Yeast-MUC1 immunotherapy composition and its use

Abstract

Disclosed are yeast-based immunotherapeutic compositions comprising mucin-1 (MUC1), and methods for preventing and / or treating cancers characterized by mucin-1 (MUC1) expression or overexpression.

Description

[0001] The present invention is a divisional application based on a patent application with an application date of August 17, 2012, a priority date of August 17, 2011, an application number of "201810735110.0", and an invention name of "Yeast-MUC1 immunotherapy composition and its use".

[0002] Government Rights

[0003] This invention was made under a cooperative research and development agreement with the National Institutes of Health, an agency of the Department of Health and Human Services. The U.S. Government has certain rights in this invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 61 / 524,407, filed on August 17, 2011. The entire disclosure of U.S. Provisional Patent Application No. 61 / 524,407 is incorporated herein by reference.

[0006] Statement on Joint Research Agreement

[0007] This invention was made by or on behalf of the parties to a research and development cooperative agreement executed on May 8, 2008 between Globe Immune, Inc. and the U.S. Department of Health and Human Services represented by the National Cancer Institute, an institute, center, or division of the National Institutes of Health.

[0008] Reference sequence list

[0009] This application contains a sequence listing submitted electronically as a text file via EFS-Web. The text file, called "3923-40-PCT_ST25", has a byte size of 89 KB and was entered on August 16, 2012. Pursuant to 37 CFR § 1.52 (e) (5), the information contained in the text file is incorporated herein by reference in its entirety. Field of the Invention

[0010] The present invention generally relates to yeast-based immunotherapeutic compositions and methods for preventing and / or treating cancers characterized by expression or overexpression of Mucin-1 (MUC1). Background of the Invention

[0011] Cancer is the number one cause of death worldwide, and developing effective treatments for cancer remains one of the most active areas of research and clinical development. Although many new approaches have been proposed to treat and prevent cancer, many cancers still have high mortality rates and can be difficult to treat or relatively unresponsive to conventional therapies.

[0012] Most cancers and hematological malignancies in humans are characterized, at least in part, by the abnormal overexpression of a protein called mucin-1 (MUC1), whose normal function is to help protect epithelial cells from toxins, microorganisms, and other types of external environmental stresses (Kufe et al., Hybridoma 1984; 3: 223-32). MUC1 is a heterodimeric protein formed from the non-covalent interaction of two subunits that are encoded by a single transcript and then post-translationally processed into subunits, called MUC1-N and MUC1-C. MUC1 is normally found at the apical border of secretory endothelial cells, and when cells lose polarity in response to stress, a reversible process in normal cells, MUC1 can interact with molecules that are normally located at the basolateral border. In addition, in response to stressful environments, the MUC1-N subunit, a large protein containing variable number tandem repeats (VNTRs) that are heavily glycosylated with O-linked polysaccharides, can be shed. Another subunit of MUC1, called MUC1-C, has an extracellular domain, a transmembrane domain, and a cytoplasmic tail and is capable of binding ligands that are responsible for the physical association of MUC1 with the epidermal growth factor receptor (EGFR) (Li et al., J Biol Chem 2001; 276:35239-42; Schroeder et al., J Biol Chem 2001; 276:13057-64) and other receptor tyrosine kinases such as ErbB2-4, 20FGFR321, and PDGFR (Li et al., Mol Cancer Res 2003; 1:765-75; Ren et al., Mol Cancer Res 2006; 4:873-83; Singh et al., Cancer Res 2007; 67:5201-10). Additionally, MUCl-C has been linked to a variety of signaling pathways involving ErbB receptors, c-Src, β-catenin, transcription factors (p53, ERα), and other effectors such as Grb2 / SOS (Pandey et al., Cancer Res 1995;55:4000-3; Kinlough et al., J Biol Chem 2004;279:53071-7).

[0013] In transformed epithelial cells, membrane polarity is irreversible and MUCl expression is upregulated across the surface of cancer cells (Kufe et al., 1984, supra). MUCl overexpression is associated with reduced MUCl-N O-glycosylation, and MUCl-N spatially blocks cell-cell and cell-extracellular matrix interactions at high levels on the cell surface, which is associated with a malignant phenotype (Ligtenberg et al., Cancer Res 1992; 52: 223-32; van de Wiel-van Kemenade et al., J Immunol 1993; 151: 767-76; Wesseling et al., Mol Biol Cell 1996; 7: 565-77). The MUCl-C subunit is now considered an oncoprotein based on its involvement in multiple signaling pathways associated with tumorigenesis, and its overexpression has been shown to be involved in blocking the induction of apoptosis in response to DNA damage (Ren et al., Cancer Cell 2004; 5: 163-75; Raina et al., J Biol Chem 2004; 279: 20607-12), oxidative stress (Yin and Kufe, J Biol Chem 2003; 278: 35458-64; Yin et al., J Biol Chem 2004; 279: 45721-7), and hypoxia (Yin et al., J Biol Chem 2007; 282: 257-66), as well as conferring anchorage-independent growth and tumorigenicity (Li et al., Oncogene 2003; 22: 6107-10; Huang et al., Cancer Biol Ther. 2003;2:702-6; Huang et al., Cancer Res 2005;65:10413-22; Schroeder et al., Oncogene 2004;23:5739-47).

[0014] As discussed above, data from various laboratories indicate that MUCl-N (α subunit) plays a role in cancer by conferring cellular properties that allow immune evasion and potential metastatic spread. MUCl-C (β subunit) is implicated in signaling pathways responsible for tumor initiation and progression. These dual functions of MUCl may explain the different roles that this antigen appears to play in different cancer indications. For example, MUC1 appears to be an early marker in cancers such as breast and colon cancer (see, e.g., Kretschmer et al., Mol Cancer. 2011 Feb 11; 10(1):15; Mukhopadhyay et al., Biochim Biophys Acta. 2011 Apr; 1815(2):224-40; Saeki et al., Gastroenterology. 2011 Mar; 140(3):892-902), while MUC1 has been implicated in the epithelial-mesenchymal transition (EMT) pathway and metastatic spread in cancers such as pancreatic and esophageal cancer (see, e.g., Xu et al., Life Sci. 2011 Jun 6; 88(23-24):1063-9; Besmer et al., Cancer Res. 2011 Jul 1; 71(13):4432-42; Roy et al., Oncogene 2011 Mar; 141(2):154-157; Roy et al., Oncogene 2011 Mar; 142(3):155-158; 24;30(12):1449-59; Ye et al., Lab Invest. 2011 May;91(5):778-87), and in acute myeloid leukemia (AML) terminal differentiation is prevented by reactive oxygen species (see, e.g., Yin et al., Blood. 2011 May 5;117(18):4863-70; Fatrai et al., Exp Hematol. 2008 Oct;36(10):1254-65), thereby allowing unrestricted self-regeneration of these cancer cells.

[0015] Given the apparent role of MUC1 in the malignant phenotype of cancer cells, MUC1, and in particular MUC1-N, has been the focus of anticancer therapeutic approaches. In fact, most therapeutic approaches target MUC1-N, the extracellular portion of the MUC1 heterodimer. However, such approaches targeting MUC1-N have not been successful in the clinic to date, possibly due to interference from MUC1-N shed from cells. More recent studies have proposed targeting the MUC1-C subunit with antibodies directed against the extracellular domain, or with peptides, peptides conjugated to glycopolymers, small molecules, with preparations of tumor cells expressing MUC1, and with dendritic cell / tumor cell fusions. However, there are currently no approved cancer therapies that specifically target MUC1. Therefore, there is still a need in the art for new products that effectively treat and / or prevent cancers associated with MUC1 expression or overexpression. SUMMARY OF THE INVENTION

[0016] One embodiment of the present invention relates to a yeast-MUC1 immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein expressed by the yeast vehicle and comprising at least one MUC1 antigen. In one aspect, the MUC1 antigen consists of, in order from N to C terminus: a MUC1 SEA / extracellular domain (ED), wherein the MUC1 SEA / ED domain comprises a MUC1 ED flanked at the N terminus by one or more amino acids from the non-ED portion of the MUC1 SEA domain; at least two variable number of tandem repeat (VNTR) domains; a MUC1 transmembrane (TM) domain; and a MUC1 cytoplasmic domain (CD).

[0017] In one aspect, the antigen comprises two VNTR domains. In one aspect, the VNTR domains have an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the following sequences: positions 126-145 of SEQ ID NO: 11; any consecutive 20 amino acids between positions 61 to 1020 of SEQ ID NO: 11; any consecutive 20 amino acids between positions 126 to 965 of SEQ ID NO: 11; SEQ ID NO: 12; any consecutive 20 amino acids between positions 90 to 130 of SEQ ID NO: 14; any consecutive 20 amino acids between positions 60 to 100 of SEQ ID NO: 15; and corresponding sequences from different human MUC1 proteins. In one aspect, the VNTR domain has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to any consecutive 20 amino acids between positions 90 to 130 of SEQ ID NO: 14 or any consecutive 20 amino acids between positions 60 to 100 of SEQ ID NO: 15. In one aspect, the fusion protein has two VNTR domains, and the amino acid sequences of the two VNTR domains are positions 90 to 130 of SEQ ID NO: 14 or positions 60 to 100 of SEQ ID NO: 15.

[0018] In one aspect, the MUCl ED has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of: positions 116-173 of SEQ ID NO:2; positions 107-164 of SEQ ID NO:4; positions 107-164 of SEQ ID NO:6; positions 98-155 of SEQ ID NO:8; positions 1098-1155 of SEQ ID NO:11; positions 32-89 of SEQ ID NO:14; positions 2-59 of SEQ ID NO:15; and corresponding sequences from different human MUCl proteins. In one aspect, the MUCl ED has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to positions 32-894 of SEQ ID NO:14 or positions 2-59 of SEQ ID NO:15. In one aspect, the MUC1 ED has an amino acid sequence of positions 32-89 of SEQ ID NO:14 or positions 2-59 of SEQ ID NO:15. In one aspect, the MUC1 SEA / ED has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of positions 115-173 of SEQ ID NO:2; positions 106-164 of SEQ ID NO:4; positions 106-164 of SEQ ID NO:6; positions 97-155 of SEQ ID NO:8; positions 1097-1155 of SEQ ID NO:11; positions 31-89 of SEQ ID NO:14; positions 1-59 of SEQ ID NO:15; and corresponding sequences from different human MUC1 proteins. In one aspect, the MUC1 SEA / ED has an amino acid sequence of positions 31-89 of SEQ ID NO:14 or positions 1-59 of SEQ ID NO:15.

[0019] In one aspect, the MUCl TM domain has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from positions 174-201 of SEQ ID NO:2, positions 165-192 of SEQ ID NO:4, positions 165-192 of SEQ ID NO:6, positions 156-183 of SEQ ID NO:8, positions 1156-1183 of SEQ ID NO:11, positions 131-158 of SEQ ID NO:14, positions 101-128 of SEQ ID NO:15, and corresponding sequences from different human MUCl proteins. In one aspect, the MUCl TM domain has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to positions 131-158 of SEQ ID NO:14 or positions 101-128 of SEQ ID NO:15. In one aspect, the MUCl TM domain has the amino acid sequence of positions 131-158 of SEQ ID NO:14 or positions 101-128 of SEQ ID NO:15.

[0020] In one aspect, the MUCl CD domain has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of: positions 202-273 of SEQ ID NO:2, positions 193-264 of SEQ ID NO:4, positions 193-264 of SEQ ID NO:6, positions 184-255 of SEQ ID NO:8, positions 1184-1255 of SEQ ID NO:11, positions 159-230 of SEQ ID NO:14, positions 129-200 of SEQ ID NO:15, positions 7-78 of SEQ ID NO:17, positions 79-150 of SEQ ID NO:17, positions 151-222 of SEQ ID NO:17; positions 1-72 of SEQ ID NO:18, positions 73-144 of SEQ ID NO:18, positions 145-216 of SEQ ID NO:18, and corresponding sequences from different human MUCl proteins. In one aspect, the MUCl CD domain has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence at positions 159-230 of SEQ ID NO: 14 or positions 129-200 of SEQ ID NO: 15. In one aspect, the MUCl CD domain has an amino acid sequence at positions 159-230 of SEQ ID NO: 14 or positions 129-200 of SEQ ID NO: 15.

[0021] In one aspect of this embodiment of the invention, the MUCl antigen has an amino acid sequence that is at least 95%, 96%, 97%, or 98% identical to SEQ ID NO: 15. In one aspect, the MUCl antigen comprises SEQ ID NO: 15 or an amino acid sequence that is at least 99% identical to SEQ ID NO: 15. In one aspect, the MUCl antigen has the amino acid sequence of SEQ ID NO: 15.

[0022] In one aspect of this embodiment of the invention, the fusion protein further comprises a MUC1 signal sequence attached to the N-terminus of MUC1 SEA / ED. In one aspect, the MUC1 signal sequence has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from positions 1-27 of SEQ ID NO:2, positions 1-32 of SEQ ID NO:4, positions 1-32 of SEQ ID NO:6, positions 1-27 of SEQ ID NO:8, positions 1-23 of SEQ ID NO:11, positions 1-30 of SEQ ID NO:14, and corresponding sequences from different human MUC1 proteins. In one aspect, the MUC1 signal sequence has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence at positions 1-30 of SEQ ID NO:14. In one aspect, the MUC1 signal sequence has an amino acid sequence at positions 1-30 of SEQ ID NO:14. In one aspect, the fusion protein has an amino acid sequence that is at least 95%, 96%, 97%, or 98% identical to SEQ ID NO: 14. In one aspect, the fusion protein comprises SEQ ID NO: 14 or an amino acid sequence that is at least 99% identical to SEQ ID NO: 14. In one aspect, the fusion protein has the amino acid sequence of SEQ ID NO: 14.

[0023] In one aspect of this embodiment of the invention, the MUC1 antigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acid substitutions compared to the wild-type MUC1 sequence to form 1 to 11 agonist epitopes within the MUC1 antigen (also referred to herein as MUC1 agonist antigens). In one aspect of this embodiment of the invention, for the MUC1 antigen portion of SEQ ID NO: 14 or SEQ ID NO: 15, the amino acid substitutions are selected from the group consisting of: A96Y, P97L, G104V, S105Y, T106L, A147Y, C161V, T199L, D200F, S215Y and T239L. In one aspect, the MUC1 agonist antigen has an amino acid sequence that is at least 95%, 96%, 97%, or 98% identical to SEQ ID NO: 23. In one aspect, the MUC1 antigen comprises SEQ ID NO: 23 or an amino acid sequence that is at least 99% identical to SEQ ID NO: 23. In one aspect, the MUCl antigen has the amino acid sequence of SEQ ID NO: 23. In one aspect, the MUCl antigen comprises 1 to 11 amino acid substitutions to create a MUCl agonist antigen.

[0024] Another embodiment of the present invention relates to a yeast-MUC1 immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein expressed by the yeast vehicle and comprising at least one MUC1 antigen. The MUC1 antigen consists of two or more cytoplasmic domains (CDs) of MUC1. In one aspect, the MUC1 antigen consists of three cytoplasmic domains (CDs) of MUC1. In one aspect, the three CDs are from the same MUC1 protein. In one aspect, each CD domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of positions 202-273 of SEQ ID NO:2, positions 193-264 of SEQ ID NO:4, positions 193-264 of SEQ ID NO:6, positions 184-255 of SEQ ID NO:8, positions 1184-1255 of SEQ ID NO:11, positions 159-230 of SEQ ID NO:14, positions 129-200 of SEQ ID NO:15, positions 7-78 of SEQ ID NO:17, positions 79-150 of SEQ ID NO:17, positions 151-222 of SEQ ID NO:17, positions 1-72 of SEQ ID NO:18, positions 73-144 of SEQ ID NO:18, positions 145-216 of SEQ ID NO:18, and corresponding sequences from different human MUCl proteins. In one aspect, each CD domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of positions 129-200 of SEQ ID NO: 15, positions 7-78 of SEQ ID NO: 17, positions 79-150 of SEQ ID NO: 17, positions 151-222 of SEQ ID NO: 17, positions 1-72 of SEQ ID NO: 18, positions 73-144 of SEQ ID NO: 18, and positions 145-216 of SEQ ID NO: 18.

[0025] In one aspect of this embodiment, the MUC1 antigen has an amino acid sequence that is at least 95%, 96%, 97%, or 98% identical to SEQ ID NO: 18. In one aspect, the MUC1 antigen has an amino acid sequence 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 MUC1 antigen has an amino acid sequence of SEQ ID NO: 18. In one aspect, the fusion protein has an amino acid sequence that is at least 95%, 96%, 97%, or 98% identical to SEQ ID NO: 17. In one aspect, the fusion protein has an amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that is at least 99% identical to SEQ ID NO: 17. In one aspect, the fusion protein has an amino acid sequence of SEQ ID NO: 17.

[0026] Another embodiment of the invention relates to a yeast-MUC1 immunotherapeutic composition comprising: (a) a yeast vehicle; and (b) a fusion protein expressed by the yeast vehicle and comprising at least one MUC1 agonist antigen. In one aspect of this embodiment of the invention, the MUC1 agonist antigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acid substitutions compared to the wild-type MUC1 sequence to form 1 to 11 agonist epitopes within the MUC1 antigen (also referred to herein as MUC1 agonist antigens). In one aspect, the MUC1 agonist antigen has an amino acid sequence that is at least 95%, 96%, 97%, or 98% identical to SEQ ID NO:23 or SEQ ID NO:25. In one aspect, the MUC1 antigen comprises SEQ ID NO:23 or SEQ ID NO:25 or an amino acid sequence that is at least 99% identical to SEQ ID NO:23 or SEQ ID NO:25. In one aspect, the MUC1 antigen has an amino acid sequence of SEQ ID NO:23 or SEQ ID NO:25.

[0027] Yet another embodiment of the invention relates to a method of reducing tumor burden, inhibiting tumor growth, and / or increasing survival in an individual having a cancer that expresses MUC1. The method comprises administering to an individual a yeast-MUC1 immunotherapy composition as described above or elsewhere herein. In one aspect, MUC1 expression is detected in the individual's cancer upon first administration of the composition. In one aspect, the individual has stage I cancer. In one aspect, the individual has stage II cancer. In one aspect, the individual has stage III cancer. In one aspect, the individual has stage IV cancer.

[0028] Another embodiment of the invention relates to the use of any of the yeast-MUCl immunotherapeutic compositions described herein to treat a disease. In one aspect, the disease is cancer.

[0029] Yet another embodiment of the invention relates to the use of any of the Yeast-MUCl immunotherapeutic compositions described herein to reduce, arrest, reverse, or prevent metastatic progression of cancer in an individual suffering from cancer.

[0030] Yet another embodiment of the present invention relates to the use of any of the yeast-MUCl immunotherapeutic compositions described herein to prevent or delay the development of a MUCl -expressing cancer.

[0031] Another embodiment of the present invention relates to the use of a combination immunotherapeutic composition for treating cancer, the immunotherapeutic composition comprising: (a) a first composition, which is any yeast-MUC1 immunotherapeutic composition described herein; and (b) at least one additional immunotherapeutic composition, which comprises a yeast vehicle and an antigen that is not a MUC1 antigen. In one aspect, the antigen that is not a MUC1 antigen is selected from mutant Ras, carcinoembryonic antigen (CEA), and / or brachyura malformation protein.

[0032] In one aspect relating to any of the embodiments of the methods or uses of the yeast-MUCl immunotherapeutic compositions described herein, the individual is receiving or has been treated with another therapy for cancer, including, but not limited to, chemotherapy, targeted cancer therapy, radiation therapy, adoptive T cell transfer, surgical removal of a tumor from the individual, and / or administration of one or more additional immunotherapeutic compositions. In one aspect, the additional immunotherapeutic composition comprises a second cancer antigen that is a MUCl antigen or a cancer antigen that is not a MUCl antigen. In one aspect, the additional immunotherapeutic composition comprises a yeast vehicle and a second cancer antigen that does not include a MUCl antigen. In one aspect, the second cancer antigen included in the additional immunotherapy composition includes, but is not limited to, mutant Ras, carcinoembryonic antigen (CEA), Brachyury, 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, CA-125, 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, BRCA-2, pax3-fkhr, ews-fli-1, HERV-H, HERV-K, TWIST, mesothelin, and / or NGEP. In one aspect, the second cancer antigen is selected from the group consisting of mutated Ras, carcinoembryonic antigen (CEA), and brachyura malformation protein. In one aspect, the additional immunotherapeutic composition is a viral vector vaccine. In one aspect, the additional immunotherapeutic composition is a dendritic cell / tumor cell fusion.

[0033] Yet another embodiment of the invention relates to a method of preventing or delaying the development of a cancer that expresses MUC1. The method comprises the step of administering to an individual any of the yeast-MUC1 immunotherapy compositions described herein. In one aspect, the cancer has not been detected in the individual. In one aspect, the individual is at high risk for developing cancer. In one aspect, the individual has a precancerous lesion. In one aspect, the individual has cancer, but cancer cells expressing MUC1 have not been detected in the cancer.

[0034] In any of the methods or uses involving the yeast-MUCl immunotherapy compositions described herein, in one aspect, the cancer is of epithelial origin. In one aspect, the cancer may include, but is not limited to, breast cancer, small intestinal cancer, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, melanoma, multiple myelogenous leukemia (MML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), Burkitt's lymphoma, Hodgkin's lymphoma, cancer of secretory tissues, and metastatic cancers thereof. In one aspect, the cancer is selected from breast cancer and colon cancer. In one aspect, the cancer is selected from breast cancer, colon cancer, pancreatic cancer, ovarian cancer, esophageal cancer, and AML. In one aspect, the cancer is AML and the yeast-MUCl immunotherapeutic composition is administered to both the donor and recipient of a bone marrow transplant (BMT) therapy.In one aspect, the cancer is AML and the yeast-MUCl immunotherapeutic composition is administered to the individual in conjunction with cytarabine and anthracycline therapy.

[0035] In any of the embodiments of the invention described above and elsewhere herein, in one aspect, the yeast vehicle is whole yeast. In one aspect, the yeast vehicle is heat inactivated. In one aspect, the yeast vehicle is from a mutant yeast strain that produces hypoglycosylated proteins compared to a wild-type yeast strain. In one aspect, the MUC1 antigen is expressed on the cell wall of the yeast vehicle. In one aspect, the MUC1 antigen is expressed in the periplasm or cytoplasm of the yeast vehicle. In one aspect, the yeast vehicle is from Saccharomyces. In one aspect, the yeast vehicle is from Saccharomyces cerevisiae.

[0036] In any of the embodiments of the invention described above and elsewhere herein, in one aspect, the immunotherapeutic composition is produced by culturing whole yeast expressing the MUCl antigen in a medium maintained at a pH level of 5.5 to 8. In one aspect, the medium is buffered with a buffer. In one aspect, the yeast is cultured in a medium maintained at a pH level of 6 to 8.

[0037] In any of the embodiments of the invention described above and elsewhere herein, in one aspect, the composition further comprises at least one biological response modifier.

[0038] In any of the embodiments of the invention described above and elsewhere herein, in one aspect, the composition further comprises a pharmaceutically acceptable excipient.

[0039] In any of the embodiments of the invention described above and elsewhere herein, in one aspect, the immunotherapeutic composition is formulated for injection.

[0040] The present invention relates to the following technical solutions:

[0041] 1. A yeast-MUC1 immunotherapeutic composition, wherein the immunotherapeutic composition comprises:

[0042] a) a yeast vehicle; and

[0043] b) a fusion protein expressed by the yeast vehicle and comprising at least one MUC1 antigen, wherein the MUC1 antigen consists of, in order from N to C terminus: a MUC1 SEA / extracellular domain (ED), wherein the MUC1 SEA / ED domain comprises a MUC1 ED flanked at the N-terminus by one or more amino acids from the non-ED portion of the MUC1 SEA domain; at least two variable number of tandem repeat (VNTR) domains; a MUC1 transmembrane (TM) domain; and a MUC1 cytoplasmic domain (CD).

[0044] 2. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the antigen comprises two VNTR domains.

[0045] 3. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the MUC1 antigen has an amino acid sequence that is at least 95% identical to SEQ ID NO:15.

[0046] 4. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the MUC1 ED has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of: positions 2-59 of SEQ ID NO:15; positions 32-89 of SEQ ID NO:14; positions 116-173 of SEQ ID NO:2; positions 107-164 of SEQ ID NO:4; positions 107-164 of SEQ ID NO:6; positions 98-155 of SEQ ID NO:8; positions 1098-1155 of SEQ ID NO:11; and corresponding sequences from different human MUC1 proteins.

[0047] 5. The yeast-MUCl immunotherapeutic composition of claim 1, wherein the MUCl ED has an amino acid sequence that is at least 95% identical to positions 2-59 of SEQ ID NO:15 or positions 32-89 of SEQ ID NO:14.

[0048] 6. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the MUC1 ED has the amino acid sequence of positions 2-59 of SEQ ID NO:15 or positions 32-89 of SEQ ID NO:14.

[0049] 7. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the MUC1 SEA / ED has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the following groups: positions 1-59 of SEQ ID NO:15; positions 31-89 of SEQ ID NO:14; positions 115-173 of SEQ ID NO:2; positions 106-164 of SEQ ID NO:4; positions 106-164 of SEQ ID NO:6; positions 97-155 of SEQ ID NO:8; positions 1097-1155 of SEQ ID NO:11; and corresponding sequences from different human MUC1 proteins.

[0050] 8. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the MUC1 SEA / ED has the amino acid sequence of positions 1-59 of SEQ ID NO:15 or positions 31-89 of SEQ ID NO:14.

[0051] 9. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the VNTR domain has an amino acid sequence that is at least 95% identical to: any consecutive 20 amino acids between positions 60 and 100 of SEQ ID NO:15; any consecutive 20 amino acids between positions 90 and 130 of SEQ ID NO:14; positions 126-145 of SEQ ID NO:11; any consecutive 20 amino acids between positions 61 and 1020 of SEQ ID NO:11; any consecutive 20 amino acids between positions 126 and 965 of SEQ ID NO:11; SEQ ID NO:12; and corresponding sequences from different human MUC1 proteins.

[0052] 10. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the VNTR domain has an amino acid sequence that is at least 95% identical to any consecutive 20 amino acids between positions 60 and 100 of SEQ ID NO:15 or any consecutive 20 amino acids between positions 90 and 130 of SEQ ID NO:14.

[0053] 11. The yeast-MUC1 immunotherapeutic composition of item 1, wherein the fusion protein has two VNTR domains, and wherein the amino acid sequence of the two VNTR domains is positions 60 to 100 of SEQ ID NO:15 or positions 90 to 130 of SEQ ID NO:14.

[0054] 12. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the MUC1 TM domain has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of positions 101-128 of SEQ ID NO:15, positions 131-158 of SEQ ID NO:14, positions 174-201 of SEQ ID NO:2, positions 165-192 of SEQ ID NO:4, positions 165-192 of SEQ ID NO:6, positions 156-183 of SEQ ID NO:8, positions 1156-1183 of SEQ ID NO:11, and corresponding sequences from different human MUC1 proteins.

[0055] 13. The yeast-MUCl immunotherapeutic composition of claim 1, wherein the MUCl TM domain has an amino acid sequence that is at least 95% identical to positions 101-128 of SEQ ID NO:15 or positions 131-158 of SEQ ID NO:14.

[0056] 14. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the MUC1 TM domain has the amino acid sequence of positions 101-128 of SEQ ID NO:15 or positions 131-158 of SEQ ID NO:14.

[0057] 15. The yeast-MUCl immunotherapeutic composition of claim 1, wherein the MUC1 CD domain has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of positions 129-200 of SEQ ID NO:15, positions 159-230 of SEQ ID NO:14, positions 202-273 of SEQ ID NO:2, positions 193-264 of SEQ ID NO:4, positions 193-264 of SEQ ID NO:6, positions 184-255 of SEQ ID NO:8, positions 1184-1255 of SEQ ID NO:11, positions 7-78 of SEQ ID NO:17, positions 79-150 of SEQ ID NO:17, positions 151-222 of SEQ ID NO:17, positions 1-72 of SEQ ID NO:18, positions 73-144 of SEQ ID NO:18, positions 145-216 of SEQ ID NO:18, and corresponding sequences from different human MUC1 proteins.

[0058] 16. The yeast-MUCl immunotherapeutic composition of claim 1, wherein the MUCl CD domain has an amino acid sequence that is at least 95% identical to the amino acid sequence at positions 129-200 of SEQ ID NO:15 or positions 159-230 of SEQ ID NO:14.

[0059] 17. The yeast-MUCl immunotherapeutic composition of claim 1, wherein the MUCl CD domain has the amino acid sequence of positions 129-200 of SEQ ID NO:15 or positions 159-230 of SEQ ID NO:14.

[0060] 18. The yeast-MUCl immunotherapeutic composition of claim 1, wherein the MUCl antigen comprises SEQ ID NO:15 or an amino acid sequence that is at least 99% identical to SEQ ID NO:15.

[0061] 19. The yeast-MUC1 immunotherapeutic composition of claim 1, wherein the MUC1 antigen has the amino acid sequence of SEQ ID NO:15.

[0062] 20. The yeast-MUCl immunotherapeutic composition of claim 1, wherein the fusion protein further comprises a MUCl signal sequence attached to the N-terminus of MUCl SEA / ED.

[0063] 21. The yeast-MUC1 immunotherapeutic composition of claim 20, wherein the MUC1 signal sequence has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of positions 1-27 of SEQ ID NO:2, positions 1-32 of SEQ ID NO:4, positions 1-32 of SEQ ID NO:6, positions 1-27 of SEQ ID NO:8, positions 1-23 of SEQ ID NO:11, positions 1-30 of SEQ ID NO:14, and corresponding sequences from different human MUC1 proteins.

[0064] 22. The yeast-MUC1 immunotherapeutic composition of claim 20, wherein the MUC1 signal sequence has an amino acid sequence that is at least 95% identical to the amino acid sequence at positions 1-30 of SEQ ID NO:14.

[0065] 23. The yeast-MUC1 immunotherapeutic composition of claim 20, wherein the MUC1 signal sequence has the amino acid sequence of positions 1-30 of SEQ ID NO:14.

[0066] 24. The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 23, wherein the fusion protein has an amino acid sequence that is at least 95% identical to SEQ ID NO:14.

[0067] 25. The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 23, wherein the fusion protein comprises SEQ ID NO: 14 or an amino acid sequence that is at least 99% identical to SEQ ID NO: 14.

[0068] 26. The yeast-MUCl immunotherapeutic composition of any one of items 1 to 23, wherein the fusion protein has the amino acid sequence of SEQ ID NO:14.

[0069] 27. The yeast-MUCl immunotherapeutic composition of claim 1, wherein the MUCl antigen comprises 1 to 11 amino acid substitutions to create a MUCl agonist antigen.

[0070] 28. The yeast-MUCl immunotherapeutic composition of claim 27, wherein the amino acid substitutions for SEQ ID NO:23 are selected from the group consisting of: A96Y, P97L, G104V, S105Y, T106L, A147Y, C161V, T199L, D200F, S215Y, and T239L.

[0071] 29. The yeast-MUCl immunotherapeutic composition of claim 27, wherein the MUCl agonist antigen comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:23.

[0072] 30. The yeast-MUCl immunotherapeutic composition of claim 27, wherein the MUCl agonist antigen comprises SEQ ID NO:23 or an amino acid sequence that is at least 99% identical to SEQ ID NO:23.

