NZ616696B2 - Yeast-brachyury immunotherapeutic compositions - Google Patents

Abstract

Disclosed are immunotherapeutic compositions comprising: a) a yeast vehicle; and b) a cancer antigen comprising at least one Brachyury antigen, wherein the Brachyury 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 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine, wherein the sequences are as defined in the complete specification. Also disclosed is use the composition in treating, delaying onset of, or preventing cancer. nce that is at least 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine, wherein the sequences are as defined in the complete specification. Also disclosed is use the composition in treating, delaying onset of, or preventing cancer.

Description

Yeast-Brachyury Immunotherapeutic Compositions REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §ll9(e) to US.

Provisional Application Serial No. 61/453,656, filed March 17, 2011. The entire disclosure of US. Provisional Application Serial No. 61/453,656, filed March 17, 2011 is incorporated herein by reference.

GOVERNMENT RIGHTS This invention was created in the performance of a Cooperative Research and Development Agreement with the National Institutes of , an Agency of the ment of Health and Human Services. The ment of the United States has certain rights in this invention.

STATEMENT REGARDING JOINT RESEARCH AGREEMENT This ion was made by or on behalf of s to a Cooperative Research and Development Agreement, executed May 8, 2008. The parties to the Cooperative Research and Development Agreement are: GlobeImmune, Inc. and the US. Department of Health and Human Services, as represented by National Cancer Institute, an Institute, Center or Division of the National utes of Health.

REFERENCE TO A SEQUENCE LISTING This application 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 ned in the text file is incorporated herein by reference in its ty pursuant to 37 CFR § l.52(e)(5).

FIELD OF THE INVENTION The present invention generally relates to yeast-based immunotherapeutic itions and methods for the prevention and/or treatment of cancers characterized by the expression 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 Brachyury gene in humans) was initially fied in 1927 by Nadine Dobrovolskaia-Zavadskai'a through a mutation 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 343:6l7-622) and in humans in 1996 by Edwards and colleagues (Edwards et al., 1996, Genome Res. 6:226-223), who also described the deduced amino acid sequence for human Brachyury.

As a member of the T-box family of transcription factors, Brachyury ns the highly conserved DNA-binding domain motif, called “T-box” or T-domain, which binds to a palindromic consensus sequence. Brachyury, 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 rates (see, e.g., Wilkinson et al., 1990, Nature 343(6259):657—659); Beddington 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 gues have trated 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. 13(8):2471-2478). Studies by do 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, ury is involved in metastatic progression of cancer.

Cancer is a leading cause of death worldwide, and the development of effective therapies for cancer continues to be one of the most active areas of research and clinical development. Although a variety of innovative approaches to treat and prevent cancers have been proposed, many cancers continue to have a high rate of mortality and may be difficult to treat or relatively unresponsive to conventional therapies. Cancers associated with Brachyury expression may be found in a variety of tissues, ing breast, small intestine, stomach, kidney, bladder, uterus, ovary, testes, lung, colon and prostate, and includes metastatic and late-stage cancers. In addition, Brachyury is sed in tumors of B cell origin, such as chronic lymphocytic leukemia (CLL), Epstein-Barr virus ormed B cells, Burkitt’s and Hodgkin’s mas. 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 , there s a need in the art for new immunotherapeutic products that effectively treat and/or t cancers associated with ury expression or overexpression.

SUMMARY OF THE INVENTION One ment of the invention s to a method to reduce, arrest, reverse, delay or prevent 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 undergoing metastatic progression, or is ted to begin undergoing metastatic progression, an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a cancer antigen sing 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, , reverse or prevent the metastatic progression of cancer in an individual who has .

In one aspect, of these embodiments of the invention, Brachyury is not ed 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 .

Another embodiment of the invention 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 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 prevent or delay the onset of a Brachyury-expressing .

In one aspect of these ments, cancer has not been detected in the individual. In one aspect, the individual is at high risk for developing cancer (e.g., via a genetic predisposition). In one aspect, the dual 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 , 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 ion-resistance of tumor cells in a patient with cancer.

The method includes the steps of administering to an individual who has cancer and is receiving chemotherapy and/or radiation therapy an immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a cancer antigen comprising at least one Brachyury antigen. Another embodiment of the ion 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 cancer. In one aspect of this embodiment of the ion, Brachyury is not detected in the dual’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.

Yet another embodiment of the invention s to a method to treat cancer.

The method includes the steps of: (a) administering 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, concurrently with, sequentially with, or subsequent to, administration of the first immunotherapeutic composition a second therapeutic composition comprising a yeast vehicle and a second cancer antigen comprising a Brachyury n. In one aspect, the method filrther comprises, in step (a), stering one or more additional immunotherapeutic compositions, wherein the each of the one or more onal immunotherapeutic compositions comprises an additional cancer n. In one aspect of either embodiment above, the cancer antigen is selected from: mutated Ras, carcinoembryonic n (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, an3-fl<hr, ews-fli-l, HERV-H, HERV-K, TWIST, elin, and NGEP. In one aspect, the cancer antigen is selected from the group consisting of: mutated Ras, carcinoembryonic antigen (CEA), and MUC-l. Another ment 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 first cancer antigen that does not comprise a Brachyury antigen; and (b) a second therapeutic composition comprising a yeast vehicle and a second cancer antigen comprising a Brachyury antigen.

Yet another embodiment of the invention relates 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 vehicle and a mutated Ras antigen; (b) stering to the individual of (a) a second immunotherapeutic composition comprising a yeast vehicle and an antigen selected from the group ting 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 administration 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 e and a mutated Ras antigen; (b) 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) a third immunotherapeutic composition sing a yeast vehicle and a Brachyury antigen.

In any of the ments or aspects of the invention described above or elsewhere herein, where the individual has cancer or a cerous lesion, in one aspect of the invention, the individual is being treated or has been treated with another therapy for cancer. For example, such a therapy can include, but is not d to, chemotherapy, targeted cancer therapy, ion therapy, adoptive T cell transfer, and/or administration of one or more additional immunotherapeutic compositions. In one aspect, an onal immunotherapeutic ition comprises a yeast vehicle and a second cancer antigen that does not include Brachyury antigen. The second cancer antigen can include, 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-Z, 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, an3-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 antigen (CEA), and MUC-l.

In one aspect of any of the embodiments or aspects of the ion bed above or ere herein, the method or use s 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 further 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 lial cell origin. In one , the cancer can include, but is not d to, breast cancer, small intestine cancer, stomach cancer, pancreatic cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, chronic lymphocytic leukemia (CLL), Epstein-Barr virus transformed B cells, Burkitt’s lymphoma, Hodgkin’s lymphoma, or metastatic cancers thereof.

In one aspect of any of the embodiments or s of the ion described above or elsewhere herein, the Brachyury antigen is full-length human Brachyury. In one , the Brachyury antigen is not fiJll-length Brachyury. In one aspect, the Brachyury antigen has an amino acid sequence represented by SEQ ID NO:6, SEQ ID NOilS, SEQ ID N02, or an amino acid sequence that is at least 95% cal to SEQ ID NO:6, SEQ ID NOilS, or SEQ ID NO:2. In one , the ury antigen comprises from at least on 1 or 2 to between position 255 and the C-terminus of SEQ ID NO:6, SEQ ID NOilS, 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 NOilS, or SEQ ID NO:2. In one aspect, the Brachyury antigen comprises positions 246 to 254 of SEQ ID NO:6, SEQ ID NOilS, or 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 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:18, or an amino acid sequence that is at least 95% 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 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:18, positions 2-435 of SEQ ID NO:18, or an amino acid sequence that is at least 99% identical to SEQ ID NO:18. In one aspect, the Brachyury n 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 at least 25 amino acids in . In one aspect, the Brachyury antigen is at least 25 amino acids in . In one aspect, the Brachyury antigen 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 ion described above or elsewhere herein, the cancer antigen is a fusion protein. In one aspect, the fusion protein 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 , the fusion protein has an amino acid sequence represented by SEQ ID NO:20, or an amino acid sequence that is at least 95% identical to SEQ ID NO:20. r embodiment of the ion relates to a Brachyury immunotherapeutic composition, wherein the immunotherapeutic composition comprises: (a) a yeast e; and (b) an antigen expressed by the yeast vehicle and comprising at least one Brachyury antigen, wherein the ury antigen ses 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 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 NO:l8 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 NO:l8 or SEQ ID NO:2. In one aspect, the Brachyury antigen ses positions 246 to 254 of SEQ ID NO:6, SEQ ID NO:18 or 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 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:18, or an amino acid sequence that is at least 95% identical to SEQ ID NO:18.

In one aspect, the Brachyury antigen ses 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% identical to SEQ ID NO:6. In one aspect, the ury antigen comprises SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, 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 , the fusion protein has an amino acid sequence of SEQ ID NO:20 or an amino acid sequence that is at least 95% cal 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 ion s to a yeast-Brachyury immunotherapeutic composition comprising: (a) a whole, inactivated yeast; and (b) a Brachyury fusion protein sing 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 composition elicits a Brachyury-speciflc 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 NO:18. 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 ury-speciflc T cell response. In one , the fusion protein comprises the amino acid sequence of SEQ ID NO:20.

In one aspect of any of the embodiments or aspects of the invention described above or ere herein, the yeast e 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 aspect, the yeast is from a genus selected from the group consisting of: Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces 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 . In one aspect, the disease is associated with an infectious agent. In one aspect, the disease is associated with a virus or viral infection. Such a virus can include, but is not limited to, Epstein Barr Virus (EBV).

Another embodiment of the ion relates to a method to treat or prevent a disease or ion 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 r embodiment of the invention relates to a method to produce a yeast-Brachyury therapeutic composition. The method includes the steps of: (a) culturing yeast that have been ormed with a recombinant nucleic acid le encoding a Brachyury antigen under the control of a CUP1 promoter in a suitable 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 between 6 and 8 hours; and (d) harvesting the yeast. In one aspect, the yeast in step (a) are cultured to a cell y of between 1.0 and 2.0 Y.U. per milliliter total culture volume. In one aspect, the yeast in step (a) are ed 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 maintained at pH 5.5 or higher. In one aspect, the method additionally includes a step of heatinactivating the yeast after step (d). For example, in one aspect, the yeast are heatinactivated at about 56°C for about 1 hour. In a further aspect of this embodiment, the yeast can be formulated for injection with a pharmaceutically acceptable excipient. In one , the yeast are from Saccharomyces . In one aspect, the yeast are from Saccharomyces cerevisiae. [0030A] Another embodiment of the invention relates to use of an immunotherapeutic composition comprising: a) a yeast vehicle; and b) a cancer n comprising at least one Brachyury antigen, wherein the Brachyury antigen comprises SEQ ID NO:6, ons 2-435 of SEQ ID NO:6, SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, or an amino acid ce that is at least 95% cal to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine; in the manufacture of a medicament for reducing, arresting, reversing, delaying or preventing the metastatic progression of cancer in an individual who has a cancer that is undergoing metastatic progression, is at risk of undergoing metastatic progression, or is predicted to begin undergoing metastatic progression. [0030B] Another embodiment of the invention s to use of an immunotherapeutic composition comprising: a) a yeast vehicle; and b) a cancer n comprising at least one Brachyury antigen, wherein the Brachyury 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 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at on 254 with a in the manufacture of a medicament for ting or delaying the onset of a Brachyury-expressing cancer. [0030C] Another embodiment of the invention s to use of an immunotherapeutic composition comprising: a) a yeast vehicle; and b) a cancer antigen comprising at least one Brachyury n, wherein the Brachyury 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 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine; in the manufacture of a medicament for reducing or preventing chemotherapy resistance or radiation resistance of tumor cells in a patient with cancer. [0030D] Another embodiment of the invention relates to a Brachyury immunotherapeutic composition, wherein the immunotherapeutic composition comprises: a) a yeast vehicle; b) an antigen expressed by the yeast vehicle and comprising at least one ury antigen, wherein the Brachyury antigen comprises an amino acid sequence represented by 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 95% identical to SEQ ID NO:18 and comprises a tution of a e at position 254 with a valine, or wherein the Brachyury antigen comprises from at least position 1 or 2 to between position 255 and the C-terminus of SEQ ID NO:6 or SEQ ID NO:18; and c) a pharmaceutically acceptable excipient suitable for administration to a human. [0030E] Another embodiment of the invention relates to a yeast-Brachyury immunotherapeutic composition comprising; a) a whole, inactivated yeast; and b) a ury fusion n comprising the amino acid sequence of positions 2-435 of SEQ ID NO:6, wherein expression of the Brachyury fusion protein is under the control of the promoter CUP1 ; wherein the ury fusion protein was expressed by the yeast; and wherein the composition elicits a ury-specific T cell response. [0030F] Another embodiment of the invention relates to a yeast-Brachyury immunotherapeutic composition comprising: a) a whole, inactivated yeast; and b) a Brachyury fusion protein sing the amino acid sequence of positions 2-435 of SEQ ID NO:18, wherein expression of the Brachyury fusion protein is under the control of the promoter CUP1 ; wherein the Brachyury fusion protein was expressed by the yeast; and wherein the composition elicits a Brachyury-specific T cell response. [0030G] Another embodiment of the invention s to use of a yeast-Brachyury immunotherapeutic composition according to the present invention in the manufacture of a medicament for treating or preventing a disease or condition associated with Epstein Barr Virus (EBV) infection. [0030H] Another embodiment of the ion relates to a method to produce a yeast- Brachyury immunotherapeutic composition, comprising: a) culturing yeast that have been transformed with a recombinant c acid molecule encoding a Brachyury antigen under the control of a CUP1 promoter in a suitable medium in the absence of CuSO4 until the yeast reach mid-log growth phase, wherein the Brachyury 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 95% cal to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine; b) inducing expression of the Brachyury antigen in the yeast by adding CuSO 4 to the ; c) culturing the yeast after step (b) for up to between 6 and 8 hours; and d) harvesting the yeast. [0030I] Another embodiment of the invention relates to a use of a combination of immunotherapeutic compositions in the manufacture of a medicament to treat cancer, the therapeutic compositions comprising: a) a first immunotherapeutic composition comprising a yeast vehicle and a first cancer antigen that does not comprise a Brachyury n; and b) a second immunotherapeutic composition comprising a yeast vehicle and a second cancer antigen sing a Brachyury antigen, wherein the Brachyury 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 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine. [0030J] Another embodiment of the invention relates to a use of a combination of immunotherapeutic compositions in the cture of a medicament 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 ition comprising a yeast vehicle and an antigen selected from the group consisting of carcinoembryonic n (CEA) and mucin-1 (MUC-1); and c) a third immunotherapeutic composition comprising a yeast vehicle and a ury antigen, wherein the Brachyury antigen comprises SEQ ID NO:6, ons 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 95% identical to SEQ ID NO:18 and ses a substitution of a leucine at position 254 with a valine.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a digitized image of a Western blot showing detection by anti- Brachyury of sion of Brachyury in a Brachyury immunotherapeutic composition, with both U2 and UL2 media.

