WO2015123532A1

WO2015123532A1 - Ras g12r immunotherapy for ras mutation-positive cancers

Info

Publication number: WO2015123532A1
Application number: PCT/US2015/015840
Authority: WO
Inventors: Claire Coeshott; Timothy C. Rodell
Original assignee: Globeimmune, Inc.
Priority date: 2014-02-16
Filing date: 2015-02-13
Publication date: 2015-08-20

Abstract

Disclosed are Ras G12R antigens in immunotherapeutic compositions for the treatment or prevention of Ras mutation-positive cancers, including yeast-based immunotherapeutic compositions comprising a Ras G12R antigen for the prevention and/or treatment of Ras mutation-positive cancers. Also disclosed is the use of the Ras mutation, G12R, as a biomarker for the positive prediction of survival in cancer patients with Ras mutation-positive tumors, and to the use of an HLA-A3 allele as a biomarker for the negative prediction of survival in cancer patients with Ras mutation-positive tumors.

Description

Ras G12R Immunotherapy for Ras Mutation-Positive Cancers

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to copending U.S. Provisional Patent Application No. 61,940,471, filed February 16, 2014, and to copending U.S. Provisional Patent Application No. 61/942,724, filed February 21, 2014. The entire disclosure of each of U.S. Provisional Patent Application No. 61,940,471 and U.S. Provisional Patent Application No. 61/942,724 is incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing submitted electronically as a text file by EFS-Web. The text file, named "3923-47-PCT_ST25", has a size in bytes of 48KB, and was recorded on 10 February 2015. The information contained in the text file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

FIELD OF THE INVENTION

[0003] The present invention generally relates to the use of Ras G12R antigens in immunotherapeutic compositions for the treatment or prevention of Ras mutation-positive cancers, and includes yeast-based immunotherapeutic compositions comprising a Ras G12R antigen for the prevention and/or treatment of Ras mutation-positive cancers. The present invention also relates to the use of the Ras mutation, G12R, as a biomarker for the positive prediction of survival in cancer patients with Ras mutation-positive tumors, and to the use of an HLA-A3 allele as a biomarker for the negative prediction of survival in cancer patients with Ras mutation-positive tumors.

BACKGROUND OF THE INVENTION

[0004] A number of human cancers have been shown to be associated with characteristic mutations in genes governing the production of proteins involved in cell division. The ras oncogene and its Ras protein gene product are mutated in many solid tumors. It is estimated that ras mutations are found in approximately 180,000 new cancer cases each year in the United States across a spectrum of tumor types, including pancreas, non-small cell lung cancers (NSCLC), colorectal, endometrial and ovarian cancers, melanoma and multiple myeloma.

[0005] Ras-mutated pancreas cancer has a particularly poor prognosis. The American Cancer Society predicted that in the United States in 2013 there would be 45,220 new cases of pancreas cancer diagnosed and 38,460 deaths from pancreas cancer. Pancreas cancer is rarely curable, with a median survival of 9 to 12 months and an overall five-year survival rate of three percent. A patient's eligibility to undergo resection is an important factor in the patient's prognosis. Only 15% to 20% of patients with pancreas cancer are candidates for resection. Pancreas cancer is particularly aggressive with non-specific initial symptoms, which frequently results in a delayed diagnosis. Therefore, the majority of patients are frequently not aware they have the disease until the cancer has metastasized.

[0006] There are over 420,000 new cases of NSCLC annually in the United States, Western Europe and Japan. Studies suggest 20%> to 25% of NSCLCs have Ras mutations. There are numerous treatments for NSCLC, including multiple chemotherapies, EGFR targeted molecular therapies and biologic therapies. A significant unmet medical need continues to exist for patients with NSCLCs containing a Ras mutation. Studies have shown that NSCLCs with Ras mutations are associated with poorer recurrence free survival and overall survival with adjuvant chemotherapy, as well as a lack of recurrence and survival benefit with EGFR targeted tyrosine kinase inhibitors, such as erlotinib and gefitinib. The five-year survival rate for patients with NSCLC is approximately 15%.

[0007] Studies including those discussed above have shown that tumors with Ras mutations may be less responsive than tumors with normal Ras to conventional chemotherapy as well as targeted agents. For some cancers, such as NSCLC or colorectal cancer, therapies that target epidermal growth factor receptor, or EGFR, have improved clinical outcomes. However, the presence of a Ras mutation in the tumor has been associated with poor prognosis despite use of EGFR targeted therapies. For example, studies have shown that NSCLC with a Ras mutation is associated with a lack of response to tyrosine kinase inhibitors, such as erlotinib and gefitinib, while these therapies result in better survival rates for patients without a Ras mutation. Similarly, other studies have shown that patients with Ras mutated colorectal tumors do not benefit from cetuximab therapy, another EGFR targeted agent, compared to patients with normal Ras, who have improved survival rates when treated with the same therapy. As a result, patients with Ras mutations have fewer available effective treatment options, and therapies targeting mutated Ras in late-stage clinical trials are lacking.

[0008] Therefore, there is a need in the art for therapies that specifically target cancers associated with mutated Ras. SUMMARY OF THE INVENTION

[0009] One embodiment of the invention relates to a method to treat a Ras mutation- positive cancer. The method includes the step of administering an immunotherapeutic composition to a subject who has a Ras mutation-positive cancer that has been selected, prior to the step of administering, to be negative for a Ras G12R mutation. The immunotherapeutic composition includes: (a) a yeast vehicle; and (b) a Ras G12R antigen. In one aspect of this embodiment, the subject is HLA-A3 -positive or has been pre-selected as being HLA-A3 positive. In one aspect of this embodiment, the subject has also been pre-selected as an immune responder in vitro to a Ras G12R antigen.

[0010] Another embodiment of the invention relates to a method to treat a Ras mutation-positive cancer. The method includes the step of administering an immunotherapeutic composition to a subject who has a Ras mutation-positive cancer and who has been pre-selected as being HLA-A3-positive. The immunotherapeutic composition includes: (a) a yeast vehicle; and (b) a Ras G12R antigen. In one aspect of this embodiment, the subject has also been pre-selected as an immune responder in vitro to a Ras G12R antigen.

[0011] Yet another embodiment of the invention relates to a method to treat a Ras mutation-positive cancer. The method includes the steps of: (a) testing cancer cells from subjects with a Ras mutation-positive cancer in vitro to identify the specific Ras mutation or mutations in the cancer of the subjects; and (b) selecting subjects with a Ras mutation- positive cancer that are identified in (a) as being Ras G12R mutation-negative; and (c) administering to the subjects selected in (b) an immunotherapeutic composition. The immunotherapeutic composition includes: (i) a yeast vehicle; and (ii) a Ras G12R antigen. In one aspect of this embodiment, the subject has been further selected as being HLA- A3 - positive.

[0012] Another embodiment of the invention relates to a method to treat a Ras mutation-positive cancer. The method includes the steps of: (a) testing a biological sample from a subject with a Ras-mutation positive cancer to identify the HLA type of the subject; and (b) administering to subjects who are HLA- A3 -positive an immunotherapeutic composition that includes: (i) a yeast vehicle; and (ii) a Ras G12R antigen.

[0013] Yet another embodiment of the invention relates to a method to treat Ras mutation-positive cancer. The method includes the step of administering an immunotherapeutic composition comprising a Ras G12R antigen to a pre-selected subject, wherein the subject has been pre-selected as having a Ras mutation-positive cancer that does not express a Ras G12R mutation. In one aspect of this embodiment, the subject is HLA-A3 -positive or has been pre-selected as being HLA-A3 positive. In one aspect of this embodiment, the subject has also been pre-selected as an immune responder in vitro to a Ras G12R antigen.

[0014] Another embodiment of the invention relates to a method to treat a Ras mutation-positive cancer. The method includes the steps of administering an immunotherapeutic composition comprising a Ras G12R antigen to a pre-selected subject, wherein the subject has been pre-selected as having a Ras mutation-positive cancer and as being HLA-A3-positive. In one aspect of this embodiment, the subject has also been preselected as an immune responder in vitro to a Ras G12R antigen.

[0015] In any of the embodiments of the invention related to methods or uses described above or elsewhere herein, in one aspect, the Ras G12R antigen comprises at least 9 amino acids of any one of SEQ ID NOs:3, 5, 7, 9, 11, or 13, wherein the Ras G12R antigen includes the amino acid at position 12 of SEQ ID NO:3, 5, 7, 9, 11 or 13, except that the glycine at that position in SEQ ID NO:3, 5, 7, 9, 11 or 13 has been substituted for an arginine. In one aspect of the embodiments of the invention described above or elsewhere herein, the Ras G12R antigen comprises the amino acid sequence of SEQ ID NO: 15.

[0016] In any of the embodiments of the invention related to methods or uses described above or elsewhere herein, in one aspect, the method further includes administering to the subject a mutated Ras antigen that has the same mutation or mutations as the Ras mutation in the subject's cancer. In one aspect, the mutated Ras antigen is contained within the same immunogenic composition as the Ras G12R antigen. In one aspect, the mutated Ras antigen is part of a fusion protein comprising the mutated Ras antigen and the Ras G12R antigen. In one aspect, the mutated Ras antigen comprises a G12 mutation that is not G12R. The G12 mutation that is not G12R can include, but is not limited to, G12V, G12D, G12C, G12S, or G12A. In one aspect, the mutated Ras antigen also includes a Q61 mutation. The Q61 mutation can include, but is not limited to, Q61L, Q61R or Q61H.

[0017] In any of the embodiments of the invention related to methods or uses described above or elsewhere herein, in one aspect, the subject is being treated or has been treated with another therapy for cancer. Such a therapy can include, but is not limited to, radiation therapy, tumor resection, or chemotherapy. Such a therapy can also or alternatively include administration of one or more additional immunotherapeutic compositions. In one aspect, the additional immunotherapeutic composition is a yeast vehicle and a cancer antigen that is not a Ras G12R antigen. In one aspect, the additional immunotherapeutic composition is an immune checkpoint inhibitor.

[0018] In any of the above-described embodiments of the invention related to methods or uses, in one aspect, the yeast vehicle is a whole yeast. In one aspect any of the above-described embodiments of the invention, the Ras G12R antigen has been expressed by the whole yeast. In one aspect any of the above-described embodiments of the invention, the whole yeast has been heat-inactivated. In one aspect any of the above- described embodiments of the invention, the yeast or yeast vehicle is from Saccharomyces.

[0019] Yet another embodiment of the invention relates to an immunotherapeutic composition. The immunotherapeutic composition includes (a) a yeast vehicle; and (b) a mutated Ras antigen, wherein the mutated Ras antigen comprises a G12R mutation and at least one additional Ras G12 mutation that is not a G12R mutation. In one aspect, the mutated Ras antigen comprises at least 9 amino acids of any one of SEQ ID NOs:3, 5, 7, 9, 11, or 13, wherein the antigen includes the amino acid at position 12 of SEQ ID NO:3, 5, 7, 9, 11 or 13, except that the glycine at that position in SEQ ID NO:3, 5, 7, 9, 11 or 13 has been substituted for an arginine. In one aspect, the at least one additional Ras G12 mutation is a mutation that has been detected in a subject who has a mutated Ras-positive cancer. In one aspect, the mutated Ras antigen is a fusion protein comprising the Ras G12R mutation and the at least one additional Ras G12 mutation. The at least one additional Ras G12 mutation can include, but is not limited to, G12V, G12D, G12C, G12S, or G12A. In one aspect, the mutated Ras antigen further comprises at least one additional Ras mutation that is not a G12 mutation. In one aspect, the at least one additional Ras mutation that is not a G12 mutation is a Q61 mutation. The Q61 mutation can include, but is not limited to, Q61L, Q61R or Q61H.

[0020] Yet another embodiment of the invention relates to a combination of immunotherapeutic compositions, wherein the combination comprises a mixture of: (a) a first yeast expressing a fusion protein having the amino acid sequence of SEQ ID NO: 15; and (b) a second yeast expressing a fusion protein having an amino acid sequence encoding a mutated Ras antigen, wherein the mutated Ras antigen does not include a G12R mutation. In one aspect of this embodiment, the second yeast expresses a fusion protein having an amino acid sequence selected from: SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:21. [0021] Another embodiment of the invention relates to an immunotherapeutic composition comprising a yeast expressing a fusion protein having a first amino acid sequence selected from SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:21, wherein the fusion protein additionally includes a second amino acid sequence from a Ras protein comprising a G12R mutation. In one aspect of this embodiment, the second amino acid sequence is appended to the N- or C-terminus of the first amino acid sequence. In one aspect, the second amino acid sequence comprises at least 9 amino acids of any one of SEQ ID NOs:3, 5, 7, 9, 11, or 13, wherein the antigen includes the amino acid at position 12 of SEQ ID NO:3, 5, 7, 9, 11 or 13, except that the glycine at that position in SEQ ID NO:3, 5, 7, 9, 11 or 13 has been substituted for an arginine.

[0022] In any of the embodiments related to an immunotherapeutic composition or a combination of immunotherapeutic compositions described above or elsewhere herein, in one aspect, the yeast vehicle is a whole yeast. In one aspect, the Ras G12R antigen has been expressed by the whole yeast. In one aspect, the whole yeast has been heat- inactivated. In one aspect, the yeast or yeast vehicle is from Saccharomyces.

[0023] Yet another embodiment of the invention relates to any of the immunotherapeutic compositions or combinations of immunotherapeutic compositions described above or elsewhere herein for use to treat a Ras mutation-positive cancer, wherein the Ras mutation-positive cancer has been pre-selected as being Ras G12R- negative.

[0024] Another embodiment of the invention relates to any of the immunotherapeutic compositions or combinations of immunotherapeutic compositions described above or elsewhere herein for use to treat a Ras mutation-positive cancer in an HLA- A3 -positive subject.

[0025] Another embodiment of the invention relates to the use of any of the immunotherapeutic compositions or combinations of immunotherapeutic compositions described above or elsewhere herein in the preparation of a medicament for treating a Ras mutation-positive cancer, wherein the Ras mutation-positive cancer has been pre-selected as being Ras G12R-negative.