[0073] 31. A Yeast-MUC1 immunotherapeutic composition, wherein the immunotherapeutic composition comprises:

[0074] a) a yeast vehicle; and

[0075] b) a fusion protein expressed by the yeast vehicle and comprising at least one MUCl agonist antigen, wherein the MUCl agonist antigen comprises an amino acid sequence at least 95% identical to SEQ ID NO:25.

[0076] 32. The yeast-MUCl immunotherapeutic composition of claim 31, wherein the MUCl agonist antigen comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:25.

[0077] 33. The yeast-MUCl immunotherapeutic composition of claim 31, wherein the MUCl agonist antigen comprises the amino acid sequence of SEQ ID NO:25.

[0078] 34. A Yeast-MUCl immunotherapeutic composition, wherein the immunotherapeutic composition comprises:

[0079] a) a yeast vehicle; and

[0080] b) a fusion protein expressed by the yeast vehicle and comprising at least one MUCl antigen, wherein the MUCl antigen consists of two or more cytoplasmic domains (CD) of MUCl.

[0081] 35. The yeast-MUC1 immunotherapeutic composition of claim 34, wherein the MUC1 antigen consists of three cytoplasmic domains (CD) of MUC1.

[0082] 36. The yeast-MUC1 immunotherapeutic composition of claim 34, wherein the three CDs are derived from the same MUC1 protein.

[0083] 37. The yeast-MUC1 immunotherapeutic composition of claim 34, wherein each CD domain comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of positions 1-72 of SEQ ID NO:18, positions 73-144 of SEQ ID NO:18, positions 145-216 of SEQ ID NO:18, positions 7-78 of SEQ ID NO:17, positions 79-150 of SEQ ID NO:17, positions 151-222 of SEQ ID NO:17, positions 202-273 of SEQ ID NO:2, positions 193-264 of SEQ ID NO:4, positions 193-264 of SEQ ID NO:6, positions 184-255 of SEQ ID NO:8, positions 1184-1255 of SEQ ID NO:11, positions 159-230 of SEQ ID NO:14, positions 129-200 of SEQ ID NO:15, and corresponding sequences from different human MUC1 proteins.

[0084] 38. The yeast-MUC1 immunotherapeutic composition of claim 34, wherein each of the CD domains comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of positions 1-72 of SEQ ID NO:18, positions 73-144 of SEQ ID NO:18, and positions 145-216 of SEQ ID NO:18, positions 7-78 of SEQ ID NO:17, positions 79-150 of SEQ ID NO:17, positions 151-222 of SEQ ID NO:17, and positions 129-200 of SEQ ID NO:15.

[0085] 39. The yeast-MUCl immunotherapeutic composition of any one of items 34 to 38, wherein the MUCl antigen has an amino acid sequence that is at least 95% identical to SEQ ID NO:18.

[0086] 40. The yeast-MUCl immunotherapeutic composition of any one of items 34 to 38, wherein the MUCl antigen has the amino acid sequence of SEQ ID NO:18 or an amino acid sequence that is at least 99% identical to SEQ ID NO:18.

[0087] 41. The yeast-MUCl immunotherapeutic composition of any one of items 34 to 38, wherein the MUCl antigen has the amino acid sequence of SEQ ID NO:18.

[0088] 42. The yeast-MUCl immunotherapeutic composition of any one of items 34 to 41, wherein the fusion protein has an amino acid sequence that is at least 95% identical to SEQ ID NO:17.

[0089] 43. The yeast-MUCl immunotherapeutic composition of any one of items 34 to 41, wherein the fusion protein has the amino acid sequence of SEQ ID NO:17 or an amino acid sequence that is at least 99% identical to SEQ ID NO:17.

[0090] 44. The yeast-MUCl immunotherapeutic composition of any one of items 34 to 41, wherein the fusion protein has the amino acid sequence of SEQ ID NO:17.

[0091] 45. The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 44, wherein the yeast vehicle is whole yeast.

[0092] 46. ​​The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 44, wherein the yeast vehicle is heat-inactivated.

[0093] 47. The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 44, wherein the yeast vehicle is from a mutant yeast strain that produces hypoglycosylated proteins compared to a wild-type yeast strain.

[0094] 48. The yeast-MUCl immunotherapeutic composition of any one of items 1 to 44, wherein the MUCl antigen is expressed on the cell wall of the yeast vehicle.

[0095] 49. The yeast-MUCl immunotherapeutic composition of any one of items 1 to 44, wherein the MUCl antigen is expressed in the periplasm or cytoplasm of the yeast vehicle.

[0096] 50. The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 49, wherein the yeast vehicle is from the genus Saccharomyces.

[0097] 51. The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 49, wherein the yeast vehicle is from Saccharomyces cerevisiae.

[0098] 52. The yeast-MUCl immunotherapeutic composition of any one of items 1 to 51, wherein the immunotherapeutic composition is produced by culturing whole yeast expressing the MUCl antigen in a culture medium maintained at a pH level of 5.5 to 8.

[0099] 53. The yeast-MUCl immunotherapeutic composition of claim 52, wherein the culture medium is buffered with a buffer.

[0100] 54. The yeast-MUC1 immunotherapeutic composition of claim 52, wherein the yeast is cultured in a culture medium maintained at a pH level of 6 to 8.

[0101] 55. The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 54, further comprising at least one biological response modifier.

[0102] 56. The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 54, further comprising a pharmaceutically acceptable excipient.

[0103] 57. The Yeast-MUCl immunotherapeutic composition of any one of items 1 to 56, wherein the immunotherapeutic composition is formulated for injection.

[0104] 58. A method of reducing tumor burden, inhibiting tumor growth and / or increasing survival in an individual suffering from a cancer expressing MUCl, comprising administering to the individual the immunotherapeutic composition of any one of items 1 to 57.

[0105] 59. The method of claim 58, wherein MUCl expression is detected in the individual's cancer upon first administration of the composition.

[0106] 60. The method of claim 58, wherein the individual has stage I cancer.

[0107] 61. The method of claim 58, wherein the individual has stage II cancer.

[0108] 62. The method of claim 58, wherein the individual has stage III cancer.

[0109] 63. The method of claim 58, wherein the individual has stage IV cancer.

[0110] 64. The method of any one of items 58 to 63, wherein the individual is receiving or has received another therapy for cancer.

[0111] 65. The method of claim 64, wherein the therapy is chemotherapy.

[0112] 66. The method of claim 64, wherein the therapy is a targeted cancer therapy.

[0113] 67. The method of claim 64, wherein the therapy is radiation therapy.

[0114] 68. The method of claim 64, wherein the therapy is adoptive T cell transfer.

[0115] 69. The method of claim 64, wherein the therapy is administration of one or more additional immunotherapeutic compositions.

[0116] 70. The method of claim 69, wherein the additional immunotherapeutic composition comprises a second cancer antigen, which is or is not a MUCl antigen.

[0117] 71. The method of claim 69, wherein the additional immunotherapeutic composition comprises a yeast vehicle and a second cancer antigen excluding the MUC1 antigen.

[0118] 72. The method of item 70 or 71, wherein the second cancer antigen is selected from the following group: mutant Ras, carcinoembryonic antigen (CEA), brachyura malformation protein, 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, CA-125, 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, BRCA-2, pax3-fkhr, ews-fli-1, HERV-H, HERV-K, TWIST, mesothelin, and NGEP.

[0119] 73. The method of item 70 or 71, wherein the second cancer antigen is selected from the group consisting of mutated Ras, carcinoembryonic antigen (CEA) and brachyura malformation protein.

[0120] 74. The method of claim 69, wherein the additional immunotherapeutic composition is a viral vector vaccine.

[0121] 75. The method of claim 69, wherein the additional immunotherapeutic composition is a dendritic cell / tumor cell fusion.

[0122] 76. The method of any one of items 58 to 75, wherein the method further comprises surgically removing the tumor from the individual.

[0123] 77. A method of preventing or delaying the development of a cancer expressing MUCl comprising administering to an individual the immunotherapeutic composition of any one of items 1 to 57.

[0124] 78. The method of claim 77, wherein the cancer has not been detected in the individual.

[0125] 79. The method of claim 78, wherein the individual has a high risk of developing cancer.

[0126] 80. The method of claim 77, wherein the individual has a precancerous lesion.

[0127] 81. The method of claim 77, wherein the individual has cancer, but cancer cells expressing MUCl have not been detected in the cancer.

[0128] 82. The method of any one of items 58 to 81, wherein the cancer is of epithelial origin.

[0129] 83. The method of any one of items 58 to 81, wherein the cancer is selected from the group consisting of breast cancer, small intestine cancer, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, melanoma, multimyelocytic leukemia (MML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), Burkitt's lymphoma, Hodgkin's lymphoma, cancer of secretory tissue, and metastatic cancer thereof.

[0130] 84. The method of any one of items 77 to 81, wherein the cancer is selected from the group consisting of breast cancer and colon cancer.

[0131] 85. The method of any one of items 58 to 75, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, ovarian cancer, esophageal cancer, and AML.

[0132] 86. The method of any one of items 58 to 75, wherein the cancer is AML, and wherein the yeast-MUCl immunotherapeutic composition is administered to both a donor and a recipient of a bone marrow transplant (BMT) therapy.

[0133] 87. The method of any one of items 58 to 75, wherein the cancer is AML, and wherein the yeast-MUCl immunotherapeutic composition is administered to the individual in conjunction with cytarabine and anthracycline antibiotic therapy.

[0134] 88. Use of the yeast-MUCl immunotherapeutic composition of any one of items 1 to 57 for treating a disease.

[0135] 89. The use of claim 88, wherein the disease is cancer.

[0136] 90. Use of the immunotherapeutic composition of any one of items 1 to 57 to reduce, arrest, reverse or prevent metastatic progression of cancer in an individual suffering from cancer.

[0137] 91. Use of the immunotherapeutic composition of any one of items 1 to 57 to prevent or delay the onset of cancer expressing MUC1.

[0138] 92. Use of a combination of multiple immunotherapeutic compositions for treating cancer, the immunotherapeutic composition comprising:

[0139] a) a first immunotherapeutic composition according to any one of items 1 to 57; and

[0140] b) at least one additional immunotherapeutic composition comprising a yeast vehicle and an antigen other than a MUCl antigen.

[0141] 93. The use of claim 92, wherein the antigen that is not a MUC1 antigen is selected from mutated Ras, carcinoembryonic antigen (CEA), and / or brachyura malformation protein.

[0142] The present invention further relates to the following technical solutions:

[0143] 1. Use of a yeast-MUC1 immunotherapy composition in the preparation of a medicament for treating cancer, wherein the immunotherapy composition comprises:

[0144] a) a yeast vehicle; and

[0145] b) a fusion protein expressed by the yeast vehicle, wherein the fusion protein comprises a MUCl antigen consisting of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 17.

[0146] 2. The use of embodiment 1, wherein the cancer is selected from the group consisting of breast cancer, small intestine cancer, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, melanoma, multimyelocytic leukemia (MML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), Burkitt's lymphoma, Hodgkin's lymphoma, cancer of secretory tissue, and metastatic cancer thereof.

[0147] 3. Use of a yeast-MUC1 immunotherapeutic composition in the preparation of a medicament for reducing, arresting, reversing or preventing metastatic progression of cancer in an individual suffering from cancer, wherein the immunotherapeutic composition comprises:

[0148] a) a yeast vehicle; and

[0149] b) a fusion protein expressed by the yeast vehicle, wherein the fusion protein comprises a MUCl antigen consisting of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 17.

[0150] 4. The use of embodiment 3, wherein the cancer is selected from the group consisting of breast cancer, small intestine cancer, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, melanoma, multimyelocytic leukemia (MML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), Burkitt's lymphoma, Hodgkin's lymphoma, cancer of secretory tissue, and metastatic cancer thereof.

[0151] 5. Use of a yeast-MUC1 immunotherapeutic composition in the preparation of a medicament for preventing or delaying the occurrence of a cancer expressing MUC1, wherein the immunotherapeutic composition comprises:

[0152] a) a yeast vehicle; and

[0153] b) a fusion protein expressed by the yeast vehicle, wherein the fusion protein comprises a MUCl antigen consisting of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 17.

[0154] 6. The use of embodiment 5, wherein the cancer is selected from the group consisting of breast cancer, small intestine cancer, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, melanoma, multimyelocytic leukemia (MML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), Burkitt's lymphoma, Hodgkin's lymphoma, cancer of secretory tissue, and metastatic cancer thereof.

[0155] 7. Use of the yeast-MUC1 immunotherapeutic composition of embodiment 1 in combination with an immunotherapeutic composition for preparing a medicament for treating cancer, wherein the immunotherapeutic composition comprises:

[0156] a) a yeast vehicle; and

[0157] b) a fusion protein expressed by the yeast vehicle, wherein the fusion protein comprises a MUC1 antigen consisting of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 17;

[0158] And wherein the combination further comprises at least one additional immunotherapeutic composition comprising a yeast vehicle and an antigen that is not a MUCl antigen.

[0159] 8. The use of embodiment 7, wherein the antigen that is not a MUC1 antigen is selected from the group consisting of: mutant Ras, carcinoembryonic antigen (CEA), brachyura malformation protein, 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, CA-125, 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, BRCA-2, pax3-fkhr, ews-fli-1, HERV-H, HERV-K, TWIST, mesothelin, and NGEP.

[0160] 9. The use according to embodiment 7, wherein the antigen other than the MUCl antigen is selected from mutated Ras, carcinoembryonic antigen (CEA), and / or Brachyury protein.

[0161] 10. The use of embodiment 7, wherein the cancer is selected from the group consisting of breast cancer, small intestine cancer, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, melanoma, multimyelocytic leukemia (MML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), Burkitt's lymphoma, Hodgkin's lymphoma, cancer of secretory tissue, and metastatic cancer thereof.

[0162] 11. Use of a yeast-MUC1 immunotherapy composition in the preparation of a medicament for activating dendritic cells and inducing the production of cytokines and chemokines, wherein the immunotherapy composition comprises:

[0163] a) a yeast vehicle; and

[0164] b) a fusion protein expressed by the yeast vehicle, wherein the fusion protein comprises a MUCl antigen consisting of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 17.

[0165] 12. The use according to any one of embodiments 1 to 11, wherein the fusion protein comprises a MUC1 antigen consisting of SEQ ID NO: 15 or SEQ ID NO: 14.

[0166] 13. The use according to any one of embodiments 1 to 11, wherein the fusion protein comprises a MUC1 antigen consisting of SEQ ID NO: 18 or SEQ ID NO: 17.

[0167] 14. The use according to any one of embodiments 1 to 11, wherein the yeast vehicle is whole yeast.

[0168] 15. The use according to any one of embodiments 1 to 11, wherein the yeast vehicle is heat-inactivated.

[0169] 16. The use according to any one of embodiments 1 to 11, wherein the yeast vehicle is from Saccharomyces cerevisiae.

[0170] 17. The use according to any one of embodiments 1-11, wherein the immunotherapeutic composition is produced by culturing whole yeast expressing the MUCl antigen in a culture medium maintained at a pH level of 5.5 to 8. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Figure 1A is a schematic diagram showing the structure of the full-length MUCl protein.

[0172] Figure 1B is a schematic diagram showing the structure of the fusion protein expressed in the yeast-based immunotherapeutic composition designated GI-6101.

[0173] Figure 1C is a schematic diagram showing the structure of the fusion protein expressed in the yeast-based immunotherapeutic composition designated GI-6104.

[0174] Figure 2A is a digitized image showing the expression of the MUC1 fusion protein of GI-6101.

[0175] Figure 2B are digitized images showing expression of MUCl fusion proteins from GI-6101 and 6104 before and after deglycosylation.

[0176] Figure 2C is a digitized image showing the expression of the MUC1 fusion protein of GI-6104.

[0177] Figure 3 is a digitized image showing the expression of the MUC1 fusion protein of GI-6105. DETAILED DESCRIPTION OF THE INVENTION

[0178] The present invention generally relates to yeast-based immunotherapeutic compositions and methods for preventing and / or treating cancers characterized by expression or overexpression of mucin-1 (which may be generally referred to herein as "MUC1", and which is also referred to or known as "DF3 antigen" or "HMFG"). The present invention includes the use of yeast-based immunotherapeutic compositions (also referred to as yeast-based immunotherapeutic compositions or products) (also referred to herein as "yeast-MUC1 immunotherapeutic compositions", "yeast-MUC1 immunotherapeutic products" or "yeast-MUC1 immunotherapeutic compositions") comprising a yeast vehicle and a novel MUC1 antigen. The inventors describe herein the construction and production of novel yeast-MUC1 immunotherapeutic products and show that yeast-MUC1 immunotherapy matures human dendritic cells (DCs), increases cytokine production from DCs (which is associated with immune responses expected to be beneficial in tumor treatment), and induces activation of MUC1-specific T cell lines. In summary, the data presented herein show that yeast-MUC1 immunotherapy can be used to induce MUC1-specific cellular immune responses (CD4 + and CD8 + ) and it is expected that yeast-MUC1 immunotherapy can be used to prevent and treat tumors expressing MUC1.

[0179] Yeast-MUCl immunotherapy can be readily adapted to use additional tumor antigens within the same yeast composition, or in combination with other yeast-based immunotherapeutics (sequentially or simultaneously) or other immunotherapeutics and treatments / therapies targeting other tumor antigens for cancer. Thus, the yeast-MUCl immunotherapy can be adapted to the cancer type, cancer stage, cancer grade, antigens expressed by the tumor, and the overall medical status of the individual (i.e., the therapy is easily individualized), and for individuals who already have cancer, its use can be altered as the cancer progresses in the individual to provide maximum efficacy at a variety of tumor stages. Yeast-MUCl immunotherapy provides the opportunity for broad-based preventive and / or therapeutic treatment of a wide range of cancers.

[0180] In fact, yeast-MUC1 immunotherapy can be used in a flexible manner to treat a variety of MUC1-positive cancers by tailoring yeast-MUC1 immunotherapy to the specific role played by this antigen in each type of cancer indication. For example, since MUC1 has been described as an early marker in cancers such as breast cancer or colon cancer, yeast-based immunotherapy can be used prophylactically in patients with MUC1-positive premalignant breast hyperplasia or colon polyps. As another example, since MUC1 has been associated with the epithelial-mesenchymal transition (EMT) pathway and metastatic spread in cancers such as pancreatic cancer, ovarian cancer, and esophageal cancer, yeast-MUC1 immunotherapy can be used as a therapeutic add-on to standard of care treatment in these cancers to promote the arrest of metastatic spread in MUC1-positive stage 3 pancreatic cancer, ovarian cancer, and esophageal cancer. As another example, since MUC1 has been shown to prevent terminal differentiation by reactive oxygen species in acute myeloid leukemia (AML), thereby allowing unrestricted self-regeneration of these cancer cells, yeast-MUC1 immunotherapy can be used as an add-on to standard therapy in MUC1-positive AML to promote apoptosis and prevent unrestricted self-regeneration of malignant cells. A clinical trial using yeast-MUC1 immunotherapy in AML patients is described in the Examples.

[0181] The yeast-MUC1 compositions described herein induce innate immune responses, as well as adaptive immune responses against the target antigen (MUC1), including CD4-dependent TH17 and TH1 T cell responses and antigen-specific CD8 +T cell responses, including cytotoxic T lymphocyte (CTL) responses, all without the use of exogenous adjuvants, cytokines or other immunostimulatory molecules, many of which have toxicity issues. In addition, the yeast-MUCl immunotherapeutic compositions suppress regulatory T cell (Treg) numbers and / or function, thereby enhancing effector T cell responses that might normally be suppressed by the presence of a tumor, for example. Moreover, the antigen-specific, broad-based and potent cellular immune responses elicited by yeast-MUCl immunotherapy are believed to be particularly effective in targeting tumor cells, compared to immunotherapeutic compositions that immunize by generating an antibody response. In fact, a number of studies have shown that when CD8+ T cells that recognize tumor peptides in the context of class I MHC molecules are activated, the immune response is enhanced. + Immunotherapy approaches are enhanced when CTLs are used to target tumor cells. Yeast-MUC1 immunotherapy is highly adept at activating antigen-presenting cells and has a unique ability to cross-prime immune responses, generating CD8 + CTL responses, even in the face of what may be an inhibitory environment. Since this type of immunotherapy exploits the natural ability of antigen presenting cells to present relevant immunogens, it is not necessary to know the exact identity of the CTL epitopes or class II MHC epitopes of MUC1 in order to produce an effective immunotherapy in accordance with the present invention. In fact, multiple CD4+ T cells can be targeted in a single yeast-MUC1 immunotherapeutic composition. + and CD8 + T cell epitopes, and therefore the yeast-MUC1 immunotherapy of the present invention is not limited to the use of short peptides. Indeed, the use of longer polypeptides and fusion proteins containing multiple domains of the target antigen is effective in these compositions. Thus, by using yeast-MUC1 immunotherapy, the use of algorithms and complex formulas to identify putative T cell epitopes is avoided.

[0182] Yeast-MUCl can be effectively utilized in immunization protocols (prophylactic or therapeutic) without the use of exogenous adjuvants, immunostimulatory agents or molecules, co-stimulatory assays, or cytokines, although such agents can be included when desired. Moreover, Yeast-MUCl immunotherapy can be administered repeatedly without loss of efficacy, which can be problematic for other types of immunotherapy.

[0183] Compositions of the present invention

[0184] One embodiment of the present invention relates to a yeast-based immunotherapeutic composition that can be used to prevent and / or treat cancers characterized by MUC1 expression or overexpression (including cancers that may not initially contain cells expressing detectable MUC1, but may or will contain cells expressing MUC1 at later stages of cancer progression). The composition is a yeast-MUC1 immunotherapeutic composition that comprises: (a) a yeast vehicle; and (b) a cancer antigen comprising one or more MUC1 antigens and / or immunogenic domains thereof. The MUC1 antigens or immunogenic domains thereof are most typically expressed as recombinant proteins by a yeast vehicle (e.g., by whole yeast or yeast spheroplasts, which optionally can be further processed into yeast cytoplasts, yeast ghosts, or yeast membrane extracts or fractions thereof), although as one embodiment of the present invention, one or more MUC1 antigens are loaded into a yeast vehicle or complexed, attached, mixed, or administered together with a yeast vehicle described herein to form a composition of the present invention.

[0185] A "yeast-MUC1 immunotherapeutic composition" is a specific type of "yeast-based immunotherapeutic composition" that contains at least one MUC1 antigen or an immunogenic domain thereof. The term "yeast-based immunotherapeutic composition" may be used interchangeably with "yeast-based immunotherapeutic product," "yeast-based immunotherapy composition," "yeast-based composition," "yeast-based immunotherapeutic," "yeast-based vaccine," or derivatives of these terms. 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, a yeast-based immunotherapeutic composition refers to a composition that comprises a yeast vehicle component and elicits an immune response sufficient to achieve at least one therapeutic benefit in a subject. More specifically, a yeast-based immunotherapeutic composition is a composition that comprises a yeast vehicle component and, typically, an antigen component, and is capable of eliciting or inducing an immune response, such as a cellular immune response, including, but not limited to, a T cell-mediated cellular immune response. In one aspect, a yeast-based immunotherapeutic composition useful in the present invention is capable of inducing CD8 + and / or CD4 + T cell mediated immune response, and in one aspect, CD8 + and CD4 + T cell-mediated immune response, especially against target antigens (e.g. cancer antigens). CD4 + The immune response may include a TH1 immune response, a TH2 immune response, a TH17 immune response, or any combination thereof. Yeast-based immunotherapeutics are particularly capable of generating TH1 and TH17 responses. +The immune response may include a cytotoxic T lymphocyte (CTL) response, and yeast-based immunotherapeutics can generate such responses. In one aspect, yeast-based immunotherapeutic compositions regulate the number and / or functionality of regulatory T cells (Tregs) in a subject. Yeast-based immunotherapies can also be modified to promote one type of response relative to another, such as by adding cytokines, antibodies, and / or regulating the yeast manufacturing process. Optionally, yeast-based immunotherapeutic compositions can elicit a humoral immune response.

[0186] The yeast-MUCl immunotherapeutic compositions of the invention may be "prophylactic" or "therapeutic." When provided prophylactically, the compositions of the invention are provided before a MUC1-expressing cancer develops or is detected to develop, with the goal of preventing, inhibiting, or delaying the development of a MUC1-expressing tumor; and / or preventing, inhibiting, or delaying the metastasis of such a tumor and / or generally preventing or inhibiting the progression of cancer in an individual. As discussed herein, MUC1 is expressed in several cancers. Thus, the prophylactic compositions may be administered to individuals who appear to be free of cancer (healthy or normal individuals), individuals in the precancerous stage (premalignant lesions), and individuals with cancer in which MUC1 has not yet been detected (i.e., before the tumor cells in the cancer express MUC1). Individuals at high risk for developing cancer, particularly cancers that are generally associated with MUC1 expression and / or metastasis, may be treated prophylactically with the compositions of the invention. When provided therapeutically, the immunotherapeutic compositions are provided to an individual with a MUC1-expressing cancer, with the goal of ameliorating the cancer, such as by reducing the tumor burden in the individual; inhibiting tumor growth in the individual; increasing survival in the individual; and / or preventing, inhibiting, reversing, or delaying the progression of cancer in the individual.

[0187] Generally speaking, a yeast-MUC1 immunotherapy composition comprises a yeast vehicle and at least one cancer antigen comprising a MUC1 antigen or an immunogenic domain thereof, wherein the cancer antigen is expressed by, attached to, loaded into, or mixed with the yeast vehicle. In some embodiments, the cancer antigen, the MUC1 antigen, or an immunogenic domain thereof is provided as a fusion protein. Several MUC1 proteins and fusion proteins suitable for use in the compositions and methods of the present invention are described below. In some embodiments, the cancer antigen and the MUC1 antigen are the same element. In some embodiments, the cancer antigen comprises other antigens, including other cancer antigens in addition to the MUC1 antigen. In one aspect of the present invention, a fusion protein useful as a cancer antigen may comprise two or more antigens, such as a MUC1 antigen and another cancer antigen that is not a MUC1 antigen, or two different MUC1 antigens. In one aspect, the fusion protein may comprise two or more immunogenic domains of one or more antigens, such as two or more immunogenic domains of a MUC1 antigen, or two or more epitopes of one or more antigens, such as two or more epitopes of a MUC1 antigen.

[0188] According to the present invention, the yeast vehicle for use in the yeast-MUCl immunotherapeutic composition is any yeast cell (e.g., whole cell or intact cell) or derivative thereof (see below) that can be used in the composition (e.g., therapeutic or prophylactic composition) of the present invention in conjunction with one or more antigens, immunogenic domains thereof, or epitopes thereof. Thus, the yeast vehicle may include, but is not limited to, a live intact (whole) yeast microorganism (i.e., a yeast cell with all its components including the cell wall), a killed (dead) or inactivated intact yeast microorganism, or a derivative of intact yeast, including: yeast spheroplasts (i.e., yeast cells lacking a cell wall), yeast cytoplasts (i.e., yeast cells lacking a cell wall and a nucleus), yeast ghosts (i.e., yeast cells lacking a cell wall, a nucleus, and a cytoplasm), a subcellular yeast membrane extract or fraction thereof (also referred to as a yeast membrane particle or formerly as a subcellular yeast particle), any other yeast particle, or a yeast cell wall preparation.

[0189] Yeast spheroplasts are typically produced by enzymatic digestion of the yeast cell wall. Such methods are described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674, incorporated herein by reference in its entirety.

[0190] Yeast cytoplasts are usually generated by enucleation of yeast cells. Such methods are described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55, which is incorporated herein by reference in its entirety.

[0191] Yeast ghosts are usually produced by resealing permeabilized or lysed cells and may, but need not, contain at least some of the organelles of the cell. Such methods are described, for example, in Franzusoff et al., 1983, J. Biol. Chem. 258, 3608-3614 and Bussey et al., 1979, Biochim. Biophys. Acta 553, 185-196, each of which is incorporated herein by reference in its entirety.

[0192] Yeast membrane particles (subcellular yeast membrane extracts or fractions thereof) refer to yeast membranes lacking a native nucleus or cytoplasm. The particles can be of any size, including sizes ranging from native yeast membranes to microparticles produced by ultrasonic treatment or other membrane disruption methods known to those skilled in the art followed by resealing. Methods for producing subcellular yeast membrane extracts are described in, for example, Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674. Yeast membrane particle fractions can also be used, which contain yeast membrane portions and the antigen or other proteins of interest (when the antigen or other proteins are recombinantly expressed by yeast before preparing yeast membrane particles). Antigens or other proteins of interest can be carried on the inside of the membrane, on any surface of the membrane, or in combination thereof (i.e., the protein can be on the inside and outside of the membrane and / or across the membrane of the yeast membrane particles). In one embodiment, the yeast membrane particles are recombinant yeast membrane particles, which can be intact, damaged, or damaged and resealed yeast membranes, containing at least one desired antigen or other protein of interest on the surface of the membrane or at least partially embedded in the membrane.

[0193] An example of a yeast cell wall preparation is an isolated preparation of yeast cell walls carrying an antigen on its surface or at least partially embedded within the cell wall, thereby stimulating a desired immune response against a disease target when the yeast cell wall preparation is administered to an animal.

[0194] Any yeast strain can be used to produce the yeast vehicle of the present invention. Yeast is a unicellular microorganism belonging to one of the following three classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. One consideration for selecting a type of yeast for use as an immunomodulator is the pathogenicity of the yeast. In one embodiment, the yeast is a non-pathogenic strain such as Saccharomyces cerevisiae. The selection of non-pathogenic yeast strains minimizes any adverse effects of the individual to which the yeast vehicle is applied. However, if the pathogenicity of the yeast can be offset by any means known to those skilled in the art (e.g., mutant strains), pathogenic yeast can be used. According to one aspect of the present invention, non-pathogenic yeast strains are used.

[0195] The genus of yeast strains that can be used in the present invention include, but are not limited to, Saccharomyces, Candida (which can be pathogenic), Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces, and Yarrowia. In one aspect, the genus of yeast is selected from Saccharomyces, Candida, Hansenula, Pichia, or Schizosaccharomyces, and in one aspect, the genus Saccharomyces is used. Species of yeast strains that can be used in the present invention include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica. lipolytica). It is to be appreciated that many of these species include multiple subspecies, types, subtypes, etc., which are intended to be included in the aforementioned species. In one aspect, yeast species used in the present invention include Saccharomyces cerevisiae, Candida albicans, Hansenula polymorpha, Pichia pastoris, and Schizosaccharomyces pombe. Saccharomyces cerevisiae can be used because it is easy to handle and is "generally recognized as safe" or "GRAS" for use as a food additive (GRAS, rule 62 FR18938 proposed by FDA, April 17, 1997).One embodiment of the present invention is a yeast strain capable of replicating plasmids to a particularly high copy number, such as a Saccharomyces cerevisiae cir° strain. The Saccharomyces cerevisiae strain is one such strain that can support expression vectors that allow for high-level expression of one or more target antigens and / or antigen fusion proteins and / or other proteins. Another yeast strain that can be used in the present invention is Saccharomyces cerevisiae W303α. In addition, any mutant yeast strain can be used in the present invention, including those that exhibit reduced post-translational modifications of the expressed target antigen or other protein, such as mutations in enzymes that expand N-linked glycosylation. In one aspect of the present invention, a yeast-MUC1 immunotherapeutic composition is produced using a mutant yeast strain that produces a MUC1 antigen as a protein that is hypoglycosylated compared to the same antigen produced by a wild-type strain (with normal glycosylation). Such MUC1 antigens may be more similar to the MU1 antigen expressed by tumor cells, which can then be processed by antigen presenting cells into unique T cell epitopes, thus enhancing specific anti-tumor responses.

