NZ724797A

NZ724797A - Yeast-brachyury immunotherapeutic compositions

Info

Publication number: NZ724797A
Application number: NZ724797A
Authority: NZ
Inventors: David Apelian; Zhimin Guo; Claudia Palena; Jeffrey Schlom
Original assignee: Globeimmune Inc; Us Health
Priority date: 2011-03-17
Filing date: 2012-03-19
Publication date: 2020-06-26

Abstract

Discloses the use of an immunotherapeutic composition comrpising a yeast vehicle, a Brachyury antigen and a further agent selected from the group consisting of an immunotherapeutic virus-based vaccine, a cytokine, a T-cell co-stimulator, a immunomodulator, CD40, anti-CTLA-4 antibody, anti-PD-1, anti-PD-L1, anti-PD-L2 in the manufacture of a medicament for treating cancer. Further discloses a recombinant nucleic acid molecule encoding a fusion protein comprising at least one Brachyury antigen.

Description

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 \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ 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

Claims

claims defining the invention

1. Use of an immunotherapeutic composition comprising: a) a yeast vehicle; b) a cancer antigen comprising a Brachyury antigen, wherein the ury antigen comprises SEQ ID NO:6, positions 2-435 of SEQ ID NO:6, SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, or an amino acid sequence that is at least 85% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine; and c) an agent selected from the group consisting of an immunotherapeutic virusbased vaccine, a cytokine, a T-cell mulator, a immunomodulator, CD40, anti-CTLA-4 antibody, anti-PD-1, D-L1, anti-PD-L2 and combinations thereof; in the manufacture of a medicament for treating cancer in an individual.

2. The use according to claim 1, wherein the Brachyury antigen comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine.

3. The use according to claim 1 or 2, wherein the Brachyury antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine.

4. The use according to any one of claims 1 to 3, wherein the individual has a stage I cancer, stage II cancer, stage III cancer, or stage IV cancer.

5. The use ing to any one of claims 1 to 4, n the cancer is ed from the group consisting of: breast cancer, small ine cancer, stomach cancer, pancreatic cancer, kidney cancer, r cancer, uterine cancer, ovarian cancer, testicular , lung cancer, colon cancer, prostate , chronic lymphocytic leukemia (CLL), Epstein-Barr virus transformed B cells, Burkitt’s lymphoma, Hodgkin’s lymphoma, and metastatic cancers thereof.

6. The use according to any one of claims 1 to 5, wherein the yeast vehicle is a whole yeast.

7. The use according to claim 6, wherein the whole yeast is killed.

8. The use according to claim 6 or 7, wherein the whole yeast is heat-inactivated.

9. The use according to any one of claims 1 to 8, wherein the yeast e expresses the antigen.

10. The use according to any one of claims 1 to 9, wherein the yeast is from a genus selected from the group consisting of: romyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia.

11. The use according to Claim 10, wherein the yeast is from Saccharomyces cerevisiae.

12. The use according to any one of claims 1 to 11, wherein the composition is formulated in a pharmaceutically acceptable excipient suitable for administration by injection to a t.

13. The use ing to claim 12, wherein the composition is formulated for stration in a dose from about 10 Y.U. to about 100 Y.U.

14. The use according to claim 12, wherein the composition is formulated for administration in a dose from about 10 Y.U. to about 40 Y.U.

15. The use according to claim 12, wherein the composition is formulated for administration weekly, every other week, and/or monthly.

16. The use according to claim 12, wherein the immunotherapeutic composition is formulated for administration weekly for 5 weeks followed by monthly.

17. The use according to claim 12, wherein the composition is ated for administration at two week intervals for seven rounds of treatment, followed by monthly.

18. The use according to claim 12, wherein the composition is formulated as a single dose.

19. The use according to claim 12, wherein the composition is formulated for administration at more than one site on the subject.

20. The use according to claim 12, wherein the immunotherapeutic composition is formulated for concurrent administration with another y for cancer.

21. A recombinant c acid molecule encoding a fusion protein comprising at least one Brachyury antigen, wherein the Brachyury n comprises SEQ ID NO:6, positions 2-435 of SEQ ID NO:6, SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, or an amino acid sequence that is at least 85% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine, and wherein the fusion protein was expressed by a yeast vehicle.

22. The recombinant nucleic acid molecule of claim 21, wherein the Brachyury n comprises SEQ ID NO:6.

23. The recombinant nucleic acid molecule of claim 21 or 22, n the Brachyury antigen comprises positions 2-435 of SEQ ID NO:6.

24. The recombinant nucleic acid molecule of claim 21, wherein the Brachyury n comprises SEQ ID NO:18.

25. The recombinant nucleic acid molecule of claim 21 or 22, wherein the Brachyury antigen ses positions 2-435 of SEQ ID NO:18.

26. The recombinant nucleic acid molecule of claim 21, wherein the Brachyury antigen comprises an amino acid sequence that is at least 85% cal to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine.

27. The recombinant nucleic acid molecule of any one of claims 21-26, wherein expression of the Brachyury fusion protein is under the control of a CUP1 promoter.

28. The recombinant nucleic acid le of claim 21, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO:8, or an amino acid sequence that is at least 85% identical to SEQ ID NO:8.

29. The recombinant nucleic acid molecule of claim 21, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO:20, or an amino acid sequence that is at least 85% identical to SEQ ID NO:20.

30. The recombinant nucleic acid le of claim 28, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO:8.

31. The recombinant nucleic acid le of claim 21 or 29, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO:20.

32. The inant nucleic acid molecule of claim 21, wherein the yeast vehicle is a whole yeast.

33. The recombinant nucleic acid le of claim 32, wherein the yeast is from a genus ed from the group consisting of: Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia.

34. The recombinant nucleic acid le of claim 33, wherein the yeast is from Saccharomyces cerevisiae.