[0026] Another embodiment of the invention relates to the use of any of the immunotherapeutic compositions or combinations of immunotherapeutic compositions described above or elsewhere herein in the preparation of a medicament for treating a Ras mutation-positive cancer in an HLA- A3 -positive subject. [0027] Yet another embodiment of the invention relates to a method to pre-select a subject with a Ras mutation-positive cancer for treatment with an immunotherapy composition comprising a G12R antigen. The method includes the steps of: (a) testing a biological sample from the subject to identify the HLA allele or alleles expressed by the subject; (b) testing a T cell-containing biological sample from the subject for an immune response to a Ras G12R antigen in vitro; and (c) pre-selecting subjects with at least one HLA- A3 allele and whose T cells respond to the Ras G12R antigen as subjects to be treated with an immunotherapy composition comprising a Ras G12R antigen. In one aspect of this embodiment, the T-cell containing biological sample is a sample of peripheral blood mononuclear cells isolated from the subject.

BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION

[0028] Fig. 1A is a schematic drawing showing the yeast-based immunotherapeutic known as GI-4014.

[0029] Fig. IB is a schematic drawing showing the yeast-based immunotherapeutic known as GI-4015.

[0030] Fig. 1C is a schematic drawing showing the yeast-based immunotherapeutic known as GI-4016.

[0031] Fig. ID is a schematic drawing showing the yeast-based immunotherapeutic known as GI-4020.

[0032] Fig. 2 shows Kaplan-Meier estimates of the duration of overall survival from randomization by Ras mutation type (intent-to-treat population with R0 resection) (circles represent patient censors).

[0033] Fig. 3 shows Kaplan-Meier estimates of the duration of overall survival from randomization (G12R mutation compared to all other mutations; intent-to-treat population with R0 resection) (circles represent patient censors).

[0034] Fig. 4 shows Kaplan-Meier estimates of the duration of overall survival from randomization by Ras mutation type (intent-to-treat population with R0 resection, GI-4000 group only) (circles represent patient censors).

[0035] Fig. 5 shows Kaplan-Meier estimates of the duration of overall survival from randomization by Ras mutation type (intent-to-treat population with R0 resection, placebo group only) (circles represent patient censors).

[0036] Fig. 6 shows Kaplan-Meier estimates of the duration of overall survival from randomization by treatment group (intent-to-treat population with R0 resection and G12R status) (circles represent patient censors). [0037] Fig. 7A is a bar graph showing the interferon-γ (IFN-γ) ELISpot response of one Ras G12R-positive subject who was treated with GI-4000 and was a categorical responder on treatment.

[0038] Fig. 7B is a bar graph showing the IFN-γ ELISpot response of one Ras G12R- positive subject who was treated with GI-4000 and was a categorical responder on treatment.

[0039] Fig. 7C is a bar graph showing the IFN-γ ELISpot response of one Ras G12R- positive subject who was a categorical baseline responder and who was treated with GI- 4000.

[0040] Fig. 7D is a bar graph showing the IFN-γ ELISpot response of one Ras G12R- positive subject who was a categorical baseline responder and who was treated with GI- 4000.

[0041] Fig. 7E is a bar graph showing the IFN-γ ELISpot response of one Ras G12R- positive subject who was treated with placebo and was a categorical responder on treatment.

[0042] Fig. 7F is a bar graph showing the IFN-γ ELISpot response of one Ras G12R- positive subject who was treated with placebo and was a categorical responder on treatment.

[0043] Fig. 7G is a bar graph showing the IFN-γ ELISpot response of one Ras G12R- positive subject who was treated with placebo and was a categorical responder on treatment.

[0044] Fig. 7H is a bar graph showing the IFN-γ ELISpot response of one Ras G12R- positive subject who was treated with placebo and was a categorical responder on treatment.

[0045] Fig. 71 is a bar graph showing the IFN-γ ELISpot response of one Ras G12R- positive subject who was treated with placebo and was a categorical responder on treatment.

[0046] Fig. 7 J is a bar graph showing the IFN-γ ELISpot response of one Ras G12R- positive subject who was a categorical baseline responder and who was treated with placebo.

DETAILED DESCRIPTION OF THE INVENTION

[0047] This invention generally relates to Ras G12R immunotherapeutic compositions, including yeast-based Ras G12R immunotherapeutic compositions, and the use of such immunotherapeutic compositions for the prevention and/or treatment of Ras mutation-positive cancers (i.e., cancers having tumor cells with one or more mutations in the ras oncogene, expressed as a mutation in the Ras protein), particularly in Ras mutation-positive cancers where the mutation(s) in Ras do not include a G12R mutation. In addition, the present invention relates to the use of Ras G12R mutation as a biomarker for the positive prediction of survival in Ras mutation-positive cancers. The present invention also relates to the use of HLA-A3 alleles (HLA-A*0301 and HLA-A*0302) as biomarkers for the negative prediction of survival in Ras mutation-positive cancers, and the use of Ras G12R immunotherapeutic compositions to improve survival in Ras mutation-positive cancer patients with HLA-A3 alleles, including in patients whose tumors do not express the Ras G12R mutation. A subject with at least one allele of HLA- A*0301 or HLA-A*0302, or other rare HLA-A3 alleles with similar peptide anchoring properties, can be generally referred to as an "HLA-A3 subject" or "HLA-A3 (or "HLA- A3 positive") according to the present invention.

[0048] Previous studies in patients with Ras-mutation positive cancers, have reported T cell responses to G12V, G12C, G12D and G12R mutations (Khleif S. N. et al, J. Immunother. 22: 155-165, 1999; Gjertsen, M.K. et al, Int. J. Cancer. 72:784-790, 1997; Gjertsen, M.K. et al, Int. J. Cancer. 92:441-50, 2001; Gjertsen, M.K. et al, J Mol Med. 81 :43-50, 2003; Kubuschok B., et al, Clin. Cancer Res 12: 1365- 1372, 2006; Carbone, D.P. et al, J. Clin. Oncology 23: 5099- 5107, 2005; Qin H., et al, Cancer Res. 55:2984-7, 1995; Abrams, S.I. et al. Generation of stable CD4⁺ and CD8⁺ T cell lines from patients immunized with ras oncogene-derived peptides reflecting codon 12 mutations. Cellular Immunology 182: 137-151, 1997).

[0049] However, the inventors disclose here for the first time their discovery that, in a recent clinical study evaluating resected pancreas cancer patients with tumors expressing mutated Ras, those patients having a G12R Ras mutation (i.e., a mutation in codon 12 of ras, where the WT glycine (G) residue of the encoded Ras protein has mutated to an arginine (R) residue), regardless of the type of treatment received by the patient, had a pronounced improvement in overall median survival as compared to patients having all other Ras mutations (1167 vs. 832 days; increase of 335 days). It is believed that this is the first time the Ras G12R mutation has been associated with improved survival in pancreas cancer or Ras mutation-positive cancer. Moreover, the inventors discovered that the improvement in survival was further associated with an immune response to mutated Ras, since the positive survival effect was enhanced among patients with the G12R mutation who also received an immunotherapy composition targeting this specific mutated Ras (referred to herein as GI-4020), as compared to patients with the G12R mutation who received placebo (1517 vs. 949 days; increase of 568 days), or as compared to all other patients (with non-G12R mutations) receiving the immunotherapy composition (1517 vs. 744 days; increase of 773 days). Among placebo subjects, there was only a 94 day improvement in overall median survival for patients with G12R mutations versus all non- G12R subjects.

[0050] Taken together, the data presented herein indicates that the presence of the G12R mutation affords a survival advantage in patients with cancers having mutated Ras, including pancreas cancer patients, and that treatment with immunotherapy directed against this Ras mutation can further improve survival, presumably by generating a G12R- specific tumor-directed immune response. Accordingly, and without being bound by theory, the present inventors believe that the G12R mutation in Ras affords a survival advantage linked, at least in part, to an ability to induce a more robust T cell immune response than other Ras mutations.

[0051] In addition, the inventors have further discovered that the human HLA- A3 allele is a negative predictor of survival in pancreas cancer patients with tumors expressing mutated Ras, regardless of the treatment received by the patient. It is believed that this is the first time an HLA-A3 allele, as compared to other HLA types, has been specifically associated with poor survival in pancreas cancer or Ras mutation-positive cancer. However, in patients having both an HLA- A3 allele and a G12R Ras mutation, improved survival resulted, indicating that these subjects were "rescued" from the HLA-A3 poor prognosis by the presence of the G12R mutation, likely due to an immune response to the G12R mutation, either as a naturally derived response or by stimulation using an immunotherapeutic composition targeting the G12R Ras mutation.

[0052] The present invention also discloses the discovery that patients with Ras mutation-positive cancer who did not have a G12R mutation, but still received immunotherapy targeting Ras G12R, had improved survival as compared to in the absence of immunotherapy targeting Ras G12R. Taken together with the discovery described above that immune responses to G12R improved survival in patients with the mutation, the present inventors believe that antigens comprising a Ras antigen having the G12R mutation can act as agonists, improving the immune response against other Ras mutations, thereby improving survival outcomes in Ras mutation-positive cancer.

[0053] Therefore, embodiments of the present invention relate to the use of Ras G12R antigens and immunotherapy compositions comprising Ras G12R antigens as agonist immunotherapeutics for all Ras mutation-positive cancers, including all Ras mutation- positive cancers comprising a G12R mutation (which may also be referred to as Ras G12R-positive cancers) and all Ras mutation-positive cancers that do not comprise a G12R mutation (which may also be referred to as Ras G12R-negative cancers), including without limitation all Ras mutation-positive pancreas cancers, and all Ras mutation- positive pancreas cancers comprising a G12R mutation, as well as all Ras mutation- positive pancreas cancers that do not comprise a G12R mutation. In addition, an embodiment of the present invention relates to the use of Ras G12R as a biomarker for the positive prediction of survival in patients with a Ras mutation-positive cancer (e.g., extended survival as compared to patients with Ras mutation-positive cancer who do not have a G12R mutation, i.e., are Ras G12R-negative), including Ras mutation-positive pancreas cancer. A further embodiment of the invention includes the use of HLA-A3 as a biomarker for the negative prediction of survival in patients with a Ras mutation-positive cancer, including Ras mutation-positive pancreas cancer, as well as a biomarker to select patients for treatment with Ras G12R immunotherapy. For patients with HLA-A3, but non-G12R mutations in particular, the G12R immunotherapy is expected to improve survival by overcoming an ineffective immune response restricted to HL-A3 and non- G12R Ras peptides from the tumor.

[0054] In one embodiment, the invention includes the use of a Ras G12R immunotherapeutic composition. An "immunotherapeutic composition" is a composition that elicits an immune response in a subject when administered to the subject. In one embodiment, administration of an immunotherapeutic composition to a subject additionally results in at least one therapeutic benefit in a subject. A "Ras G12R immunotherapeutic composition" is an immunotherapeutic composition that specifically and/or selectively targets (elicits an immune response against) G12R mutations in Ras, typically by including a Ras G12R antigen in the immunotherapeutic composition (i.e., a Ras antigen comprising a G12R mutation). A "target antigen" is an antigen that is specifically targeted by an immunotherapeutic composition of the invention (i.e., an antigen against which elicitation of an immune response is desired). A "cancer antigen" is an antigen that comprises at least one antigen that is associated with or expressed by a cancer (e.g., cancer) such as an antigen expressed by a tumor cell, such that targeting the antigen also targets the cancer. A cancer antigen can include one or more antigens from one or more proteins, including one or more tumor-associated proteins. A "mutated Ras antigen" is an antigen derived, designed, or produced from a mutated Ras protein, and a "Ras G12R antigen" is an antigen derived, designed or produced from a Ras protein having a G12R mutation.

[0055] In one aspect, the immunotherapeutic composition elicits a CD8⁺ T cell response. In one aspect, the immunotherapeutic composition elicits a CD4⁺ T cell response. In one aspect, the immunotherapeutic composition elicits a CD4⁺ T cell response and a CD8⁺ T cell response. In one aspect, the immunotherapeutic composition has one or more of the following characteristics: (a) stimulates one or more pattern recognition receptors effective to activate an antigen presenting cell; (b) upregulates adhesion molecules, co-stimulatory molecules, and MHC Class I and/or Class II molecules on antigen presenting cells; (c) induces production of proinflammatory cytokines by antigen presenting cells; (d) induces production of Thl-type cytokines by T cells; (e) induces production of Thl7-type cytokines by T cells; (f) inhibits or downregulates regulatory T cells (Tregs); and/or (g) elicits MHC Class I- and/or MHC Class II-restricted, antigen-specific immune responses. Suitable immunotherapeutic compositions can include yeast-based immunotherapy compositions, viral-based immunotherapy compositions, antibody-based immunotherapy compositions, DNA immunotherapy compositions, subunit vaccines, and any components or adjuvants useful for stimulating or modulating an immune response, such as toll-like receptor (TLR) agonists, cytokines, immune potentiators, and other similar agents.

[0056] In one embodiment, the invention includes the use of a yeast-based Ras G12R immunotherapeutic composition (also referred to herein as "yeast-Ras G12R immunotherapy" or a "yeast-Ras G12R immunotherapeutic composition" or variations thereof), including, but not limited to, yeast-based immunotherapy compositions comprising a yeast vehicle and a Ras antigen that has a glycine (G) to arginine (R) mutation at codon 12 of Ras {i.e., a "G12R" mutation) to treat cancer, and in particular, cancers expressing a Ras G12 mutation that is not a G12R mutation, and including cancers expressing a transforming Ras mutation that is at a position other than codon 12 {e.g., codon 13, 59, 61 or 76). The Ras antigen expressed by a yeast-Ras G12R immunotherapeutic composition can also contain, in addition to the G12R mutation, mutations other than the G12R mutation {e.g., other mutations at codon 12, and/or mutations at codons 13, 59, 61 or 76, described in more detail below). Yeast-based immunotherapy compositions are described in more detail below.

[0057] One embodiment of the invention relates to the use of yeast-based Ras G12R immunotherapy as an agonist for Ras-mutation positive cancers. In one aspect of this embodiment, the Ras-mutation positive cancer does not have a G12R mutation (i.e., the cancer cells are Ras G12R-negative, and the mutation(s) in Ras expressed by the cancer cells are different than G12R). Another embodiment of the invention relates to the use of yeast-based Ras G12R immunotherapy for the treatment of HLA-A3 cancer patients (cancer patients having at least one HLA-A3 allele). Yet another embodiment of the invention relates to use of the Ras mutation, G12R, as a biomarker to positively predict survival in cancer patients. Another embodiment relates to the use of HLA-A3 as a biomarker to negatively predict survival in cancer patients. Yet another embodiment relates to the use of HLA-A3 as a biomarker to negatively predict survival in cancer patients, with the exception of HLA-A3⁺ cancer patients who also have a G12R Ras mutation.