Fig. 1B is a digitized image of a Western blot showing detection 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 composition where the cell density at antigen induction and the time to harvest after antigen induction were .

Figs. 3A-3C are graphs g that peripheral blood mononuclear cells (PBMCs) from two out of three healthy donors pulsed with yeast-Brachyury for two cycles of stimulation, followed by pulsing with Brachyury CTL peptide, were capable of generating CD8+ CTLs that could kill SW480 oma cells (HLA-A2 positive/Brachyury high), with l lysis of MCF7 carcinoma (HLA-A2 positive/Brachyury low); (Fig. 3A, donor 07706; Fig. 3B, donor 17663; Fig. 3C, donor 26532).

Fig. 4A is a graph g that Brachyury-specific T cells from healthy donor PBMCs stimulated 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 (GAPDH) 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, les), in response to indicated doses of purified Brachyury protein or l β-gal protein.

Fig. 6 graph showing that administration of a yeast-Brachyury immunotherapeutic composition (GI-6301, circles) of the ion shows a trend towards reducing Brachyury-expressing tumors in mice compared to mice receiving yeast alone (no Brachyury antigen).

Figs. 7A and 7B are flow cytometry analyses showing that the Brachyuryspecific T cell line, TBR-A, binds to a Brachyury-specific HLA-A2 er (Fig. 7B) and not to a l tetramer (Fig. 7A).

Fig. 8 is a flow cytometry analysis showing the expression of perforin in the Brachyury-specific T cell line, TBR-A, after stimulation with ury t peptide-pulsed autologous B cells.

DETAILED DESCRIPTION OF THE INVENTION ] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an ion that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application. [0040B] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

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 ion includes 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 thereof (also referred to herein as “yeast-Brachyury immunotherapy” or “yeast-Brachyury immunotherapeutic compositions”). The inventors describe herein the uction and production of novel yeast-Brachyury therapy products, and demonstrate that yeast-Brachyury immunotherapy expands Brachyury-specific T cells, including CD8+ CTLs, from normal duals and from cancer patients. In addition, mice immunized with yeast-Brachyury immunotherapeutic compositions generated Brachyury-specific T cell responses in vivo, and Brachyury-expressing tumor growth was inhibited 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, ng novel therapy for the prevention and/or ent of metastatic cancers and associated conditions.

Yeast-Brachyury immunotherapeutic compositions useful in the present invention are uniquely adapted to effectively target Brachyury-expressing s for several reasons. First, Brachyury is involved in EMT processes, and therefore, without being bound by theory, the inventors believe that it plays a role in late-stage tumors and metastatic processes. ingly, in one aspect of the invention, yeast-Brachyury immunotherapy is effective at targeting tumor cells before or at the time during which they begin to acquire ty and invade other tissues, thereby preventing, inhibiting, arresting, reversing or delaying the onset of metastatic cancer and/or the ssion of cancer, and especially metastatic cancer. There is a great need for effective therapies for late stage s, especially metastatic cancers, which may have few options for treatment once conventional cancer therapy has failed. Yeast-Brachyury presents a novel approach to treat such cancers, or to delay, inhibit, reverse, or prevent 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 ment, in cancers that have a high rate of metastatic progression, and may be useful to arrest ssion of the cancer. Furthermore, yeast-Brachyury immunotherapy is useful in duals who have a cerous (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 duals as a prophylactic agent for the prevention of , 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 chemotherapy and radiation therapy.

More particularly, metastatic cancers are known in some cases to be more resistant to chemotherapy and/or radiation therapy than the primary cancers. Therefore, the yeast- Brachyury immunotherapy compositions of the invention can be used to inhibit or reduce or eliminate chemotherapy resistance or radiation resistance that may occur in metastatic cancer by inhibiting Brachyury expression in the cancer (and thereby inhibiting anti- proliferative influences), and compositions of the invention may e the performance of chemotherapy or radiation y in an individual.

Yeast-Brachyury therapy 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 disease or condition ated with Brachyury expression, ing, 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 y adaptable 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 Brachyury therapy can be adapted to the cancer type, the cancer stage, the cancer grade, the antigens expressed by the tumor, and the overall medical 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 e maximum efficacy at a variety of tumor stages. Yeast- Brachyury immunotherapy offers the opportunity to design sophisticated and effective, individualized approaches for the 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 ses against the target antigen (Brachyury), ing 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 fianctionality, thereby enhancing or T cell responses that might normally be suppressed by the presence of the tumor, for example. Moreover, as compared to immunotherapeutic itions that immunize by ting antibody responses, the n-specific, broad-based, and potent cellular immune responses elicited by yeast-Brachyury immunotherapy are ed to be particularly effective in targeting tumor cells. Indeed, numerous studies have shown that immunotherapeutic approaches 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 cross-prime the immune response, generating CD8+ CTL responses that are typically ive against tumors, even in the face of what may ise be a suppressive environment. Since this type of immunotherapy utilizes the natural ability of the n presenting cell to present relevant immunogens, it is not necessary to know the precise identity of CTL epitopes or MHC Class II epitopes of Brachyury to produce an ive immunotherapeutic according to the present invention.

In fact, multiple CD4+ and CD8+ T cell epitopes can be targeted in a single yeast- Brachyury immunotherapeutic ition, and so the yeast-Brachyury immunotherapeutics of the invention are not limited to the use of short es 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 as to identify 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 target” effects related to normal tissues are not of concern. As mentioned above, yeast-Brachyury can be effectively utilized in an immunization protocol (prophylactic or therapeutic) t the use of exogenous adjuvants, immunostimulatory agents or molecules, costimulatory molecules, or cytokines, although such agents may be included, if desired. Moreover, yeast-Brachyury immunotherapy can be administered repeatedly without losing efficacy, as may be problematic with other types of immunotherapy.

Comgosz'tz'ons at the Invention One embodiment of the t invention s 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 Brachyury at later stages of the development of the cancer). The composition is a yeast-Brachyury immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a cancer antigen sing one or more Brachyury antigen(s) and/or immunogenic domain(s) thereof. The Brachyury antigen or immunogenic domain thereof is most typically sed as a recombinant protein by the yeast vehicle (e.g., by an intact yeast or yeast spheroplast, which can optionally be further sed to a yeast cytoplast, yeast ghost, or yeast membrane extract or fraction thereof), although it is an embodiment of the invention that one or more Brachyury antigens are loaded into a yeast vehicle or otherwise complexed with, attached to, mixed with or administered with a yeast vehicle as described herein to form a composition of the present ion.

A “yeast-Brachyury immunotherapeutic composition” is a specific type of “yeast-based immunotherapeutic composition3, that contains at least one Brachyury antigen or immunogenic domain thereof. The phrase, -based immunotherapeutic composition” may be used interchangeably with “yeast-based immunotherapy product”, “yeast-based immunotherapy composition”, “yeast-based ition”, “yeast-based immunotherapeutic”, “yeast-based vaccine”, or tives of these phrases. An “immunotherapeutic composition” is a composition that elicits an immune response sufficient to achieve at least one therapeutic benefit in a subject. As used herein, yeast- based immunotherapeutic composition refers to a composition that includes a yeast vehicle component and that s an immune response sufficient to achieve at least one therapeutic benefit in a subject. More particularly, a yeast-based immunotherapeutic composition is a composition that includes a yeast vehicle component and typically, an n component, and can elicit or induce an immune response, such as a cellular immune response, 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 , a CD8+ and a CD4+ T cell-mediated immune se, 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 ation of the above. Yeast-based immunotherapeutics are ularly capable of generating THl and THl7 responses. A CD8+ immune response can e a xic 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 fianctionality of regulatory T cells (Tregs) in a subject.

Yeast-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 immunotherapeutic composition is e of eliciting a humoral immune response.

Yeast-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 ion 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 (metastases) 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 ed in the EMT process, which is a process associated with invasiveness and migration of , such as in metastatic cancer. Therefore, lactic compositions can be administered to individuals that appear to be cancer-free (healthy, or , individuals), to individuals with pre-cancerous (pre-malignant lesions), and also to individuals who have cancer, but in which Brachyury has not yet been detected (tie. 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 ases 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 ameliorating the cancer, such as by reducing tumor burden in the individual; inhibiting tumor growth in the individual; increasing survival of the dual; preventing, inhibiting, ing or delaying development of tumor migration and/or tumor invasion of other tissues (metastatic cancer) and/or preventing, inhibiting, ing or delaying progression of the cancer in the dual. In one aspect, yeast-Brachyury immunotherapy is used therapeutically to inhibit, reduce or eliminate 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 ion therapy in an individual.

Typically, a yeast-Brachyury immunotherapy composition includes a yeast vehicle and at least one cancer n comprising a ury 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 antigen, or immunogenic domain thereof is provided as a fusion protein. Several Brachyury proteins and fusion proteins suitable for use in the compositions and methods of the invention are described below. In some embodiments, 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 antigen. 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 r cancer n that is not a Brachyury antigen, or two different ury antigens. In one aspect, the fusion n can include two or more genic s of one or more antigens, such as two or more genic 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.

According to the present invention, a yeast vehicle 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 epitopes thereof in a composition of the invention (e.g., a therapeutic or prophylactic composition). The yeast vehicle can therefore include, but is not limited to, a live intact (whole) yeast microorganism , 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 (z'.e., a yeast cell lacking a cell wall and nucleus), a yeast ghost (i.e., a yeast cell g a cell wall, s 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 tic digestion of the yeast cell wall. Such a method is described, for e, 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 entirety.

Yeast ghosts are typically produced by resealing a permeabilized or lysed cell and can, but need not, contain at least some of the organelles of that cell. Such a method is described, for e, in Franzusoff et al., 1983, J. Biol. Chem. 258, 3608-3614 and Bussey et al., 1979, m. Biophys. Acta 553, 185-196, each of which is incorporated herein by reference in its entirety.

A yeast membrane particle (subcellular yeast membrane extract or fraction thereof) refers to a yeast ne that lacks a natural nucleus or cytoplasm. The particle can be of any size, including sizes g from the size of a natural yeast membrane to articles produced by sonication or other membrane tion methods known to those skilled in the art, followed by resealing. A method for producing subcellular yeast membrane extracts is bed, for example, in Franzusoff et al., 1991, Math. 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 preparation 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 membrane, or combinations thereof (i.e., the protein can be both inside and e the membrane and/or spanning the membrane of the yeast membrane particle). In one embodiment, a yeast membrane particle is a recombinant yeast membrane particle that can be an intact, disrupted, or disrupted and resealed yeast membrane that includes at least one desired antigen or other protein of st on the surface of the membrane or at least partially embedded within the membrane.

