Yeast-Brachyury therapeutic Compositions
CROSS-REFERENCE TO D APPLICATIONS
This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S.
Provisional Application Serial No. 61/453,656, filed March 17, 2011. The entire disclosure of
U.S. Provisional Application Serial No. 61/453,656, filed March 17, 2011 is incorporated
herein by reference.
[0001A] This application is a divisional of New Zealand Patent Application No. 711188, in
turn, a divisional application of New Zealand Patent Application No. 616696, the entire
contents of which are herein incorporated by reference.
GOVERNMENT RIGHTS
This invention was created in the mance of a Cooperative Research and
Development Agreement with the National Institutes of Health, an Agency of the Department
of Health and Human Services. The Government of the United States has certain rights in this
invention.
STATEMENT REGARDING JOINT RESEARCH AGREEMENT
This invention was made by or on behalf of parties to a Cooperative Research and
Development Agreement, ed May 8, 2008. The parties to the Cooperative Research and
Development Agreement are: mmune, Inc. and the U.S. Department of Health and
Human es, as represented by National Cancer Institute, an Institute, Center or Division
of the National Institutes of Health.
REFERENCE TO A SEQUENCE LISTING
This ation contains a Sequence Listing submitted electronically as a text file
by EFS-Web. The text file, named "3923PCT_ST25", has a size in bytes of 76 KB, and
was recorded on 13 March 2012. The information contained in the text file is incorporated
herein by nce in its entirety pursuant to 37 CFR § 1.52(e)(5).
FIELD OF THE INVENTION
The present invention generally relates to yeast-based immunotherapeutic
compositions and methods for the prevention and/or treatment of cancers terized by the
sion or overexpression of Brachyury.
BACKGROUND OF THE INVENTION
Brachyury, also known as “T”, is a mesodermal transcription factor and member
of the T-box complex of genes. The gene encoding Brachyury (denoted as either T gene or
ury gene in humans) was initially identified in 1927 by Nadine Dobrovolskaïa-
Zavadskaïa through a on in mice that affected tail length and sacral vertebrae in
heterozygous animals. The Brachyury gene was cloned in mice in 1990 by Hermann and
colleagues (Herrmann et al., 1990, Nature 343:617-622) and in humans in
1996 by Edwards and gues (Edwards et al., 1996, Genome Res. 6:226-223), who
also described the d amino acid sequence for human Brachyury.
As a member of the T-box family of transcription factors, Brachyury contains
the highly conserved DNA-binding domain motif, called “T-box” or in, which
binds to a palindromic consensus sequence. ury, like other T-box proteins, has
been shown to play a role in early development, and is vital for the formation and
differentiation of posterior mesoderm and axial development in vertebrates (see, e.g.,
son et al., 1990, Nature 343(6259):657—659); gton et al., 1992, Development
(Suppl.):157-165; Schulte-Merker et al., 1994, Development 120: 1009-1015; Kispert and
Herrmann, 1994, Dev. Biol. 161:179-193; Showell et al., 2004, Dev Dyn 229:201-218).
More recently, Palena and colleagues have demonstrated that Brachyury is expressed in a
variety of human tumor s and cancer cell lines and have shown that peptides of
Brachyury can be used to generate Brachyury-specific T cell lines in normal donors and
cancer patients (Palena et al., 2007, Clin. Cancer Res. 2471-2478). Studies by
Fernando et al. have shown that Brachyury promotes the epithelial-mesenchymal
transition (EMT) in human tumor cells, conferring on tumor cells a mesenchymal
phenotype, as well as migratory and invasive abilities, while attenuating tumor cell cycle
progression (Fernando et al., 2010, J. Clin. Invest. 120(2):533-544). Accordingly,
Brachyury is involved in metastatic progression of cancer.
Cancer is a leading cause of death worldwide, and the development of
effective ies for cancer continues to be one of the most active areas of research and
clinical pment. Although a variety of innovative approaches to treat and prevent
cancers have been proposed, many cancers continue to have a high rate of ity and
may be difficult to treat or relatively unresponsive to conventional therapies. Cancers
associated with Brachyury expression may be found in a variety of s, including
breast, small intestine, stomach, kidney, bladder, uterus, ovary, testes, lung, colon and
prostate, and includes metastatic and late-stage cancers. In addition, Brachyury is
expressed in tumors of B cell origin, such as chronic lymphocytic ia (CLL),
Epstein-Barr virus transformed B cells, Burkitt’s and Hodgkin’s lymphomas. Therefore,
Brachyury appears to play a role in a large number of human cancers. While Brachyury
has been proposed to be a target for cancer immunotherapy (see, e. g., Palena et al., supra,
Fernando et al., supra, and WC 2008/106551), since this is a relatively new cancer target,
there remains a need in the art for new immunotherapeutic products that effectively treat
and/or prevent cancers ated with Brachyury expression or overexpression.
SUMMARY OF THE INVENTION
One embodiment of the invention relates to a method to reduce, arrest, reverse,
delay or t the metastatic progression of cancer in an individual Who has cancer. The
method includes the step of administering to an individual Who has a cancer that is
undergoing metastatic progression, is at risk of oing metastatic progression, or is
predicted to begin undergoing metastatic progression, an therapeutic composition
comprising: (a) a yeast vehicle; and (b) a cancer antigen comprising at least one Brachyury
antigen. Another embodiment of the invention relates to the use of an immunotherapeutic
composition comprising a yeast vehicle and a cancer antigen comprising at least one
Brachyury antigen to reduce, arrest, reverse or t the metastatic progression of
cancer in an individual who has cancer.
In one aspect, of these ments of the ion, Brachyury is not
detected in the individual’s cancer at the time the composition is first administered. In one
aspect, Brachyury expression is detected in the individual’s cancer at the time the
composition is first administered. The individual may have stage I cancer, stage II cancer,
stage III cancer, or stage IV cancer.
Another ment of the ion relates to a method to prevent or delay
the onset of a Brachyury-expressing cancer. The method includes the step of
administering to an individual an immunotherapeutic composition comprising: (a) a yeast
vehicle; and (b) a cancer antigen comprising at least one Brachyury n. Another
embodiment of the invention relates to the use of an immunotherapeutic composition
sing a yeast vehicle and a cancer antigen comprising at least one Brachyury antigen
to prevent or delay the onset of a Brachyury-expressing cancer.
In one aspect of these ments, cancer has not been detected in the
individual. In one aspect, the individual is at high risk for ping cancer (e.g., via a
genetic predisposition). In one aspect, the individual has a pre-cancerous lesion.
In one aspect of these embodiments, the individual has cancer, but Brachyury-
expressing cancer cells have not been detected in the cancer. In one aspect, the cancer is
not yet metastatic. In one aspect, the cancer has a high risk of metastasizing. In one
aspect, the subject has stage I cancer. In one aspect, the subject has stage II cancer.
Another embodiment of the invention relates to a method to reduce or prevent
chemotherapy-resistance or radiation-resistance of tumor cells in a t With cancer.
The method includes the steps of administering to an individual Who has cancer and is
receiving herapy and/or radiation y an immunotherapeutic composition
comprising: (a) a yeast vehicle; and (b) a cancer antigen comprising at least one Brachyury
antigen. Another embodiment of the invention relates to the use of an immunotherapeutic
composition comprising a yeast vehicle and a cancer antigen comprising at least one
Brachyury antigen to reduce or prevent chemotherapy-resistance or radiation-resistance of
tumor cells in a patient With . In one aspect of this embodiment of the invention,
Brachyury is not detected in the individual’s cancer at the time the composition is first
administered. In one aspect, Brachyury sion is detected in the individual’s cancer at
the time the composition is first administered.
Yet another embodiment of the invention relates to a method to treat cancer.
The method includes the steps of: (a) stering to an individual Who has cancer in
Which Brachyury expression has not been detected, a first immunotherapeutic composition
comprising a yeast vehicle and a first cancer antigen that does not comprise a Brachyury
antigen; and (b) administering to the individual, prior to, rently With, sequentially
With, or subsequent to, administration of the first immunotherapeutic composition a
second immunotherapeutic composition comprising a yeast vehicle and a second cancer
n comprising a Brachyury antigen. In one aspect, the method further comprises, in
step (a), administering one or more additional immunotherapeutic compositions, wherein
the each of the one or more additional immunotherapeutic itions comprises an
additional cancer n. In one aspect of either embodiment above, the cancer antigen is
selected from: mutated Ras, carcinoembryonic antigen (CEA), MUC-l, EGFR, BCR-Abl,
MART-l, MAGE-l, MAGE-3, GAGE, GP-lOO, MUC-2, PSMA, tyrosinase, TRP-l
(gp75), NY-ESO-l, TRP-2, TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2, hTERT,
p73, B-RAF, adenomatous polyposis coli (APC), Myc, von Hippel-Lindau protein (VHL),
Rb-l, Rb-2, androgen receptor (AR), Smad4, MDRl, Flt-3, BRCA-l, BRCA-2, pax3-fl<hr,
ews-fii-l, HERV-H, HERV-K, TWIST, Mesothelin, and NGEP. In one aspect, the cancer
antigen is selected from the group consisting of: mutated Ras, carcinoembryonic antigen
(CEA), and MUC-l. Another embodiment of the invention relates to the use of a
combination of immunotherapeutic itions to treat cancer, the immunotherapeutic
itions comprising: (a) a first therapeutic composition comprising a yeast
vehicle and a first cancer antigen that does not se a Brachyury antigen; and (b) a
second immunotherapeutic composition comprising a yeast vehicle and a second cancer
antigen comprising a Brachyury n.
Yet another embodiment of the invention s to a method treat cancer. The
method includes the steps of: (a) administering to an individual Who has cancer a first
immunotherapeutic composition comprising a yeast e and a mutated Ras n; (b)
stering to the individual of (a) a second immunotherapeutic composition
comprising a yeast vehicle and an antigen selected from the group consisting of
carcinoembryonic antigen (CEA) and mucin-l (MUC-l); and (c) administering to the
individual of (a) and (b) a third immunotherapeutic composition comprising a yeast
vehicle and a Brachyury antigen. In one aspect, the steps of stration in (a), (b) and
(c) are concurrent. Another embodiment of the invention relates to the use of a
combination of immunotherapeutic compositions to treat cancer, the immunotherapeutic
compositions comprising: (a) a first immunotherapeutic composition comprising a yeast
vehicle and a mutated Ras antigen; (b) a second immunotherapeutic composition
comprising a yeast vehicle and an antigen selected from the group ting of
carcinoembryonic antigen (CEA) and mucin-l ); and (c) a third
therapeutic composition comprising a yeast vehicle and a Brachyury antigen.
In any of the embodiments or aspects of the invention described above or
elsewhere herein, where the individual has cancer or a precancerous lesion, in one aspect
of the invention, the individual is being treated or has been d with another therapy
for cancer. For example, such a therapy can include, but is not limited to, herapy,
targeted cancer therapy, radiation therapy, adoptive T cell transfer, and/or administration
of one or more additional immunotherapeutic compositions. In one aspect, an additional
immunotherapeutic ition comprises a yeast vehicle and a second cancer antigen
that does not include Brachyury antigen. The second cancer antigen can e, but is not
limited to, mutated Ras, carcinoembryonic antigen (CEA), MUC-l, EGFR, BCR-Abl,
MART-l, MAGE-l, MAGE-3, GAGE, GP-lOO, MUC-2, PSMA, nase, TRP-l
, NY-ESO-l, TRP-2, TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2, hTERT,
p73, B-RAF, adenomatous polyposis coli (APC), Myc, von -Lindau protein (VHL),
Rb-l, Rb-2, androgen receptor (AR), Smad4, MDRl, Flt-3, BRCA-l, BRCA-2, pax3-fl<hr,
ews-fli-l, HERV-H, HERV-K, TWIST, Mesothelin, and NGEP. In one aspect, the second
cancer antigen is selected from: mutated Ras, carcinoembryonic n (CEA), and
MUC-l.
In one aspect of any of the embodiments or aspects of the invention described
above or elsewhere herein, the method or use reduces tumor burden in the individual,
increases survival of the individual, and/or inhibits tumor growth in the individual.
In one aspect of any of the embodiments or aspects of the invention described
above or elsewhere herein, the method r comprises surgical resection of a tumor
from the individual.
In one aspect of any of the embodiments or aspects of the invention described
above or elsewhere herein, the cancer is of epithelial cell origin. In one aspect, the cancer
can e, but is not d to, breast cancer, small intestine cancer, stomach cancer,
pancreatic cancer, kidney cancer, bladder cancer, e cancer, ovarian cancer, testicular
cancer, lung cancer, colon cancer, prostate cancer, chronic cytic leukemia (CLL),
Epstein-Barr virus ormed B cells, Burkitt’s lymphoma, Hodgkin’s lymphoma, or
metastatic cancers f.
In one aspect of any of the embodiments or aspects of the invention described
above or elsewhere herein, the Brachyury antigen is full-length human Brachyury. In one
aspect, the Brachyury antigen is not full-length Brachyury. In one aspect, the Brachyury
antigen has an amino acid sequence represented by SEQ ID NO:6, SEQ ID NOil8, SEQ
ID N02, or an amino acid sequence that is at least 95% identical to SEQ ID NO:6, SEQ
ID NOil8, or SEQ ID NO:2. In one aspect, the Brachyury antigen comprises from at least
position 1 or 2 to between position 255 and the C-terminus of SEQ ID NO:6, SEQ ID
NOil8, or SEQ ID NO:2. In one aspect, the Brachyury antigen comprises from at least
position 1 or 2 to between position 430 and the C-terminus of SEQ ID NO:6, SEQ ID
NOil8, or SEQ ID NO:2. In one aspect, the ury n comprises positions 246 to
254 of SEQ ID NO:6, SEQ ID NOil8, or SEQ ID NO:2. In one aspect, the Brachyury
n comprises SEQ ID NO:6, positions 2-435 of SEQ ID NO:6, or an amino acid
sequence that is at least 95% identical to SEQ ID NO:6. In one aspect, the Brachyury
antigen comprises SEQ ID NO:l8, positions 2-435 of SEQ ID NO:l8, or an amino acid
sequence that is at least 95% cal to SEQ ID NO:l8. In one aspect, the Brachyury
antigen comprises SEQ ID NO:2, positions 2-435 of SEQ ID N02, or an amino acid
sequence that is at least 95% identical to SEQ ID NO:2. In one aspect, the Brachyury
antigen comprises SEQ ID NO:6, positions 2-435 of SEQ ID NO:6, or an amino acid
sequence that is at least 99% cal to SEQ ID NO:6. In one aspect, the Brachyury
antigen comprises SEQ ID NO:l8, positions 2-435 of SEQ ID NO:l8, or an amino acid
sequence that is at least 99% cal to SEQ ID NO:l8. In one aspect, the Brachyury
antigen comprises SEQ ID NO:2, ons 2-435 of SEQ ID N02, or an amino acid
sequence that is at least 99% identical to SEQ ID NO:2. In one aspect, the cancer antigen
is at least 25 amino acids in length. In one aspect, the Brachyury antigen is at least 25
amino acids in length. In one aspect, the Brachyury n is greater than 30 amino acids
in length. In one aspect, the cancer antigen comprises two or more immunogenic domains
of Brachyury.
In one aspect of any of the embodiments or aspects of the invention described
above or elsewhere herein, the cancer antigen is a fusion protein. In one aspect, the fusion
n has an amino acid sequence represented by SEQ ID NO:8, or an amino acid
sequence that is at least 95% identical to SEQ ID NO:8. In one aspect, the fusion protein
has an amino acid sequence represented by SEQ ID NO:20, or an amino acid ce
that is at least 95% identical to SEQ ID NO:20.
Another embodiment of the ion relates to a yeast-Brachyury
immunotherapeutic composition, wherein the immunotherapeutic composition comprises:
(a) a yeast vehicle; and (b) an antigen expressed by the yeast vehicle and comprising at
least one Brachyury antigen, wherein the Brachyury antigen comprises greater than 30
amino acids of an amino acid sequence represented by SEQ ID NO:6, SEQ ID NO:l8 or
SEQ ID NO:2. In one aspect, the Brachyury antigen comprises an amino acid sequence
that is at least 95% identical to SEQ ID NO:6, SEQ ID NO:l8 or SEQ ID NO:2. In one
, the Brachyury antigen comprises from at least on 1 or 2 to n position
255 and the C-terminus of SEQ ID NO:6, SEQ ID NO:l8 or SEQ ID NO:2. In one aspect,
the Brachyury antigen comprises from at least position 1 or 2 to n position 430 and
the C-terminus of SEQ ID NO:6, SEQ ID NO:l8 or SEQ ID NO:2. In one aspect, the
Brachyury antigen comprises positions 246 to 254 of SEQ ID NO:6, SEQ ID NOil8 or
SEQ ID NO:2. In one aspect, the Brachyury n comprises SEQ ID NO:6, ons
2-435 of SEQ ID NO:6, or an amino acid sequence that is at least 95% identical to SEQ ID
NO:6. In one aspect, the Brachyury antigen comprises SEQ ID NO:18, positions 2-435 of
SEQ ID NO:l8, or an amino acid sequence that is at least 95% identical to SEQ ID NOil8.
In one aspect, the Brachyury antigen comprises SEQ ID NO:2, ons 2-435 of SEQ ID
N02, or an amino acid sequence that is at least 95% identical to SEQ ID NO:2. In one
aspect, the Brachyury antigen comprises SEQ ID NO:6, positions 2-435 of SEQ ID NO:6,
or an amino acid sequence that is at least 99% identical to SEQ ID NO:6. In one aspect,
the Brachyury antigen comprises SEQ ID NO:l8, ons 2-435 of SEQ ID NOil8, or an
amino acid sequence that is at least 99% identical to SEQ ID NO:18. In one aspect, the
Brachyury antigen comprises SEQ ID NO:2, positions 2-435 of SEQ ID N02, or an
amino acid sequence that is at least 99% identical to SEQ ID NO:2. In one aspect, the
cancer antigen is a fusion protein. In one aspect, the fusion protein has an amino acid
sequence that is SEQ ID NO:8 or an amino acid sequence that is at least 95% identical to
SEQ ID NO:8. In one aspect, the fusion protein has an amino acid sequence of SEQ ID
NO:20 or an amino acid sequence that is at least 95% identical to SEQ ID NO:20. In one
aspect, the yeast vehicle is a whole yeast. In one aspect, the whole yeast is heat-
inactivated.
Yet another embodiment of the invention relates to a yeast-Brachyury
therapeutic composition comprising: (a) a whole, inactivated yeast; and (b) a
Brachyury fusion protein comprising the amino acid sequence of positions 2-435 of SEQ
ID NO:6. The expression of the Brachyury fusion protein is under the control of the
promoter CUPI , the Brachyury fusion protein is expressed by the yeast, and the
ition elicits a ury-specific T cell response. In one aspect, the fusion protein
comprises the amino acid sequence of SEQ ID NO:8.
Yet another embodiment of the invention relates to a yeast-Brachyury
immunotherapeutic composition comprising: (a) a whole, inactivated yeast; and (b) a
Brachyury fusion protein comprising the amino acid sequence of positions 2-435 of SEQ
ID NOil8. The expression of the Brachyury fusion protein is under the control of the
promoter CUPI , the Brachyury fusion protein is expressed by the yeast, and the
composition elicits a Brachyury-specific T cell response. In one aspect, the fusion protein
comprises the amino acid sequence of SEQ ID NO:20.
In one aspect of any of the embodiments or s of the invention described
above or ere herein, the yeast vehicle is a whole yeast. In one aspect, the whole
yeast is killed. In one aspect, the whole yeast is heat-inactivated. In one aspect, the yeast
expresses the antigen. In one , the yeast is from a genus selected from the group
consisting of: Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces,
Pichia, Rhodotorula, saccharomyces and Yarrowz'a. In one aspect, the yeast is from
Saccharomyces. In one aspect, the yeast is from Saccharomyces cerevisiae.
In one aspect of any of the embodiments of the invention described above or
elsewhere herein, the composition is formulated in a pharmaceutically acceptable
excipient suitable for administration to a subject.
Yet another embodiment of the invention relates to the use of any of the yeast-
Brachyury immunotherapeutic itions described herein to treat a disease. In one
, the disease is cancer. In one aspect, the disease is ated with an infectious
agent. In one aspect, the e is associated with a virus or viral ion. Such a virus
can e, but is not limited to, Epstein Barr Virus (EBV).
Another embodiment of the invention relates to a method to treat or prevent a
disease or condition associated with Epstein Barr Virus (EBV) infection. The method
includes the step of administering to an individual any of the yeast-Brachyury
immunotherapeutic compositions described herein.
Yet another embodiment of the invention relates to a method to produce a
yeast-Brachyury immunotherapeutic composition. The method includes the steps of: (a)
culturing yeast that have been transformed with a recombinant nucleic acid molecule
encoding a Brachyury antigen under the control of a CUPI er in a le medium
in the absence of CuSO4 until the yeast reach mid-log growth phase; (b) inducing
expression of the Brachyury antigen in the yeast by adding CuSO4 to the medium; (c)
culturing the yeast after step (b) for up to n 6 and 8 hours; and (d) harvesting the
yeast. In one aspect, the yeast in step (a) are cultured to a cell density of between 1.0 and
2.0 Y.U. per milliliter total culture volume. In one aspect, the yeast in step (a) are cultured
to a cell density of between 1.0 and 1.5 Y.U. per milliliter total culture volume. In one
aspect, the yeast are cultured in steps (a)-(c) in a medium where the pH is ined at
pH 5.5 or higher. In one aspect, the method additionally includes a step of heat-
inactivating the yeast after step (d). For e, in one aspect, the yeast are heat-
inactivated at about 56°C for about 1 hour. In a further aspect of this ment, the
yeast can be formulated for injection with a pharmaceutically acceptable excipient. In one
aspect, the yeast are from Saccharomyces. In one aspect, the yeast are from
Saccharomyces cerevisiae.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1A is a digitized image of a n blot showing detection by anti-
Brachyury of expression of Brachyury in a yeast-Brachyury immunotherapeutic
composition, with both U2 and UL2 media.