[0058] With respect to the use of Ras G12R antigens as an agonist, studies in cancer patients have indicated that peptides derived from other cancer-specific antigens e.g. mesothelin (Yokokawa, J. et al, Clin. Cancer Research 11 :6342-6351, 2005), carcinoembryonic antigen (CEA) (Salazar E., et al., Int. J. Cancer 15:829-838, 2000; Zaremba, S. et al, Cancer Res. 57:4570-4577, 1997), can act as agonists for either HLA binding or T cell receptor engagement. For mesothelin (Yokokawa et al. 2005, supra), agonist peptide epitopes were defined that more efficiently activated T cells than WT sequences and that showed improved binding to HLA-A2 molecules. Improved binding to HLA-A2 was achieved by the replacement of residues in putative anchor positions of the wild-type (WT) sequence with residues with known increased potency as anchor residues. For CEA, an agonist epitope was identified that harbored a single amino acid substitution at a non-MHC anchor residue and thus was proposed to exert its effects at the level of the T-cell receptor (TCR). The agonist peptide was found to increase specific T cell activation by 2-3 logio compared to the WT peptide (Salazar et al 2000, supra; Zaremba et al. 1997, supra).

[0059] The present inventors believe that the G12R containing peptide can act as an agonist peptide for G12V-, D- or C- (or other G12 mutations, e.g., G12S, G12A, etc.) specific immune responses. The non-G12R mutations may generate less effective or ineffective immune responses that can be reversed by G12R-containing immunotherapy. Alternatively, and without being bound by theory, Ras mutations other than G12R may not, in some patients, break the immune tolerance directed towards the self-antigen, WT Ras. These ineffective or less effective immune responses may explain in part the very poor prognosis usually associated with Ras-mutated pancreas cancers, for example. This may particularly apply to patients having at least one HLA-A3 allele based on the data described herein. Accordingly, immunization with a G12R-containing immunotherapy composition is expected to provide an opportunity to reverse the poor survival associated with this HLA allele and with Ras-mutated pancreas cancers in general, and the data herein provide evidence of such an effect.

[0060] Yeast-based immunotherapy compositions described for use in the methods of the invention induce innate immune responses, as well as adaptive immune responses against the target antigen (e.g., mutated Ras), including CD4-dependent Thl7 and Thl T cell responses and antigen- specific CD8⁺ T cell responses, which include 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, yeast-based immunotherapeutic compositions inhibit Treg numbers and/or functionality, thereby enhancing effector T cell responses that might normally be suppressed by the presence of the tumor, for example. Moreover, as compared to immunotherapeutic compositions that immunize by generating antibody responses, the antigen-specific, broad- based, and potent cellular immune responses elicited by yeast-based immunotherapy are believed to be particularly effective in targeting tumor cells. Indeed, numerous studies have shown that immunotherapeutic approaches are enhanced when tumor cells are targeted via CD8⁺ CTLs which recognize tumor peptides in the context of MHC Class I molecules.

[0061] Yeast-based immunotherapy compositions can be effectively utilized in an immunization protocol (prophylactic or therapeutic) without the use of exogenous adjuvants, immunostimulatory agents or molecules, costimulatory molecules, or cytokines, although such agents may be included, if desired. Moreover, yeast-based immunotherapy can be administered repeatedly without losing efficacy, as may be problematic with other types of immunotherapy.

Methods for the Treatment or Prevention of Ras Mutation-Positive Cancers

[0062] One embodiment of the invention relates to a method to treat or prevent a Ras mutation-positive cancer by administering a Ras G12R immunotherapy composition, such as a yeast-based Ras G12R immunotherapy composition, to an individual with a Ras mutation-positive cancer (i.e., a cancer in which the cancer cells express at least one mutation in Ras). In one aspect, the individual has a Ras mutation-positive cancer that does not have a G12R mutation (i.e., the cancer expresses one or more non-G12R mutations, including, but not limited to, a different codon 12 mutation and/or a mutation in codon 13, 59, 69 and/or 76). In one aspect, the individual has a Ras-mutation-positive cancer that has a G12R mutation, along with one or more additional Ras mutations. In another aspect, the individual has a cancer that does not include cancer cells expressing a Ras G12R mutation. In one aspect, the individual has a Ras mutation-positive cancer and the individual expresses at least one HLA-A3 allele (i.e., is HLA-A3 -positive). In one aspect, the individual has a Ras mutation-positive cancer that includes a G12R mutation, and the individual expresses at least one HLA-A3 allele. In another aspect, the individual has a Ras mutation-positive cancer that does not have a G12R mutation, and the individual expresses at least one HLA-A3 allele.

[0063] In one aspect, in addition to the Ras G12R immunotherapy composition, the method of invention includes the administration of an immunotherapy composition to the subject that includes a Ras antigen with a mutation corresponding to the actual Ras mutation(s) in the subject's tumor. For example, a subject with tumor cells having a Ras G12C mutation, but no Ras G12R mutation, is administered at least one immunotherapy composition comprising both a Ras G12R mutation and a Ras G12C mutation. The immunotherapy composition can be administered as two separate compositions concurrently or sequentially (i.e., one composition comprising the Ras G12R antigen and one composition comprising the Ras antigen with a mutation other than G12R), or as a single immunotherapy composition comprising a Ras G12R antigen and the one or more different mutated Ras antigens (e.g., an antigen comprising the above-mentioned G12C mutation), including the Ras mutation corresponding to the mutation(s) found in the subject's tumor. Ras G12R immunotherapy compositions, yeast-based Ras G12R immunotherapy compositions and Ras G12R antigens useful in these compositions are described in detail below.

[0064] In another aspect, a subject that has a non-G12R mutation in Ras is administered an immunotherapy composition comprising only the Ras G12R antigen, and is not administered an antigen corresponding to the actual Ras mutation in their tumor.

[0065] In yet another aspect, a subject with one or more Ras mutations is administered an immunotherapeutic composition comprising a Ras G12R antigen, and in the same composition or a different immunotherapy composition, one or more additional non-Ras antigens, such as a different tumor antigen that is expressed by the subject's tumor or that is likely to be expressed by the subject's tumor. Such non-Ras antigens include, but are not limited to, carcinoembryonic antigen (CEA), brachyury, mucin- 1 (MUC-1), EGFR, BCR-Abl, MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-2, PSMA, tyrosinase, TRP-1 (gp75), NY-ESO-1 , TRP-2, TAG72, KSA, 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.

[0066] The method of the invention may treat cancer, for example, by ameliorating at least one symptom of cancer, such as by reducing tumor burden in the individual; inhibiting tumor growth in the individual; increasing (improving, extending, enhancing) survival of the individual; preventing, inhibiting, reversing or delaying development of tumor migration and/or tumor invasion of other tissues (metastatic cancer) and/or preventing, inhibiting, reversing or delaying progression of the cancer in the individual. The method of the invention may also prevent cancer, inhibit or delay the onset of cancer, or improve outcomes for cancer (including metastatic cancer or tumor progression) that develops subsequent to the administration of the immunotherapy (e.g., by increasing survival, by inhibiting/reducing/slowing cancer progression, by reducing or controlling tumor burden over time and/or increasing sensitivity of the tumor to chemotherapy or radiation therapy). This method includes the step of administering a Ras G12R immunotherapy composition to an individual who does not presently have cancer, but who is or may be predisposed to develop cancer. For example, an individual who is predisposed to develop cancer can include an individual who has a family history of cancer (familial cancer) and therefore is at a higher risk of developing cancer as compared to the population as a whole.

[0067] In one aspect, a cancer to be treated or prevented using the compositions or methods of the invention can include, but is not limited to: squamous cell carcinoma, head and neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas, breast cancer, small intestine cancer, stomach cancer, pancreas cancer, kidney cancer, bladder cancer, uterine cancer, brain cancer, angiosarcomas, hemangiosarcomas, primary hepatic cancers, ovarian cancer, testicular cancer, lung cancer, colon cancer, prostate cancer, melanoma or other skin cancer, renal cell carcinomas, bone cancer, hematopoietic neoplasias, multiple myelogenous leukemia (MML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), Burkitt's lymphoma, Hodgkin's lymphoma, cancers of secretory tissues, and metastatic cancers thereof.

[0068] In general, as used herein, to "treat" a cancer, or any permutation thereof (e.g., "treated for cancer", etc.) generally refers to administering a composition of the invention once the cancer has occurred (e.g., once the cancer has been diagnosed or detected in an individual), with at least one therapeutic goal of the treatment (as compared to in the absence of this treatment) including: reduction in tumor burden; inhibition of tumor growth; increase in survival of the individual; delaying, inhibiting, arresting or preventing the recurrence of the tumor; delaying, inhibiting, arresting or preventing onset or development of metastatic cancer; delaying or arresting cancer progression; improvement of immune responses against the tumor; improvement of long term memory immune responses against the tumor antigens; and/or improved general health of the individual. To "prevent" or "protect" from cancer, or any permutation thereof (e.g., "prevention of cancer", etc.), generally refers to administering a composition of the invention before cancer has occurred or developed, with at least one goal of the treatment (as compared to in the absence of this treatment) including: preventing or delaying the onset or development of cancer, or, should cancer occur after the treatment, at least improving the outcomes in the individual, including, but not limited to, improving survival, delaying the onset of the cancer, reducing or slowing the level of tumor growth, arresting cancer progression, improving the immune response against the cancer, and/or inhibiting metastatic processes.

[0069] The present invention includes the delivery (administration, immunization) of an immunotherapeutic composition of the invention, which may include, but is not limited to, a yeast-based Ras G1R immunotherapeutic composition, to a subject or individual. The administration process can be performed ex vivo or in vivo, but is typically performed in vivo. Ex vivo administration refers to performing part of the regulatory step outside of the patient, such as administering a composition of the present invention to a population of cells (e.g., dendritic cells) removed from a patient under conditions such that a yeast vehicle, antigen(s) and any other agents or compositions are loaded into the cell, and returning the cells to the patient. The therapeutic composition of the present invention can be returned to a patient, or administered to a patient, by any suitable mode of administration.

[0070] Administration of a composition can be systemic, mucosal and/or proximal to the location of the target site (e.g., near a site of a tumor). Suitable routes of administration will be apparent to those of skill in the art. Various acceptable methods of administration include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (e.g., into a carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), intracranial, intraspinal, intraocular, aural, intranasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue. In one aspect, routes of administration include: intravenous, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, inhaled, intranasal, oral, intraocular, intraarticular, intracranial, and intraspinal. Parenteral delivery can include intradermal, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheter and venal catheter routes. Aural delivery can include ear drops, intranasal delivery can include nose drops or intranasal injection, and intraocular delivery can include eye drops. Aerosol (inhalation) delivery can also be performed using methods standard in the art (see, for example, Stribling et al, Proc. Natl. Acad. Sci. USA 189: 11277-11281, 1992). In one aspect, a Ras G12R immunotherapeutic composition of the invention is administered subcutaneous ly. In one aspect, the Ras G12R immunotherapeutic composition is administered directly into a tumor milieu.

[0071] With respect to the yeast-based immunotherapy compositions of the invention, in general, a suitable single dose is a dose that is capable of effectively providing a yeast vehicle and an antigen (if included) to a given cell type, tissue, or region of the patient body in an amount effective to elicit an antigen-specific immune response against one or more mutated Ras antigens or epitopes, when administered one or more times over a suitable time period. For example, in one embodiment, a single dose of a yeast vehicle of the present invention is from about 1 x 10⁵ to about 5 x 10⁷ yeast cell equivalents (or Yeast Units, Y.U.) per kilogram body weight of the organism being administered the composition. One Yeast Unit (Y.U.) is 1 x 10⁷ yeast cells or yeast cell equivalents. In one aspect, a single dose of a yeast vehicle of the present invention is from about 0.1 Y.U. (1 x 10⁶ cells) to about 100 Y.U. (1 x 10⁹ cells) per dose (i.e., per organism), including any interim dose, in increments of 0.1 x 10⁶ cells (i.e., 1.1 x 10⁶, 1.2 x 10⁶, 1.3 x 10⁶...etc). In one embodiment, doses include doses between 1 Y.U and 40 Y.U., doses between 1 Y.U. and 50 Y.U., doses between 1 Y.U. and 60 Y.U., doses between 1 Y.U. and 70 Y.U., or doses between 1 Y.U. and 80 Y.U., and in one aspect, between 10 Y.U. and 40 Y.U., 50 Y.U., 60 Y.U., 70 Y.U., or 80 Y.U. In one embodiment, the doses are administered at different sites on the individual but during the same dosing period. For example, a 40 Y.U. dose may be administered by injecting 10 Y.U. doses to four different sites on the individual during one dosing period, or a 20 Y.U. dose may be administered by injecting 5 Y.U. doses to four different sites on the individual, or by injecting 10 Y.U. doses to two different sites on the individual, during the same dosing period. The invention includes administration of an amount of the yeast-based immunotherapy composition (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 Y.U. or more) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different sites on an individual to form a single dose.

[0072] "Boosters" or "boosts" of a therapeutic composition are administered, for example, when the immune response against the antigen has waned or as needed to provide an immune response or induce a memory response against a particular antigen or antigen(s). Boosters can be administered about 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart, or monthly, bimonthly, quarterly, annually, and/or in a few or several year increments after the original administration, depending on the status of the individual being treated and the goal of the therapy at the time of administration (e.g., prophylactic, active treatment, maintenance). In one embodiment, an administration schedule is one in which doses of yeast-based immunotherapeutic composition are administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times over a time period of from weeks, to months, to years. In one embodiment, the doses are administered weekly or biweekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses, followed by biweekly or monthly doses as needed to achieve the desired preventative or therapeutic treatment for cancer. Additional boosters can then be given at similar or longer intervals (months or years) as a maintenance or remission therapy, if desired.

[0073] In one aspect of the method to treat cancer, the individual is additionally treated with at least one other therapeutic compound or therapeutic protocol useful for the treatment of cancer. Such therapy can include any of the therapeutic protocols or use of any therapeutic compound or agent that is useful for treating cancer, particularly Ras mutation-positive cancers, or that may be useful generally for treating cancer, including, but not limited to, surgical resection, radiation therapy (including, but not limited to, stand alone radiation therapy and adjuvant radiation therapy), chemotherapy (e.g., gemcitabine, fluorouracil (5-FU), mitomycin, platinum-based chemotherapeutics (cisplatin, oxaliplatin and carboplatin), mitotic inhibitors (paclitaxel and vinorelbine)), targeted cancer therapy, stem cell transfer, cytokine therapy, adoptive T cell transfer, and/or administration of a second immunotherapeutic composition. In the case of administration of a second immunotherapeutic composition, such compositions may include, but are not limited to, additional yeast-based immunotherapy, recombinant virus-based immunotherapy (viral vectors), immunostimulant therapy (including chemotherapy with immunostimulating properties), DNA vaccines, antibody-based immunotherapy compositions, subunit vaccines, components or adjuvants useful for stimulating or modulating an immune response, such as TLR agonists, cytokines, immune potentiators, and other immunotherapy compositions.