An example of a yeast cell wall preparation is a ation of isolated yeast cell walls carrying an antigen on its surface or at least partially embedded within the cell wall such that the yeast cell wall preparation, when administered to an animal, stimulates a d immune response against a disease .

Any yeast strain can be used to e 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 cerevisiae. The ion of a non-pathogenic yeast strain minimizes any adverse effects to the individual to whom the yeast vehicle is administered. However, enic 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 present invention, non- enic 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), Cryptococcas, Hansenala, Kluyveromyces, Pichia, orula, Schizosaccharomyces and Yarrowz'a. In one aspect, yeast genera are selected from romyces, Candida, Hansenala, Pichia or saccharomyces, and in one , Saccharomyces is used. Species 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, Cryptococcas laurentii, Cryptococcas neoformans, Hansenala a, Hansenala polymorpha, Klayveromycesfragilis, Klayveromyces lactis, Klayveromyces marxianas var. lactis, Pichia pastoris, Rhodotorala rabra, Schizosaccharomyces pombe, and Yarrowia lipolytica. It is to be iated that a number of these species include a y of subspecies, types, subtypes, 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. is and S. pombe. S. cerevisiae is useful as it is relatively easy to manipulate and being "Generally Recognized As Safe" or "GRAS" for use as food additives (GRAS, FDA proposed Rule 62FR18938, April 17, 1997). One embodiment 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 e of supporting expression s that allow one or more target antigen(s) and/or n 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 0t. In on, any mutant yeast strains can be used in the t invention, including those that exhibit reduced post-translational modifications of expressed target antigens or other ns, such as mutations in the enzymes that extend N—linked glycosylation.

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 n of a protein (e.g., peptide, partial protein, fiJll-length protein), wherein the protein is naturally occurring or synthetically d 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 portion 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 larger, and can include multiple epitopes or immunogenic 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 filll length protein, a multimer, a fiasion protein, a chimeric protein, a whole cell, a whole microorganism, or any portions f (e.g., protein fragments (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 chimeric proteins. In addition, antigens can include carbohydrates, which can be loaded into a yeast vehicle or into a composition of the ion. It will be iated 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 thereof, rather than an entire cell or microorganism. For sion in yeast, an antigen is of a minimum size capable of being expressed recombinantly in yeast if the n is the entire protein to be expressed by the yeast, and is typically at least or greater than 25 amino acids in length, 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 greater 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 greater than 42, at least or greater than 43, at least or greater than 44, at least or r 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 greater than 25-50 amino acids in length, or at least or r than 30-50 amino acids in length, or at least or greater 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 expressed, and considerably larger ns (e.g., hundreds of amino acids in length or even a few thousand amino acids in ) may be expressed. In one aspect, a ength 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 ns that may be expressed in the invention. A “target antigen” is an antigen that is specifically targeted by an immunotherapeutic ition 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 n that is associated with a cancer such as an antigen expressed by a tumor cell, such that targeting the antigen also targets the . A cancer antigen can include one or more antigens 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 en". An immunogen, as used herein, bes an antigen which elicits a l and/or cell-mediated immune response (i.e., is immunogenic), such that administration of the immunogen to an individual mounts an antigen-specific immune response against the same or similar antigens that are encountered by the immune system of the individual. In one embodiment, the immunogen 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, nt or epitope of an antigen (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 epitope that can act as an gen. For example, a single protein can n le ent immunogenic domains. Immunogenic domains need not be linear ces within a protein, such as in the case of a humoral immune response, where conformational domains are contemplated.

An epitope is defined herein as a single immunogenic site within a given antigen that is sufficient to elicit an immune response when ed to the immune system in the context of appropriate costimulatory signals and/or activated cells of the immune system. 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 ize that T cell epitopes are different in size and composition from B cell or antibody epitopes, and that es 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 molecules are less restricted in length and may be up to 25 amino acids or longer. In addition, T cell epitopes have predicted structural characteristics depending on the specific MHC molecules bound by the epitope. Epitopes can be linear sequence es or mational epitopes rved g regions). Most antibodies recognize conformational epitopes.

Brachyury (which may also be referred to as “T”) is a highly conserved n among multiple different animal species and is a transcription factor that contains a “T-box” domain or ain”, a DNA-binding domain motif shared among several different proteins, collectively called the T-box family of proteins. Human ury 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:l9743811). SEQ ID NO:l encodes a 435 amino acid human Brachyury protein, the amino acid ce of which is represented here as SEQ ID NO:2 (also found in GENBANK® Accession No. NP_003 172; GI:4507339).

Another human Brachyury protein disclosed herein is a variant of the human Brachyury protein ented 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 d 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 full-length of the protein. SEQ ID NO:6 differs 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 disclosed herein is an agonist of the human Brachyury protein represented 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, peptides, antibodies, nucleic acid g agents, etc., that binds to a receptor or ligand and produces or triggers a response, which may include 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” antigen or protein refers to an antigen or protein that ses at least one T cell t 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 fianction) of the natural 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 n is represented here by SEQ ID NO:l8.

SEQ ID NO:l8 is a 435 amino acid protein is encoded by a nucleotide sequence ented herein by SEQ ID NO:l7. SEQ ID NO:l8 is identical to SEQ ID NO:6, except for a substitution of a leucine at position 254 with t to SEQ ID NO:6 with a valine in SEQ ID NO:l8. This substitution creates a T cell t epitope in SEQ ID NO:l8 at positions 246 to 254 that, without being bound by theory, is believed to induce enhanced T cell responses against Brachyury as compared to the wild-type epitope ions 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 ces from other species, can be readily fied by comparison to these ces. As used herein, reference to a T-box domain of any Brachyury protein described herein or known in the art and utilized in the invention may include an onal 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 ury, including the two human Brachyury 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 discussed above, SEQ ID NO:l8 comprises an t epitope of SEQ ID NO: 12, represented 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 ces of Brachyury from other organisms in the preparation of a yeast-Brachyury immunotherapeutic composition of the invention, particularly where these sequences are identical, substantially homologous, and elicit an effective immune response against the target antigen (e.g., native Brachyury expressed by a tumor cell). For example, murine Brachyury, which was 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 respect to Brachyury from other animals, at the amino acid level, human Brachyury is 99.5% identical to Brachyury from Pan dytes, 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 , mouse and human Brachyury 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 ury protein, the amino acid sequence of which is represented here as SEQ ID NO:4. Positions 41 to 223 of SEQ ID NO:4 ent the T-box DNA binding domain of murine Brachyury.

In one embodiment of the invention, a Brachyury antigen ses or consists of the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:l8, or at least one immunogenic domain f. In one embodiment, a Brachyury antigen comprises or consists of two, three, four, five, or more immunogenic s of Brachyury. In one ment of the invention, a Brachyury antigen comprises or consists of the amino acid ce ented by amino acid positions 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 (126., 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 antigen 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 antigen useful in the invention includes from at least amino acid positions 1 to 85 to between on 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 on 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 fiJll-length fianctional domain or fiJll-length immunological domain) includes the full-length amino acid sequence of the protein or fianctional domain or immunological domain, as described herein or as ise known or described in a publicly available sequence. A protein or domain that is “near filll-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 ength protein or full-length domain. By way of example, l of the fusion proteins described herein comprise a “near full-length” Brachyury antigen since the antigen omits the methionine at position 1 and substitutes an inal peptide. General nce to a protein or domain or antigen can include both fiJll-length and near full-length ns, as well as other homologues thereof.

In one aspect of any embodiments related to a Brachyury n, 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 sed, and considerably larger proteins may be expressed by yeast. For example, a Brachyury antigen useful in the invention is a fragment of a Brachyury protein that can be expressed recombinantly by yeast and that contains at least one genic 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 , and contains at least one immunogenic domain of Brachyury. In one aspect, such an antigen is greater than 30 amino acids in length, and contains at least one genic domain of Brachyury. In one aspect, such an antigen is at least 25-50 amino acids in length, and contains at least one immunogenic domain of Brachyury. 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 antigen is at least 35-50 amino acids in length, and contains at least one immunogenic domain of ury. 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: 18.

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 ses an amino acid sequence of STL (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 . 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 ces) is substituted with a serine (S), a threonine (T), an cine (I), or a valine (V).

In one aspect, the Brachyury antigen comprises an amino acid sequence of SQYPSLWSV (SEQ ID NO:l4). In one aspect, the amino acid at position 2 of SEQ ID NO:14 (a glutamine or Q in this sequence) is substituted with a e (L). In one aspect, 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 aspect, 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 , the amino acid at on 9 of SEQ ID NO: 14 (a valine or V in this sequence) is tuted with a leucine (L). An antigen comprising a sequence haVing any combination of one or more of these substitutions 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 sequence 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 methionine or M in this sequence) is substituted with a e (L).

In one 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:8. The fusion protein of SEQ ID NO:8 is a single polypeptide with the ing sequence elements fused in frame from N— to C-terminus: (1) an inal peptide to impart resistance to proteasomal degradation and stabilize expression in yeast (positions 1- 6 of SEQ ID NOS); (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 stidine tag (positions 441- 446 of SEQ ID NO:8). The amino acid ce of SEQ ID NO:8 is d by the polynucleotide sequence of SEQ ID NO:7.

In another embodiment of the ion, a Brachyury antigen comprises, consists essentially of, or consists of a fiasion protein having the amino acid sequence of SEQ ID NOilO. The filsion protein of SEQ ID NO:lO 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 ation and ize expression in yeast (positions 1-6 of SEQ ID NO:lO); (2) a murine Brachyury antigen 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 ury antigen ses, consists essentially of, or consists of a fiasion protein having the amino acid sequence of SEQ ID NO:20. The filsion protein of SEQ ID NO:20 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 somal 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:18 (positions 7-440 of SEQ ID NO:20), SEQ ID NO:18 representing a full-length human Brachyury agonist protein; and (3) a hexahistidine tag (positions 6 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 sequence of SEQ ID NO:20 is encoded by the polynucleotide sequence of SEQ ID NO: 19.

A Brachyury antigen useful in the present invention 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 bed herein over the full length of the protein, or with respect to a defined fragment or domain thereof (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 ion, 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 n, when used as an antigen in a yeast-Brachyury immunotherapeutic ition of the invention, elicits an immune response against a native Brachyury protein as the wild-type or reference Brachyury protein, which may include an enhanced immune response, a diminished immune response, or a ntially similar immune response. For example, the invention includes the use of ury agonist antigens, which may include one or more T cell epitopes that have been mutated to enhance the T cell response against the ury agonist, such as by improving the avidity or ty of the e for an MHC molecule or for the T cell receptor that recognizes the epitope in the context of MHC presentation. Brachyury agonists may ore improve the potency or efficiency of a T cell se against native Brachyury expressed by a tumor cell. The Brachyury antigen having the amino acid sequence of SEQ ID NO:l8 is a non-limiting example of a Brachyury agonist (or a Brachyury antigen comprising an agonist epitope).

In addition, N—terminal expression sequences and the C-terminal tags, such as those described above with respect to the fusion proteins of SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO:20 are optional, 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 le 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 cleavage sites for host phagosomal ses, to accelerate protein or antigen processing, and for future manipulation of the constructs.

Optionally, proteins, including fusion proteins, which are used as a component of the yeast-Brachyury immunotherapeutic composition of the invention are produced using antigen constructs that are particularly useful for ing or stabilizing the expression of heterologous antigens in yeast. In one embodiment, the desired antigenic n(s) or peptide(s) are fused at their amino-terminal end to: (a) a specific synthetic peptide that stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein (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 entirety); (b) at least a portion of an endogenous yeast protein, including but not limited to yeast alpha factor leader ce, n 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 detail, 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 sed on the surface of the yeast (e.g., an Aga protein, described in more detail herein). In addition, 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 tic 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 invention can be used with or t 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 expressed in a yeast is linked to the N—terminus of the antigen, the peptide consisting of at least two amino acid ons 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 expressed fusion protein. The synthetic peptide and N—terminal portion of the antigen together form a fusion protein that has the ing requirements: (1) the amino acid residue at position one of the qulOI‘l protein is a methionine (z'.e., the first amino acid in the synthetic e is a methionine); (2) the amino acid residue at position two of the fusion protein is not a glycine or a e (z'.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 (z'.e., the amino acids at positions 2-6, whether part of the tic 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 (z'.e., the amino acids at positions 2-6, whether part of the synthetic peptide or the protein, if the tic peptide is shorter than 5 amino acids, do not include a lysine or an arginine).