Fig. 1B is a digitized image of a Western blot showing ion by anti-His of
expression of Brachyury in a yeast-Brachyury immunotherapeutic composition, with both
U2 and UL2 media.
Fig. 2 is a digitized image of a Western blot showing expression of Brachyury
in a yeast-Brachyury immunotherapeutic ition where the cell density at antigen
induction and the time to harvest after n induction were varied.
Figs. 3A-3C are graphs showing that peripheral blood mononuclear cells
(PBMCs) from two out of three healthy donors pulsed with yeast-Brachyury for two
cycles of ation, followed by g with Brachyury CTL peptide, were capable of
generating CD8+ CTLs that could kill SW480 carcinoma cells (HLA-A2
positive/Brachyury high), with minimal lysis of MCF7 carcinoma 2
positive/Brachyury low); (Fig. 3A, donor 07706; Fig. 3B, donor 17663; Fig. 3C, donor
26532).
Fig. 4A is a graph showing that Brachyury-specific T cells from healthy donor
PBMCs ated with a yeast-Brachyury immunotherapeutic composition specifically
lyse tumor cells that have the appropriate MHC (SW480, HLA-A2 positive/Brachyury
high) versus H226 carcinoma cells (HLA-A2 negative/Brachyury high).
Fig. 4B is a graph showing the expression of Brachyury mRNA relative to that
of a control gene ) in the SW480 and H226 tumor cells used in the experiment
shown in Fig. 4A.
Fig. 5 is a graph showing proliferation of CD4+ T cells isolated from the
spleen of mice that were vaccinated with yeast-Brachyury (GI-6301, circles) or control
yeast (Yeast control, triangles), in response to indicated doses of purified Brachyury
n or control B-gal protein.
Fig. 6 graph showing that administration of a yeast-Brachyury
immunotherapeutic composition (GI-6301, circles) of the invention shows a trend towards
reducing ury-expressing tumors in mice compared to mice receiVing yeast alone
(no Brachyury antigen).
Figs. 7A and 7B are flow try analyses showing that the Brachyuryspecif1c
T cell line, TBR-A, binds to a Brachyury-specific HLA-A2 tetramer (Fig. 7B)
and not to a control tetramer (Fig. 7A).
Fig. 8 is a flow cytometry analysis showing the expression of in in the
Brachyury-specific T cell line, -A, after stimulation with Brachyury agonist
peptide-pulsed autologous B cells.
DETAILED DESCRIPTION OF THE INVENTION
This ion generally relates to yeast-based immunotherapeutic
compositions and methods for the prevention and/or treatment of cancers that express or
overexpress Brachyury. The invention es the use of a yeast-based
immunotherapeutic composition (also referred to as yeast-based immunotherapy)
comprising a yeast vehicle and Brachyury antigens or immunogenic domains f (also
referred to herein as “yeast-Brachyury immunotherapy” or “yeast-Brachyury
therapeutic compositions”). The inventors describe herein the construction and
production of novel yeast-Brachyury immunotherapy products, and demonstrate that
yeast-Brachyury therapy expands Brachyury-specific T cells, ing CD8+
CTLs, from normal individuals and from cancer patients. In addition, mice immunized
with yeast-Brachyury immunotherapeutic itions generated Brachyury-specific T
cell responses in vivo, and Brachyury-expressing tumor growth was ted in these mice.
Taken together, the data presented herein show that yeast-Brachyury immunotherapy is
useful for the elicitation of Brachyury-specific cellular immune responses (CD4+ and
CD8+) and for the prevention and treatment of Brachyury-expressing tumors, offering
novel therapy for the prevention and/or treatment of metastatic cancers and associated
ions.
Yeast-Brachyury immunotherapeutic compositions useful in the present
invention are uniquely adapted to effectively target Brachyury-expressing cancers for
several reasons. First, Brachyury is involved in EMT processes, and ore, without
being bound by theory, the ors believe that it plays a role in late-stage tumors and
metastatic processes. Accordingly, in one aspect of the invention, yeast-Brachyury
immunotherapy is ive at targeting tumor cells before or at the time during which they
begin to acquire motility and invade other tissues, thereby preventing, inhibiting, arresting,
reversing or delaying the onset of metastatic cancer and/or the progression of cancer, and
especially metastatic cancer. There is a great need for effective therapies for late stage
cancers, especially metastatic s, which may have few options for ent once
conventional cancer therapy has failed. Yeast-Brachyury presents a novel approach to
treat such cancers, or to delay, inhibit, reverse, or t them altogether. In addition,
yeast-Brachyury immunotherapy can be used to prevent or delay metastatic cancer or
progression of cancer in individuals who have early stage cancer. The therapy is useful, in
one embodiment, in cancers that have a high rate of metastatic progression, and may be
useful to arrest progression of the . Furthermore, yeast-Brachyury immunotherapy
is useful in individuals who have a precancerous (pre-malignant) lesion or tumor, in
individuals who are at a high risk for developing a cancer, particularly one that has a high
rate of metastases, and even in normal individuals as a prophylactic agent for the
prevention of cancer, which may be used in conjunction with other prophylactic
immunotherapy for cancer, such as described herein.
Yeast-Brachyury immunotherapy also provides a benefit to individuals who
are undergoing other therapy for cancer, including herapy and radiation therapy.
More particularly, metastatic cancers are known in some cases to be more resistant to
chemotherapy and/or radiation y than the primary s. Therefore, the yeast-
ury immunotherapy compositions of the invention can be used to inhibit or reduce
or ate chemotherapy resistance or radiation ance that may occur in atic
cancer by inhibiting Brachyury sion in the cancer (and thereby inhibiting anti-
proliferative influences), and compositions of the ion may enhance the mance
of chemotherapy or radiation therapy in an individual.
Yeast-Brachyury immunotherapy can also be used to treat conditions or
diseases associated with Brachyury expression that may be non-oncological in nature, or
that may precede ant transformation. For example, Brachyury may be upregulated
in cells that are infected with an infectious agent, e.g., a Virus such as Epstein Barr Virus
(EBV). Accordingly, yeast-Brachyury immunotherapy can be used to treat or prevent any
e or condition associated with ury expression, including, but not limited to,
infectious diseases, such as Viral infection, including, but not limited to, EBV-associated
conditions (e.g., mononucleosis).
Yeast-Brachyury immunotherapy is also readily ble to the use of
additional tumor antigens within the same yeast composition, or to use in combination
with other yeast-based immunotherapeutics that target other tumor antigens (sequentially
or concurrently) or other immunotherapeutics and treatments/therapies for cancer.
Accordingly, the yeast-Brachyury immunotherapy can be adapted to the cancer type, the
cancer stage, the cancer grade, the antigens expressed by the tumor, and the overall
l status of the indiVidual (i.e., the therapy is easily personalized), and for the
individual who already has cancer, its use can be modified as cancer progresses in an
indiVidual, in order to provide maximum cy at a variety of tumor stages. Yeast-
Brachyury therapy offers the opportunity to design sophisticated and effective,
individualized approaches for the broad-based prophylactic and/or therapeutic treatment of
a wide range of cancers.
Yeast-Brachyury compositions described herein induce innate immune
responses, as well as adaptive immune responses t the target antigen (Brachyury),
including CD4-dependent THl7 and THl T cell responses and antigen-specific CD8+ T
cell responses, which include xic T lymphocyte (CTL) responses, all without the use
of exogenous adjuvants, cytokines, or other immunostimulatory molecules, many of which
have toxicity issues. In addition, yeast-Brachyury immunotherapeutic compositions
inhibit regulatory T cell (Treg) numbers and/or functionality, thereby enhancing effector T
cell responses that might normally be suppressed by the presence of the tumor, for
example. Moreover, as compared to immunotherapeutic compositions that immunize by
generating antibody responses, the n-specific, broad-based, and potent cellular
immune responses elicited by Brachyury immunotherapy are believed to be
particularly effective in targeting tumor cells. , numerous studies have shown that
immunotherapeutic ches are enhanced when tumor cells are targeted via CD8+
CTLs which recognize tumor peptides in the context ofMHC Class I molecules.
Yeast-Brachyury immunotherapy is highly adept at ting antigen
presenting cells, and has a unique ability to prime the immune response, generating
CD8+ CTL responses that are typically effective against tumors, even in the face of what
may otherwise be a suppressive environment. Since this type of immunotherapy utilizes
the natural ability of the antigen presenting cell to t relevant immunogens, it is not
necessary to know the precise identity of CTL epitopes or MHC Class II epitopes of
Brachyury to produce an effective immunotherapeutic according to the present invention.
In fact, multiple CD4+ and CD8+ T cell epitopes can be targeted in a single yeast-
Brachyury immunotherapeutic composition, and so the yeast-Brachyury
immunotherapeutics of the invention are not limited to the use of short peptides and in fact,
the use of longer ptides and fusion proteins in these compositions is efficacious.
Accordingly, by using yeast-Brachyury immunotherapy, the use of algorithms and
complex formulas to fy putative T cell epitopes is eliminated.
Furthermore, since Brachyury is not expressed by most normal (non-tumor)
tissues, and is typically over-expressed in tumor cells, any “off ” effects related to
normal tissues are not of n. As mentioned above, yeast-Brachyury can be
effectively utilized in an immunization protocol (prophylactic or therapeutic) without the
use of exogenous adjuvants, immunostimulatory agents or molecules, costimulatory
molecules, or cytokines, gh such agents may be included, if desired. Moreover,
Brachyury immunotherapy can be administered repeatedly without losing efficacy,
as may be problematic with other types of immunotherapy.
Comgositl'ons 0: the ion
One embodiment of the present invention relates to a yeast-based
immunotherapy composition which can be used to prevent and/or treat cancers
characterized by Brachyury expression or overexpression (including cancers that may not
contain cells expressing detectable Brachyury initially, but which may or will contain cells
expressing ury at later stages of the development of the cancer). The composition
is a Brachyury immunotherapeutic composition comprising: (a) a yeast vehicle; and
(b) a cancer antigen comprising one or more Brachyury antigen(s) and/or immunogenic
domain(s) thereof. The Brachyury antigen or immunogenic domain thereof is most
typically expressed as a recombinant protein by the yeast vehicle (e.g., by an intact yeast
or yeast spheroplast, which can ally be further processed to a yeast cytoplast, yeast
ghost, or yeast membrane extract or fraction thereof), although it is an ment of the
invention that one or more Brachyury antigens are loaded into a yeast vehicle or otherwise
complexed with, ed to, mixed with or administered with a yeast vehicle as described
herein to form a composition of the present invention.
A “yeast-Brachyury therapeutic composition” is a specific type of
“yeast-based immunotherapeutic ition3, that contains at least one Brachyury
antigen or immunogenic domain thereof. The phrase, “yeast-based immunotherapeutic
composition” may be used interchangeably with -based immunotherapy product”,
-based therapy composition”, “yeast-based composition”, “yeast-based
immunotherapeutic”, “yeast-based vaccine”, or derivatives of these phrases. An
“immunotherapeutic composition” is a composition that elicits an immune response
ent to achieve at least one therapeutic benefit in a subject. As used , yeast-
based immunotherapeutic composition refers to a ition that es a yeast vehicle
component and that elicits an immune response sufficient to achieve at least one
eutic benefit in a subject. More particularly, a yeast-based immunotherapeutic
composition is a composition that includes a yeast vehicle component and typically, an
antigen component, and can elicit or induce an immune response, such as a cellular
immune se, including without limitation a T cell-mediated cellular immune
response. In one aspect, a yeast-based immunotherapeutic composition useful in the
invention is capable of inducing a CD8+ and/or a CD4+ T cell-mediated immune response
and in one aspect, a CD8+ and a CD4+ T cell-mediated immune response, particularly
against a target antigen (e. g., a cancer antigen). A CD4+ immune response can include
THl immune responses, TH2 immune responses, THl7 immune responses, or any
combination of the above. Yeast-based immunotherapeutics are particularly capable of
generating THl and THl7 responses. A CD8+ immune response can include a cytotoxic T
lymphocyte (CTL) response, and yeast-based immunotherapeutics are capable of
generating such responses. In one aspect, a yeast-based immunotherapeutic composition
modulates the number and/or functionality of regulatory T cells (Tregs) in a subject.
based immunotherapy can also be modified to promote one type of response over
another, e.g., by the addition of cytokines, antibodies, and/or modulating the
manufacturing process for the yeast. Optionally, a yeast-based therapeutic
composition is capable of eliciting a humoral immune response.
Brachyury immunotherapeutic compositions of the invention may be
either "prophylactic" or "therapeutic". When provided prophylactically, the compositions
of the present invention are provided in advance of the development of, or the detection of
the development of, a cancer that expresses Brachyury, with the goal of preventing,
inhibiting or delaying the development of Brachyury-expressing tumors; and/or preventing,
inhibiting or delaying tumor migration and/or tumor invasion of other tissues tases)
and/or generally preventing or inhibiting ssion of cancer in an individual. As
discussed herein, Brachyury is expressed in several cancers, including late-stage cancers,
and has been shown to be involved in the EMT process, which is a process associated with
veness and migration of tumors, such as in metastatic cancer. ore,
prophylactic compositions can be administered to individuals that appear to be cancer-free
(healthy, or normal, individuals), to duals with pre-cancerous (pre-malignant lesions),
and also to individuals who have cancer, but in which Brachyury has not yet been ed
(Le. prior to the expression of Brachyury by tumor cells in the cancer). Individuals who
are at high risk for developing a cancer, particularly a cancer with which Brachyury
expression and/or metastases are typically associated, may be treated prophylactically with
a composition of the invention. When provided therapeutically, the immunotherapy
compositions are provided to an individual with a Brachyury-expressing cancer, with the
goal of rating the cancer, such as by reducing tumor burden in the individual;
inhibiting tumor growth in the individual; increasing survival of the individual; preventing,
inhibiting, reversing or delaying development of tumor ion and/or tumor invasion of
other tissues (metastatic cancer) and/or preventing, ting, reversing or delaying
progression of the cancer in the individual. In one aspect, yeast-Brachyury
immunotherapy is used therapeutically to inhibit, reduce or ate chemotherapy
resistance or radiation ance that may occur in metastatic cancer by inhibiting
Brachyury expression in the cancer, and compositions of the invention may enhance the
performance of herapy or radiation therapy in an individual.
Typically, a yeast-Brachyury immunotherapy composition includes a yeast
vehicle and at least one cancer antigen comprising a Brachyury antigen or immunogenic
domain thereof, where the cancer antigen is expressed by, attached to, loaded into, or
mixed with the yeast vehicle. In some embodiments, the cancer antigen, Brachyury
n, or immunogenic domain thereof is provided as a fusion protein. Several
Brachyury proteins and fusion proteins le for use in the itions and s
of the invention are described below. In some ments, the cancer antigen and the
Brachyury antigen are the same element. In some embodiments, the cancer antigen
includes other antigens, including other cancer antigens, in addition to the Brachyury
n. In one aspect of the invention, a fusion protein useful as a cancer antigen can
include two or more antigens, e.g., a Brachyury antigen and another cancer antigen that is
not a Brachyury antigen, or two different Brachyury antigens. In one aspect, the fusion
protein can include two or more immunogenic domains of one or more antigens, such as
two or more immunogenic domains of a Brachyury antigen, or two or more epitopes of
one or more antigens, such as two or more epitopes of a Brachyury antigen.
ing to the present invention, a yeast e used in a yeast-Brachyury
immunotherapy composition is any yeast cell (e.g., a whole or intact cell) or a derivative
thereof (see below) that can be used in conjunction with one or more antigens,
immunogenic domains thereof or es thereof in a composition of the invention (e.g.,
a therapeutic or prophylactic composition). The yeast vehicle can therefore e, but is
not limited to, a live intact (whole) yeast rganism (i.e., a yeast cell having all its
components including a cell wall), a killed (dead) or inactivated intact yeast
microorganism, or derivatives of intact yeast including: a yeast spheroplast (i.e., a yeast
cell lacking a cell wall), a yeast cytoplast (i.e., a yeast cell lacking a cell wall and nucleus),
a yeast ghost (i.e., a yeast cell lacking a cell wall, nucleus and cytoplasm), a subcellular
yeast membrane extract or fraction thereof (also referred to as a yeast membrane particle
and previously as a subcellular yeast particle), any other yeast particle, or a yeast cell wall
preparation.
Yeast spheroplasts are typically produced by enzymatic digestion of the yeast
cell wall. Such a method is described, for example, in Franzusoff et al., 1991, Meth.
Enzymol. 194, 662-674., incorporated herein by reference in its entirety.
Yeast cytoplasts are typically produced by enucleation of yeast cells. Such a
method is described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55
incorporated herein by reference in its ty.
Yeast ghosts are typically produced by resealing a bilized or lysed cell
and can, but need not, contain at least some of the organelles of that cell. Such a method
is described, for example, in Franzusoff et al., 1983, J. Biol. Chem. 258, 3608-3614 and
Bussey et al., 1979, Biochim. s. Acta 553, 185-196, each of which is incorporated
herein by reference in its entirety.
A yeast membrane particle (subcellular yeast membrane t or fraction
thereof) refers to a yeast membrane that lacks a natural nucleus or cytoplasm. The particle
can be of any size, ing sizes ranging from the size of a natural yeast membrane to
microparticles produced by sonication or other membrane disruption methods known to
those d in the art, followed by resealing. A method for producing subcellular yeast
ne extracts is described, for example, in Franzusoff et al., 1991, Meth. Enzymol.
194, 662-674. One may also use fractions of yeast membrane particles that contain yeast
membrane portions and, when the antigen or other protein was expressed recombinantly
by the yeast prior to ation of the yeast membrane particles, the antigen or other
protein of interest. Antigens or other proteins of interest can be carried inside the
membrane, on either surface of the ne, or combinations thereof (i.e., the protein
can be both inside and outside the membrane and/or ng the membrane of the yeast
membrane particle). In one embodiment, a yeast ne particle is a recombinant
yeast membrane le that can be an intact, disrupted, or disrupted and resealed yeast
membrane that includes at least one desired antigen or other protein of interest on the
surface of the membrane or at least partially embedded within the membrane.
An example of a yeast cell wall preparation is a preparation of isolated yeast
cell walls carrying an antigen on its surface or at least partially ed within the cell
wall such that the yeast cell wall preparation, when administered to an animal, stimulates a
desired immune se against a disease target.
Any yeast strain can be used to produce a yeast vehicle of the present
invention. Yeast are unicellular microorganisms that belong to one of three classes:
Ascomycetes, Basidiomycetes and Fungi Imperfecti. One consideration for the selection
of a type of yeast for use as an immune modulator is the pathogenicity of the yeast. In one
embodiment, the yeast is a non-pathogenic strain such as Saccharomyces siae. The
selection of a non-pathogenic yeast strain minimizes any adverse effects to the individual
to whom the yeast vehicle is administered. However, pathogenic yeast may be used if the
pathogenicity of the yeast can be negated by any means known to one of skill in the art
(e.g., mutant strains). In accordance with one aspect of the t invention, non-
pathogenic yeast strains are used.
Genera of yeast strains that may be used in the invention include but are not
limited to Saccharomyces, Candida (which can be pathogenic), coccus, Hansemda,
Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowz'a. In one aspect,
yeast genera are selected from Saccharomyces, Candida, Hansemda, Pichia or
Schizosaccharomyces, and in one , Saccharomyces is used. s of yeast strains
that may be used in the invention include but are not limited to Saccharomyces cerevisiae,
Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis,
Cryptococcus laurentii, Cryptococcus neoformans, Hansenula a, Hansenula
polymorpha, Kluyveromycesfragilis, Kluyveromyces lactis, Kluyveromyces nus var.
, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia
lipolytica. It is to be appreciated that a number of these species include a variety of
subspecies, types, es, etc. that are intended to be included within the
aforementioned species. In one aspect, yeast species used in the invention include S.
cerevisiae, C. albicans, H. polymorpha, P. pastoris and S. pombe. S. siae is useful
as it is relatively easy to late and being "Generally ized As Safe" or
"GRAS" for use as food additives (GRAS, FDA proposed Rule 62FR18938, April 17,
1997). One ment of the present invention is a yeast strain that is capable of
replicating plasmids to a particularly high copy number, such as a S. cerevisiae cir° strain.
The S. cerevisiae strain is one such strain that is capable of supporting expression vectors
that allow one or more target antigen(s) and/or antigen fusion protein(s) and/or other
proteins to be expressed at high levels. Another yeast strain is useful in the invention is
Saccharomyces cerevisiae W303 or. In addition, any mutant yeast strains can be used in the
present invention, including those that exhibit reduced post-translational ations of
expressed target antigens or other proteins, such as mutations in the enzymes that extend
N—linked ylation.