[0074] In one aspect of the invention, the one or more additional therapeutic agents or therapeutic protocols are administered or performed sequentially and/or concurrently with the administration of the Ras G12R immunotherapy composition (e.g., surgical resection of the tumor, administration of radiation therapy, administration of chemotherapy or targeted cancer therapy, administration of another immunotherapy composition or protocol, cytokine therapy, adoptive T cell transfer, or stem cell transplantation). For example, one or more therapies can be administered or performed prior to the first dose of Ras G12R immunotherapy composition or after the first dose is administered. As one example, for individuals who have a cancer that can be resected, surgical resection, optionally followed by chemotherapy, and then by yeast-based Ras G12R immunotherapy, may be representative of the course of therapy. In one embodiment, one or more therapies can be administered or performed in an alternating manner with the dosing of Ras G12R immunotherapy composition, such as in a protocol in which the Ras G12R composition is administered at prescribed intervals in between one or more consecutive doses of chemotherapy or other targeted therapy. In one embodiment, the Ras G12R immunotherapy composition is administered in one or more doses over a period of time prior to commencing additional therapies. In other words, the Ras G12R immunotherapeutic composition is administered as a monotherapy for a period of time, and then an additional therapy is added (e.g., chemotherapy), either concurrently with new doses of Ras G12R immunotherapy, or in an alternating fashion with Ras G12R immunotherapy. Alternatively or in addition, another therapy may be administered for a period of time prior to beginning administration of the Ras G12R immunotherapy composition, and the concepts may be combined (e.g., surgical resection of a tumor, followed by monotherapy with Ras G12R immunotherapy for several weeks, followed by alternating doses of chemotherapy or targeted therapy and Ras G12R immunotherapy for weeks or months, optionally followed by monotherapy using Ras G12R immunotherapy or another therapy, or by a new protocol of combinations of therapy provided sequentially, concurrently, or in alternating fashion). Various protocols for the treatment of cancer using Ras G12R immunotherapy are contemplated by the invention, and these examples should be considered to be non- limiting examples of various possible protocols. [0075] In the method of the present invention, compositions and therapeutic compositions can be administered to animals, including any vertebrate, including, without limitation, primates, rodents, birds, livestock and domestic pets. Livestock include animals to be consumed or that produce useful products (e.g., sheep for wool production). Mammals to treat or protect utilizing the invention include humans, non-human primates, dogs, cats, mice, rats, goats, sheep, cattle, horses and pigs. The term "individual" can be used interchangeably with the term "animal", "subject" or "patient".

Compositions for Use in the Methods of the Invention

[0076] The methods of the present invention utilize a Ras G12R antigen, a Ras G12R immunotherapy composition, and in one embodiment, the Ras G12R immunotherapy composition is a yeast-based Ras G12R immunotherapy composition. According to the present invention, a Ras G12R immunotherapy composition useful in the present invention is a composition comprising: (a) an immunotherapy vector component (e.g., a yeast vehicle, a viral vector, DNA, an adjuvant, etc.); and (b) a cancer antigen, where the cancer antigen contains a Ras antigen comprising an amino acid sequence that includes position 12 of WT Ras, except that the glycine (G) at position 12 in the WT Ras is substituted with an arginine (R) (i.e., a G12R mutation). According to the present invention, a yeast-based Ras G12R immunotherapy composition useful in the present invention is a composition comprising: (a) a yeast vehicle (described in detail below); and (b) a cancer antigen, where the cancer antigen contains a Ras antigen comprising an amino acid sequence that includes position 12 of WT Ras, except that the glycine (G) at position 12 in the WT Ras is substituted with an arginine (R) (i.e., a G12R mutation). With respect to yeast-based immunotherapy, the cancer antigen is most typically expressed as a recombinant protein by the yeast vehicle (e.g., by an intact yeast or yeast spheroplast, which can optionally be further processed to a yeast cytoplast, yeast ghost, or yeast membrane extract or fraction thereof), although it is an embodiment of the invention that one or more cancer antigens are loaded into a yeast vehicle or otherwise complexed with, attached to, mixed with or administered with a yeast vehicle as described herein to form a composition of the present invention.

[0077] Although the following discussion focuses particularly on yeast-based Ras G12R immunotherapy compositions, elements of the discussion, such as elements regarding the Ras G12R antigen, are also applicable to other types of Ras G12R immunotherapy. [0078] A "yeast-based Ras G12R immunotherapeutic composition" is a specific type of "yeast-based immunotherapeutic composition" that contains at least one Ras G12R antigen. The phrase, "yeast-based immunotherapeutic composition" may be used interchangeably with "yeast-based immunotherapy product", "yeast-based immunotherapy composition", "yeast-based composition", "yeast-based immunotherapeutic", "yeast-based vaccine", or derivatives of these phrases.

[0079] According to the present invention, a yeast vehicle used in a yeast-based immunotherapy composition for the treatment or prevention of Ras mutation-positive cancer is any yeast cell (e.g., a whole or intact cell) or a derivative thereof (see below) that can be used in conjunction with one or more antigens, immunogenic domains thereof or epitopes thereof in a composition of the invention (e.g., a therapeutic or prophylactic composition). The yeast vehicle can therefore include, but is not limited to, a live intact (whole) yeast microorganism (i.e., a yeast cell having all its components including a cell wall), a killed (dead) or inactivated intact yeast microorganism, or derivatives of intact yeast including: a yeast spheroplast (i.e., a yeast cell lacking a cell wall), a yeast cytoplast (i.e., a yeast cell lacking a cell wall and nucleus), a yeast ghost (i.e., a yeast cell lacking a cell wall, nucleus and cytoplasm), a subcellular yeast membrane extract or fraction thereof (also referred to as a yeast membrane particle and previously as a subcellular yeast particle), any other yeast particle, or a yeast cell wall preparation.

[0080] Yeast spheroplasts are typically produced by enzymatic digestion of the yeast cell wall. Such a method is described, for example, in Franzusoff et al, 1991, Meth. Enzymol. 194, 662-674., incorporated herein by reference in its entirety.

[0081] Yeast cytoplasts are typically produced by enucleation of yeast cells. Such a method is described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55 incorporated herein by reference in its entirety.

[0082] Yeast ghosts are typically produced by resealing a permeabilized or lysed cell and can, but need not, contain at least some of the organelles of that cell. Such a method is described, for 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.

[0083] A yeast membrane particle (subcellular yeast membrane extract or fraction thereof) refers to a yeast membrane that lacks a natural nucleus or cytoplasm. The particle can be of any size, including sizes ranging from the size of a natural yeast membrane to microparticles produced by sonication or other membrane disruption methods known to those skilled in the art, followed by resealing. A method for producing subcellular yeast membrane extracts is described, for example, in Franzusoff et al, 1991, Meth. Enzymol. 194, 662-674. One may also use fractions of yeast membrane particles that contain yeast membrane portions and, when the antigen or other protein was expressed recombinantly by the yeast prior to preparation of the yeast membrane particles, the antigen or other protein of interest. Antigens or other proteins of interest can be carried inside the membrane, on either surface of the membrane, or combinations thereof (i.e., the protein can be both inside and outside the membrane and/or spanning the membrane of the yeast membrane particle). In one embodiment, a yeast membrane particle is a recombinant yeast membrane particle that can be an intact, disrupted, or disrupted and resealed yeast membrane that includes at least one desired antigen or other protein of interest on the surface of the membrane or at least partially embedded within the membrane.

[0084] An example of a yeast cell wall preparation is a preparation of isolated yeast cell walls carrying an antigen on its surface or at least partially embedded within the cell wall such that the yeast cell wall preparation, when administered to an animal, stimulates a desired immune response against a disease target.

[0085] Any yeast strain can be used to produce a yeast vehicle for use in the present invention. Yeast are unicellular microorganisms that belong to one of three classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. One consideration for the selection of a type of yeast for use as an immune modulator is the pathogenicity of the yeast. In one embodiment, the yeast is a non-pathogenic strain such as Saccharomyces cerevisiae. The selection of a non-pathogenic yeast strain minimizes any adverse effects to the individual to whom the yeast vehicle is administered. However, pathogenic yeast may be used if the pathogenicity of the yeast can be negated by any means known to one of skill in the art (e.g., mutant strains). In accordance with one aspect of the present invention, nonpathogenic yeast strains are used.

[0086] Genera of yeast strains that may be used in the invention include but are not limited to Saccharomyces, Candida (which can be pathogenic), Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one aspect, yeast genera are selected from Saccharomyces, Candida, Hansenula, Pichia or Schizosaccharomyces, and in one aspect, Saccharomyces is used. Species of yeast strains that may be used in the invention include but are not limited to Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, 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. It is to be appreciated that a number of these species include a variety of subspecies, types, subtypes, etc. that are intended to be included within the aforementioned species. In one aspect, yeast species used in the invention include S. cerevisiae, C. albicans, H. polymorpha, P. pastoris and S. pombe. S. cerevisiae is useful as it is relatively easy to manipulate and being "Generally Recognized As Safe" or "GRAS" for use as food additives (GRAS, FDA proposed Rule 62FR18938, April 17, 1997). One embodiment of the present invention is a yeast strain that is capable of replicating plasmids to a particularly high copy number, such as a S. cerevisiae cir° strain. The S. cerevisiae strain is one such strain that is capable of supporting expression vectors that allow one or more target antigen(s) and/or antigen fusion protein(s) and/or other proteins to be expressed at high levels. Another yeast strain that is useful in the invention is Saccharomyces cerevisiae W303a. In addition, any mutant yeast strains can be used in the present invention, including those that exhibit reduced post-translational modifications of expressed target antigens or other proteins, such as mutations in the enzymes that extend N-linked glycosylation.

[0087] The immunotherapy compositions of the invention, including the yeast-based immunotherapy compositions, include a Ras G12R antigen. In addition, various methods of the invention related to the use of G12R as a biomarker for the positive prediction of increased (e.g., longer term) cancer survival, and therefore, various G12R antigens or G12R peptides, and nucleic acid molecules encoding such antigens and peptides (including oligonucleotides), or antibodies that selectively and specifically bind to Ras antigens comprising G12R (e.g., sufficiently to identify the G12R mutation), are also included in the present invention for use as tools for identifying the G12R biomarker.

[0088] According to the present invention, the general use herein of the term "antigen" refers: to any portion of a protein (e.g., peptide, partial protein, full-length protein), wherein the protein is naturally occurring or synthetically derived or designed, to a cellular composition (whole cell, cell lysate or disrupted cells), to an organism (whole organism, lysate or disrupted cells) or to a carbohydrate, or other molecule, or a portion thereof. An antigen may elicit an antigen-specific immune response (e.g., a humoral and/or a cell-mediated immune response) against the same or similar antigens that are encountered by an element of the immune system (e.g., T cells, B cells, antibodies). [0089] An antigen can be as small as a single epitope, a single immunogenic domain or larger, and can include multiple epitopes or immunogenic domains. As such, the size of an antigen can be as small as about 8-11 amino acids (i.e., a peptide) and as large as: a full length protein, a multimer, a fusion protein, a chimeric protein, a whole cell, a whole microorganism, or any portions thereof (e.g., protein fragments (polypeptides) lysates of whole cells or extracts of microorganisms). Antigens useful in the yeast-based immunotherapeutic of the present invention are peptides, polypeptides, full-length proteins, multimers, fusion proteins and chimeric proteins. In addition, antigens can include carbohydrates, which can be loaded into a yeast vehicle or into a composition of the invention. For expression in yeast, an antigen is of a minimum size capable of being expressed recombinantly in yeast if the antigen is the entire protein to be expressed by the yeast, and is typically at least or greater than 25 amino acids in length, or at least or greater than 26, at least or greater than 27, at least or greater than 28, at least or greater than 29, at least or greater than 30, at least or greater than 31, at least or greater than 32, at least or greater than 33, at least or greater than 34, at least or greater than 35, at least or greater than 36, at least or greater than 37, at least or greater than 38, at least or greater than 39, at least or greater than 40, at least or greater than 41, at least or greater than 42, at least or greater than 43, at least or greater than 44, at least or greater than 45, at least or greater than 46, at least or greater than 47, at least or greater than 48, at least or greater than 49, or at least or greater than 50 amino acids in length, or at least or greater than 25-50 amino acids in length, or at least or greater than 30-50 amino acids in length, or at least or greater than 35-50 amino acids in length, or at least or greater than 40-50 amino acids in length, or at least or greater than 45-50 amino acids in length, although smaller proteins may be expressed, and considerably larger proteins (e.g., hundreds of amino acids in length or even a few thousand amino acids in length) may be expressed. In one embodiment, the Ras G12R antigen useful in the present invention is at least 25 amino acids in length, or at least: 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, or 430 amino acids in length.

[0090] When referring to stimulation of an immune response, the term "immunogen" is a subset of the term "antigen", and therefore, in some instances, can be used interchangeably with the term "antigen". An immunogen, as used herein, describes an antigen which elicits a humoral and/or cell-mediated immune response (i.e., is immunogenic), such that administration of the immunogen to an individual mounts an antigen-specific immune response against the same or similar antigens that are encountered by the immune system of the individual. In one embodiment, the immunogen elicits a cell-mediated immune response, including a CD4⁺ T cell response (e.g., Thl, Th2 and/or Thl 7) and/or a CD8⁺ T cell response (e.g., a CTL response).

[0091] An "immunogenic domain" of a given antigen can be any portion, fragment or epitope of an antigen (e.g., a peptide fragment or subunit or an antibody epitope or other conformational epitope) that contains at least one epitope that can act as an immunogen when administered to an animal. Therefore, an immunogenic domain is larger than a single amino acid and is at least of a size sufficient to contain at least one epitope that can act as an immunogen. For example, a single protein can contain multiple different immunogenic domains. Immunogenic domains need not be linear sequences within a protein, such as in the case of a humoral immune response, where conformational domains are contemplated.