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 arginine; wherein X4 is any amino acid except methionine, lysine or arginine; wherein X5 is any amino acid except methionine, lysine or arginine; and wherein X6 is any amino acid except nine, lysine or arginine. In one embodiment, the X6 residue is a proline. An exemplary synthetic sequence that enhances the stability of sion 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 (represented herein by SEQ ID NO:11). In addition to the enhanced stability of the sion product, this fusion partner does not appear to negatively impact the immune response against the immunizing antigen in the construct. In addition, the synthetic fusion es can be designed to provide an epitope that can be recognized by a selection agent, such as an antibody.

In one aspect of the invention, the yeast vehicle is manipulated such that the antigen is sed or provided by delivery or ocation of an expressed protein product, partially 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 e, one can use a spacer arm to create a filSlOI‘l n of the antigen(s) or other protein of st 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 surface of the yeast. Proteins other than yeast proteins may be used for the spacer arm; however, for any spacer arm protein, it is most desirable to have the immunogenic response be directed against the target antigen 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 se to the spacer arm protein itself such that the immune se 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 ve 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 antigen to be y removed or processed away from the yeast, if desired. Any known method of determining the magnitude of immune responses can be used (e.g., antibody production, lytic assays, etc.) and are y known to one of skill in the art.

Another method for positioning the target n(s) or other proteins to be exposed on the yeast surface is to use signal sequences such as ylphosphatidyl 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 asmic 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 n. 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 e 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 trimer, or even more units connected together. This use of monomers, dimers, trimers, etc. allows for appropriate spacing or folding of the antigen 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 anslational 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, ary yeast proteins to use e, 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 l phase of daughter cell formation; Flolp; Pir2p; and Pir4p.

Other ces 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 endoplasmic reticulum or the nucleus. Examples of le 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. s 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 yeast-based immunotherapy compositions are contemplated by the invention.

According to the present invention, the term "yeast vehicle-antigen complex" or "yeast-antigen complex" is used generically to describe any association of a yeast vehicle with an antigen, and can be used interchangeably with “yeast-based immunotherapy composition” when such composition is used to elicit an immune response as described above. Such association includes expression of the antigen by the yeast (a recombinant yeast), introduction of an antigen into a yeast, physical attachment of the antigen to the yeast, and mixing of the yeast and n together, such as in a buffer or other solution or ation. These types of complexes are described in detail below.

In one embodiment, a yeast cell used to prepare the yeast vehicle is transfected with a heterologous nucleic acid molecule encoding a protein (e.g., the antigen) such that the n is expressed by the yeast cell. Such a yeast is also referred to herein as a recombinant yeast or a recombinant yeast vehicle. The yeast cell can then be formulated with a pharmaceutically acceptable excipient and stered directly to a patient, stored for later administration, or loaded into a dendritic cell as an intact cell. The yeast cell can also be killed, or it can be derivatized such as by formation of yeast spheroplasts, asts, ghosts, or subcellular particles, any of which may be followed by storing, stering, or loading of the derivative into the dendritic cell. Yeast spheroplasts can also be directly transfected with a recombinant nucleic acid molecule (e.g., the plast is produced from a whole yeast, and then transfected) in order to produce a inant spheroplast that expresses the antigen. Yeast cells or yeast spheroplasts that recombinantly express the antigen(s) may be used to produce a yeast vehicle sing a yeast cytoplast, a yeast ghost, or a yeast membrane particle or yeast cell wall le, or on thereof.

In general, the yeast vehicle and antigen(s) and/or other agents can be ated by any technique described herein. In one aspect, the yeast vehicle was loaded intracellularly with the antigen(s) and/or agent(s). In another aspect, the antigen(s) and/or agent(s) was covalently or valently attached to the yeast vehicle. In yet another aspect, the yeast vehicle and the antigen(s) and/or agent(s) were associated by mixing. In another aspect, and in one ment, 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 proteins to be produced by a yeast vehicle of the present invention is any number of ns 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 proteins.

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. , a nucleic acid molecule encoding 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 sequence in order to be capable of effecting either constitutive or regulated expression of the nucleic acid molecule when transformed into a host yeast cell.

Nucleic acid molecules encoding one or more antigens and/or other ns can be on one or more expression s operatively linked to one or more expression control sequences.

Particularly important expression l sequences are those which control transcription initiation, such as promoter and upstream activation sequences. Any suitable yeast promoter can be used in the present invention and a variety of such promoters are known to those skilled in the art. Promoters for expression in Saccharomyces cerevisiae include, but are not limited to, ers of genes encoding the following yeast proteins: alcohol dehydrogenase I (ADHl) or II (ADH2), CUPl, phosphoglycerate kinase (PGK), triose phosphate ase (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 ), 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 ces (UASs), also referred to as enhancers, are known.

Upstream activation sequences for expression in Saccharomyces siae e, 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 t, 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 operatively 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 control sequences to express genes in methyltrophic yeast include the transcription l regions of the genes encoding alcohol oxidase and formate dehydrogenase.

Transfection of a nucleic acid le into a yeast cell according to the present invention can be accomplished 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, ction, adsorption, and last fusion. Transfected c acid molecules can be integrated into a yeast chromosome or maintained 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 sed above, yeast cytoplast, yeast ghost, and yeast ne particles or cell wall ations can also be produced recombinantly by transfecting intact yeast microorganisms or yeast spheroplasts with desired nucleic acid molecules, producing the antigen therein, and then fiarther manipulating the microorganisms or spheroplasts using techniques known to those skilled in the art to produce cytoplast, ghost or subcellular yeast membrane extract or fractions thereof containing desired ns or other proteins.

Effective conditions for the production of recombinant yeast vehicles and expression of the antigen and/or other protein by the yeast vehicle include an effective medium in which a yeast strain can be cultured. An effective medium is typically an s medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, ls, metals and other nutrients, such as ns and growth factors. The medium may comprise complex nutrients or may be a defined minimal medium. Yeast s of the present invention can be ed 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 temperature, 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 logy, vol. 194, Academic Press, San Diego). For example, under one protocol, liquid cultures containing a suitable medium can be inoculated using cultures obtained from starter plates and/or starter cultures of yeast-Brachyury therapy compositions, and are grown for approximately 20h at 30°C, with ion at 250 rpm. Primary es can then be expanded into larger cultures as desired. Protein expression from vectors with which the yeast were transformed (e.g., Brachyury expression) may be constitutive if the promoter utilized is a constitutive promoter, 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 e 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 ition of the invention is U2 medium. U2 medium comprises the following ents: 20g/L of glucose, 6.7 g/L of Yeast nitrogen base containing ammonium sulfate, and 0.04 mg/mL each of histidine, leucine, phan, and adenine. Another non-limiting example of a medium suitable for the culture of yeast- Brachyury immunotherapy ition of the invention is UL2 medium. UL2 medium comprises the following ents: 20g/L of glucose, 6.7 g/L of Yeast nitrogen base containing ammonium sulfate, and 0.04 mg/mL each of histidine, tryptophan, and adenine.

In one embodiment of the invention, when an inducible promoter is used (e.g. the CUPI promoter) to express a Brachyury protein in a yeast vehicle according to the invention, induction of n expression is initiated at a higher cell density 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 n expression driven by the CUP] promoter 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 Y.U./ml, 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 ng expression of the Brachyury antigen in the yeast. The present inventors have discovered that subsequent to induction of ury 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 ies higher than about 2.0 Y.U./ml, or for longer than about 6-8 hours, results in decreased ity of the cultures, while not substantially improving antigen accumulation in the yeast. Therefore, in one ment of the invention, a yeast-Brachyury immunotherapy composition having antigen sion 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 n 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 ion 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 n about pH 5.5 and about pH 8, and in one aspect, between about pH 6 and about 8.

One of skill the art will appreciate that minor tions (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 l pH for the majority of the time that they are in culture. 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 biological effects that are desirable characteristics for using the yeast as vehicles for immunomodulation. For example,, 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 t 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 ion of nes by antigen presenting cells that have phagocytosed the yeast (e.g., THl-type cytokines including, but not limited to, IFN-y, interleukin-12 (IL- 12), and IL-2, as well as proinflammatory nes such as IL-6). In addition, greater accessibility to the antigens located in the cell wall is afforded by such culture methods. In another aspect, the use of neutral pH for some antigens 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 sion of antigens by the yeast, particularly on the e. The amount of yeast glycosylation can affect the immunogenicity and nicity of the n, ularly one sed on the surface, since sugar moieties tend to be bulky. As such, the existence of sugar moieties on the surface of yeast and its impact on the three- dimensional space around the target antigen(s) should be considered in the modulation of yeast according 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 invention, as an alternative to expression of an antigen or other protein inantly in the yeast vehicle, a yeast vehicle is loaded intracellularly with the protein or peptide, or with carbohydrates or other molecules that serve as an n and/or are useful as immunomodulatory agents or biological response 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 vehicles of the present ion by techniques known to those skilled in the art, such as by diffusion, active transport, liposome fusion, electroporation, phagocytosis, freeze-thaw cycles and bath sonication. Yeast vehicles that can be directly loaded with peptides, proteins, carbohydrates, or other les 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 lular particles can be prepared therefrom. 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 r up to hundreds or thousands of antigens and/or other , such as would be provided by the loading of a microorganism or portions thereof, for example.

In another embodiment of the present invention, an antigen and/or other agent is physically attached to the yeast e. Physical attachment of the antigen and/or other agent to the yeast vehicle can be accomplished by any method suitable in the art, ing covalent and non-covalent association methods which include, but are not limited to, chemically inking the antigen and/or other agent to the outer e 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 antibody or other binding partner. Chemical cross- linking can be achieved, for e, by methods including glutaraldehyde linkage, photoaffinity labeling, treatment with carbodiimides, treatment with chemicals capable of linking di-sulf1de bonds, and treatment with other cross-linking chemicals standard in the art. Alternatively, a chemical can be contacted with the yeast vehicle that alters the charge of the lipid bilayer of yeast ne 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. ing agents such as antibodies, binding es, 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 n to the yeast vehicle.

When the antigen or other protein is expressed on or physically 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 surface. The size of the spacer arm(s) can affect how much of the antigen or other protein is exposed for g on the surface of the yeast. Thus, ing 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 e. In one embodiment, the spacer arm is a yeast protein 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 mechanism, 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 t expression of heterologous antigens or other proteins) can be ground up or processed in a manner to produce yeast cell wall preparations, yeast membrane particles 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 e 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 adjuvant.

] In one embodiment of the invention, yeast vehicles useful in the invention e yeast vehicles that have been killed or inactivated. Killing or inactivating of yeast can be accomplished by any of a variety of suitable s 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 n, if desired, by standard methods known in the art. Alternatively, 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 culturing textbooks such as Methods of Enzymology, Vol. 194, Cold Spring Harbor Publishing . Any of the inactivation strategies used should take the ary, tertiary or nary ure of the target n into consideration 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 lyophilization.

Formulations comprising yeast vehicles 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 es can be mixed with a pharmaceutically acceptable excipient, such as an isotonic buffer that is tolerated by a host or host cell. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl , or triglycerides may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, ol, glycerol or dextran. Excipients can also contain minor amounts of ves, such as substances that enhance isotonicity and chemical stability. es of s include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives e 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 example, dextrose, human serum albumin, and/or preservatives to which e water or saline can be added prior to administration.

] In one embodiment of the present invention, a composition can e additional agents, which may also be referred to as biological response modifier nds, 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 ses, which may be referred to as immunomodulatory compounds, as well as compounds that modify the biological ty of another compound or agent, such as a yeast-based immunotherapeutic, such biological activity not being limited to immune system effects.

Certain immunomodulatory compounds can stimulate a protective immune se whereas others can suppress a harmful immune response, and whether an immunomodulatory is useful in combination with a given based therapeutic 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 response modifiers preferentially enhance a cell-mediated immune response whereas others preferentially enhance a humoral immune response (z'.e., can stimulate an immune se in which there is an increased level of cell-mediated compared 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 biological properties of yeast-based immunotherapeutics. There are a number of techniques known to those skilled in the art to measure stimulation or suppression of immune responses, as well as to differentiate cell-mediated immune ses 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 modifiers useful in the invention may include, but are not limited to, cytokines, chemokines, hormones, lipidic derivatives, es, proteins, polysaccharides, 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, nucleic acid binding es, aptamers, 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 nists; 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 antagonists; IL-22 or agonists of IL-22 or of IL-22R, anti-IL-22 or other IL- 22 antagonists; 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 antagonists; 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 eron or a receptor thereof; anti-CD40, CD40L, lymphocyte- activation gene 3 (LAG3) protein 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®); D4; 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 tory cells); Flt3 ligand, imiquimod (AldaraTM), ocyte- macrophage colony stimulating factor (GM-CSF); granulocyte-colony stimulating factor (G-CSF), sargramostim (Leukine®); hormones including t limitation prolactin and growth hormone; Toll-like receptor (TLR) agonists, including but not limited to TLR-2 agonists, TLR-4 agonists, TLR-7 agonists, and TLR-9 ts; TLR antagonists, including but not d to TLR-2 antagonists, TLR-4 antagonists, TLR-7 antagonists, and TLR-9 nists; anti-inflammatory agents and immunomodulators, including but not limited to, COX-2 inhibitors (e.g., Celecoxib, NSAIDS), glucocorticoids, statins, and thalidomide and analogues thereof including IMiDTMs (which are structural and functional analogues of thalidomide (e.g., ID® (lenalidomide), ACTIMID® (pomalidomide)); proinflammatory agents, such as fiJngal or bacterial ents or any proinflammatory ne or chemokine; immunotherapeutic vaccines including, but not limited to, virus-based es, bacteria-based vaccines, or antibody-based vaccines; and any other immunomodulators, immunopotentiators, nflammatory 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 protocol with (e.g., concurrently, sequentially, or in other formats with) a yeast-based therapeutic is a ition 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 n 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 g , etc., that binds to a receptor or ligand and produces or triggers a response, which may include agents that mimic or e the action of a lly occurring nce 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 agents, etc., that blocks or inhibits or reduces the action of an agonist.