[0196] The yeast-MUCl immunotherapeutic composition of the present invention comprises at least one cancer antigen comprising a MUCl antigen. According to the present invention, the term "antigen" is generally used herein to refer to any portion of a protein (e.g., a peptide, a partial protein, a full-length protein), wherein the protein is naturally present or synthetically obtained or designed for a cell composition (whole cell, cell lysate, or disrupted cell), for an organism (whole organism, lysate, or disrupted cell), or for a carbohydrate, or other molecule, or a portion thereof. Antigens can elicit an antigen-specific immune response (e.g., a humoral and / or cell-mediated immune response) by immune system elements (e.g., T cells, antibodies) directed against the same or similar antigens encountered in vitro, in vivo, or ex vivo.

[0197] Antigens can be as small as a single epitope, a single immunogenic domain, or larger, and can contain multiple epitopes or immunogenic domains. Thus, the size of an antigen can be as small as about 8-11 amino acids (i.e., a peptide) and as large as: a domain of a protein, a full-length protein, a multimer, a fusion protein, a chimeric protein, a whole cell, a whole organism, or any portion thereof (e.g., a protein fragment (polypeptide), a lysate of a whole cell, or an extract of a microorganism). Antigens that can be used in the yeast-MUC1 immunotherapeutics of the present invention are peptides, polypeptides, protein domains, protein subunits, full-length proteins, multimers, fusion proteins, and chimeric proteins. In addition, the antigen can contain carbohydrates, which can be loaded into the yeast vehicle or composition of the present invention. It will be appreciated that in some embodiments (e.g., when the antigen is expressed by a yeast vehicle from a recombinant nucleic acid molecule), the antigen is a protein (including fragments, domains, subunits, and full-length proteins), a fusion protein, a chimeric protein, or a fragment thereof, rather than a complete cell or microorganism. For expression in yeast, in one embodiment, if the antigen is a complete protein to be expressed by yeast, the antigen has a minimum size that can be recombinantly expressed in yeast (in other words, the protein expressed by yeast that comprises or consists of the antigen is preferably at least 25 amino acids in length), and is typically at least or more than 25 amino acids, or at least or more than 26 amino acids, at least or more than 27 amino acids, at least or more than 28 amino acids, at least or more than 29 amino acids, at least or more than 30 amino acids, at least or more than 31 amino acids, at least or more than 32 amino acids, at least or more than 33 amino acids, at least or more than 34 amino acids, at least or more than 35 amino acids, at least or more than 36 amino acids, at least or more than 37 amino acids, at least or more than 38 amino acids, at least or more than 39 amino acids, at least or more than 40 amino acids, at least or more than 41 amino acids, at least or more than 42 amino acids, at least or more than 43 amino acids, at least or more than 44 amino acids, at least or more than 45 amino acids, at least or more than 46 amino acids, at least or more than 47 amino acids, at least or more than 48 amino acids, at least or more than 49 amino acids, at least or more than 50 amino acids, at least or more than 51 amino acids, at least or more than 52 amino acids, at least or more than 53 amino acids, at least or more than 54 amino acids, at least or more than 55 amino acids, at least or more than 56 amino acids, at least or more than 57 amino acids, at least or more than 58 amino acids, at least or more than 59 amino acids, at least or more than 60 amino acids, at least or more than 61 amino acids, at least or more than 62 amino acids, at least or more than 63 amino acids, at least or more than 64 amino acids More than 39 amino acids, at least or more than 40 amino acids, at least or more than 41 amino acids, at least or more than 42 amino acids, at least or more than 43 amino acids, at least or more than 44 amino acids, at least or more than 45 amino acids, at least or more than 46 amino acids, at least or more than 47 amino acids, at least or more than 48 amino acids, at least or more than 49 amino acids, or at least or more than 50 amino acids in length, or at least 25-50 amino acids in length, at least 30-50 amino acids in length, or at least 35-50 amino acids in length, or at least 40-50 amino acids in length, or at least 45-50 amino acids in length, although smaller proteins can be expressed, and much larger proteins (e.g., hundreds of amino acids in length or even thousands of amino acids in length) can be expressed. In one aspect, full-length proteins or domains of proteins can be expressed, which lack 1 to 20 amino acids at the N and / or C termini. Fusion proteins and chimeric proteins are also antigens that can be expressed in the present invention. A "target antigen" is an antigen that is specifically targeted by the immunotherapeutic compositions of the invention (ie, an antigen, typically a native antigen, against which it is desired to elicit an immune response).A "cancer antigen" is an antigen that contains at least one antigen associated with cancer (e.g., an antigen expressed by a tumor cell), such that targeting the antigen also targets the tumor cell and / or cancer. A cancer antigen may contain one or more antigens from one or more proteins (including one or more tumor-associated proteins). A "MUC1 antigen" is an antigen that is derived, designed, or generated from the MUC1 protein (including MUC1-N, MUC1-C, or both MUC1-N and MUC1-C).

[0198] When referring to the stimulation of an immune response, the term "immunogen" is a subset of the term "antigen" and, therefore, in some cases, may be used interchangeably with the term "antigen". As used herein, an immunogen describes an antigen that elicits a humoral and / or cell-mediated immune response (i.e., is immunogenic), such that administration of the immunogen to an individual results in an antigen-specific immune response to the same or similar antigen encountered by the individual's immune system. In one embodiment, the immunogen elicits a cell-mediated immune response, including CD4 + T cell responses (e.g. TH1, TH2 and / or TH17) and / or CD8 + T cell response (eg, CTL response).

[0199] An "immunogenic domain" or "immunological domain" of a given antigen can be any portion, fragment or epitope of the antigen that contains at least one epitope (e.g., a peptide fragment or subunit or an antibody epitope or other conformational epitope) that can act as an immunogen when administered to an animal. Thus, an immunogenic domain is larger than a single amino acid and is at least large enough to contain at least one epitope that can act as an immunogen. For example, a single protein may contain multiple different immunogenic domains. Immunogenic domains need not be linear sequences within a protein, as in the case of a humoral immune response, where conformational domains are encompassed.

[0200] Epitope is defined herein as a single immunogenic site within a given antigen, which is sufficient to induce an immune response when provided to the immune system in the context of suitable costimulatory signals and / or activated cells of the immune system. In other words, an epitope is an antigenic portion recognized by an immune system component, which may also be referred to as an antigenic determinant. Those skilled in the art will recognize that T cell epitopes are different from B cell or antibody epitopes in size and composition, and that epitopes presented via class I MHC approaches are different in size and structural attributes from epitopes presented via class II MHC approaches. For example, T cell epitopes presented by class I MHC molecules are generally 8 to 11 amino acids in length, while epitopes presented by class II MHC molecules are less limited in length and may be up to 25 amino acids or longer. In addition, T cell epitopes have predicted structural features according to the specific MHC molecules to which the epitope is bound. An epitope may be a linear sequence epitope or a conformational epitope (conserved binding region). Most antibodies recognize conformational epitopes.

[0201] MUC1 (which may also be referred to as "mucin-1" and also "DF3 antigen" or "HMFG1") is a large glycoprotein expressed at the basal level by most epithelial secretory tissues and at high levels by malignancies of epithelial origin. MUC1 is most commonly found as a polymorphic, type I transmembrane protein with a large extracellular domain (also referred to as the MUC1-N subunit) that contains a variable number of tandem repeats (VNTRs; typically 20 to 125 repeats) that are heavily glycosylated via O-linkages. The MUC1 protein is encoded as a single transcript and then processed post-translationally into subunits, referred to as MUC1-N and MUC1-C, or α and β subunits, respectively, which then form a heterodimeric protein through strong non-covalent interactions of the two subunits. MUC1 is cleaved into its N- and C-subunits within the "sea urchin sperm protein, enterokinase, and agrin" (SEA) domain, a highly conserved protein domain named based on its initial identification in sperm proteins, enterokinase, and agrin, and found in many heavily glycosylated mucin-like proteins that are often membrane-bound. The MUC1 protein is cleaved between glycine and serine residues present in the sequence GSVVV (e.g., positions 1097-1101 of SEQ ID NO: 11) within the SEA domain (Lillehoj et al., 2003, Biochem. Biophys. Res. Commun. 307:743-749; Parry et al., 2001, Biochem. Biophys. Res. Commun. 283:715-720; Wreschner et al., 2002, Protein Sci. 11:698-706).

[0202] The MUC1-C subunit contains an extracellular domain (ED) that is glycosylated and binds to a galectin-3 ligand, which in turn acts as a bridge that physically associates MUC1 with the epidermal growth factor receptor (EGFR) and possibly other receptor tyrosine kinases. MUC1-C also contains a transmembrane (TM) domain and a cytoplasmic domain (CD) containing several tyrosine residues that, when phosphorylated, can serve as a binding motif for proteins with SH2 domains (for a detailed discussion of MUC1 proteins and known and putative functions, see Kufe, 2008, Cancer Biol. & Ther. 7: 81-84). Alternative splice variants of MUC1 (e.g., called MUC1 / Y and MUC1 / X) are "short" forms of MUC1 that lack most of MUC1-N (including the larger VNTR region), but contain ED, TM, and CD regions as well as portions of the SEA domain and N-terminal region signal sequence region. Cleavage within the SEA domain may not occur in these short forms.

[0203] The isolation and sequencing of DNA and cDNA encoding human MUC1 have been reported (see, e.g., Siddiqui et al., 1998, PNAS 85:2320-2323; Abe and Kufe, 1993, PNAS 90:282-286; Hareuveni et al., 1990, Eur. J. Biochem. 189(3)475-486; Gendler et al., 1990, J. Biol. Chem. 265(25)15286-15293; Lan et al., 1990, J. Biol. Chem 265(25)15294-15299; Tsarfaty et al., 1990, Genet. 93(2)313-318; Lancaster, 1990, Biochem. Biophys. Res. Commun. 173(3)1019-1029). An example of a full-length human MUCl precursor protein containing the MUCl-N and MUCl-C regions is described in SwissProt Accession No. P15941.3 (GI:296439295) and is represented herein by SEQ ID NO:11. Ten different MUCl isoforms can be created from the gene encoding SEQ ID NO:11 by alternative transcript splicing. For example, the isoform referred to as MUC1 / Y lacks positions 54-105 of SEQ ID NO:11. Various other isoforms are described in the database description of the protein.

[0204] To illustrate the identification of domains within a MUCl protein (which can be readily applied to any human MUCl protein as well as other mammalian MUCl proteins), the following domains can be readily identified in SEQ ID NO: 11. The MUCl signal sequence, also referred to herein as the leader sequence, is located at approximately positions 1-23 of SEQ ID NO: 11 (the MUCl signal sequence is identified in some MUCl variants as being longer and may include additional amino acids such as positions 1-32). The MUCl-N subunit or α subunit comprises approximately positions 24-1097 of SEQ ID NO: 11, while the MUCl-C subunit or β subunit comprises approximately positions 1098-1255 of SEQ ID NO: 11.

[0205] In the MUCl-N subunit, a VNTR (variable number of tandem repeats) domain can be found, which contains multiple repeats within this particular protein, including a region from approximately positions 126-965, which contains 42 20 amino acid repeats of the sequence PAPGSTAPPAHGVTSAPDTR (e.g., positions 126-145 of SEQ ID NO: 11), which is a commonly recognized VNTR sequence (see also SEQ ID NO: 12 below, which indicates common polymorphisms within this sequence). Since these are repeated sequences, counting can be started at any of the 20 amino acids in a VNTR, and then the numbering can be restarted at the repeat of the first amino acid. More specifically, since a single VNTR domain is a sequence of approximately 20 amino acids preceded and / or followed by another identical, nearly identical or homologous 20 amino acid sequence, which may be within a large number of such repeated sequences, for purposes of describing a single VNTR within a VNTR region, "position 1" of a given VNTR can be considered to be any of the 20 amino acids in the VNTR, and the preceding and following flanking amino acids are then numbered accordingly, wherein the amino acid upstream (preceding) of position 1 is the last amino acid of the preceding VNTR (position 20) or the last amino acid of the sequence attached to the VNTR (if such preceding sequence is not also a VNTR), and the amino acid downstream of position 1 is position 2 of the VNTR, followed by position 3, and so on, until the sequence is repeated in the next VNTR.

[0206] Positions 61-1120 of SEQ ID NO: 11 include the VNTR regions discussed above, plus additional regions referred to as “repeat regions.” For example, positions 81-100, positions 101-120, positions 121-140, positions 141-160, positions 161-180, positions 181-200, positions 201-220, positions 221-240, positions 241-260, positions 261-180, positions 281-300, and so forth, in increments of 20 amino acids up to positions 1001-1120 of SEQ ID NO: 11, represent repeat regions in the protein.

[0207] In the full-length MUCl protein represented by SEQ ID NO: 11 (before cleavage into subunits), the SEA domain spans positions 1034-1152 of SEQ ID NO: 11. Cleavage of the SEA domain at amino acids 1097 to 1098 of SEQ ID NO: 11 generates the MUCl-C domain. Within the MUCl-C domain, the extracellular domain (ED) is found at approximately positions 1098-1155 of SEQ ID NO: 11; the transmembrane (TM) domain is found at approximately positions 1156-1183 of SEQ ID NO: 11; and the cytoplasmic domain (CD, also known as the cytoplasmic tail) is found at approximately positions 1184-1255 of SEQ ID NO: 11.

[0208] The number of VNTRs in a given MUCl-N subunit is highly polymorphic and can vary, for example, from 20 to 125 repeats. The tandemly repeated twenty-peptide often has polymorphisms in one or more of three positions (positions 9, 18, and 19 of SEQ ID NO: 12): PAPGSTAP[P / A / Q / T]AHGVTSAP[DT / ES]R (SEQ ID NO: 12, bracketed regions indicate common polymorphisms), wherein the polymorphisms at positions 18 and 19 of SEQ ID NO: 12 occur with a preference of DT>ES, and wherein the single substitution at position 9 occurs with the following preference: P>A, P>Q, and P>T. The most frequent substitution, DT>ES, occurs in up to 50% of the repeats.

[0209] A variety of transcript variants of MUC1 are known, but the MUC1 subunits, domains or regions described in the exemplary SEQ ID NO: 11 above can be easily identified in the variants, so that MUC1 antigens useful in the present invention can be designed or produced based on a given MUC1 sequence or a corresponding sequence from another MUC1 protein. For example, a nucleotide sequence encoding a human MUC1 protein is represented herein by SEQ ID NO: 1, which corresponds to Accession number NM_002456.4 (GI: 65301116). SEQ ID NO: 1 encodes the 273 amino acid human MUC1 protein (transcript variant 1, also known as MUC1 / ZD), the amino acid sequence of which is represented herein as SEQ ID NO: 2 (also found in Accession No. NP_002447.4; GI:65301117). Within SEQ ID NO:2, the following domains are present: signal sequence (positions 1-27 of SEQ ID NO:2); SEA domain (positions 55-170 of SEQ ID NO:2); ED (positions 116-173 of SEQ ID NO:2); TM domain (positions 174-201 of SEQ ID NO:2); and CD (positions 202-273 of SEQ ID NO:2). The proteolytic cleavage site within the SEA domain that cleaves the ED domain from the N-terminal portion of the SEA domain is at positions 115 to 116 of SEQ ID NO:2. This transcript variant does not contain the VNTR region as shown in SEQ ID NO:11.

[0210] Another nucleotide sequence encoding another human MUCl protein is represented herein by SEQ ID NO:3, which corresponds to Accession number NM_001018016.1 (GI: 67189006). SEQ ID NO: 3 encodes the 264 amino acid human MUC1 protein (transcript variant 2, also known as "MUC1 / Y"), the amino acid sequence of which is represented herein as SEQ ID NO: 4 (also found in Accession No. NP_001018016.1; GI:67189007). Within SEQ ID NO:4, the following domains are present: signal sequence (positions 1-32 of SEQ ID NO:4); SEA domain (positions 45-161 of SEQ ID NO:4); ED (107-164 of SEQ ID NO:4); TM domain (positions 165-192 of SEQ ID NO:4); and CD (positions 193-264 of SEQ ID NO:4). The proteolytic cleavage site within the SEA domain that cleaves the ED domain from the N-terminal portion of the SEA domain is at positions 106 to 107 of SEQ ID NO:4. This transcript variant does not contain the VNTR region as shown in SEQ ID NO:11.

[0211] Another nucleotide sequence encoding another human MUCl protein is represented herein by SEQ ID NO:5, which corresponds to Accession No. AY327587.1 (GI: 33150003). SEQ ID NO: 5 encodes the 264 amino acid human MUC1 protein (transcript variant 2, also known as "MUC1 / Y"), the amino acid sequence of which is represented herein as SEQ ID NO: 6 (also found in Accession number AAP97018.1 (GI:33150004)). Within SEQ ID NO:6, the following domains are present: signal sequence (positions 1-32 of SEQ ID NO:6); SEA domain (positions 45-161 of SEQ ID NO:6); ED (positions 107 to 164 of SEQ ID NO:6); TM domain (positions 165-192 of SEQ ID NO:6); and CD (positions 193 to 264 of SEQ ID NO:6). The proteolytic cleavage site within the SEA domain that cleaves the ED domain from the N-terminal portion of the SEA domain is at positions 106 to 107 of SEQ ID NO:6. This transcript variant does not contain the VNTR region as shown in SEQ ID NO:11. SEQ ID NO:6 is 99% identical to SEQ ID NO:4, exemplifying the high degree of homology among MUC1 sequences from different sources.

[0212] Another nucleotide sequence encoding another human MUCl protein is represented herein by SEQ ID NO:7, which corresponds to Accession number NM_001018017 (GI: 324120954). SEQ ID NO: 7 encodes the 255 amino acid human MUCl protein (transcript variant 3), the amino acid sequence of which is represented herein as SEQ ID NO: 8 (also found in Accession No. NP_001018017.1; GI:67189069). Within SEQ ID NO:8, the following domains are present: signal sequence (positions 1-27 of SEQ ID NO:8); SEA domain (positions 36-152 of SEQ ID NO:8); ED (positions 98-155 of SEQ ID NO:8); TM domain (positions 156-183 of SEQ ID NO:8); and CD (positions 184-255 of SEQ ID NO:8). The proteolytic cleavage site within the SEA domain that cuts off the ED domain from the N-terminal portion of the SEA domain is at positions 97 to 98 of SEQ ID NO:6. This transcript variant does not contain the VNTR region as shown in SEQ ID NO:11.

[0213] Human MUC1 has a high degree of homology with MUC1 from other animal species, and therefore, it is expected that domains within a given MUC1 protein can be identified based on sequence comparison. In addition, specific sequences of MUC1 from other animal species, particularly mammalian species, can be utilized in the preparation of the yeast-MUC1 immunotherapeutic compositions of the present invention, especially when the sequences are identical or substantially homologous and when the sequences elicit an effective immune response against the target antigen (e.g., native MUC1 expressed by tumor cells). For example, the mouse MUC1 protein is represented herein by the amino acid sequence of SEQ ID NO:9. SEQ ID NO:9 corresponds to Accession number NM_013605 (GI:7305292). SEQ ID NO:9 encodes the 631 amino acid mouse MUC1 protein, the amino acid sequence of which is represented herein as SEQ ID NO:10 (also found in Accession No. NP_038633; GI:7305293). Within SEQ ID NO:10, the following domains are present: a signal sequence (approximately positions 1-20 of SEQ ID NO:10); a VNTR (identifiable in positions 21-425 of SEQ ID NO:10); a SEA domain (positions 426-528 of SEQ ID NO:10); an ED (positions 475-536 of SEQ ID NO:10); a TM domain (positions 531-559 of SEQ ID NO:10); and a CD (positions 560-631 of SEQ ID NO:10). The proteolytic cleavage site within the SEA domain that cleaves the ED domain from the N-terminal portion of the SEA domain is at positions 474 to 475 of SEQ ID NO:10. To illustrate the level of conservation of MUC1 sequences within domains, the murine MUC1 SEA domain of SEQ ID NO: 10 is 62% identical and 68% homologous or positive (as defined by BLAST) to the human MUC1 SEA domain of SEQ ID NO: 11. The murine MUC1 ED of SEQ ID NO: 10 is 56% identical and 73% homologous to the human MUC1 ED of SEQ ID NO: 11. The murine MUC1 TM domain of SEQ ID NO: 10 is 89% identical and 93% homologous to the human MUC1 TM domain of SEQ ID NO: 11. The murine MUC1 CD of SEQ ID NO: 10 is 88% identical and 88% homologous to the human MUC1 CD of SEQ ID NO: 11.

[0214] Human MUC1, including the human MUC1 proteins and MUC1 antigens described herein, contains various CD4 + and CD8 +T cell epitopes. Such T cell epitopes have been described, for example, in U.S. Patent No. 6,546,643; U.S. Patent No. 7,118,738; U.S. Patent No. 7,342,094; U.S. Patent No. 7,696,306; and U.S. Patent Application Publication No. 2008 / 0063653.

[0215] In one embodiment of the invention, the MUC1 antigen comprises or consists of a fusion protein comprising multiple domains of the MUC1 protein. In one embodiment, the MUC1 antigen is obtained or designed from a portion of the MUC1-C subunit. In one embodiment, the MUC1 antigen is obtained or designed from a portion of the MUC1-N subunit. In one embodiment, the MUC1 antigen is obtained or designed from a portion of both the MUC1-C and MUC1-N subunits.

[0216] In one embodiment of the invention, a fusion protein useful in a yeast-based immunotherapeutic composition of the invention comprises at least two, at least three, at least four, or at least five of the following MUCl antigens, arranged in any order within the fusion protein, and any of which may be repeated two or more times within the fusion protein (e.g., a combination of two or more CD segments): (1) a MUCl signal sequence; (2) at least a portion of a MUCl SEA domain and / or at least a portion of a MUCl extracellular domain (ED) or an immunogenic domain thereof, which in one aspect may comprise a majority or complete ED plus one or more flanking amino acids flanking the SEA domain; (3) at least two VNTR domains; (4) at least one MUCl transmembrane domain or an immunogenic domain thereof; and (5) at least one MUCl cytoplasmic domain (CD) or an immunogenic domain thereof. Such a fusion protein does not comprise a full-length MUCl protein (i.e., it does not comprise a complete MUCl-N subunit and a complete MUCl-C subunit), and such a fusion protein does not comprise a full-length MUCl-N subunit. In one aspect, such fusion proteins do not comprise a full-length MUCl-C subunit.In one aspect, the segments (eg, MUCl protein or domains, including immunogenic domains) of the fusion protein are arranged in an order different from that in which the segments would be present in a native or wild-type MUCl protein.

[0217] The MUC1 signal sequence (or leader sequence) useful in the fusion proteins described above or elsewhere herein can be a signal sequence from any MUC1 protein, and in one aspect, is from a human MUC1 protein. In one aspect of the invention, the MUC1 signal sequence used in the fusion proteins of the invention has an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of positions 1-27 of SEQ ID NO:2, positions 1-32 of SEQ ID NO:4, positions 1-32 of SEQ ID NO:6, positions 1-27 of SEQ ID NO:8, positions 1-23 of SEQ ID NO:11, positions 1-30 of SEQ ID NO:14, a corresponding sequence from a different MUC1 protein, such as another human MUC1 protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences. In one aspect of the invention, the MUC1 signal sequence is placed at the N-terminus of a fusion protein useful in the invention. In one aspect of the invention, a non-MUCl sequence is used in place of the MUCl signal sequence, such as any of the N-terminal synthetic peptides and yeast-derived peptides described below for use with the fusion proteins of the invention.

[0218] The MUCl sea urchin sperm protein, enterokinase and agrin (SEA) domain that can be used in the fusion proteins described above or elsewhere herein can be a SEA domain or a portion thereof that contains at least one immunogenic domain from any MUCl protein, and in one aspect, is from a human MUCl protein. In one aspect of the invention, a portion of a MUCl SEA domain that can be used in a fusion protein of the invention contains at least an amino acid sequence from the MUCl extracellular domain (ED), but can exclude sequences upstream of the ED domain. In one aspect of the invention, a MUC1 SEA domain useful for a fusion protein of the invention has an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of positions 55-170 or positions 116-170 of SEQ ID NO:2, or at least one immunogenic domain thereof, positions 45-161 or positions 107-161 of SEQ ID NO:4, or at least one immunogenic domain thereof, positions 45-161 or positions 107-161 of SEQ ID NO:6, or at least one immunogenic domain thereof, positions 36-152 or positions 98-152 of SEQ ID NO:8, positions 1034-1152 or positions 1098-1152 of SEQ ID NO:11, or at least one immunogenic domain thereof, positions 31-86 of SEQ ID NO:14, or at least one immunogenic domain thereof. Positions 1-56 of NO:15, or at least one immunogenic domain thereof, are from the corresponding sequence of a different MUCl protein, such as another human MUCl protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences.

[0219] The MUCl extracellular domain (ED) useful in the fusion proteins described above or elsewhere herein can be an ED or a portion thereof comprising at least one immunogenic domain from any MUCl protein, and in one aspect, is from a human MUCl protein. In one aspect of the invention, the MUC1 ED used in the fusion protein of the invention has an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of positions 116-173 of SEQ ID NO:2 or at least one immunogenic domain thereof, positions 107-164 of SEQ ID NO:4 or at least one immunogenic domain thereof, positions 107-164 of SEQ ID NO:6 or at least one immunogenic domain thereof, positions 98-155 of SEQ ID NO:8 or at least one immunogenic domain thereof, positions 1098-1155 of SEQ ID NO:11 or at least one immunogenic domain thereof, positions 32-89 of SEQ ID NO:14 or at least one immunogenic domain thereof, positions 2-59 of SEQ ID NO:15 or at least one immunogenic domain thereof, a corresponding sequence from a different MUC1 protein, such as another human MUC1 protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences.

[0220]

[00136] A MUCl single variable number tandem repeat (VNTR) domain useful in a fusion protein described above or elsewhere herein can be a VNTR domain from any MUCl protein, and in one aspect, is from a human MUCl protein. In one aspect of the invention, the MUC1 VNTR domain used in the fusion protein of the invention has an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of: positions 126-145 of SEQ ID NO:11, any consecutive 20 amino acids between positions 61 to 1020 of SEQ ID NO:11, or any consecutive 20 amino acids between positions 126 to 965 of SEQ ID NO:11; SEQ ID NO:12 (including any polymorphism within SEQ ID NO:12 as described above), any consecutive 20 amino acids between positions 90 to 130 of SEQ ID NO:14, any consecutive 20 amino acids between positions 60 to 100 of SEQ ID NO:15, a corresponding VNTR sequence from a different MUC1 protein, such as another human MUC1 protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences.

[0221] A MUCl transmembrane (TM) domain useful in a fusion protein described above or elsewhere herein can be a TM domain or portion thereof comprising at least one immunogenic domain from any MUCl protein, and in one aspect, is from a human MUCl protein. In one aspect of the invention, the MUC1 TM domain used in the fusion protein of the invention has an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of positions 174-201 of SEQ ID NO:2, or at least one immunogenic domain thereof, positions 165-192 of SEQ ID NO:4, or at least one immunogenic domain thereof, positions 165-192 of SEQ ID NO:6, or at least one immunogenic domain thereof, positions 156-183 of SEQ ID NO:8, positions 1156-1183 of SEQ ID NO:11, or at least one immunogenic domain thereof, positions 131-158 of SEQ ID NO:14, or at least one immunogenic domain thereof, positions 101-128 of SEQ ID NO:15, or at least one immunogenic domain thereof, the corresponding sequence from a different MUC1 protein, such as another human MUC1 protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences.

[0222] The MUCl cytoplasmic domain (CD) useful in the fusion proteins described above or elsewhere herein can be a CD or portion thereof comprising at least one immunogenic domain from any MUCl protein, and in one aspect, is from a human MUCl protein. In one aspect of the invention, the MUC1 CD used in the fusion protein of the invention has an amino acid sequence comprising or consisting of an amino acid sequence selected from the group consisting of: positions 202-273 of SEQ ID NO:2 or at least one immunogenic domain thereof, positions 193-264 of SEQ ID NO:4 or at least one immunogenic domain thereof, positions 193-264 of SEQ ID NO:6 or at least one immunogenic domain thereof, positions 184-255 of SEQ ID NO:8 or at least one immunogenic domain thereof, positions 1184-1255 of SEQ ID NO:11 or at least one immunogenic domain thereof, positions 159-230 of SEQ ID NO:14 or at least one immunogenic domain thereof, positions 129-200 of SEQ ID NO:15 or at least one immunogenic domain thereof, positions 7-78 or positions 79-150 or positions 151-222 of SEQ ID NO:17 or at least one immunogenic domain thereof; Positions 1-72 or positions 73-144 or positions 145-216 of NO:18, or an immunogenic domain thereof, are from the corresponding sequence of a different MUC1 protein, such as another human MUC1 protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences.

[0223] In one embodiment of the present invention, a fusion protein useful in a yeast-based immunotherapy composition of the present invention comprises the following MUC1 antigens, in order from N to C terminus: (1) a MUC1 SEA domain and a portion of a MUC1 extracellular domain (ED) that includes most or all of the MUC1 ED; (2) at least two VNTR domains; (3) a MUC1 transmembrane domain; and (4) a MUC1 cytoplasmic domain (CD). In one embodiment, the fusion protein comprises the following MUC1 antigens, in order from N to C terminus: (1) a MUC1 signal sequence; (2) a MUC1 SEA domain and a portion of a MUC1 extracellular domain (ED) that includes most or all of the MUC1 ED; (3) at least two VNTR domains; (4) a MUC1 transmembrane domain; and (5) a MUC1 cytoplasmic domain (CD). Additional or alternative elements to be incorporated into fusion proteins of the invention may include N-terminal and / or C-terminal peptides that improve or assist in the expression or stability of the fusion protein, and / or allow identification and / or purification of the fusion protein, and / or short intervening linker sequences (e.g., peptides of 1, 2, 3, 4, or 5 amino acids) between fusion protein segments that can be used to introduce restriction enzyme sites to assist in cloning, as cleavage sites for host phagosomal proteases, to accelerate protein or antigen processing, and for subsequent manipulation of the construct. Such elements are described in detail below.