The yeast-Brachyury immunotherapy composition of the invention includes at
least one cancer antigen comprising a Brachyury antigen. According to the present
invention, the general use herein of the term "antigen" refers: to any portion of a protein
(e.g., e, partial protein, full-length protein), wherein the protein is naturally
occurring or synthetically derived or designed, to a cellular composition (whole cell, cell
lysate or disrupted cells), to an organism (whole organism, lysate or disrupted cells) or to a
carbohydrate, or other molecule, or a n thereof. An antigen may elicit an antigenspecific
immune response (e. g., a humoral and/or a cell-mediated immune response)
against the same or similar antigens that are encountered by an element of the immune
system (e.g., T cells, antibodies).
An antigen can be as small as a single epitope, a single immunogenic domain
or , and can include multiple epitopes or genic domains. As such, the size of
an antigen can be as small as about 8-11 amino acids (i.e., a peptide) and as large as: a full
length protein, a multimer, a fusion protein, a chimeric n, a Whole cell, a Whole
microorganism, or any portions thereof (e.g., n nts (polypeptides) lysates of
Whole cells or extracts of microorganisms). Antigens useful in the yeast-Brachyury
immunotherapeutic of the present invention are peptides, polypeptides, full-length proteins,
multimers, fusion proteins and ic proteins. In addition, antigens can include
carbohydrates, Which can be loaded into a yeast vehicle or into a composition of the
invention. It Will be appreciated that in some embodiments (e.g., When the antigen is
expressed by the yeast vehicle from a recombinant nucleic acid molecule), the antigen is a
protein, fusion protein, chimeric protein, or fragment f, rather than an entire cell or
microorganism. For expression in yeast, an antigen is of a minimum size capable of being
expressed recombinantly in yeast if the antigen is the entire protein to be expressed by the
yeast, and is typically at least or greater than 25 amino acids in , or at least or greater
than 26, at least or greater than 27, at least or greater than 28, at least or greater than 29, at
least or greater than 30, at least or greater than 31, at least or greater than 32, at least or
r than 33, at least or greater than 34, at least or greater than 35, at least or greater
than 36, at least or greater than 37, at least or greater than 38, at least or greater than 39, at
least or greater than 40, at least or greater than 41, at least or r than 42, at least or
greater than 43, at least or greater than 44, at least or greater than 45, at least or greater
than 46, at least or greater than 47, at least or greater than 48, at least or greater than 49, or
at least or greater than 50 amino acids in length, or at least or r than 25-50 amino
acids in length, or at least or greater than 30-50 amino acids in length, or at least or r
than 35-50 amino acids in length, or at least or greater than 40-50 amino acids in length, or
at least or greater than 45-50 amino acids in length, although smaller proteins may be
sed, and considerably larger proteins (e.g., hundreds of amino acids in length or
even a few thousand amino acids in ) may be expressed. In one , a full-length
protein or a protein that is lacking between 1 and 20 amino acids from the N— and/or the C-
terminus may be expressed. Fusion proteins and chimeric proteins are also antigens that
may be expressed in the invention. A “target antigen” is an antigen that is specifically
targeted by an immunotherapeutic composition of the invention (i.e., an antigen against
Which elicitation of an immune response is desired). A “cancer antigen” is an antigen that
comprises at least one antigen that is associated With a cancer such as an antigen expressed
by a tumor cell, such that targeting the n also targets the cancer. A cancer antigen
can include one or more ns from one or more proteins, including one or more tumor-
associated proteins. A “Brachyury antigen” is an antigen derived, designed, or produced
from a Brachyury protein.
When referring to stimulation of an immune response, the term “immunogen”
is a subset of the term “antigen”, and therefore, in some instances, can be used
interchangeably with the term "antigen". An gen, as used herein, describes an
antigen which elicits a humoral and/or cell-mediated immune response (i.e., is
immunogenic), such that stration of the immunogen to an individual mounts an
n-specific immune response against the same or similar antigens that are
encountered by the immune system of the individual. In one ment, the gen
elicits a cell-mediated immune response, including a CD4+ T cell response (e.g., THl,
TH2 and/or THl7) and/or a CD8+ T cell response (e.g., a CTL response).
An “immunogenic domain” of a given antigen can be any portion, fragment or
epitope of an n (e.g., a peptide fragment or subunit or an antibody epitope or other
mational epitope) that contains at least one epitope that can act as an immunogen
when administered to an animal. Therefore, an immunogenic domain is larger than a
single amino acid and is at least of a size sufficient to contain at least one e that can
act as an immunogen. For example, a single protein can contain multiple different
immunogenic s. Immunogenic domains need not be linear sequences within a
protein, such as in the case of a humoral immune response, where conformational domains
are plated.
An epitope is defined herein as a single immunogenic site within a given
antigen that is sufficient to elicit an immune response when provided to the immune
system in the context of appropriate costimulatory signals and/or activated cells of the
immune . In other words, an epitope is the part of an antigen that is recognized by
components of the immune system, and may also be referred to as an antigenic
determinant. Those of skill in the art will recognize that T cell epitopes are different in
size and composition from B cell or antibody epitopes, and that epitopes presented through
the Class I MHC pathway differ in size and structural attributes from epitopes presented
through the Class II MHC pathway. For example, T cell epitopes presented by Class I
MHC molecules are typically between 8 and 11 amino acids in length, whereas epitopes
presented by Class II MHC les are less restricted in length and may be up to 25
amino acids or longer. In addition, T cell epitopes have ted structural characteristics
depending on the specific MHC les bound by the epitope. Epitopes can be linear
sequence epitopes or conformational epitopes (conserved binding regions). Most
antibodies recognize conformational epitopes.
Brachyury (which may also be referred to as “T”) is a highly conserved
protein among multiple different animal species and is a transcription factor that contains a
“T-box” domain or “T-domain”, a DNA-binding domain motif shared among l
different proteins, collectively called the T-box family of proteins. Human Brachyury was
first cloned in 1996 (Edwards et al., supra). One nucleotide sequence encoding human
Brachyury is represented herein by SEQ ID NO:l, which is an mRNA sequence that was
obtained from GENBANK® Accession No. NM_003l8l (GI:l97438l l). SEQ ID NO:l
encodes a 435 amino acid human Brachyury n, the amino acid sequence of which is
represented here as SEQ ID NO:2 (also found in GENBANK® Accession No. NP_003 172;
GI:4507339).
Another human Brachyury n disclosed herein is a variant of the human
Brachyury protein represented by SEQ ID N02, and has the amino acid sequence of SEQ
ID NO:6. SEQ ID NO:6, also a 435 amino acid protein, is encoded by a nucleotide
sequence represented herein by SEQ ID NO:5. SEQ ID NO:6 is approximately 99%
identical to SEQ ID NO:2 over the ength of the protein. SEQ ID NO:6 s from
SEQ ID NO:2 at position 177 (Asp vs. Gly, respectively), position 368 (Thr vs. Ser,
respectively) and position 409 (Asn vs. Asp, respectively).
Another human Brachyury protein sed herein is an t of the human
Brachyury protein ented by SEQ ID N02 or SEQ ID NO:6. As generally used
herein, an “agonist” is any compound or agent, including without limitation small
molecules, proteins, es, antibodies, nucleic acid binding agents, etc., that binds to a
receptor or ligand and produces or triggers a response, which may e agents that
mimic or enhance the action of a naturally occurring substance that binds to the receptor or
ligand. When used in the context of a Brachyury antigen of the invention, an “agonist”
n or protein refers to an antigen or protein that comprises at least one T cell agonist
epitope, which may also be referred to as a “mimotope”. A mimotope peptide is a peptide
that mimics the structure of a wild-type epitope and as an agonist, the mimotope mimics or
enhances the action (biological function) of the l epitope. For example, the amino
acid sequence of SEQ ID NO:12 (WLLPGTSTL) is a T cell epitope of a wild-type
Brachyury protein. The amino acid sequence of SEQ ID NOil3 (WLLPGTSTV) is a
mimotope or t of the T cell epitope of SEQ ID NO: 12.
One human Brachyury agonist antigen is represented here by SEQ ID NO:l8.
SEQ ID NO:l8 is a 435 amino acid protein is encoded by a nucleotide sequence
represented herein by SEQ ID NO:l7. SEQ ID NO:l8 is identical to SEQ ID NO:6,
except for a tution of a leucine at position 254 with respect to SEQ ID NO:6 with a
valine in SEQ ID NO:l8. This substitution creates a T cell agonist e in SEQ ID
NO:l8 at positions 246 to 254 that, without being bound by theory, is believed to induce
enhanced T cell ses against Brachyury as compared to the ype epitope
(positions 246 to 254 of SEQ ID NO:6).
Positions 41 to 223 of any of SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:l8
represent the T-box DNA binding domain of human Brachyury, and the T-box domain in
other Brachyury sequences, including Brachyury sequences from other species, can be
readily identified by comparison to these sequences. As used herein, reference to a T-box
domain of any Brachyury protein described herein or known in the art and utilized in the
ion may include an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, l4, l5, l6, l7,
l8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40
consecutive amino acids of the Brachyury sequence on the N—terminal and/or the C-
terminal end of the defined T-box domain (e.g., on either side of positions 41-223 of SEQ
ID NOs:2, 6 or 18). Human Brachyury, including the two human ury proteins
described herein, also contains various CD4+ and CD8+ T cell epitopes. Such epitopes
have been described, for example, in WC 2008/106551, and include a CD8+ CTL epitope,
WLLPGTSTL (also referred to herein as Tp2, SEQ ID NO:l2), at positions 246 to 254 of
SEQ ID NO:2 or SEQ ID NO:6. As sed above, SEQ ID NO:l8 comprises an
agonist epitope of SEQ ID NO: 12, ented herein by SEQ ID NO: 13.
Human Brachyury has very high homology with Brachyury from other animal
species and therefore, one is able to utilize the sequences of Brachyury from other
organisms in the preparation of a yeast-Brachyury therapeutic composition of the
invention, particularly where these sequences are identical, substantially gous, and
elicit an effective immune response against the target n (e.g., native Brachyury
expressed by a tumor cell). For example, murine Brachyury, which was first cloned by
Hermann and colleagues in l990 (Hermann et al., supra) is approximately 85% identical
to human Brachyury at the nucleotide level, and approximately 91% identical at the amino
acid level. With t to Brachyury from other animals, at the amino acid level, human
Brachyury is 99.5% identical to Brachyury from Pan troglodytes, 90.1% identical to
Brachyury from Cam's lupus familiaris, 88.5% identical to Brachyury from Bos Taurus,
92.2% identical to Brachyury from Rattus norvegicus, and 80.9% identical to Brachyury
from Gallus gallus. Within amino acids 1-223 of Brachyury, which contains the T-box
domain, mouse and human ury differ by only two amino acids (at positions 26 and
96). A nucleotide sequence encoding murine Brachyury is represented herein by SEQ ID
NO:3, which is an mRNA sequence that was obtained from GENBANK® Accession No.
NM_009309 (GI:ll8l30357). SEQ ID NO:3 encodes a 436 amino acid murine Brachyury
protein, the amino acid ce of which is represented here as SEQ ID NO:4. Positions
41 to 223 of SEQ ID NO:4 represent the T-box DNA binding domain of murine
Brachyury.
In one embodiment of the invention, a Brachyury antigen comprises or
consists of the amino acid ce represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:l8, or at least one immunogenic domain thereof. In one embodiment,
a Brachyury antigen comprises or ts of two, three, four, five, or more immunogenic
domains of Brachyury. In one ment of the invention, a Brachyury n
comprises or consists of the amino acid sequence represented by amino acid ons 1 or
2 through one of the last 25 amino acids at the C-terminus of SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6 or SEQ ID NO:l8 (1'. 6., through any one of positions 441 to 435 of SEQ ID
NO:2 or SEQ ID NO:6 or SEQ ID NO:l8, or through any one of positions 442 to 436 of
SEQ ID NO:4). Another Brachyury n useful in the invention also includes at least
amino acid positions 1-223 of Brachyury (e.g., positions 1-223 of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6 or SEQ ID NO:l8) or positions 41-223 of Brachyury (e.g., positions
41-223 of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:l8). Another
Brachyury antigen useful in the invention includes from at least amino acid positions 1 to
85 to between position 255 and the C-terminus of SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6 or SEQ ID NO: 1 8. Another Brachyury n useful in the invention includes from
at least amino acid positions 1 to 85 to between position 430 and the C-terminus of SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:l8. Another Brachyury antigen
useful in the invention includes from at least amino acid positions 1, 2, 3, 4, 5, 6, 7, 8, 9 or
to between position 255 and the C-terminus of SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6 or SEQ ID NO:l8.
According to any embodiment of the present invention, reference to a “full-
length” protein (or a ength functional domain or full-length immunological )
includes the full-length amino acid sequence of the protein or functional domain or
immunological domain, as described herein or as otherwise known or described in a
publicly available sequence. A protein or domain that is “near full-length”, which is also a
type of homologue of a protein, differs from a full-length n or domain, by the
addition or deletion or omission of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N-
and/or C-terminus of such a full-length protein or full-length domain. By way of e,
several of the fusion proteins described herein comprise a “near full-length” Brachyury
antigen since the n omits the methionine at position 1 and substitutes an inal
peptide. General reference to a protein or domain or antigen can include both full-length
and near full-length proteins, as well as other homologues thereof.
In one aspect of any ments related to a Brachyury antigen, a cancer
antigen or a Brachyury antigen is of a minimum size sufficient to allow the antigen to be
expressed by yeast. For expression in yeast, a protein is typically at least about 25 amino
acids in length, although smaller proteins may be expressed, and considerably larger
proteins may be expressed by yeast. For example, a Brachyury antigen useful in the
invention is a fragment of a ury protein that can be expressed recombinantly by
yeast and that contains at least one immunogenic domain of Brachyury, which could
include at least one immunogenic domain of any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6 or SEQ ID NO:18. In one aspect, such an antigen is at least 25 amino acids in
length, and ns at least one immunogenic domain of ury. In one aspect, such
an n is greater than 30 amino acids in length, and contains at least one genic
domain of Brachyury. In one , such an antigen is at least 25-50 amino acids in
length, and contains at least one immunogenic domain of ury. In one aspect, such
an antigen is at least 30-50 amino acids in length, and contains at least one immunogenic
domain of Brachyury. In one aspect, such an n is at least 35-50 amino acids in
length, and contains at least one immunogenic domain of Brachyury. In one aspect, such
an antigen is at least 40-50 amino acids in length, and contains at least one immunogenic
domain of Brachyury. In one aspect, such an antigen is at least 45-50 amino acids in
length, and contains at least one immunogenic domain of Brachyury. In one embodiment,
the Brachyury antigen useful in the present invention is at least 25 amino acids in length,
or at least: 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,
125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,
215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300,
305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390,
395, 400, 405, 410, 415, 420, 425, or 430 amino acids in length, which can include any
fragment of at least any of these lengths of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or
SEQ ID NO: 1 8.
In one aspect, a Brachyury antigen comprises one or more CTL epitopes,
Which may include two or more copies of any one, two, three, or more of the CTL
epitopes described herein. In one aspect, the Brachyury antigen comprises one or more
CD4+ T cell epitopes. The T cell In one aspect, the Brachyury antigen comprises one or
more CTL epitopes and one or more CD4+ T cell epitopes. In one aspect, the T cell
epitope is an agonist epitope.
In one aspect, a Brachyury antigen comprises an amino acid sequence of
WLLPGTSTL (SEQ ID NO:l2, also represented by positions 245 to 254 of SEQ ID N02
or SEQ ID NO:6). In one aspect, the Brachyury antigen comprises an amino acid
sequence of WLLPGTSTV (SEQ ID NO:l3, also represented by positions 245 to 254 of
SEQ ID NO:l8). In one aspect, the amino acid at on 4 of either SEQ ID NO:l2 or
SEQ ID NO:13 (a proline or P in these sequences) is substituted With a serine (S), a
ine (T), an isoleucine (I), or a valine (V).
In one aspect, the Brachyury antigen comprises an amino acid sequence of
SQYPSLWSV (SEQ ID . In one aspect, the amino acid at position 2 of SEQ ID
NO:14 (a glutamine or Q in this ce) is substituted With a e (L). In one ,
the amino acid at position 4 of SEQ ID NO:l4 (a proline or P in this sequence) is
substituted With a serine (S), threonine (T), leucine (L), or valine (V). In one , the
amino acid at position 7 of SEQ ID NO:l4 (a tryptophan or W in this sequence) is
substituted With a valine (V), leucine (L), isoleucine (I), serine (S), or threonine (T). In
one aspect, the amino acid at on 9 of SEQ ID NO: 14 (a valine or V in this sequence)
is substituted With a leucine (L). An antigen comprising a sequence haVing any
combination of one or more of these tutions in SEQ ID NO:l4 is contemplated by
the invention.
In one aspect, the Brachyury antigen comprises an amino acid sequence of
RLIASWTPV (SEQ ID NO:l5). In one aspect, the amino acid at position 1 of SEQ ID
NO:15 (an arginine or R in this sequence) is substituted With a tyrosine (Y) or a
tryptophan (W). In one aspect, the amino acid at position 6 of SEQ ID NO:15 (a
tryptophan or W in this sequence) is substituted With a valine (V), a lysine (L), an
isoleucine (I), a serine (S), or a threonine (T). An antigen comprising a ce haVing
any combination of one or both of these substitutions in SEQ ID NO:15 is contemplated
by the invention.
In one aspect, the Brachyury antigen comprises an amino acid sequence of
AMYSFLLDFV (SEQ ID NO:l6). In one aspect, the amino acid at position 2 of SEQ ID
NO: 16 (a nine or M in this sequence) is substituted with a leucine (L).
In one embodiment of the invention, a Brachyury antigen ses, consists
essentially of, or consists of a fusion protein having the amino acid sequence of SEQ ID
NO:8. The fusion n of SEQ ID NO:8 is a single polypeptide with the following
sequence elements fused in frame from N— to C-terminus: (1) an N—terminal peptide to
impart resistance to proteasomal degradation and stabilize expression in yeast (positions 1-
6 of SEQ ID NO:8); (2) a human Brachyury antigen consisting of positions 2-435 of SEQ
ID NO:6 (positions 7-440 of SEQ ID NO:8); and (3) a hexahistidine tag (positions 441-
446 of SEQ ID NO:8). The amino acid sequence of SEQ ID NO:8 is encoded by the
polynucleotide sequence of SEQ ID NO:7.
In another embodiment of the invention, a Brachyury n comprises,
consists essentially of, or consists of a fusion protein having the amino acid sequence of
SEQ ID NOilO. The fusion protein of SEQ ID NO:lO is a single polypeptide with the
ing sequence elements fused in frame from N- to inus: (1) an N—terminal
peptide to impart resistance to proteasomal degradation and stabilize expression in yeast
ions 1-6 of SEQ ID NO:lO); (2) a murine Brachyury n consisting of positions
2-436 of SEQ ID NO:4 (positions 7-441 of SEQ ID NO:lO); and (3) a hexahistidine tag
(positions 442-447 of SEQ ID NO:10). The amino acid sequence of SEQ ID NO:lO is
encoded by the polynucleotide sequence of SEQ ID NO:9.
In another embodiment of the invention, a Brachyury antigen comprises,
consists essentially of, or consists of a fusion protein having the amino acid sequence of
SEQ ID NO:20. The fusion protein of SEQ ID NO:20 is a single ptide with the
following sequence elements fused in frame from N- to inus: (1) an N—terminal
peptide to impart resistance to proteasomal degradation and stabilize expression (positions
1 to 6 of SEQ ID NO:20, the peptide sequence also represented herein by SEQ ID NO: 1 l);
2) amino acids 2-435 of SEQ ID NO:l8 (positions 7-440 of SEQ ID NO:20), SEQ ID
NO:l8 representing a full-length human Brachyury agonist protein; and (3) a hexahistidine
tag ions 6 of SEQ ID NO:20). The t epitope (SEQ ID NO:l3) is
located at positions 251 to 259 of SEQ ID NO:20 (positions 246 to 254 of SEQ ID NO:l8).
The amino acid sequence of SEQ ID NO:20 is encoded by the polynucleotide sequence of
SEQ ID NO: 19.
A Brachyury antigen useful in the present ion also includes proteins
having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of
any of the Brachyury proteins or antigens described herein over the full length of the
protein, or with respect to a defined fragment or domain f (e.g., an immunological
domain or functional domain (domain with at least one ical actiVity)) that forms part
of the protein. For example, a domain of the Brachyury protein described herein includes
the T-box domain. An immunological domain has been described in detail above.
In some aspects of the invention, amino acid insertions, deletions, and/or
substitutions can be made for one, two, three, four, five, six, seven, eight, nine, ten, or
more amino acids of a wild-type or reference Brachyury protein, provided that the
resulting Brachyury protein, when used as an antigen in a yeast-Brachyury
immunotherapeutic composition of the invention, elicits an immune response against a
native Brachyury protein as the wild-type or reference Brachyury protein, which may
e an enhanced immune response, a diminished immune response, or a substantially
similar immune response. For example, the invention includes the use of Brachyury
t ns, which may include one or more T cell epitopes that have been mutated to
enhance the T cell response against the Brachyury agonist, such as by improving the
aVidity or y of the epitope for an MHC le or for the T cell receptor that
recognizes the epitope in the context of MHC presentation. Brachyury agonists may
therefore improve the potency or ncy of a T cell response against native Brachyury
expressed by a tumor cell. The Brachyury antigen haVing the amino acid sequence of
SEQ ID NO:l8 is a miting example of a Brachyury agonist (or a Brachyury antigen
comprising an t epitope).
