowlpox, in addition to the TK region, other insertion regions
include, for example, the BamHI J fragment (Jenkins et al., 1991,
AIDS Research and Human Retroviruses 7:991-998) the ECORI-HindIII
fragment, EcoRV-HindIII fragment, BamHI fragment and the HindIII
fragment set forth in EPO Application No. 0 308220 A1 (see also
Calvert et al., 1993, J. Virol. 67:3069-3076; Taylor et al., 1988,
Vaccine 6:497-503; Spehner et al., 1990; Boursnell et al., 1990, J.
Gen. Virol. 71:621-628).
[0140] In swinepox, insertion sites include the thymidine kinase
gene region. In addition to the requirement that the gene be
inserted into an insertion region, successful expression of the
inserted gene by the modified poxvirus requires the presence of a
promoter operably linked to the desired gene. Generally, the
promoter is placed so that it is located upstream from the gene to
be expressed. Promoters are well known in the art and can readily
be selected depending on the host and the cell type you wish to
target. In one example, in poxviruses, pox viral promoters are
used, such as the vaccinia 7.5K, 40K or fowlpox promoters such as
FPV CIA. Enhancer elements can also be used in combination to
increase the level of expression. Furthermore, inducible promoters
can be utilized.
[0141] 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 a pox virus are chick
embryo fibroblasts, HuTK143 (human) cells, and CV-1 and BSC-40
(both monkey kidney) cells. Infection of cells with pox 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).
[0142] Following in vivo recombination, recombinant viral progeny
can be identified by one of several techniques. For example, if the
DNA donor vector is designed to insert foreign genes into the
parent virus thymidine kinase (TK) gene, viruses containing
integrated DNA will be TK- and can be selected on this basis
(Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415).
Alternatively, 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 sequence 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).
[0143] This disclosure encompasses a recombinant virus comprising
more than one antigen of interest for the purpose of having a
multivalent vaccine. For example, the recombinant virus may
comprise the virus genome or portions thereof, the nucleic acid
sequence encoding the Brachyury polypeptide and a nucleic acid
sequence encoding a hepatitis B surface antigen.
[0144] In one embodiment, a composition is provided that includes a
recombinant virus comprising a vaccinia virus genome or portions
thereof, the nucleic acid sequence encoding a Brachyury polypeptide
and a recombinant virus comprising the nucleic acid sequence
encoding the immunostimulatory molecule, B 7.1 alone or in
combination with the nucleic acid sequence encoding the
immunostimulatory molecule, B7-2, or a recombinant virus containing
both the genes for a tumor antigen and an immunostimulatory
molecule. This disclosure also encompasses a recombinant virus
comprising the Brachyury polypeptide that is administered with a
second recombinant virus comprising the virus genome or portion
thereof, and one or more nucleic acid sequences encoding one or
more B7 molecules, such as a recombinant vaccinia virus expressing
B7-1 and/or B7-2. It is disclosed in U.S. Pat. No. 893,869
(incorporated by reference herein) that the rapid infection of
tumor cells with these recombinant viruses demonstrates that
vaccinia can authentically express these proteins and that they are
functional molecules. Following transfer of the nucleic acids,
weakly immunogenic syngeneic tumors expressing these recombinant
molecules are rejected by immunocompetent hosts.
[0145] Thus, in one example, recombinant virus is disclosed that is
a recombinant vaccinia virus containing B7-1 and a recombinant
vaccinia virus containing B7-2 (designated rV-B7-1 and rV-B7-2,
respectively); the composition can include rV-B7-1 and/or rV-B7-2
in combination with an immunogenic Brachyury polypeptide.
[0146] The B7 molecule includes but is not limited to B7-1, B7-2
and analogs thereof. The B7 gene may be cloned from mammalian
sources, including but not limited to mammalian tissues, genomic
libraries or cDNA libraries, such as from murine or human sources.
Without being bound by theory, co-stimulatory molecules of the B7
family (namely B7-1, B7-2, and possibly B7.3) are believed to be
members of the immunoglobulin gene superfamily. These molecules are
present on macrophages, dendritic cells, monocytes (antigen
presenting cells (APCs)). Significant amplification of the immune
response against a given antigen generally does not occur without
co-stimulation (June et al. (Immunology Today 15:321-331, 1994);
Chen et al. (Immunology Today 14:483-486); Townsend et al. (Science
259:368-370)). Freeman et al. (J. Immunol. 143:2714-2722, 1989)
report cloning and sequencing of B7-1 gene. Azuma et al. Nature
366:76-79, 1993) report cloning and sequencing B7-2 gene. Thus, in
one embodiment the B7-1 gene or the B7-2 genes are administered in
conjunction with the Brachyury polypeptide. The insertion of
nucleic acids encoding B7-1 and B7-2 into vaccinia virus has been
disclosed (see for example, U.S. Pat. No. 6,893,869, incorporated
herein by reference; this U.S. Patent also discloses the use of a
nucleic acid encoding IL-2 in a vaccinia virus). Several vectors
including IL-2, B7-1 and B7-2 have been deposited with the American
Type Culture Collection (ATCC) on Oct. 3, 1994 under the terms of
the Budapest Treaty (for example, rV-CEA/.sub.nIL-2 (ATCC
Designation VR 2480), rV-.sub.mB7-2 (ATCC Designation VR 2482); and
rV-.sub.mB7-1 (ATCC Designation VR 2483)).
[0147] DNA sequences encoding a Brachyury polypeptide can be
expressed in vitro by DNA transfer into a suitable host cell. The
cell may be prokaryotic or eukaryotic. 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. Methods of stable
transfer, meaning that the foreign DNA is continuously maintained
in the host, are known in the art.
[0148] As noted above, a polynucleotide sequence encoding a
Brachyury 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 perrrrit proper
translation of mRNA, and stop codons.
[0149] Hosts cells can include microbial, yeast, 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 bacteria,
archea, insect, fungi (for example, yeast), plant, and animal cells
(for example, mammalian cells, such as human). Exemplary cells of
use include Escherichia coli, Bacillus subtilis, Saccharomyces
cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293
cells, Neurospora, and immortalized mammalian myeloid and lymphoid
cell lines. 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. As discussed above,
techniques for the transformation of yeast cells, such as
polyethylene glycol transformation, protoplast transformation and
gene guns are also known in the art (see Gietz and Woods Methods in
Enzymology 350: 87-96, 2002).
[0150] 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.
[0151] 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 virus vectors can be used.
Eukaryotic cells can also be co-transformed with polynucleotide
sequences encoding a Brachyury polypeptide, and a second foreign
DNA molecule encoding a selectable phenotype, such as the herpes
simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein (see for example, Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
Therapeutic Methods and Pharmaceutical Compositions
[0152] The Brachyury polypeptides disclosed herein, or nucleic
acids encoding the Brachyury polypeptides, can be used to generate
an immune response in a subject. In several examples, the subject
has a tumor that expresses Brachyury. Thus, in several embodiments,
the methods include administering to a subject with cancer a
therapeutically effective amount of one or more of the Brachyury
polypeptides disclosed herein, in order to generate an immune
response.
[0153] The methods can include selecting a subject in need of
treatment, such as a subject with a tumor that expresses Brachyury.
In several examples, the methods include selecting a subject with a
tumor of the small intestine, stomach, kidney, bladder, uterus,
ovaries, testes lung, colon or prostate. In additional examples,
the method includes selecting a subject with a tumor of B cell
origin, such as chronic lymphocytic leukemia (CLL), a B cell
lymphoma, Burkitt's lymphoma or a Hodgkin's lymphoma.
[0154] In exemplary applications, compositions are administered to
a subject having a disease, such as cancer (for example, small
intestine, stomach, kidney, bladder, uterus, ovary, testes, lung
colon, or prostate cancer), in an amount sufficient to raise an
immune response to Brachyury-expressing cells. Administration
induces a sufficient immune response to slow the proliferation of
such cells or to inhibit their growth, or to reduce a sign or a
symptom of the tumor. 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
compound 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.
[0155] A Brachyury polypeptide 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) 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. To extend the time during
which the peptide or protein is available to stimulate a response,
the peptide or protein can be provided as an implant, an oily
injection, 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. Aluminum salts can also be used as adjuvants to
produce an immune response.
[0156] In one specific, non-limiting example, the Brachyury
polypeptide is administered in a manner to direct the immune
response to a cellular response (that is, a cytotoxic T lymphocyte
(CTL) response), rather than a humoral (antibody) response.
[0157] Optionally, one or more cytokines, such as IL-2, IL-6,
IL-12, RANTES, GM-CSF, TNF-.alpha., or IFN-.gamma., one or more
growth factors, such as GM-CSF or G-CSF, one or more costimulatory
molecules, such as ICAM-1, LFA-3, CD72, B7-1, B7-2, or other B7
related molecules; one or more molecules such as 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):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 ICAM-1 are
administered.
[0158] 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 CTL 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. 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 CTL when covalently attached to an appropriate
peptide (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 peptide which displays an appropriate
epitope, two compositions can be combined to elicit both humoral
and cell-mediated responses where that is deemed desirable.