[0224] An example of such a fusion protein that can be used in the yeast-based immunotherapy composition of the present invention comprises or includes the following MUCl antigens in the order from N- to C-terminus:

[0225] (1) a MUC1 extracellular domain (ED) which may be attached at the N-terminus by 1, 2, 3, 4, 5 or more flanking amino acids from the non-ED portion of the SEA domain located upstream of the ED in the wild-type protein, wherein the ED segment comprises or consists of an amino acid sequence selected from the group consisting of: positions 116-173 of SEQ ID NO:2; or positions 107-164 of SEQ ID NO:4; positions 107-164 of SEQ ID NO:6; positions 98-155 of SEQ ID NO:8; positions 1098-1155 of SEQ ID NO:11; positions 32-89 of SEQ ID NO:14; positions 2-59 of SEQ ID NO:15; a corresponding sequence from a different MUC1 protein, such as another human MUC1 protein; or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences;

[0226] (2) at least two VNTR domains, wherein each VNTR domain comprises or consists of an amino acid sequence selected from the group consisting of: positions 126-145 of SEQ ID NO:11, any consecutive 20 amino acids between positions 61 to 1020 of SEQ ID NO:11, or any consecutive 20 amino acids between positions 126 to 965 of SEQ ID NO:11; SEQ ID NO:12 (including any polymorphism within SEQ ID NO:12 as described above), any consecutive 20 amino acids between positions 90 to 130 of SEQ ID NO:14, any consecutive 20 amino acids between positions 60 to 100 of SEQ ID NO:15, a corresponding VNTR sequence from a different MUC1 protein, such as another human MUC1 protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences;

[0227] (3) a MUC1 transmembrane (TM) domain, wherein the TM domain comprises or consists of an amino acid sequence selected from the group consisting of positions 174-201 of SEQ ID NO:2, positions 165-192 of SEQ ID NO:4, positions 165-192 of SEQ ID NO:6, positions 156-183 of SEQ ID NO:8, positions 1156-1183 of SEQ ID NO:11, positions 131-158 of SEQ ID NO:14, positions 101-128 of SEQ ID NO:15, a corresponding sequence from a different MUC1 protein, such as another human MUC1 protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences; and

[0228] (4) a MUC1 cytoplasmic domain (CD), wherein the CD comprises or consists of an amino acid sequence selected from the group consisting of positions 202-273 of SEQ ID NO:2, positions 193-264 of SEQ ID NO:4, positions 193-264 of SEQ ID NO:6, positions 184-255 of SEQ ID NO:8, positions 1184-1255 of SEQ ID NO:11, positions 159-230 of SEQ ID NO:14, positions 129-200 of SEQ ID NO:15, positions 7-78 or positions 79-150 or positions 151-222 of SEQ ID NO:17; positions 1-72 or positions 73-144 or positions 145-216 of SEQ ID NO:18, a corresponding sequence from a different MUC1 protein, such as another human MUC1 protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences.

[0229] In one aspect of this embodiment, the fusion protein useful in the yeast-based immunotherapy composition of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. SEQ ID NO: 14 comprises the following MUC1 antigens in the following order from N- to C-terminus: (1) MUC1 signal sequence (positions 1-30 of SEQ ID NO: 14); (2) MUC1 SEA / ED segment (positions 31-89 of SEQ ID NO: 14); (3) VNTR segment comprising two VNTR domains (positions 90-130 of SEQ ID NO: 14); (4) MUC1 TM domain (positions 131-158 of SEQ ID NO: 14); (5) MUC1 CD (positions 159-230 of SEQ ID NO: 14); and (6) hexa-histidine tag (positions 231-236 of SEQ ID NO: 14). SEQ ID NO: 14 is encoded by the nucleotide sequence represented by SEQ ID NO: 13. SEQ ID NO: 15 comprises the following MUC1 antigens, in order from N to C terminus: (1) MUC1 SEA / ED segment (positions 1-59 of SEQ ID NO: 15); (2) VNTR segment comprising two VNTR domains (positions 60-100 of SEQ ID NO: 15); (3) MUC1 TM domain (positions 101-128 of SEQ ID NO: 15); and (4) MUC1 CD (positions 129-200 of SEQ ID NO: 15). Optionally, the fusion protein of SEQ ID NO: 14 or SEQ ID NO: 15 comprises an N-terminal peptide, which is a synthetic N-terminal peptide designed to confer resistance to proteasomal degradation and stable expression, representatively SEQ ID NO: 21, or an N-terminal peptide from a yeast alpha leader sequence such as SEQ ID NO: 19 or SEQ ID NO: 20, or another N-terminal peptide suitable for use with the yeast-based immunotherapeutics described herein. Also optionally, one or more linker peptides having 1, 2, 3 or more amino acids, such as a two amino acid linker of Thr-Ser, can be inserted between the segments of the fusion protein. A 6-histidine tag at the C-terminus of the fusion protein is also optional.

[0230] In one embodiment of the invention, a fusion protein useful in a yeast-based immunotherapy composition of the invention comprises the following MUCl antigens, in order from N to C terminus: (1) at least two VNTR domains; and (2) a portion of the MUCl SEA domain and the MUCl extracellular domain (ED), which comprises most or all of the MUCl ED. Such fusion proteins may comprise additional portions of the MUCl-N region flanking the VNTR domains. Such fusion proteins do not comprise the complete MUCl-N subunit.

[0231] In another embodiment of the invention, a fusion protein useful in a yeast-based immunotherapeutic composition of the invention comprises the following MUC1 antigens in the following order from N to C terminus: (1) a first MUC1 cytoplasmic domain (CD); (2) a second MUC1 cytoplasmic domain (CD); and (3) a third MUC1 cytoplasmic domain (CD). In one embodiment, additional MUC1 cytoplasmic domains may be incorporated into such fusion proteins. In one embodiment, at least one MUC1 CD is from a different source than the other MUC1 CDs (e.g., one MUC1 CD is from a first human MUC1 protein and one MUC1 CD is from a second MUC1 protein, wherein there may be sequence differences between the first and second MUC1 CDs). In one embodiment, the fusion protein has a MUC1 signal sequence attached to the N-terminus. In another embodiment, the fusion protein may comprise an N-terminal and / or C-terminal peptide that improves or facilitates expression or stability of the fusion protein, and / or allows identification and / or purification of the fusion protein, and / or a short intervening linker sequence (e.g., a peptide of 1, 2, 3, 4, or 5 amino acids) between the fusion protein segments, which can be used to introduce restriction enzyme sites to facilitate cloning, serve as cleavage sites for host phagosomal proteases, accelerate protein or antigen processing, and for subsequent manipulation of the construct.

[0232] An example of such a fusion protein that can be used in the yeast-based immunotherapeutic composition of the present invention comprises or includes the following MUCl antigens in the following order from N- to C-terminus:

[0233] (1) a first MUCl cytoplasmic domain (CD), wherein the CD comprises or consists of an amino acid sequence selected from the group consisting of positions 202-273 of SEQ ID NO:2, positions 193-264 of SEQ ID NO:4, positions 193-264 of SEQ ID NO:6, positions 184-255 of SEQ ID NO:8, positions 1184-1255 of SEQ ID NO:11, positions 159-230 of SEQ ID NO:14, positions 129-200 of SEQ ID NO:15, positions 7-78 or positions 79-150 or positions 151-222 of SEQ ID NO:17; positions 1-72 or positions 73-144 or positions 145-216 of SEQ ID NO:18, a corresponding sequence from a different MUCl protein, such as another human MUCl protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences;

[0234] (2) a second MUCl cytoplasmic domain (CD), wherein the CD comprises or consists of an amino acid sequence selected from the group consisting of positions 202-273 of SEQ ID NO:2, positions 193-264 of SEQ ID NO:4, positions 193-264 of SEQ ID NO:6, positions 184-255 of SEQ ID NO:8, positions 1184-1255 of SEQ ID NO:11, positions 159-230 of SEQ ID NO:14, positions 129-200 of SEQ ID NO:15, positions 7-78 or positions 79-150 or positions 151-222 of SEQ ID NO:17; positions 1-72 or positions 73-144 or positions 145-216 of SEQ ID NO:18, a corresponding sequence from a different MUCl protein, such as another human MUCl protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences;

[0235] (3) a third MUC1 cytoplasmic domain (CD), wherein the CD comprises or consists of an amino acid sequence selected from the group consisting of positions 202-273 of SEQ ID NO:2, positions 193-264 of SEQ ID NO:4, positions 193-264 of SEQ ID NO:6, positions 184-255 of SEQ ID NO:8, positions 1184-1255 of SEQ ID NO:11, positions 159-230 of SEQ ID NO:14, positions 129-200 of SEQ ID NO:15, positions 7-78 or positions 79-150 or positions 151-222 of SEQ ID NO:17; positions 1-72 or positions 73-144 or positions 145-216 of SEQ ID NO:18, a corresponding sequence from a different MUC1 protein, such as another human MUC1 protein, or an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of these amino acid sequences.

[0236] In one aspect of this embodiment, the fusion protein useful in the yeast-based immunotherapy composition of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18. SEQ ID NO: 17 comprises the following MUC1 antigens in the following order from N- to C-terminus: (1) a synthetic peptide represented by SEQ ID NO: 21 (positions 1-6 of SEQ ID NO: 17); (2) a first MUC1 CD (positions 7-78 of SEQ ID NO: 17); (3) a second MUC1 CD (positions 79-150 of SEQ ID NO: 17); (4) a third MUC1 CD (positions 151-222 of SEQ ID NO: 17); and (5) a 6-histidine tag (positions 223-228 of SEQ ID NO: 17). SEQ ID NO: 17 is encoded by the nucleotide sequence represented by SEQ ID NO: 16. SEQ ID NO: 18 comprises the following MUC1 antigens, in order from N to C terminus: (1) a first MUC1 CD (positions 1-72 of SEQ ID NO: 18); (2) a second MUC1 CD (positions 73-144 of SEQ ID NO: 18); (3) a third MUC1 CD (positions 145-216 of SEQ ID NO: 18). Optionally, the fusion protein of SEQ ID NO: 17 or SEQ ID NO: 18 comprises an N-terminal peptide, which is a synthetic N-terminal peptide designed to confer resistance to proteasomal degradation and stable expression, representatively SEQ ID NO: 21, or an N-terminal peptide from a yeast alpha leader sequence such as SEQ ID NO: 19 or SEQ ID NO: 20, or another N-terminal peptide suitable for use with the yeast-based immunotherapeutics described herein. Still optionally, one or more linker peptides having 1 to 3 or more amino acids, linker sequences having 1, 2, 3 or more amino acids, such as a two amino acid linker of Thr-Ser, can be inserted between the segments of the fusion protein. A 6-histidine tag at the C-terminus of the fusion protein is also optional.

[0237] MUC1 antigens useful in the present invention also include 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 MUC1 proteins, domains, fusion proteins or antigens described herein, over the full length of the protein, or for a defined fragment or domain thereof forming a portion of the protein, such as an immunological domain or a functional domain (domain having at least one biological activity). For example, the domains of the MUC1 proteins described herein include a signal sequence, a VNTR domain, a SEA domain, an extracellular domain (ED), a TM domain and / or a cytoplasmic domain (CD). The immunological domain has been described in detail above. The MUC1 fusion proteins described herein include those represented by SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18. Thus, the MUCl antigen useful in the yeast-based immunotherapeutics of the present invention comprises or consists of a MUCl antigen of any one of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18, an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:18, and / or a corresponding sequence from a different MUCl protein (e.g., a fusion protein in which one or more fusion segments are from a corresponding sequence of a different MUCl protein or MUCl protein agonist sequence, such that there may be minor sequence differences compared to the sequences presented herein).

[0238] It is straightforward to create fusion proteins having similar or identical overall structures to the fusion proteins described herein using corresponding portions of any MUCl protein or domain derived or obtained from sequences other than those exemplified herein (and particularly from sequences of the same animal species or source). For example, the corresponding sequence corresponding to positions 1034-1152 of SEQ ID NO: 11 can be readily identified in a given human MUCl protein from any source using simple sequence alignment tools or processes. Thus, sequences having minor and / or conservative differences from the sequences exemplified herein are expressly contemplated by the present invention.

[0239] In some aspects of the invention, amino acid insertions, deletions and / or substitutions may be made to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acids of a wild-type or reference MUC1 protein, so long as the resulting MUC1 protein, when used as an antigen in a yeast-MUC1 immunotherapeutic composition of the invention, elicits an immune response to a native MUC1 protein, such as a wild-type or reference MUC1 protein, which may include an enhanced immune response, a reduced immune response, or a substantially similar immune response. For example, the invention includes the use of MUC1 agonist antigens, which may include one or more T cell epitopes that have been mutated to enhance a T cell response to the MUC1 agonist, such as by improving the affinity or avidity of the epitope for an MHC molecule or for a T cell receptor that recognizes the epitope in the context of MHC presentation. Thus, a MUC1 agonist may improve the potency or efficiency of a T cell response to a native MUC1 expressed by a tumor cell. A variety of MUC1 T cell epitopes, including agonist epitopes, are described in: U.S. Patent No. 6,546,643; U.S. Patent No. 7,118,738; U.S. Patent No. 7,342,094; U.S. Patent No. 7,696,306; and U.S. Patent Application Publication No. 2008 / 0063653, and any one or more of these epitopes can be used in the MUC1 antigen of the present invention, including by adding, deleting or substituting one or more amino acids within the sequence described herein so that the sequence conforms to the epitope sequence disclosed at that position.

[0240] Examples of MUCl agonist antigens are provided herein (see Examples 3 and 4). In one embodiment, a MUCl agonist antigen suitable for use in the present invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11 of the following substitutions, wherein the substituted positions are provided relative to the wild-type MUCl represented by Accession No. NP_001191214 (although the same or equivalent positions can be readily identified in any other wild-type MUCl sequence): T93, A161, P162, G169, S170, T171, A392, C406, T444, D445, or S460. In one embodiment, a MUCl agonist antigen useful in a yeast-based immunotherapy composition of the present invention comprises or consists of the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:25. SEQ ID NO:23 comprises the following MUC1 antigens, in order from N to C terminus: (1) MUC1 signal sequence (positions 1-30 of SEQ ID NO:23); (2) MUC1 SEA / ED segment (positions 31-89 of SEQ ID NO:23); (3) VNTR segment comprising two VNTR domains (positions 90-130 of SEQ ID NO:23); (4) MUC1 TM domain (positions 131-158 of SEQ ID NO:23); (5) MUC1 CD (positions 159-230 of SEQ ID NO:23); (6) MUC1 agonist epitope (positions 231-246 of SEQ ID NO:23) and (7) hexa-histidine tag (positions 247-252 of SEQ ID NO:23). SEQ ID NO:23 is encoded by the nucleotide sequence represented by SEQ ID NO:22 (codons optimized for yeast expression). The MUC1 signal sequence (positions 1-30 of SEQ ID NO: 23) can be replaced with a different N-terminal sequence designed to confer resistance to proteasomal degradation and / or stabilize expression (such as the peptide represented by SEQ ID NO: 21), or an N-terminal peptide from a yeast alpha leader sequence such as SEQ ID NO: 19 or SEQ ID NO: 20. A 6-histidine C-terminal tag is optional and facilitates identification and / or purification of the protein.Compared to the MUC1 antigen in SEQ ID NO: 14 or SEQ ID NO: 15, SEQ ID NO: 23 contains the following amino acid substitutions to create various agonist epitopes (see SEQ ID NO: IDNO:23 gives the substitution positions and further references the substitution positions in wild-type MUC1 represented by accession number NP_001191214): A96Y (position 161 in wild-type MUC1), P97L (position 162 in wild-type MUC1), G104V (position 169 in wild-type MUC1), S105Y (position 170 in wild-type MUC1), T106L (position 171 in wild-type MUC1), A147Y (position 392 in wild-type MUC1), C161V (position 406 in wild-type MUC1), T199L (position 444 in wild-type MUC1), D200F (position 445 in wild-type MUC1), S215Y (position 460 in wild-type MUC1) and T239L (position 93 in wild-type MUC1).

[0241] SEQ ID NO:25 comprises the following MUC1 antigens, in order from N to C terminus: (1) the alpha factor leader sequence disclosed elsewhere herein by SEQ ID NO:19 (positions 1-89 of SEQ ID NO:25); (2) a Thr-Ser linker sequence (positions 90-91 of SEQ ID NO:25); (3) a full-length MUC1 agonist protein corresponding to the wild-type protein except that 11 agonist epitopes are introduced (positions 92-566 of SEQ ID NO:25) and (7) a hexa-peptide histidine tag (positions 567-572 of SEQ ID NO:25). SEQ ID NO:25 is encoded by the nucleotide sequence represented by SEQ ID NO:24 (codons optimized for yeast expression). The alpha leader sequence (positions 1-89 of SEQ ID NO:25) can be replaced with a different N-terminal sequence designed to confer resistance to proteasomal degradation and / or stabilize expression (such as the peptide represented by SEQ ID NO:21), or an N-terminal peptide from a different yeast alpha leader sequence such as SEQ ID NO:20, or the MUC1 signal sequence. A 6-histidine C-terminal tag is optional and facilitates identification and / or purification of the protein. The sequence in GI-6106 contains the following amino acid substitutions compared to the wild-type MUC1 protein used as a template to create various agonist epitopes (see SEQ ID NO:21). NO:25 gives the substitution positions, and further references the substitution positions in wild-type MUC1 represented by accession number NP_001191214): T184L (position 93 in wild-type MUC1), A232Y (position 161 in wild-type MUC1), P233L (position 162 in wild-type MUC1), G240V (position 169 in wild-type MUC1), S241Y (position 170 in wild-type MUC1), T242L (position 171 in wild-type MUC1), A483Y (position 392 in wild-type MUC1), C497V (position 406 in wild-type MUC1), T535L (position 444 in wild-type MUC1), D536F (position 445 in wild-type MUC1) and S551Y (position 460 in wild-type MUC1).

[0242] Thus, MUC1 agonist antigens useful in the yeast-based immunotherapeutics of the present invention include MUC1 antigens comprising or consisting of SEQ ID NO:23 or SEQ ID NO:25, or MUC1-specific sequences in these larger fusion proteins, or amino acid sequences that are at least about 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO:23, SEQ ID NO:25, or MUC1-specific sequences in these larger fusion proteins, and / or corresponding sequences from different MUC1 proteins (e.g., a fusion protein in which one or more fusion segments are derived from corresponding sequences of different MUC1 proteins, such that there may be minor sequence differences compared to the sequences presented herein).

[0243] As discussed above, N-terminal expression sequences and C-terminal tags, such as those described above for the fusion proteins of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO: 25, are optional, but may be selected from several different sequences described elsewhere herein to improve or facilitate expression, stability, and / or allow identification and / or purification of the protein. Also, many different promoters suitable for use in yeast are known in the art. In addition, short intervening linker sequences (e.g., peptides having 1, 2, 3, 4, or 5 amino acids) may be introduced between the portions of the fusion protein comprising the MUC1 antigen for a variety of reasons, including introduction of restriction enzyme sites to facilitate cloning, as cleavage sites for host phagosomal proteases, to accelerate protein or antigen processing, and subsequent manipulation of the construct.

[0244] Optionally, antigenic constructs that are particularly useful for improving or stabilizing heterologous antigen expression in yeast are used to generate proteins, including fusion proteins, for use as components of the yeast-MUCl immunotherapeutic compositions of the invention. In one embodiment, the desired antigenic protein or peptide is fused at its amino-terminal end to: (a) a specific synthetic peptide that stabilizes expression of the fusion protein in a yeast vehicle or prevents post-translational modification of the expressed fusion protein (such peptides are described in detail, e.g., U.S. Patent Publication No. 2004-0156858A1, published on August 12, 2004, and incorporated herein by reference in its entirety); (b) at least a portion of an endogenous yeast protein, wherein either fusion partner provides improved stability of the protein expressed in yeast and / or prevents post-translational modification of the protein by the yeast cell (such proteins are also described in detail, e.g., U.S. Patent Publication No. 2004-0156858A1, supra); and / or (c) at least a portion of a yeast protein that results in expression of the fusion protein on the surface of the yeast (e.g., Aga protein, described in more detail herein). In addition, the present invention optionally includes the use of peptides fused to the C-terminal of the construct encoding the antigen, specifically for the selection and identification of proteins. Such peptides include, but are not limited to, any synthetic or natural peptides, such as peptide tags (e.g., 6X His or 6 peptides) or any other short epitope tags. The peptide attached to the C-terminal of the antigen according to the present invention can be used with or without the addition of the above-mentioned N-terminal peptide, and vice versa.

[0245] In one embodiment, a synthetic peptide useful in a fusion protein to be expressed in yeast is linked to the N-terminus of an antigen, the peptide comprising at least two amino acid positions heterologous to the antigen, wherein the peptide stabilizes expression of the fusion protein in a yeast vehicle or prevents post-translational modification of the expressed fusion protein. The synthetic peptide and the N-terminal portion of the antigen together form a fusion protein having the following requirements: (1) the amino acid residue at position 1 of the fusion protein is methionine (i.e., the first amino acid in the synthetic peptide is methionine); (2) the amino acid residue at position 2 of the fusion protein is not glycine or proline (i.e., the second amino acid in the synthetic peptide is not glycine or proline); (3) none of the amino acid positions 2-6 of the fusion protein is methionine (i.e., the amino acids at positions 2-6, whether they are part of a synthetic peptide or a protein (if the synthetic peptide is shorter than 6 amino acids), do not contain methionine); and (4) none of the amino acids at positions 2-6 of the fusion protein is lysine or arginine (i.e., the amino acids at positions 2-6, whether they are part of a synthetic peptide or a protein (if the synthetic peptide is shorter than 5 amino acids), do not contain lysine or arginine). Synthetic peptides can be as short as 2 amino acids, but in one aspect, are 2-6 amino acids (including 3, 4, 5 amino acids), and can be longer than 6 amino acids, up to about 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids or longer in full integers.

[0246] In one embodiment, the fusion protein comprises an amino acid sequence of M-X2-X3-X4-X5-X6, wherein M is methionine; wherein X2 is any amino acid other than glycine, proline, lysine or arginine; wherein X3 is any amino acid other than methionine, lysine or arginine; wherein X4 is any amino acid other than methionine, lysine or arginine; wherein X5 is any amino acid other than methionine, lysine or arginine; and wherein X6 is any amino acid other than methionine, lysine or arginine. In one embodiment, the X6 residue is proline. An exemplary synthetic sequence that enhances the stability of antigen expression in yeast cells and / or prevents post-translational modification of the protein in yeast includes the sequence MADEAP (represented herein by SEQ ID NO: 21). In addition to the enhanced stability of the expression product, the fusion partner does not appear to adversely affect the immune response to the immunizing antigen in the construct. In addition, the synthetic fusion peptide can be designed to provide an epitope that can be recognized by a selection agent such as an antibody.

[0247] In one embodiment, the MUCl antigen is linked at the N-terminus to a yeast protein, such as the alpha factor prepro sequence (also known as the alpha factor signal leader sequence), the amino acid sequence of which is exemplified herein by SEQ ID NO: 19 or SEQ ID NO: 20. Other sequences of yeast alpha factor prepro sequences are known in the art and are contemplated for use in the present invention.

[0248] In one aspect of the invention, a yeast vehicle is manipulated so that an antigen is expressed or provided partially or completely on the surface of the yeast vehicle (extracellular expression) by delivery or translocation of the expressed protein product. One method of achieving this aspect of the invention is to use a spacer arm to position one or more proteins on the surface of a yeast vehicle. For example, a spacer arm can be used to create a fusion protein of an antigen or other protein of interest with a protein that targets the antigen 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 Flo1 protein) that enables antigens or other proteins to be targeted to the yeast cell wall so that the antigen or other protein is located on the surface of the yeast. Proteins other than yeast proteins can be used for the spacer arm; however, for any spacer arm protein, it is most desirable to have an immunogenic response to the target antigen rather than the spacer arm protein. Thus, if other proteins are used for the spacer arm, the spacer arm protein used should not generate a larger immune response to the spacer arm protein itself and cover the immune response to the target antigen. Those skilled in the art should aim for an immune response to the spacer arm protein that is smaller than the immune response to the target antigen. The spacer arm can be constructed with a cleavage site (e.g., a protease cleavage site) that allows the antigen to be easily removed from the yeast or processed away as desired. Any known method for determining the magnitude of the immune response (e.g., antibody production, lysis assays, etc.) can be used and is readily known to those skilled in the art.

[0249] Another method for localizing a target antigen or other protein to be exposed on the surface of yeast is to use a signal sequence such as glycosylphosphatidylinositol (GPI) to anchor the target to the yeast cell wall. Alternatively, localization can be achieved by attaching a signal sequence that targets the antigen or other protein of interest to the secretory pathway (via translocation into the endoplasmic reticulum (ER)), thereby allowing the antigen to bind to a cell wall-bound protein (e.g., cwp).

[0250] In one aspect, the spacer arm protein is a yeast protein. The yeast protein can be composed of yeast proteins of about 2 to about 800 amino acids. In one embodiment, the yeast protein is about 10 to about 700 amino acids. In another embodiment, the yeast protein is about 40 to 600 amino acids. Other embodiments of the present invention include yeast proteins of 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 long. Another consideration for optimizing antigen surface expression (if desired) is whether the antigen and spacer arm combined epitopes should be monomers, dimers, trimers, or even more connected units. The use of monomers, dimers, trimers, etc. allows for suitable spacing or folding of antigens, so that some parts of the antigen (if not all) are displayed on the surface of the yeast vehicle in a manner that makes it more immunogenic.

[0251] The use of yeast proteins can stabilize the expression of the fusion protein in the yeast vehicle, prevent post-translational modification of the expressed fusion protein, and / or target the fusion protein to a specific compartment in the yeast (e.g., to be expressed on the surface of the yeast cell). For delivery into the yeast secretory pathway, exemplary yeast proteins used include, but are not limited to: Aga (including but not limited to Agal and / or Aga2); SUC2 (yeast invertase); alpha factor signal leader sequence; CPY; Cwp2p (because of its localization and retention in the cell wall); BUD gene (because it is located at the yeast cell bud during the initial stages of daughter cell formation); Flo1p; Pir2p; and Pir4p.

[0252] Other sequences that target, retain and / or stabilize proteins to other parts of the yeast vehicle (e.g., in the cytosol or mitochondria or endoplasmic reticulum or nucleus) may be used. Examples of suitable yeast proteins that can be used in any of the above embodiments include, but are not limited to, TK, AF, SEC7; phosphoenolpyruvate carboxykinase PCK1, phosphoglycerol kinase PGK, and triosephosphate isomerase TPI gene products (for their repressible expression in glucose and cytosol localization); heat shock proteins SSA1, SSA3, SSA4, SSC1, whose expression is induced and whose proteins are more thermostable when cells are exposed to heat treatment; mitochondrial protein CYC1 (for import into mitochondria); ACT1.

[0253] The present invention encompasses methods of producing yeast vehicles and expressing, combining and / or associating yeast vehicles with antigens and / or other proteins and / or agents of interest to produce yeast-based immunotherapeutic compositions.

[0254] According to the present invention, the term "yeast vehicle-antigen complex" or "yeast-antigen complex" is generally used to describe any association of a yeast vehicle with an antigen, and is used interchangeably with "yeast-based immunotherapy composition" when such composition is used to elicit an immune response as described above. Such associations include expression of the antigen by yeast (recombinant yeast), introduction of the antigen into yeast, physical attachment of the antigen to yeast, and mixing yeast and antigen together, such as in a buffer or other solution or formulation. These types of complexes are described in detail below.

[0255] In one embodiment, the yeast cells used to prepare yeast vehicles are transfected with heterologous nucleic acid molecules encoding a certain protein (e.g., an antigen) so that the protein is expressed by the yeast cells. Such yeasts are also referred to herein as recombinant yeast or recombinant yeast vehicles. Then, the yeast cells can be formulated with pharmaceutically acceptable excipients and directly administered to patients, stored for later administration, or loaded into dendritic cells as complete cells. The yeast cells can also be killed, or they can be derivatized such as by forming yeast spheroplasts, cytoplasm, ghosts, or subcellular particles, any of which can be followed by storage, administration, or loading derivatives into dendritic cells. Yeast spheroplasts can also be directly transfected with recombinant nucleic acid molecules (e.g., spheroplasts are produced from whole yeast and then transfected) to produce recombinant spheroplasts expressing the antigen. Yeast cells or yeast spheroplasts of recombinantly expressed antigens can be used to produce yeast vehicles comprising yeast cytoplasm, yeast ghosts, or yeast membrane particles or yeast cell wall particles, or their fractions.

[0256] Generally, the yeast vehicle and antigen and / or other agent can be combined by any technique described herein. In one aspect, the yeast vehicle is loaded intracellularly with antigen and / or agent. In another aspect, the antigen and / or agent is covalently or non-covalently attached to the yeast vehicle. In yet another aspect, the yeast vehicle and antigen and / or agent are combined by mixing. In another aspect, and in one embodiment, the antigen and / or agent is recombinantly expressed by the yeast vehicle or by the yeast cell or yeast spheroplast from which the yeast vehicle is derived.

[0257] The plurality of antigens and / or other proteins to be produced by the yeast vehicles of the invention is any number of antigens and / or other proteins that can reasonably be produced by a yeast vehicle, and generally ranges from at least 1 to at least about 6 or more, including from about 2 to about 6 antigens and / or other proteins.

[0258] The expression of antigen or other protein in yeast vehicle of the present invention is realized using technology known to those skilled in the art. In short, the nucleic acid molecule encoding at least one desired antigen or other protein is inserted into the expression vector in such a way that the nucleic acid molecule is operably connected to a transcriptional regulatory sequence, thereby causing the constitutive or regulated expression of the nucleic acid molecule when transformed into the host yeast cell. The nucleic acid molecule encoding one or more antigens and / or other proteins can be operably connected to one or more expression regulatory sequences on one or more expression vectors. Particularly important expression regulatory sequences are those that regulate transcription initiation, such as promoters and upstream activation sequences. Any suitable yeast promoter can be used for 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, promoters of genes encoding the following yeast proteins: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), translation elongation factor EF-1alpha (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as TDH3 for triose phosphate dehydrogenase), galactokinase (GAL1), galactose-1-phosphate uridyltransferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome c1 (CYC1), Sec7 protein (SEC7), and acid phosphatase (PHO5), including hybrid promoters such as the ADH2 / GAPDH and CYC1 / GAL10 promoters, and including the ADH2 / GAPDH promoter, which is induced when the glucose concentration in the cell is low (e.g., about 0.1 to about 0.2 percent), as well as the CUP1 promoter and the TEF2 promoter. Similarly, many upstream activation sequences (UAS), also known as enhancers, are known. Upstream activation sequences for expression in Saccharomyces cerevisiae include, but are not limited to, UASs of genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7, and GAL10, as well as other UASs activated by the GAL4 gene product, wherein in one aspect ADH2 UAS is used. Since ADH2 UAS is activated by the ADR1 gene product, when a heterologous gene is operably linked to ADH2 UAS, it is preferred that the ADR1 gene can be overexpressed. Transcription termination sequences for expression in Saccharomyces cerevisiae include transcription termination sequences of α-factor, GAPDH, and CYC1 genes.

[0259] Transcriptional regulatory sequences for expressing genes in methylotrophic yeast include transcriptional regulatory regions of genes encoding alcohol oxidase and formate dehydrogenase.

[0260] Transfection of nucleic acid molecules into yeast cells according to the present invention can be achieved by any method in which nucleic acid molecules can be introduced into cells, including, but not limited to, diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Using techniques known to those skilled in the art, transfected nucleic acid molecules can be integrated into yeast chromosomes or maintained on extrachromosomal vectors. Examples of yeast vehicles carrying such nucleic acid molecules are disclosed in detail herein. As discussed above, complete yeast microorganisms or yeast spheroplasts can also be transfected with desired nucleic acid molecules, wherein antigens are produced, and then further using techniques known to those skilled in the art to operate microorganisms or spheroplasts to recombinantly produce yeast cytoplasm, yeast ghosts, and yeast membrane particles or cell wall preparations, to produce cytoplasm, ghosts, and subcellular yeast membrane extracts or fractions thereof containing desired antigens or other proteins.