Compositions of the invention can fiarther include or can be administered with (concurrently, sequentially, or intermittently with) any other agents or compositions or protocols that are useful for preventing or ng cancer or any compounds that treat or ameliorate any symptom of cancer, and particularly cancers associated with Brachyury expression or overexpression. In addition, compositions of the invention can be used together with other immunotherapeutic compositions, including prophylactic and/or therapeutic immunotherapy. Indeed, the compositions of the invention can be used to inhibit or reduce herapy resistance or radiation resistance that may occur in metastatic cancer by inhibiting Brachyury 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 ols (e.g., therapeutic ols) that are useful for the treatment of cancer include, but are not limited to, chemotherapy, surgical resection of a tumor, radiation y, allogeneic or autologous stem cell transplantation, and/or ed cancer therapies (e.g., small molecule drugs, biologics, or monoclonal antibody therapies that specifically target les involved in tumor growth and progression, including, but not limited to, selective estrogen receptor modulators (SERMs), aromatase inhibitors, tyrosine kinase inhibitors, serine/threonine kinase inhibitors, histone deacetylase (HDAC) tors, retinoid receptor activators, apoptosis stimulators, angiogenesis inhibitors, poly (ADP-ribose_) polymerase (PARP) inhibitors, or immunostimulators). Any of these additional therapeutic agents and/or eutic protocols may be administered , concurrently with, alternating with, or after the immunotherapy compositions of the invention, or at different time . For example, when given to an dual in conjunction with chemotherapy or a targeted cancer therapy, it may be ble to administer the yeast-Brachyury immunotherapy compositions during the “holiday” between doses of chemotherapy or targeted cancer therapy, in order to maximize the efficacy of the immunotherapy compositions. Surgical resection of a tumor may frequently precede administration of a yeast-Brachyury immunotherapy composition, but onal or primary surgery may occur during or after administration of a yeast- Brachyury immunotherapy composition.

The invention also includes a kit comprising any of the compositions described herein, or any of the individual components of the compositions bed herein. Kits may include additional reagents and written instructions or directions for using any of the itions of the invention to prevent or treat cancer associated with Brachyury expression or overexpression.

Methods for Administration or Use of Coonsitions of the Invention Yeast-Brachyury immunotherapeutic compositions of the invention are designed for use to prevent or treat cancers that are associated with or terized by Brachyury expression or overexpression, ing by preventing emergence of such cancers, arresting progression of such cancers or eliminating such s. More particularly, 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 progression of cancer in an individual. Yeast-Brachyury immunotherapeutic itions 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 al of the individual; preventing, inhibiting, reversing or delaying pment 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 individual. Yeast-Brachyury immunotherapy can also be used therapeutically to t, reduce or eliminate chemotherapy resistance or radiation resistance that may occur in metastatic cancer by ting Brachyury expression in the cancer, and compositions of the invention may enhance the mance of chemotherapy or radiation therapy in an dual.

] Cancers that are relevant to the compositions and methods of the invention are any cancer that expresses, or may express, Brachyury, or cancers in ity to cancers that express or may express Brachyury, and include, but are not limited to, cancer of the breast, small intestine, h, kidney, bladder, , ovary, testes, 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 chronic lymphocytic leukemia (CLL), Epstein-Barr virus ormed 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 progression of cancer in an individual who has cancer, or to e the development of atic events in a . As discussed above, 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. ore, 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 effective to prevent or treat metastatic , including arresting progression of the cancer, in the face of escape (or ted escape) of the cancer from traditional therapy, such as chemotherapy and radiation. The method includes the steps of administering to the individual who has cancer an immunotherapeutic composition a yeast-Brachyury immunotherapeutic composition of the invention as described herein, including, but not limited to: (a) a yeast vehicle; and (b) a cancer antigen comprising at least one ury antigen.

In one aspect, Brachyury is not detected in the dual’s cancer at the time the composition is first administered. In general, when Brachyury is not detected in the individual’s cancer, the individual 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 methods). In this aspect of the invention, the development of Brachyury-expressing tumor cells is prevented, delayed or inhibited by use of the yeast- Brachyury immunotherapeutic composition. As a result, tumor migration and/or other metastatic processes leading to metastatic progression of the tumor are prevented, delayed or inhibited 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 composition 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 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 experience an arrest, g or reversal in metastatic processes, improving survival and health of the patient, and filrthermore, allowing other therapeutic protocols to treat the cancer.

Indeed, metastatic cancer can be associated with resistance, or increased resistance, to cancer therapies such as chemotherapy, ion, or targeted cancer therapy, y the cancer es” from the therapy or is simply less impacted by the therapy and progresses. Accordingly, there is a need to reduce or eliminate resistance to such therapies to improve or enhance the efficacy of the therapy and improve t health and survival. Accordingly, one embodiment 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 ion therapy for the cancer, a yeast-Brachyury immunotherapeutic composition as described herein, which may include a composition comprising: (a) a yeast vehicle; 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 therapy.

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 therapeutic composition prevents or inhibits the onset of resistance to herapy or ion therapy by inhibiting the development of ury-expressing tumor cells in the cancer. In another , Brachyury expression is ed in the individual’s cancer at the time the composition is first administered. In this aspect, the dual may or may not already be encing resistance to chemotherapy or radiation. In either case, administration of the yeast-Brachyury immunotherapeutic composition 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 immunotherapeutic composition bed 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 al of the patient. In one aspect, the method inhibits tumor growth in the individual.

In one , the method prevents, arrests or reverses metastatic ssion of the tumor.

Since Brachyury sion 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 metastatic processes, it is an embodiment of the invention to provide a method to t or delay the onset of a Brachyury-expressing cancer, or to arrest the cancer at a pre-metastatic or pre-malignant stage. Such a method includes stering to an individual in whom Brachyury- expressing cancer cells are not detected a yeast-Brachyury immunotherapeutic composition described herein, which can e a composition comprising: (a) a yeast vehicle; and (b) a cancer antigen comprising at least one Brachyury antigen. In one aspect of this embodiment, the cancer is known to express or believed to be susceptible to expressing Brachyury at some stage of the cancer in at least a subset of individuals with the cancer. In one aspect of this embodiment, 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 r stage cancer in which Brachyury sion has not yet manifested, or in which Brachyury sion 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 ion 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 Brachyury immunotherapeutic composition prevents, delays or ts 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 another 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 marker, or because the individual has shown signs of cerous cells or s or has precancerous (premalignant) cells or lesions.

In one aspect, the individual is additionally treated with at least one other therapeutic nd or therapeutic ol useful for the treatment of cancer. Such therapeutic agents and protocols have been discussed in detail ere herein. For example, in any of the embodiments regarding methods of the invention described herein, in one , when the individual has cancer dless of the status of detectable Brachyury expression in tumor cells) the dual is being treated or has been d with another therapy for cancer. Such therapy can include any of the therapeutic protocols or use of any therapeutic compound or agent bed previously herein, including, but not limited to, chemotherapy, radiation therapy, targeted cancer therapy, surgical resection of a tumor, stem cell transfer, cytokine therapy, adoptive T cell transfer, and/or administration of a second immunotherapeutic composition. In the case of administration of a second immunotherapeutic composition, such compositions may include, but are not limited to, additional yeast-based immunotherapy, recombinant virus-based immunotherapy (viral vectors), ne therapy, immunostimulant therapy (including chemotherapy with immunostimulating properties), DNA vaccines, and other immunotherapy compositions.

In one aspect, the second immunotherapeutic composition includes a second cancer antigen that does not include Brachyury antigen. For example, a second immunotherapeutic composition useful in combination with a Brachyury immunotherapeutic composition is a yeast-immunotherapeutic composition comprising another cancer antigen. Such cancer antigens may include, but are not limited to, oembryonic antigen (CEA), point mutated Ras oncoprotein, MUC-l, EGFR, BCR- Abl, MART-l, MAGE-l, MAGE-3, GAGE, GP-lOO, MUC-2, normal and point mutated p53 oncoproteins, 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, -l, HERV-H, HERV-K, TWIST, Mesothelin, NGEP, modifications of such ns, splice variants of such antigens, and epitope agonists of such antigens, as well as combinations of such antigens, and/or immunogenic domains thereof, modifications thereof, variants thereof, and/or epitope agonists thereof As used herein, to “treat” 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, delaying, inhibiting, arresting or preventing the onset or development of metastatic cancer (such as by ng, inhibiting, 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, improvement of long term memory immune responses t the tumor antigens, and/or improved general health of the dual. To “prevent” or “protect” from a cancer, or any permutation thereof (e.g., “prevention of cancer”, etc.), lly refers to stering a composition of the invention before a cancer has occurred, or before a ic 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, ing cancer progression, improving the immune response t the , inhibiting metastatic processes) or improving es in the individual (e.g., improving survival).

The present invention includes the delivery (administration, immunization) of a yeast-Brachyury immunotherapeutic composition of the invention to a subject or individual. The administration s can be performed ex vivo or in viva, but is lly performed in vivo. Ex vivo administration refers to performing part of the regulatory step outside of the patient, such as administering a ition of the present invention to a population of cells (dendritic cells) removed from a t under ions such that a yeast vehicle, antigen(s) and any other agents or compositions are loaded into the cell, and returning the cells to the patient. The therapeutic composition of the present invention can be returned to a patient, or administered to a patient, by any suitable mode of administration. stration 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 . Various acceptable methods of administration include, but are not limited to, intravenous stration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, rterial administration (e.g., into a carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), intracranial, intraspinal, cular, aural, intranasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue. In one aspect, routes of administration include: intravenous, intraperitoneal, aneous, intradermal, intranodal, intramuscular, transdermal, inhaled, intranasal, oral, intraocular, intraarticular, ranial, and intraspinal. Parenteral delivery can include intradermal, intramuscular, intraperitoneal, intrapleural, intrapulmonary, enous, subcutaneous, atrial catheter and venal catheter routes. Aural delivery can include ear drops, intranasal delivery can include nose drops or intranasal injection, and intraocular delivery can include eye drops. Aerosol (inhalation) delivery can also be med 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 composition of the invention is administered subcutaneously. In one aspect, the yeast-Brachyury immunotherapeutic composition is stered directly into a tumor milieu.

In general, a suitable single dose of a yeast-Brachyury immunotherapeutic composition is a dose that is capable 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 se against 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 am body weight of the sm being administered the composition. In one , 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 suitable dose includes doses between 1 Y.U. and 40 Y.U. and in one aspect, 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 different sites on the dual during one dosing period. The invention includes administration of an amount of the yeast-Brachyury therapy 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 lents.

"Boosters" or "boosts" of a therapeutic composition are administered, for example, when the immune response against the antigen has waned or as needed to provide an immune response or induce a memory response against a particular n 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 dual 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 yeast-Brachyury immunotherapeutic composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times over a time period of from weeks, to months, 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 ly or monthly doses as needed to achieve the desired tative 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 ition (e.g., surgical ion of the tumor, administration of chemotherapy, administration of radiation therapy, administration of r immunotherapy composition or protocol, cytokine therapy, adoptive T cell transfer, or stem cell transplantation). For example, one or more ies can be administered or performed prior to the first dose of yeast-Brachyury immunotherapy ition or after the first dose is administered. In one ment, one or more therapies can be stered or performed in an alternating manner with the dosing of yeast-Brachyury immunotherapy composition, such as in a protocol in which the yeast-Brachyury composition is administered 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 ies. In other words, the yeast- ury immunotherapeutic composition is administered as a monotherapy for a period of time, and then an additional therapy is added (e.g., chemotherapy), either rently with new doses of yeast-Brachyury immunotherapy, or in an alternating fashion with yeast-Brachyury therapy. 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 ts 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 immunotherapy for weeks or months, optionally followed by monotherapy using yeast- Brachyury immunotherapy or another therapy, or by a new protocol of combinations of therapy ed sequentially, concurrently, or in alternating fashion). Various protocols for the treatment of cancer using yeast-Brachyury immunotherapy are plated by the invention, and these examples should be considered to be miting examples of various possible ols.