[0092] An epitope is defined herein as a single immunogenic site within a given antigen that is sufficient to elicit an immune response when provided to the immune system in the context of appropriate costimulatory signals and/or activated cells of the immune system. In other words, an epitope is the part of an antigen that is recognized by components of the immune system, and may also be referred to as an antigenic determinant. Those of skill in the art will recognize that T cell epitopes are different in size and composition from B cell or antibody epitopes, and that epitopes presented through the MHC Class I pathway differ in size and structural attributes from epitopes presented through the MHC Class II pathway. For example, T cell epitopes presented by MHC Class I molecules are typically between 8 and 11 amino acids in length (e.g., 8, 9, 10 or 11 amino acids), whereas epitopes presented by MHC Class II molecules are less restricted in length and may be up to 25 amino acids or longer. In addition, T cell epitopes have predicted structural characteristics depending on the specific MHC molecules bound by the epitope. Epitopes can be linear sequence epitopes or conformational epitopes (conserved binding regions). Most antibodies recognize conformational epitopes.

[0093] In one aspect, an antigen useful in the methods of the invention comprises one or more CTL epitopes, which may include two or more copies of any one, two, three, or more of the CTL epitopes described herein. In one aspect, the antigen comprises one or more CD4⁺ T cell epitopes. In one aspect, the antigen comprises one or more CTL epitopes and one or more CD4 T cell epitopes. In one aspect, the T cell epitope is an agonist epitope.

[0094] According to any embodiment of the present invention, reference to a "full- length" protein (or a full-length functional domain or full-length immunological domain) includes the full-length amino acid sequence of the protein or functional domain or immunological domain, as described herein or as otherwise known or described in a publicly available sequence. A protein or domain that is "near full-length", which is also a type of homolog of a protein, differs from a full-length protein or domain, by the addition or deletion or omission of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the N- and/or C- terminus of such a full-length protein or full-length domain. General reference to a protein or domain or antigen can include both full-length and near full-length proteins, as well as other homologs thereof.

[0095] With respect to a Ras G12R antigen, a "Ras G12R antigen" is an antigen derived, designed or produced from a Ras protein that comprises an amino acid sequence containing amino acid position 12 of Ras (codon 12), where the glycine (G) at position 12 has been substituted with an arginine (R) (i.e., a "G12R" mutation). A Ras G12R antigen therefore comprises at least a minimum amino acid sequence of a WT Ras protein sufficient to form at least one immunogenic domain of Ras that includes position 12 of Ras, except that, as compared to the WT protein, the glycine (G) at position 12 has been substituted with an arginine (R). A Ras G12 antigen can be an immunogenic domain of a Ras protein, several consecutive immunogenic domains of a Ras protein, a full-length Ras protein, or a fusion protein comprising at least one immunogenic domain of Ras fused to another immunogenic domain of Ras or another cancer antigen, and in all cases, at least one immunogenic domain of Ras in the antigen includes position 12 of Ras (and amino acid sequence surrounding this position sufficient to form at least one T cell epitope), but as compared to the WT protein, the glycine at position 12 is substituted with an arginine.

[0096] A Ras G12R antigen can include or be derived from a Ras protein encoded by a ras gene selected from: K-ras, N-ras or H-ras genes. In one aspect, the Ras G12R antigen is a Ras protein with the single mutation at position 12, where the WT glycine at this position is substituted with an arginine. In another aspect, the Ras G12R antigen includes one or more further mutations in addition to the mutation at position 12, including, but not limited to, additional mutations at position 12 (in this case, the antigen would include more than one immunogenic domain of Ras with an amino acid sequence that includes position 12, where one immunogenic domain contains a G12R mutation and the other domain(s) contain the additional mutation(s) at position 12), position 13, position 59, position 61 , and/or position 76, the positions being relative to a WT K-, H- or N-Ras amino acid sequence. In one embodiment, a preferred fragment of a Ras protein includes between about 4 and 9 amino acids of the natural Ras amino acid sequence (the WT sequence, or the sequence that is associated with cellular, non-oncogenic Ras) flanking either side of the mutation at position 12 (e.g., a 9 amino acid fragment of Ras comprising the amino acid at position 12 flanked by four amino acids on either side of position 12, wherein the amino acid position at position 12 is mutated to an arginine as compared to a WT, non-tumorigenic, Ras protein). In one embodiment, a Ras protein useful in the invention includes a fragment of at least 9 contiguous amino acids that includes amino acid position 12 of Ras, or at least 10 contiguous amino acids, or at least 1 1 , or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 50, or at least 60, or at least 75, or at least 100, or at least 125, or at least 150, or at least 175 contiguous amino acids, and up to the full-length size of the Ras protein, including any intervening size fragment of Ras of at least 9 amino acids, in whole number increments (9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, . ..39, 40, 41 , . ..46, 47, 48. .. etc.).

[0097] As mentioned above, reference to positions with regard to WT Ras proteins herein are generally made with reference to the position in mammalian WT Ras proteins, or at least with regard to the position in human or murine K-Ras, H-Ras or N-Ras, and particularly human K-Ras, H-Ras or N-Ras. It is noted that the amino acid sequences in the region of the protein flanking position 12 for human or murine K-Ras, H-Ras or N-Ras are identical, as can be determined by comparing the sequences provided below. The nucleotide and amino acid sequence for a variety of Ras family members are well known in the art. SEQ ID NO:2 is the nucleic acid sequence encoding human K-Ras (also known in GenBank Accession No. NM 033360). SEQ ID NO:2 encodes human K-Ras, represented herein as SEQ ID NO:3. SEQ ID NO:4 is the nucleic acid sequence encoding murine K-Ras (also known in GenBank Accession No. NM 021284). SEQ ID NO:4 encodes murine K-Ras, represented herein as SEQ ID NO:5. SEQ ID NO:6 is the nucleic acid sequence encoding human H-Ras (also known in GenBank Accession No. NM 005343). SEQ ID NO:6 encodes human H-Ras, represented herein as SEQ ID NO:7. SEQ ID NO: 8 is the nucleic acid sequence encoding murine H-Ras (also known in GenBank Accession No. NM 008284). SEQ ID NO:8 encodes murine H-Ras, represented herein as SEQ ID NO:9. SEQ ID NO: 10 is the nucleic acid sequence encoding human N-Ras (also known in GenBank Accession No. NM 002524). SEQ ID NO: 10 encodes human N-Ras, represented herein as SEQ ID NO: l l . SEQ ID NO: 12 is the nucleic acid sequence encoding murine N-Ras (also known in GenBank Accession No. NM 010937). SEQ ID NO: 12 encodes human N-Ras, represented herein as SEQ ID NO: 13. SEQ ID NOs:3, 5, 7, 9, 11 and 13 are representative of "wild-type" Ras amino acid sequences.

[0098] In one aspect, a Ras fusion protein suitable for use as a Ras G12R antigen in the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 15 (encoded by a nucleic acid sequence represented herein as SEQ ID NO: 14). SEQ ID NO: 15 is the fusion protein expressed by a yeast-based immunotherapeutic known as "GI- 4020" which is a member of yeast-based immunotherapeutics targeting mutated Ras known collectively as "GI-4000" (see Example 1). In another aspect, a Ras G12R antigen useful in the invention can include an antigen comprising at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids of any one of SEQ ID NOs:3, 5, 7, 9, 11, or 13, wherein the antigen includes the amino acid at position 12 of SEQ ID NO:3, 5, 7, 9, 11 or 13, except that the glycine at that position in SEQ ID NO:3, 5, 7, 9, 11 or 13 has been substituted for an arginine. In one aspect, this antigen can be one domain of a multi-domain fusion protein in which the other domain(s) are additional immunogenic domains of Ras comprising one or more mutations, or in which the other domain(s) are a different cancer antigen. SEQ ID NO: 15 represents the amino acid sequence of such a fusion protein. In one aspect, the Ras G12R antigen includes at least from position 4-12, position 5-13, position 6-14, position 7-15, position 8-16, position 9-17, position 10-18, position 11-19 or position 12-20, of the WT Ras sequence, except that the glycine at position 12 is substituted with an arginine.

[0099] In one embodiment of the invention, a Ras G12R immunotherapeutic composition, such as GI-4020 (antigen represented by SEQ ID NO: 15) described above, is administered concurrently or sequentially with another immunotherapeutic composition that comprises a Ras antigen with one or more mutations that do not include the G12R mutation. The one or more mutations that are not G12R are, in a preferred embodiment, one or more mutations that are present in cells of a tumor from a subject to be treated. In other words, the Ras G12R immunotherapy composition and the composition comprising a Ras mutation other than G12R are used to specifically target the subject's tumor (by including the Ras mutation expressed by the subject's tumor) and to further provide an agonist antigen to enhance the immune response against the tumor (by including the Ras G12R antigen). Alternatively, the RasG12R immunotherapeutic composition may also include additional mutated Ras antigens having a mutation other than G12R, or additional non-G12R mutations can be included together with the G12R mutation in the same Ras antigen construct.

[00100] Some exemplary Ras fusion proteins having non-G12R mutations (Ras antigens with mutations other than G12R) useful in this aspect of the invention are described in Example 1, and have been described, for example, in U.S. Patent Nos. 7,465,454 and 7,563,447. Example 1 describes four yeast immunotherapy compositions (including the Ras G12R composition known as GI-4020 discussed above), each of which expresses a fusion protein including three different Ras mutations {i.e., SEQ ID NO: 15 (GI-4020), SEQ ID NO: 17 (GI-4014), SEQ ID NO: 19 (GI-4015) and SEQ ID NO:21 (GI- 4016)). Specifically, each protein product expressed in the yeast contains: (A) two mutations at codon 61 (glutamine to arginine [Q61R] (GI-4014, GI-4015, GI-4016) or glutamine to histidine [Q61H] (GI-4020), and glutamine to leucine [Q61L] (GI-4014, GI- 4015, GI-4016, GI-4020); plus (B) one of four different mutations at codon 12 (glycine to valine [G12V] (GI-4014), glycine to cysteine [G12C] (GI-4015), glycine to aspartate [G12D] (GI-4016), or glycine to arginine [G12R] (GI-4020)). Other similar fusion proteins can be created for use in an immunotherapy composition, and can be designed expressly to provide the mutation expressed by the tumor of the subject to be treated.

[00101] Accordingly, the immunotherapeutic composition known as GI-4020 can be used in combination (admixed, administered concurrently, or administered sequentially) in a "spice rack" approach with any of the other compositions described herein, where the other compositions are selected based on the Ras mutation in the tumor of the subject to be treated. For example, if the subject has a cancer with a G12V mutation, one could select the product known as GI-4014 (SEQ ID NO: 17) and administer it together with, concurrently with, or sequentially with the product known as GI-4020 (SEQ ID NO: 15; the Ras G12R product). Similarly, and again by way of example, if the subject has a cancer with a Q61L mutation, one could select any of the products known as GI-4014, GI- 4015 or GI-4016 described above, and administer it together with, concurrently with, or sequentially with the product known as GI-4020. The invention is not limited to these particular Ras fusion proteins and immunotherapeutic compositions described herein.

[00102] In one embodiment, the Ras fusion proteins of any of SEQ ID NO: 17 (encoded by SEQ ID NO: 16), SEQ ID NO: 19 (encoded by SEQ ID NO: 18) , or SEQ ID NO:21 (encoded by SEQ ID NO:20) can be modified to include a G12R mutation within the same fusion protein. This can be achieved, for example, by introducing into the fusion protein a new immunogenic domain of Ras that is at least nine amino acids in length and that contains the G12R mutation. The new domain can be introduced at the N-terminus, the C-terminus, or between other existing immunogenic domains in the fusion protein. In another embodiment, a Ras protein having a G12R mutation can be further modified to introduce a mutation at amino acid position 61 , such as a Q61L mutation, a Q61R mutation, or a Q61H mutation. In another embodiment, one can create a fusion protein comprising an immunogenic domain of a Ras protein that includes the G12R mutation (e.g., at least 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more amino acids of Ras that include amino acid position 12, where the WT glycine residue has been substituted with an arginine residue), this immunogenic domain being fused at the N- or C-terminus to a second immunogenic domain of Ras, which may be a repeat of the same domain, except that in the repeated domain, the WT glycine at position 12 is substituted with a non-arginine residue (e.g., a valine, cysteine, aspartic acid, serine or alanine). In this manner, an immunogenic composition of the invention can provide both a G12R mutation and a second Ras G12 mutation that is expressed by a subject's tumor in the same fusion protein, and the protein may be expected to be efficiently processed and relevant epitopes presented to the immune system. Other permutations of these embodiments will be apparent to those of skill in the art given this disclosure.

[00103] The present invention also includes, in some embodiments, the provision of one or more additional tumor antigens in the same or a different immunotherapy composition, in addition to the Ras G12R antigen. Various mutated Ras antigens can be constructed using the guidance provided herein regarding Ras sequences and transforming mutations. Additional tumor antigens that can be used in an immunotherapy composition and immunotherapy compositions comprising such antigens have been described, for example, in WO 07/133835, WO 08/1 15610, WO 2010/121 180, WO 2012/125998, or WO 2013/025972, each of which is incorporated by reference in its entirety. Such additional antigens include, but are not limited to, carcinoembryonic antigen (CEA), brachyury, mucin- 1 (MUC-1), EGFR, BCR-Abl, MART-1 , MAGE-1 , MAGE-3, GAGE, GP-100, MUC-2, PSMA, tyrosinase, TRP-1 (gp75), NY-ESO-1 , TRP-2, TAG72, KSA, 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. [00104] An antigen useful in a composition for use in a method according to the present invention also includes proteins having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any of the cancer proteins or cancer antigens described herein over the full length of the protein, or with respect to a defined fragment or domain thereof (e.g., an immunological domain or functional domain (domain with at least one biological activity)) that forms part of the protein. An immunological domain has been described in detail above.

[00105] In some aspects of the invention, amino acid insertions, deletions, and/or substitutions can be made for one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids of a WT or reference protein, provided that the resulting protein, when used as an antigen in a Ras G12R immunotherapeutic composition of the invention, elicits an immune response against a native protein, which can be a mutated Ras protein with a G12R mutation, or a mutated Ras protein with a different, non-G12R mutation, which for agonist antigens may include an enhanced immune response. For example, the invention includes the use of Ras G12R antigens as agonist antigens with respect to the treatment of Ras mutation-positive cancers that do not include a G12R mutation. The agonist may improve the avidity or affinity of the epitope for an MHC molecule or for the T cell receptor that recognizes the epitope in the context of MHC presentation. Antigen agonists may therefore improve the potency or efficiency of a T cell response against native mutated antigen expressed by a tumor cell, even if the mutation is different than G12R.