[0159] In yet another embodiment, to induce a CTL response to an
immunogenic Brachyury polypeptide, a MHC Class II-restricted
T-helper epitope is added to the immunogenic Brachyury polypeptide
to induce T-helper cells to secrete cytokines in the
microenvironment to activate CTL precursor cells. The technique
further involves adding short lipid molecules to retain the
construct at the site of the injection for several days to localize
the antigen at the site of the injection and enhance its proximity
to dendritic cells or other "professional" antigen presenting cells
over a period of time (see Chesnut et al., "Design and Testing of
Peptide-Based Cytotoxic T-Cell-Mediated Immunotherapeutics to Treat
Infectious Diseases and Cancer," in Powell et al., eds., Vaccine
Design, the Subunit and Adjuvant Approach, Plenum Press, New York,
1995).
[0160] A pharmaceutical composition including a Brachyury
polypeptide is thus provided. These compositions are use to
generate an immune response, such as for immunotherapy. In one
embodiment, 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; U.S. Pat. No. 5,709,860; U.S. Pat. No. 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%.
[0161] 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%.
[0162] 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.
[0163] 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.). An adjuvant
can also be an immunostimulatory nucleic acid, such as a nucleic
acid including a CpG motif, or a biological adjuvant (see
above).
[0164] 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).
[0165] 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. No. 5,055,303; U.S. Pat. No.
5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S.
Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; and U.S. Pat. No.
5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S.
Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat. No.
5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S.
Pat. No. 5,271,961; U.S. Pat. No. 5,254,342; and U.S. Pat. No.
5,534,496).
[0166] In another embodiment, a pharmaceutical composition includes
a nucleic acid encoding an Brachyury polypeptide. A therapeutically
effective amount of the Brachyury polynucleotide 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 Brachyury polynucleotide is administered to a subject
to treat prostate cancer or breast cancer.
[0167] Optionally, one or more cytokines, such as IL-2, IL-6,
IL-12, RANTES, GM-CSF, TNF-.alpha., or IFN-.gamma., one or more
growth factors, such as GM-CSF or G-CSF, one or more costimulatory
molecules, such as ICAM-1, LFA-3, CD72, B7-1, B7-2, or other B7
related molecules; one or more molecules such as 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):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 to the host. It should be noted that
these molecules can be co-administered via insertion of a nucleic
acid encoding the molecules into a vector, for example, a
recombinant pox vector (see, for example, U.S. Pat. No. 6,045,802).
In various embodiments, the nucleic acid encoding the biological
adjuvant can be cloned into same vector as the 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.
[0168] 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 polypeptide can be placed under the control of a promoter
to increase expression of the molecule.
[0169] 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).
[0170] In another approach to using nucleic acids for immunization,
a Brachyury polypeptide can also be expressed by attenuated viral
hosts or vectors or bacterial vectors. Recombinant vaccinia virus,
adeno-associated virus (AAV), herpes virus, retrovirus, or other
viral vectors can be used to express the peptide or protein,
thereby eliciting a CTL response. For example, vaccinia vectors and
methods useful in immunization protocols are described in U.S. Pat.
No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another
vector for expression of the peptides (see Stover, Nature
351:456-460, 1991).
[0171] A first recombinant virus, such as a poxvirus (for example,
vaccine virus) encoding a Brachyury immunogenic polypeptide can be
used in conjunction with a second recombinant 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.
[0172] When a 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.sup.5 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 viral
vector, carrying the nucleic acid sequence of one or more Brachyury
polypeptides 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.
[0173] In the embodiment where a combination of a first recombinant
viral vector carrying a nucleic acid sequence of one or more
Brachyury polypeptides and a second recombinant viral vector
carrying the nucleic acid sequence of one or more 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).
[0174] In one embodiment the recombinant viruses have been
constructed to express cytokines (such as TNF-.alpha., IL-6,
GM-CSF, and IL-2), and co-stimulatory and accessory molecules
(B7-1, B7-2) alone and in a variety of combinations. Simultaneous
production of an immunostimulatory molecule and the 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, interferon, tumor necrosis factor, or a
nucleic acid encoding these molecules, can be administered in
conjunction with a Brachyury immunogenic polypeptide, or a nucleic
acid encoding a Brachyury polypeptide. The co-expression of a
Brachyury polypeptide together with at least one immunostimulatory
molecule can be effective in an animal model to show anti-tumor
effects. In one embodiment, a nucleic acid encoding a Brachyury
polypeptide is introduced directly into cells. For example, 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).
[0175] 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 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.
[0176] 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 treat or ameliorate symptoms
or signs of disease without producing unacceptable toxicity to the
subject. Systemic or local administration can be utilized.
[0177] In another method, antigen presenting cells (APCs), such as
dendritic cells, are pulsed or co-incubated with peptides
comprising a 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 can then be administered to a subject.
[0178] The 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 polypeptide. The Brachyury polypeptide 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.
[0179] In one embodiment, mature antigen presenting cells are
generated to present the immunogenic Brachyury polypeptide. These
dendritic cells are then administered alone (or in combination with
another agent) to a subject with a tumor that expresses Brachyury,
such as a small intestine, stomach, kidney, bladder, uterus, ovary,
testis, lung colon and/or prostate cancer. The dendritic cells can
also be administered to a subject with a tumor of B cell origin,
such as chronic lymphocytic leukemia (CLL), a B cell lymphoma,
Burkitt's lymphoma or a Hodgkin's lymphoma.
[0180] In another embodiment, the mature dendritic cells are
administered in conjunction with a chemotherapeutic agent.
[0181] Alternatively, the APCs are used to sensitize CD8 cells,
such as tumor infiltrating lymphocytes (TILs) from tumors or
peripheral blood lymphocytes (PBLs). The TILs or PBLs can be from
the same subject (autologous) that is to be treated. Alternatively,
the TILs or PBLs can be heterologous. However, they should at least
be MHC Class-I restricted to the HLA types the subject possesses.
An effective amount of the sensitized cells are then administered
to the subject.
[0182] Peripheral blood mononuclear cells (PBMCs) can be used as
the responder cell source of CTL precursors. The appropriate
antigen-presenting cells are incubated with peptide, after which
the peptide-loaded antigen-presenting cells are then incubated with
the responder cell population under optimized culture conditions.
Positive CTL activation can be determined by assaying the culture
for the presence of CTLs that kill radio-labeled target cells, both
specific peptide-pulsed targets as well as target cells expressing
endogenously processed forms of the antigen from which the peptide
sequence was derived, such as Brachyury (for example, SEQ ID NO:
1).
[0183] The cells can be administered to a subject to inhibit the
growth of cells of Brachyury expressing tumors. In these
applications, a therapeutically effective amount of activated
antigen presenting cells, or activated lymphocytes, 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.
[0184] 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.
[0185] In a further method, any of these immunotherapies is
augmented by administering an additional chemotherapeutic agent. In
one example, this administration is sequential. Examples of such
agents 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).
Methods of Treatment Using Specific Binding Agents
[0186] Expression of Brachyury is associated with tumor cell
migration and invasion. Brachyury is expressed in small intestine,
stomach, kidney, bladder, uterus, ovaries, testes lung, colon and
prostate tumors but not in most normal tissues. In addition,
expression of Brachyury is associated with epithelial-to
mesenchymal transition. Moreover, Brachyury is expressed in tumors
of B cell origin, such as chronic lymphocytic leukemia (CLL),
Epstein-Barr virus transformed B cells, Burkitt's and Hodgkin's
lymphomas. Reagents that can reduce the expression of Brachyury
also can be used to treat tumors that express Brachyury. The
reagents can be used alone, or can be used in combination with the
immunogenic Brachyury polypeptides disclosed herein.
[0187] The methods can include selecting a subject in need of
treatment, such as a subject with a tumor that expresses Brachyury.
In several examples, the methods include selecting a subject with a
tumor of the small intestine, stomach, kidney, bladder, uterus,
ovaries, testes lung, colon or prostate. In additional examples,
the method includes selecting a subject with a tumor of B cell
origin, such as chronic lymphocytic leukemia (CLL), a B cell
lymphoma, Burkitt's lymphoma or a Hodgkin's lymphoma.
[0188] In one example, the method includes administering a
therapeutically effective amount of a specific binding agent that
preferentially binds to Brachyury. The specific binding agent can
be an inhibitor such as a siRNA or an antisense molecule that
specifically binds Brachyury mRNA (such as an mRNA encoding SEQ ID
NO: 1). Inhibition of Brachyury does not require 100% inhibition,
but can include at least a reduction if not a complete inhibition
of cell growth or differentiation associated with a specific
pathological condition. Treatment of a tumor by reducing Brachyury
expression can include delaying the development of the tumor in a
subject (such as preventing metastasis of a tumor) by altering the
ability of the tumor to metastasize. Treatment of a tumor also
includes reducing signs or symptoms associated with the presence of
such a tumor (for example by reducing the size or volume of the
tumor or a metastasis thereof) by decreasing the number of
metastases. In some examples decrease or slowing metastasis of the
tumor, or reducing the size or volume of the tumor, is an
alteration of at least 10%, at least 20%, at least 50%, or at least
75. In some examples, treatment using the methods disclosed herein
prolongs the time of survival of the subject. Treatment can also
result in a down-regulation of mesenchymal markers (such as
fibronectin, vimentin and/or N-cadherin) and an up-regulation of
epithelial markers (such as E-cadherin or g-catenin).