[0261] Effective conditions for the production of recombinant yeast vehicles and the expression of antigens and / or other proteins by yeast vehicles include effective culture media in which yeast strains can be cultured. Effective culture media are generally aqueous cultures containing assimilable sugars, nitrogen and phosphate sources, as well as suitable salts, minerals, metals and other nutrients such as vitamins and growth factors. The culture medium may contain complex nutrients or may be a limited minimum culture medium. The yeast strains of the present invention can be cultured in a variety of containers, including but not limited to bioreactors, Erlenmeyer bottles, test tubes, microtiter dishes and Petri plates. Cultivation is carried out at a temperature, pH and oxygen content suitable for the yeast strain. Such culture conditions are completely within the skill range of ordinary technicians in the art (see, for example, Guthrie et al. (ed.), 1991, Methods in Enzymology, vol. 194, Academic Press, San Diego). For example, in one approach, a culture obtained from a starter plate and / or starter culture of a yeast-MUC1 immunotherapy composition can be used to inoculate a liquid culture containing a suitable culture medium and grown at 30° C. for about 20 hours, accompanied by stirring at 250 rpm. The primary culture can then be expanded into more cultures when desired. Protein expression (e.g., MUC1 expression) from the vector of the transformed yeast can be constitutive (if the promoter utilized is a constitutive promoter), or can be induced by adding appropriate inducing conditions for the promoter (if the promoter utilized is an inducible promoter) (e.g., copper sulfate in the case of the CUP1 promoter). In the case of an inducible promoter, induction of protein expression can be initiated after the culture has grown to an appropriate cell density, which can be at about 0.2 YU / ml or higher.

[0262] A non-limiting example of a culture medium suitable for culturing the yeast-MUCl immunotherapeutic composition of the present invention is U2 medium. U2 medium comprises the following components: 15 g / 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, and 0.06 mg / ml of leucine. Another non-limiting example of a culture medium suitable for culturing the yeast-MUCl immunotherapeutic composition of the present invention is UL2 medium. UL2 medium comprises the following components: 15 g / 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.

[0263] In some embodiments of the present invention, yeast is grown under neutral pH conditions. As used herein, the general use of the term "neutral pH" refers to a pH range of about pH 5.5 to about pH 8, and in one aspect, about pH 6 to about 8. Those skilled in the art will appreciate that there may be slight fluctuations (e.g., in tens of tenths or hundreds of percents) when measured with a pH meter. Thus, using neutral pH to grow yeast cells means that yeast cells grow in neutral pH for most of the time in culture. In one embodiment, yeast is grown in a culture medium maintained at a pH level of at least 5.5 (i.e., the pH of the culture medium is not allowed to fall below pH 5.5). In one other aspect, yeast is grown in a pH level maintained at about 6, 6.5, 7, 7.5, or 8. In one aspect, neutral pH is maintained by creating a buffered culture or growth medium using a suitable buffer. Using neutral pH in culturing yeast promotes several biological effects, which are desirable features for using yeast as a medium for immunomodulation. For example, yeast is cultured at a neutral pH to allow good growth of yeast without adversely affecting cell generation time (e.g., delaying doubling time). Yeast can be grown sustainably to higher density without losing its cell wall flexibility. Using a neutral pH allows the production of yeast with a flexible cell wall and / or a yeast that is more sensitive to enzymes (e.g., glucanases) that digest the cell wall at all harvest densities. This character is desired because yeast with a flexible cell wall can induce different or improved immune responses compared to yeast grown under more acidic conditions, such as by promoting the secretion of cytokines (e.g., TH1 type cytokines, including but not limited to IFN-γ, interleukin-12 (IL-12) and IL-2, and proinflammatory cytokines such as IL-6) of antigen-presenting cells that have engulfed yeast. In addition, this type of culture method provides greater accessibility to antigens located in the cell wall. On the other hand, using a neutral pH for some antigens allows the release of disulfide bonded antigens by processing with dithiothreitol (DTT), which is impossible when culturing yeast expressing antigens in a culture medium at a lower pH (e.g., pH 5). In one non-limiting example of using neutral pH conditions to culture yeast for use in the present invention, 4.2 g / L Bis-Tris is used, for example, to buffer the UL2 medium described above.

[0264] The inventors show herein that yeast-MUC1 immunotherapeutic compositions of the invention grown using neutral pH conditions are more potent activators of dendritic cells and activate MUC-1-specific T cells to produce higher levels of IFN-γ than the same yeast-MUC1 immunotherapeutic compositions grown under standard conditions (where neutral pH is not maintained) (see Examples).

[0265] In one embodiment, control of the amount of yeast glycosylation is used to control the expression of yeast antigens (especially on the surface). The amount of yeast glycosylation can affect the immunogenicity and antigenicity of antigens, especially antigens expressed on the surface, because sugar modules tend to be larger. Thus, the presence of sugar modules on the yeast surface and their effect on the three-dimensional space around the target antigen should be considered in the regulation of yeast according to the present invention. Any method can be used to reduce or increase the amount of yeast glycosylation, as desired. For example, selected yeast mutant strains with lower glycosylation (e.g., mnn1, och1, and mnn9 mutants) can be used, or glycosylation acceptor sequences on the target antigen can be eliminated by mutation. Alternatively, yeast with a short glycosylation pattern, such as Pichia pastoris, can be used. Yeast can also be treated using methods that reduce or alter glycosylation.

[0266] In one embodiment of the invention, as an alternative to antigen or other protein recombinant expression in yeast vehicles, yeast vehicles are loaded intracellularly with proteins or peptides, or with carbohydrates or other molecules that serve as antigens and / or can be used as immunomodulators or bioreaction modifiers according to the present invention. Thereafter, the yeast vehicles containing antigens and / or other proteins in the present cell can be administered to individuals or loaded into carriers such as dendritic cells. Peptides and proteins can be directly inserted into the yeast vehicles of the present invention, which are processed by techniques known to those skilled in the art, such as diffusion, active transport, liposome fusion, electroporation, phagocytosis, freeze-thaw cycles, and bath-type ultrasonic treatment. Yeast vehicles directly loaded with peptides, proteins, carbohydrates or other molecules include complete yeast, as well as spheroplasts, ghosts or cytoplasm, which can be loaded with antigens and other agents after production. Alternatively, complete yeast can be loaded with antigens and / or agents, and spheroplasts, ghosts, cytoplasm or subcellular particles can then be prepared therefrom. Any number of antigens and / or other agents may be loaded into the yeast vehicle of this embodiment, from at least 1, 2, 3, 4 or any full integer up to hundreds or thousands of antigens and / or other agents, as would be provided by loading, for example, a microorganism or portion thereof.

[0267] In another embodiment of the present invention, antigen and / or other agent are physically attached to yeast vehicle. Antigen and / or other agent physical attachment to yeast vehicle can be realized by any method suitable in the art, including covalent and non-covalent combination methods, including but not limited to, antigen and / or other agent chemical cross-linking to the external surface of yeast vehicle or antigen and / or other agent biological connection to the external surface of yeast vehicle, such as by using antibodies or other binding partners. Chemical cross-linking can be realized, for example, by methods including glutaraldehyde connection, photoaffinity labeling, carbodiimide treatment, treatment with chemicals that can connect disulfide bonds, and treatment with other cross-linking chemicals that are standard in the art. Alternatively, the lipid bilayer or cell wall composition of the yeast membrane can be changed to contact the yeast vehicle, so that the external surface of the yeast is more likely to fuse or bind to antigens and / or other agents with specific charge attributes. Targeted agents such as antibodies, binding peptides, soluble receptors and other ligands can also be included in antigens as fusion proteins or associated with antigens for antigens to bind to yeast vehicles.

[0268] When an antigen or other protein is expressed on the surface of yeast or physically attached to the surface, in one aspect, the spacer arm can be carefully selected to optimize the expression or content of the antigen or other protein on the surface. The size of the spacer arm can affect how much the antigen or other protein is exposed for binding on the yeast surface. Thus, depending on which antigen or other protein is used, one skilled in the art will select a spacer arm that achieves appropriate spacing for the antigen or other protein on the yeast surface. In one embodiment, the spacer arm is a yeast protein of at least 450 amino acids. The spacer arm has been discussed in detail above.

[0269] In yet another embodiment, the yeast vehicle and antigen or other protein are associated with each other through a more passive, non-specific or non-covalent binding mechanism, such as by gently mixing the yeast vehicle and antigen or other protein together in a buffer or other suitable formulation (e.g., an admixture).

[0270] In one embodiment, whole yeast (with or without expression of heterologous antigens or other proteins) can be ground or processed to produce yeast cell wall preparations, yeast membrane particles, or yeast fragments (i.e., incomplete), and in some embodiments, yeast fragments can be provided or administered together with other compositions comprising antigens (e.g., DNA vaccines, protein subunit vaccines, killed or inactivated pathogens, viral vector vaccines) to enhance the immune response. For example, enzymatic treatment, chemical treatment, or physical force (e.g., mechanical shearing or sonication) can be used to break yeast into parts for use as adjuvants.

[0271] In one embodiment of the invention, yeast vehicles useful in the invention include killed or inactivated yeast vehicles. The killing or inactivation of yeast can be achieved by any of a variety of suitable methods known in the art. For example, heat inactivation of yeast is a standard approach to inactivating yeast, and those skilled in the art can monitor structural changes in the target antigen by standard methods known in the art if desired. Alternatively, other methods of inactivating yeast can be used, such as chemical, electrical, radioactive or UV methods. See, for example, the methods disclosed in standard yeast culture textbooks such as Methods of Enzymology , Vol. 194, Cold Spring Harbor Publishing (1990). Any inactivation strategy used should take into account the secondary, tertiary, or quaternary structure of the target antigen and preserve such structure to optimize its immunogenicity.

[0272] Yeast vehicles can be formulated into yeast-based immunotherapy compositions or products of the present invention using many techniques known to those skilled in the art. For example, yeast vehicles can be dried by lyophilization. Formulations containing yeast vehicles can also be prepared by packaging the yeast into cakes or tablets, such as for yeast used in baking or brewing operations. In addition, the yeast vehicle can be mixed with pharmaceutically acceptable excipients such as isotonic buffers tolerated by the host or host cells. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other physiologically balanced aqueous salt solutions. Non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides can also be used. Other available formulations include suspensions containing viscosity enhancers such as sodium carboxymethylcellulose, sorbitol, glycerol, or dextran. Excipients can also contain a small amount of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffers, bicarbonate buffers, and Tris buffers, while examples of preservatives include thimerosal, m-cresol or o-cresol, formalin, and benzyl alcohol. Standard formulations may be liquid injections or solids that can be absorbed into a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient may include, for example, dextrose, human serum albumin, and / or a preservative, to which sterile water or saline may be added prior to administration.

[0273] In one embodiment of the invention, the composition may include additional agents, which may also be referred to as biological response modifier compounds, or the ability to produce such agents / modifiers. For example, a yeast vehicle may be transfected or loaded with at least one antigen and at least one agent / biological response modifier compound, or the composition of the invention may be administered together with at least one agent / biological response modifier. Biological response modifiers include adjuvants and other compounds that can modulate immune responses (which may be referred to as immunomodulatory compounds), as well as compounds that modify the biological activity of other compounds or agents such as yeast-based immunotherapeutics, such biological activities being not limited to immune system effects. Certain immunomodulatory compounds can stimulate protective immune responses, while others can inhibit harmful immune responses, and whether immunomodulation is useful in combination with a given yeast-based immunotherapeutic may depend at least in part on the disease state or condition to be treated or prevented, and / or the individual to be treated. Certain biological response modifiers preferentially enhance cell-mediated immune responses while others preferentially enhance humoral immune responses (i.e., can stimulate immune responses in which there is an increased level of cell-mediated immunity 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. Many techniques are known to those skilled in the art to measure stimulation or inhibition of an immune response, as well as to distinguish between a cell-mediated immune response and a humoral immune response, and to distinguish between one type of cell-mediated response and another type of cell-mediated response (e.g., a TH17 response versus a TH1 response).

[0274] The pharmaceutical biological response modifiers that can be used in the present invention may include, but are not limited to, cytokines, chemokines, hormones, lipid derivatives, peptides, proteins, polysaccharides, small molecule drugs, antibodies and their antigen-binding fragments (including but not limited to anti-cytokine antibodies, anti-cytokine receptor antibodies, anti-chemokine antibodies), vitamins, polynucleotides, nucleic acid binding modules, aptamers, and growth regulators. Some suitable agents include, but are not limited to, IL-1 or an agonist of IL-1 or IL-1R, anti-IL-1 or other IL-1 antagonists; IL-6 or an agonist of IL-6 or IL-6R, anti-IL-6 or other IL-6 antagonists; IL-12 or an agonist of IL-12 or IL-12R, anti-IL-12 or other IL-12 antagonists; IL-17 or an agonist of IL-17 or IL-17R, anti-IL-17 or other IL-17 antagonists; IL-21 or an agonist of IL-21 or IL-21R, anti-IL-21 or other IL-21 antagonists; IL-22 or an agonist of IL-22 or IL-22R, anti-IL-22 or other IL-22 antagonists; IL-23 or an agonist of IL-23 or IL-23R, anti-IL-23 or other IL-23 antagonists antagonists; IL-25 or IL-25 or IL-25R agonists, anti-IL-25 or other IL-25 antagonists; IL-27 or IL-27 or IL-27R agonists, anti-IL-27 or other IL-27 antagonists; type I interferons (including IFN-α) or agonists or antagonists of type I interferons or their receptors; type II interferons (including IFN-γ) or agonists or antagonists of type II interferons or their receptors; anti-CD40, CD40L, lymphocyte activation gene 3 (LAG3) protein and / or IMP321 (a soluble form of T cell immunostimulatory factor derived from LAG3), anti-CTLA-4 antibodies (e.g., to release anergic T cells); T cell co-stimulators (e.g., anti-CD137, anti-CD28, anti-CD40); alemtuzumab (e.g., ), denileukin diftitox (e.g. ); anti-CD4; anti-CD25; anti-PD-1, anti-PD-L1, anti-PD-L2; agents that block FOXP3 (e.g., abolish active / kill CD4+ / CD25+ T regulatory cells); Flt3 ligand, imiquimod (Aldara TM ), granulocyte-macrophage colony-stimulating factor (GM-CSF); granulocyte colony-stimulating factor (G-CSF), sargramostim Hormones include, but are not limited to, 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 agonists; TLR antagonists, including, but not limited to, TLR-2 antagonists, TLR-4 antagonists, TLR-7 antagonists, and TLR-9 antagonists; anti-inflammatory agents and immunomodulators, including, but not limited to, COX-2 inhibitors (e.g., Celecoxib, NSAIDs), glucocorticoids, statins, and thalidomide and its analogs, including IMiDs TM (which is a structural and functional analog of thalidomide (e.g. (lenalidomide), (pomalidomide)); pro-inflammatory agents, such as fungal or bacterial components or any pro-inflammatory cytokines or chemokines; immunotherapeutic vaccines, including but not limited to virus-based vaccines, bacteria-based vaccines, or antibody-based vaccines; and any other immunomodulators, immunopotentiators, anti-inflammatory agents, pro-inflammatory agents, and any agents that regulate the number of antigen presenting cells or TH17, TH1 and / or T regulatory cells, regulate their activation state, and / or regulate their survival. Any combination of such agents is encompassed by the present invention, and any such agent that is combined with a yeast-based immunotherapeutic or administered together in a regimen (e.g., simultaneously, sequentially, or otherwise together) is a composition encompassed by the present invention. Such agents are well known in the art. These agents may be used alone or in combination with other agents described herein.

[0275] Agents may include agonists and antagonists of a given protein or its peptides or domains. As used herein, an "agonist" is any compound or agent that binds to a receptor or ligand and produces or triggers a response, including but not limited to small molecules, proteins, peptides, antibodies, nucleic acid binders, etc., which may include agents that simulate or enhance the effects of naturally occurring substances that bind to receptors or ligands. An "antagonist" is any compound or agent that blocks or inhibits or reduces the effects of an agonist, including but not limited to small molecules, proteins, peptides, antibodies, nucleic acid binders, etc.

[0276] The compositions of the present invention may also include, or be administered together (simultaneously, sequentially or intermittently) with any other agent or composition or regimen useful for preventing or treating cancer, or any compound for treating or ameliorating any symptom of cancer (particularly cancer associated with MUC1 expression or overexpression). In addition, the compositions of the present invention may be used together with other immunotherapy compositions, including preventive medication and / or therapeutic immunotherapy. Additional agents, compositions or regimens (e.g., treatment regimens) that can be used to treat cancer include, but are not limited to, chemotherapy, surgical resection of tumors, radiotherapy, allogeneic or autologous stem cell transplantation, T cell adoptive transfer, other types of immunotherapy, including viral vector-based immunotherapy and dendritic cell / tumor fusion immunotherapy, and / or targeted cancer therapy (e.g., small molecule drugs, biologics, or monoclonal antibody therapy that specifically targets molecules 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) inhibitors, retinoid receptor activators, apoptosis stimulators, angiogenesis inhibitors, poly (ADP-ribose) polymerase (PARP) inhibitors, or immunostimulants). Any of these additional therapeutic agents and / or treatment regimens can be administered before, simultaneously, alternately, or after the immunotherapy composition of the present invention, or at different time points. For example, when administered to an individual in conjunction with chemotherapy or targeted cancer therapy, it may be desirable to administer the Yeast-MUCl immunotherapeutic composition during a "holiday" period between doses of the chemotherapy or targeted cancer therapy to maximize the efficacy of the immunotherapeutic composition. Surgical resection of a tumor may often precede administration of a Yeast-MUCl immunotherapeutic composition, but additional or initial surgery may be performed during or after administration of a Yeast-MUCl immunotherapeutic composition.

[0277] The present invention also includes kits comprising any of the compositions described herein, or any of the individual components of the compositions described herein. The kits may include additional reagents and written instructions or directions for using any of the compositions of the present invention to prevent or treat cancer associated with or characterized by MUC1 expression or overexpression.

[0278] Methods for applying or using the compositions of the present invention

[0279] The yeast-MUCl immunotherapeutic compositions of the present invention are designed to prevent or treat cancers associated with or characterized by MUC1 expression or overexpression, including by preventing the appearance of such cancers, arresting the progression of such cancers, or eliminating such cancers. More specifically, the yeast-MUCl immunotherapeutic compositions can be used to prevent, inhibit, or delay the formation of tumors that express MUC1, and / or prevent, inhibit, or delay tumor migration and / or tumor invasion (metastasis) of other tissues and / or generally prevent or inhibit the progression of cancer in an individual. The yeast-MUCl immunotherapeutic compositions can also be used to ameliorate at least one symptom of cancer, such as by reducing tumor burden in an individual; inhibiting tumor growth in an individual; increasing survival of an individual; and / or preventing, inhibiting, reversing, or delaying the progression of cancer in an individual.

[0280] Cancers relevant to the compositions and methods of the present invention are any cancer that expresses or can express MUC1, or cancers that are adjacent to cancers that express or can express MUC1, including cancers of epithelial tissues, and include, but are not limited to, cancers of the breast, small intestine, stomach, kidney, bladder, uterus, ovary, testis, lung, colon, pancreas, prostate, testis, and metastatic cancers thereof. In addition, MUC1 is or can be expressed in blood cancers, such as lymphomas, leukemias, and myelomas, including, but not limited to, chronic lymphocytic leukemia (CLL), multimyelocytic lymphoma (MML), acute myeloid leukemia (AML), Epstein-Barr virus (EBV) transformed B cells, Burkitt's lymphoma, and Hodgkin's lymphoma.

[0281] In one aspect, MUC1 is not detected in the individual's cancer when the composition is first administered. When MUC1 is not detected in the individual's cancer, the individual may have an earlier stage of cancer in which MUC1 expression has not yet been demonstrated (e.g., stage I or stage II), or in which MUC1 expression cannot be detected at any event (i.e., MUC1 may or may not be expressed at low levels or in a small number of tumor cells, but is not yet readily detectable using standard detection methods). Alternatively, the individual may have a precancerous lesion or tumor, or may be known to be predisposed to developing cancer (e.g., known by family history, genetic markers, etc.). In these aspects of the invention, the formation of tumor cells expressing MUC1 is prevented, delayed, or inhibited by using the yeast-MUC1 immunotherapy composition.

[0282] In another aspect, MUC1 expression is or can be detected in a cancer of the individual upon first administration of the composition. In this aspect of the invention, the individual may have stage I, stage II, stage III, or stage IV cancer. In this aspect, use of the yeast-MUCl immunotherapeutic composition reduces, eliminates, or delays or arrests the growth of a tumor expressing MUC1, which can result in a reduction in tumor burden in the individual, inhibition of growth of a tumor expressing MUC1, and / or increased survival of the individual.

[0283] Another embodiment of the invention relates to a method of treating cancer, particularly a cancer that expresses MUC1. The method comprises administering to an individual suffering from a cancer that expresses MUC1 a yeast-MUC1 immunotherapy composition described herein, which may include a composition comprising: (a) a yeast vehicle; and (b) a cancer antigen comprising at least one MUC1 antigen. In one aspect, the method reduces tumor burden in a patient. In one aspect, the method increases survival of a patient. In one aspect, the method inhibits tumor growth in an individual.

[0284] In one aspect, the individual is additionally treated with at least one other therapeutic compound or therapeutic regimen that can be used to treat cancer. Such therapeutic agents and regimens have been discussed in detail elsewhere herein. For example, in any embodiment of the inventive methods described herein, in one aspect, when the individual suffers from cancer (regardless of the state of detectable MUC1 expression in tumor cells), the individual is receiving or has received another therapy for cancer. Such therapy may include any therapeutic regimen or use of any therapeutic compound or agent previously described herein, including, but not limited to, chemotherapy, radiotherapy, targeted cancer therapy, surgical resection of tumors, stem cell transfer, cytokine therapy, adoptive T cell transfer, and / or administration of a second immunotherapy composition. In cases where a second immunotherapeutic composition is administered, such compositions may include, but are not limited to, additional yeast-based immunotherapies, recombinant virus-based immunotherapies (viral vectors, see, e.g., PCT Publication No. WO / 00 / 34494), cytokine therapy, immunostimulatory therapy (including chemotherapy with immunostimulatory properties), DNA vaccines, dendritic cell / tumor fusion immunotherapies (see, e.g., PCT Publication No. WO / 2009 / 062001), and other immunotherapeutic compositions.

[0285] In one aspect, the second immunotherapeutic composition comprises a second cancer antigen that is not a MUC1 antigen. For example, a second immunotherapeutic composition that can be used in combination with a yeast-MUC1 immunotherapeutic composition is a yeast-immunotherapeutic composition that comprises another cancer antigen expressed by the same tumor type or by other tumors that the individual to be treated has or may develop. Such cancer antigens include, but are not limited to, any one or more of the following: carcinoembryonic antigen (CEA), point mutated Ras oncoprotein, Brachyura malformation protein, EGFR, BCR-Abl, MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-2, normal and point mutated p53 oncoprotein, PSMA, tyrosinase, TRP-1 (gp75), NY-ESO-1, 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-1, Rb-2, androgen receptor (AR), Smad4, MDR1, Flt-3, BRCA-1, BRCA-2, pax3-fkhr, ews-fli-1, 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 epitope agonists thereof.

[0286] As used herein, "treating" cancer or any variation thereof (e.g., "treatment for cancer," etc.) generally refers to administering a composition of the invention once cancer has occurred (e.g., once cancer has been diagnosed or detected in an individual), with a therapeutic goal of at least one of the following treatments (e.g., compared to without such treatment): reduction of tumor burden; inhibition of tumor growth; increase in individual survival; delaying, inhibiting, arresting, or preventing the onset or formation of metastatic cancer (e.g., by delaying, inhibiting, arresting, or preventing the onset of tumor migration formation and / or tumor invasion of tissues other than the primary cancer and / or other processes associated with the metastatic progression of cancer); delaying or arresting the progression of primary cancer; improving immune responses against tumors; improving long-term memory immune responses against tumor antigens; and / or improving the general health of the individual. "Preventing" or "protecting against" cancer, or any variation thereof (e.g., "preventing cancer," etc.) generally refers to administering a composition of the invention before cancer develops, when precancerous cells are detected, or before a particular stage of cancer or tumor antigen expression in a cancer occurs (e.g., before MUC1 expression is detected in a cancer), with at least one therapeutic goal (e.g., compared to without such treatment) including: preventing or delaying the onset or development of cancer, or, if cancer develops after treatment, at least reducing the severity of the cancer (e.g., reducing the level of tumor growth, arresting cancer progression, improving immune response against cancer, inhibiting metastatic processes, etc.) or improving outcomes in an individual (e.g., improving survival).

[0287] The present invention includes delivering (administering, immunizing, vaccinating) a yeast-MUC1 immunotherapeutic composition of the present invention to a subject or individual. The administration process can be performed ex vivo or in vivo, but is usually performed in vivo. Ex vivo administration refers to performing some regulatory steps outside the patient, such as administering the composition of the present invention to a cell population (dendritic cells) removed from the patient under conditions that allow the yeast vehicle, antigen, and any other agent or composition to be loaded into the cells, and returning the cells to the patient. The therapeutic composition of the present invention can then be returned to the patient, or administered to the patient by any suitable mode of administration.

[0288] The administration of the composition can be systemic, permucosal and / or in a position close to the target site (e.g., near the tumor site). According to the cancer type and / or target cell colony or tissue to be prevented or treated, suitable administration routes will be obvious to those skilled in the art. Various acceptable administration methods include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intrasegmental administration, intracoronary administration, intraarterial administration (e.g., to the carotid artery), subcutaneous administration, percutaneous administration, intratracheal administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), intracranial, intraspinal, intraocular, ear, intranasal, oral, pulmonary administration, catheter injection, and direct injection into tissue. In one aspect, administration routes include: intravenous, intraperitoneal, subcutaneous, intracutaneous, intrasegmental, intramuscular, percutaneous, inhalation, intranasal, oral, intraocular, intraarticular, intracranial, and intraspinal. Parenteral delivery may include intracutaneous, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous, arterial catheter, and intravenous catheter routes. Auris delivery may include ear drops, intranasal delivery may include nasal drops or intranasal injection, and intraocular delivery may include eye drops. Aerosol (inhalation) delivery may also be performed using standard methods in the art (see, e.g., Stribling et al., Proc. Natl. Acad. Sci. USA 189: 11277-11281, 1992). In one aspect, the yeast-MUC1 immunotherapeutic composition of the invention is administered subcutaneously. In one aspect, the yeast-MUC1 immunotherapeutic composition is administered directly into the tumor environment.

[0289] Generally, a suitable single dose of a yeast-MUC1 immunotherapeutic composition is one that, when administered one or more times over an appropriate period of time, is effective to provide a dose of the yeast vehicle and the MUC1 antigen to a given cell type, tissue, or area of ​​a patient's body in an amount effective to elicit an antigen-specific immune response to one or more MUC1 antigens or epitopes. For example, in one embodiment, a single dose of the yeast-MUC1 composition of the invention is about 1 x 10 5 About 5x10 7 Yeast cell equivalents per kg of body weight of the organism to which the composition is administered. One Yeast Unit (YU) is 1x10 7 yeast cells or yeast cell equivalents. In one aspect, a single dose of the yeast vehicle of the present invention is about 0.1 YU (1 x 10 6 yeast cells or yeast cell equivalent) to about 100 Y.U. (1x10 9 cells) per dose (i.e., per organism), including any intermediate doses, in increments of 0.1x10 6 cells (i.e. 1.1x10 6 , 1.2x10 6 , 1.3x10 6...). In one embodiment, suitable doses include doses of 1 Y.U. to 40 Y.U. and in one aspect, 10 Y.U. to 40 Y.U. or 10 Y.U. to 80 Y.U. In one embodiment, doses are administered at different sites on an individual but within the same administration period. For example, a 40 Y.U. dose can be administered by injecting a 10 Y.U. dose into 4 different sites on an individual within one administration period. The present invention includes administering an amount of the yeast-MUCl immunotherapeutic 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) to form a single dose at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites on an individual.

[0290] "Boosters" of therapeutic compositions are administered, for example, when the immune response to an antigen is waning or when it is necessary to provide an immune response or induce a memory response to a specific antigen. Boosters may be administered about 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart, or monthly, bimonthly, quarterly, annually, and / or in increments of several years or years after initial administration, depending on the state of the individual being treated and the therapeutic goal at the time of administration (e.g., preventive, active treatment, maintenance). In one embodiment, the administration regimen is one in which a dose of the yeast-MUCl immunotherapeutic composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times over a period of from several weeks to several months to several years. In one embodiment, for doses of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, the dose is administered weekly or biweekly, followed by biweekly or monthly administration as needed to achieve the desired preventive or therapeutic treatment of cancer. Additional booster doses may then be given at similar or longer intervals (months or years) as maintenance or remission therapy, as desired.

[0291] In one aspect of the invention, one or more additional therapeutic agents or treatment regimens are administered or performed sequentially and / or simultaneously with the administration of the yeast-MUCl immunotherapeutic composition, such as surgical resection of a tumor, administration of chemotherapy, administration of radiation therapy, administration of other immunotherapeutic compositions or regimens, including viral vector therapy and dendritic cell / tumor fusion therapy, cytokine therapy, adoptive T cell transfer (including adoptive transfer of T cells that have been stimulated ex vivo by antigen and / or immunotherapeutic composition), or stem cell transplantation. In one example, yeast-MUCl immunotherapy is administered in conjunction with a therapy utilizing a viral vector-based immunotherapy, such as described in PCT Publication No. WO / 00 / 34494. In another example, yeast-MUCl immunotherapy is administered in conjunction with dendritic cell / tumor cell fusion therapy or immune system cells (e.g., T cells) stimulated with such dendritic cell / tumor cell fusions, such as described in PCT Publication No. WO / 2009062001. In such embodiments, non-yeast-based immunotherapy may target MUCl or a different tumor antigen, and such therapy may include the additional administration of other agents, such as cytokines, antibodies, or other agents.

[0292] In one aspect, one or more therapies for cancer (including any of those described herein or known in the art) may be administered prior to or after administration of the first dose of the yeast-MUC1 immunotherapeutic composition. In one embodiment, one or more therapies may be administered or implemented in a manner alternating with administration of the yeast-MUC1 immunotherapeutic composition, such as in one protocol, administering the yeast-MUC1 composition at specified intervals between one or more consecutive doses of chemotherapy or other therapy. In one embodiment, one or more doses of the yeast-MUC1 immunotherapeutic composition are administered over a period of time prior to initiating an additional therapy. In other words, the yeast-MUC1 immunotherapeutic composition is administered as a monotherapy over a period of time, and then an additional therapy (e.g., chemotherapy) is added, either concurrently with a new yeast-MUC1 immunotherapeutic dose or in a manner alternating with the yeast-MUC1 immunotherapy. Alternatively, or additionally, another therapy may be administered for a period of time prior to initiating administration of the Yeast-MUCl immunotherapy composition, and concepts may be combined (e.g., surgical resection of a tumor, followed by monotherapy with Yeast-MUCl immunotherapy for several weeks, followed by alternating chemotherapy and Yeast-MUCl immunotherapy doses for several weeks or months, optionally followed by a new regimen of monotherapy with Yeast-MUCl immunotherapy and / or another therapy, or a combination of therapies given sequentially, simultaneously, or in an alternating manner). Various regimens for treating cancer using Yeast-MUCl immunotherapy are contemplated by the present invention, and these examples should be considered non-limiting examples of various possible regimens.

[0293] Virus-based immunotherapeutic compositions typically include a viral vector (e.g., a recombinant virus) comprising a viral genome or portion thereof and a nucleic acid sequence encoding at least one antigen from a pathogenic agent or disease state (e.g., a cancer antigen, an infectious disease antigen, and / or at least one immunogenic domain thereof). In some embodiments, a virus-based immunotherapeutic composition further includes at least one viral vector comprising one or more nucleic acid sequences encoding one or more immunostimulatory molecules. In some embodiments, genes encoding immunostimulatory molecules and antigens are inserted into the same viral vector (same recombinant virus).