In addition, N—terminal expression sequences and the C-terminal tags, such as
those described above with respect to the fusion ns of SEQ ID NO:8, SEQ ID NO: 10,
or SEQ ID NO:20 are al, but may be selected from several different sequences
described elsewhere herein to improve or assist with expression, stability, and/or allow for
identification and/or purification of the protein. Also, many different promoters suitable
for use in yeast are known in the art. Furthermore, short intervening linker sequences (e.g.,
l, 2, 3, 4, or 5 amino acid peptides) may be introduced between portions of a fusion
protein comprising a Brachyury antigen for a variety of reasons, including the introduction
of restriction enzyme sites to facilitate cloning, as ge sites for host phagosomal
proteases, to accelerate protein or antigen processing, and for future manipulation of the
constructs.
Optionally, ns, ing fusion proteins, which are used as a component
of the yeast-Brachyury immunotherapeutic composition of the invention are produced
using n constructs that are particularly useful for improving or stabilizing the
expression of heterologous antigens in yeast. In one embodiment, the desired antigenic
protein(s) or peptide(s) are fused at their amino-terminal end to: (a) a ic synthetic
peptide that stabilizes the expression of the fusion protein in the yeast vehicle or prevents
posttranslational modification of the expressed fusion n (such peptides are described
in detail, for example, in US. Patent Publication No. 2004-0156858 Al, published August
12, 2004, incorporated herein by reference in its ty); (b) at least a portion of an
endogenous yeast protein, including but not limited to yeast alpha factor leader sequence,
wherein either fusion partner provides improved stability of expression of the protein in
the yeast and/or a prevents post-translational modification of the proteins by the yeast cells
(such proteins are also described in , for example, in US. Patent Publication No.
2004-0156858 Al, supra); and/or (c) at least a portion of a yeast protein that causes the
fusion protein to be expressed on the surface of the yeast (e.g., an Aga protein, described
in more detail herein). In on, the present invention optionally includes the use of
peptides that are fused to the C-terminus of the antigen-encoding construct, particularly for
use in the selection and identification of the protein. Such peptides include, but are not
limited to, any synthetic or natural peptide, such as a peptide tag (e.g., 6X His or
hexapeptide) or any other short epitope tag. Peptides attached to the C-terminus of an
antigen ing to the ion can be used with or without the addition of the N-
terminal peptides discussed above, and vice versa.
In one embodiment, a synthetic peptide useful in a fusion protein to be
sed in a yeast is linked to the N—terminus of the antigen, the peptide consisting of at
least two amino acid positions that are heterologous to the antigen, wherein the e
stabilizes the expression of the fusion protein in the yeast vehicle or prevents
posttranslational modification of the sed fusion protein. The synthetic peptide and
N—terminal portion of the antigen together form a fusion protein that has the following
requirements: (1) the amino acid residue at position one of the fusion protein is a
methionine (z'.e., the first amino acid in the synthetic peptide is a nine); (2) the
amino acid residue at position two of the fusion n is not a glycine or a proline (i.e.,
the second amino acid in the synthetic peptide is not a glycine or a proline); (3) none of
the amino acid positions at positions 2-6 of the fusion protein is a methionine (i.e., the
amino acids at positions 2-6, whether part of the synthetic peptide or the protein, if the
synthetic peptide is shorter than 6 amino acids, do not include a methionine); and (4) none
of the amino acids at positions 2-6 of the fusion protein is a lysine or an arginine (i.e., the
amino acids at positions 2-6, whether part of the synthetic peptide or the protein, if the
synthetic peptide is r than 5 amino acids, do not include a lysine or an ne).
The synthetic peptide can be as short as two amino acids, but in one aspect, is 2-6 amino
acids (including 3, 4, 5 amino acids), and can be longer than 6 amino acids, in whole
integers, up to about 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids,
or more.
In one embodiment, a fusion protein comprises an amino acid sequence of M-
X2-X3-X4-X5-X6, wherein M is nine; wherein X2 is any amino acid except
glycine, proline, lysine or arginine; wherein X3 is any amino acid except methionine,
lysine or ne; wherein X4 is any amino acid except methionine, lysine or arginine;
wherein X5 is any amino acid except methionine, lysine or ne; and wherein X6 is
any amino acid except methionine, lysine or arginine. In one ment, the X6 residue
is a proline. An exemplary synthetic sequence that enhances the stability of expression of
an antigen in a yeast cell and/or prevents post-translational modification of the protein in
the yeast includes the sequence M-A-D-E-A-P sented herein by SEQ ID NO:11). In
addition to the enhanced stability of the expression product, this fusion partner does not
appear to negatively impact the immune response against the immunizing antigen in the
uct. In addition, the synthetic fusion peptides can be designed to provide an e
that can be recognized by a selection agent, such as an dy.
In one aspect of the invention, the yeast vehicle is manipulated such that the
antigen is expressed or ed by delivery or translocation of an expressed protein
product, lly or wholly, on the surface of the yeast vehicle (extracellular sion).
One method for accomplishing this aspect of the invention is to use a spacer arm for
positioning one or more protein(s) on the surface of the yeast vehicle. For example, one
can use a spacer arm to create a fusion protein of the antigen(s) or other protein of interest
with a protein that targets the antigen(s) or other protein of interest to the yeast cell wall.
For example, one such protein that can be used to target other proteins is a yeast protein
(e.g., cell wall protein 2 (cwp2), Aga2, Pir4 or Flol protein) that enables the antigen(s) or
other protein to be targeted to the yeast cell wall such that the antigen or other protein is
located on the e of the yeast. Proteins other than yeast proteins may be used for the
spacer arm; however, for any spacer arm protein, it is most ble to have the
immunogenic response be ed against the target n rather than the spacer arm
protein. As such, if other proteins are used for the spacer arm, then the spacer arm protein
that is used should not generate such a large immune response to the spacer arm protein
itself such that the immune response to the target antigen(s) is overwhelmed. One of skill
in the art should aim for a small immune response to the spacer arm protein relative to the
immune response for the target antigen(s). Spacer arms can be constructed to have
cleavage sites (e.g., protease cleavage sites) that allow the n to be readily removed
or processed away from the yeast, if desired. Any known method of determining the
magnitude of immune responses can be used (e.g., dy production, lytic assays, etc.)
and are readily known to one of skill in the art.
r method for positioning the target antigen(s) or other proteins to be
exposed on the yeast surface is to use signal sequences such as glycosylphosphatidyl
inositol (GPI) to anchor the target to the yeast cell wall. Alternatively, positioning can be
accomplished by appending signal sequences that target the antigen(s) or other proteins of
interest into the secretory pathway Via translocation into the endoplasmic reticulum (ER)
such that the antigen binds to a protein which is bound to the cell wall (e.g., cwp).
In one aspect, the spacer arm protein is a yeast protein. The yeast protein can
consist of between about two and about 800 amino acids of a yeast protein. In one
embodiment, the yeast protein is about 10 to 700 amino acids. In another embodiment, the
yeast protein is about 40 to 600 amino acids. Other embodiments of the invention include
the yeast protein being at least 250 amino acids, at least 300 amino acids, at least 350
amino acids, at least 400 amino acids, at least 450 amino acids, at least 500 amino acids, at
least 550 amino acids, at least 600 amino acids, or at least 650 amino acids. In one
embodiment, the yeast protein is at least 450 amino acids in length. Another consideration
for optimizing antigen surface expression, if that is desired, is whether the antigen and
spacer arm combination should be expressed as a monomer or as dimer or as a , or
even more units connected together. This use of monomers, dimers, trimers, etc. allows
for appropriate spacing or g of the n such that some part, if not all, of the
antigen is displayed on the e of the yeast vehicle in a manner that makes it more
immunogenic.
Use of yeast proteins can stabilize the expression of fusion proteins in the
yeast vehicle, prevents posttranslational modification of the expressed fusion protein,
and/or targets the fusion protein to a particular compartment in the yeast (e.g., to be
expressed on the yeast cell surface). For delivery into the yeast secretory pathway,
exemplary yeast proteins to use include, but are not limited to: Aga (including, but not
limited to, Agal and/or Aga2); SUC2 (yeast invertase); alpha factor signal leader
sequence; CPY; pr2p for its localization and retention in the cell wall; BUD genes for
localization at the yeast cell bud during the initial phase of daughter cell formation; Flolp;
Pir2p; and Pir4p.
Other sequences can be used to target, retain and/or stabilize the protein to
other parts of the yeast vehicle, for example, in the cytosol or the mitochondria or the
asmic reticulum or the nucleus. Examples of suitable yeast protein that can be used
for any of the embodiments above include, but are not limited to, TK, AF, SEC7;
phosphoenolpyruvate carboxykinase PCKl, oglycerokinase PGK and triose
phosphate isomerase TPI gene products for their repressible expression in glucose and
cytosolic localization; the heat shock proteins SSAl, SSA3, SSA4, SSCl, whose
expression is induced and whose proteins are more thermostable upon exposure of cells to
heat treatment; the mitochondrial protein CYCl for import into mitochondria; ACTl.
Methods of producing yeast vehicles and expressing, combining and/or
associating yeast vehicles with antigens and/or other proteins and/or agents of interest to
produce based immunotherapy compositions are contemplated by the invention.
According to the present ion, the term "yeast vehicle-antigen complex"
or "yeast-antigen complex" is used generically to describe any association of a yeast
e with an antigen, and can be used interchangeably with “yeast-based
immunotherapy composition” when such composition is used to elicit an immune response
as bed above. Such ation includes expression of the antigen by the yeast (a
recombinant yeast), introduction of an n into a yeast, physical attachment of the
antigen to the yeast, and mixing of the yeast and antigen together, such as in a buffer or
other on or formulation. These types of complexes are described in detail below.
In one embodiment, a yeast cell used to e the yeast vehicle is transfected
with a heterologous nucleic acid le encoding a protein (e.g., the antigen) such that
the protein is expressed by the yeast cell. Such a yeast is also ed to herein as a
recombinant yeast or a recombinant yeast vehicle. The yeast cell can then be formulated
with a pharmaceutically acceptable excipient and administered directly to a patient, stored
for later administration, or loaded into a tic cell as an intact cell. The yeast cell can
also be killed, or it can be derivatized such as by formation of yeast spheroplasts,
cytoplasts, ghosts, or lular particles, any of which may be followed by storing,
administering, or loading of the derivative into the dendritic cell. Yeast spheroplasts can
also be ly transfected with a recombinant nucleic acid molecule (e.g., the spheroplast
is ed from a whole yeast, and then transfected) in order to produce a recombinant
spheroplast that expresses the antigen. Yeast cells or yeast spheroplasts that
recombinantly express the antigen(s) may be used to produce a yeast vehicle comprising a
yeast cytoplast, a yeast ghost, or a yeast membrane particle or yeast cell wall particle, or
fraction f.
In general, the yeast vehicle and antigen(s) and/or other agents can be
associated by any technique described herein. In one aspect, the yeast e was loaded
intracellularly with the antigen(s) and/or agent(s). In another aspect, the n(s) and/or
agent(s) was covalently or non-covalently attached to the yeast e. In yet another
aspect, the yeast vehicle and the antigen(s) and/or agent(s) were associated by mixing. In
r aspect, and in one embodiment, the antigen(s) and/or agent(s) are expressed
recombinantly by the yeast vehicle or by the yeast cell or yeast spheroplast from which the
yeast vehicle was derived.
A number of antigens and/or other ns to be produced by a yeast vehicle
of the present ion is any number of antigens and/or other proteins that can be
reasonably produced by a yeast vehicle, and typically ranges from at least one to at least
about 6 or more, including from about 2 to about 6 antigens and or other ns.
Expression of an antigen or other protein in a yeast vehicle of the present
invention is accomplished using techniques known to those skilled in the art. Briefly, a
nucleic acid molecule ng at least one desired antigen or other protein is inserted into
an expression vector in such a manner that the nucleic acid molecule is operatively linked
to a transcription control ce in order to be capable of effecting either constitutive or
ted expression of the nucleic acid molecule when transformed into a host yeast cell.
Nucleic acid molecules encoding one or more antigens and/or other proteins can be on one
or more expression vectors operatively linked to one or more expression control sequences.
Particularly important expression control sequences are those which control transcription
initiation, such as er and upstream activation sequences. Any le yeast
promoter can be used in the present invention and a variety of such ers are known
to those skilled in the art. Promoters for expression in Saccharomyces cerevisiae include,
but are not limited to, promoters of genes encoding the following yeast proteins: alcohol
dehydrogenase I (ADHl) or II (ADH2), CUPl, phosphoglycerate kinase (PGK), triose
phosphate isomerase (TPI), translational elongation factor EF-l alpha (TEF2),
glyceraldehydephosphate dehydrogenase (GAPDH; also referred to as TDH3, for triose
phosphate dehydrogenase), galactokinase (GALl), galactose-l-phosphate uridyl-
transferase (GAL7), UDP-galactose epimerase (GALlO), cytochrome cl (CYCl), Sec7
protein (SEC7) and acid phosphatase (PHOS), including hybrid promoters such as
ADH2/GAPDH and CYC]/GALI0 promoters, and including the ADH2/GAPDH promoter,
which is induced when glucose concentrations in the cell are low (e.g., about 0.1 to about
0.2 percent), as well as the CUPI promoter and the TEF2 promoter. Likewise, a number
of upstream activation sequences (UASs), also referred to as enhancers, are known.
Upstream activation ces for expression in romyces cerevisiae include, but
are not limited to, the UASs of genes encoding the following proteins: PCKl, TPI, TDH3,
CYCl, ADHl, ADH2, SUC2, GALl, GAL7 and GALlO, as well as other UASs activated
by the GAL4 gene product, with the ADH2 UAS being used in one aspect. Since the
ADH2 UAS is activated by the ADRl gene product, it may be preferable to overexpress
the ADRl gene when a heterologous gene is ively linked to the ADH2 UAS.
Transcription termination sequences for expression in Saccharomyces cerevisiae include
the termination sequences of the u-factor, GAPDH, and CYCl genes.
Transcription l sequences to express genes in methyltrophic yeast
include the transcription control regions of the genes encoding alcohol oxidase and
e dehydrogenase.
Transfection of a nucleic acid le into a yeast cell ing to the
present invention can be lished by any method by which a nucleic acid molecule
can be introduced into the cell and includes, but is not limited to, diffusion, active
transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and
protoplast fusion. Transfected nucleic acid molecules can be ated into a yeast
chromosome or ined on extrachromosomal vectors using techniques known to those
skilled in the art. Examples of yeast vehicles carrying such nucleic acid molecules are
disclosed in detail herein. As discussed above, yeast cytoplast, yeast ghost, and yeast
membrane particles or cell wall preparations can also be produced recombinantly by
ecting intact yeast microorganisms or yeast spheroplasts with desired nucleic acid
molecules, producing the n therein, and then r manipulating the
microorganisms or spheroplasts using techniques known to those skilled in the art to
produce ast, ghost or subcellular yeast membrane extract or fractions thereof
containing desired antigens or other proteins.
Effective conditions for the production of recombinant yeast vehicles and
expression of the antigen and/or other n by the yeast vehicle include an effective
medium in which a yeast strain can be cultured. An effective medium is typically an
aqueous medium comprising assimilable carbohydrate, en and phosphate sources, as
well as appropriate salts, minerals, metals and other nutrients, such as Vitamins and growth
factors. The medium may comprise complex nutrients or may be a defined minimal
medium. Yeast s of the present invention can be cultured in a variety of containers,
including, but not limited to, bioreactors, Erlenmeyer flasks, test tubes, microtiter dishes,
and Petri plates. Culturing is carried out at a ature, pH and oxygen content
appropriate for the yeast strain. Such culturing conditions are well within the expertise of
one of ordinary skill in the art (see, for example, Guthrie et al. (eds.), 1991, Methods in
Enzymology, vol. 194, Academic Press, San Diego). For example, under one protocol,
liquid es containing a suitable medium can be inoculated using es obtained
from starter plates and/or starter cultures of yeast-Brachyury immunotherapy compositions,
and are grown for approximately 20h at 30°C, with agitation at 250 rpm. Primary cultures
can then be expanded into larger cultures as desired. Protein expression from vectors with
which the yeast were transformed (e.g., Brachyury sion) may be constitutive if the
promoter utilized is a constitutive er, or may be induced by addition of the
appropriate induction conditions for the promoter if the promoter utilized is an inducible
promoter (e.g., copper sulfate in the case of the CUP] promoter). In the case of an
inducible promoter, induction of protein expression may be initiated after the culture has
grown to a suitable cell density, which may be at about 0.2 Y.U./ml or higher densities.
One non-limiting example of a medium suitable for the culture of a yeast-
ury immunotherapy composition of the invention is U2 medium. U2 medium
comprises the following components: 20g/L of glucose, 6.7 g/L of Yeast nitrogen base
containing um sulfate, and 0.04 mg/mL each of histidine, leucine, tryptophan, and
adenine. r non-limiting example of a medium suitable for the culture of yeast-
Brachyury immunotherapy composition of the invention is UL2 medium. UL2 medium
comprises the following components: 20g/L of glucose, 6.7 g/L of Yeast en base
containing um sulfate, and 0.04 mg/mL each of histidine, tryptophan, and adenine.
In one embodiment of the invention, when an ble promoter is used (e.g.
the CUPI promoter) to express a Brachyury protein in a yeast e according to the
invention, induction of protein expression is initiated at a higher cell y as compared
to the cell density that would be suitable for most proteins expressed by yeast using such a
promoter. More specifically, the present inventors have discovered that optimal
Brachyury antigen expression driven by the CUP] er occurs when the yeast
expressing the Brachyury antigen are allowed to grow to a cell density of between at least
0.5 Y.U/ml and approximately 2.0 Y.U./ml, and in one aspect, to between 0.5 Y.U./ml and
approximately 1.5 l, and in one aspect, to between at least 1.0 Y.U./ml and about
2.0 Y.U./ml, and in another aspect, to at least about 1.0 Y.U./ml, prior to inducing
sion of the Brachyury antigen in the yeast. The present ors have discovered
that subsequent to induction of Brachyury expression, the yeast will double only about 1X
to l.5X. Moreover, after induction of Brachyury expression, the inventors have
discovered that growth of the yeast to cell densities higher than about 2.0 Y.U./ml, or for
longer than about 6-8 hours, results in sed viability of the cultures, while not
ntially improving antigen accumulation in the yeast. Therefore, in one embodiment
of the invention, a Brachyury immunotherapy composition having antigen
expression under the control of an inducible promoter, such as the CUP] promoter, is
grown to mid-log phase prior to inducing antigen expression. In one aspect, the cells are
grown to between about 1 and 2 Y.U./ml prior to induction of antigen expression. In one
aspect, antigen expression is induced (e.g., by the addition of copper sulfate) and continues
for up to 6, 6.5, 7, 7.5, or 8 hours. In one aspect, the induction occurs at a temperature of
about 30°C and agitation rate of 250 rpm.
In some embodiments of the invention, the yeast are grown under neutral pH
conditions. As used herein, the general use of the term “neutral pH” refers to a pH range
between about pH 5.5 and about pH 8, and in one , between about pH 6 and about 8.
One of skill the art will appreciate that minor fluctuations (e.g., tenths or hundredths) can
occur when measuring with a pH meter. As such, the use of neutral pH to grow yeast cells
means that the yeast cells are grown in neutral pH for the majority of the time that they are
in e. In one embodiment, yeast are grown in a medium maintained at a pH level of
at least 5.5 (i.e., the pH of the culture medium is not allowed to drop below pH 5.5). In
another aspect, yeast are grown at a pH level maintained at about 6, 6.5, 7, 7.5 or 8. The
use of a neutral pH in culturing yeast promotes several ical effects that are desirable
characteristics for using the yeast as vehicles for immunomodulation. For e,,
culturing the yeast in neutral pH allows for good growth of the yeast without negative
effect on the cell generation time (e.g., slowing of doubling time). The yeast can continue
to grow to high densities without losing their cell wall lity. The use of a neutral pH
allows for the production of yeast with pliable cell walls and/or yeast that are more
sensitive to cell wall digesting enzymes (e.g., glucanase) at all harvest densities. This trait
is desirable because yeast with flexible cell walls can induce different or improved
immune responses as compared to yeast grown under more acidic conditions, e. g., by
promoting the secretion of cytokines by antigen presenting cells that have phagocytosed
the yeast (e.g., THl-type cytokines including, but not limited to, IFN-y, eukin-12 (IL-
12), and IL-2, as well as proinflammatory cytokines such as IL-6). In addition, greater
accessibility to the antigens located in the cell wall is ed by such culture methods. In
another aspect, the use of neutral pH for some ns allows for release of the di-sulfide
bonded antigen by treatment with dithiothreitol (DTT) that is not possible when such an
antigen-expressing yeast is cultured in media at lower pH (e.g., pH 5).
In one embodiment, control of the amount of yeast glycosylation is used to
control the expression of antigens by the yeast, particularly on the surface. The amount of
yeast glycosylation can affect the immunogenicity and antigenicity of the antigen,
particularly one sed on the surface, since sugar moieties tend to be bulky. As such,
the existence of sugar moieties on the e of yeast and its impact on the three-
dimensional space around the target antigen(s) should be considered in the modulation of
yeast ing to the invention. Any method can be used to reduce the amount of
glycosylation of the yeast (or increase it, if desired). For example, one could use a yeast
mutant strain that has been selected to have low glycosylation (e. g. mnnl, ochl and mnn9
mutants), or one could eliminate by mutation the glycosylation acceptor sequences on the
target antigen. Alternatively, one could use yeast with abbreviated glycosylation patterns,
e.g., Pichia. One can also treat the yeast using methods that reduce or alter the
glycosylation.