In one aspect of the ion, additional antigens other than Brachyury are also targeted using yeast-based immunotherapy, in addition to targeting Brachyury. Such additional target antigens can be included within the same yeast-vehicle as the Brachyury antigens, or onal yeast-based immunotherapy compositions targeting different antigens can be produced and then combined as desired depending on the individual to be treated, the antigens expressed by the type of cancer or by the individual’s ular tumor, and/or depending on the stage of cancer in the individual, or the stage of treatment of the individual. For examples a combination 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) ury, which is involved in metastatic processes. For example, on or more other yeast-based immunotherapy compositions may s one or more antigens including, but 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), NY-ESO-l, TRP-2, TAG72, KSA, CA-lZS, 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, hr, ews-fli-l, HERV-H, HERV-K, TWIST, Mesothelin, NGEP, modifications of such antigens, splice variants of such antigens, and epitope agonists of such antigens, as well as combinations of such antigens, and/or immunogenic domains thereof, modifications thereof, variants thereof, and/or e 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 yeast-Brachyury immunotherapy composition, in order to ze targeting of antigens in the dual’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) administering 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, concurrently with, or subsequent to, administration of the first immunotherapeutic composition a second immunotherapeutic composition comprising a yeast vehicle and a second cancer n sing a Brachyury antigen. In additional embodiments, the method can include administering one or more additional immunotherapeutic compositions, n the each of the one or more additional immunotherapeutic itions comprises an additional cancer antigen. The additional antigen can be any of those known in the art or described herein, including, but not limited to, mutated Ras, oembryonic n (CEA), and MUC-l.

In another embodiment of the invention, a method to treat cancer includes the ing steps: (a) administering to an individual who has cancer a first immunotherapeutic composition sing 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 (MUC-l); and (c) administering to the dual of (a) and (b) a third immunotherapeutic composition comprising a yeast vehicle and a Brachyury n. One or more of the steps of administration in (a), (b) and (c) can be performed concurrently, or sequentially. Steps may be repeated as needed to treat a particular individual’s , and the cancer antigens can be modified before or during treatment to specifically address the particular individual’s cancer.

In the method of the present invention, compositions and therapeutic compositions can be stered to animal, including any vertebrate, and particularly to any member of the Vertebrate class, Mammalia, ing, 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 invention include humans, non-human primates, dogs, cats, mice, rats, goats, sheep, cattle, horses and pigs.

An “individual” is a vertebrate, such as a , including without tion a human. Mammals include, but are not d 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 invention will , unless otherwise ted, 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, Methods of Enzymology, Vol. 194, Guthrie et al., eds., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts, Skinner, et al., eds., Academic Press (1980); Methods in yeast genetics : a laboratory 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 Laboratory Press (1997); T_he Yeast romyces: Gene Expression, Jones et al., eds., Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces: Genome Dynamics, n Synthesis, and Energetics, Broach et al., eds., Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: A Laboratogy Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), (jointly ed to herein as “Sambrook”); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain on, (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 tory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (jointly referred to herein as “Harlow and Lane”), Beaucage et al. eds., Current Protocols in c Acid tgy, John Wiley & Sons, Inc., New York, 2000); tt and Doull’s Toxicology The Basic Science of Poisons, C. Klaassen, ed., 6th edition , and Vaccines, S. Plotkin, W. Orenstein, and P. Off1t, eds., Fifth Edition (2008).

General ions ] A “TARMOGEN®” (GlobeImmune, Inc., Louisville, Colorado) generally refers to a yeast vehicle expressing one or more heterologous antigens extracellularly (on its surface), intracellularly (internally or cytosolically) or both extracellularly and intracellularly. TARMOGEN®s have been generally described (see, e. g., US. Patent No. ,830,463). Certain yeast-based therapy compositions, and methods of making and lly 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., e 2008 Jan 24;26(4):509-21, each of which is orated herein by reference in its entirety.

As used , 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 fianctional group, or the replacement of one functional group by another functional group). Thus, an analog is a nd 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 ed with a different atom or a chemical moiety.

Although a derivative has a similar al structure to the parent compound, the derivative may have different chemical and/or biological properties than the parent compound. Such ties can include, but are not limited to, increased or decreased activity of the parent nd, new activity as compared to the parent compound, enhanced or decreased bioavailability, enhanced or decreased eff1cacy, enhanced or decreased stability in vitro and/or in vivo, and/or enhanced or decreased absorption properties.

] In general, the term "biologically active" indicates that a compound (including a protein or peptide) has at least one detectable actiVity that has an effect on the metabolic, physiological, chemical, or other processes of a cell, a tissue, or an organism, as measured or ed in viva (i.e., in a l 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 generally to upregulation or downregulation of a particular activity. As used herein, the term “upregulate” can be used generally to describe any of: elicitation, initiation, increasing, augmenting, ng, improving, enhancing, amplifying, promoting, or ing, with respect to a particular activity. rly, the term egulate” can be used generally to describe any of: decreasing, reducing, inhibiting, ameliorating, diminishing, lessening, blocking, or preventing, with respect to a particular activity.

In one ment of the present invention, any of the amino acid sequences described 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 polypeptide 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 fianction of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid ce 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. rly, the phrase "consisting essentially of', when used with reference to a nucleic acid sequence herein, refers to a c acid sequence ng a specified amino acid ce 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 c 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 natural gene or do not encode a n that imparts any onal filnction to the protein or changes the function of the protein having the specified amino acid sequence.

According to the present invention, the phrase "selectively binds to" refers to the ability of an antibody, antigen-binding fragment or binding partner of the present ion 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, controls typically include a on well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of vity (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.). l reference to a protein or polypeptide used in the present invention includes fiJll-length ns, near full-length proteins ed above), or any fragment, domain (structural, functional, or immunogenic), conformational epitope, or a gue or t of a given protein. A filSlOI‘l protein may also be generally referred 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 l milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for example. As such, "isolated" does not reflect the extent to which the protein has been ed. Preferably, an isolated protein of the present ion is produced recombinantly. According to the present ion, the terms "modification" and "mutation" can be used interchangeably, particularly with regard to the modifications/mutations to the amino acid sequence of proteins or portions thereof (or nucleic acid sequences) described herein.

As used , 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 n 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, including deletions (e. g., a truncated version of the protein or peptide) insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, ylation, phosphorylation, acetylation, oylation, prenylation, palmitation, amidation and/or on 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 protein. gues or variants can be ed using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated reference protein, direct protein synthesis, or modifications to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or ed mutagenesis, resulting in the ng of a protein variant. In addition, naturally occurring variants of a reference protein 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 essentially 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% cal, 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% cal (or any t identity between 45% and 99%, in whole r increments), to the amino acid sequence of the nce protein (e.g, an amino acid sequence specified herein, or the amino acid sequence of a specified protein). In one embodiment, the gue or variant comprises, consists 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% cal to the amino acid ce of the reference protein.

As used herein, unless otherwise specified, reference to a percent (%) identity refers to an tion of homology which is med 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, wherein the query sequence 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 programs." Nucleic Acids Res. 25:3389-3402, incorporated herein by reference in its entirety); (2) a BLAST alignment of two sequences (e.g, using the parameters described ; (3) and/or AST with the standard default parameters (Position- Specif1c Iterated BLAST. It is noted that due to some differences in the standard parameters between Basic BLAST and BLAST for two ces, 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, easy-to-use version of a "profile" search, which is a sensitive way to look for sequence homologues. The program f1rst ms a gapped BLAST se search. The PSI-BLAST m uses the ation from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined 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 protein and tide sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein by reference 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 ces allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For es of y herein, a BLAST sequence ent for two sequences is performed using the standard default parameters 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 , 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 (z'.e., that has been subject to human manipulation), its natural milieu being the genome or chromosome in which the nucleic 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 molecule can include a complete gene. An isolated nucleic acid molecule that includes a gene is not a fragment of a chromosome that es such gene, but rather includes the coding region and regulatory s associated with the gene, but no onal genes that are naturally found on the same chromosome. An isolated nucleic acid molecule may also include portions of a gene. An isolated nucleic acid le can also include a specified nucleic acid sequence flanked by (2'.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 (2'.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 le, the two phrases can be used interchangeably, especially with respect to a c acid molecule, or a c acid sequence, being capable of encoding a n or domain of a protein.

A recombinant 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 effectively regulating expression of the nucleic acid molecule(s) in the cell to be transfected. 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 le, the two phrases can be used interchangeably, especially with respect to a c acid molecule, or a nucleic acid sequence, being capable of ng 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 inant vector, which is any nucleic acid sequence, typically a heterologous sequence, which is operatively linked to the isolated nucleic acid molecule encoding a fusion protein of the present invention, which is capable of enabling recombinant production of the fiasion protein, and which is capable of delivering the nucleic acid molecule into a host cell according to the present invention. Such a vector can contain nucleic acid sequences that are not naturally found nt 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 d useful for transfecting yeast. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating 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 vectors 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 ected host cell, such as a yeast.

In a recombinant molecule of the present ion, nucleic acid molecules are operatively linked to expression vectors containing regulatory sequences such as ription control ces, ation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of c acid molecules of the present invention. In particular, recombinant molecules of the present invention include c acid molecules that are operatively linked to one or more sion control ces. The phrase "operatively linked" refers to linking a nucleic acid le to an expression control sequence in a manner such that the molecule is expressed when transfected (i.e., transformed, transduced 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 uction 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.’ Therefore, transfection techniques include, but are not limited to, transformation, chemical treatment of cells, particle bombardment, electroporation, njection, 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 example describes the production of a yeast-Brachyury immunotherapeutic composition.

In this ment, yeast (Saccharomyces cerevisiae) were ered to express human Brachyury under the control of the copper-inducible promoter, CUPI , or the constitutive er, TEF2 , producing yeast-Brachyury immunotherapy compositions.

In each case, a fusion protein comprising a Brachyury antigen was produced as a single polypeptide with the following sequence elements fused in frame from N— to C-terminus, represented by SEQ ID NOS (1) an N—terminal peptide to impart resistance to proteasomal degradation and stabilize expression (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 ions 441-446 of SEQ ID NO:8). 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:8 (codon optimized for yeast expression) is represented herein by SEQ ID NO:7.

Briefly, DNA ng a full length human ury protein from a Brachyury-PCRII plasmid provided by the National 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 pGI-172) in yeast 2 um expression vectors. Nucleotide sequences encoding the N—terminal ization e, MADEAP (SEQ ID NO: 1 l) and a inal hexahistidine peptide were also added to the d vector to encode the complete fusion protein represented by SEQ ID NO:8. The resulting plasmids were transformed into DHSu for plasmid storage, and into Saccharomyces cerevisiae W3030L for production of the yeast-Brachyury immunotherapeutic compositions.

Transformation into Saccharomyces cerevisiae was performed by lithium acetate/polyethylene glycol transfection, and primary ectants were selected on solid minimal plates lacking Uracil (UDM; uridine dropout ). Colonies were selected by g in U2 (uridine dropout medium) or UL2 ne 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] er is also referred 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 ition comprising a polynucleotide encoding the human Brachyury fusion protein represented by SEQ ID NO:8 under the control of the TEF2 promoter (in vector 2) is also referred to herein as GI-6303.

Liquid cultures lacking uridine (U2) or lacking uridine and leucine (UL2) were inoculated using the plates and starter cultures bed 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 ated to evaluate yeast-Brachyury immunotherapeutics produced under neutral pH manufacturing conditions (data not shown). Primary es were used to inoculate final cultures of the same formulation.

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 ers, CUPI-driven ible expression) yeast-Brachyury expression was initiated by the addition of 0.5 mM copper sulfate after the yeast-Brachyury culture reached a density of approximately 0.2 Y.U./ml, and was continued until the culture d a density of 0.5-1.5 Y.U. -Brachyury doubled only about 1-1.5 after the addition of copper sulfate, but a large amount of ury protein was produced by the cells). TEF2-driven yeast-Brachyury sion is tutive, 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 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, dge, 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 immunotherapy compositions of the invention expressed the Brachyury fusion protein, z'.e., using either the CUPI promoter or the TEF2 promoter, and expression was detected using either media (U2 and UL2). In addition, n 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 comprising the CUP] promoter (GI-6301) and so this composition was selected for further studies, including expression optimization and for in vitro and in vivo ments (see Examples below).

Fig. 1A shows expression of Brachyury in GI-6301 using both U2 and UL2 media using the rachyury antibody for detection. Control yeast expressing a non- Brachyury antigen did not stain with the dy. Fig. 1B shows sion 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 Brachyury 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, GI-6301.