[0294] Dendritic cell / tumor cell fusion immunotherapy compositions are generally hybrid cells generated by fusion between dendritic cells and non-dendritic cells (including tumor cells) expressing tumor antigens using fusion methods known in the art. The fused cells have dendritic cell characteristics and also express and present tumor antigens from tumor cells. The composition can be administered to an individual, or used to stimulate T cells in vitro for use in T cell transfer methods.

[0295] In one aspect of the invention, yeast-based immunotherapy is used to target antigens other than MUC1 in addition to MUC1. Such other target antigens can be incorporated into the same yeast vehicle as the MUC1 antigen, or additional yeast-based immunotherapy compositions can be generated that target different antigens and then combined as desired depending on the individual to be treated, the antigens expressed by the cancer type or the individual's particular tumor, and / or depending on the stage of cancer in the individual, or the stage of treatment for the individual. For example, a combination of antigens can be selected to cover: (1) antigens involved in important events in the development of cancer, such as mutated Ras, (2) antigens involved in or associated with dysregulated cellular processes, such as CEA or MUC1, and (3) Brachyury protein, which is involved in metastatic processes. For example, one or more other yeast-based immunotherapeutic compositions can express one or more antigens, including but not limited to carcinoembryonic antigen (CEA), point mutated Ras oncoprotein, Brachyura malformation protein, EGFR, BCR-Abl, MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-2, normal and point mutated p53 oncoprotein, PSMA, tyrosinase, TRP-1 (gp75), NY-ESO-1, 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-1, Rb-2, androgen receptor (AR), Smad4, MDR1, Flt-3, BRCA-1, BRCA-2, pax3-fkhr, ews-fli-1, HERV-H, HERV-K, TWIST, mesothelin, NGEP, modifications of such antigens, splice variants of such antigens, and epitope agonists of such antigens, and combinations of such antigens, and / or immunogenic domains thereof, modifications thereof, variants thereof, and / or epitope agonists thereof. One, two, three or more of these yeast-based immunotherapeutic compositions may be administered prior to, concurrently with, or alternating with, and / or after administration of a yeast-MUCl immunotherapeutic composition to optimize targeting of antigens in an individual's tumor. As above, additional therapies (e.g., surgical resection of the tumor, chemotherapy, targeted cancer therapy, radiation therapy, etc.) may also be used in such regimens.

[0296] In one embodiment of the present invention, a method for treating cancer is provided. The method comprises the following steps: (a) administering a first immunotherapeutic composition comprising a yeast vehicle and a MUC1 antigen as described herein to an individual with a cancer or precancerous tumor; and (b) administering one or more additional immunotherapeutic compositions to the individual before, simultaneously with, or after the administration of the first immunotherapeutic composition, each of which comprises a yeast vehicle and each of which comprises a different cancer antigen that is not a MUC1 antigen. The additional cancer antigens may be any known in the art or described herein, including but not limited to mutated Ras, carcinoembryonic antigen (CEA), brachyura malformation protein, EGFR, and the like. The steps may be repeated as needed to treat cancer in a particular individual, and the cancer antigens may be modified before or during treatment to specifically target cancer in a particular individual.

[0297] In the methods of the present invention, compositions and therapeutic compositions may be administered to any animal, including any vertebrate, and specifically to any member of the vertebrate class Mammalia, including, but not limited to, primates, rodents, livestock, and domesticated pets. Livestock include mammals that are consumed or produce usable products (e.g., sheep for wool production). Mammals to be treated or protected using the present invention include humans, non-human primates, dogs, cats, mice, rats, goats, sheep, cattle, horses, and pigs.

[0298] An "individual" is a vertebrate, such as a mammal, including, but not limited to, a human. Mammals include, but are not limited to, farm animals, sports animals, pets, primates, mice, and rats. The term "individual" can be used interchangeably with the terms "animal," "subject," or "patient."

[0299] General techniques that can be used in the present invention

[0300] Unless otherwise indicated, the practice of the present invention will employ 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 fully explained in the literature, such as Methods of Enzymology , Vol.194, Guthrie et al., 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, edited by Pringle et al., Cold Spring Harbor Laboratory Press (1997); The Yeast Saccharomyces:Gene Expression , Jones et al., eds., Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces:Genome Dynamics,Protein Synthesis, and Energetics , Broach et al., eds., Cold Spring Harbor Laboratory Press (1992); Molecular Cloning:A Laboratory Manual , second edition (Sambrook et al., 1989) and Molecular Cloning:A Laboratory Manual ,third edition(Sambrook and Russel, 2001),(collectively referred to as “Sambrook” in this paper); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987, including supplements to 2001); PCR:The Polymerase Chain Reaction , (Mullis et al., 1994); Harlow and Lane (1988), Antibodies,A Laboratory Manual , ColdSpring Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (collectively referred to herein as "Harlow and Lane"), Beaucage et al., eds. Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000); Casarett and Doull Toxicology Basic Science of Poisons , edited by C.Klaassen, 6th edition (2001), and Vaccines , S. Plotkin, W. Orenstein, and P. Offit, eds., Fifth Edition (2008).

[0301] General Definition

[0302] “ "(GlobeImmune, Inc., Louisville, Colorado) generally refers to a yeast vehicle that expresses one or more heterologous antigens extracellularly (on its surface), intracellularly (inside or in the cytosol), or both. (See, e.g., U.S. Pat. No. 5,830,463.) Certain yeast-based immunotherapeutic compositions, and methods of making and generally using the same, are also described in detail in, e.g., U.S. Pat. No. 5,830,463, U.S. Pat. No. 7,083,787, U.S. Pat. No. 7,736,642, Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research 64:5084-5088 (2004), and Bernstein et al., Vaccine 2008 Jan 24; 26(4):509-21, each of which is incorporated herein by reference in its entirety.

[0303] As used herein, the term "analog" refers to a chemical compound that is similar in structure to another compound but slightly different in composition (such as in that one atom is replaced by an atom of a different element or, where a particular functional group is present, one functional group is replaced by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance to a reference compound, but has a different structure or origin.

[0304] The terms "substituted," "substituted derivative," and "derivative," when used to describe a compound, mean that at least one hydrogen bound to the unsubstituted compound is replaced with a different atom or chemical moiety.

[0305] Although the derivative has a physical structure similar to the parent compound, the derivative may have chemical and / or biological properties that are different from the parent compound. Such properties may include, but are not limited to, increased or decreased activity of the parent compound, new activity compared to the parent compound, enhanced or decreased bioavailability, enhanced or decreased efficacy, enhanced or decreased in vitro and / or in vivo stability, and / or enhanced or decreased absorption characteristics.

[0306] Generally, the term "biological activity" indicates that a compound (including a protein or peptide) has at least one detectable activity that has an effect on the metabolism, physiology, chemical or other process of a cell, tissue or organism, as measured or observed in vivo (i.e., in a natural physiological environment) or in vitro (i.e., under laboratory conditions).

[0307] According to the present invention, the term "regulate" can be used interchangeably with "adjust" and generally refers to the upregulation or downregulation of a specific activity. As used herein, the term "upregulate" can be generally used to describe any of the following: elicitation, initiation, increase, increase, strengthen, improve, enhance, amplify, promote or provide for a specific activity. Similarly, the term "downregulate" can be generally used to describe any of the following: reduction, decrease, inhibition, improvement, reduction, decrease, blocking or prevention of a specific activity.

[0308] In one embodiment of the invention, any of the amino acid sequences described herein may be produced with at least one up to about 20 additional heterologous amino acids flanking each C- and / or N-terminal end of the specified amino acid sequence. The resulting protein or polypeptide may be referred to as "consisting essentially of" the specified amino acid sequence. According to the present invention, the heterologous amino acids are sequences of amino acids that are not naturally found (i.e., not found in vivo in nature) flanking the specified amino acid sequence, or are not involved in the function of the specified amino acids, or would not be encoded by nucleotides flanking the naturally occurring nucleic acid sequence encoding the specified amino acid sequence (such as those present in a gene), provided that such nucleotides in the naturally occurring sequence are translated using the standard codon usage of the organism from which the given amino acid sequence is derived. Similarly, the term "consisting essentially of" when referring to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding the specified amino acid sequence, which may be flanked by at least one, and up to about 60 additional heterologous nucleotides at each 5' and / or 3' end of the nucleic acid sequence encoding the specified amino acid sequence. The heterologous nucleotide is not found naturally (i.e., not found in vivo in nature) flanking the nucleic acid sequence encoding the specified amino acid sequence (as it occurs in a natural gene) and does not encode a protein that confers any additional function to the protein or alters the function of the protein having the specified amino acid sequence.

[0309] According to the present invention, the term "selective binding" refers to the ability of an antibody, antigen-binding fragment or binding partner of the present invention to preferentially bind to a specified protein. More specifically, the term "selective binding" refers to the specific binding of one protein to another (e.g., an antibody, its fragment or binding partner to an antigen), wherein the binding level measured by any standard assay (e.g., immunoassay) is statistically significantly higher than the background control of the assay. For example, when an immunoassay is performed, the control typically includes a reaction well / tube containing a single antibody or antigen-binding fragment (i.e., without antigen), wherein the amount of reactivity of the antibody or its antigen-binding fragment (e.g., non-specific binding to the well) in the absence of antigen is considered as background. Binding can be measured using a variety of methods standard in the art, including enzyme immunoassays (e.g., ELISA, immunoblotting assays, etc.).

[0310] General references to proteins or polypeptides used in the present invention include the full-length protein, or any fragment, domain (structural, functional or immunogenic), conformational epitope, or homologue or variant of a given protein. Fusion proteins may also be generally referred to as proteins or polypeptides. According to the present invention, an isolated protein is a protein (including a polypeptide or peptide) that has been removed from its natural environment (i.e., has been manipulated by humans) and may include, for example, purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins. Thus, "isolated" does not reflect the degree of protein purification. Preferably, the isolated protein of the present invention is recombinantly produced. According to the present invention, the terms "modification" and "mutation" are used interchangeably, particularly with respect to modifications / mutations of the amino acid sequences (or nucleic acid sequences) of proteins or portions thereof described herein.

[0311] As used herein, the term "homolog" or "variant" is used to refer to a protein or peptide that differs from a reference protein or polypeptide by minor modifications to the reference protein or peptide (i.e., a "prototype" or "wild-type" protein), but maintains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to: changes in one or several amino acid side chains; changes in one or several amino acids, including deletions (e.g., truncated forms of proteins or peptides), insertions and / or substitutions; changes in the stereochemistry of one or several atoms; and / or minor derivatizations, including, but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and / or addition of glycosylphosphatidylinositol. A homolog or variant may have enhanced, reduced, or nearly similar properties compared to a reference protein or peptide. A homolog or variant may include an agonist of a protein or an antagonist of a protein. Homologues or variants can be produced using techniques known in the art for protein production, including but not limited to, direct modification of an isolated reference protein, direct protein synthesis, or modification of a nucleic acid sequence encoding a protein using, for example, classical or recombinant DNA techniques to cause random or targeted mutagenesis, resulting in encoded protein variants. In addition, naturally occurring variants of the reference protein (e.g., isoforms, allelic variants, or other natural variants that may exist between individuals) may exist and may be isolated, produced, and / or utilized in the present invention.

[0312] A homolog 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% identical, or at least about 93% identical, or at least about 94% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least about 99% identical (or any percentage identity from 45% to 99% in whole integer increments) to the amino acid sequence of a reference protein (e.g., an amino acid sequence specified herein, or an amino acid sequence of a specified protein). In one embodiment, the homolog 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% identical to the amino acid sequence of the reference protein.

[0313] As used herein, unless otherwise specified, references to percent (%) identity refer to homology estimates performed using the following methods: (1) Basic Local Alignment Search Tool (BLAST) basic homology searches using blastp for amino acid searches and blastn for nucleic acid searches, using standard default parameters, wherein the search sequences are filtered by default for low complexity regions (as described in Altschul, SF, Madden, TL, AA, Zhang, J., Zhang, Z., Miller, W. & Lipman, DJ (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25: 3389-3402, incorporated herein by reference in its entirety); (2) BLAST alignment of the two sequences (e.g., using the parameters described below); (3) and / or PSI-BLAST (Position-Specific Iterated BLAST) using standard default parameters. Note that due to some differences in the standard parameters between Basic BLAST and BLAST for two sequences, two specific sequences may be identified as having significant homology when using the BLAST program, while a search using Basic BLAST using one of the sequences as a search sequence may not identify the second sequence in a superior match. In addition, PSI-BLAST provides an automated, easy-to-use "profile" search format, which is a sensitive method for finding sequence homologs. The program first performs a BLAST database search with a gap. The PSI-BLAST program utilizes information from any returned significant comparisons to construct a position-specific scoring matrix, which replaces the search sequence for the next round of database searches. Therefore, it should be understood that percentage identity can be measured using any of these programs.

[0314] Two specific sequences can be aligned to each other using BLAST, as described in Tatusova and Madden, (1999), "Blast 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250, which is incorporated herein by reference in its entirety. Such sequence alignments are performed with blastp or blastn, which use the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between two sequences, allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For clarity, the BLAST sequence alignment of two sequences is performed using the following standard default parameters.

[0315] For blastn, use the 0BLOSUM62 matrix:

[0316] Match bonus points = 1

[0317] Mismatch Penalty = -2

[0318] Open gap (5) and extended gap (2) penalties

[0319] Gap x_dropoff (50) expect (10)Word length (11)Filter (on)

[0320] For blastp, use the 0BLOSUM62 matrix:

[0321] Open gap (11) and extended gap (1) penalties

[0322] Gap x_No selection (50) expect (10)Word length (3)Filter (on).

[0323] An isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural environment (i.e., has been artificially manipulated), and its natural environment refers to the genome or chromosome where the nucleic acid molecule is found in nature. Therefore, "isolated" does not necessarily reflect the degree to which the nucleic acid molecule has been purified, but indicates that the molecule does not include the entire genome or entire chromosome where the nucleic acid molecule is found in nature, or a genomic segment containing more than one gene. An isolated nucleic acid molecule can include a complete gene. An isolated nucleic acid molecule containing a gene is not a chromosome fragment containing such a gene, but rather contains coding regions and regulatory regions associated with the gene, but no other genes naturally found on the same chromosome. An isolated nucleic acid molecule can also include a portion of a gene. An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and / or 3' end of the sequence) another nucleic acid, which is not usually flanked by the specified nucleic acid sequence in nature (i.e., a heterologous sequence). An isolated nucleic acid molecule can include DNA, RNA (e.g., mRNA), or a derivative of either DNA or RNA (e.g., cDNA). Although the term "nucleic acid molecule" mainly refers to the physical nucleic acid molecule, and the term "nucleic acid sequence" mainly refers to the nucleotide sequence on the nucleic acid molecule, the two terms can be used interchangeably, especially for nucleic acid molecules or nucleic acid sequences that can encode proteins or protein domains.

[0324] A recombinant nucleic acid molecule is a molecule that may contain at least one of any nucleic acid sequences encoding any one or more proteins described herein operably linked to at least one of any transcriptional regulatory sequences that are effective to regulate the expression of the nucleic acid molecule in the cell to be transfected. Although the term "nucleic acid molecule" refers primarily to a physical nucleic acid molecule and the term "nucleic acid sequence" refers primarily to a nucleotide sequence on a nucleic acid molecule, the two terms are used interchangeably, especially for nucleic acid molecules or nucleic acid sequences that can encode a protein. In addition, the term "recombinant molecule" refers primarily to a nucleic acid molecule operably linked to a transcriptional regulatory sequence, but is used interchangeably with the term "nucleic acid molecule" (administered to an animal).

[0325] Recombinant nucleic acid molecules include recombinant vectors, which are any nucleic acid sequences (usually heterologous sequences) that are operably linked to an isolated nucleic acid molecule encoding a fusion protein of the present invention, which enables the recombinant production of the fusion protein, and which enables the delivery of the nucleic acid molecule to a host cell according to the present invention. Such vectors may contain nucleic acid sequences that are not naturally visible adjacent to the isolated nucleic acid molecule to be inserted into the vector. The vector may be RNA or DNA, prokaryotic or eukaryotic, and preferably in the present invention, is a plasmid that can be used to transfect yeast. Recombinant vectors can be used for cloning, sequencing and / or manipulating nucleic acid molecules, and can be used to deliver such molecules (e.g., as in DNA compositions or compositions based on viral vectors). Preferably, the recombinant vector is used to express the nucleic acid molecule, which may also be referred to as an expression vector. Preferred recombinant vectors can be expressed in transfected host cells such as yeast.

[0326] In the recombinant molecules of the present invention, the nucleic acid molecules are operably connected to an expression vector containing a regulatory sequence, such as a transcriptional regulatory sequence, a translational regulatory sequence, a replication origin, and other regulatory sequences that are compatible with the host cell and control the expression of the nucleic acid molecules of the present invention. Specifically, the recombinant molecules of the present invention include nucleic acid molecules that are operably connected to one or more expression regulatory sequences. The term "operably connected" refers to a nucleic acid molecule that is connected to an expression regulatory sequence in a manner that the molecule is expressed when transfected (i.e., transformed, transduced, or transfected) into a host cell.

[0327] 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. When such terms are used to refer to the introduction of nucleic acid molecules into microbial cells, such as algae, bacteria and yeast, the term "transformation" can be used interchangeably with the term "transfection". In microbial systems, the term "transformation" is used to describe the genetic changes caused by the acquisition of exogenous nucleic acids by microorganisms 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, microinjection, lipofection, adsorption, infection and protoplast fusion.

[0328] The following experimental results are provided to illustrate but are not intended to limit the scope of the present invention. Example

[0329] Example 1

[0330] The following example describes the production of a yeast-MUCl immunotherapeutic composition designated GI-6101.

[0331] In this experiment, yeast (Saccharomyces cerevisiae) was engineered to express human MUC1 antigen under the control of the copper-inducible promoter CUP1 or the constitutive promoter TEF2 to produce yeast-MUC1 immunotherapeutic compositions. In each case, a fusion protein comprising the MUC1 antigen was produced as a single polypeptide having the following sequence elements fused in frame from the N- to the C-terminus, represented by SEQ ID NO: 15: (1) MUC1 SEA / ED segment (positions 1-59 of SEQ ID NO: 15); (2) a VNTR segment comprising two VNTR domains (positions 60-100 of SEQ ID NO: 15); (3) MUC1 TM domain (positions 101-128 of SEQ ID NO: 15); and (4) MUC1 CD (positions 129-200 of SEQ ID NO: 15). The fusion protein also comprises a MUC1 signal sequence (positions 1-30 of SEQ ID NO:14), which may be substituted with a different N-terminal sequence designed to confer resistance to proteasomal degradation and / or stabilize expression, such as the peptide represented by SEQ ID NO:21, or an N-terminal peptide from a yeast alpha leader sequence such as SEQ ID NO:19 or SEQ ID NO:20. The complete fusion protein comprising an N-terminal MUC1 signal sequence and a hexa-histidine C-terminal tag to facilitate identification and / or purification of the protein is a single polypeptide having the following sequence elements fused in frame from the N to the C terminus, represented by SEQ ID NO: 14: (1) MUC1 signal sequence (positions 1-30 of SEQ ID NO: 14); (2) MUC1 SEA / ED segment (positions 31-89 of SEQ ID NO: 14); (3) VNTR segment comprising two VNTR domains (positions 90-130 of SEQ ID NO: 14); (4) MUC1 TM domain (positions 131-158 of SEQ ID NO: 14); (5) MUC1 CD (positions 159-230 of SEQ ID NO: 14); and (6) hexa-histidine tag (positions 231-236 of SEQ ID NO: 14). SEQ ID NO: 14 is encoded by the nucleotide sequence represented by SEQ ID NO: 13 (codons optimized for yeast expression).

[0332] Briefly, DNA encoding the MUC1 antigen was synthesized and then inserted into the yeast 2μm expression vector at the EcoRI and NotI cloning sites behind the CUP1 promoter (vector pGI-100) or the TEF2 promoter (vector plu011). A nucleotide sequence encoding a C-terminal 6-histidine peptide was added to the plasmid vector to encode the complete fusion protein represented by SEQ ID NO: 14. The resulting plasmid was transformed into DH5α for plasmid storage and into Saccharomyces cerevisiae W303α for production of yeast-MUC1 immunotherapy compositions.

[0333] Transformation into Saccharomyces cerevisiae was performed by lithium acetate / polyethylene glycol transfection, and initial transfectants were selected on solid minimal plates lacking uracil (UDM; uridine deletion medium). Colonies were selected by growth at 30°C in U2 (uridine deletion medium) or UL2 (uridine and leucine deletion medium) medium.

[0334] The yeast-MUCl immunotherapeutic composition, also referred to herein as GI-6101, comprises a polynucleotide encoding the human MUCl fusion protein represented by SEQ ID NO: 14 under the control of the TEF2 promoter.

[0335] Liquid cultures lacking uridine (U2) or lacking uridine and leucine (UL2) were inoculated using the plates and starter cultures described above and grown at 30°C, 250rpm for approximately 24h. pH-buffered UL2 medium (BT-UL2) containing 4.2g / L Bis-Tris was also inoculated from a frozen yeast bank to evaluate yeast-MUC1 immunotherapeutics produced under neutral pH production conditions (data not shown). Cultivation in pH-buffered UL2 medium exposed the β-glucan on the yeast cell wall and was thought to alter the yeast-induced cellular immune response, which is a result of altered interaction with the dectin receptor on antigen presenting cells. Other culture conditions were the same regardless of whether U2, UL2 or BT-UL2 was used. The primary culture was used to inoculate the final culture with the same formulation.

[0336] U2 liquid medium formula:

[0337] 15g / L glucose

[0338] 6.7g / L Yeast Nitrogen Base with Ammonium Sulfate

[0339] 0.04g / L each of histidine, tryptophan, adenine and 0.06g / L leucine

[0340] UL2 liquid medium formula:

[0341] 15g / L glucose

[0342] 6.7g / L Yeast Nitrogen Base with Ammonium Sulfate

[0343] 0.04 g / L each of histidine, tryptophan and adenine

[0344] In initial experiments comparing yeast-MUC1 immunotherapeutic compositions under the control of different promoters, CUP1-driven (inducible expression) yeast-MUC1 expression was generated in 2-step or 3-step cultures; after the start or intermediate culture reached mid-log phase (1.5-4 Y.U. / ml), the intermediate culture was finally diluted to a culture of 0.1 or 0.2 YU / mL, and expression was initiated by adding 0.375 mM copper sulfate and continued until the culture reached a density of 1.5-3 Y.U. TEF2-driven yeast-MUC1 expression was constitutive, and growth of these cells continued until the culture reached a density of 1.1 to 4.0 YU / ml. Cells from each culture were then harvested, washed in PBS and heat killed at 56°C for 1 hour.

[0345] After heat killing of the cultures, cells were washed 3 times in PBS. Total protein expression was measured by TCA precipitation / nitrocellulose binding assay and by Western blotting using anti-his tag monoclonal antibody and anti-MUC1 (VNTR) antibody (sc-7313, Santa Cruz). Protein content was quantified using a semi-quantitative digital imaging method. GI-6101 is expected to express the MUC1 fusion protein as a membrane-associated protein of approximately 25 kDa.

[0346] Figure 2A The expression of MUCl antigen in GI-6101 is shown, which was detected using anti-MUCl (VNTR) and anti-His antibodies. These results show good expression of MUCl protein. Figure 2B Shown is the MUCl antigen of GI-6101 after deglycosylation with EdoH or PNGase F. The figure shows that the GI-6101 fusion protein is expressed as a product of glycosylation, as the size of the fusion protein is larger than estimated before deglycosylation, but is reduced to the expected size (25 kDa) after deglycosylation by EdoH or PNGase F.

[0347] Quantification of antigen expression in GI-6101 grown under standard culture conditions was compared to GI-6101 grown under neutral conditions, as described above. Antigen expression levels were approximately the same using either process (data not shown), demonstrating that the neutral pH process does not alter the level of MUCl antigen expression by this yeast.

[0348] Example 2

[0349] The following example describes the production of a yeast-MUCl immunotherapeutic composition designated GI-6104.

[0350] In this experiment, yeast (Saccharomyces cerevisiae) was engineered to express human MUC1 antigen under the control of the copper-inducible promoter CUP1 or the constitutive promoter TEF2 to produce yeast-MUCl immunotherapeutic compositions. In each case, a fusion protein comprising a MUC1 antigen was produced as a single polypeptide having the following sequence elements fused in frame from the N- to the C-terminus, represented by SEQ ID NO:18: SEQ ID NO:18 comprises the following multiple MUC1 antigens in the following order from N- to C-terminus: (1) a first MUC1 CD (positions 1-72 of SEQ ID NO:18); (2) a second MUC1 CD (positions 73-144 of SEQ ID NO:18); and (3) a third MUC1 CD (positions 145-216 of SEQ ID NO:18). The fusion protein also comprises an N-terminal sequence designed to confer resistance to proteasomal degradation and / or stabilize expression (represented in this fusion protein by SEQ ID NO:21). Alternatively, a fusion protein can be designed using the MUCl signal sequence (eg, positions 1-30 of SEQ ID NO:14), another synthetic N-terminal peptide as described herein, or using an N-terminal peptide from a yeast alpha leader sequence such as SEQ ID NO:19 or SEQ ID NO:20. The complete fusion protein, including an N-terminal peptide and a hexa-histidine C-terminal tag (to aid in identification and / or purification of the protein), is a single polypeptide having the following sequence elements (represented by SEQ ID NO: 17) fused in frame from the N to the C terminus: (1) a synthetic peptide represented by SEQ ID NO: 21 (positions 1-6 of SEQ ID NO: 17); (2) a first MUCl CD (positions 7-78 of SEQ ID NO: 17); (3) a second MUCl CD (positions 79-150 of SEQ ID NO: 17); (4) a third MUCl CD (positions 151-222 of SEQ ID NO: 17); and (5) a hexa-histidine tag (positions 223-228 of SEQ ID NO: 17). SEQ ID NO: 17 is encoded by the nucleotide sequence represented by SEQ ID NO: 16 (codons optimized for yeast expression).

[0351] Briefly, DNA encoding the MUC1 antigen was synthesized and then inserted into the yeast 2μm expression vector at the EcoRI and NotI cloning sites behind the CUP1 promoter (vector pGI-100) or the TEF2 promoter (vector plu011). A nucleotide sequence encoding a C-terminal 6-histidine peptide was added to the plasmid vector to encode the complete fusion protein represented by SEQ ID NO: 17. The resulting plasmid was transformed into DH5α for plasmid storage and into Saccharomyces cerevisiae W303α for production of yeast-MUC1 immunotherapy compositions.

[0352] Transformation into Saccharomyces cerevisiae was performed by lithium acetate / polyethylene glycol transfection, and initial transfectants were selected on solid minimal plates lacking uracil (UDM; uridine deletion medium). Colonies were selected by growth at 30°C in U2 (uridine deletion medium) or UL2 (uridine and leucine deletion medium) medium.

[0353] The yeast-MUCl immunotherapeutic composition, also referred to herein as GI-6104, comprises a polynucleotide encoding the human MUCl fusion protein represented by SEQ ID NO: 17 under the control of the CUP1 promoter.

[0354] Liquid cultures lacking uridine (U2) or lacking uridine and leucine (UL2) (media formulations are provided in Example 1) were inoculated using the plates and starter cultures described above and grown at 30°C, 250 rpm for approximately 24 h. pH-buffered UL2 medium (BT-UL2) containing 4.2 g / L Bis-Tris was also inoculated from a frozen yeast bank to evaluate yeast-MUC1 immunotherapeutics produced under neutral pH production conditions (data not shown). Cultivation in pH-buffered UL2 medium exposes the β-glucan on the yeast cell wall and is believed to alter the yeast-induced cellular immune response as a result of altered interaction with the dectin receptor on antigen presenting cells. Other culture conditions were the same regardless of whether U2, UL2 or BT-UL2 was used. The primary culture was used to inoculate the final culture with the same formulation.

[0355] In initial experiments comparing yeast-MUC1 immunotherapeutic compositions under the control of different promoters, CUP1-driven (inducible expression) yeast-MUC1 expression was generated in 2-step or 3-step cultures; after the start or intermediate culture reached mid-log phase (1.5-4 Y.U. / ml), the intermediate culture was diluted to a final culture of 0.1 or 0.2 YU / mL, and expression was initiated by adding 0.375 mM copper sulfate and continued until the culture reached a density of 1.5-3 Y.U. CUP1-driven (inducible expression) yeast-MUC1 expression was also initiated by adding 0.375 mM copper sulfate after the final yeast-MUC1 culture reached a density of approximately 2 Y.U. / ml and induced for 4-6 hours. TEF2-driven yeast-MUC1 expression was constitutive, and growth of these cells continued until the culture reached a density of 1.1 to 4.0 YU / ml. Cells from each culture were then harvested, washed in PBS and heat killed at 56°C for 1 hour.

[0356] After heat killing of the cultures, cells were washed three times in PBS. Total protein expression was measured by TCA precipitation / nitrocellulose binding assay and by Western blotting using anti-his tag monoclonal antibody and anti-MUC1 (C-terminal) antibody (sc-6827, Santa Cruz). Protein content was quantified using a semi-quantitative digital imaging method. GI-6104 is expected to express the MUC1 fusion protein as a cytosolic protein of approximately 25 kDa.

[0357] Figure 2C The expression of MUCl antigen in GI-6104 is shown, which was detected using anti-MUCl (C-terminal) and anti-His antibodies. These results show good expression of MUCl protein. Figure 2B Shown is the MUCl antigen of GI-6104 after deglycosylation with EdoH. This figure shows that the GI-6104 fusion protein is not expressed as a glycosylation product, as the size of the fusion protein is the same before and after deglycosylation with EdoH.

[0358] Quantification of antigen expression in GI-6104 grown under standard culture conditions was compared to GI-6104 grown under neutral conditions, as described above. Antigen expression levels were approximately the same using either process (data not shown), demonstrating that the neutral pH process does not alter the level of MUCl antigen expression by this yeast.

[0359] Example 3

[0360] The following example describes the production of a yeast-MUCl immunotherapeutic composition designated GI-6105.