In one embodiment of the present ion, as an alternative to expression of
an antigen or other n recombinantly in the yeast vehicle, a yeast vehicle is loaded
intracellularly with the protein or e, or with carbohydrates or other molecules that
serve as an antigen and/or are useful as immunomodulatory agents or ical se
modifiers according to the invention. Subsequently, the yeast vehicle, which now contains
the antigen and/or other proteins intracellularly, can be administered to an individual or
loaded into a carrier such as a dendritic cell. Peptides and proteins can be inserted directly
into yeast es of the present invention by techniques known to those skilled in the art,
such as by diffusion, active transport, liposome fusion, electroporation, phagocytosis,
-thaw cycles and bath sonication. Yeast vehicles that can be directly loaded with
peptides, proteins, carbohydrates, or other molecules include intact yeast, as well as
spheroplasts, ghosts or cytoplasts, which can be loaded with antigens and other agents
after production. Alternatively, intact yeast can be loaded with the antigen and/or agent,
and then spheroplasts, ghosts, cytoplasts, or subcellular particles can be prepared
rom. Any number of antigens and/or other agents can be loaded into a yeast vehicle
in this embodiment, from at least 1, 2, 3, 4 or any whole integer up to hundreds or
thousands of antigens and/or other agents, such as would be provided by the loading of a
microorganism or portions thereof, for e.
In another embodiment of the t invention, an antigen and/or other agent
is physically attached to the yeast vehicle. Physical attachment of the antigen and/or other
agent to the yeast e can be accomplished by any method suitable in the art, including
covalent and non-covalent ation methods which include, but are not limited to,
chemically crosslinking the antigen and/or other agent to the outer surface of the yeast
vehicle or biologically linking the antigen and/or other agent to the outer surface of the
yeast vehicle, such as by using an dy or other binding partner. Chemical cross-
linking can be achieved, for example, by methods including glutaraldehyde linkage,
photoaffinity labeling, ent with carbodiimides, ent with chemicals capable of
linking di-sulf1de bonds, and treatment with other cross-linking chemicals rd in the
art. Alternatively, a chemical can be contacted with the yeast vehicle that alters the charge
of the lipid bilayer of yeast membrane or the composition of the cell wall so that the outer
surface of the yeast is more likely to fuse or bind to antigens and/or other agent haVing
particular charge characteristics. Targeting agents such as dies, binding peptides,
soluble receptors, and other ligands may also be incorporated into an antigen as a fusion
protein or otherwise associated with an antigen for binding of the antigen to the yeast
vehicle.
When the antigen or other protein is expressed on or ally attached to the
surface of the yeast, spacer arms may, in one aspect, be carefully selected to optimize
antigen or other protein expression or content on the e. The size of the spacer arm(s)
can affect how much of the antigen or other protein is exposed for binding on the surface
of the yeast. Thus, depending on which antigen(s) or other protein(s) are being used, one
of skill in the art will select a spacer arm that effectuates appropriate spacing for the
antigen or other protein on the yeast surface. In one ment, the spacer arm is a yeast
n of at least 450 amino acids. Spacer arms have been discussed in detail above.
In yet another embodiment, the yeast vehicle and the antigen or other protein
are associated with each other by a more passive, non-specific or non-covalent binding
ism, such as by gently mixing the yeast vehicle and the antigen or other protein
together in a buffer or other suitable formulation (e.g., admixture).
] In one embodiment, intact yeast (with or without expression of heterologous
antigens or other proteins) can be ground up or processed in a manner to produce yeast
cell wall preparations, yeast membrane les or yeast fragments (i.e., not intact) and
the yeast fragments can, in some embodiments, be provided with or administered with
other compositions that include antigens (e.g., DNA vaccines, protein subunit vaccines,
killed or inactivated pathogens, viral vector vaccines) to enhance immune responses. For
example, enzymatic treatment, chemical treatment or physical force (e.g., mechanical
shearing or tion) can be used to break up the yeast into parts that are used as an
In one embodiment of the ion, yeast vehicles useful in the invention
include yeast vehicles that have been killed or inactivated. Killing or inactivating of yeast
can be accomplished by any of a variety of suitable methods known in the art. For
example, heat inactivation of yeast is a standard way of inactivating yeast, and one of skill
in the art can monitor the structural changes of the target antigen, if desired, by standard
methods known in the art. atively, other methods of inactivating the yeast can be
used, such as chemical, electrical, radioactive or UV methods. See, for example, the
methodology disclosed in standard yeast ing textbooks such as Methods of
Enzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of the inactivation
strategies used should take the secondary, tertiary or quaternary structure of the target
antigen into eration and preserve such structure as to optimize its immunogenicity.
Yeast vehicles can be formulated into yeast-based immunotherapy
compositions or products of the present invention using a number of techniques known to
those skilled in the art. For example, yeast vehicles can be dried by lization.
Formulations comprising yeast es can also be prepared by packing yeast in a cake or
a tablet, such as is done for yeast used in baking or brewing operations. In addition, yeast
vehicles can be mixed with a pharmaceutically acceptable excipient, such as an isotonic
buffer that is ted by a host or host cell. Examples of such excipients include water,
saline, Ringer's solution, dextrose solution, Hank's on, and other aqueous
physiologically balanced salt solutions. Nonaqueous es, such as fixed oils, sesame
oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include
suspensions containing ity-enhancing agents, such as sodium
carboxymethylcellulose, sorbitol, glycerol or n. Excipients can also contain minor
amounts of ves, such as substances that enhance isotonicity and chemical stability.
Examples of s include phosphate buffer, bicarbonate buffer and Tris buffer, while
examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol.
Standard formulations can either be liquid injectables or solids which can be taken up in a
le liquid as a suspension or solution for injection. Thus, in a non-liquid formulation,
the excipient can comprise, for e, dextrose, human serum albumin, and/or
vatives to which e water or saline can be added prior to administration.
In one ment of the present invention, a ition can include
additional agents, which may also be referred to as biological response r
compounds, or the ability to produce such agents/modifiers. For example, a yeast vehicle
can be transfected with or loaded with at least one antigen and at least one agent/biological
response modifier compound, or a composition of the invention can be administered in
conjunction with at least one agent/biological response modifier. Biological response
modifiers include adjuvants and other compounds that can modulate immune responses,
which may be referred to as immunomodulatory compounds, as well as compounds that
modify the biological activity of another compound or agent, such as a yeast-based
immunotherapeutic, such biological activity not being limited to immune system effects.
n immunomodulatory compounds can stimulate a protective immune response
whereas others can suppress a harmful immune response, and whether an
immunomodulatory is useful in combination with a given yeast-based immunotherapeutic
may depend, at least in part, on the disease state or condition to be treated or prevented,
and/or on the individual who is to be treated. Certain biological se modifiers
preferentially enhance a cell-mediated immune response whereas others preferentially
enhance a l immune response (i.e., can ate an immune response in which
there is an increased level of cell-mediated ed to humoral immunity, or vice versa.).
Certain biological response modifiers have one or more properties in common with the
biological properties of yeast-based immunotherapeutics or enhance or complement the
ical properties of yeast-based immunotherapeutics. There are a number of
techniques known to those skilled in the art to e stimulation or suppression of
immune responses, as well as to differentiate cell-mediated immune responses from
humoral immune responses, and to differentiate one type of cell-mediated response from
another (e. g., a THl7 response versus a THl response).
Agents/biological response rs useful in the invention may include, but
are not limited to, nes, chemokines, hormones, lipidic derivatives, peptides, proteins,
ccharides, small molecule drugs, dies and antigen binding fragments thereof
(including, but not limited to, anti-cytokine antibodies, anti-cytokine receptor antibodies,
anti-chemokine antibodies), Vitamins, polynucleotides, c acid binding moieties,
rs, and growth modulators. Some suitable agents include, but are not limited to,
IL-1 or agonists of IL-1 or of IL-lR, anti-IL-l or other IL-l antagonists; IL-6 or agonists
of IL-6 or of IL-6R, anti-IL-6 or other IL-6 antagonists; IL-12 or agonists of IL-12 or of
IL-l2R, anti-IL-12 or other IL-12 antagonists; IL-17 or agonists of IL-17 or of IL-l7R,
anti-IL-l7 or other IL-l7 antagonists; IL-21 or agonists of IL-21 or of IL-21R, anti-IL-21
or other IL-21 nists; IL-22 or agonists of IL-22 or of IL-22R, anti-IL-22 or other IL-
22 nists; IL-23 or agonists of IL-23 or of IL-23R, anti-IL-23 or other IL-23
antagonists; IL-25 or agonists of IL-25 or of IL-25R, anti-IL-25 or other IL-25
nists; IL-27 or agonists of IL-27 or of IL-27R, anti-IL-27 or other IL-27
antagonists; type I interferon (including IFN—u) or agonists or antagonists of type I
interferon or a receptor thereof; type II interferon (including IFN—y) or agonists or
antagonists of type II interferon or a receptor thereof; anti-CD40, CD40L, lymphocyte-
activation gene 3 (LAG3) n and/or IMP321 (T-cell immunostimulatory factor
derived from the soluble form of LAG3), anti-CTLA-4 antibody (e.g., to release anergic T
cells); T cell co-stimulators (e.g., anti-CD137, anti-CD28, anti-CD40); alemtuzumab (e.g.,
CamPath®), denileukin diftitox (e.g., ONTAK®); anti-CD4; anti-CD25; anti-PD-l, anti-
PD-Ll, anti-PD-L2; agents that block FOXP3 (e.g., to abrogate the activity/kill
CD4+/CD25+ T regulatory cells); Flt3 ligand, imiquimod (AldaraTM), granulocyte-
macrophage colony stimulating factor (GM-CSF); granulocyte-colony stimulating factor
(G-CSF), mostim (Leukine®); es ing Without limitation prolactin and
growth hormone; ike receptor (TLR) agonists, including but not limited to TLR-2
agonists, TLR-4 agonists, TLR-7 agonists, and TLR-9 agonists; TLR antagonists,
including but not d to TLR-2 antagonists, TLR-4 antagonists, TLR-7 antagonists,
and TLR-9 antagonists; anti-inflammatory agents and immunomodulators, including but
not limited to, COX-2 inhibitors (e.g., Celecoxib, NSAIDS), glucocorticoids, statins, and
thalidomide and analogues f including IMiDTMs (Which are structural and functional
analogues of thalidomide (e.g., REVLIMID® idomide), ACTIMID®
(pomalidomide)); proinflammatory agents, such as fungal or bacterial components or any
proinflammatory cytokine or chemokine; therapeutic vaccines including, but not
d to, Virus-based vaccines, bacteria-based vaccines, or antibody-based vaccines; and
any other immunomodulators, immunopotentiators, anti-inflammatory agents, pro-
inflammatory agents, and any agents that modulate the number of, modulate the activation
state of, and/or modulate the survival of antigen-presenting cells or of THl7, THl, and/or
Treg cells. Any combination of such agents is contemplated by the invention, and any of
such agents combined with or administered in a ol with (e.g., concurrently,
sequentially, or in other formats with) a yeast-based immunotherapeutic is a composition
encompassed by the invention. Such agents are well known in the art. These agents may
be used alone or in combination with other agents described herein.
Agents can include agonists and antagonists of a given protein or peptide or
domain thereof. As used herein, an “agonist” is any compound or agent, including without
limitation small molecules, proteins, peptides, antibodies, nucleic acid binding agents, etc.,
that binds to a receptor or ligand and produces or triggers a response, which may e
agents that mimic or enhance the action of a naturally occurring substance that binds to the
receptor or ligand. An “antagonist” is any compound or agent, including without
limitation small molecules, proteins, peptides, antibodies, nucleic acid binding , etc.,
that blocks or inhibits or reduces the action of an agonist.
Compositions of the invention can further include or can be administered with
(concurrently, sequentially, or intermittently with) any other agents or compositions or
protocols that are useful for ting or treating cancer or any compounds that treat or
ameliorate any symptom of , and particularly cancers associated with Brachyury
expression or overexpression. In addition, compositions of the ion can be used
together with other immunotherapeutic compositions, including prophylactic and/or
therapeutic immunotherapy. , the itions of the invention can be used to
inhibit or reduce chemotherapy resistance or ion resistance that may occur in
atic cancer by inhibiting ury expression in the cancer (and thereby inhibiting
anti-proliferative influences) or compositions of the invention may enhance the
performance of chemotherapy or radiation therapy in an individual. Additional agents,
compositions or protocols (e.g., therapeutic protocols) that are useful for the ent of
cancer include, but are not limited to, chemotherapy, surgical resection of a tumor,
radiation therapy, allogeneic or autologous stem cell transplantation, and/or ed
cancer therapies (e.g., small molecule drugs, biologics, or monoclonal antibody therapies
that cally target molecules involved in tumor growth and ssion, including, but
not limited to, selective estrogen receptor modulators (SERMs), aromatase tors,
tyrosine kinase inhibitors, serine/threonine kinase inhibitors, histone deacetylase (HDAC)
inhibitors, retinoid receptor activators, apoptosis stimulators, angiogenesis inhibitors, poly
(ADP-ribose_) polymerase (PARP) inhibitors, or immunostimulators). Any of these
additional therapeutic agents and/or therapeutic protocols may be administered before,
concurrently with, ating with, or after the immunotherapy compositions of the
invention, or at ent time points. For example, when given to an individual in
conjunction with chemotherapy or a targeted cancer y, it may be desirable to
administer the yeast-Brachyury immunotherapy compositions during the “holiday”
between doses of chemotherapy or ed cancer therapy, in order to maximize the
cy of the immunotherapy compositions. Surgical resection of a tumor may
frequently precede administration of a yeast-Brachyury immunotherapy composition, but
additional or primary y may occur during or after administration of a yeast-
ury immunotherapy composition.
The invention also includes a kit comprising any of the compositions
described herein, or any of the individual components of the itions described
herein. Kits may include additional reagents and written instructions or directions for
using any of the compositions of the invention to prevent or treat cancer associated with
ury expression or overexpression.
Methods for Administration or Use of Coonsitions o: the Invention
Brachyury therapeutic compositions of the invention are
designed for use to prevent or treat cancers that are associated with or characterized by
Brachyury expression or overexpression, including by preventing emergence of such
cancers, arresting progression of such cancers or ating such cancers. More
ularly, yeast-Brachyury immunotherapeutic compositions can be used to prevent,
inhibit or delay the development of Brachyury-expressing tumors, and/or to prevent,
inhibit or delay tumor migration and/or tumor invasion of other tissues (metastases) and/or
to generally prevent or inhibit ssion of cancer in an individual. Yeast-Brachyury
immunotherapeutic compositions can also be used to ameliorate at least one symptom of
the cancer, such as by reducing tumor burden in the individual; inhibiting tumor growth in
the individual; increasing survival of the individual; preventing, inhibiting, reversing or
delaying development of tumor migration and/or tumor invasion of other tissues
(metastatic cancer) and/or preventing, inhibiting, reversing or delaying progression of the
cancer in the dual. Yeast-Brachyury immunotherapy can also be used
therapeutically to inhibit, reduce or eliminate chemotherapy resistance or radiation
resistance that may occur in atic cancer by inhibiting Brachyury expression in the
cancer, and compositions of the ion may enhance the performance of chemotherapy
or radiation therapy in an individual.
Cancers that are relevant to the compositions and methods of the invention are
any cancer that ses, or may express, Brachyury, or cancers in proximity to cancers
that express or may express Brachyury, and include, but are not limited to, cancer of the
breast, small ine, stomach, kidney, bladder, uterus, ovary, , lung, colon,
pancreas, or prostate, and include metastatic and late-stage cancers. In addition,
Brachyury is expressed in tumors of B cell origin, such as c lymphocytic leukemia
(CLL), Epstein-Barr virus transformed B cells, Burkitt’s and Hodgkin’s lymphomas, as
well as metastatic cancers thereof.
One embodiment of the invention relates to a method to inhibit tumor
migration and/or to reduce, halt (arrest), reverse or prevent the metastatic ssion of
cancer in an individual who has cancer, or to reverse the development of metastatic events
in a cancer. As discussed above, ury promotes the epithelial-mesenchymal
transition (EMT) in human tumor cells, conferring on tumor cells a mesenchymal
phenotype, as well as migratory and invasive abilities, while attenuating tumor cell cycle
ssion. Therefore, the involvement of Brachyury in metastatic processes makes it an
ideal target for the prevention or inhibition of metastatic processes, including arresting
cancer at a pre-metastatic stage. Use of a yeast-Brachyury immunotherapeutic
composition of the invention can be ive to prevent or treat metastatic cancer,
including arresting progression of the , in the face of escape (or attempted escape)
of the cancer from traditional y, such as chemotherapy and ion. The method
includes the steps of administering to the individual who has cancer an immunotherapeutic
composition a Brachyury immunotherapeutic composition of the invention as
described herein, including, but not limited to: (a) a yeast vehicle; and (b) a cancer
antigen sing at least one Brachyury antigen.
In one aspect, Brachyury is not detected in the individual’s cancer at the time
the composition is first administered. In general, when Brachyury is not detected in the
individual’s cancer, the dual may have an earlier stage cancer in which Brachyury
expression has not yet manifested (e.g., stage I or stage II), or in which Brachyury
expression is not yet detectable in any event (i.e., Brachyury may or may not be expressed
at a low level or in a small number of tumor cells, but is not yet readily detectable using
standard detection s). In this aspect of the invention, the development of
Brachyury-expressing tumor cells is prevented, delayed or inhibited by use of the yeast-
Brachyury therapeutic composition. As a result, tumor migration and/or other
metastatic processes leading to metastatic progression of the tumor are prevented, d
or ted and/or l arrest of tumor progression occurs in the individual.
In another aspect, Brachyury expression is or can be detected in the
individual’s cancer at the time the ition is first administered. The individual may
have stage I, stage II, stage III, or stage IV cancer in this aspect of the invention. In this
aspect, use of the yeast-Brachyury immunotherapeutic composition reduces, eliminates or
slows or arrests the growth of tumors expressing Brachyury, which can result in reduction
in tumor burden in the individual, inhibition of Brachyury-expressing tumor growth,
and/or increased survival of the individual. The individual may ence an arrest,
slowing or reversal in metastatic processes, improving survival and health of the patient,
and rmore, allowing other therapeutic protocols to treat the cancer.
Indeed, metastatic cancer can be associated with resistance, or increased
ance, to cancer therapies such as chemotherapy, radiation, or targeted cancer therapy,
whereby the cancer “escapes” from the therapy or is simply less impacted by the therapy
and sses. Accordingly, there is a need to reduce or eliminate resistance to such
therapies to improve or enhance the efficacy of the therapy and improve patient health and
survival. Accordingly, one ment of the invention relates to a method to reduce or
prevent chemotherapy-resistance, targeted cancer therapy-resistance, or radiation-
resistance in a patient with cancer. The method comprises administering to an individual
who has cancer and is receiving chemotherapy and/or radiation therapy for the cancer, a
yeast-Brachyury immunotherapeutic composition as described herein, which may include
a composition comprising: (a) a yeast e; and (b) a cancer antigen comprising at least
one Brachyury antigen. This method of the invention may also be used to treat resistance
associated with other therapeutic treatments for cancer, including, but not limited to,
targeted cancer y.
In one aspect of this embodiment, Brachyury is not detected in the individual’s
cancer at the time the composition is first administered. In this aspect, administration of a
yeast-Brachyury immunotherapeutic composition prevents or inhibits the onset of
resistance to herapy or ion therapy by inhibiting the development of
Brachyury-expressing tumor cells in the cancer. In another aspect, Brachyury expression
is detected in the individual’s cancer at the time the ition is first administered. In
this aspect, the individual may or may not already be experiencing resistance to
chemotherapy or radiation. In either case, administration of the yeast-Brachyury
therapeutic ition of the ion prevents or inhibits the resistance to
chemotherapy or radiation therapy or enhances the ability of the chemotherapy or
radiation therapy to treat the individual, by reducing or eliminating Brachyury-expressing
tumor cells in the patient.
Another embodiment of the invention relates to a method to treat cancer, and
particularly, a Brachyury-expressing cancer. The method includes administering to an
indiVidual who has a Brachyury-expressing cancer a yeast-Brachyury therapeutic
composition described herein, which can include a composition comprising: (a) a yeast
vehicle; and (b) a cancer antigen comprising at least one Brachyury antigen. In one aspect,
the method reduces tumor burden in the patient. In one aspect, the method increases
survival of the patient. In one aspect, the method inhibits tumor growth in the indiVidual.
In one aspect, the method prevents, arrests or reverses metastatic progression of the tumor.
Since Brachyury expression is believed to be more prevalent as a cancer
advances or progresses into higher stages (e.g., from stage I to stage II to stage III to stage
IV, depending on the particular cancer) and is associated with atic processes, it is an
embodiment of the ion to provide a method to prevent or delay the onset of a
Brachyury-expressing , or to arrest the cancer at a pre-metastatic or pre-malignant
stage. Such a method includes administering to an indiVidual in whom Brachyury-
expressing cancer cells are not detected a yeast-Brachyury immunotherapeutic
composition described herein, which can include a composition sing: (a) a yeast
e; and (b) a cancer n comprising at least one Brachyury antigen. In one aspect
of this embodiment, the cancer is known to express or believed to be tible to
expressing Brachyury at some stage of the cancer in at least a subset of indiViduals with
the cancer. In one aspect of this ment, the indiVidual already has a cancer, but
Brachyury is not detected in the cancer at the time the composition is first administered,
meaning that the indiVidual may have an earlier stage cancer in which Brachyury
expression has not yet manifested, or in which Brachyury expression is not yet able
in any event (i.e., Brachyury may or may not be expressed at a low level or in a small
number of tumor cells, but is not yet readily detectable using standard detection methods).