[00106] In addition, N-terminal expression sequences and the C-terminal tags are optional, but may be selected from several different sequences described elsewhere herein to improve or assist with expression, stability, and/or allow for identification and/or purification of the protein. Also, many different promoters suitable for use in yeast are known in the art. Furthermore, short intervening linker sequences (e.g., 1 , 2, 3, 4, or 5 amino acid peptides) may be introduced between portions of a fusion protein comprising a cancer antigen for a variety of reasons, including the introduction of restriction enzyme sites to facilitate cloning, as cleavage sites for host phagosomal proteases, to accelerate protein or antigen processing, and for future manipulation of the constructs.

[00107] Optionally, proteins, including fusion proteins, which are used as a component of the yeast-based immunotherapeutic composition of the invention, are produced using antigen constructs that are particularly useful for improving or stabilizing the expression of heterologous antigens in yeast. In one embodiment, the desired antigenic protein(s) or peptide(s) are fused at their amino-terminal end to: (a) a specific synthetic peptide that stabilizes the expression of the fusion protein in the yeast vehicle or prevents posttranslational modification of the expressed fusion protein (such peptides are described in detail, for example, in U.S. Patent Publication No. 2004-0156858 Al, published August 12, 2004, incorporated herein by reference in its entirety); (b) at least a portion of an endogenous yeast protein, including but not limited to yeast alpha factor leader sequence, wherein either fusion partner provides improved stability of expression of the protein in the yeast and/or a prevents post-translational modification of the proteins by the yeast cells (such proteins are also described in detail, for example, in U.S. Patent Publication No. 2004-0156858 Al, supra); and/or (c) at least a portion of a yeast protein that causes the fusion protein to be expressed on the surface of the yeast {e.g., an Aga protein, described in more detail herein). In addition, the present invention optionally includes the use of peptides that are fused to the C-terminus of the antigen-encoding construct, particularly for use in the selection and identification of the protein. Such peptides include, but are not limited to, any synthetic or natural peptide, such as a peptide tag {e.g., 6X His or hexapeptide) or any other short epitope tag. Peptides attached to the C-terminus of an antigen according to the invention can be used with or without the addition of the N- terminal peptides discussed above, and vice versa.

[00108] In one embodiment, a fusion protein comprises an amino acid sequence of M- X2-X3-X4-X5-X6, wherein M is methionine; wherein X2 is any amino acid except glycine, proline, lysine or arginine; wherein X3 is any amino acid except methionine, lysine or arginine; wherein X4 is any amino acid except methionine, lysine or arginine; wherein X5 is any amino acid except methionine, lysine or arginine; and wherein X6 is any amino acid except methionine, lysine or arginine. In one embodiment, the X6 residue is a proline. An exemplary synthetic sequence that enhances the stability of expression of an antigen in a yeast cell and/or prevents post-translational modification of the protein in the yeast includes the sequence M-A-D-E-A-P (SEQ ID NO: l). In addition to the enhanced stability of the expression product, this fusion partner does not appear to negatively impact the immune response against the immunizing antigen in the construct. In addition, the synthetic fusion peptides can be designed to provide an epitope that can be recognized by a selection agent, such as an antibody.

[00109] 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 immunotherapy compositions are contemplated by the invention. [00110] According to the present invention, the term "yeast vehicle-antigen complex" or "yeast-antigen complex" is used generically to describe any association of a yeast vehicle with an antigen, and can be used interchangeably with "yeast-based immunotherapy composition" when such composition is used to elicit an immune response as described above. Such association includes expression of the antigen by the yeast (a recombinant yeast), introduction of an antigen into a yeast, physical attachment of the antigen to the yeast, and mixing of the yeast and antigen together, such as in a buffer or other solution or formulation. These types of complexes are described in detail below.

[00111] In one embodiment, a yeast cell used to prepare the yeast vehicle is transfected with a heterologous nucleic acid molecule encoding a protein (e.g., the antigen) such that the protein is expressed by the yeast cell. Such a yeast is also referred to herein as a recombinant yeast or a recombinant yeast vehicle. The yeast cell can then be formulated with a pharmaceutically acceptable excipient and administered directly to a patient, stored for later administration, or loaded into a dendritic cell as an intact cell. The yeast cell can also be killed, or it can be derivatized such as by formation of yeast spheroplasts, cytoplasts, ghosts, or subcellular particles, any of which may be followed by storing, administering, or loading of the derivative into the dendritic cell. Yeast spheroplasts can also be directly transfected with a recombinant nucleic acid molecule (e.g., the spheroplast is produced from a whole yeast, and then transfected) in order to produce a recombinant spheroplast that expresses the antigen. Yeast cells or yeast spheroplasts that recombinantly express the antigen(s) may be used to produce a yeast vehicle comprising a yeast cytoplast, a yeast ghost, or a yeast membrane particle or yeast cell wall particle, or fraction thereof.

[00112] In general, the yeast vehicle and antigen(s) and/or other agents can be associated by any technique described herein. In one aspect, the yeast vehicle was loaded intracellularly with the antigen(s) and/or agent(s). In another aspect, the antigen(s) and/or agent(s) was covalently or non-covalently attached to the yeast vehicle. In yet another aspect, the yeast vehicle and the antigen(s) and/or agent(s) were associated by mixing. In another aspect, and in one embodiment, the antigen(s) and/or agent(s) are expressed recombinantly by the yeast vehicle or by the yeast cell or yeast spheroplast from which the yeast vehicle was derived.

[00113] A number of antigens and/or other proteins to be produced by a yeast vehicle of the present invention is any number of antigens and/or other proteins that can be reasonably produced by a yeast vehicle, and typically ranges from at least one to at least about 6 or more, including from about 2 to about 6 antigens and/or other proteins.

[00114] Expression of an antigen or other protein in a yeast vehicle of the present invention is accomplished using techniques known to those skilled in the art. Briefly, a nucleic acid molecule encoding at least one desired antigen or other protein is inserted into an expression vector in such a manner that the nucleic acid molecule is operatively linked to a transcription control sequence in order to be capable of effecting either constitutive or regulated expression of the nucleic acid molecule when transformed into a host yeast cell. Nucleic acid molecules encoding one or more antigens and/or other proteins can be on one or more expression vectors operatively linked to one or more expression control sequences. Particularly important expression control sequences are those which control transcription initiation, such as promoter and upstream activation sequences. Any suitable yeast promoter can be used in the present invention and a variety of such promoters are known to those skilled in the art. Promoters for expression in S. cerevisiae include, but are not limited to, promoters of genes encoding the following yeast proteins: alcohol dehydrogenase I (ADHl) or II (ADH2), copper-inducible yeast metallothionein (CUP1), phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), translational elongation factor EF-1 alpha (TEF2), glyceraldehyde-3 -phosphate dehydrogenase (GAPDH; also referred to as TDH3, for triose phosphate dehydrogenase), galactokinase (GALl), galactose- 1 -phosphate uridyl-transferase (GAL7), UDP-galactose epimerase (GAL 10), cytochrome cl (CYCl), Sec7 protein (SEC7) and acid phosphatase (PH05), including hybrid promoters such as ADH2/GAPDH and CYCl /GAL 10 promoters, and including the ADH2/GAPDH promoter, which is induced when glucose concentrations in the cell are low (e.g., about 0.1 to about 0.2 percent). Likewise, a number of upstream activation sequences (UASs), also referred to as enhancers, are known. Upstream activation sequences for expression in S. cerevisiae include, but are not limited to, the UASs of genes encoding the following proteins: PCKl, TPI, TDH3, CYCl, ADHl, ADH2, SUC2, GALl, GAL7 and GAL 10, as well as other UASs activated by the GAL4 gene product, with the ADH2 UAS being used in one aspect. Since the ADH2 UAS is activated by the ADR1 gene product, it may be preferable to overexpress the ADR1 gene when a heterologous gene is operatively linked to the ADH2 UAS. Transcription termination sequences for expression in S. cerevisiae include the termination sequences of the a-factor, GAPDH, and CYCl genes. [00115] Transcription control sequences to express genes in methyltrophic yeast include the transcription control regions of the genes encoding alcohol oxidase and formate dehydrogenase.

[00116] Transfection of a nucleic acid molecule into a yeast cell according to the present invention can be accomplished by any method by which a nucleic acid molecule can be introduced into the cell and includes, but is not limited to, diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Transfected nucleic acid molecules can be integrated into a yeast chromosome or maintained on extrachromosomal vectors using techniques known to those skilled in the art. Examples of yeast vehicles carrying such nucleic acid molecules are disclosed in detail herein. As discussed above, yeast cytoplast, yeast ghost, and yeast membrane particles or cell wall preparations can also be produced recombinantly by transfecting intact yeast microorganisms or yeast spheroplasts with desired nucleic acid molecules, producing the antigen therein, and then further manipulating the microorganisms or spheroplasts using techniques known to those skilled in the art to produce cytoplast, ghost or subcellular yeast membrane extract or fractions thereof containing desired antigens or other proteins.

[00117] Effective conditions for the production of recombinant yeast vehicles and expression of the antigen and/or other protein by the yeast vehicle include an effective medium in which a yeast strain can be cultured. An effective medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins and growth factors. The medium may comprise complex nutrients or may be a defined minimal medium. Yeast strains of the present invention can be cultured in a variety of containers, including, but not limited to, bioreactors, Erlenmeyer flasks, test tubes, microtiter dishes, and Petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the yeast strain. Such culturing conditions are well within the expertise of one of ordinary skill in the art (see, for example, Guthrie et al. (eds.), 1991, Methods in Enzvmology, vol. 194, Academic Press, San Diego). For example, under one protocol, liquid cultures containing a suitable medium can be inoculated using cultures obtained from starter plates and/or starter cultures of yeast-mutated Ras immunotherapy compositions, and are grown for approximately 20h at 30°C, with agitation at 250 rpm. Primary cultures can then be expanded into larger cultures as desired. Protein expression from vectors with which the yeast were transformed may be constitutive if the promoter utilized is a constitutive promoter, or may be induced by addition of the appropriate induction conditions for the promoter if the promoter utilized is an inducible promoter (e.g., copper sulfate in the case of the CUP I promoter). In the case of an inducible promoter, induction of protein expression may be initiated after the culture has grown to a suitable cell density, which may be at about 0.2 Y.U./ml or higher densities.

[00118] In one embodiment of the present invention, as an alternative to expression of an antigen or other protein recombinantly in the yeast vehicle, a yeast vehicle is loaded intracellularly with the protein or peptide, or with carbohydrates or other molecules that serve as an antigen and/or are useful as immunomodulatory agents or biological response modifiers according to the invention. Subsequently, the yeast vehicle, which now contains the antigen and/or other proteins intracellularly, can be administered to an individual or loaded into a carrier such as a dendritic cell. Peptides and proteins can be inserted directly into yeast vehicles of the present invention by techniques known to those skilled in the art, such as by diffusion, active transport, liposome fusion, electroporation, phagocytosis, freeze-thaw cycles and bath sonication. Yeast vehicles that can be directly loaded with peptides, proteins, carbohydrates, or other molecules include intact yeast, as well as spheroplasts, ghosts or cytoplasts, which can be loaded with antigens and other agents after production. Alternatively, intact yeast can be loaded with the antigen and/or agent, and then spheroplasts, ghosts, cytoplasts, or subcellular particles can be prepared therefrom. Any number of antigens and/or other agents can be loaded into a yeast vehicle in this embodiment, from at least 1, 2, 3, 4 or any whole integer up to hundreds or thousands of antigens and/or other agents, such as would be provided by the loading of a microorganism or portions thereof, for example.

[00119] In another embodiment of the present invention, an antigen and/or other agent is physically attached to the yeast vehicle. Physical attachment of the antigen and/or other agent to the yeast vehicle can be accomplished by any method suitable in the art, including covalent and non-covalent association methods which include, but are not limited to, chemically crosslinking the antigen and/or other agent to the outer surface of the yeast vehicle or biologically linking the antigen and/or other agent to the outer surface of the yeast vehicle, such as by using an antibody or other binding partner. Chemical cross- linking can be achieved, for example, by methods including glutaraldehyde linkage, photoaffinity labeling, treatment with carbodiimides, treatment with chemicals capable of linking di-sulfide bonds, and treatment with other cross-linking chemicals standard in the art. Alternatively, a chemical can be contacted with the yeast vehicle that alters the charge of the lipid bilayer of yeast membrane or the composition of the cell wall so that the outer surface of the yeast is more likely to fuse or bind to antigens and/or other agent having particular charge characteristics. Targeting agents such as antibodies, binding peptides, soluble receptors, and other ligands may also be incorporated into an antigen as a fusion protein or otherwise associated with an antigen for binding of the antigen to the yeast vehicle.

[00120] When the antigen or other protein is expressed on or physically attached to the surface of the yeast, spacer arms may, in one aspect, be carefully selected to optimize antigen or other protein expression or content on the surface. The size of the spacer arm(s) can affect how much of the antigen or other protein is exposed for binding on the surface of the yeast. Thus, depending on which antigen(s) or other protein(s) are being used, one of skill in the art will select a spacer arm that effectuates appropriate spacing for the antigen or other protein on the yeast surface. In one embodiment, the spacer arm is a yeast protein of at least 450 amino acids. Spacer arms have been discussed in detail above.

[00121] In yet another embodiment, the yeast vehicle and the antigen or other protein are associated with each other by a more passive, non-specific or non-covalent binding mechanism, such as by gently mixing the yeast vehicle and the antigen or other protein together in a buffer or other suitable formulation (e.g., admixture).

[00122] In one embodiment, intact yeast (with or without expression of heterologous antigens or other proteins) can be ground up or processed in a manner to produce yeast cell wall preparations, yeast membrane particles or yeast fragments (i.e., not intact) and the yeast fragments can, in some embodiments, be provided with or administered with other compositions that include antigens (e.g., DNA vaccines, protein subunit vaccines, killed or inactivated pathogens, viral vector vaccines) to enhance immune responses. For example, enzymatic treatment, chemical treatment or physical force (e.g., mechanical shearing or sonication) can be used to break up the yeast into parts that are used as an adjuvant.

[00123] In one embodiment of the invention, yeast vehicles useful in the invention include yeast vehicles that have been killed or inactivated. Killing or inactivating of yeast can be accomplished by any of a variety of suitable methods known in the art. For example, heat inactivation of yeast is a standard way of inactivating yeast, and one of skill in the art can monitor the structural changes of the target antigen, if desired, by standard methods known in the art. Alternatively, other methods of inactivating the yeast can be used, such as chemical, electrical, radioactive or UV methods. See, for example, the methodology disclosed in standard yeast culturing textbooks such as Methods of Enzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of the inactivation strategies used should take the secondary, tertiary or quaternary structure of the target antigen into consideration and preserve such structure as to optimize its immunogenicity.