[0361] In this experiment, yeast (Saccharomyces cerevisiae) was engineered to express human MUC1 antigen under the control of the copper-inducible promoter CUP1 to generate a yeast-MUC1 immunotherapeutic composition. Antigens from the yeast-MUC1 immunotherapeutic composition of GI-6101 (see Example 1) were used as templates to design MUC1 agonist antigens. Briefly, a fusion protein comprising a MUC1 agonist antigen is produced as a single polypeptide having the following sequence elements fused in frame from the N- to the C-terminus, represented by SEQ ID NO:23: (1) a MUC1 signal sequence (positions 1-30 of SEQ ID NO:23); (2) a MUC1 SEA / ED segment (positions 31-89 of SEQ ID NO:23); (3) a VNTR segment comprising two VNTR domains (positions 90-130 of SEQ ID NO:23); (4) a MUC1 TM domain (positions 131-158 of SEQ ID NO:23); (5) a MUC1 CD (positions 159-230 of SEQ ID NO:23); (6) a MUC1 agonist epitope (positions 231-246 of SEQ ID NO:23) and (7) a hexa-histidine tag (positions 247-252 of SEQ ID NO:23). SEQ ID NO:23 is encoded by the nucleotide sequence represented by SEQ ID NO:22 (codons optimized for yeast expression). The MUC1 signal sequence (positions 1-30 of SEQ ID NO:23) can be substituted with a different N-terminal sequence designed to confer resistance to proteasomal degradation and / or stabilize expression, such as the peptide represented by SEQ ID NO:21, or an N-terminal peptide from a yeast alpha leader sequence such as SEQ ID NO:19 or SEQ ID NO:20. A 6-histidine C-terminal tag is optional and facilitates identification and / or purification of the protein.Compared to the antigen in GI-6101 (e.g., SEQ ID NO: 14 or 15), the sequence in GI-6105 contains the following amino acid substitutions to create various agonist epitopes (the substitution positions refer to SEQ ID NO: NO:23, with further reference to the substituted positions in wild-type MUC1 represented by accession number NP_001191214): A96Y (position 161 in wild-type MUC1), P97L (position 162 in wild-type MUC1), G104V (position 169 in wild-type MUC1), S105Y (position 170 in wild-type MUC1), T106L (position 171 in wild-type MUC1), A147Y (position 392 in wild-type MUC1), C161V (position 406 in wild-type MUC1), T199L (position 444 in wild-type MUC1), D200F (position 445 in wild-type MUC1), S215Y (position 460 in wild-type MUC1), and T239L (position 93 in wild-type MUC1).

[0362] A plasmid containing the MUC1 agonist antigen of GI-6105 (SEQ ID NO: 23) was transfected into W303α yeast and transformants were selected after 3 days of growth on uridine dropout agar (UDA) at 30° C. Single colonies were restreaked onto uridine and leucine dropout agar (ULDA) plates and incubated at 30° C. for another 4 days to select for cells with increased plasmid copy number.

[0363] A single colony of GI-6105 was removed from the ULDA plate and used to inoculate 25 mL of UL2 liquid medium (starter culture). The starter culture was incubated at 30°C with shaking to a density of 3.7 YU / mL and then used to inoculate an intermediate culture to 0.3 YU / mL. The intermediate culture was grown to a density of 3.0 YU / mL and then used to inoculate a final culture to a density of 0.04 YU / mL. The final culture was grown to a density of 3.6 YU / mL and then treated with 0.5 mM copper sulfate at 30°C for 3 h to induce Muc1 agonist v1.0 antigen expression.

[0364] The induced cells were washed once with PBS, heat killed at 56°C for 1 h, and then washed three times in PBS. The total protein content of the heat-killed cells was measured by the Amidoschwarz assay, and the antigen content was measured by western blotting using a monoclonal antibody that recognizes the C-terminal 6-histidine epitope tag. The amount of antigen was determined by interpolation of a standard curve consisting of HCV NS3 protein with a his-tagged protein. Figure 3 As shown in , yeast expressed the antigen, and the antigen content of GI-6105 was estimated to be about 2801 Ng / YU.

[0365] Example 4

[0366] The following example describes the production of a yeast-MUCl agonist immunotherapeutic composition designated GI-6106.

[0367] In this experiment, yeast (Saccharomyces cerevisiae) was engineered to express human MUC1 agonist antigen under the control of the copper-inducible promoter CUP1 to produce a yeast-MUC1 agonist immunotherapeutic composition. The full-length wild-type MUC1 antigen with accession number NP_001191214 was used to design the MUC1 agonist antigen, although other wild-type MUC1 proteins can also be used to design similar agonists. Briefly, a fusion protein comprising a MUC1 agonist antigen is produced as a single polypeptide having the following sequence elements fused in frame from the N to the C terminus, represented by SEQ ID NO:25: (1) the alpha factor leader sequence disclosed elsewhere herein by SEQ ID NO:19 (positions 1-89 of SEQ ID NO:25); (2) a Thr-Ser linker sequence (positions 90-91 of SEQ ID NO:25); (3) a full-length MUC1 agonist protein corresponding to the wild-type protein except for the introduction of 11 agonist epitopes (positions 92-566 of SEQ ID NO:25) and (7) a hexa-peptide histidine tag (positions 567-572 of SEQ ID NO:25). SEQ ID NO:25 is encoded by the nucleotide sequence represented by SEQ ID NO:24 (codons optimized for yeast expression). The alpha leader sequence (positions 1-89 of SEQ ID NO:25) can be substituted with different N-terminal sequences designed to confer resistance to proteasomal degradation and / or stabilize expression, such as the peptide represented by SEQ ID NO:21, or N-terminal peptides from different yeast alpha leader sequences such as SEQ ID NO:20, or the MUC1 signal sequence. A 6-histidine C-terminal tag is optional and facilitates identification and / or purification of the protein. Compared to the wild-type MUC1 protein used as a template, the sequence in GI-6106 contains the following amino acid substitutions to create various agonist epitopes (the substitution positions are referred to in SEQ ID NO:21). NO:25, with further reference to the substituted positions in wild-type MUC1 represented by accession number NP_001191214): T184L (position 93 in wild-type MUC1), A232Y (position 161 in wild-type MUC1), P233L (position 162 in wild-type MUC1), G240V (position 169 in wild-type MUC1), S241Y (position 170 in wild-type MUC1), T242L (position 171 in wild-type MUC1), A483Y (position 392 in wild-type MUC1), C497V (position 406 in wild-type MUC1), T535L (position 444 in wild-type MUC1), D536F (position 445 in wild-type MUC1), and S551Y (position 460 in wild-type MUC1).

[0368] A plasmid containing the MUC1 agonist antigen of GI-6106 was transfected into W303α yeast and transformants were selected after 3 days of growth on uridine dropout agar (UDA) at 30° C. Single colonies were restreaked onto uridine and leucine dropout agar (ULDA) plates and incubated at 30° C. for another 4 days to select for cells with increased plasmid copy number.

[0369] A single colony of GI-6106 was removed from the ULDA plate and used to inoculate 25 mL of UL2 liquid medium (starter culture). The starter culture was incubated at 30°C with shaking to a density of ~3 YU / mL and then used to inoculate the intermediate culture to 0.3 YU / mL. The intermediate culture was grown to a density of 3 YU / mL and then used to inoculate the final culture to a density of 0.04 YU / mL. The final culture was grown to a density of 3 YU / mL and then treated with 0.5 mM copper sulfate at 30°C for 3 h to induce Muc1 agonist v2.0 antigen expression.

[0370] The induced cells were washed once with PBS, heat killed at 56°C for 1 h, and then washed three times in PBS. The total protein content of heat-killed cells was measured by Amidoschwarz assay, and the agonist antigen content was measured by western blotting, which used a monoclonal antibody that recognizes the C-terminal 6-histidine epitope tag. The amount of antigen was determined by interpolation of a standard curve consisting of HCV NS3 protein with a his tag. The results showed that GI-6106 yeast expressed antigen (data not shown); the antigen content of GI-6106 was estimated to be approximately 2940 Ng / YU.

[0371] Example 5

[0372] The following examples describe phenotypic and functional analyses of the effects of yeast-MUCl immunotherapeutic compositions on human dendritic cells.

[0373] To evaluate the effects of the yeast-MUCl immunotherapeutic compositions described in Examples 1 and 2 on dendritic cell phenotype and function, the following experiments were performed.

[0374] In the first experiment, human dendritic cells (DCs) were cultured for 48 hours with: (1) medium alone (untreated), (2) CD40L (1 μg / ml) plus an enhancer of the ligand (1 μg / ml) as a positive control; (3) control yeast (control yeast; yeast containing an empty vector (no MUC1 antigen insert)); (4) a yeast-MUC1 immunotherapeutic composition called GI-6101, grown under standard growth conditions as described in Example 1 (GI-6101); and (5) a yeast-MUC1 immunotherapeutic composition called GI-6101. -6101, grown under neutral pH growth conditions as described in Example 1 (GI-6101(DEC)); (6) a yeast-MUC1 immunotherapeutic composition designated GI-6104 (GI-6104), grown under standard growth conditions as described in Example 2; or (7) a yeast-MUC1 immunotherapeutic composition designated GI-6104, grown under neutral pH growth conditions as described in Example 2 (GI-6104(DEC)). Dendritic cells and yeast were mixed at a ratio of 1:10 (DC:yeast). DCs were harvested and analyzed for DC surface marker expression by flow cytometry. The results are shown in Table 1 below as the percentage of positive cells and MFI (in parentheses).

[0375] Table 1

[0376]

[0377] The results showed that both yeast (control yeast and yeast expressing MUC1 antigen) upregulated the expression of CD80 and CD83 on dendritic cells compared to untreated cells. CD80, or B7.1, is a co-stimulatory molecule required for T cell activation and survival, which is upregulated on activated dendritic cells. CD83 is a marker of dendritic cell maturation. Therefore, this experiment shows that the yeast-MUC1 immunotherapy composition can upregulate DC maturation markers.

[0378] In a second experiment, cytokine and chemokine production by dendritic cells was assessed following culture with yeast-MUC1 immunotherapeutics. Briefly, human DCs from normal donors (donors considered cancer-free) were cultured with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5 days, or treated with CD40L (1 μg / ml, Enzo Life Sciences) plus an enhancer of the ligand (1 μg / ml, Enzo Life Sciences) for 24 hours, or treated with control yeast (empty vector), GI-6101 cultured under standard growth conditions (GI-6101), GI-6101 cultured under neutral pH conditions (GI-6101(DEC)), GI-6104 cultured under standard growth conditions (GI-6104), or GI-6104 cultured under neutral pH conditions (GI-6104(DEC)) for 48 hours (DC:yeast ratio = 1:10). The culture supernatants were collected and screened for cytokine and chemokine production by a multiplex cytokine / chemokine kit. The results were expressed as pg / ml and are shown in Table 2.

[0379] Table 2

[0380]

[0381] The results in Table 2 show that treatment of dendritic cells from normal donors with the yeast-MUC1 immunotherapeutic compositions described in Examples 1 and 2 increased cytokine and chemokine production by these cells. In particular, interferon-γ (IFN-γ) production was increased following exposure to yeast-MUC1 immunotherapeutic compositions grown under neutral pH conditions, which are expected to enhance TH1 and CD8+ T cell responses. In addition, yeast-MUC1 immunotherapeutic compositions grown under neutral pH conditions induced higher levels of cytokine and chemokine production by DCs, with the yeast-MUC1 immunotherapeutic composition designated GI-6101 (neutral pH) showing the highest stimulation of DC cytokine and chemokine production. Numbers highlighted in bold show statistically significantly improved cytokine / chemokine production over untreated controls (data not shown).

[0382] The experiments of Table 2 were repeated using dendritic cells isolated from another normal donor. The results (data not shown) were comparable to those shown in Table 2 and confirmed that the yeast-MUC1 immunotherapeutic composition induced the production of cytokines and chemokines by dendritic cells, and that of the two yeast-MUC1 compositions grown under each condition, the yeast-MUC1 composition called GI-6101 grown under neutral pH growth conditions induced the highest levels of cytokine and chemokine production by dendritic cells.

[0383] Taken together, these results demonstrate that the yeast-MUCl immunotherapeutic composition can activate dendritic cells and induce the production of cytokines and chemokines associated with a productive immune response.

[0384] Example 6

[0385] The following examples show that the yeast-MUCl immunotherapeutic compositions of the present invention can activate MUCl-specific T cells.

[0386] T-3-P93L is a MUC-1 specific T cell line that specifically recognizes a MUC1 agonist peptide (called P93L) in the context of HLA-A2. P93L is a peptide spanning positions 92-101 of the full-length MUC1 protein (e.g., ATWGQDVTSV; positions 92-101 of SEQ ID NO: 11), except that the threonine at position 2 of the peptide (position 93 in positions 92-101 of SEQ ID NO: 11) is substituted with leucine, thereby creating an agonist peptide. P93L binds to HLA-A2 at a higher level than the native (wild-type) peptide, and is a better inducer of MUC1-specific T cells than the native peptide (higher levels of TH1 cytokine production (see U.S. Patent Application Publication No. 2008 / 0063653)). The T cell line T-3-P93L can specifically cleave HLA-A2-positive, MUC1-positive tumor targets in vitro (data not shown). This T cell line is specific for a portion of MUCl within the MUCl-N subunit.

[0387] In this experiment, DCs from normal donors prepared as described in Example 5 were treated with the yeast-immunotherapeutic composition described in Examples 1 and 2, or with control yeast (empty vector), CD40L (positive control), or untreated (negative control) using the conditions described in Example 5 above. DCs treated with control yeast or CD40L were pulsed with or without P93 L peptide (P93L was used at 10 μg / ml). The treated DCs were then used as antigen presenting cells (APCs) to assess their ability to stimulate the MUC1-specific T cell line T-3-P93L (T cell: DC ratio = 10: 1). 24-hour culture supernatants were collected and screened for secretion of interferon-γ (IFN-γ). The results are shown in Table 3, expressed as the amount of IFN-γ produced by T cells, in pg / ml.

[0388] Table 3

[0389]

[0390]

[0391] The results showed that dendritic cells treated with GI-6101, which was produced under standard and neutral pH conditions and expressed the VNTR domain of the MUCl-N subunit, could stimulate MUCl-N-specific T cells to produce significant amounts of IFN-γ. GI-6104, which does not express antigens from the MUCl-N protein (GI-6104 only expresses MUCl antigens from the cytoplasmic domain (CD)), did not stimulate MUCl-N-specific T cells.

[0392] In a second experiment, a MUCl-C specific T cell line designated T-15-P1240(1Y) was stimulated with DCs that had been treated in the above experiments to determine whether the yeast-MUCl immunotherapeutic composition of the present invention could stimulate these T cells. The T-15-P1240(1Y) cell line is a MUC-1 specific T cell line that specifically recognizes a MUC1 agonist peptide (designated P1240(1Y), which is a MUC1-C peptide) in the context of HLA-A2. P1240(1Y) is a peptide that spans positions 1240-1248 of the full-length MUC1 protein (e.g., SLSYTNPAV; positions 1240-1248 of SEQ ID NO: 11), except that the serine at position 1 of the peptide (position 1240 in positions 1240-1248 of SEQ ID NO: 11) is substituted with a lysine, thereby creating an agonist peptide. P1240(1Y) binds HLA-A2 as well or better than the native (wild-type) peptide and is a better inducer of MUC1-specific T cells (higher TH1 cytokine production levels) than the native peptide. The T cell line T-15-P1240(1Y) is able to specifically lyse HLA-A2-positive, MUC1-positive tumor targets in vitro (data not shown). This T cell line is specific for a portion of MUC1 within the MUC1-C subunit, specifically the cytoplasmic domain (CD).

[0393] In this experiment, DCs were generated from PBMCs of healthy HLA-A2 positive donors and prepared as described in Example 5. DCs were treated with the yeast-immunotherapeutic composition described in Examples 1 and 2, or with CD40L (positive control) using the conditions described in Example 5 above or were left untreated (negative control). DCs treated with CD40L were pulsed with or without P1240 (1Y) peptide (the peptide was used at 10 μg / ml). The treated DCs were then used as antigen presenting cells (APCs) to assess their ability to stimulate the MUC1-specific T cell line T-15-P1240 (1Y) (T cell: DC ratio = 10: 1). 24 hour culture supernatants were collected and screened for secretion of interferon-γ (IFN-γ). The results are shown in Table 4, expressed as the amount of IFN-γ produced by T cells, in pg / ml.

[0394] Table 4

[0395]

[0396] The results showed that both GI-6101 and GI-6104 grown under standard or neutral pH conditions stimulated MUCl-C specific T cells to produce significant amounts of IFN-γ. Yeast-MUCl immunotherapeutic compositions grown under neutral pH conditions (both GI-6101 and GI-6104) stimulated T cells to produce higher levels of IFN-γ than yeast-MUCl immunotherapeutic compositions grown under standard conditions.

[0397] In a third experiment, the experiment described in Table 4 above was repeated but using different DC:T cell ratios for DCs treated with the yeast-MUCl immunotherapeutic compositions GI-6101 and GI-6104. The results are presented in Table 5 below.

[0398] Table 5

[0399]

[0400]

[0401] The results again showed that both GI-6101 and GI-6104 grown under standard or neutral pH conditions stimulated MUCl-C specific T cells to produce significant amounts of IFN-γ, and that the composition grown under neutral pH conditions stimulated T cells to produce higher levels of IFN-γ than the yeast-MUCl immunotherapy composition grown under standard conditions. The results also showed a dose response as the number of DCs increased relative to the number of T cells.

[0402] In summary, these results show that the yeast-MUC1 immunotherapeutic composition can activate MUC1-specific T cells in an antigen-specific manner, as exemplified by IFN-γ release from T cells stimulated by DC treated with the yeast-MUC1 immunotherapeutic composition. These results also show the advantage in IFN-γ production by T cells resulting from yeast-MUC1 immunotherapeutic compositions grown under neutral pH conditions.

[0403] Example 7

[0404] The following examples show that the yeast-MUCl compositions of the invention can expand and stimulate MUCl -specific T cells from cancer patients.

[0405] In this experiment, DCs were prepared from PBMCs of cancer patients (after treatment with cancer therapy, which may include chemotherapy or viral vaccine treatment, and / or before treatment). DCs were prepared by culturing for 5 days in the presence of GM-CSF and IL-4, followed by incubation in the presence of yeast (GI-6101 and / or GI-6104, cultured under standard or neutral pH conditions). After 48 hours of co-culture, DCs were used as APCs to stimulate autologous T cells, and cytokine production and / or proliferation of CD4+ T cells were measured. Yeast-MUC1 immunotherapy compositions are expected to expand and activate T cells from cancer patients.

[0406] In a second experiment, DCs were prepared from PBMCs of cancer patients (after treatment with cancer therapy, which may include chemotherapy or viral vaccine treatment, and / or before treatment). DCs were prepared by culturing for 5 days in the presence of GM-CSF and IL-4, followed by incubation in the presence of yeast (GI-6101 and / or GI-6104, cultured under standard or neutral pH conditions). After 48 hours of co-culture, DCs were used as APCs to stimulate autologous T cells. Each round of IVS consisted of 3 days in the absence of IL-2, followed by another 4 days in the presence of 20 U / ml recombinant IL-2. Tetramers specific for the MUC1 peptide were used to detect the percentage of CD8+T cells expanded by treatment with the yeast-MUC1 immunotherapy composition. The yeast-MUC1 immunotherapy composition is expected to expand and activate T cells from cancer patients.

[0407] In a third experiment, a yeast-MUC1 immunotherapeutic composition was used to generate MUC1-specific CTLs from PBMCs that lyse MUC1-expressing targets. In this experiment, MUC1-specific T cells from normal donors and / or cancer patients (after and / or before treatment with cancer therapy, which may include chemotherapy or viral vaccine therapy) were expanded in vitro and subjected to two rounds of in vitro stimulation (IVS) using DCs incubated with a yeast-MUC1 immunotherapeutic composition (GI-6101 or GI-6104). On day 5, CD8 + T cells and used in an overnight CTL assay against MUCl expressing tumor cells. These experiments are expected to demonstrate that the yeast-MUCl immunotherapeutic composition can generate MUCl -specific CTLs capable of killing MUCl expressing tumor cells.

[0408] Example 8

[0409] The following examples demonstrate that immunization with a yeast-MUCl immunotherapeutic composition reduces MUCl -expressing tumors in vivo.

[0410] In this experiment, mice received tumor cells expressing recombinant human MUC1 protein via the tail vein (day 0). Four days after tumor implantation, animals received weekly vaccinations of yeast-MUC1 (GI-6101 or GI-6104) with yeast control (YVEC, or empty vector yeast) at a dose of 1 YU per site administered at 4 different sites (4 YU per dose total). At 40 days after tumor implantation, animals were killed and the number of lung tumor nodules was assessed. The yeast-MUC1 immunotherapy composition is expected to reduce MUC1-expressing tumors in mice compared to mice that received yeast alone (no MUC1 antigen).

[0411] Example 9

[0412] The following example describes a Phase 1 clinical trial in subjects with MUCl -positive cancer.

[0413] An open-label, dose-expansion Phase 1 clinical trial was conducted using either the yeast-MUC1 immunotherapeutic composition called GI-6101 described in Example 1 or GI-6104 described in Example 2 (grown under standard growth conditions or neutral pH conditions). The yeast-MUC1 immunotherapeutic composition was administered subcutaneously in a sequential dose-group expansion schedule to 12-24 subjects with MUC1-positive tumors, utilizing a dose range of 4Y.U. (1Y.U x 4 sites), 16Y.U. (4Y.U x 4 sites), and 40Y.U. (10Y.U x 4 sites). Yeast-MUC1 immunotherapy was administered at 2-week intervals for 3 months and then monthly. Expansion patient groups (n=10) at the maximum tolerated dose (MTD) or best observed dose were selected for additional studies. Results Safety was monitored as the primary endpoint, antigen-specific T cell responses (e.g., MUC1-specific CD8 + T cells) and clinical activity were secondary endpoints.

[0414] GI-6101 and GI-6104 are expected to be safe and well tolerated without significant toxicity. In addition, GI-6101 or GI-6104 is expected to produce treatment-emergent MUCl-specific T cell responses or improvements in pre-existing MUCl-specific baseline T cell responses in a statistically significant number of patients. Some patients are also expected to have disease stabilization.

[0415] Example 10

[0416] The following examples describe clinical trials (P1 / P2) using yeast-MUCl immunotherapeutic compositions.

[0417] Increased MUCl expression is observed in -70% of acute myeloid leukemia (AML) cases, suggesting that elevated MUCl levels may be involved in regulating the proliferative potential of the immature leukemic compartment.

[0418] In the first clinical trial, the first-line use of a yeast-based immunotherapy product called GI-6101 (see Example 1) was performed in the setting of MUC1-positive AML (this clinical design is also applicable to other yeast-MUC1 immunotherapy compositions such as GI-6104). The use of GI-6101 is designed to supplement the existing standard of care regimen of cytarabine and anthracycline antibiotics (e.g., daunorubicin) cytotoxicity as an additional approach by promoting immune killing of MUC1-positive leukemia cells and eliminating MUC1 leukemia cells that can escape the terminal differentiation (apoptosis) pathway. Endpoints include improvements in remission induction and overall survival (pre- and post-transplant).

[0419] In the second trial, GI-6101 is used in the setting of a bone marrow transplant (BMT) to prevent relapse of AML in patients with MUC1-positive disease (this trial design is also applicable to other yeast-MUC1 immunotherapy compositions such as GI-6104). Clinical strategies evaluating vaccination of bone marrow donors (adoptive transfer) and / or vaccination of bone marrow recipients in the post-transplant period are being used to reduce relapse rates after BMT.

[0420] (A) Clinical study design for first-line treatment of patients with MUC1-positive AML with GI-6101 plus cytarabine and daunorubicin versus standard of care alone

[0421] Patients received induction chemotherapy consisting of continuous intravenous infusion of cytarabine (cytosine arabinoside) at 100-200 mg / m2 per day for 7 days, plus intravenous daunomycin (or daunomycin cerubidine) at 45 mg / m2 on days 1, 2, and 3 of cytarabine therapy, or a recognized variant of this regimen, followed by GI-6101 (or placebo) for 14 days after completing the induction cycle of chemotherapy. GI-6101 (or placebo) was then administered 14 days after re-induction therapy or 14 days after each subsequent consolidation cycle of chemotherapy. GI-6101 (or placebo) was administered monthly for up to 3 years after induction, re-induction, and consolidation therapy, with the primary goal of preventing relapse of remission.

[0422] Use of GI-6101 is expected to enhance relapse in patients as compared to those receiving placebo.

[0423] (B) Clinical study design for post-BMT treatment of MUC1-positive AML with GI-6101 versus placebo

[0424] For patients with MUC1-positive AML who require myelo-ablative therapy followed by a bone marrow transplant, GI-6101 (or placebo) is administered to bone marrow donors 7-14 days prior to donating bone marrow, and to bone marrow recipients on a monthly basis for up to 3 years after the bone marrow transplant occurs. The primary goal is to reduce the relapse rate of AML.

[0425] Use of GI-6101 is expected to reduce relapse rates in AML patients as compared to those patients administered a placebo.