In some cases, the type of cancer may be known to have a high rate of metastatic
progression, In this aspect, administration of the yeast-Brachyury immunotherapeutic
composition prevents, delays or inhibits the development of Brachyury-expressing tumor
cells in the patient’s cancer, and therefore prevents, arrests, delays or inhibits metastatic
processes that accompany Brachyury expression. In r aspect, the indiVidual does
not have cancer when the composition is administered. Such an individual may be
“predisposed” or likely to develop cancer, perhaps because of family history or a genetic
, or because the individual has shown signs of precancerous cells or lesions or has
precancerous (premalignant) cells or lesions.
In one aspect, the individual is additionally treated with at least one other
therapeutic compound or therapeutic protocol useful for the treatment of cancer. Such
therapeutic agents and protocols have been discussed in detail elsewhere herein. For
example, in any of the embodiments regarding methods of the invention described herein,
in one aspect, when the individual has cancer (regardless of the status of detectable
Brachyury expression in tumor cells) the individual is being treated or has been treated
with another therapy for cancer. Such therapy can include any of the therapeutic protocols
or use of any therapeutic compound or agent described previously herein, including, but
not limited to, herapy, radiation therapy, ed cancer therapy, surgical resection
of a tumor, stem cell transfer, cytokine therapy, adoptive T cell transfer, and/or
administration of a second therapeutic composition. In the case of administration
of a second therapeutic composition, such compositions may include, but are not
limited to, onal yeast-based immunotherapy, recombinant virus-based
immunotherapy (viral vectors), cytokine therapy, immunostimulant therapy (including
chemotherapy with immunostimulating properties), DNA es, and other
immunotherapy compositions.
In one aspect, the second immunotherapeutic composition includes a second
cancer antigen that does not include Brachyury n. For example, a second
immunotherapeutic composition useful in ation with a yeast-Brachyury
immunotherapeutic composition is a immunotherapeutic composition comprising
another cancer antigen. Such cancer antigens may include, but are not limited to,
carcinoembryonic antigen (CEA), point mutated Ras oncoprotein, MUC-l, EGFR, BCR-
Abl, MART-l, MAGE-l, MAGE-3, GAGE, , MUC-2, normal and point mutated
p53 oncoproteins, PSMA, tyrosinase, TRP-l (gp75), -l, TRP-2, TAG72, KSA,
, PSA, HER-2/neu/c-erb/B2, hTERT, p73, B-RAF, adenomatous polyposis coli
(APC), Myc, von Hippel-Lindau protein (VHL), Rb-l, Rb-2, androgen receptor (AR),
Smad4, MDRl, Flt-3, BRCA-l, BRCA-2, pax3-fl<hr, ews-fli-l, HERV-H, HERV-K,
TWIST, Mesothelin, NGEP, modifications of such antigens, splice variants of such
antigens, and epitope ts of such antigens, as well as combinations of such antigens,
and/or immunogenic s thereof, modifications thereof, variants thereof, and/or
epitope agonists thereof
As used herein, to ” a cancer, or any permutation thereof (e.g., “treated
for cancer”, etc.) generally refers to administering a composition of the invention once the
cancer has occurred (e.g., once the cancer has been diagnosed or detected in an individual),
with at least one therapeutic goal of the treatment (as compared to in the absence of this
treatment) including: reduction in tumor burden, inhibition of tumor growth, increase in
survival of the individual, ng, inhibiting, arresting or preventing the onset or
pment of metastatic cancer (such as by delaying, ting, arresting or preventing
the onset of development of tumor migration and/or tumor invasion of tissues outside of
primary cancer and/or other processes associated with metastatic progression of cancer),
delaying or arresting cancer progression, improvement of immune responses against the
tumor, ement of long term memory immune responses t the tumor antigens,
and/or improved general health of the individual. To “prevent” or “protect” from a cancer,
or any permutation thereof (e.g., ntion of cancer”, etc.), generally refers to
administering a composition of the invention before a cancer has occurred, or before a
specific stage of cancer or tumor antigen expression in a cancer has occurred (e.g., before
Brachyury expression is detected in the cancer), with at least one goal of the treatment (as
compared to in the absence of this treatment) including: preventing or delaying the onset
or development of a cancer, or, should the cancer occur after the treatment, at least
reducing the severity of the cancer (e. g., reducing the level of tumor growth, arresting
cancer progression, improving the immune response against the cancer, inhibiting
metastatic processes) or improving outcomes in the individual (e.g., improving survival).
The present invention includes the delivery istration, zation) of
a yeast-Brachyury immunotherapeutic ition of the invention to a subject or
individual. The administration process can be performed ex vivo or in viva, but is typically
performed in viva. Ex vivo administration refers to performing part of the regulatory step
outside of the patient, such as administering a composition of the present invention to a
tion of cells (dendritic cells) removed from a patient under conditions such that a
yeast vehicle, n(s) and any other agents or compositions are loaded into the cell, and
returning the cells to the patient. The eutic composition of the present invention can
be returned to a t, or administered to a patient, by any suitable mode of
administration.
Administration of a composition can be systemic, mucosal and/or proximal to
the location of the target site (e. g., near a site of a tumor). Suitable routes of
administration will be apparent to those of skill in the art, depending on the type of cancer
to be prevented or treated and/or the target cell population or tissue. Various acceptable
methods of administration include, but are not limited to, intravenous administration,
intraperitoneal stration, intramuscular administration, odal administration,
intracoronary administration, intraarterial stration (e.g., into a carotid artery),
subcutaneous administration, transdermal delivery, intratracheal administration,
intraarticular administration, intraventricular administration, inhalation (e.g., aerosol),
intracranial, intraspinal, intraocular, aural, intranasal, oral, pulmonary administration,
impregnation of a catheter, and direct injection into a tissue. In one aspect, routes of
administration include: enous, eritoneal, subcutaneous, intradermal, intranodal,
intramuscular, transdermal, d, intranasal, oral, intraocular, intraarticular, intracranial,
and intraspinal. Parenteral delivery can include intradermal, intramuscular, intraperitoneal,
intrapleural, ulmonary, intravenous, subcutaneous, atrial er and venal catheter
routes. Aural delivery can e ear drops, intranasal delivery can include nose drops or
intranasal injection, and intraocular delivery can include eye drops. Aerosol (inhalation)
delivery can also be performed using methods standard in the art (see, for example,
Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992). In one aspect, a
yeast-Brachyury immunotherapeutic ition of the invention is administered
subcutaneously. In one aspect, the yeast-Brachyury immunotherapeutic composition is
administered directly into a tumor milieu.
] In general, a suitable single dose of a yeast-Brachyury immunotherapeutic
composition is a dose that is e of effectively providing a yeast vehicle and the
Brachyury antigen to a given cell type, tissue, or region of the patient body in an amount
effective to elicit an antigen-specific immune response t one or more Brachyury
antigens or epitopes, when administered one or more times over a suitable time period.
For example, in one embodiment, a single dose of a yeast-Brachyury of the present
invention is from about 1 x 105 to about 5 x 107 yeast cell equivalents per kilogram body
weight of the organism being administered the composition. In one aspect, a single dose
of a yeast vehicle of the present invention is from about 0.1 Yeast Units (Y.U., which is 1
x 106 yeast cells or yeast cell lents) to about 100 Y.U. (1 x 109 cells) per dose (i.e.,
per organism), including any interim dose, in increments of 0.1 x 106 cells (i.e., 1.1 x 106,
1.2 x 106, 1.3 x 106...). In one embodiment, a le dose includes doses between 1 Y.U.
and 40 Y.U. and in one , between 10 Y.U. and 40 Y.U. In one embodiment, the
doses are administered at different sites on the individual but during the same dosing
period. For example, a 40 Y.U. dose may be administered by injecting 10 Y.U. doses to
four ent sites on the individual during one dosing period. The ion includes
administration of an amount of the yeast-Brachyury immunotherapy composition (e.g., 1,
2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 Y.U. or more) at 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more different sites on an individual to form a single dose. One Yeast Unit
(Y.U.) is 1 x 107 yeast cells or yeast cell equivalents.
"Boosters" or "boosts" of a therapeutic ition are administered, for
example, when the immune se against the antigen has waned or as needed to
provide an immune response or induce a memory response against a ular antigen or
antigen(s). Boosters can be administered about 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart, or
monthly, bimonthly, quarterly, annually, and/or in a few or several year increments after
the original administration, depending on the status of the individual being treated and the
goal of the therapy at the time of administration (e.g., prophylactic, active treatment,
maintenance). In one embodiment, an administration schedule is one in which doses of
Brachyury immunotherapeutic composition is stered at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, or more times over a time period of from weeks, to , to years. In one
embodiment, the doses are administered weekly or biweekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more doses, followed by biweekly or monthly doses as needed to achieve the desired
preventative or therapeutic treatment for cancer. Additional boosters can then be given at
similar or longer intervals (months or years) as a maintenance or remission therapy, if
desired.
In one aspect of the invention, one or more additional therapeutic agents or
therapeutic protocols are administered or performed sequentially and/or concurrently with
the administration of the yeast-Brachyury immunotherapy composition (e.g., surgical
resection of the tumor, administration of chemotherapy, administration of radiation
therapy, administration of another immunotherapy composition or protocol, cytokine
therapy, adoptive T cell er, or stem cell transplantation). For example, one or more
therapies can be administered or performed prior to the first dose of yeast-Brachyury
immunotherapy ition or after the first dose is administered. In one embodiment,
one or more therapies can be administered or med in an alternating manner with the
dosing of yeast-Brachyury immunotherapy composition, such as in a protocol in which the
yeast-Brachyury composition is stered at prescribed intervals in between one or
more consecutive doses of chemotherapy or other therapy. In one embodiment, the yeast-
Brachyury immunotherapy composition is administered in one or more doses over a
period of time prior to commencing additional therapies. In other words, the yeast-
Brachyury immunotherapeutic composition is administered as a monotherapy for a period
of time, and then an onal therapy is added (e.g., chemotherapy), either concurrently
with new doses of yeast-Brachyury immunotherapy, or in an alternating fashion with
yeast-Brachyury immunotherapy. Alternatively or in addition, another therapy may be
administered for a period of time prior to beginning administration of the yeast-Brachyury
immunotherapy composition, and the concepts may be combined (e.g., surgical resection
of a tumor, followed by monotherapy with yeast-Brachyury immunotherapy for several
weeks, followed by alternating doses of chemotherapy and yeast-Brachyury
therapy for weeks or months, optionally followed by monotherapy using yeast-
Brachyury immunotherapy or another therapy, or by a new protocol of combinations of
therapy provided sequentially, concurrently, or in ating fashion). Various ols
for the treatment of cancer using yeast-Brachyury immunotherapy are plated by the
invention, and these examples should be considered to be non-limiting examples of
various possible protocols.
In one aspect of the invention, additional antigens other than Brachyury are
also targeted using yeast-based immunotherapy, in on to targeting Brachyury. Such
additional target antigens can be included within the same vehicle as the Brachyury
ns, or additional yeast-based immunotherapy compositions targeting different
ns can be produced and then combined as desired depending on the individual to be
treated, the ns expressed by the type of cancer or by the individual’s particular tumor,
and/or depending on the stage of cancer in the individual, or the stage of treatment of the
individual. For es a ation of antigens may be ed that cover: (1)
antigens involved in seminal events in cancer development, such as mutated Ras, antigens
involved in or associated with dysregulation of cellular processes, such as CEA, and (3)
Brachyury, which is involved in metastatic processes. For example, on or more other
yeast-based immunotherapy compositions may express one or more antigens including,
but not limited to, carcinoembryonic antigen (CEA), point mutated Ras oncoprotein,
MUC-l, EGFR, BCR—Abl, , , MAGE-3, GAGE, GP-lOO, MUC-2,
normal and point mutated p53 oncoproteins, PSMA, nase, TRP-l (gp75), NY-ESO-l,
TRP-2, TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2, hTERT, p73, B-RAF,
adenomatous polyposis coli (APC), Myc, von Hippel-Lindau protein (VHL), Rb-l, Rb-2,
androgen receptor (AR), Smad4, MDRl, Flt-3, BRCA-l, BRCA-2, an3-fl<hr, ews-fii-l,
HERV-H, HERV-K, TWIST, Mesothelin, NGEP, modifications of such antigens, splice
variants of such antigens, and epitope agonists of such ns, as well as ations
of such antigens, and/or immunogenic domains thereof, modifications f, variants
thereof, and/or epitope agonists thereof. One, two, three, or more of these yeast-based
immunotherapy compositions may be administered to an individual prior to, concurrently
or alternating with, and/or after administration of a Brachyury immunotherapy
composition, in order to optimize targeting of antigens in the individual’s tumor. As
above, additional therapies can also be used in such protocols (e.g., surgical resection of
tumor, chemotherapy, targeted cancer therapy, radiation therapy, etc.).
In one embodiment of the invention, a method to treat cancer is provided. The
method includes the steps of: (a) stering to an individual who has cancer in which
Brachyury expression has not been detected, a first therapeutic composition
comprising a yeast vehicle and a first cancer antigen that does not comprise a ury
antigen; and (b) administering to the individual, prior to, concurrently with, or uent
to, administration of the first immunotherapeutic composition a second
immunotherapeutic composition comprising a yeast vehicle and a second cancer antigen
comprising a Brachyury antigen. In additional embodiments, the method can include
stering one or more onal immunotherapeutic compositions, wherein the each
of the one or more additional immunotherapeutic compositions comprises an additional
cancer antigen. The additional antigen can be any of those known in the art or described
herein, including, but not d to, mutated Ras, carcinoembryonic antigen (CEA), and
MUC-l.
In another embodiment of the invention, a method to treat cancer includes the
following steps: (a) administering to an individual who has cancer a first
immunotherapeutic composition comprising a yeast vehicle and a mutated Ras antigen; (b)
administering to the individual of (a) a second immunotherapeutic composition
comprising a yeast vehicle and an antigen selected from the group consisting of
carcinoembryonic antigen (CEA) and mucin-l ); and (c) administering to the
individual of (a) and (b) a third immunotherapeutic composition comprising a yeast
vehicle and a Brachyury antigen. One or more of the steps of administration in (a), (b) and
(c) can be med concurrently, or sequentially. Steps may be repeated as needed to
treat a ular individual’s cancer, and the cancer antigens can be modified before or
during treatment to cally address the particular dual’s cancer.
In the method of the present invention, compositions and therapeutic
compositions can be administered to animal, ing any vertebrate, and particularly to
any member of the Vertebrate class, Mammalia, including, without limitation, primates,
rodents, livestock and domestic pets. Livestock include mammals to be consumed or that
produce useful products (e.g., sheep for wool production). Mammals to treat or protect
utilizing the ion include humans, non-human primates, dogs, cats, mice, rats, goats,
sheep, , horses and pigs.
An “individual” is a vertebrate, such as a mammal, including without
limitation a human. Mammals include, but are not limited to, farm animals, sport animals,
pets, primates, mice and rats. The term “individual” can be used interchangeably with the
term “animal”, “subject” or “patient”.
General Techniques Useful in the Invention
The practice of the present ion will , unless otherwise indicated,
conventional techniques of molecular biology (including recombinant techniques),
microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which
are well known to those skilled in the art. Such techniques are explained fully in the
literature, such as, s of Enzymology, Vol. 194, Guthrie et al., eds., Cold Spring
Harbor tory Press (1990); Biology and activities of yeasts, Skinner, et al., eds.,
Academic Press (1980); Methods in yeast genetics : a tory course manual, Rose et
al., Cold Spring Harbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle
and Cell Biology, Pringle et al., eds., Cold Spring Harbor tory Press (1997); T_he
Yeast Saccharomyces: Gene Expression, Jones et al., eds., Cold Spring Harbor Laboratory
Press ; The Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and
Energetics, Broach et al., eds., Cold Spring Harbor Laboratory Press (1992); lar
Cloning: A Laboratogy Manual, second edition (Sambrook et al., 1989) and Molecular
Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly
referred to herein as “Sambrook”); Current Protocols in Molecular Biology (F.M. Ausubel
et al., eds., 1987, including supplements through 2001); PCR: The rase Chain
Reaction, (Mullis et al., eds., 1994); Harlow and Lane (1988), Antibodies, A Laboratogy
Manual, Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Mg
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY ly ed to herein as “Harlow and Lane”), Beaucage et al. eds.,
Current Protocols in Nucleic Acid Chemistgy, John Wiley & Sons, Inc., New York, 2000);
Casarett and s Toxicology The Basic Science of s, C. Klaassen, ed., 6th
edition (2001), and Vaccines, S. Plotkin, W. Orenstein, and P. Offlt, eds., Fifth Edition
(2008).
General Definitions
A “TARMOGEN®” (GlobeImmune, Inc., Louisville, do) generally
refers to a yeast vehicle expressing one or more heterologous antigens extracellularly (on
its e), intracellularly (internally or lically) or both extracellularly and
intracellularly. TARMOGEN®s have been generally described (see, e. g., US. Patent No.
463). Certain yeast-based immunotherapy compositions, and methods of making
and generally using the same, are also described in detail, for example, in US. Patent No.
,830,463, US. Patent No. 7,083,787, US. Patent No. 7,736,642, Stubbs et al., Nat. Med.
7:625-629 (2001), Lu et al., Cancer Research 64:5084-5088 (2004), and in Bernstein et al.,
Vaccine 2008 Jan 24;26(4):509-21, each of Which is incorporated herein by reference in its
As used herein, the term "analog" refers to a chemical compound that is
structurally similar to r compound but differs slightly in composition (as in the
replacement of one atom by an atom of a different element or in the presence of a
particular functional group, or the replacement of one onal group by another
functional group). Thus, an analog is a compound that is similar or comparable in function
and appearance, but has a different structure or origin With respect to the reference
compound.
The terms "substituted", "substituted derivative" and "derivative", When used
to describe a compound, means that at least one hydrogen bound to the unsubstituted
compound is replaced with a different atom or a chemical moiety.
Although a derivative has a similar physical structure to the parent compound,
the derivative may have different chemical and/or biological properties than the parent
compound. Such properties can e, but are not d to, increased or decreased
activity of the parent compound, new ty as compared to the parent compound,
enhanced or decreased bioavailability, ed or decreased efficacy, ed or
decreased stability in vitro and/or in vivo, and/or enhanced or decreased absorption
properties.
In general, the term "biologically active" tes that a nd (including
a protein or peptide) has at least one detectable actiVity that has an effect on the metabolic,
physiological, al, or other processes of a cell, a tissue, or an organism, as measured
or observed in viva (i.e., in a natural physiological environment) or in vitro (i.e., under
laboratory conditions).
According to the present invention, the term “modulate” can be used
interchangeably with “regulate” and refers lly to lation or downregulation of
a particular activity. As used herein, the term “upregulate” can be used generally to
describe any of: elicitation, tion, increasing, augmenting, boosting, ing,
enhancing, amplifying, promoting, or ing, with respect to a ular activity.
Similarly, the term “downregulate” can be used generally to describe any of: decreasing,
reducing, inhibiting, ameliorating, diminishing, lessening, blocking, or preventing, with
t to a particular activity.
In one embodiment of the present invention, any of the amino acid sequences
bed herein can be produced with from at least one, and up to about 20, additional
heterologous amino acids flanking each of the C- and/or N—terminal ends of the specified
amino acid sequence. The resulting protein or ptide can be referred to as
"consisting essentially of‘ the specified amino acid sequence. According to the present
invention, the heterologous amino acids are a sequence of amino acids that are not
naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid
sequence, or that are not related to the function of the specified amino acid ce, or
that would not be encoded by the nucleotides that flank the naturally occurring nucleic
acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such
nucleotides in the naturally occurring sequence were translated using standard codon
usage for the organism from which the given amino acid sequence is derived. Similarly,
the phrase "consisting essentially of', when used with reference to a nucleic acid sequence
herein, refers to a nucleic acid sequence encoding a specified amino acid sequence that can
be flanked by from at least one, and up to as many as about 60, additional heterologous
nucleotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the
specified amino acid sequence. The heterologous nucleotides are not naturally found (i.e.,
not found in nature, in viva) flanking the nucleic acid sequence encoding the specified
amino acid sequence as it occurs in the l gene or do not encode a protein that
imparts any additional function to the protein or changes the function of the protein having
the ed amino acid sequence.
According to the present invention, the phrase tively binds to" refers to
the ability of an antibody, n-binding fragment or binding partner of the t
invention to preferentially bind to specified proteins. More specifically, the phrase
"selectively binds" refers to the specific binding of one protein to another (e.g., an
antibody, fragment thereof, or binding partner to an n), wherein the level of binding,
as measured by any standard assay (e.g., an assay), is statistically significantly
higher than the background control for the assay. For example, when performing an
assay, ls typically e a reaction well/tube that contain antibody or
antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of
reactiVity (e.g., non-specific binding to the well) by the antibody or antigen-binding
fragment thereof in the absence of the antigen is considered to be background. Binding
can be measured using a variety of methods standard in the art including enzyme
immunoassays (e.g., ELISA, immunoblot assays, etc.).