] 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 diluted to 0.5 Y.U./ml, 1.0 Y.U./ml 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 ury expression, and culturing was ued. 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 (Y.U.lm|) As shown in Table l, yeast only doubled about 1 time after copper induction (other ments showed up to 1.5X doubling), and cell density 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 densities. However, additional experiments using ion starting densities of 2.1 Y.U./ml and 2.8 Y.U./ml and 375 uM CuSO4 showed that n expression began to decrease as the density of the es at the start of copper induction increased, and did not significantly improve after about 6-8 hours (data not shown).

Next, the effect of the amount of CuSO4 on Brachyury expression was investigated. GI-6301 was grown from starter and intermediate cultures 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 n 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 n blots as described above. While both concentrations of CuSO4 resulted 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 ion of antigen expression at mid-log phase growth of the yeast was optimal for antigen production. For production of the yeast-Brachyury therapeutic composition (GI-6301) used in the following Examples, cells were grown in UL2 media as bed 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 following example describes the construction and tion of an additional yeast-Brachyury immunotherapeutic composition, where the Brachyury antigen contains a T cell agonist epitope.

In this experiment, yeast (Saccharomyces cerevisiae) were ered to express a human Brachyury antigen that is a near-filll-length Brachyury protein comprising the T cell epitope WLLPGTSTV (SEQ ID NO: 13), which is an t epitope.

The native Brachyury 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 sed under the control of the copper-inducible er, CUP], ing a yeast-Brachyury immunotherapy composition. More particularly, a fusion protein comprising a Brachyury agonist antigen (z'.e., a Brachyury antigen containing at least one agonist epitope) was ed as a single polypeptide with the following ce 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 degradation and ize 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:18 represents a full-length human Brachyury agonist protein having a single amino acid substitution at position 254 as compared to wild-type Brachyury protein); and (3) a stidine tag (positions 441-446 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 sequences 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 shorter portions of the Brachyury antigen may also be used. A c 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 Example 1 (z'.e., full-length ury minus the N-terminal methionine), modified by site ed mutagenesis to introduce a substitution of a valine for the leucine 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. Nucleotide sequences encoding the N—terminal stabilization e, 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:20. The resulting plasmids were transformed into DHSu for plasmid storage, and into Saccharomyces cerevisiae W3030L for production of the Brachyury immunotherapeutic 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 ). Colonies were ed by growing in UL2 ne and leucine dropout medium) medium at 30° C.

The yeast-Brachyury immunotherapy composition comprising a polynucleotide encoding the human Brachyury agonist fiasion 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 l, and were then induced by the addition of 0375-05 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 bed 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 itation/nitrocellulose binding assay and by Western blot using an is tag monoclonal antibody and an anti-Brachyury dy (Abcam, Cambridge, MA). Protein content was quantified using semi-quantitative digital imaging methods.

Fig. 1C shows the robust expression of Brachyury t antigen in GI-6305 using anti-His to identify the hexahistidine tag on the Brachyury fusion protein. The approximate antigen t for 5 grown in UL2 medium in this ment was >22615 ng/Y.U.

Example 4 The following example demonstrates the expansion of Brachyury-specific T cells using a yeast-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, dendritic 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 “YVEC”, which is a Saccharomyces cerevisiae yeast that is transformed with an empty vector, or vector that does not n an antigen-encoding insert) or Brachyury Yeast (GI-6301, described in Examples 1 and 2 above), at a ratio of yeast:DCs= l:l. After 48-hours co-culture, the DCs were used as APCs for stimulation of autologous T cells. Each cycle of stimulation, designated as IVS (in vitro stimulation), consisted of 3 days culture 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 er 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 ury er Tetramer Control Yeast 07706 ury 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 ation 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 Brachyury Yeast Brachyury Yeast Brachyury Yeast The results in Tables 2 and 3 show that stimulation of normal donor T cells with a yeast-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 gen.

Example 5 The ing 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, ury-specific T cells from three of the normal donors from Table 2 above were expanded in vitro using DCs ted 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 cytotoxic T lymphocyte (CTL) assay against SW480 (HLA-A2+ / Brachyury high) and MCF7 (HLA-A2+ / 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 ury-specific Tp2 tetramer.

Table 4 Brachyury Yeast/Tp2 17663 Brachyury 0.11 0.65 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 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 performed 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 Example 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 negative/Brachyury high) tumor cells, at an effector:target (ET) ratio of 15:1. Fig. 4A shows the percentage of specific 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 ments r trate that yeast-Brachyury immunotherapeutic composition can generate Brachyury-specific CTLs that are capable of g a Brachyury-expressing tumor cell.

Example 6 The ing example demonstrates that a yeast-Brachyury composition of the invention can expand Brachyury-specific T cells from cancer patients.

In this experiment, DCs were ed from the PBMCs of two breast cancer patients, post-vaccination with viral vector vaccines comprising CEA and MUC-l ns.

The DCs were prepared in a 5-day culture in presence of GM-CSF and IL-4 as described in Example 4, ed by tion 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, following 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 Brachyury peptide Tp2.

Table 5 Breast Pt 01 Brachyury Yeast 0.11 0.42 Breast Pt 10 ury Yeast 0.23 0.91 ] The s in Table 5 demonstrate that stimulation of T cells from breast cancer donors with a yeast-Brachyury immunotherapeutic of the ion increases the percentage of tetramer-positive CD8+ T cells in a majority of the donors, as compared to ls, indicating that T cells from donors with ongoing cancer have the capacity to recognize ury as an immunogen.

Example 7 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 protein (obtained from insect cells). As a l, 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 (GI-6301) are shown in Fig. 5. Fig. 5 shows that immunization with yeast-Brachyury (GI- 6301) generates Brachyury-specific CD4+ T cells.

The following example demonstrates that immunization with yeast-Brachyury immunotherapeutic composition reduces Brachyury-expressing tumors in vivo.

In this experiment, C57BL/6 mice received 1 x 106 MC38-phBrachyury cells (MC38 tumor cells expressing 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 post-tumor implantation, animals were sacrificed and the number of lung tumor nodules was evaluated. Results from two ed experiments are shown in Fig. 6. Table 6 shows the mean lung tumor number (:: SEM) and the number (and percentage) of animals 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 therapeutic ition of the invention is capable of reducing Brachyury-expressing tumors in mice, as compared to mice receiving yeast alone (no Brachyury n).

Example 9 The following example demonstrates the generation of Brachyury-specific CD4+ T cell responses in vitro using yeast-Brachyury immunotherapy in human peripheral blood mononuclear cells (PBMCs) obtained from y donors.

In the following experiments, a full-length human Brachyury protein was sed in insect cells via baculovirus expression and subsequently purif1ed.

] Dendritic cells (DCs) were prepared from PBMCs of healthy donors by 5-day e with GM-CSF and IL-4 and subsequently treated in vitro with yeast-control (YVEC, see Example 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, ated T cells were harvested and subsequently 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 ted, with 3/9 donors demonstrating Brachyury-specific CD4+ T-cell responses post-stimulation in vitro with 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 background 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 AIEms 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, ing in vitro stimulation with yeast-Brachyury (GI-630l)-treated DCs by intracellular cytokine ng of IFN—y in CD4+ cells. tic cells were prepared from PBMCs of y donors by 5-day culture with GMCSF and IL-4 and subsequently 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 DC:PBMCs = 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 production in response to autologous PBMCs (ratio T cells: PBMCs = 1:3) alone or in the presence of 10 ug/ml of purified ury protein or l human serum albumin protein. Following 2 hours ation, BD GOLGISTOPTM 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 Cy5.5 and anti-IFN-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 ve for CD4 and intracellular IFN—y in response to control human serum n (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 \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ e 10 The following example trates 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 herent cells at an effector-to-APC ratio of 10:1. Cultures were incubated for 3 days at 37°C, in a fied 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- 6305-exposed autologous DCs were used as APCs for three IVS . After the third IVS, irradiated (23,000 rads) autologous EBV-transformed B cells, pulsed with an agonist Brachyury peptide, WLLPGTSTV (SEQ ID NO:13), were used as APCs. A Brachyury- specific T cell line, denoted, TBR-A, was established. This T cell line was used in the immunoassays bed below.

Table 9 demonstrates that Brachyury-specific T cells (TBR-A) release significant levels of IFN-y after stimulation with allogeneic DCs treated with GI-6305, whereas l yeast (YVEC, see Example 4) did not stimulate 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 ated in Table 9) and then used to stimulate Brachyury agonist epitope- specific T cells (TBR-A). In this ment, 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 effectively lyse MDA-MB-23l breast cancer cells that are HLA- A2 ve/Brachyury positive, but do not lyse ASPC-l pancreatic cancer cells that are HLA-A2 negative/Brachyury positive. Briefly, the Brachyury-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 MDA-MB-231 mzam 649m Baum 02am nsmm zsam In another experiment, the ability of the TBR-A cell line to bind to ury-specific HLA-A2 tetramers was ted. Briefly, TBR-A cells (used at IVS-4) were stained with a control tetramer or a tetramer specific for the Brachyury agonist 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 (perforin is a mediator of the cytolytic ty of cytotoxic T lymphocytes (CTLs)). Briefly, T cells were tested on day 5 after ulation with Brachyury agonist peptide-pulsed autologous EBV transformed B cells. Fig. 8 shows the expression of perforin in the TBR-A cell line after stimulation with Brachyury agonist peptide-pulsed autologous B cells, further demonstrating the cytotoxic capability of this Brachyuryspecific T cell line, which was generated using GItreated DCs.

Example 11 The ing example describes a phase 1 al trial in subjects with Brachyury-positive cancer.

An open-label, sequential dose-escalation, phase 1 al trial has been initiated using the yeast-Brachyury therapy 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 sequential 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 stered at 2 week intervals for a total of 7 visits (~3 months), and then monthly thereafter until the patients meet udy criteria. An ion cohort of patients (n=lO) at maximum tolerated dose (MTD) or the observed best dose are selected for additional study. The s are monitoring safety and tolerability as a primary endpoint, and in the expanded cohort, r a significant change in T cell precursors is detectable as ed by an increase in Brachyury-specific T cells in ELISpot assay and proliferation in se to Brachyury protein (e.g., Brachyury-specific CD8+ or CD4+ T cells emerging or expanding on treatment). As secondary 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 ters of general immune activation, including ncy 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 nes (e.g, IFN-y, IL-10, IL-12, IL-2, IL-4, TGF-B, etc.). l is expected to be safe and well-tolerated with no significant toxicities.

In addition, GI-630l is expected to produce treatment-emergent Brachyury-specific T cell responses or an improvement in pre-existing Brachyury-speciflc baseline T cell responses at least some or a majority of patients. Some ts 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 additional cohort of patients, utilizing the maximum tolerated dose or observed best dose determined above, and the same primary and secondary endpoints are measured. GI-6305 is also expected to be safe and well-tolerated with no significant toxicities, as well as produce treatment-emergent Brachyury-specific T cell responses or an improvement 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. e 12 The following example describes a phase 2 clinical trial using yeast-Brachyury immunotherapeutic compositions.

A randomized phase 2 al trial in patients with breast cancer is run using a yeast-Brachyury immunotherapeutic 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. t inclusion criteria can include subjects with Grade 1, 2 or 3 cancers. Subject including criteria can also include 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 patients 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 ent arm patients receiving several serial injections of yeast-Brachyury immunotherapeutic composition during treatment. The primary endpoint is recurrence free survival or overall survival.

Additional endpoints can include antigen-specific T cell responses (e. g., Brachyuryspecific CD8+ T cells emerging or expanding on treatment), maintenance of lymph node negativity, progression to ases, and Brachyury expression in tumor cells.

The yeast-Brachyury immunotherapeutic composition is ed 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 responses in at least some or a majority of patients. Some or a majority of patients are also expected to have stabilized disease, in lymph node negativity, and/or prevention, reduction or arrest in metastatic progression.

While s embodiments of the present invention have been described in detail, it is apparent that ations and tions 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.

Claims (89)

The claims defining the invention are as follows:

1. Use of an immunotherapeutic composition comprising: a) a yeast vehicle; and b) a cancer antigen comprising at least one Brachyury antigen, n the Brachyury 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 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine; in the manufacture of a medicament for reducing, arresting, reversing, delaying or ting the metastatic progression of cancer in an dual who has a cancer that is undergoing metastatic progression, is at risk of undergoing metastatic progression, or is predicted to begin undergoing metastatic progression.

2. The use of Claim 1, wherein Brachyury expression is not detected in the individual’s cancer at the time the composition is first administered.

3. The use of Claim 1, wherein Brachyury expression is ed in the individual’s cancer at the time the composition is first administered.