[0189] Specific binding agents are agents that bind with higher
affinity to Brachyury, than to other molecules. For example, a
specific binding agent can be one that binds with high affinity to
Brachyury but does not substantially bind to another gene or gene
product. For example, the specific binding agent interferes with
gene expression (transcription, processing, translation,
post-translational modification), such as, by interfering with
Brachyury mRNA and blocking translation into protein.
[0190] A reduction of Brachyury protein expression in a target cell
may be obtained by introducing into cells an antisense or other
suppressive construct based on the Brachyury coding sequence. For
antisense suppression, a nucleotide sequence from a Brachyury
encoding sequence, e.g. all or a portion of the Brachyury cDNA or
gene, is arranged in reverse orientation relative to the promoter
sequence in the transformation vector.
[0191] The introduced sequence need not be the full length
Brachyury gene, and need not be exactly homologous to the
equivalent sequence found in the cell type to be transformed. Thus,
portions or fragments of a nucleic acid encoding Brachyury (SEQ ID
NO: 2) could also be used to knock out or suppress expression.
Generally, however, where the introduced sequence is of shorter
length, a higher degree of identity to the native Brachyury
sequence will be needed for effective antisense suppression. The
introduced antisense sequence in the vector may be at least 15
nucleotides in length, and improved antisense suppression typically
will be observed as the length of the antisense sequence increases.
The length of the antisense sequence in the vector advantageously
may be greater than 100 nucleotides, and can be up to about the
full length of the Brachyury cDNA or gene. For suppression of the
Brachyury gene itself, transcription of an antisense construct
results in the production of RNA molecules that are the reverse
complement of mRNA molecules transcribed from the endogenous
Brachyury gene in the cell.
[0192] Although the exact mechanism by which antisense RNA
molecules interfere with gene expression has not been elucidated,
it is believed that antisense RNA molecules bind to the endogenous
mRNA molecules and thereby inhibit translation of the endogenous
mRNA. Expression of Brachyury can also be reduced using small
inhibitory RNAs, for instance using techniques similar to those
described previously (see, e.g., Tuschl et al., Genes Dev 13,
3191-3197, 1999; Caplen et al., Proc. Nat'l Acad. Sci. U.S.A. 98,
9742-9747, 2001; and Elbashir et al., Nature 411, 494-498, 2001).
Methods of making siRNA that can be used clinically are known in
the art. Exemplary siRNAs are commercially available from several
sources, such as Sigma Aldrich and Dharmacon, and therapeutic
siRNAs can readily be produced using methods known in the art.
[0193] Suppression of endogenous Brachyury expression can also be
achieved using ribozymes. Ribozymes are synthetic RNA molecules
that possess highly specific endoribonuclease activity. The
production and use of ribozymes are disclosed in U.S. Pat. No.
4,987,071 to Cech and U.S. Pat. No. 5,543,508 to Haselhoff. The
inclusion of ribozyme sequences within antisense RNAs may be used
to confer RNA cleaving activity on the antisense RNA, such that
endogenous mRNA molecules that bind to the antisense RNA are
cleaved, which in turn leads to an enhanced antisense inhibition of
endogenous gene expression.
[0194] In certain examples, expression vectors are employed to
express the inhibitor nucleic acid, such as the antisense, ribozyme
or siRNA molecule (see above for additional information on vectors
and expression systems). For example, an expression vector can
include a nucleic acid sequence encoding the antisense, ribozyme or
siRNA molecule. In a particular example, the vector contains a
sequence(s) encoding both strands of a siRNA molecule comprising a
duplex. In another example, the vector also contains sequence(s)
encoding a single nucleic acid molecule that is self-complementary
and thus forms a siRNA molecule. Non-limiting examples of such
expression vectors are described in Paul et al., Nature
Biotechnology 19:505, 2002; Miyagishi and Taira, Nature
Biotechnology 19:497, 2002; Lee et al., Nature Biotechnology
19:500, 2002; and Novina et al., Nature Medicine, online
publication Jun. 3, 2003, and additional vectors are described
above.
[0195] In other examples, inhibitory nucleic acids, such as siRNA
molecules include a delivery vehicle, including inter alia
liposomes, for administration to a subject, carriers and diluents
and their salts, and can be present in pharmaceutical compositions.
Nucleic acid molecules can be administered to cells by a variety of
methods known to those of skill in the art, including, but not
restricted to, encapsulation in liposomes, by iontophoresis, or by
incorporation into other delivery vehicles, such as hydrogels,
cyclodextrins, biodegradable nanocapsules, and bioadhesive
microspheres, or by proteinaceous vectors (see, for example, O'Hare
and Normand, International PCT Publication No. WO 00/53722, see
also the additional methods described above).
[0196] Alternatively, the nucleic acid/vehicle combination can be
locally delivered such as into a tumor by direct injection or by
use of an infusion pump. Direct injection of the nucleic acid
molecules of the disclosure, whether subcutaneous, intramuscular,
or intradermal, can take place using standard needle and syringe
methodologies, or by needle-free technologies such as those
described by Barry et al., International PCT Publication No. WO
99/31262. Other delivery routes include, but are not limited to,
oral delivery (such as in tablet or pill form), intrathecal or
intraperitoneal delivery (see below). For example, intraperitoneal
delivery can take place by injecting the treatment into the
peritoneal cavity of the subject in order to directly deliver the
molecules to the tumor site. More detailed descriptions of nucleic
acid delivery and administration are provided in Sullivan et al.,
PCT WO 94/02595, Draper et al., PCT Publication No. WO93/23569,
Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT
Publication No. WO 99/04819, all of which are incorporated by
reference herein.
[0197] Alternatively, certain siRNA molecules can be expressed
within cells from eukaryotic promoters. Those skilled in the art
will recognize that any nucleic acid can be expressed in eukaryotic
cells using the appropriate DNA/RNA vector (see above). The
activity of such nucleic acids can be augmented by their release
from the primary transcript by an enzymatic nucleic acid (Draper et
al., PCT Publication No. WO 93/23569, and Sullivan et al., PCT
Publication No. WO 94/02595).
[0198] In other examples, siRNA molecules can be expressed from
transcription units (see for example, Couture et al., 1996, TIG
12:510) inserted into DNA or RNA vectors. The recombinant vectors
can be DNA plasmids or viral vectors. siRNA expressing viral
vectors can be constructed based on, for example, but not limited
to, adeno-associated virus, retrovirus, adenovirus, lentivirus or
alphavirus. In another example, pol III based constructs are used
to express nucleic acid molecules of the invention (see for
example, Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886 and
others described above).
[0199] The recombinant vectors capable of expressing the siRNA
molecules can be delivered as described above, and persist in
target cells. Alternatively, viral vectors can be used that provide
for transient expression of nucleic acid molecules. Such vectors
can be repeatedly administered as necessary. Once expressed, the
siRNA molecule interacts with the target mRNA and generates an RNAi
response. Delivery of siRNA molecule expressing vectors can be
systemic, such as by intravenous or intramuscular administration,
by administration to target cells explanted from a subject followed
by reintroduction into the subject, or by any other means that
would allow for introduction into the desired target cell.
Diagnostic Methods
[0200] A method is also provided herein for detecting Brachyury in
a biological sample. The method includes contacting the sample with
one or more of an antibody that specifically binds Brachyury to
form an antibody-Brachyury complex. The presence or absence of the
complex is detected. The methods are of use to improve the
confidence of a tissue diagnosis, such as to confirm a diagnosis,
or to determine the origin of a tumor. Thus, the method disclosed
herein can be used to confirm the diagnosis of a tumor of the small
intestine, stomach, kidney, bladder, uterus, ovaries, testes lung,
colon and prostate tumors. The methods disclosed herein can be used
to confirm the diagnosis of a B cell tumor, such as chronic
lymphocytic leukemia (CLL), Epstein-Barr virus transformed B cells,
Burkitt's and Hodgkin's lymphomas. The methods disclosed herein can
also be used to determine if the origin of a tumor, such as to
determine if a metastatic cancer is of small intestine, stomach,
kidney, bladder, uterus, ovaries, testes lung, colon, prostate or B
cell origin.
[0201] In addition, expression of Brachyury is associated with
epithelial-to mesenchymal transition. Thus, the methods disclosed
herein can be used to determine the likelihood of tumor cell
migration and invasion.
[0202] The methods can include selecting a subject in need of
diagnosis, such as a subject with a tumor, and obtaining a sample
from this subject. In several examples, the methods include
selecting a subject with a tumor of the small intestine, stomach,
kidney, bladder, uterus, ovaries, testes lung, colon or prostate,
and obtaining a sample from this subject. In additional examples,
the method includes selecting a subject with a tumor of B cell
origin, such as chronic lymphocytic leukemia (CLL), a B cell
lymphoma, Burkitt's lymphoma or a Hodgkin's lymphoma, and obtaining
a sample from this subject.
[0203] The sample can be any sample, including, but not limited to,
tissue from biopsies, autopsies and pathology specimens. Biological
samples also include sections of tissues, such as frozen sections
taken for histological purposes. Biological samples further include
body fluids, such as blood, serum, spinal fluid or urine. A
biological sample is typically obtained from a mammal, such as a
rat, mouse, cow, dog, guinea pig, rabbit, or primate. In one
embodiment, the primate is macaque, chimpanzee, or a human. In some
embodiments, a histological section is utilized, and an
immunohistochemical assay is performed.