General reference to a protein or polypeptide used in the t invention
includes full-length proteins, near full-length proteins (defined above), or any nt,
domain (structural, functional, or immunogenic), conformational e, or a homologue
or variant of a given protein. A fusion protein may also be generally ed to as a
protein or polypeptide. An isolated protein, according to the present invention, is a protein
(including a polypeptide or peptide) that has been removed from its natural milieu (i.e.,
that has been subject to human lation) and can include purified proteins, partially
d proteins, recombinantly produced ns, and synthetically produced proteins,
for example. As such, "isolated" does not reflect the extent to which the protein has been
purified. Preferably, an isolated protein of the present invention is produced
recombinantly. According to the present invention, the terms "modification" and
"mutation" can be used interchangeably, particularly with regard to the
ations/mutations to the amino acid sequence of proteins or portions thereof (or
nucleic acid sequences) bed herein.
As used herein, the term "homologue" or “variant” is used to refer to a protein
or peptide which differs from a reference protein or peptide (i.e., the "prototype" or "wild-
type" protein) by minor modifications to the reference protein or peptide, but which
maintains the basic protein and side chain structure of the naturally occurring form. Such
changes include, but are not limited to: changes in one or a few amino acid side chains;
changes one or a few amino acids, ing deletions (e. g., a truncated version of the
protein or peptide) insertions and/or substitutions; s in stereochemistry of one or a
few atoms; and/or minor derivatizations, including but not limited to: methylation,
glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation,
amidation and/or addition of glycosylphosphatidyl inositol. A homologue or variant can
have enhanced, decreased, or substantially similar properties as compared to the reference
protein or peptide. A homologue or variant can include an agonist of a protein or an
antagonist of a n. Homologues or variants can be produced using techniques known
in the art for the production of proteins including, but not d to, direct modifications
to the isolated nce protein, direct protein synthesis, or modifications to the nucleic
acid sequence encoding the n using, for example, classic or recombinant DNA
techniques to effect random or ed mutagenesis, resulting in the encoding of a protein
variant. In addition, naturally occurring variants of a reference n may exist (e.g.,
isoforms, allelic variants, or other natural variants that may occur from individual to
individual) and may be isolated, produced and/or utilized in the invention.
A homologue or variant of a given protein may comprise, consist ially
of, or consist of, an amino acid sequence that is at least about 45%, or at least about 50%,
or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%,
or at least about 75%, or at least about 80%, or at least about 85%, or at least about 86%
identical, or at least about 87% identical, or at least about 88% identical, or at least about
89% identical, or at least about 90%, or at least about 91% identical, or at least about 92%
identical, or at least about 93% identical, or at least about 94% identical, or at least about
95% identical, or at least about 96% identical, or at least about 97% identical, or at least
about 98% identical, or at least about 99% identical (or any percent identity between 45%
and 99%, in whole integer increments), to the amino acid sequence of the reference
protein (e. g., an amino acid sequence specified herein, or the amino acid sequence of a
specified protein). In one embodiment, the homologue or variant comprises, ts
essentially of, or consists of, an amino acid sequence that is less than 100% identical, less
than about 99% identical, less than about 98% identical, less than about 97% identical, less
than about 96% identical, less than about 95% identical, and so on, in increments of 1%, to
less than about 70% identical to the amino acid ce of the reference protein.
As used herein, unless otherwise specified, reference to a percent (%) identity
refers to an evaluation of gy which is performed using: (1) a Basic Local
Alignment Search Tool (BLAST) basic homology search using blastp for amino acid
searches and blastn for nucleic acid searches with standard default parameters, n the
query ce is filtered for low complexity regions by default (such as described in
Altschul, S.F., Madden, T.L., Schaaffer, A.A., Zhang, J ., Zhang, 2., Miller, W. & Lipman,
D]. (1997) "Gapped BLAST and PSI-BLAST: a new tion of protein database
search ms." Nucleic Acids Res. 25:3389-3402, orated herein by reference in
its entirety); (2) a BLAST alignment of two sequences (e. g., using the parameters
described below); (3) and/or PSI-BLAST with the standard default parameters (Position-
Specific Iterated BLAST. It is noted that due to some differences in the standard
parameters between Basic BLAST and BLAST for two sequences, two specific sequences
might be recognized as having significant homology using the BLAST program, whereas a
search performed in Basic BLAST using one of the sequences as the query sequence may
not identify the second sequence in the top matches. In addition, PSI-BLAST provides an
automated, o-use version of a "profile" search, which is a sensitive way to look for
sequence gues. The program first ms a gapped BLAST database search. The
PSI-BLAST program uses the information from any significant alignments returned to
uct a position-specific score matrix, which replaces the query sequence for the next
round of database searching. ore, it is to be understood that percent identity can be
ined by using any one of these programs.
Two specific sequences can be aligned to one another using BLAST as
described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing
n and tide sequences", FEMS Microbiol Lett. 7-250, incorporated
herein by nce in its entirety. Such a sequence alignment is performed in blastp or
blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0)
between the two sequences allowing for the introduction of gaps ions and insertions)
in the resulting alignment. For purposes of clarity herein, a BLAST sequence alignment
for two sequences is performed using the standard default ters as follows.
For blastn, using 0 BLOSUM62 matrix:
Reward for match = l
Penalty for mismatch = -2
Open gap (5) and extension gap (2) penalties
gap x_dropoff (50) expect (10) word size (ll) filter (on)
For blastp, using 0 BLOSUM62 matrix:
Open gap (ll) and extension gap (1) penalties
gap x_dropoff (50) expect (10) word size (3) filter (on).
An isolated nucleic acid molecule is a nucleic acid molecule that has been
removed from its natural milieu (i.e., that has been subject to human manipulation), its
natural milieu being the genome or chromosome in which the c acid molecule is
found in nature. As such, "isolated" does not necessarily reflect the extent to which the
nucleic acid le has been purified, but indicates that the molecule does not include
an entire genome or an entire chromosome or a segment of the genome containing more
than one gene, in which the nucleic acid le is found in . An isolated nucleic
acid le can include a complete gene. An isolated nucleic acid molecule that
includes a gene is not a fragment of a some that includes such gene, but rather
includes the coding region and regulatory regions associated with the gene, but no
additional genes that are naturally found on the same chromosome. An isolated nucleic
acid molecule may also include portions of a gene. An ed nucleic acid molecule can
also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end of
the sequence) additional nucleic acids that do not normally flank the specified nucleic acid
sequence in nature (i.e., heterologous sequences). Isolated nucleic acid molecule can
include DNA, RNA (e. g., mRNA), or derivatives of either DNA or RNA (e. g., cDNA).
Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid
molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of
nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably,
especially with respect to a nucleic acid le, or a nucleic acid sequence, being
capable of encoding a n or domain of a n.
A inant nucleic acid molecule is a molecule that can include at least
one of any nucleic acid sequence encoding any one or more proteins described herein
operatively linked to at least one of any transcription control sequence capable of
ively regulating expression of the nucleic acid molecule(s) in the cell to be
transfected. Although the phrase "nucleic acid le" primarily refers to the al
nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the
sequence of nucleotides on the nucleic acid molecule, the two phrases can be used
interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid
sequence, being capable of encoding a protein. In addition, the phrase "recombinant
molecule" primarily refers to a nucleic acid molecule operatively linked to a ription
control sequence, but can be used interchangeably with the phrase "nucleic acid molecule"
which is administered to an animal.
A recombinant nucleic acid molecule includes a recombinant vector, which is
any nucleic acid sequence, typically a heterologous sequence, which is operatively linked
to the isolated nucleic acid molecule ng a fusion protein of the present ion,
which is capable of enabling recombinant production of the fusion protein, and which is
capable of delivering the nucleic acid le into a host cell according to the t
invention. Such a vector can contain nucleic acid sequences that are not naturally found
adjacent to the isolated nucleic acid molecules to be inserted into the vector. The vector
can be either RNA or DNA, either prokaryotic or eukaryotic, and preferably in the present
invention, is a plasmid useful for transfecting yeast. Recombinant vectors can be used in
the cloning, sequencing, and/or otherwise lating of nucleic acid molecules, and can
be used in delivery of such molecules (e.g., as in a DNA composition or a viral vector-
based composition). Recombinant s are preferably used in the expression of nucleic
acid molecules, and can also be referred to as expression vectors. Preferred recombinant
vectors are capable of being expressed in a transfected host cell, such as a yeast.
In a inant molecule of the present invention, nucleic acid les are
operatively linked to expression s containing regulatory sequences such as
ription control sequences, translation control sequences, origins of ation, and
other regulatory sequences that are compatible with the host cell and that control the
expression of nucleic acid molecules of the present invention. In particular, recombinant
molecules of the present ion include nucleic acid molecules that are operatively
linked to one or more expression control sequences. The phrase "operatively linked"
refers to linking a nucleic acid molecule to an expression control sequence in a manner
such that the molecule is expressed when transfected (i.e., transformed, uced or
transfected) into a host cell.
According to the present invention, the term “transfection” is used to refer to
any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid
molecule) can be inserted into a cell. The term "transformation" can be used
interchangeably with the term "transfection" when such term is used to refer to the
introduction of nucleic acid molecules into microbial cells, such as algae, bacteria and
yeast. In microbial systems, the term "transformation" is used to describe an inherited
change due to the acquisition of exogenous nucleic acids by the microorganism and is
essentially synonymous with the term "transfection.’ ore, transfection techniques
include, but are not limited to, transformation, al ent of cells, particle
bombardment, electroporation, microinjection, lipofection, adsorption, infection and
protoplast fusion.
The following experimental results are provided for purposes of illustration
and are not intended to limit the scope of the invention.
EXAMPLES
Example 1
] The following e describes the production of a yeast-Brachyury
immunotherapeutic composition.
] In this experiment, yeast aromyces cerevisiae) were engineered to
s human Brachyury under the control of the -inducible promoter, CUPI , or
the constitutive promoter, TEF2
, producing yeast-Brachyury immunotherapy compositions.
In each case, a fusion protein sing a Brachyury antigen was produced as a single
polypeptide with the following ce elements fused in frame from N— to C-terminus,
represented by SEQ ID NO:8 (1) an N—terminal peptide to impart resistance to
proteasomal degradation and stabilize sion (positions 1 to 6 of SEQ ID NO:8, the
peptide sequence also represented herein by SEQ ID NO:11); 2) amino acids 2-435 of
SEQ ID NO:6, SEQ ID NO:6 representing a near full-length human Brachyury protein
(positions 7-440 of SEQ ID NO:8); and (3) a hexahistidine tag (positions 441-446 of SEQ
ID NO:8). The amino acid ces used in this fusion protein can be modified by the
use of additional or alternate amino acids flanking either end of the Brachyury antigen, if
desired, and r portions of the Brachyury antigen may also be used. A nucleic acid
sequence encoding the fusion protein of SEQ ID NO:8 (codon optimized for yeast
expression) is represented herein by SEQ ID NO:7.
Briefly, DNA encoding a full length human Brachyury protein from a
Brachyury-PCRII plasmid provided by the al Cancer Institute (Dr. Jeffrey Schlom)
was amplified using PCR, and then inserted at EcoRI and Spel cloning sites behind the
CUP] promoter (vector pGI-lOO) or the TEF2 promoter (vectors pluOll or 2) in
yeast 2 um expression vectors. tide sequences encoding the N—terminal
stabilization peptide, MADEAP (SEQ ID NO: 1 l) and a C-terminal hexahistidine peptide
were also added to the plasmid vector to encode the complete fusion protein represented
by SEQ ID NO:8. The resulting plasmids were transformed into DH50L for plasmid
storage, and into Saccharomyces cerevisiae W3030L for production of the yeast-Brachyury
immunotherapeutic compositions.
Transformation into Saccharomyces cerevisiae was performed by m
acetate/polyethylene glycol transfection, and primary transfectants were selected on solid
minimal plates lacking Uracil (UDM; uridine dropout medium). es were selected
by growing in U2 (uridine dropout ) or UL2 (uridine and leucine dropout medium)
medium at 30° C.
The yeast-Brachyury immunotherapy composition comprising a
polynucleotide encoding the human Brachyury fusion protein represented by SEQ ID
NO:8 under the control of the CUP] promoter is also ed to herein as GI-6301. The
yeast-Brachyury immunotherapy composition comprising a polynucleotide encoding the
human Brachyury fusion protein represented by SEQ ID NO:8 under the control of the
TEF2 promoter (in vector plu011) is also referred to herein as GI-6302. The yeast
Brachyury immunotherapy composition comprising a polynucleotide encoding the human
Brachyury fusion protein ented by SEQ ID NO:8 under the control of the TEF2
promoter (in vector pGI-172) is also ed to herein as GI-6303.
Liquid cultures lacking uridine (U2) or lacking uridine and leucine (UL2)
were inoculated using the plates and starter cultures described above, and were grown for
20h at 30°C, 250 rpm. pH buffered media containing 4.2g/L of Bis-Tris (BT-U2; BT-
UL2) were also inoculated to evaluate yeast-Brachyury immunotherapeutics produced
under l pH manufacturing conditions (data not shown). Primary cultures were used
to inoculate final es of the same ation.
Recipe for U2 liguid media:
0 15g/L of glucose
0 6.7 g/L of Yeast nitrogen base containing ammonium sulfate
0 0.04 g/L each of histidine, tryptophan, adenine and 0.06 g/L of leucine
Recipe for UL2 liguid media:
0 15g/L of glucose
0 6.7 g/L of Yeast nitrogen base containing ammonium sulfate
0 0.04 g/L each of histidine, tryptophan, and adenine
In initial experiments comparing yeast-Brachyury immunotherapeutic
compositions under the control of different promoters, CUPI-driven (inducible
sion) yeast-Brachyury sion was initiated by the addition of 0.5 mM copper
sulfate after the yeast-Brachyury culture d a density of approximately 0.2 l,
and was continued until the culture reached a density of 0.5-1.5 Y.U. (yeast-Brachyury
doubled only about 1-1.5 after the addition of copper sulfate, but a large amount of
Brachyury protein was produced by the cells). riven yeast-Brachyury expression
is constitutive, and growth of these cells was continued until the cultures reached a density
of between 1.1 to 4.0 Y.U./ml. The cells from each culture were then harvested, washed
and heat-killed at 56°C for 1 hour in PBS. Live cells from each culture were also
processed for comparison.
After ill of the cultures, the cells were washed three times in PBS. Total
protein expression was measured by a TCA precipitation/nitrocellulose binding assay and
by Western blot using an anti-his tag monoclonal antibody and an anti-Brachyury antibody
(Abcam, Cambridge, MA). Protein content was quantified using semi-quantitative digital
imaging methods.
The results of the initial expression experiments (data not shown)
demonstrated that each of the yeast-Brachyury therapy compositions of the
invention sed the Brachyury fusion protein, z'.e., using either the CUPI er or
the TEF2 promoter, and sion was detected using either media (U2 and UL2). In
on, antigen expression was detectable in both heat-killed and live yeast cells (data
not shown). Brachyury expression was significantly higher in the yeast-Brachyury
immunotherapeutic composition sing the CUP] er (GI-6301) and so this
composition was selected for further studies, including expression optimization and for in
vitro and in vivo experiments (see Examples below).
Fig. 1A shows expression of Brachyury in GI-6301 using both U2 and UL2
media using the anti-Brachyury antibody for detection. Control yeast expressing a non-
Brachyury antigen did not stain with the antibody. Fig. 1B shows expression of
Brachyury in the same GI-6301 preparations, using anti-His to identify the hexahistidine
tag on the Brachyury fusion n. Control yeast expressing a non-Brachyury antigen
but having a hexahistidine tag is also shown. These results showed good ury
expression using either media, although expression in UL2 media was significantly higher.
Example 2
The following example describes the identification of conditions for antigen
expression and manufacturing of the yeast-Brachyury immunotherapeutic composition,
To determine the optimum density for copper induction of GI-6301 antigen
expression, starter and intermediate cultures of GI-6301 were prepared using the standard
growth conditions in UL2 media described in Example 1 above. Aliquots of the culture
were then d to 0.5 Y.U./ml, 1.0 l and 1.5 Y.U./ml and incubated at 30°C for 1
hour. 0.5 mM CuSO4 was added to the cultures to induce Brachyury expression, and
culturing was continued. Cells were collected and counted at 6 hours and 14 hours for
measurement of cell density. 20 Y.U. of heat-killed yeast from each condition was lysed,
total protein was measured, and Western blots were generated using anti-His.
Cell Density
As shown in Table l, yeast only doubled about 1 time after copper induction
(other experiments showed up to 1.5X doubling), and cell y and viability (not
shown) decreased after 6 hours of copper induction. Fig. 2 shows that all three induction
densities resulted in significant expression of Brachyury, with a trend toward higher
Brachyury expression at the higher induction ies. However, onal experiments
using induction starting densities of 2.1 Y.U./ml and 2.8 Y.U./ml and 375 uM CuSO4
showed that protein expression began to decrease as the density of the cultures at the start
of copper induction increased, and did not significantly e after about 6-8 hours
(data not .
Next, the effect of the amount of CuSO4 on Brachyury expression was
investigated. GI-6301 was grown from starter and intermediate es in UL2 media as
described in Example 1. Aliquots of the culture were then diluted to 1.0 Y.U./ml and
incubated at 30°C for 1 hour. CuSO4 was added to each culture at a concentration of
either 375 uM or 500 uM, and induction of protein expression was allowed to proceed to
various time points (2 hrs, 4 hrs, 6 hrs, 8 hrs, 24 hrs), at which point the cells were
harvested, heat-killed, and processed for evaluation of protein expression using anti-His
Western blots as described above. While both concentrations of CuSO4 ed in good
expression of Brachyury, protein expression using 375 uM appeared to be slightly better,
particularly at later time points (data not shown).
Accordingly, for CUPI-driven Brachyury (inducible expression), the
inventors discovered that induction of n expression at mid-log phase growth of the
yeast was optimal for antigen production. For production of the yeast-Brachyury
immunotherapeutic composition 01) used in the following Examples, cells were
grown in UL2 media as described in Example 1 to between 1 and 2 Y.U./ml, and were
then induced by the addition of 0375-05 mM copper sulfate for up to 6-8 hours at 30°C,
250 rpm. Cells were harvested, washed and heat killed at 56°C for 1h in PBS.
Example 3
The ing example describes the construction and production of an
additional yeast-Brachyury immunotherapeutic composition, where the Brachyury antigen
contains a T cell t epitope.
In this experiment, yeast (Saccharomyces cerevisiae) were engineered to
s a human Brachyury antigen that is a ull-length Brachyury protein
comprising the T cell epitope WLLPGTSTV (SEQ ID NO: 13), which is an agonist epitope.
The native ury T cell epitope, present in SEQ ID NO:6 or 8, for example, is
WLLPGTSTL (SEQ ID NO:l2). The human Brachyury agonist antigen was expressed
under the control of the copper-inducible promoter, CUP], producing a yeast-Brachyury
immunotherapy composition. More particularly, a fusion protein comprising a Brachyury
agonist n (i.e., a ury antigen containing at least one agonist epitope) was
produced as a single polypeptide with the following sequence ts fused in frame
from N- to C-terminus, represented by SEQ ID NO:20 (1) an N-terminal peptide to impart
resistance to proteasomal ation and stabilize expression (positions 1 to 6 of SEQ ID
NO:20, the peptide sequence also represented herein by SEQ ID NO:ll); 2) amino acids
2-435 of SEQ ID NO:l8 (represented by positions 7-440 of SEQ ID NO:20; SEQ ID
NO:l8 represents a full-length human ury t protein having a single amino
acid substitution at position 254 as compared to wild-type Brachyury protein); and (3) a
hexahistidine tag (positions 441-446 of SEQ ID NO:20). The agonist epitope (SEQ ID
NO:l3) is located at positions 251 to 259 of SEQ ID NO:20 (positions 246 to 254 of SEQ
ID NO:l8). The amino acid sequences used in this fusion n can be modified by the
use of additional or alternate amino acids flanking either end of the Brachyury antigen, if
desired, and shorter portions of the Brachyury antigen may also be used. A nucleic acid
sequence encoding the fusion protein of SEQ ID NO:20 (codon optimized for yeast
expression) is represented herein by SEQ ID NO: 19.
Briefly, DNA encoding the near full-length human Brachyury protein as
described in e 1 (i.e., full-length Brachyury minus the N-terminal methionine),
modified by site directed mutagenesis to introduce a substitution of a valine for the e
at position 254 with respect to the full-length Brachyury protein, was amplified using PCR,
and then inserted at EcoRI and Spel cloning sites behind the CUP] promoter (vector pGI-
100) in yeast 2 um expression vectors. tide sequences encoding the N—terminal
stabilization peptide, MADEAP (SEQ ID NO: 1 l) and a C-terminal hexahistidine peptide
were also added to the plasmid vector to encode the te fusion protein represented
by SEQ ID NO:20. The resulting plasmids were ormed into DHSu for plasmid
storage, and into Saccharomyces cerevisiae W3030L for production of the yeast-Brachyury
therapeutic composition.
Transformation into Saccharomyces cerevisiae was performed by lithium
acetate/polyethylene glycol transfection, and primary transfectants were selected on solid
minimal plates lacking Uracil (UDM; uridine dropout medium). Colonies were selected
by growing in UL2 (uridine and leucine dropout medium) medium at 30° C.
The yeast-Brachyury immunotherapy composition comprising a
cleotide encoding the human Brachyury agonist fusion protein represented by SEQ
ID NO:20 under the control of the CUP] promoter is also referred to herein as GI-6305.
GI-6305 cells were grown in UL2 media as described in Example 1 to
between 1 and 2 Y.U./ml, and were then induced by the addition of 5 mM copper
sulfate for up to 6-8 hours at 30°C, 250 rpm, using the conditions developed by the
inventors for GI-6301 as described in Example 2. Cells were harvested, washed and heat
killed at 56°C for lh in PBS.