[0426] Although various embodiments of the present invention have been described in detail, it is apparent that modifications and changes to those embodiments will occur to those skilled in the art. However, it must be clearly understood that these modifications and changes are within the scope of the present invention as set forth in the appended claims. Sequence Listing <110> GlobeImmune, Inc. The United States of America, represented by the Secretary of Health and Human Services <120> Yeast-MUC1 immunotherapy composition and its use <130> 3923-40-PCT <150> 61 / 524,407 <151> 2011-08-17 <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 1209 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (67)..(888) <400> 1 acctctcaag cagccagcgc ctgcctgaat ctgttctgcc ccctccccac ccatttcacc 60 accacc atg aca ccg ggc acc cag tct cct ttc ttc ctg ctg ctg ctc 108 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu 1 5 10 ctc aca gtg ctt aca gtt gtt acg ggt tct ggt cat gca agc tct acc 156 Leu Thr Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr 15 20 25 30 cca ggt gga gaa aag gag act tcg gct acc cag aga agt tca gtg ccc 204 Pro Gly Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro 35 40 45 agc tct act gag aag aat gct ttg tct act ggg gtc tct ttc ttt ttc 252 Ser Ser Thr Glu Lys Asn Ala Leu Ser Thr Gly Val Ser Phe Phe Phe 50 55 60 ctg tct ttt cac att tca aac ctc cag ttt aat tcc tct 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gtt ctg gtt gcg 636 Pro Gly Trp Gly Ile Lion Wing Lion Val Lion Val Cys Val Lion Wing 175 180 185 190 ctg gcc att gtc tat ctc att gcc ttg gct gtc tgt cag tgc cgc cga 684 Lion Wing Valley Tyr Lion Wing Lion Wing Valley Cys Gln Cys Arg Arg 195 200 205 aag aac tac ggg cag ctg gac atc ttt cca gcc cgg gat acc tac cat 732 Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His 210 215 220 cct atg agc gag tac ccc acc tac cac acc cat ggg cgc tat gtg ccc 780 Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro 225 230 235 cct agc agt acc gat cgt agc ccc tat gag aag gtt tct gca gca ggt aat 828 Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn 240 245 250 ggt ggc agc agc ctc tct tac aca aac cca gca gtg gca gcc act tct 876 Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser 255 260 265 270 gcc aac ttg tag gggcacgtcg cccgctgagc tgagtggcca gccagtgcca 928 Ala Asn Leu ttccactcca ctcaggttct tcagggccag agcccctgca ccctgtttgg gctggtgagc 988 tgggagttca ggtgggctgc tcacagcctc cttcagaggc cccaccaatt tctcggacac 1048 ttctcagtgt gtggaagctc atgtgggccc ctgagggctc atgcctggga agtgttgtgg 1108 tgggggctcc caggaggact ggcccagaga gccctgagat agcggggatc ctgaactgga 1168 ctgaataaaa cgtggtctcc cactgcgcca aaaaaaaaaa a 1209 <210> 2 <211> 273 <212> PRT <213> Homo sapiens <400> 2 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Leu Ser Thr Gly Val Ser Phe Phe Phe Leu Ser 50 55 60 Phe His Ile Ser Asn Leu Gln Phe Asn Ser Ser Leu Glu Asp Pro Ser 65 70 75 80 Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Phe Leu 85 90 95 Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe 100 105 110 Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly 115 120 125 Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr 130 135 140 Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser 145 150 155 160 Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly 165 170 175 Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala 180 185 190 Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys Asn 195 200 205 Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met 210 215 220 Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser 225 230 235 240 Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly 245 250 255 Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn 260 265 270 Leu <210> 3 <211> 1182 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (67)..(861) <400> 3 acctctcaag cagccagcgc ctgcctgaat ctgttctgcc ccctccccac ccatttcacc 60 accacc atg aca ccg ggc acc cag tct cct ttc ttc ctg ctg ctg ctc 108 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu 1 5 10 ctc aca gtg ctt aca gct acc aca gcc cct aaa ccc gca aca gtt gtt 156 Leu Thr Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val 15 20 25 30 acg ggt tct ggt cat gca agc tct acc cca ggt gga gaa aag gag act 204 Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr 35 40 45 tcg gct acc cag aga agt tca gtg ccc agc tct act gag aag aat gct 252 Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala 50 55 60 ttt aat tcc tct ctg gaa gat ccc agc acc gac tac tac caa gag ctg 300 Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu 65 70 75 cag aga gac att tct gaa atg ttt ttg cag att tat aaa caa ggg ggt 348 Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly 80 85 90 ttt ctg ggc ctc tcc aat att aag ttc agg cca gga tct gtg gtg gta 396 Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val 95 100 105 110 caa ttg act ctg gcc ttc cga gaa ggt acc atc aat gtc cac gac gtg 444 Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val 115 120 125 gag aca cag ttc aat cag tat aaa acg gaa gca gcc tct cga tat aac 492 Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn 130 135 140 ctg acg atc tca gac gtc agc gtg agt gat gtg cca ttt cct ttc tct 540 Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser 145 150 155 gcc cag tct ggg gct ggg gtg cca ggc tgg ggc atc gcg ctg ctg gtg 588 Ala Gln Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val 160 165 170 ctg gtc tgt gtt ctg gtt gcg ctg gcc att gtc tat ctc att gcc ttg 636 Leu Val Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu 175 180 185 190 gct gtc tgt cag tgc cgc cga aag aac tac ggg cag ctg gac atc ttt 684 Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe 195 200 205 cca gcc cgg gat acc tac cat cct atg agc gag tac ccc acc tac cac 732 Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His 210 215 220 acc cat ggg cgc tat gtg ccc cct agc agt acc gat cgt agc ccc tat 780 Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr 225 230 235 gag aag gtt tct gca ggt aat ggt ggc agc agc ctc tct tac aca aac 828 Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn 240 245 250 cca gca gtg gca gcc act tct gcc aac ttg tag gggcacgtcg cccgctgagc 881 Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 255 260 tgagtggcca gccagtgcca ttccactcca ctcaggttct tcagggccag agcccctgca 941 ccctgtttgg gctggtgagc tgggagttca ggtgggctgc tcacagcctc cttcagaggc 1001 cccaccaatt tctcggacac ttctcagtgt gtggaagctc atgtgggccc ctgagggctc 1061 atgcctggga agtgttgtgg tgggggctcc caggaggact ggcccagaga gccctgagat 1121 agcggggatc ctgaactgga ctgaataaaa cgtggtctcc cactgcgcca aaaaaaaaaa 1181 a 1182 <210> 4 <211> 264 <212> PRT <213> Homo sapiens <400> 4 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Phe Asn 50 55 60 Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg 65 70 75 80 Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu 85 90 95 Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu 100 105 110 Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr 115 120 125 Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr 130 135 140 Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln 145 150 155 160 Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val 165 170 175 Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val 180 185 190 Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala 195 200 205 Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His 210 215 220 Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys 225 230 235 240 Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala 245 250 255 Val Ala Ala Thr Ser Ala Asn Leu 260 <210> 5 <211> 1032 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (51)..(845) <400> 5 gcgcctgcct gaatctgttc tgccccctcc ccacccattt caccaccacc atg aca 56 Met Thr 1 ccg ggc acc cag tct cct ttc ttc ctg ctg ctg ctc ctc aca gtg ctt 104 Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr Val Leu 5 10 15 aca gct acc aca gcc cct aaa ccc gca aca gtt gtt aca ggt tct ggt 152 Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly Ser Gly 20 25 30 cat gca agc tct acc cca ggt gga gaa aag gag act tcg gct acc cag 200 His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala Thr Gln 35 40 45 50 aga agt tca gtg ccc agc tct act gag aag aat gct ttt aat tcc tct 248 Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Phe Asn Ser Ser 55 60 65 ctg gaa gat ccc agc acc gac tac tac caa gag ctg cag aga gac att 296 Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile 70 75 80 tct gaa atg ttt ttg cag att tat aaa caa ggg ggt ttt ctg ggc ctc 344 Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu 85 90 95 tcc aat att aag ttc agg cca gga tct gtg gtg gta caa ttg act ctg 392 Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu 100 105 110 gcc ttc cga gaa ggt acc atc aat gtc cac gac atg gag aca cag ttc 440 Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Met Glu Thr Gln Phe 115 120 125 130 aat cag tat aaa acg gaa gca gcc tct cga tat aac ctg acg atc tca 488 Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser 135 140 145 gac gtc agc gtg agt ggt gtg cca ttt cct ttc tct gcc cag tct ggg 536 Asp Val Ser Val Ser Gly Val Pro Phe Pro Phe Ser Ala Gln Ser Gly 150 155 160 gct ggg gtg cca ggc tgg ggc atc gcg ctg ctg gtg ctg gtc tgt gtt 584 Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val 165 170 175 ctg gtt gcg ctg gcc att gtc tat ctc att gcc ttg gct gtc tgt cag 632 Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln 180 185 190 tgc cgc cga aag aac tac ggg cag ctg gac atc ttt cca gcc cgg gat 680 Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp 195 200 205 210 acc tac cat cct atg agc gag tac ccc acc tac cac acc cat ggg cgc 728 Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg 215 220 225 tat gtg ccc cct agc agt acc gat cgt agc ccc tat gag aag gtt tct 776 Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 230 235 240 gca ggt aat ggt ggc agc agc ctc tct tac aca aac cca gca gtg gca 824 Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala 245 250 255 gcc act tct gcc aac ttg tag gggcacgtcg cccgctgagc tgagtggcca 875 Ala Thr Ser Ala Asn Leu 260 gccagtgcca ttccactcca ctcaggttct tcagggccag agcccctgca ccctgtttgg 935 gctggtgagc tgggagttca ggtgggctgc tcacagcctc cttcagaggc cccaccaatt 995 tctcggacac ttctcagtgt gtggaagctc atgtggg 1032 <210> 6 <211> 264 <212> PRT <213> Homo sapiens <400> 6 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Thr Gly 20 25 30 Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala 35 40 45 Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Phe Asn 50 55 60 Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg 65 70 75 80 Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu 85 90 95 Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu 100 105 110 Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Met Glu Thr 115 120 125 Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr 130 135 140 Ile Ser Asp Val Ser Val Ser Gly Val Pro Phe Pro Phe Ser Ala Gln 145 150 155 160 Ser Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val 165 170 175 Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val 180 185 190 Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala 195 200 205 Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His 210 215 220 Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys 225 230 235 240 Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala 245 250 255 Val Ala Ala Thr Ser Ala Asn Leu 260 <210> 7 <211> 1166 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (73)..(840) <400> 7 cgctccacct ctcaagcagc cagcgcctgc ctgaatctgt tctgccccct ccccacccat 60 ttcaccacca cc atg aca ccg ggc acc cag tct cct ttc ttc ctg ctg ctg 111 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu 1 5 10 ctc ctc aca gtg ctt aca gtt gtt acg ggt tct ggt cat gca agc tct 159 Leu Leu Thr Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser 15 20 25 acc cca ggt gga gaa aag gag act tcg gct acc cag aga agt tca gtg 207 Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val 30 35 40 45 ccc agc tct act gag aag aat gct ttt aat tcc tct ctg gaa gat ccc 255 Pro Ser Ser Thr Glu Lys Asn Ala Phe Asn Ser Ser Leu Glu Asp Pro 50 55 60 agc acc gac tac tac caa gag ctg cag aga gac att tct gaa atg ttt 303 Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Phe 65 70 75 ttg cag att tat aaa caa ggg ggt ttt ctg ggc ctc tcc aat att aag 351 Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys 80 85 90 ttc agg cca gga tct gtg gtg gta caa ttg act ctg gcc ttc cga gaa 399 Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu 95 100 105 ggt acc atc aat gtc cac gac gtg gag aca cag ttc aat cag tat aaa 447 Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys 110 115 120 125 acg gaa gca gcc tct cga tat aac ctg acg atc tca gac gtc agc gtg 495 Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val 130 135 140 agt gat gtg cca ttt cct ttc tct gcc cag tct ggg gct ggg gtg cca 543 Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro 145 150 155 ggc tgg ggc atc gcg ctg ctg gtg ctg gtc tgt gtt ctg gtt gcg ctg 591 Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu 160 165 170 gcc att gtc tat ctc att gcc ttg gct gtc tgt cag tgc cgc cga aag 639 Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys 175 180 185 aac tac ggg cag ctg gac atc ttt cca gcc cgg gat acc tac cat cct 687 Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro 190 195 200 205<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atg agc gag tac ccc acc tac cac acc cat ggg cgc tat gtg ccc cct 735<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 210 215 220<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agc agt acc gat cgt agc ccc tat gag aag gtt tct gca ggt aat ggt 783<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 225 230 235<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggc agc agc ctc tct tac aca aac cca gca gtg gca gcc act tct gcc 831<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 240 245 250<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aac ttg tag gggcacgtcg cccgctgagc tgagtggcca gccagtgcca 880<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Asn Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 255<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttccactcca ctcaggttct tcaggggccag agcccctgca ccctgtttgg gctggtgagc 940<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgggagttca ggtgggctgc tcacagcctc cttcagaggc cccaccaatt tctcggacac 1000<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttctcagtgt gtggaagctc atgtgggccc ctgagggctc atgcctggga agtgttgtgg 1060<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tggggggctcc caggaggact ggcccagaga gccctgagat agcggggatc ctgaactgga 1120<h2 style=";text-align:left;direction:ltr"> ctgaataaaa cgtggtctcc cactgcgcca aaaaaaaaaa aaaaaa 1166 <210> 8 <211> 255 <212> PRT <213> Homo sapiens <400> 8 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp 50 55 60 Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile 65 70 75 80 Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro 85 90 95 Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile 100 105 110 Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala 115 120 125 Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val 130 135 140 Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly Trp Gly 145 150 155 160 Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu Ala Ile Val 165 170 175 Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg Lys Asn Tyr Gly 180 185 190 Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu 195 200 205 Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr 210 215 220 Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser 225 230 235 240 Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 245 250 255 <210> 9 <211> 2235 <212> DNA <213> Mus musculus <220> <221> CDS <222> (69)..(1964) <400> 9 ccacctcaca cacggagcgc cagccttgag tttgttttct agccccttcc cgcctgttca 60 ccaccacc atg acc ccg ggc att cgg gct cct ttc ttc ctg ctg cta ctt 110 Met Thr Pro Gly Ile Arg Ala Pro Phe Phe Leu Leu Leu Leu 1 5 10 cta gca agt cta aaa ggt ttt ctt gcc ctt cca agt gag gaa aac agt 158 Leu Ala Ser Leu Lys Gly Phe Leu Ala Leu Pro Ser Glu Glu Asn Ser 15 20 25 30 gtc acc tca tct cag gac acc agc agt tcc tta gca tcg act acc act 206 Val Thr Ser Ser Gln Asp Thr Ser Ser Ser Leu Ala Ser Thr Thr Thr 35 40 45 cca gtc cac agc agc aac tca gac cca gcc acc aga cct cca ggg gac 254 Pro Val His Ser Ser Asn Ser Asp Pro Ala Thr Arg Pro Pro Gly Asp 50 55 60 tcc acc agc tct cca gtc cag agt agc acc tct tct cca gcc acc aga 302 Ser Thr Ser Ser Pro Val Gln Ser Ser Thr Ser Ser Pro Ala Thr Arg 65 70 75 gct cct gaa gac tct acc agt act gca gtc ctc agt ggc acc tcc tcc 350 Ala Pro Glu Asp Ser Thr Ser Thr Ala Val Leu Ser Gly Thr Ser Ser 80 85 90 cca gcc acc aca gct cca gtg aac tcc gcc agc tct cca gta gcc cat 398 Pro Ala Thr Thr Ala Pro Val Asn Ser Ala Ser Ser Pro Val Ala His 95 100 105 110 ggt gac acc tct tcc cca gcc act agc ctt tca aaa gac tcc aac agc 446 Gly Asp Thr Ser Ser Pro Ala Thr Ser Leu Ser Lys Asp Ser Asn Ser 115 120 125 tct cca gta gtc cac agt ggc acc tct tca gct ccg gcc acc aca gct 494 Ser Pro Val Val His Ser Gly Thr Ser Ser Ala Pro Ala Thr Thr Ala 130 135 140 cca gtg gat tcc acc agc tct cca gta gtc cac ggt ggt acc tcg tcc 542 Pro Val Asp Ser Thr Ser Ser Pro Val Val His Gly Gly Thr Ser Ser 145 150 155 cca gcc acc agc cct cca ggg gac tcc acc agc tct cca gac cat agt 590 Pro Ala Thr Ser Pro Pro Gly Asp Ser Thr Ser Ser Pro Asp His Ser 160 165 170 agc acc tct tct cca gcc acc aga gct ccc gaa gac tct acc agt act 638 Ser Thr Ser Ser Pro Ala Thr Arg Ala Pro Glu Asp Ser Thr Ser Thr 175 180 185 190 gca gtc ctc agt ggc acc tcc tcc cca gcc acc aca gct cca gtg gac 686 Ala Val Leu Ser Gly Thr Ser Ser Pro Ala Thr Thr Ala Pro Val Asp 195 200 205 tcc acc agc tct cca gta gcc cat gat gac acc tct tcc cca gcc act 734 Ser Thr Ser Ser Pro Val Ala His Asp Asp Thr Ser Ser Pro Ala Thr 210 215 220 agc ctt tca gaa gac tcc gcc agc tct cca gta gcc cac ggt ggc acc 782 Ser Leu Ser Glu Asp Ser Ala Ser Ser Pro Val Ala His Gly Gly Thr 225 230 235 tct tct cca gcc acc agc cct cta agg gac tcc acc agt tct cca gtc 830 Ser Ser Pro Ala Thr Ser Pro Leu Arg Asp Ser Thr Ser Ser Pro Val 240 245 250 cac agt agt gcc tcc atc caa aac atc aag act aca tca gac tta gct 878 His Ser Ser Ala Ser Ile Gln Asn Ile Lys Thr Thr Ser Asp Leu Ala 255 260 265 270 agc act cca gac cac aat ggc acc tca gtc aca act acc agc tct gca 926 Ser Thr Pro Asp His Asn Gly Thr Ser Val Thr Thr Thr Ser Ser Ala 275 280 285 ctg ggc tca gcc acc agt cca gac cac agt ggt acc tca act aca act 974 Leu Gly Ser Ala Thr Ser Pro Asp His Ser Gly Thr Ser Thr Thr Thr 290 295 300 aac agc tct gaa tca gtc ttg gcc acc act cca gtt tac agt agc atg 1022 Asn Ser Ser Glu Ser Val Leu Ala Thr Thr Pro Val Tyr Ser Ser Met 305 310 315 cca ttc tct act acc aaa gtg acg tca ggc tca gct atc att cca gac 1070 Pro Phe Ser Thr Thr Lys Val Thr Ser Gly Ser Ala Ile Ile Pro Asp 320 325 330 cac aat ggc tcc tcg gtg cta cct acc agt tct gtg ttg ggc tca gct 1118 His Asn Gly Ser Ser Val Leu Pro Thr Ser Ser Val Leu Gly Ser Ala 335 340 345 350 acc agt cta gtc tat aat acc tct gca ata gct aca act cca gtc agc 1166 Thr Ser Leu Val Tyr Asn Thr Ser Ala Ile Ala Thr Thr Pro Val Ser 355 360 365 aat ggc act cag cct tca gtg cca agt caa tac cct gtt tct cct acc 1214 Asn Gly Thr Gln Pro Ser Val Pro Ser Gln Tyr Pro Val Ser Pro Thr 370 375 380 atg gcc acc acc tcc agc cac agc act att gcc agc agc tct tac tat 1262 Met Ala Thr Thr Ser Ser His Ser Thr Ile Ala Ser Ser Ser Tyr Tyr 385 390 395 agc aca gta cca ttt tct acc ttc tcc agt aac agt tca ccc cag ttg 1310 Ser Thr Val Pro Phe Ser Thr Phe Ser Ser Asn Ser Ser Pro Gln Leu 400 405 410 tct gtt ggg gtc tcc ttc ttc ttc ttg tct ttt tac att caa aac cac 1358 Ser Val Gly Val Ser Phe Phe Phe Leu Ser Phe Tyr Ile Gln Asn His 415 420 425 430 cca ttt aat tct tct ctg gac gac ccc agc tcc aac tac tac caa gaa 1406 Pro Phe Asn Ser Ser Leu Glu Asp Pro Ser Asn Tyr Tyr Gln Glu 435 440 445 ctg aag agg aac att tct gga ttg ttt ctg cag attt aac gga gat 1454 Lys Arg Asn Ile Gly Leu Phe Leu Gln Ile Asn Gly Asp 450 455 460 ttt ctg ggg atc tct agc atc aag ttc agg tca ggc tcc gtg gtg gta 1502 Phe Leu Gly Ile Ser Ser Ile Lys Phe Arg Ser Gly Ser Val Val Val 465 470 475 gaa tcg act gtg gtt ttc cgg gag ggt act ttt agt gcc tct gac gtg 1550 Glu Ser Thr Val Val Phe Arg Glu Gly Thr Phe Ser Ala Ser Asp Val 480 485 490 aag tca cag ctt ata cag cat aag aag gag gca gat gac tat aat ctg Lys Ser Gln Leu And Gln His Lys Glu Ala Asp Asp Tyr Asn Leu 495,500,505,510 act att tca gaa gtc aaa gtg aat gag atg cag ttc cct ccc tct gcc 1646 Thr Ile Ser Glu Val Lys Val Asn Glu Met Gln Phe Pro Pro Ser Ala 515 520 525 cag tcc cgg ccg ggg gta cca ggc tgg ggc to gcc ctg ctg gtg ctg 1694 Gln Ser Arg Pro Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu 530 535 540 gtc tgt to ttg gtt gct ttg gct atc gtc tat ttc ctt gcc ctg gca 1742 Val Cys Ile Leu Val Ala Leu Ala Ile Val Tyr Phe Leu Ala Leu Ala 545 550 555 gtg tgc cag tgc cgc cga aag agc tat ggg cag ctg gac atc ttt cca 1790 Val Cys Gln Cys Arg Arg Lys Ser Tyr Gly Gln Leu Asp Ile Phe Pro 560 565 570 acc cag gac acc tac cat cct atg agt gaa tac cct acc tac cac act 1838 Thr Gln Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr 575 580 585 590 cac gga cgc tac gtg ccc cct ggc agt acc aag cgt agc ccc tat gag 1886 His Gly Arg Tyr Val Pro Pro Gly Ser Thr Lys Arg Ser Pro Tyr Glu 595 600 605 gag gtt tcg gca ggt aat ggc agt agc agc ctc tct tat acc aac cca 1934 Glu Val Ser Ala Gly Asn Gly Ser Ser Leu Ser Tyr Thr Asn Pro 610 615 620 gct gtg gtg acc act tct gcc aac tg tg gagcaagtca cccaccac 1984 Ala Val Thr Thr Ser Ala Asn Leu 625,630 2044 ctgggctggt gagccaggac ttctgtagg ctgttcatgc ccttgtca gcgcctcac 2104 tacgtaagcc tggtgaagcc cagccctgcc ctgggggaca ctggggcagt tagtggtggc 2164 tctcagaagg actggcctgg aaaactggag acagggatg gaacccaac atagctgaat 2224 aaagatggc c 2235 <210> 10 <211> 631 <212> PRT <213> Muscles <400> 10 Met Thr Pro Gly Ile Arg Ala Pro Phe Phe Leu Leu Leu Leu Ala 1 5 10 15 Ser Leu Lys Gly Phe Leu Ala Leu Pro Ser Glu Glu Asn Ser Val Thr 20 25 30 Ser Ser Gln Asp Thr Ser Ser Ser Leu Ala Ser Thr Thr Thr Pro Val 35 40 45 His Ser Ser Asn Ser Asp Pro Ala Thr Arg Pro Pro Gly Asp Ser Thr 50 55 60 Be Be Pro Val Gln Be Be Thr Be Be Pro Ala Thr Arg Ala Pro 65 70 75 80 Glu Asp Ser Thr Ser Thr Ala Val Leu Ser Gly Thr Ser Ser Pro Ala 85 90 95 Thr Thr Ala Pro Val Asn Ser Ala Ser Ser Pro Val Ala His Gly Asp 100 105 110 Thr Ser Ser Pro Ala Thr Ser Leu Ser Lys Asp Ser Asn Ser Ser Pro 115 120 125 Val Val His Ser Gly Thr Ser Ser Ala Pro Ala Thr Thr Ala Pro Val 130 135 140 Asp Ser Thr Ser Ser Pro Val Val His Gly Gly Thr Ser Ser Pro Ala 145 150 155 160 Thr Ser Pro Pro Gly Asp Ser Thr Ser Ser Pro Asp His Ser Ser Thr 165 170 175 Ser Ser Pro Ala Thr Arg Ala Pro Glu Asp Ser Thr Ser Thr Ala Val 180 185 190 Leu Ser Gly Thr Ser Ser Pro Ala Thr Thr Ala Pro Val Asp Ser Thr 195 200 205 Ser Ser Pro Val Ala His Asp Asp Thr Ser Ser Pro Ala Thr Ser Leu 210 215 220 Ser Glu Asp Ser Ala Ser Ser Pro Val Ala His Gly Gly Thr Ser Ser 225 230 235 240 Pro Ala Thr Ser Pro Leu Arg Asp Ser Thr Ser Ser Pro Val His Ser 245 250 255 Ser Ala Ser Ile Gln Asn Ile Lys Thr Thr Ser Asp Leu Ala Ser Thr 260 265 270 Pro Asp His Asn Gly Thr Ser Val Thr Thr Thr Ser Ser Ala Leu Gly 275 280 285 Ser Ala Thr Ser Pro Asp His Ser Gly Thr Ser Thr Thr Thr Asn Ser 290 295 300 Ser Glu Ser Val Leu Ala Thr Thr Pro Val Tyr Ser Ser Met Pro Phe 305 310 315 320 Ser Thr Thr Lys Val Thr Ser Gly Ser Ala Ile Ile Pro Asp His Asn 325 330 335 Gly Ser Ser Val Leu Pro Thr Ser Ser Val Leu Gly Ser Ala Thr Ser 340 345 350 Leu Val Tyr Asn Thr Ser Ala Ile Ala Thr Thr Pro Val Ser Asn Gly 355 360 365 Thr Gln Pro Ser Val Pro Ser Gln Tyr Pro Val Ser Pro Thr Met Ala 370 375 380 Thr Thr Ser Ser His Ser Thr Ile Ala Ser Ser Ser Tyr Tyr Ser Thr 385 390 395 400 Val Pro Phe Ser Thr Phe Ser Ser Asn Ser Ser Pro Gln Leu Ser Val 405 410 415 Gly Val Ser Phe Phe Phe Leu Ser Phe Tyr Ile Gln Asn His Pro Phe 420 425 430 Asn Ser Ser Leu Glu Asp Pro Ser Ser Asn Tyr Tyr Gln Glu Leu Lys 435 440 445 Arg Asn Ile Ser Gly Leu Phe Leu Gln Ile Phe Asn Gly Asp Phe Leu 450 455 460 Gly Ile Ser Ser Ile Lys Phe Arg Ser Gly Ser Val Val Val Glu Ser 465 470 475 480 Thr Val Val Phe Arg Glu Gly Thr Phe Ser Ala Ser Asp Val Lys Ser 485 490 495 Gln Leu Ile Gln His Lys Lys Glu Ala Asp Asp Tyr Asn Leu Thr Ile 500 505 510 Ser Glu Val Lys Val Asn Glu Met Gln Phe Pro Pro Ser Ala Gln Ser 515 520 525 Arg Pro Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys 530 535 540 Ile Leu Val Ala Leu Ala Ile Val Tyr Phe Leu Ala Leu Ala Val Cys 545 550 555 560 Gln Cys Arg Arg Lys Ser Tyr Gly Gln Leu Asp Ile Phe Pro Thr Gln 565 570 575 Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly 580 585 590 Arg Tyr Val Pro Pro Gly Ser Thr Lys Arg Ser Pro Tyr Glu Glu Val 595 600 605 Ser Ala Gly Asn Gly Ser Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val 610 615 620 Val Thr Thr Ser Ala Asn Leu 625 630 <210> 11 <211> 1255 <212> PRT <213> Homo sapiens <400> 11 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30 Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45 Thr Glu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His 50 55 60 Ser Pro Gly Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu 65 70 75 80 Ala Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gln 85 90 95 Asp Val Thr Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr 100 105 110 Pro Pro Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro 115 120 125 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 130 135 140 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 145 150 155 160 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 165 170 175 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 180 185 190 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 195 200 205 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 210 215 220 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 225 230 235 240 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 245 250 255 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 260 265 270 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 275 280 285 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 290 295 300 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 305 310 315 320 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 325 330 335 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 340 345 350 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 355 360 365 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 370 375 380 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 385 390 395 400 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 405 410 415 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 420 425 430 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 435 440 445 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 450 455 460 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 465 470 475 480 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 485 490 495 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 500 505 510 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 515 520 525 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 530 535 540 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 545 550 555 560 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 565 570 575 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 580 585 590 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 595 600 605 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 610 615 620 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 625 630 635 640 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 645 650 655 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 660 665 670 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 675 680 685 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 690 695 700 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 705 710 715 720 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 725 730 735 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 740 745 750 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 755 760 765 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 770 775 780 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 785 790 795 800 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 805 810 815 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 820 825 830 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 835 840 845 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 850 855 860 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 865 870 875 880 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 885 890 895 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 900 905 910 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 915 920 925 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn 930 935 940 Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser 945 950 955 960 Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly 965 970 975 Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe 980 985 990 Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His 995 1000 1005 Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro 1010 1015 1020 Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr 1025 1030 1035 Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln 1040 1045 1050 Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu 1055 1060 1065 Leu Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln 1070 1075 1080 Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser 1085 1090 1095 Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn 1100 1105 1110 Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala 1115 1120 1125 Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp 1130 1135 1140 Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly 1145 1150 1155 Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu 1160 1165 1170 Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg 1175 1180 1185 Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr 1190 1195 1200 His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr 1205 1210 1215 Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 1220 1225 1230 Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val 1235 1240 1245 Ala Ala Thr Ser Ala Asn Leu 1250 1255 <210> 12 <211> 20 <212> PRT <213> Homo sapiens <220> <221> VARIANT <222> (9)..(9) <223> X = P, A, Q or T <220> <221> VARIANT <222> (18) <223> X = D or E <220> <221> VARIANT <222> (19)..(19) <223> X = T or S <400> 12 Pro Ala Pro Gly Ser Thr Ala Pro Xaa Ala His Gly Val Thr Ser Ala 1 5 10 15 Pro Xaa Xaa Arg 20 <210> 13 <211> 711 <212> DNA <213> Artificial <220> <223> Synthetic constructs <220> <221> CDS <222> (1)..(711) <400> 13 atg aca ccg ggc acc cag tct cct ttc ttc ctg ctg ctg ctc ctc aca 48 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 gtg ctt aca gct acc aca gcc cct aaa ccc gca aca gtt gtt gga tct 96 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Gly Ser 20 25 30 gtg gtg gta caa ttg act ctg gcc ttc cga gaa ggt acc atc aat gtc 144 Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val 35 40 45 cac gac gtg gag aca cag ttc aat cag tat aaa acg gaa gca gcc tct 192 His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser 50 55 60 cga tat aac ctg acg atc tca gac gtc agc gtg agt gat gtg cca ttt 240 Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe 65 70 75 80 cct ttc tct gcc cag tct ggg gct ggg gcc cac ggt gtc acc agc gcc 288 Pro Phe Ser Ala Gln Ser Gly Ala Gly Ala His Gly Val Thr Ser Ala 85 90 95 cct gac acc agg ccg gcc ccg ggc tcc acc gcg ccc cca gcc cat ggt 336 Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly 100 105 110 gtc acc tcg gcc ccg gag agc agg ccc gcc cct ggg agc acc gcc ccc 384 Val Thr Ser Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr Ala Pro 115 120 125 cct gca gtg cca ggc tgg ggc atc gcg ctg ctg gtg ctg gtc tgt gtt 432 Pro Ala Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val 130 135 140 ctg gtt gcg ctg gcc att gtc tat ctc att gcc ttg gct gtc tgt cag 480 Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln 145 150 155 160 tgc cgc cga aag aac tac ggg cag ctg gac atc ttt cca gcc cgg gat 528 Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp 165 170 175 acc tac cat cct atg agc gag tac ccc acc tac cac acc cat ggg cgc 576 Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg 180 185 190 tat gtg ccc cct agc agt acc gat cgt agc ccc tat gag aag gtt tct 624 Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 195 200 205 gca ggt aat ggt ggc agc agc ctc tct tac aca aac cca gca gtg gca 672 Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala 210 215 220 gcc act tct gcc aac ttg cat cat cac cac cat cac tag 711 Ala Thr Ser Ala Asn Leu His His His His His His 225 230 235 <210> 14 <211> 236 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 14 Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr 1 5 10 15 Val Leu Thr Ala Thr Thr Ala Pro Lys Pro Ala Thr Val Val Gly Ser 20 25 30 Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val 35 40 45 His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser 50 55 60 Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe 65 70 75 80 Pro Phe Ser Ala Gln Ser Gly Ala Gly Ala His Gly Val Thr Ser Ala 85 90 95 Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly 100 105 110 Val Thr Ser Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr Ala Pro 115 120 125 Pro Ala Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val 130 135 140 Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln 145 150 155 160 Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp 165 170 175 Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg 180 185 190 Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 195 200 205 Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala 210 215 220 Ala Thr Ser Ala Asn Leu His His His His His His 225 230 235 <210> 15 <211> 200 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 15 Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile 1 5 10 15 Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala 20 25 30 Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val 35 40 45 Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Ala His Gly Val Thr 50 55 60 Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala 65 70 75 80 His Gly Val Thr Ser Ala Pro Glu Ser Arg Pro Ala Pro Gly Ser Thr 85 90 95 Ala Pro Pro Ala Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val 100 105 110 Cys Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val 115 120 125 Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala 130 135 140 Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His 145 150 155 160 Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys 165 170 175 Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala 180 185 190 Val Ala Ala Thr Ser Ala Asn Leu 195 200 <210> 16 <211> 687 <212> DNA <213> Artificial <220> <223> Synthetic construct <220> <221> CDS <222> (1)..(687) <400> 16 atg gcc gat gaa gct ccg tgt cag tgc cgc cga aag aac tac ggg cag 48 Met Ala Asp Glu Ala Pro Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln 1 5 10 15 ctg gac atc ttt cca gcc cgg gat acc tac cat cct atg agc gag tac 96 Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr 20 25 30 ccc acc tac cac acc cat ggg cgc tat gtg ccc cct agc agt acc gat 144 Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp 35 40 45 cgt agc ccc tat gag aag gtt tct gca ggt aat ggt ggc agc agc ctc 192 Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu 50 55 60 tct tac aca aac cca gca gtg gca gcc act tct gcc aac ttg tgc caa 240 Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu Cys Gln 65 70 75 80 tgc aga aga aaa at tat ggt caa ctt gac at ttt cct gca aga gat Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp and Phe Pro Ala Arg Asp 85 90 95 aca tat cat cca atg agt gaa tat cca act tat cac act cat ggc aga Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg 100 105 110 tat gtc cca cca tcg tct aca gat aga tcg cca tac gaa aaa gta tcg 384 Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 115 120 125 gct gga aat gga ggt agt tca ttg tca tac act aat cct gca gtc gct 432 Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala 130 135 140 gct acg tcc gct aat cta tgt caa tgt cgt agg aaa aac tac ggt caa 480 Ala Thr Ser Ala Asn Leu Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln 145 150 155 160 ttg gat ata ttc cca gcc agg gac act tat cat cct atg tcc gaa tac 528 Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr 165 170 175 cca acg tac cat aca cat ggc agg tat gtt cct cct tct tca act gat 576 Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp 180 185 190 aga tcc ccg tat gaa aag gta tca gct gga aac ggt gga agt tcc tta 624 Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu 195 200 205 tca tat aca aat ccg gct gtt gcg gcg aca tct gca aat tta cat cat 672 Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu His His 210 215 220 cac cac cat cac tga 687 His His His His 225 <210> 17 <211> 228 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 17 Met Ala Asp Glu Ala Pro Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln 1 5 10 15 Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr 20 25 30 Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp 35 40 45 Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu 50 55 60 Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu Cys Gln 65 70 75 80 Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp 85 90 95 Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg 100 105 110 Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 115 120 125 Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala 130 135 140 Ala Thr Ser Ala Asn Leu Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln 145 150 155 160 Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser Glu Tyr 165 170 175 Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp 180 185 190 Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser Ser Leu 195 200 205 Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu His His 210 215 220 His His His His 225 <210> 18 <211> 216 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 18 Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala 1 5 10 15 Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His 20 25 30 Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys 35 40 45 Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala 50 55 60 Val Ala Ala Thr Ser Ala Asn Leu Cys Gln Cys Arg Arg Lys Asn Tyr 65 70 75 80 Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr His Pro Met Ser 85 90 95 Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr Val Pro Pro Ser Ser 100 105 110 Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser Ala Gly Asn Gly Gly Ser 115 120 125 Ser Leu Ser Tyr Thr Asn Pro Ala Val Ala Ala Thr Ser Ala Asn Leu 130 135 140 Cys Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala 145 150 155 160 Arg Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His 165 170 175 Gly Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys 180 185 190 Val Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala 195 200 205 Val Ala Ala Thr Ser Ala Asn Leu 210 215 <210> 19 <211> 89 <212> PRT <213> Saccharomyces cerevisiae <400> 19 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Ser Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Asp Lys Arg Glu Ala Glu Ala 85 <210> 20 <211> 89 <212> PRT <213> Saccharomyces cerevisiae <400> 20 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 ...

Claims

1. A yeast-MUC1 immunotherapeutic composition, wherein the immunotherapeutic composition comprises: a) a yeast vehicle; and b) at least one MUCl antigen expressed by the yeast vehicle, wherein the MUCl antigen comprises an amino acid sequence identical to SEQ ID NO:

25.

2. A yeast-MUC1 immunotherapeutic composition, wherein the immunotherapeutic composition comprises: a) a yeast vehicle; and b) at least one MUCl antigen expressed by the yeast vehicle, wherein the MUCl antigen comprises an amino acid sequence identical to positions 92-566 of SEQ ID NO:

25.

3. The yeast-MUCl immunotherapeutic composition of claim 1 or 2, wherein the yeast vehicle is whole yeast.

4. The yeast-MUCl immunotherapeutic composition of claim 3, wherein the whole yeast is heat-inactivated.

5. The yeast-MUCl immunotherapeutic composition of claim 1 or 2, wherein the yeast vehicle is from a mutant yeast strain that produces hypoglycosylated protein compared to a wild-type yeast strain.

6. The yeast-MUCl immunotherapeutic composition of claim 1 or 2, wherein the yeast vehicle is from Saccharomyces cerevisiae.

7. The Yeast-MUCl immunotherapeutic composition of claim 2, wherein the MUCl antigen consists of positions 92-566 of SEQ ID NO:25.

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