[0204] Antibodies that specifically bind Brachyury are known in the
art. Antibodies include polyclonal and monoclonal antibodies. In
some embodiments, an antibody fragment, such as an Fv fragment is
utilized. In a further embodiment, the antibody is labeled (such as
with a fluorescent, radioactive, or an enzymatic label). In
additional examples, the antibodies can be conjugated to compounds
including, but not limited to, enzymes, magnetic beads, colloidal
magnetic beads, haptens, fluorochromes, metal compounds or
radioactive compounds.
[0205] Methods of determining the presence or absence of a protein
are well known in the art. Assays of use include, but are not
limited to, radioimmunoassays (RIAs), enzyme linked immunosorbant
assays (ELISA), or immunohistochemical assays. The method for
detecting Brachyury in a biological sample generally includes the
steps of contacting the biological sample with an antibody which
specifically reacts, under immunologically reactive conditions, to
Brachyury. The antibody is allowed to specifically bind under
immunologically reactive conditions to form an immune complex, and
the presence of the immune complex (bound antibody) is detected
directly or indirectly. A control cell, such as a non-transformed
cell or section of the same tissue type, can be included as a
control.
Reagents for the Detection of Cells that Express CD8 (CD8+) Cells
that Specifically Bind Brachyury
[0206] Reagents are provided herein for the detection of CD8
expressing cells that specifically bind Brachyury. These reagents
are tetrameric MHC Class I/immunogenic Brachyury polypeptide
complexes. These tetrameric complexes include an immunogenic
Brachyury polypeptide that includes at most twelve consecutive
amino acids, wherein the isolated polypeptide comprises the amino
acid sequence set forth as WLLPGTSTX.sub.1 (SEQ ID NO: 3), wherein
X.sub.1 is a leucine (L) or a valine (V). Specific examples of
immunogenic Brachyury polypeptide that are ten amino acids in
length are disclosed above. The tetrameric complexes disclosed
herein do not include additional consecutive amino acids of
Brachyury (SEQ ID NO: 1), such that the polypeptide does not
include the full length Brachyury amino acid sequence.
[0207] Tetrameric MHC Class I/peptide complexes can be synthesized
using methods well known in the art (Altmann et al., Science
274:94, 1996, which is herein incorporated by reference). In one
specific non-limiting example, purified HLA heavy chain and
.beta.2-microglobulin (.beta.2m) can be synthesized by means of a
prokaryotic expression system. One specific, non-limiting example
of an expression system of use is the pET system (R&D Systems,
Minneapolis, Minn.). The heavy chain is modified by deletion of the
trans-membrane and cytosolic tail and COOH-telininal addition of a
sequence containing the biotin protein ligase (Bir-A) enzymatic
biotinylation site. Heavy chain, .beta.2m, and peptide are then
refolded. The refolded product can be isolated by any means known
in the art, and then biotinylated by Bir-A. A tetramer is then
produced by contacting the biotinylated product with
strepavidin.
[0208] In one embodiment, the strepavidin is labeled. Suitable
labels include, but are not limited to, enzymes, magnetic beads,
colloidal magnetic beads, haptens, fluorochromes, metal compounds,
radioactive compounds or drugs. The enzymes that can be conjugated
to strepavidin include, but are not limited to, alkaline
phosphatase, peroxidase, urease and .beta.-galactosidase. The
fluorochromes that can be conjugated to the strepavidin include,
but are not limited to, fluorescein isothiocyanate,
tetramethylrhodamine isothiocyanate, phycoerythrin,
allophycocyanins and Texas Red. For additional fluorochromes that
can be conjugated to strepavidin, see Haugland, R. P., Molecular
Probes: Handbook of Fluorescent Probes and Research Chemicals
(1992-1994). The metal compounds that can be conjugated to the
strepavidin include, but are not limited to, ferritin, colloidal
gold, and particularly, colloidal superparamagnetic beads. The
haptens that can be conjugated to the strepavidin include, but are
not limited to, biotin, digoxigenin, oxazalone, and nitrophenol.
The radioactive compounds that can be conjugated to strepavidin are
known to the art, and include but are not limited to technetium 99m
(.sup.99Tc), .sup.125I and amino acids comprising any
radionuclides, including, but not limited to, .sup.14C, .sup.3H and
.sup.35S. Generally, strepavidin labeled with a fluorochrome is
utilized in the methods disclosed herein.
[0209] In one embodiment, suspension of cells including T cells
that specifically recognize Brachyury is produced, and the cells
are reacted with the tetramer in suspension. In one embodiment,
these reagents are used to label cells, which are then analyzed by
fluorescence activated cell sorting (FACS). A machine for FACS
employs a plurality of color channels, low angle and obtuse
light-scattering detection channels, and impedance channels, among
other more sophisticated levels of detection, to separate or sort
cells. Any FACS technique can be employed as long as it is not
detrimental to the detection of the desired cells. (For exemplary
methods of FACS see U.S. Pat. No. 5,061,620.)
[0210] The disclosure is illustrated by the following non-limiting
Examples.
Examples
Example 1
Materials and Methods
[0211] Computer-Based Differential Display (CDD) Analysis.
[0212] Comparison of all EST clusters on the human Unigene Built
171 (see the NCBI Unigene website, available on the internet) was
conducted by using the HSAnalyst program as previously described
(Baronova et al., FEBS Lett 508(1):143-8, 2001). Unigene EST
cluster Hs.389457 corresponds to accession number
NM.sub.--003181.
[0213] Source of cDNA.
[0214] Expression in normal tissues was studied by using Multiple
Tissue cDNA (MTC) panels containing sets of normalized cDNAs from
pooled normal tissues from several individuals (Clontech, Mountain
View, Calif.). The following panels were used: human MTC Panel I,
Panel II, and Blood Fractions Panel. Commercially available tumor
tissue-derived cDNAs, prepared from different individuals with
different tumor types, were obtained from BioChain Institute Inc.
(Hayward, Calif.). Total RNA from human cancer cell lines and
normal CD19+ isolated B cells were prepared by using the RNAeasy
extraction kit (Qiagen Inc., Valencia, Calif.).
[0215] PCR Analysis.
[0216] PCR amplification of cDNA panels was carried out with the
following primers specific for NM.sub.--003181:
TABLE-US-00008 (SEQ ID NO: 4) E7F 5'-GGGTGGCTTCTTCCTGGAAC-3' and
(SEQ ID NO: 5) E7R 5'-TTGGAGAATTGTTCCGATGAG-3'.
[0217] G3PDH specific primers were:
TABLE-US-00009 (SEQ ID NO: 6) forward
5'-TGAAGGTCGGAGTCAACGGATTTGGT-3', (SEQ ID NO: 7) reverse
5'-CATGTGGGCCATGAGGTCCACCAC-3'.
[0218] The following conditions were used: 1 minute at 95.degree.
C., 35 cycles consisting of 30 sec at 95.degree. C., 30 sec at
58.degree., and 1 minute at 72.degree. C., and 5 minutes elongation
at 72.degree. C. The expected size for the Brachyury and G3PDH
products was 172 and 983 bp, respectively. Total RNA derived from
human cancer cell lines and normal CD19+ isolated B cells were
amplified by using the TITANIUM One-Step RT-PCR kit (Clontech),
following the manufacturer's instructions. Primer sequences were as
follow:
[0219] Brachyury, E3F 5'-ACTGGATGAAGGCTCCCGTCTCCTT-3' (SEQ ID NO:
8), and E8R 5'-CCAAGGCTGGACCAATTGTCATGGG-3' (SEQ ID NO: 9) (Edwards
et al., Genome Res 6(3):226-33, 1996); and
[0220] .beta.-actin, forward 5'-ATCTGGCACCACACCTTCTACAATGAG-3' (SEQ
ID NO: 10), and reverse 5'-CGTGGTGGTGAAGCTGTAGCCGCGCTC-3' (SEQ ID
NO: 11). The expected size of the PCR products was 568 bp and 356
bp, respectively.
[0221] RT-PCR Amplification from NCI-H460 Cells:
[0222] Total RNA was prepared from stably transfected NCI-H460
cells containing a control shRNA plasmid or a Brachyury-specific
shRNA construct (Br. shRNA clones 1 and 2) by using the RNeasy
extraction kit (Qiagen Inc., Valencia, Calif.), following the
manufacturer's recommendations. Five ng of total RNA were amplified
by using the TITANIUM One-Step RT-PCR kit (Clontech, Mountain View,
Calif.), following the manufacturer's instructions. Primer
sequences were as follows: Brachyury, E3F
5'-ACTGGATGAAGGCTCCCGTCTCCTT-3' (SEQ ID NO: 8), and E8R
5'-CCAAGGCTGGACCAATTGTCATGGG-3' (SEQ ID NO: 9); and .beta.-actin,
forward 5'-ATCTGGCACCACACCTTCTACAATGAG-3' (SEQ ID NO: 10), and
reverse 5'-CGTGGTGGTGAAGCTGTAGCCGCGCTC-3' (SEQ ID NO: 11). The
expected size of the PCR products was 568 and 356 bp,
respectively.