[00124] Yeast vehicles can be formulated into yeast-based immunotherapy compositions or products of the present invention using a number of techniques known to those skilled in the art. For example, yeast vehicles can be dried by lyophilization. Formulations comprising yeast vehicles can also be prepared by packing yeast in a cake or a tablet, such as is done for yeast used in baking or brewing operations. In addition, yeast vehicles can be mixed with a pharmaceutically acceptable excipient, such as an isotonic buffer that is tolerated by a host or host cell. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity-enhancing agents, such as sodium carboxymethylcellulose, sorbitol, glycerol or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise, for example, dextrose, human serum albumin, and/or preservatives to which sterile water or saline can be added prior to administration.

[00125] In one embodiment of the present invention, a composition can 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 can be transfected with or loaded with at least one antigen and at least one agent/biological response modifier compound, or a composition of the invention can be administered in conjunction with at least one agent/biological response modifier. Biological response modifiers include adjuvants and other compounds that can modulate immune responses, which may be referred to as immunomodulatory compounds, as well as compounds that modify the biological activity of another compound or agent, such as a yeast-based immunotherapeutic, such biological activity not being limited to immune system effects. Certain immunomodulatory compounds can stimulate a protective immune response whereas others can suppress a harmful immune response, and whether an immunomodulatory compound is useful in combination with a given yeast-based immunotherapeutic may depend, at least in part, on the disease state or condition to be treated or prevented, and/or on the individual who is to be treated. Certain biological response modifiers preferentially enhance a cell-mediated immune response whereas others preferentially enhance a humoral immune response (i.e., can stimulate an immune response in which there is an increased level of cell-mediated compared to humoral immunity, or vice versa.). Certain biological response modifiers have one or more properties in common with the biological properties of yeast-based immunotherapeutics or enhance or complement the biological properties of yeast-based immunotherapeutics. There are a number of techniques known to those skilled in the art to measure stimulation or suppression of immune responses, as well as to differentiate cell-mediated immune responses from humoral immune responses, and to differentiate one type of cell-mediated response from another (e.g., a Thl7 response versus a Thl response).

[00126] Agents/biological response modifiers useful in the invention may include, but are not limited to, cytokines, chemokines, hormones, lipidic derivatives, peptides, proteins, polysaccharides, small molecule drugs, antibodies and antigen binding fragments thereof (including, but not limited to, anti-cytokine antibodies, anti-cytokine receptor antibodies, anti-chemokine antibodies), vitamins, polynucleotides, nucleic acid binding moieties, aptamers, and growth modulators. Any combination of such agents is contemplated by the invention, and any of such agents combined with or administered in a protocol with (e.g. , concurrently, sequentially, or in other formats with) a yeast-based immunotherapeutic is a composition encompassed by the invention. Such agents are well known in the art. These agents may be used alone or in combination with other agents described herein.

[00127] Agents/biological response modifiers that are particularly useful in combination with a yeast-based immunotherapy composition in accordance with the invention include, but are not limited to: anti-CD40 antibody, CD40L, lymphocyte- activation gene 3 (LAG3) protein and/or IMP321 (T-cell immunostimulatory factor derived from the soluble form of LAG3); T cell co-stimulators (e.g., anti-CD 137, anti- CD28, anti-CD40 antibodies); alemtuzumab (e.g., CamPath®), denileukin diftitox (e.g., ONTAK®); anti-CD4 antibody; anti-CD25 antibody; immune checkpoint inhibitors (e.g., inhibitors of "immune checkpoints" which are inhibitory pathways of the immune system that maintain self-tolerance and modulate the duration and amplitude of physiological immune responses, such immune checkpoint inhibitors including but not limited to: anti- CTLA-4 antibody, such as ipilimumab (Bristol-Myers Squibb, Princeton, NJ) or tremelimumab (Medlmmune/AstraZeneca, Wilmington, DE), programmed cell death protein 1 (PD-1), programmed cell death protein 1 ligand (PD-Ll), programmed cell death protein 2 ligand (PD-L2, such as the PD-L2 fusion protein known as AMP-224 (Amplimmune, Gaithersburg, MD/GlaxoSmithKline, Philadelphia, PA)), anti-PD-1 antibody (such as nivolumab (Bristol-Myers Squibb), pembrolizumanb (Merck, Whitehouse Station, NJ), or pidilizumab (CureTech, Yavne, Israel)), anti-PD-Ll antibody (such as MPDL3280A (Genentech, South San Francisco, CA), MEDI4736 (Medlmmune/AstraZeneca), BMS-936559 (Bristol-Myers Squibb), MSB0010718C (EMD Serono, Rockland, MD)), or anti-PD-L2 antibody); indoleamine 2,3-dioxygenase (IDO) inhibitors (such as INCB24360); agents that block FOXP3 (e.g., to abrogate the activity/kill CD4⁺/CD25⁺ Treg cells); Flt3 ligand, imiquimod (Aldara™), 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 analogs thereof including IMiDs® (which are structural and functional analogues of thalidomide (e.g., REVLIMID® (lenalidomide), POMALYST® (pomalidomide)) and any agents that modulate the number of, modulate the activation state of, and/or modulate the survival of antigen-presenting cells or of Thl7, Thl , and/or Treg cells. Any combination of such agents is contemplated by the invention, and any of such agents combined with or administered in a protocol with (e.g., concurrently, sequentially, or in other formats with) a yeast-based immunotherapeutic is a composition encompassed by the invention. Such agents are well known in the art. These agents may be used alone or in combination with other agents described herein. In addition, one or more therapies can be administered or performed prior to the first dose of yeast-based immunotherapy composition or after the first dose is administered.

[00128] Agents can include agonists and antagonists of a given protein or peptide or domain thereof. As used herein, an "agonist" is any compound or agent, including without limitation small molecules, proteins, peptides, antibodies, nucleic acid binding agents, etc., that binds to a receptor or ligand and produces or triggers a response, which may include agents that mimic or enhance the action of a naturally occurring substance that binds to the receptor or ligand. An "antagonist" is any compound or agent, including without limitation small molecules, proteins, peptides, antibodies, nucleic acid binding agents, etc., that blocks or inhibits or reduces the action of an agonist.

Biomarkers Based on G12R and HLA-A3 and Uses Thereof

[00129] An additional embodiment of the present invention relates to the use of Ras G12R as a biomarker for the positive prediction of survival in patients (subjects) with a Ras mutation-positive cancer {e.g., extended survival as compared to patients with Ras mutation-positive cancer who do not have a G12R mutation), including Ras mutation- positive pancreas cancer. In this embodiment, a sample of a subject's tumor is collected and the presence of mutated Ras, and specifically, a Ras G12R mutation, is detected via laboratory analysis of DNA, cDNA, RNA or proteins isolated from the tumor. If a G12R Ras or G12R ras mutation is detected (either by detection of the nucleic acid or the protein) in a subject's tumor, this result is considered to be a positive predictor that the subject having the Ras G12R mutation has an improved likelihood of survival (or is likely to survive for longer), as compared to subjects having all other Ras mutations. This result also indicates that the subject will have an improved survival if treated with an immunotherapy product having a G12R antigen, as compared to treatment without inclusion of a G12R immunotherapy product as part of the protocol. If the method detects a non-G12R mutation in Ras (the absence of the G12R mutation) in the subject's tumor, such a result indicates that the subject has a lower likelihood of survival (shorter period of survival) as compared to a subject who has a G12R mutation in Ras. This result also indicates that the subject without a G12R mutation in Ras could or should be treated with an immunotherapeutic composition that includes a G12R antigen, in order to increase the likelihood of survival of the subject and/or enhance anti-tumor immune responses in the patient. In one embodiment in the case of a subject that has a cancer expressing mutated Ras, where the subject's tumor(s) do not have a G12R mutation {i.e., the tumor has a different mutation in Ras), the detection of a non-G12R mutation in Ras indicates that the subject should be treated with an immunogenic composition that includes a G12R antigen, and also a Ras antigen having the same mutation as in the subject's tumor.

[00130] A further embodiment of the invention includes the use of HLA-A3 as a biomarker for the negative prediction of survival in subjects with a Ras mutation-positive cancer, including Ras mutation-positive pancreas cancer, as well as a biomarker to select subjects for treatment with Ras G12R immunotherapy. As discussed previously, according to the present invention, a subject with at least one allele of HLA-A*0301 or HLA-A*0302, or other rare alleles with similar peptide anchoring properties, can be generally referred to as an "HLA-A3 subject" or "HLA-A3^+" (or "HLA-A3 positive") according to the present invention. In this embodiment, a biological sample (e.g., blood, serum, tissue, etc.) from the subject is collected and the HLA type(s) expressed by the cells of the subject is detected. If the subject expresses at least one HLA-A3 allele, but not a Ras G12R mutation in the tumor cells, then the result indicates that the subject has a lower likelihood of survival (shorter period of survival) as compared to subjects who do not express HLA- A3. In this case, administration of an immunotherapy composition comprising a Ras G12R antigen could be used to improve the likelihood of survival of the subject. If the subject expresses HLA-A3 and has a tumor that expresses a Ras G12R mutation, than the subject can be treated the same as other subjects having a Ras G12R mutation above, i.e., administration of a G12R immunotherapeutic composition can be expected to further enhance the likelihood that the subject will survive longer than a subject not treated with a G12R immunotherapeutic composition.

[00131] A further embodiment of the invention includes the detection of the ability of a subject to elicit an immune response that is specific for (directed against or targets) a G12R antigen. A subject with a Ras mutation-positive cancer whose T cells exhibit a Ras G12R-specific immune response in vitro or ex vivo, regardless of the identity of the particular Ras mutations in the subject's tumor cells, may then be selected as a subject for administration of an immunotherapeutic composition comprising a Ras G12R antigen according to the invention. Testing a subject for an immune response to Ras G12R can, in some embodiments, be combined with detection of the HLA-A3 biomarker and/or detection of the absence (or presence) of a Ras G12R mutation in the tumor of the subject. A subject whose immune system cells {e.g., T cells) demonstrate a detectable or measurable, specific immune response to a G12R antigen can be referred to herein as an "immune responder". Typically, the response is measured over a background or control level of response. In some embodiments, an immune responder is a "categorical responder" based on pre-specified criteria used to evaluate the immune response. For example, if the immune response to be detected is a T cell response as measured by interferon-γ (IFN-γ) production (or another cytokine) by peripheral blood mononuclear cells (PBMCs) isolated from a subject in response to exposure to the target antigen, or by proliferation of T cells or PBMCs isolated from a subject in response to exposure to the target antigen, in vitro, one skilled in the art may pre-define criteria to be met in order to classify a subject as an immune responder. Such criteria can include a measurable increase of a minimum magnitude in an immune response parameter {e.g., IFN-γ production or lymphocyte proliferation), over a baseline response or previously measured response, after subtraction of a control or background response. An example of a control or background response can be the response of the PBMCS in the absence of the target antigen. An example of such a pre-specified criteria-based evaluation is provided in Example 1. An immune response can be evaluated using any suitable method known in the art, including but not limited to, lymphocyte (or T cell) proliferation assay, cytokine assays, and CTL killing assays. Lymphocyte proliferation is typically measured in vitro, by obtaining T cells or PBMCs from the subject and exposing them to target antigen in the presence of antigen presenting cells, and measuring proliferation of the T cells, such as by using a radioisotope or colorimetric detection method. Cytokine assays include, but are not limited to, enzyme-linked immunospot assay (ELISpot), enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunohistochemical analysis, immunoblotting, fluorescence activated cell sorting (FACS), and flow cytometry.

[00132] Accordingly, the invention includes the use of Ras G12R (mutation in Ras protein or ras oncogene), as well as the use of HLA-A3 alleles as biomarkers in a diagnostic or prognostic assay or kit for cancer. The invention includes methods to detect the presence or absence of these biomarkers in a subject sample {e.g., a subject's tumor cells or blood sample or other cell or tissue sample), the results of which are used to indicate and prescribe a preferred treatment protocol based on the result of the detection of the biomarker, or the lack of detection of the biomarker.

[00133] Suitable methods of obtaining a subject sample are known to a person of skill in the art. A subject sample can include any bodily fluid or tissue from a subject that may contain tumor cells or proteins of tumor cells. More specifically, according to the present invention, the term "test sample" or "patient sample" or "subject sample" or "biological sample" can be used generally to refer to a sample of any type which contains cells or products that have been secreted from or is contained within cells to be evaluated by the present method, including but not limited to, a sample of isolated cells {e.g., peripheral blood mononuclear cells (PBMCs), tumor cells, etc.), a sample of isolated proteins, a tissue sample {e.g., a section or biopsy of a tumor or other tissue), a bodily fluid sample {e.g., blood, serum, saliva, etc.), or, for example, a sample of nucleic acids (DNA, cDNA, RNA) obtained from and/or produced from a cell or tissue sample isolated from the subject. A "T cell-containing biological sample" is a biological sample as described above that contains within the sample T cells {i.e., T lymphocytes, which may include CD4⁺ and/or CD8 T cells) from the subject. Such samples can include, but are not limited to, blood samples, PBMCs, or a tissue sample.

[00134] Once a sample is obtained from the subject, the sample is evaluated to detect the presence or absence of the ras gene or Ras protein with a G12R mutation (and may also include detection of other Ras mutations at positions 12, 13, 59, 61 and/or 76), or to detect the presence or absence of an HLA-A3 allele or its encoded protein. For example, the presence and/or level of the G12R ras biomarker and/or the HLA-A3 allele can be determined by conventional methods such as gene, DNA or RNA detection methods {e.g., DNA sequencing, oligonucleotide hybridization, PCR amplification with primers specific to the mutation or allele), or protein detection methods {e.g., immunoassays or biochemical assays to determine the level of the gene product). In general, the nucleic acid sequence in a subject sample can be detected by any suitable method or technique of measuring or detecting nucleic acid sequence or expression. Such methods include, but are not limited to, PCR, reverse transcriptase-PCR (RT-PCR), in situ PCR, in situ hybridization, Southern blot, Northern blot, sequence analysis, microarray analysis, detection of a reporter gene, or other DNA/RNA hybridization platforms. Expression can be evaluated simply for the presence of the mutated ras sequence(s) or the HLA allele, and/or compared to samples isolated from healthy individuals or another negative control.