4. The use of any one of Claims 1 to 3, wherein the individual has a stage I

5. The use of any one of Claims 1 to 3, wherein the individual has a stage II cancer.

6. The use of any one of Claims 1 to 3, wherein the individual has a stage III cancer.

7. The use of any one of Claims 1 to 3, wherein the dual has a stage IV cancer.

8. Use of an immunotherapeutic ition comprising: a) a yeast vehicle; and b) a cancer antigen comprising at least one Brachyury antigen, wherein the Brachyury 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 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at on 254 with a valine; in the manufacture of a medicament for preventing or delaying the onset of a Brachyury-expressing cancer.

9. The use of Claim 8, wherein cancer has not been detected in the individual.

10. The use of Claim 8 or 9, wherein the individual is at high risk for developing cancer.

11. The use of Claim 8, wherein the individual has a pre-cancerous lesion.

12. The use of Claim 8, wherein the individual has cancer, but uryexpressing cancer cells have not been detected in the cancer.

13. The use of Claim 12, n the cancer is not yet atic.

14. The use of Claim 12, wherein the cancer has a high risk of asizing.

15. The use of Claim 12, wherein the subject has a stage I cancer.

16. The use of Claim 12, wherein the subject has a stage II cancer.

17. The use of any one of Claims 1 to 16, wherein the individual has previously been treated with another therapy for cancer.

18. The use of Claim 17, wherein the therapy is chemotherapy.

19. The use of Claim 17, wherein the therapy is targeted cancer therapy.

20. The use of Claim 17, wherein the therapy is radiation therapy.

21. The use of Claim 17, n the therapy is adoptive T cell transfer.

22. The use of Claim 17, wherein the therapy is one or more additional immunotherapeutic compositions.

23. The use of Claim 22, wherein the onal immunotherapeutic itions comprise a yeast vehicle and a second cancer antigen that does not include Brachyury antigen.

24. The use of Claim 23, n the second cancer antigen is selected from the group consisting of: mutated Ras, carcinoembryonic antigen (CEA), MUC-1, EGFR, BCR-Abl, MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-2, PSMA, tyrosinase, TRP-1 (gp75), NY-ESO-1, 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-1, Rb-2, androgen receptor (AR), Smad4, MDR1, Flt-3, BRCA-1, , pax3-fkhr, ews-fli-1, HERV-H, HERV-K, TWIST, Mesothelin, and NGEP.

25. The use of Claim 23, wherein the second cancer antigen is selected from the group consisting of: mutated Ras, oembryonic antigen (CEA), and MUC-1.

26. Use of an immunotherapeutic composition comprising: a) a yeast vehicle; and b) a cancer antigen comprising at least one Brachyury antigen, wherein the Brachyury 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 ce that is at least 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine; in the manufacture of a medicament for reducing or preventing chemotherapy resistance or radiation resistance of tumor cells in a patient with cancer.

27. The use of Claim 26, wherein Brachyury is not detected in the individual’s cancer.

28. The use of Claim 26, wherein Brachyury expression is detected in the individual’s .

29. The use of any one of Claims 1 to 28, wherein the ment reduces tumor burden in the individual, increases survival of the individual, and/or inhibits tumor growth in the individual.

30. The use of any one of Claims 1 to 29, wherein the cancer is of epithelial cell origin.

31. The use of any one of Claims 1 to 29, wherein the cancer is selected from the group consisting of: breast cancer, small intestine cancer, stomach cancer, pancreatic cancer, kidney cancer, bladder cancer, uterine cancer, n cancer, testicular cancer, lung cancer, colon cancer, te , chronic lymphocytic leukemia (CLL), Epstein- Barr virus transformed B cells, Burkitt’s lymphoma, Hodgkin’s lymphoma, and metastatic cancers thereof.

32. The use of any one of Claims 1 to 31, wherein the Brachyury antigen has an amino acid ce represented by SEQ ID NO:6, SEQ ID NO:18, or 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.

33. The use of any one of Claims 1 to 31, wherein the Brachyury n comprises from at least position 1 or 2 to between position 255 and the C-terminus of SEQ ID NO:6 or SEQ ID NO:18.

34. The use of any one of Claims 1 to 31, wherein the Brachyury antigen ses from at least on 1 or 2 to between position 430 and the C-terminus of SEQ ID NO:6 or SEQ ID NO:18.

35. The use of any one of Claims 1 to 31, wherein the Brachyury antigen comprises ons 246 to 254 of SEQ ID NO:18.

36. The use of any one of Claims 1 to 31, wherein the Brachyury antigen comprises SEQ ID NO:6, or positions 2-435 of SEQ ID NO:6.

37. The use of any one of Claims 1 to 31, wherein the ury antigen ses SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, or 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.

38. The use of any one of Claims 1 to 31, wherein the Brachyury antigen comprises SEQ ID NO:6, or positions 2-435 of SEQ ID NO:6.

39. The use of any one of Claims 1 to 31, wherein the Brachyury antigen comprises SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, or an amino acid sequence that is at least 99% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a .

40. The use of any one of Claims 1 to 39, wherein the cancer antigen comprises two or more immunogenic domains of ury.

41. The use of any one of Claims 1 to 40, wherein a cancer antigen is a fusion protein.

42. The use of Claim 41, n the fusion protein 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.

43. The use of Claim 41, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO:20, or an amino acid sequence that is at least 95% identical to SEQ ID NO:20.

44. The use of Claim 41, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO:8.

45. The use of claim 41, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO:20.

46. The use of any one of Claims 1 to 45, wherein the yeast vehicle is a whole yeast.

47. The use of Claim 46, wherein the whole yeast is .

48. The use of Claim 47, wherein the whole yeast is heat-inactivated.

49. The use of any one of Claims 1 to 48, wherein the yeast e expresses the antigen.

50. The use of any one of Claims 1 to 48, wherein the yeast is from a genus selected from the group consisting of: Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, , Rhodotorula, Schizosaccharomyces and Yarrowia .

51. The use of any one of Claims 1 to 48, wherein the yeast is from romyces .

52. The use of any one of Claims 1 to 48, wherein the yeast is from Saccharomyces cerevisiae.

53. The use of any one of Claims 1 to 52, wherein the composition is formulated in a pharmaceutically acceptable excipient.

54. A yeast-Brachyury immunotherapeutic composition, wherein the immunotherapeutic composition comprises: a) a yeast vehicle; b) an antigen expressed by the yeast vehicle and comprising at least one Brachyury n, wherein the ury antigen comprises an amino acid sequence represented by 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 95% identical to SEQ ID NO:18 and comprises a substitution of a e at position 254 with a valine, or wherein the Brachyury n comprises from at least on 1 or 2 to between position 255 and the inus of SEQ ID NO:6 or SEQ ID NO:18; and c) a pharmaceutically acceptable excipient suitable for administration to a human.

55. The Brachyury immunotherapeutic composition of Claim 54, 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.

56. The yeast-Brachyury immunotherapeutic composition of Claim 54 or 55, wherein the Brachyury antigen comprises from at least position 1 or 2 to between position 430 and the C-terminus of SEQ ID NO:6, or SEQ ID NO:18.

57. The yeast-Brachyury immunotherapeutic composition of any one of Claims 54 to 56, wherein the Brachyury antigen comprises SEQ ID NO:6, or positions 2-435 of SEQ ID NO:6.

58. The yeast-Brachyury immunotherapeutic composition of any one of Claims 54 to 56, n the Brachyury antigen comprises SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, or 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.

59. The yeast-Brachyury immunotherapeutic composition of any one of Claims 54 to 57, wherein the ury antigen consists of SEQ ID NO:6, or positions 2-435 of SEQ ID NO:6.

60. The yeast-Brachyury immunotherapeutic ition of any one of Claims 54 to 56 or 58, wherein the Brachyury antigen comprises SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, or an amino acid sequence that is at least 99% cal to SEQ ID NO:18 and comprises a substitution of a leucine a position 254 with a valine.

61. The yeast-Brachyury therapeutic composition of any one of Claims 54 to 60, wherein the cancer antigen is a fusion protein.

62. The yeast-Brachyury immunotherapeutic ition of Claim 61, wherein the fusion n has an amino acid sequence that is at least 95% identical to SEQ ID NO:8.

63. The yeast-Brachyury immunotherapeutic composition of Claim 61, wherein the fusion n has an amino acid sequence that is at least 95% identical to SEQ ID NO:20.

64. The yeast-Brachyury immunotherapeutic composition of Claim 61, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO:8.

65. The yeast-Brachyury immunotherapeutic composition of claim 61, wherein the fusion protein has an amino acid ce represented by SEQ ID NO:20.

66. The yeast-Brachyury immunotherapeutic composition of any one of Claims 54 to 65, wherein the yeast vehicle is a whole yeast.

67. The yeast-Brachyury therapeutic composition of any one of Claims 54 to 65, wherein the whole yeast is heat-inactivated.

68. 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 NO:6, wherein expression of the Brachyury fusion protein is under the control of the promoter CUP1 ; wherein the Brachyury fusion protein was expressed by the yeast; and wherein the composition s a Brachyury-specific T cell response.

69. The yeast-Brachyury immunotherapeutic composition of Claim 68, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:8.

70. A yeast-Brachyury immunotherapeutic composition comprising: a) a whole, vated yeast; and b) a Brachyury fusion protein comprising the amino acid sequence of positions 2-435 of SEQ ID NO:18, wherein expression of the Brachyury fusion protein is under the control of the promoter CUP1 ; wherein the Brachyury fusion protein was expressed by the yeast; and wherein the composition elicits a Brachyury-specific T cell response.

71. The yeast-Brachyury therapeutic composition of Claim 70, n the fusion n comprises the amino acid sequence of SEQ ID NO:20.

72. The yeast-Brachyury immunotherapeutic composition of any one of Claims 54 to 71, wherein the yeast vehicle is from Saccharomyces .

73. The yeast-Brachyury therapeutic composition of any one of Claims 54 to 71, wherein the yeast vehicle is from Saccharomyces cerevisiae.

74. A Brachyury immunotherapeutic composition of any one of Claims 54 to 73 for use to treat a e.

75. The yeast-Brachyury immunotherapeutic composition of Claim 74, wherein the e is cancer.

76. The yeast-Brachyury immunotherapeutic composition of Claim 74, wherein the disease is ated with Epstein Barr Virus (EBV) infection.

77. Use of a yeast-Brachyury immunotherapeutic composition of any one of Claims 54 to 73 in the manufacture of a medicament for treating or preventing a disease or condition associated with Epstein Barr Virus (EBV) infection.

78. A method to produce a yeast-Brachyury immunotherapeutic composition, comprising: a) culturing yeast that have been transformed with a recombinant nucleic acid molecule encoding a Brachyury antigen under the control of a CUP1 promoter in a suitable medium in the absence of CuSO4 until the yeast reach mid-log growth phase, wherein the Brachyury 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 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine; b) inducing expression of the Brachyury antigen in the yeast by adding CuSO 4 to the medium; c) culturing the yeast after step (b) for up to between 6 and 8 hours; and d) harvesting the yeast.

79. The method of Claim 78, wherein the yeast in step (a) are cultured to a cell density of n 1.0 and 2.0 Y.U. per milliliter total e volume.

80. The method of Claim 78, wherein 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.

81. The method of any one of Claims 78 to 80, wherein the yeast are cultured in steps (a)-(c) in a medium where the pH is maintained at pH 5.5 or higher.

82. The method of any one of claims Claim 78 to 81, r comprising heatinactivating the yeast after step (d).

83. The method of Claim 82, wherein the yeast are heat-inactivated at about 56°C for about 1 hour.

84. The method of any one of Claims 78 to 83, further comprising formulating the yeast for injection with a pharmaceutically acceptable excipient.

85. The method of any one of Claims 78 to 84, wherein the yeast are from Saccharomyces .

86. The method of any one of Claims 78 to 84, wherein the yeast are from Saccharomyces cerevisiae.

87. Use of a combination of immunotherapeutic itions in the manufacture of a medicament to treat cancer, the immunotherapeutic compositions comprising: a) a first immunotherapeutic composition comprising a yeast vehicle and a first cancer n that does not se a Brachyury antigen; and b) a second immunotherapeutic composition comprising a yeast e and a second cancer antigen comprising a Brachyury antigen, n the Brachyury 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 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine.

88. Use of a combination of immunotherapeutic compositions in the manufacture of a medicament to treat , the immunotherapeutic compositions comprising: a) a first immunotherapeutic ition 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 consisting of carcinoembryonic antigen (CEA) and mucin-1 (MUC-1); and c) a third immunotherapeutic composition comprising a yeast vehicle and a 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 95% identical to SEQ ID NO:18 and comprises a substitution of a leucine at position 254 with a valine.

89. A use according to any one of Claims 1, 8, 26, 77, 87 or 88, the composition according to any one of Claims 54, 68 or 70, or the method according to Claim 78 substantially as described herein with reference to the