[0223] Cell cultures.
[0224] The human carcinoma cell lines were maintained free of
Mycoplasma in RPMI 1640 medium (Invitrogen, Carlsbad, Calif.)
supplemented with 10% fetal bovine serum, 2 mM glutamine, and
1.times. solution of antibiotic/antimycotic (Invitrogen).
Additional cell lines used in this study were the C1R-A2 cell line,
which is a human B-cell lymphoblastoid line transfected to express
surface HLA-A2 antigen (Shimojo et al., J Immunol 143(9):2939-47,
1989), and the T2 (HLA-A2+) transport-deletion mutant cell line
(Salter et al., Embo J 5(5):943-9, 1986).
[0225] Peptides.
[0226] The computer algorithm from the Bioinformatics and Molecular
Analysis Section of NIH (BIMAS) developed by Parker et al. was used
(Parker et al., J Immunol 152(1):163-75, 1994, incorporated by
reference herein). A panel of 9-mer and 10-mer peptides (see Table
2, below) was synthesized at >90% purity (Biosynthesis,
Lewisville, Tex.). The CEA peptide CAP1-6D (YLSGADLNL, SEQ ID NO:
12), the HIV peptide (ILKEPVHGV, SEQ ID NO: 13), and a CEA peptide
specific for HLA-A3 were used as controls.
[0227] HLA A2 Binding Assay.
[0228] Binding of Brachyury-specific peptides T-p1, T-p2, T-p3, and
T-p4 (SEQ ID NOs: 1-4) to HLA-A0201 molecules was evaluated by flow
cytometry analysis of HLA-A02 surface expression on T2 cells. T2
cells (1.times.10.sup.6) in serum-free Iscove's modified Dulbecco's
medium were incubated in the presence of various concentrations of
each peptide, in 24-well culture plates at 37.degree. C. with 5%
CO.sub.2. After 18 hours in culture, T2 cells were harvested,
washed with 1.times. phosphate buffered saline (PBS) (Invitrogen)
and stained with 20 .mu.l of a FITC-conjugated
anti-HLA-A02-specific monoclonal antibody (MAb) (One Lambda, Inc.,
Canoga Park, Calif.). A FITC-conjugated IgG2a MAb (BD Biosciences,
San Jose, Calif.) was used as an isotype control. Data acquisition
and analysis were conducted on a FACSCalibur.TM. system using the
CELLQuest.TM. software (BD Biosciences). Results were expressed as
mean fluorescence intensity (MFI) collected on a log scale. To
measure the half-life of major histocompatability complex
(MHC)-peptide complexes, T2 cells were incubated for 18 hours in
the presence of 25 .mu.M of each peptide, subsequently washed free
of unbound peptides and incubated for various time points in
presence of 10 .mu.g/ml of Brefeldin A. Flow cytometry was
conducted as described above. Assuming first order kinetics, the
log.sub.2 of MFI/MFI.sub.0 (MFI is the fluorescence at each time
point and MFI.sub.0 the initial fluorescence at time 0) was plotted
against time (minutes). The decay rate constant was calculated as
the slope of the linear regression for each curve and the half-life
of each peptide-MHC complex was calculated as the inverse of the
ratio 1/decay rate constant.
[0229] Culture of DCs from Peripheral Blood Mononuclear Cells
(PBMCs).
[0230] Peripheral blood used in this study was collected from
healthy donors and cancer patients. Peripheral blood mononuclear
cells (PBMCs) were isolated from leukapheresis samples by
centrifugation on a Ficoll density gradient (LSM Lymphocyte
Separation Medium, ICN Biochemicals Inc., Aurora, Ohio). For the
preparation of dendritic cells (DCs), PBMCs were resuspended in
AIM-V medium (Invitrogen) and allowed to adhere to the surface of
T-150 flasks (Corning Costar Corp., Cambridge, Mass.). After 2
hours at 37.degree. C., the non-adherent cell fractions were
removed and the adherent cells were cultured in AIM-V medium
containing 100 ng/ml of recombinant human GM-CSF (rhGM-CSF) and 20
ng/ml of recombinant human IL-4 (rhIL-4) for 7 days.
[0231] Generation of T-Cell Lines.
[0232] To generate Brachyury-specific cytotoxic T cells (CTLs),
peptide-pulsed irradiated (30 Gy) DCs were used as antigen
presenting cells (APCs) with autologous, non-adherent cells used as
effector cells at an effector-to-APC ratio of 10:1. Cultures were
maintained for three initial days in medium containing 10% human AB
serum, and four additional days in the same medium supplemented
with 20 U/ml of recombinant human IL-2. After a seven-day culture
period, designated as an in vitro stimulation (IVS) cycle, cells
were re-stimulated as described above.
[0233] Detection of Cytokines.
[0234] After three IVS cycles, CD8+ T cells that were negatively
isolated by using a CD8+ isolation kit (Miltenyi Biotec, Auburn,
Calif.) were stimulated for 24 hours in the presence of
peptide-pulsed autologous DCs. Culture supernatants were analyzed
for the presence of IFN-.gamma. by using an enzyme linked
immunosorbant assay (ELISA) kit (Biosource International Inc.,
Camarillo, Calif.). Results were expressed in pg/ml.
[0235] Cytotoxic Assay.
[0236] Target cells were labeled with 50 .mu.Ci of
.sup.111Indium-labeled oxyquinoline (Amersham Health, Silver
Spring, Md.) for 15 minutes at room temperature. Target cells in
medium containing 10% human AB serum were plated at
3.times.10.sup.3 cells per well, in 96-well rounded-bottom culture
plates. Labeled C1R-A2 or T2 cells were incubated with peptides at
the indicated concentrations for 60 minutes at 37.degree. C. in 5%
CO.sub.2 before the addition of effector cells. No peptide was
added when carcinoma cells or CD19+ B cells were used as targets.
CD8+ T cells negatively isolated from T-cell cultures were used as
effector cells, at various effector-to-target (E:T) cell ratios.
When target cells were C1R-A2 or T2, co-cultures were incubated at
37.degree. C. in a 5% CO2 atmosphere for 6 hours as previously
described (Tsang et al., Clin Cancer Res 11(4):1597-607, 2005);
when carcinoma cell lines were used as targets, co-cultures were
incubated as previously described (Tsang et al., supra) in the same
conditions for a period of 16 hours. Cytotoxic assays employing
normal donor CD19+ B cells as targets were conducted for 5 hours as
previously described (Palena et al., Blood 106(10):3515-23, 2005),
due to the high levels of spontaneous release observed after a
16-hour incubation period. Supernatants were harvested and the
.sup.111In released was measured by gamma counting. Spontaneous
release was determined by incubating the target cells with medium
alone, and complete lysis by incubating the target cells with 2.5%
Triton X-100. All determinations were performed in triplicate, and
standard deviations were calculated. Specific lysis was calculated
as follows: specific lysis (%)=[(observed release-spontaneous
release)/(complete release-spontaneous release)].times.100.
[0237] Generation of Stably Transfected NCI-H460 Cells:
[0238] The NCI-H460 cell line, originally derived from a patient
with a large cell carcinoma of the lung, was obtained from the
American Type Culture Collection (ATCC, Manassas, Va.) and
maintained in RPMI-1640 medium supplemented with 1.times.
Antibiotic/Antimycotic solution (Invitrogen, Carlsbad, Calif.) and
10% fetal bovine serum (FBS, Gemini Bio-Products West Sacramento,
Calif.). Cells (1.times.10.sup.6) were transfected using the
nucleofector device and technology (Amaxa Biosystems, Gaithersburg,
Md.) with 1 .mu.g of purified, linearized DNA plasmid encoding for
a non-targeting shRNA (designated as control shRNA) or two
Brachyury-specific targeting shRNA constructs (designated as Br.
shRNA clones 1 and 2), following the recommendations of the
manufacturers. After 48 hours in culture, stably transfected cells
were selected in RPMI-1640 medium containing 10% FBS and 1 .mu.g/ml
of puromycin (Sigma Aldrich, St. Louis, Mo.).
[0239] Western Blot Analysis of Mesenchymal and Epithelial
Markers:
[0240] Protein extracts were prepared from NCI-H460 cells stably
transfected with the control shRNA and Brachyury-specific shRNA
(clone 2) by using the Ripa Lysis Buffer kit (Santa Cruz Biotech,
Santa Cruz, Calif.) following the manufacturer's instructions.
Protein concentration was determined by using the BCA Protein Assay
kit (Thermo Scientifics, Rockford, Ill.).
[0241] Ten micrograms (mg) of proteins from each sample were
resolved on 4-12% polyacrylamide gradient pre-cast gels and
subsequently transferred to nitrocellulose membranes (Invitrogen).
Blots were blocked with freshly made 0.5% casein in PBS for 1 hour
at room temperature. Subsequently, blots were probed with
1:500-1:1000 dilution of primary antibodies in 0.5% casein solution
overnight at 4.degree. C. Antibodies were anti-human fibronectin,
vimentin, g-catenin, and b-actin (BD-Biosciences, San Jose,
Calif.). Blots were washed 3 times with PBS and incubated with a
1:5000 dilution of horseradish peroxidase (HRP)-conjugated
secondary anti-mouse IgG antibody (Invitrogen) for 1 hour at room
temperature. Blots were washed 5 times with PBS/Tween 20 and were
developed using the Western Lighting chemiluminescent detection
reagent (PerkinElmer, Boston Mass.) and autoradiographs were
obtained.