After heat-kill of the cultures, the cells were washed three times in PBS. Total
protein expression was measured by a TCA precipitation/nitrocellulose binding assay and
by Western blot using an anti-his tag monoclonal antibody and an anti-Brachyury antibody
(Abcam, Cambridge, MA). Protein t was quantified using semi-quantitative digital
imaging methods.
Fig. 1C shows the robust expression of Brachyury agonist antigen in GI-6305
using anti-His to identify the hexahistidine tag on the ury fusion protein. The
approximate antigen content for GI-6305 grown in UL2 medium in this experiment was
>22615 ng/Y.U.
Example 4
] The following example demonstrates the expansion of Brachyury-specific
T cells using a Brachyury immunotherapeutic composition of the invention.
To determine whether T cells from normal donors were e of generating
T cells that are specific for Brachyury antigen, tic cells (DCs) were prepared from
the peripheral blood mononuclear cells (PBMCs) of two normal donors. Briefly, isolated
PBMCs were cultured for 5-days in the presence of GM-CSF and IL-4, and were
subsequently incubated with Control Yeast (also denoted , which is a
Saccharomyces cerevisiae yeast that is transformed with an empty vector, or vector that
does not contain an n-encoding ) or ury Yeast (GI-6301, described in
Examples 1 and 2 above), at a ratio of yeast:DCs= l:l. After rs co-culture, the DCs
were used as APCs for stimulation of autologous T cells. Each cycle of stimulation,
designated as IVS (in vitro stimulation), ted of 3 days e in absence of IL-2,
following by 4 additional days in the presence of recombinant IL-2 (20 U/ml). At the end
of IVS 2, T cells were stained with a control tetramer or a tetramer specific for the
Brachyury peptide Tp2 (WLLPGTSTL, positions 246 to 254 of SEQ ID NO:2 or SEQ ID
NO:6). Table 2 shows the percentage of CD8+ T cells that stained positive with each
tetramer.
Table 2
Donor Stimulation Control Brachyury
Tetramer er
Control Yeast
07706
Brachyury Yeast
Control Yeast
17663
Brachyury Yeast
In a second experiment, dendritic cells (DCs) were prepared from PBMCs of
nine normal donors using a 5-day culture in presence of GM-CSF and IL-4, subsequently
incubated with Brachyury yeast (GI-6301), at a ratio yeast:DCs= 1:1, as described above.
After 48-hours in co-culture, the DCs were used as APCs for stimulation of autologous T
cells. Each cycle of IVS was performed as described above. At the end of IVS 2, T cells
were stained with a control tetramer or a er specific for the Brachyury peptide Tp2.
Table 3 shows the percentage of CD8+ T cells that stained positive with each Tetramer.
Table 3
Brachyury Yeast
Brachyury Yeast
Brachyury Yeast
Brachyury Yeast
Brachyury Yeast
Brachyury Yeast
ury Yeast
Brachyury Yeast
Brachyury Yeast
The results in Tables 2 and 3 show that ation of normal donor T cells
with a Brachyury immunotherapeutic of the invention increases the tage of
tetramer-positive CD8+ T cells in a majority of the normal donors, as compared to controls,
indicating that normal human T cells have the capacity to ize Brachyury as an
immunogen.
Example 5
The following example demonstrates the ability of a yeast-Brachyury
immunotherapeutic composition to generate Brachyury-specific CTLs from normal donor
PBMCs that lyse Brachyury-expressing targets.
In this experiment, Brachyury-specific T cells from three of the normal donors
from Table 2 above were expanded in vitro using DCs incubated with Brachyury yeast
(GI-6301) for 2 cycles of IVS (as described in Example 4). A third IVS was carried out
with DCs matured in presence of CD40L and pulsed with the Brachyury-specific Tp2
peptide (WLLPGTSTL, positions 246 to 254 of SEQ ID NO:2 or SEQ ID NO:6). At day
, CD8+ T cells were isolated and used in an overnight xic T lymphocyte (CTL)
assay against SW480 (HLA-A2+ / Brachyury high) and MCF7 2+ / Brachyury
low ) tumor cell targets, at the indicated effector:target (ET) ratios (see Fig. 3). Shown in
Table 4 is the percentage of CD8+ T cells that stained positive with a control tetramer
versus a Brachyury-specific Tp2 tetramer.
Table 4
Brachyury
Yeast/Tp2
17663 Brachyury 0.11 0.65
Yeast/Tp2
26532 Brachyury 0.05 0.11
Yeast/Tp2
] Figs. 3A (donor 07706), 3B (donor 17663) and 3C (donor 26532) show that
PBMCs from two out of three normal donors were capable of generating CD8+ CTLs that
could kill targets expressing Brachyury. Taken together, these data demonstrate that
yeast-Brachyury immunotherapeutic compositions can generate Brachyury-specific CTLs
that are capable of killing a Brachyury-expressing tumor cell.
In order to show that yeast-Brachyury immunotherapy can induce Brachyury-
specific CTLs in the absence of pulsing with a specific peptide (i.e., by ting CTLs
against potentially multiple different CTL epitopes), additional experiments were
med using normal donor T cells expanded in vitro using only the yeast-Brachyury
immunotherapeutic composition, GI-6301 (i.e., no peptide pulse). Briefly, Brachyuryspecific
T cells from normal donor PBMCs (donor 19063) were expanded in vitro by
using DCs incubated with GI-6301 for 2 cycles of IVS (as described in e 4). At
day 5, CD8+ T cells were isolated and used in an overnight CTL assay against SW480
(HLA-A2 positive/Brachyury high) and H226 (HLA-A2 ve/Brachyury high) tumor
cells, at an effector:target (ET) ratio of 15:1. Fig. 4A shows the percentage of c
lysis of SW480 and H226 tumor cells. Fig. 4B shows the expression of Brachyury mRNA
relative to that of GAPDH in SW480 and H226 tumor cells by real-time RT-PCR. These
experiments further demonstrate that yeast-Brachyury immunotherapeutic composition
can te Brachyury-specific CTLs that are capable of killing a Brachyury-expressing
tumor cell.
Example 6
The following example demonstrates that a yeast-Brachyury composition of
the invention can expand Brachyury-specific T cells from cancer patients.
In this experiment, DCs were prepared from the PBMCs of two breast cancer
patients, post-vaccination with viral vector vaccines comprising CEA and MUC-l antigens.
The DCs were ed in a 5-day e in presence of GM-CSF and IL-4 as described
in e 4, followed by incubation in presence of Brachyury yeast (GI-6301) at a ratio
of yeast:DCs= 1:1. After 48-hours co-culture, the DCs were used as APCs for stimulation
of autologous T cells. Each cycle of IVS consisted of 3 days in absence of IL-2, ing
by 4 additional days in presence of 20 U/ml of recombinant IL-2. Shown in Table 5 is the
percentage of CD8+ T cells (IVSl) that stained positive with a control tetramer or a
tetramer specific for the ury peptide Tp2.
Table 5
Breast Pt 01 Brachyury Yeast 0.11 0.42
Breast Pt 10 ury Yeast 0.23 0.91
The results in Table 5 demonstrate that stimulation of T cells from breast
cancer donors with a yeast-Brachyury immunotherapeutic of the invention increases the
percentage of tetramer-positive CD8+ T cells in a majority of the donors, as compared to
controls, indicating that T cells from donors with ongoing cancer have the capacity to
recognize Brachyury as an gen.
The following Example demonstrates the generation of CD4+ T cell responses
specific for Brachyury in viva using yeast-Brachyury immunotherapy.
In this experiment, C57BL/6 mice were vaccinated weekly for a total of 4
times with 4 YU of yeast-hBrachyury (GI-6301), administered at four separate injection
sites at 1 YU per site). Two weeks after the final boost, the mice were sacrificed and CD4+
T cells were purified and assayed for proliferation in presence of various concentrations of
Brachyury purified n (obtained from insect cells). As a control, B-Gal was used at
40 ug/ml.
The results showing the proliferation of CD4+ T cells isolated from the spleens
of animals vaccinated with yeast-control (YVEC, see Example 4) and yeast-hBrachyury
01) are shown in Fig. 5. Fig. 5 shows that immunization with yeast-Brachyury (GI-
6301) generates Brachyury-specific CD4+ T cells.
Example 8
The following e demonstrates that immunization with yeast-Brachyury
therapeutic composition reduces Brachyury-expressing tumors in vivo.
In this experiment, C57BL/6 mice received 1 x 106 MC38-phBrachyury cells
(MC38 tumor cells sing a recombinant human Brachyury) via the tail vein (day 0).
Four days post-tumor implantation, animals began receiving weekly vaccinations with
yeast control (YVEC, see Example 4) versus yeast-hBrachyury (GI-6301), administered at
a dose of lYU per site at four different sites (4YU total per dose). At day 40 umor
implantation, animals were sacrificed and the number of lung tumor nodules was
evaluated. Results from two combined experiments are shown in Fig. 6. Table 6 shows
the mean lung tumor number (:: SEM) and the number (and percentage) of s
bearing 2 5 lung nodules.
Table 6
Yeast-Control (YVEC) 4.1 i 1.2 7/15 (46.7%)
Yeast-Brachyury (GI-6301) 1.9 i 0.5 2/15 (13.3%)
The results in Fig. 6 and Table 6 demonstrate that administration of a yeast-
Brachyury immunotherapeutic composition of the invention is e of reducing
Brachyury-expressing tumors in mice, as compared to mice receiving yeast alone (no
Brachyury antigen).
Example 9
The following example trates the generation of Brachyury-specific
CD4+ T cell ses in vitro using yeast-Brachyury immunotherapy in human peripheral
blood mononuclear cells (PBMCs) obtained from healthy donors.
In the ing experiments, a full-length human Brachyury protein was
expressed in insect cells via baculovirus expression and subsequently purified.
] Dendritic cells (DCs) were prepared from PBMCs of healthy donors by 5-day
culture with GM-CSF and IL-4 and subsequently treated in vitro with yeast-control
(YVEC, see e 4) or yeast-Brachyury (GI-6301, see Examples 1 and 2) (ratio
yeast:DCs = 1:1). After 48 hours, DCs were harvested, irradiated (30 Gy) and used for
stimulation of autologous PBMCs, at a ratio DC:PBMCs = 1:10. On day 3, IL-2 (10
U/ml) was added to the cultures. On day 7, stimulated T cells were ted and
uently tested for IFN—y production in response to autologous, irradiated PBMCs
(ratio T cells: PBMCs = 1:3) alone or in the presence of 10 ug/ml of purified Brachyury
protein or control human serum albumin protein. Following 96 hours stimulation,
supematants were collected and evaluated for IFN—y levels by ELISA assay. A total of 9
healthy donors were evaluated, with 3/9 donors demonstrating Brachyury-specific CD4+
T-cell ses post-stimulation in vitro with yeast-Brachyury-treated DCs. Results for 3
positive cases are presented in Table 7 (values indicate the levels of IFN-y in response to
Brachyury protein, after subtracting ound levels induced by stimulation with control Human
Serum Albumin protein; for donor 3, two cycles of stimulation were performed prior to evaluating
response to Brachyury protein).
Table 7
Donor ID DC stimulation 6mm!)
1 Yeast-control 1500.0
Yeast-Brachyury 2950.0
2 Yeast-control 13.4
Yeast-Brachyury 889.0
3 Yeast-control 17.4
Yeast-Brachyury 102.8
] Six additional healthy donors were evaluated for CD4+ T cell responses to the
Brachyury protein, following in vitro stimulation with yeast-Brachyury (GI-630l)-treated
DCs by ellular cytokine staining of IFN-y in CD4+ cells. Dendritic cells were
prepared from PBMCs of healthy donors by 5-day culture with GMCSF and IL-4 and
uently treated in vitro with yeast-control (YVEC) or yeast-Brachyury (GI-6301)
(ratio DCs = 1:1). After 48 hours, the DCs were harvested, irradiated (30 Gy) and
used for stimulation of autologous PBMCs, at a ratio Cs = 1:10. On day 3, IL-2
(10 U/ml) was added to the cultures. On day 7, stimulated T cells were harvested and
subsequently tested for IFN—y tion in response to autologous PBMCs (ratio T cells:
PBMCs = 1:3) alone or in the presence of 10 ug/ml of purified Brachyury protein or
control human serum albumin protein. Following 2 hours stimulation, BD
TOPTM Protein Transport Inhibitor (BD Biosciences) was added to the cultures.
Following 4 hours stimulation, cells were harvested, permeabilized, and stained for CD4
and IFN-y utilizing anti-CD4 PerCP-Cy5.5 and FN-y FITC antibodies (BD
Biosciences). A total of 6 healthy donors were evaluated, with 2/6 donors demonstrating
Brachyury-specific CD4+ T-cell responses post-stimulation in vitro with yeast-Brachyury
treated DCs. Results for positive cases are shown in Table 8 (values indicate the percentage
of T cells that were simultaneously positive for CD4 and intracellular IFN—y in response to l
human serum albumin (HSA) or Brachyury protein, after subtracting background levels induced
by stimulation with PBMCs alone).
Table 8
% CD4+IIFN-v+ cells
Number of
Donor
stlmulatlons In Vltro. . . . HSA Brachyury
4 1 0.07 0 24
2 0.00 1.00
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Example 10
The following example demonstrates that a yeast-Brachyury immunotherapy
composition expressing a Brachyury agonist antigen generates Brachyury-specific T cells
from a prostate cancer patient.
To generate a Brachyury-specific T-cell line, immature autologous dendritic
cells (DCs) were exposed to the yeast-Brachyury immunotherapy composition known at
GI-6305 (see Example 3) at a ratio of DCs:GI-6305 = 1:1 for 48 hours, and subsequently
used as antigen presenting cells (APCs) to stimulate autologous non-adherent cells at an
or-to-APC ratio of 10:1. Cultures were incubated for 3 days at 37°C, in a humidified
atmosphere containing 5% C02, and uently supplemented with recombinant human
IL-2 at a concentration of 20 U/ml for an additional 7 days. The 10-day culture
constituted one in vitro stimulation (IVS) cycle. T cells were restimulated with GI
exposed autologous DCs as described above on day 11, to begin the next IVS cycle. GI-
xposed autologous DCs were used as APCs for three IVS cycles. After the third
IVS, irradiated (23,000 rads) autologous EBV-transformed B cells, pulsed with an agonist
ury peptide, WLLPGTSTV (SEQ ID NO:13), were used as APCs. A Brachyury-
c T cell line, denoted, -A, was established. This T cell line was used in the
immunoassays described below.
Table 9 demonstrates that Brachyury-specific T cells (TBR-A) release
significant levels of IFN-y after stimulation with neic DCs treated with GI-6305,
whereas control yeast (YVEC, see Example 4) did not ate the release of IFN—y by T-
2-BR-A cells. Results are expressed in pg/ml/ 105 T cells. Briefly, allogeneic HLA-A2
positive DCs from a normal donor were treated with GI-6305 for 48 hours at various yeast
to DC ratios (indicated in Table 9) and then used to stimulate Brachyury agonist epitope-
specific T cells (TBR-A). In this experiment, the DC to T cell ratio was 1:10.
Table 9
Dendritic Cells Yegjttilgc T Cells lFN-v
—————
—————
—————
——-——
——--m
——-——
——--m
——-——
Table 10 demonstrates that Brachyury-specific T cells established by using
GI-6305 treated DCs can ively lyse MDA-MB-23l breast cancer cells that are HLA-
A2 positive/Brachyury ve, but do not lyse ASPC-l pancreatic cancer cells that are
HLA-A2 negative/Brachyury positive. Briefly, the ury-specific T cell line TBR—
A was used at IVS-6 in an overnight cytotoxic T lymphocyte (CTL) assay against MDA-
MB-23l (HLA-A2+/Brachyury+) and ASPC-l (HLA-A2'/Brachyury+) tumor cell targets,
at the indicated effector:target (ET) ratios (see Table 10). Results are expressed as the
percentage of specific lysis.
Table 10
ET ratio -231
azam 619m
nsum 02am
nsmm zsam
In another experiment, the ability of the TBR-A cell line to bind to
Brachyury-specific HLA-A2 tetramers was evaluated. Briefly, TBR-A cells (used at
IVS-4) were stained with a control tetramer or a tetramer specific for the Brachyury
t peptide. Figs. 7A and 7B show that 10.8% of CD8+ T cells in the TBR-A cell
line generated with GItreated DCs specifically bind to a Brachyury-HLA-A2
tetramer (Fig. 7B) and not to a control tetramer (Fig. 7A).
Perforin expression of the TBR-A T cell line was analyzed by flow
cytometry rin is a mediator of the cytolytic actiVity of cytotoxic T lymphocytes
(CTLs)). Briefly, T cells were tested on day 5 after restimulation with Brachyury agonist
peptide-pulsed autologous EBV transformed B cells. Fig. 8 shows the expression of
in in the -A cell line after stimulation with Brachyury agonist peptide-pulsed
autologous B cells, further trating the cytotoxic capability of this Brachyuryspecific
T cell line, which was generated using GItreated DCs.
Example 11
The following example describes a phase 1 clinical trial in ts with
Brachyury-positive cancer.
An open-label, tial dose-escalation, phase 1 clinical trial has been
initiated using the yeast-Brachyury immunotherapy composition known as GI-6301,
described in Examples 1, 2, and 4-9. Under this clinical trial protocol, 9-18 cancer
patients (3-6 patients per dose cohort) are administered the yeast-Brachyury
immunotherapy composition known as GI-6301 in a tial dose cohort escalation
protocol utilizing dose ranges of 4 Y.U. (l Y.U. x 4 sites, meaning that l Y.U. of GI-6301
is administered at 4 different sites on the body of the patient each Visit), 16 Y.U. (4 Y.U x
4 sites) and 40 Y.U. (lO Y.U. x 4 sites), administered subcutaneously. GI-630l is
administered at 2 week intervals for a total of 7 Visits (~3 months), and then monthly
thereafter until the patients meet off-study criteria. An expansion cohort of patients
(n=lO) at maximum tolerated dose (MTD) or the observed best dose are selected for
additional study. The results are monitoring safety and tolerability as a primary endpoint,
and in the ed cohort, whether a significant change in T cell precursors is detectable
as measured by an increase in Brachyury-specific T cells in t assay and
proliferation in response to ury protein (e.g., ury-specific CD8+ or CD4+ T
cells emerging or expanding on treatment). As ary endpoints, clinical benefit, such
as progression-free survival, clinical radiographic response, reduction in serum markers,
and/or reduction in circulating tumor cells is measured, as well as parameters of general
immune activation, ing frequency of immune cell subsets in peripheral blood (CD8+
memory/effector T cells, CD4+ memory/effector T cells, Tregs, NK cells, DCs) and
changes in serum levels of cytokines (e.g., IFN-y, IL-10, IL-12, IL-2, IL-4, TGF-B, etc.).
GI-630l is expected to be safe and well-tolerated with no significant toxicities.
In addition, l is expected to produce treatment-emergent Brachyury-specific T cell
responses or an ement in pre-existing Brachyury-specific baseline T cell responses
at least some or a ty of patients. Some patients are also expected to have stabilized
disease.
In an additional study or an expansion of this study, the yeast-Brachyury
immunotherapeutic composition known as GI-6305 (see Example 3) is administered to an
onal cohort of patients, utilizing the maximum tolerated dose or observed best dose
determined above, and the same primary and secondary endpoints are measured. 5
is also expected to be safe and well-tolerated with no cant toxicities, as well as
produce treatment-emergent ury-specific T cell responses or an improvement in
pre-existing Brachyury-specific baseline T cell responses at least some or a majority of
patients. Some patients are also expected to have stabilized disease.
Example 12
The following example describes a phase 2 clinical trial using yeast-Brachyury
immunotherapeutic compositions.
A randomized phase 2 clinical trial in patients with breast cancer is run using a
yeast-Brachyury therapeutic composition as described in Example 1 and 2 (e.g.,
GI-630l) or in Example 3 (GI-6305). At least 100 or more subjects with Stage I, II or III
Brachyury-positive breast cancer are enrolled. Subject ion criteria can include
subjects with Grade 1, 2 or 3 cancers. Subject including criteria can also e subjects
with “triple negative” breast cancer (cancers that are negative for each of estrogen receptor
(ER), progesterone receptor (PR) and HER2). Subject inclusion criteria can also include
ts with lymph node-negative cancer.
The trial is run as a double-blind or open-label, placebo-controlled, multi-
center trial. All patients e standard of care therapy with treatment arm patients
receiving several serial injections of yeast-Brachyury immunotherapeutic composition
during ent. The primary endpoint is recurrence free survival or overall survival.
Additional endpoints can e antigen-specific T cell responses (e. g., Brachyuryspecific
CD8+ T cells emerging or expanding on treatment), maintenance of lymph node
vity, ssion to metastases, and Brachyury expression in tumor cells.
The yeast-Brachyury immunotherapeutic composition is expected to be safe
and well-tolerated with no significant toxicities. In addition, the yeast-Brachyury
immunotherapeutic composition is expected to produce treatment-emergent Brachyuryspecific
T cell responses and/or an improvement in pre-existing Brachyury-specific
baseline T cell ses in at least some or a majority of patients. Some or a majority of
patients are also expected to have ized disease, maintain lymph node negativity,
and/or prevention, reduction or arrest in metastatic progression.
While various embodiments of the present invention have been described in
detail, it is apparent that modifications and adaptations of those embodiments will occur to
those skilled in the art. It is to be expressly understood, however, that such modifications
and tions are within the scope of the present invention, as set forth in the following
exemplary claims.
The