[0242] Migration and Invasion Assays:
[0243] The migratory abilities of NCI-H460 cells stably transfected
with a control shRNA or a Brachyury-specific shRNA (Br.shRNA clone
2) were examined in vitro using Blind Well Chambers (Neuroprobe,
Gaithersburg, Md.) with 12 micrometer-pore size polycarbonate
filters. Briefly, RPMI-1640 medium containing 10% fetal bovine
serum (FBS) was added to the lower chambers, and cells
(1.times.10.sup.5 cells, 300 .mu.l in RPMI medium free of serum) to
the upper chambers. For the invasion assays, polycarbonate filters
were pre-coated with a 1:1 dilution of Matrigel (BD Biosciences)
and serum free RPMI-1640 medium. Experiments were conducted in
triplicate samples of each cell line. After incubation for 48 hours
at 37.degree. C., the upper side of the filters was extensively
cleaned with cotton tips, filters were removed from the chambers,
fixed, and stain with Diff-Quik stain (Dade Behring Inc., Newark,
Del.). The number of cells associated with the lower side of the
membranes was evaluated by direct counting of five random
100.times. objective fields. Each bar represents the results for
each replicate assay.+-.SEM.
Example 2
Computer-Based Prediction
[0244] In silico profiling of gene expression in the human Unigene
Built 171 was conducted as previously described (Baranova et al.,
FEBS Lett 508(1):143-8, 2001) by using the HSANALYST.TM. software
tool. An algorithm executed by the program returned a list of
candidate EST clusters that contained >10 ESTs with >90% of
the ESTs derived from tumor libraries. Among them, the cluster
Hs.389457 contained the whole mRNA sequence encoding for the human
Brachyury gene (mouse Brachyury homolog). From a total of 55 ESTs
included in this cluster, 50 ESTs corresponded to tumor-derived
libraries constructed from lung carcinoma cell lines, germ-cell
tumors, chronic lymphocytic leukemia B cells, and breast cancer.
Two normal tissue-derived ESTs found in the cluster Hs.389457
belonged to a library constructed from pooled RNA from fetal lung,
testis, and normal B cells. The other three ESTs in the cluster
were designated as "undefined," since they lacked tissue origin
descriptions.
Example 3
Confirmation of Expression
[0245] The computer-based predictions of the expression of
Brachyury mRNA were then verified by RT-PCR analysis of Brachyury
expression in a range of normal and malignant human tissues. Most
normal tissue-derived cDNA samples, as predicted by the algorithm,
showed no Brachyury mRNA expression (FIGS. 1A and 1B). Very weak
signals, however, were observed with cDNA derived from normal
testis, spleen (FIG. 1A), and resting CD19+ purified cells (FIG.
1B). These results were also in accordance with the software's
prediction that two out of 55 ESTs in the cluster belonged to a
library prepared from pooled testis, fetal lung, and normal B
lymphocytes.
[0246] The expression of Brachyury in normal B cells was further
evaluated in CD19+ samples isolated from various healthy donors;
weak amplification was observed in four out of nine samples
analyzed when using 1 microgram of total RNA and 35 cycles of PCR
amplification.
[0247] In contrast, RT-PCR amplification of cDNA samples derived
from tumor-tissues demonstrated relatively high levels of Brachyury
mRNA expression in carcinomas of the esophagus, stomach, small
intestine, kidney, bladder, uterus, ovary, and testis (FIG. 1C),
and a weak signal in a lung carcinoma-derived sample. PCR products
derived from two of the reactions were subsequently sequenced to
confirm the gene and rule oat the possibility of non-specific
amplification. The expression of Brachyury was further analyzed in
total RNA derived from 30 human carcinoma cell lines (Table 1).
TABLE-US-00010 TABLE 1 RT-PCR expression of human Brachyury in
human tumor cell lines Brachyury Tumor type Tumor cell line
mRNA.sup.1 Lung H441 ++ NCI-H460 ++ H226 + NCI-H520 + SW900 - Colon
SW480 ++ SW620 ++ Colo 201 + Colo 205 + CaCo2 + SW403 + T-84 +
SW948 - SW1463 - HT-29 - SW1116 - Prostate LNCAP + PC-3 + DU145 +
Pancreatic Capan-2 + Paca-2 - BxPC3 - PANC-1 - ASPC-1 - Breast
MCF-7 - MA-MB-231 - Ovarian SW626 ++.sup.a NIH-OVCAR3 - SK-OV3 -
Osteosarcoma U2OS - .sup.1Expression of Brachyury mRNA is shown
relative to the expression of .beta.-actin as being negative (-),
positive (+), or strongly positive (++). .sup.aThere is conflicting
evidence that this cell line may be of colonic origin. See Furlong
et al., J Natl Cancer Inst 91(15): 1327-8, 1999.
[0248] Brachyury mRNA expression was observed in most of the lung
cancer-derived, colon cancer-derived, and prostate cancer-derived
tumor cell lines (Table 1). These results thus validated the CDD
predictions through RT-PCR and confirmed expression of Brachyury in
several tumors but not in normal tissues.
[0249] Reverse transcriptase polymerase chain reaction (RT-PCR)
analysis demonstrated Brachyury expression in tumors of the small
intestine, stomach, kidney, bladder, uterus, ovary, and testis, as
well as in cell lines derived from lung, colon, and prostate
carcinomas, but not in the vast majority of the normal tissues
tested. Elevated Brachyury mRNA expression was also detected in
most of the B-cell malignancies examined, including chronic
lymphocytic leukemia (CLL) cells, Epstein-Barr virus
(EBV)-transformed B-cell lines, Burkitt's and Hodgkin's lymphoma
cell lines. Quantitative real-time PCR analysis showed elevated
expression of Brachyury mRNA in CD19.sup.+ cells isolated from
13/25 CLL patients, as compared with very low, if any, level of
expression in B lymphocytes isolated from peripheral blood from
healthy donors or a panel of normal human tissues. A time-course
infection of normal B-lymphocytes with EBV showed that Brachyury
mRNA expression is induced as early as 48 hours post-infection and
is maintained in long-term cultures of transformed B-cell lines.
EBV, a lymphotropic human herpesvirus, is linked to various
clinical disorders including human neoplasms of hematological
origin such as lymphomas in immunocompromised individuals and
posttransplant lymphoproliferative disorders, and those of
epithelial origin such as nasopharyngeal carcinomas and gastric
adenocarcinomas. These results suggest that Brachyury is a
potential tumor target for hematological malignancies of B-cell
origin and, in particular, for EBV-associated malignancies. The
results also demonstrate that elevated Brachyury expression (as
compared to a control cell of the same tissue type) serves as a
marker for confirmation of diagnosis of tumors of these types. In
addition to improving the confidence with which a tissue diagnosis
of the cancers is made, the detection of Brachyury over-expression
in a metastatic lesion helps identify potential sites of primary
tumor so that further diagnostic tests of these potential sites can
be more cost effectively conducted and therapy (such as surgical
excision of the primary tumor) more quickly achieved. Hence
detection of Brachyury can be used in methods of diagnosing and
treatment of cancers characterized by Brachyury expression.
Example 4
Production of Brachyury Immunogenic Peptides that Bind MHC
[0250] The amino acid sequence of the Brachyury protein was then
analyzed for HLA-A0201 peptide-binding prediction by using a
computer algorithm from BIMAS. The top-ranking candidate peptides
generated by the program, including three 9-mers and a 10-mer whose
amino acid sequences and algorithm scores are presented in Table 2,
were selected for further studies.
TABLE-US-00011 TABLE 2 HLA-A0201 peptide motif search using BIMAS
software Start Peptide Residues position* Sequence Score.dagger.
T-p1 9-mer 345 SQYPSLWSV 389.26 (SEQ ID NO: 14) T-p2 9-mer 246
WLLPGTSTL 363.59 (SEQ ID NO: 15) T-p3 9-mer 422 RLIASWTPV 118.24
(SEQ ID NO: 16) T-p4 10-mer 86 AMYSFLLDFV 996.36 (SEQ ID NO: 17)
*Start position corresponds to the amino acid position in the
protein sequence. .dagger.Estimate of half-time disassociation of a
molecule containing this subsequence.
[0251] In silico-predicted epitopes were then assessed for binding
to the MHC molecules in a cell-based assay. TAP-deficient T2
(HLA-A2+) cells were incubated in the presence of 25 .mu.M of each
peptide and subsequently tested for cell surface MHC-stabilization
by bound peptides. Flow cytometry staining of HLA-A02 (FIG. 2A)
demonstrated that all four candidate peptides predicted by the
algorithm efficiently bound to HLA-A02 molecules when compared to
positive and negative control peptides. Peptides with the highest
binding to T2 cells (T-p2, T-p3, and T-p4) were selected for
further studies. The half-life of each peptide-MHC complex was
determined; T2 cells were incubated overnight in the presence of 25
.mu.M of each peptide followed by the addition of brefeldin A and
subsequent evaluation of cell surface staining of HLA-A02 at
various time points. MHC-peptide complexes involving Tp-2 have a
half-life of 514 minutes, similar to that of the positive control
peptide (CAP1-6D). In contrast, MHC-peptide complexes involving
T-p3 and T-p4 showed shorter half-lives of 225 and 312 minutes,
respectively (FIG. 2B).
Example 5
Immunogenicity
[0252] Once the ability of the predicted peptides to bind HLA-A02
molecules was demonstrated, the immunogenicity of peptides T-p2,
T-p3, and T-p4 was investigated by evaluating their ability to
induce specific CTLs in vitro. Irradiated DCs pulsed with 25 .mu.M
of each peptide were used to stimulate autologous T cells from a
healthy donor's PBMCs. After three in vitro stimulations (IVS),
isolated CD8+ T cells were subsequently stimulated for 24 hours in
the presence of autologous DCs alone or DCs pulsed with each of the
"inducer" peptides (T-p2, T-p3, or T-p4) or an irrelevant
HIV-peptide. Of the three peptides tested, T-p2 and T-p3 induced
antigen-specific CTLs able to release IFN-.gamma. upon stimulation
with the specific peptide (FIG. 3A). Both CTL lines were then
tested for their cytotoxic activity against peptide-pulsed
HLA-A0201+ targets. As shown in FIG. 3B, only T cells generated
with the T-p2 peptide were able to specifically lyse peptide-pulsed
target cells, consistent with the peptide's ability to form stable
MHC complexes compared with T-p3 and T-p4. Titration of the
cytotoxic activity of the T-p2 CTLs showed cytotoxic responses at
peptide concentrations as low as 1 nM (FIG. 3C). Cytotoxic lysis of
normal B lymphocytes was also analyzed since low expression of
Brachyury was detectable in CD19+ cells isolated from various
healthy donors. No lysis was observed with any of the normal B
cells analyzed from five different healthy donors.
[0253] The cytolytic activity of the T-p2-specific CTLs was then
tested against several tumor targets. Tumor cell lines used as
targets included the lung carcinoma cells H441 (HLA-A0201+/T
antigen+) and NCI-H460 (HLA-A24, 68+/T antigen+), the colorectal
carcinoma line SW1463 (HLA-A0201+/T antigen-), and the pancreatic
carcinoma cells AsPC-1 (HLA-A02-). CTLs derived with the T-p2
epitope were highly efficient at killing H441 tumor cells, while no
lysis was observed against the other cell lines. MHC-restriction
was shown by the observation that the H460 tumor cell line that is
highly positive for Brachyury but HLA-A0201 negative was not killed
by the Tp-2 CTLs (FIG. 4A). Conversely, the tumor cell line SW 1463
served as an antigen-specific control, since it is negative for the
expression of Brachyury but positive for the expression of
HLA-A0201. Similarly, the control AsPC-1 (HLA-A0201-) cells were
also not killed by the Brachyury-specific T cells. These results
indicate that T cells that have been expanded in vitro in the
presence of the T-p2 peptide are able to specifically lyse those
tumor cells that express Brachyury within the correct MHC-class I
context. As shown in FIG. 4B, T-p2 CTLs-mediated killing of H441
tumor cells was blocked by antibodies directed against the
MHC-class I molecules but not the MHC-class II molecules, further
confirming the MHC-class I restriction of the observed lysis.
[0254] The Tp-2 peptide was then tested for in vitro expansion of
Brachyury-specific T cells from PBMC of four additional healthy
donors. Tp-2-specific CTLs were induced from two out of five
healthy donors tested.
[0255] Generation of T-p2 specific CTLs was also successfully
carried out from PBMCs of two cancer patients. T cells isolated
from PBMCs of a colorectal cancer patient (designated as patient 1)
and an ovarian cancer patient (designated as patient 2) were
stimulated in vitro for three cycles in the presence of autologous,
irradiated T-p2-pulsed DCs as described in Example 1. CD8+ T cells
negatively isolated from these cultures were assayed for cytotoxic
activity against tumor cells. As shown in FIGS. 4C and 4D, after
three IVS both CTL lines were able to lyse H441 tumor cells. After
five IVS, CTLs derived from both patients were tested for their
ability to lyse additional tumor cell lines positive for the
expression of Brachyury. As shown in FIG. 4E, T-p2-specific CTLs
derived from patient 1 were able to lyse LNCAP cells
(HLA-A2+/Brachyury+) in an HLA-A02 restricted way, as denoted by
the blocking of cytotoxic killing in presence of anti-HLA-A02 but
not in presence of a control IgG. FIG. 4F shows that T-p2 cells
expanded from the blood of patient 2 were able to lyse H441, SW620,
and SW480 tumor cells, all of them being Brachyury+ and HLA-A2+. On
the other hand, lysis of SW403 cells, which are HLA-A2+ and express
lower levels of Brachyury mRNA (FIG. 4E), was only minimal.
Altogether, Tp-2 cells derived from healthy individuals and cancer
patients were able to lyse 4/5 Brachyury positive tumors, while no
lysis was observed for control tumor cells that were (a)
HLA-A2-/Brachyury+ (NCI-H460), or (b)
HLA-A2+/Brachyury-(SW1463).
[0256] In conclusion, the results demonstrated that T-p2-specific T
cells generated from both healthy donors and cancer patients were
able to recognize and mediate cytotoxic lysis of tumor cells that
endogenously express the Brachyury protein.
[0257] As demonstrated herein, high-throughput gene expression
analysis in tumors versus normal tissues constitutes a relatively
new approach for the identification of therapeutic cancer targets.
Computer programs have been emerging for mining of EST databases
that use publicly available information from the vast collection of
ESTs (Scheurle et al., Cancer Res 2000, 60(15):4037-4043). As the
frequency of ESTs in a cDNA library appears to be proportional to
the abundance of associated transcripts in the tissue from which
the library was prepared (Audic and Claverie, Genome Res 1997;
7:986-995), data on ESTs expression can be correlated with
tissue-related or disease-related gene expression signatures. In
the present studies, data mining software tool (HSANALYST.TM.) was
successfully used for the identification of Unigene EST cluster
Hs.389457, corresponding to the human gene Brachyury, as a tumor
antigen, and validated the in silico prediction by RT-PCR in a set
of normal and tumor tissues and cancer cell lines. Expression of
Brachyury was shown to be elevated in tumors of the small
intestine, stomach, kidney, bladder, uterus, ovary, and testis, and
in the majority of cell lines derived from lung, colon, and
prostate carcinomas. Because of the high grade of conservation
among members of the T-box family, BLAST analysis of the primers
sequence was conducted to discard any possible amplification of
sequences derived from other members of the T-box family, and the
fidelity of the amplified band was confirmed by DNA sequencing. The
high levels of expression of Brachyury in tumors contrasted with
its lack of expression in most normal adult tissues, with the
exception of low levels observed in testis, spleen, and CD19+
(resting) lymphocytes. Without being bound by theory, the weak
signal in spleen could be attributable to the presence of CD19+
cells.
[0258] The affinity prediction method from BIMAS was applied in the
studies presented herein for identifying Brachyury peptides with
high affinity binding for HLA-A0201. All four top-ranked peptides
effectively bound to HLA-A0201 molecules, although peptide-MHC
complexes showed differences in their decay rate. Tp-2 was the only
peptide, however, able to expand CTLs in vitro that are capable of
releasing IFN-.gamma. in response to peptide-specific stimulation
and lysing peptide-pulsed targets with high efficiency. This
peptide also showed the maximum stability of binding to HLA-A0201,
which could result in increased immunogenicity.
[0259] The lung carcinoma cell line H441 was effectively lysed in
the presence of Brachyury-specific CTLs even at a low ratio of
effector T cells-to-targets, in an antigen-specific and
MHC-restricted manner. Furthermore, it was demonstrated that
Brachyury-T-p2-specific CTLs can be expanded in vitro from PBMCs of
a colorectal cancer patient and an ovarian carcinoma patient,
demonstrating that Brachyury is of use as a therapeutic target for
cancer vaccine regimens. Thus, it has been demonstrated that (a) a
T-box transcription factor and (b) a molecule implicated in
mesodermal development, (such as epithelial-to mesodermal
transition, EMT), can be a potential target for human T-cell
mediated cancer immunotherapy.
Example 6
Stable Knockdown of Brachyury Expression Induces a
Mesenchymal-to-Epithelial Transition in NCI-H460 Lung Carcinoma
Cancer Cells
[0260] In order to evaluate whether Brachyury plays a role on
modulating the epithelial-to-mesenchymal (EMT) program, Brachyury
expression was stably silenced in NCI-H460 lung carcinoma cells
that normally express high levels of Brachyury mRNA. RT-PCR
analysis confirmed the silencing of Brachyury expression in cells
transfected with the Brachyury-specific shRNA clones 1 and 2, as
compared with cells transfected with the non-targeting shRNA
control construct (FIG. 5A), with the clone 2 showing a higher
level of silencing of Brachyury expression.
[0261] Western blot analysis of expression of various epithelial
and mesenchymal markers showed that silencing of Brachyury
expression resulted in marked decreases on the expression of
fibronectin and vimentin, both markers of a mesenchymal phenotype.
The expression of the epithelial marker .gamma.-catenin, on the
other hand, was enhanced as a result of Brachyury's silencing.
Therefore, at the biochemical level, silencing of Brachyury
expression in NCI-H460 cells repressed expression of mesenchymal
markers and concomitantly elicited expression of epithelial
markers, changes typically observed during a
mesenchymal-to-epithelial (MET) transition.
Example 7
Loss of Brachyury Impairs the Migratory and Invasive Properties of
NCI-H460 Lung Carcinoma Cells In Vitro
[0262] Boyden-chamber transwell assays were also performed in order
to determine whether Brachyury expression in NCI-H460 cells
modulates the migratory and invasive characteristics of these
cells. As shown in FIG. 6, NCI-H460 cells stably silenced for
Brachyury expression (Br. shRNA clone 2) showed a significant
reduction on their migratory ability (FIG. 6A) as well as a marked
reduction on their ability to degrade and invade the extracellular
matrix (FIG. 6A). These results support the conclusion that the
T-box transcription factor Brachyury can serve as a modulator of
the mesenchymal phenotype of tumor cells and could program
metastasis-associated cellular qualities.
Example 8
Expression of Brachyury in Hematological Malignancies
[0263] The expression of Brachyury in chronic lymphocytic leukemia
(CLL) as well as other hematological malignancies (Hodgkin's
lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma) was
investigated. CD19+ cell were isolated from CLL patients. The
relative expression of Brachyury in CD19+ B cells was evaluated by
quantitative real-time polymerase chain reaction (PCR), using
primers to human Brachyury and GAPDH. The expression of Brachyury
in CD19+ cells isolated from CLL patients was 0.318+/-0.752, while
the expression of Brachyury in healthy donors was 0.019+/-0.023. It
was also demonstrated that Brachyury was expressed on CD5+CD19+
leukemia cells. Using reverse-transcriptase PCR (RT-PCR) Brachyury
was detected in EBV transformed B Cell lines, (B-EBV 701, B-EBV
1383, C1R) as well as in Burkitt's lymphoma (DAUDI and RAJI) and
Hodgkin's lymphoma (RPMI 6666) cell lines.
[0264] B lymphocytes from healthy donors were infected in vitro
with EBV supernatants. The RNA was isolated over time and analyzed
for Brachyury expression (by RT-PCR). Brachyury was expressed at 72
and 96 hours following EBV infection. In the tree subject tested,
Brachyury expression also could be detected six days after EBV
infection.
[0265] Thus, elevated expression of Brachyury mRNA was detected in
CD19+ cells from CLL patients as compared to CD19+ cells from
healthy donors. 13/25 CLL patients showed increased levels of
Brachyury as compared to normal B cells. No correlation was found
between the level of Brachyury expression and the white blood cell
count or Rai stage. Expression of Brachyury was also elevated in
LCL (EBV-transformed B cell) lines, Burkitt's lymphoma cell lines
and the Hodgkin's lymphoma line RPMI16666. EBV infection of normal
B lymphocytes resulted in expression of Brachyury mRNA. Brachyury
expression could be detected as early as 48 hours after infection;
Brachyury expression peaked at 72 hours post-infection.
Example 9
Inhibition of Tumor Growth or Metastasis Using a Specific Binding
Agent
[0266] This example describes methods that can be used to
significantly reduce tumor growth or metastasis in a subject with a
tumor of epithelial origin, such as small intestine, kidney,
bladder, uterus, ovary or testis, or in carcinomas, such as lung,
colon and prostate carcinomas, or in a tumor of B-cell origin, such
as chronic lymphocytic leukemia, Burkitt's lymphoma or Hodgkin's
lymphoma.
[0267] Based upon the teaching disclosed herein, tumor growth or
metastasis can be reduced or inhibited by administering a
therapeutically effective amount of a composition, wherein the
composition comprises a specific binding agent for Brachyury,
thereby reducing or inhibiting tumor growth or metastasis in the
subject.
[0268] In an example, a subject who has been diagnosed with the
tumor is identified and selected for treatment. Following subject
selection, a therapeutic effective dose of the composition
including the specific binding agent is administered to the
subject. For example, a therapeutic effective dose of a specific
binding agent for Brachyury is administered to the subject to
inhibit tumor growth and/or metastasis. In an example, the specific
binding agent is a siRNA. In a further example, the specific
binding agent is an antisense molecule. The amount of the
composition administered to prevent, reduce, inhibit, and/or treat
the tumor depends on the subject being treated, the severity of the
disorder, and the manner of administration of the therapeutic
composition. Ideally, a therapeutically effective amount of an
agent is the amount sufficient to prevent, reduce, and/or inhibit,
and/or treat the tumor in the subject without causing a substantial
cytotoxic effect in the subject.
[0269] In one specific example, siRNAs are administered at
according to the teachings of Soutschek et al. (Nature Vol. 432:
173-178, 2004) or Karpilow et al. (Pharma Genomics 32-40, 2004)
both of which are herein incorporated by reference in their
entireties. In other examples, the subject is administered the
therapeutic composition daily for a period of at least 30 days,
such as at least 2 months, at least 4 months, at least 6 months, at
least 12 months, at least 24 months, or at least 36 months.
[0270] Subjects will monitored by methods known to those skilled in
the art to determine tumor responsiveness to the siRNA or
antisense. It is contemplated that additional agents can be
administered, such as antineoplastic agents in combination.
Example 10
Retrospective Study
[0271] In order to determine the correlation of Brachyury
expression with the prognosis of a tumor, samples of lung, colon,
breast, and prostate cancer tissues, among others, are analyzed by
real-time PCR to determine the expression of Brachyury mRNA, as
described above. Commercially available cDNA panels (TissueScan
Real-Time Disease qPCR Arrays, Origene Technologies, Rockville,
Md.) containing cDNA prepared from individual tumor samples are
tested with Brachyury- and GAPDH-specific PCR primers to obtain a
quantitative expression of Brachyury mRNA in each sample. Each cDNA
panel includes information on tumor grade and stage (Samples from
stages 1A-IV for lung cancer; stages I to IV for prostate cancer;
stages I to IV for colon cancer; and stages 0 to IV for breast
cancer will be tested, for example). Brachyury mRNA is
preferentially expressed in samples of tumors from patients
diagnosed to be at higher stages than in samples of tumors from
patients diagnosed to be at lower stages.
[0272] It will be apparent that the precise details of the methods
or compositions described may be varied or modified without
departing from the spirit of the described invention. We claim all
such modifications and variations that fall within the scope and
spirit of the claims below.
Sequence CWU 1
1
221435PRTHomo sapiens 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 251839PRTHomo sapiensMISC_FEATURE(9)..(9)Xaa
can be Leu or Val 3Trp Leu Leu Pro Gly Thr Ser Thr Xaa 1 5
420DNAArtificial SequenceSynthetic oligonucleotide primer
4gggtggcttc ttcctggaac 20521DNAArtificial SequenceSynthetic
oligonucleotide primer 5ttggagaatt gttccgatga g 21626DNAArtificial
SequenceSynthetic oligonucleotide primer 6tgaaggtcgg agtcaacgga
tttggt 26724DNAArtificial SequenceSynthetic oligonucleotide primer
7catgtgggcc atgaggtcca ccac 24825DNAArtificial SequenceSynthetic
oligonucleotide primer 8actggatgaa ggctcccgtc tcctt
25925DNAArtificial SequenceSynthetic oligonucleotide primer
9ccaaggctgg accaattgtc atggg 251027DNAArtificial sequenceSynthetic
oligonucleotide primer 10atctggcacc acaccttcta caatgag
271127DNAArtificial SequenceSynthetic oligonucleotide primer
11cgtggtggtg aagctgtagc cgcgctc 27129PRTHomo sapiens 12Tyr Leu Ser
Gly Ala Asp Leu Asn Leu 1 5 139PRTHomo sapiens 13Ile Leu Lys Glu
Pro Val His Gly Val 1 5 149PRTHomo sapiens 14Ser Gln Tyr Pro Ser
Leu Trp Ser Val 1 5 159PRTHomo sapiens 15Trp Leu Leu Pro Gly Thr
Ser Thr Leu 1 5 169PRTHomo sapiens 16Arg Leu Ile Ala Ser Trp Thr
Pro Val 1 5 1710PRTHomo sapiens 17Ala Met Tyr Ser Phe Leu Leu Asp
Phe Val 1 5 10 189PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa can be Leu
or Val 18Ser Xaa Tyr Xaa Ser Leu Xaa Ser Xaa 1 5 199PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa can be Ser, Thr, Ile, or Val 19Trp
Leu Leu Xaa Gly Thr Ser Thr Xaa 1 5 209PRTHomo
sapiensMISC_FEATURE(1)..(1)Xaa can be Tyr or Trp 20Xaa Leu Ile Ala
Ser Thr Thr Pro Val 1 5 219PRTHomo sapiensMISC_FEATURE(1)..(1)Xaa
can be Arg, Tyr or Trp 21Xaa Leu Ile Ala Ser Xaa Thr Pro Val 1 5
2210PRTHomo sapiens 22Ala Leu Tyr Ser Phe Leu Leu Asp Phe Val 1 5
10
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