[00135] For example, a patient tumor biopsy sample from an embedded paraffin block may be sectioned and stained with hematoxylin, after which the pathological cells from the sample may be isolated by laser capture microdissection. The genomic DNA from the isolated cells is then used as a template for a PCR reaction to amplify the DNA fragment harboring the specified ras sequence (in this case, ras encoding a Ras protein with a G12R mutation) using primers that flank the sequence of interest. Alternatively, the sections from the tumor biopsy may be analyzed by in situ PCR, such that amplification is dependent on hybridization with primers that bind to the mutated sequence, and elongated with labeled nucleotides, such that an amplified sequence is specifically detected within the tumor cells. As yet another alternative, the sections may be probed with oligonucleotides that hybridize specifically with the G12R mutation, but not with WT ras sequence.

[00136] When the biomarker is detected as a protein {e.g., a Ras protein having a G12R mutation), protein expression can be detected in suitable tissues, such as tumor tissue, peripheral blood mononuclear cells, and cell material obtained by biopsy. For example, the patient tumor biopsy sample, which can be immobilized, can be contacted with an antibody, an antibody fragment, or an aptamer, that selectively binds to the protein to be detected and then it can be determined whether the antibody, fragment thereof or aptamer has bound to the protein. Binding can be measured using a variety of methods standard in the art, including, but not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry.

[00137] HLA type is presently most often detected using any of a variety of methods, including, but not limited to: serological methods (detection by antibody), mixed lymphocyte culture, and molecular techniques of sequence-specific priming (SSP), sequence-specific oligonucleotide probing (SSOP), sequence based typing (SBT) and reference strand-based conformation analysis (RSCA) method and similar methods. Ras mutations are presently most often detected using any of a variety of assays described above, and including hybridization assays, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), DNA sequencing, PCR pyrosequencing, amplification-refractory mutation system (ARMS), and similar methods.

[00138] The present invention also includes a kit for performing the prognostic methods of the present invention. The kit preferably contains any reagent useful for detecting the presence or absence of the Ras (protein) or ras (nucleic acid) mutation in a test sample, and must be able to detect the presence or absence of a Ras or ras G12R mutation, and so may include, for example, oligonucleotide probes, PCR primers, and/or antibodies, antigen binding peptides, or aptamers as detection tool(s). The kit can include any reagent needed to perform a detection method envisioned herein. The kit may also include any reagent useful for detecting the presence or absence of an HLA- A3 allele. The kit can also include reagents for the detection of other cancer biomarkers, such as the other Ras mutations, or any other suitable target for cancer diagnosis, even for cancers having causes or contributions unrelated to the Ras mutation described herein. The reagents can be conjugated to another unit, for example a marker, or immobilized to a solid carrier (substrate). In one embodiment, the kit can contain a reagent for detecting a control biomarker characteristic of a cell type in the test sample. The reagent may be present in free form or immobilized to a substrate such as a plastic dish, microarray plate, a test tube, a test rod and so on. The kit can also include suitable reagents for the detection of the reagent and/or for the labeling of positive or negative controls, wash solutions, dilution buffers and the like. The kit can also include a set of written instructions for using the kit and interpreting the results. In one embodiment, the kit is formulated to be a high- throughput assay.

[00139] The reagents can be conjugated to a detectable tag or detectable label. Such a tag can be any suitable tag which allows for detection of the reagents used to detect the biomarker or control marker and includes, but is not limited to, any composition or label detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the

3 125 35 14 32

like), radiolabels (e.g., H, I, S, C, or P), enzymes (e.g., horse-radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

General Techniques Useful in the Invention

[00140] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Methods of Enzvmology, Vol. 194, Guthrie et al, eds., Cold Spring Harbor Laboratory Press (1990); Biology and activities of yeasts. Skinner, et al, eds., Academic Press (1980); Methods in yeast genetics : a laboratory course manual. Rose et al, Cold Spring Harbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle and Cell Biology, Pringle et al, eds., Cold Spring Harbor Laboratory Press (1997); 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), (jointly referred to herein as "Sambrook"); Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (jointly referred to herein as "Harlow and Lane"), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000); Casarett and Doull's Toxicology The Basic Science of Poisons, C. Klaassen, ed., 6th edition (2001), and Vaccines, S. Plotkin, W. Orenstein, and P. Offit, eds., Fifth Edition (2008).

General Definitions

[00141] A "TARMOGEN^®" (Globelmmune, Inc., Louisville, Colorado) generally refers to a yeast vehicle expressing one or more heterologous antigens extracellularly (on its surface), intracellularly (internally or cytosolically) or both extracellularly and intracellularly. TARMOGEN^®s have been generally described (see, e.g., U.S. Patent No. 5,830,463). Certain yeast-based immunotherapy compositions, and methods of making and generally using the same, are also described in detail, for example, in U.S. Patent No. 5,830,463, U.S. Patent No. 7,083,787, U.S. Patent No. 7,736,642, Stubbs et al, Nature Medicine 7:625-629 (2001), Lu et al, Cancer Research 64:5084-5088 (2004), and in Bernstein et al., Vaccine 2008 Jan 24;26(4):509-21, each of which is incorporated herein by reference in its entirety.

[00142] In general, the term "biologically active" indicates that a compound (including a protein or peptide) has at least one detectable activity that has an effect on the metabolic, physiological, chemical, or other processes of a cell, a tissue, or an organism, as measured or observed in vivo {i.e., in a natural physiological environment) or in vitro {i.e., under laboratory conditions).

[00143] According to the present invention, the term "modulate" can be used interchangeably with "regulate" and refers generally to upregulation or downregulation of a particular activity. As used herein, the term "upregulate" can be used generally to describe any of: elicitation, initiation, increasing, augmenting, boosting, improving, enhancing, amplifying, promoting, or providing, with respect to a particular activity. Similarly, the term "downregulate" can be used generally to describe any of: decreasing, reducing, inhibiting, ameliorating, diminishing, lessening, blocking, or preventing, with respect to a particular activity.

[00144] In one embodiment of the present invention, any of the amino acid sequences described herein can be produced with from at least one, and up to about 20, additional heterologous amino acids flanking each of the C- and/or N-terminal ends of the specified amino acid sequence. The resulting protein or polypeptide can be referred to as "consisting essentially of the specified amino acid sequence. According to the present invention, the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Similarly, the phrase "consisting essentially of, when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a specified amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the specified amino acid sequence. The heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid sequence.

[00145] According to the present invention, the phrase "selectively binds to" refers to the ability of an antibody, antigen-binding fragment or binding partner of the present invention to preferentially bind to specified proteins. More specifically, the phrase "selectively binds" refers to the specific binding of one protein to another (e.g., an antibody, fragment thereof, or binding partner to an antigen), wherein the level of binding, as measured by any standard assay (e.g., an immunoassay), is statistically significantly higher than the background control for the assay. For example, when performing an immunoassay, controls typically include a reaction well/tube that contain antibody or antigen binding fragment alone (i.e., in the absence of antigen), wherein an amount of reactivity (e.g., non-specific binding to the well) by the antibody or antigen-binding fragment thereof in the absence of the antigen is considered to be background. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA, immunoblot assays, etc.).

[00146] General reference to a protein or polypeptide used in the present invention includes full-length proteins, near full-length proteins (defined above), or any fragment, domain (structural, functional, or immunogenic), conformational epitope, or a homolog or variant of a given protein. A fusion protein may also be generally referred to as a protein or polypeptide. An isolated protein, according to the present invention, is a protein (including a polypeptide or peptide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for example. As such, "isolated" does not reflect the extent to which the protein has been purified. Preferably, an isolated protein of the present invention is produced recombinantly. According to the present invention, the terms "modification" and "mutation" can be used interchangeably, particularly with regard to the modifications/mutations to the amino acid sequence of proteins or portions thereof (or nucleic acid sequences) described herein.

[00147] As used herein, the term "homolog" or "variant" is used to refer to a protein or peptide which differs from a reference protein or peptide (i.e., the "prototype" or "wild- type" protein) by minor modifications to the reference protein or peptide, but which maintains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to: changes in one or a few amino acid side chains; changes one or a few amino acids, including deletions (e.g., a truncated version of the protein or peptide) insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. A homolog or variant can have enhanced, decreased, or substantially similar properties as compared to the reference protein or peptide. A homolog or variant can include an agonist of a protein or an antagonist of a protein. Homologs or variants can be produced using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated reference protein, direct protein synthesis, or modifications to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis, resulting in the encoding of a protein variant. In addition, naturally occurring variants of a reference protein may exist (e.g., isoforms, allelic variants, or other natural variants that may occur from individual to individual) and may be isolated, produced and/or utilized in the invention.

[00148] 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 percent identity between 45% and 99%, in whole integer increments), to the amino acid sequence of the reference protein (e.g., an amino acid sequence specified herein, or the amino acid sequence of a specified protein). In one embodiment, the 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.

[00149] As used herein, unless otherwise specified, reference to a percent (%) identity refers to an evaluation of homology which is performed using: (1) a Basic Local Alignment Search Tool (BLAST) basic homology search using blastp for amino acid searches and blastn for nucleic acid searches with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (such as described in Altschul, S.F., Madden, T.L., Schaaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (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) a BLAST alignment of two sequences (e.g., using the parameters described below); (3) and/or PSI-BLAST with the standard default parameters (Position- Specific Iterated BLAST). It is noted that due to some differences in the standard parameters between Basic BLAST and BLAST for two sequences, two specific sequences might be recognized as having significant homology using the BLAST program, whereas a search performed in Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top matches. In addition, PSI-BLAST provides an automated, easy-to-use version of a "profile" search, which is a sensitive way to look for sequence homologs. The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined by using any one of these programs. [00150] Two specific sequences can be aligned to one another using BLAST as described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety. Such a sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For purposes of clarity herein, a BLAST sequence alignment for two sequences is performed using the standard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

Reward for match = 1

Penalty for mismatch = -2

Open gap (5) and extension gap (2) penalties

gap x dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties

gap x dropoff (50) expect (10) word size (3) filter (on).

[00151] An isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation), its natural milieu being the genome or chromosome in which the nucleic acid molecule is found in nature. As such, "isolated" does not necessarily reflect the extent to which the nucleic acid molecule has been purified, but indicates that the molecule does not include an entire genome or an entire chromosome or a segment of the genome containing more than one gene, in which the nucleic acid molecule is found in nature. An isolated nucleic acid molecule can include a complete gene. An isolated nucleic acid molecule that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes that are naturally found on the same chromosome. An isolated nucleic acid molecule may also include portions of a gene. An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end of the sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., heterologous sequences). Isolated nucleic acid molecules can include DNA, RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA). Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein or domain of a protein.

[00152] A recombinant nucleic acid molecule is a molecule that can include at least one of any nucleic acid sequence encoding any one or more proteins described herein operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transfected. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a protein. In addition, the phrase "recombinant molecule" primarily refers to a nucleic acid molecule operatively linked to a transcription control sequence, but can be used interchangeably with the phrase "nucleic acid molecule" which is administered to an animal.

[00153] A recombinant nucleic acid molecule includes a recombinant vector, which is any nucleic acid sequence, typically a heterologous sequence, which is operatively linked to the isolated nucleic acid molecule encoding a fusion protein of the present invention, which is capable of enabling recombinant production of the fusion protein, and which is capable of delivering the nucleic acid molecule into a host cell according to the present invention. Such a vector can contain nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molecules to be inserted into the vector. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and preferably in the present invention, is a plasmid useful for transfecting yeast. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of nucleic acid molecules, and can be used in delivery of such molecules (e.g., as in a DNA composition or a viral vector- based composition). Recombinant vectors are preferably used in the expression of nucleic acid molecules, and can also be referred to as expression vectors. Preferred recombinant vectors are capable of being expressed in a transfected host cell, such as a yeast.

[00154] In a recombinant molecule of the present invention, nucleic acid molecules are operatively linked to expression vectors containing regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include nucleic acid molecules that are operatively linked to one or more expression control sequences. The phrase "operatively linked" refers to linking a nucleic acid molecule to an expression control sequence in a manner such that the molecule is expressed when transfected (i.e., transformed, transduced or transfected) into a host cell.

[00155] According to the present invention, the term "transfection" is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" can be used interchangeably with the term "transfection" when such term is used to refer to the introduction of nucleic acid molecules into microbial cells, such as algae, bacteria and yeast. In microbial systems, the term "transformation" is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term "transfection." Therefore, transfection techniques include, but are not limited to, transformation, chemical treatment of cells, particle bombardment, electroporation, microinjection, lipofection, adsorption, infection and protoplast fusion.

[00156] The following experimental results are provided for purposes of illustration and are not intended to limit the scope of the invention.

EXAMPLES

Example 1

[00157] The following example describes results of a clinical study evaluating the use of yeast-based immunotherapy targeting mutated Ras in resected pancreas cancer.

[00158] In the clinical trial known as GI-4000-02 (Globelmmune, Inc., Louisville, Colorado) for patients with resected pancreas cancer, patients (n=176) enrolled in two arms at 27 centers in the United States and at five international centers. The study drug was a yeast-based immunotherapeutic (TARMOGEN^® product, Globelmmune, Inc., Louisville, Colorado) known as GI-4000. GI-4000 is a series of yeast-based immunotherapy products developed by Globelmmune, Inc. that target mutated Ras. GI- 4000 currently consists of four different heat-inactivated S. cerevisiae yeast (referred to individually as "GI-4014", "GI-4015", "GI-4016" and "GI-4020"), together expressing seven common Ras mutations in human cancers (see Fig. 1). Each of these four yeast immunotherapy compositions expresses a fusion protein including three different Ras mutations (i.e., SEQ ID NO: 15 (GI-4020), SEQ ID NO: 17 (GI-4014), SEQ ID NO: 19 (GI- 4015) and SEQ ID NO:21 (GI-4016)). Specifically, each protein product expressed in the yeast contains: (A) two mutations at codon 61 (glutamine to arginine [Q61R] (GI-4014, GI-4015, GI-4016) or glutamine to histidine [Q61H] (GI-4020), and glutamine to leucine [Q61L] (GI-4014, GI-4015, GI-4016, GI-4020); plus (B) one of four different mutations at codon 12 (glycine to valine [G12V] (GI-4014), glycine to cysteine [G12C] (GI-4015), glycine to aspartate [G12D] (GI-4016), or glycine to arginine [G12R] (GI-4020)). Patient tumors were sequenced to identify the specific Ras mutation(s) contained in their tumor, and only the specific yeast immunotherapeutic with the matching mutation was administered to the patient.

[00159] General Clinical Trial Design for GI-4000-02. Patients were administered Globelmmune's GI-4000 product matched to their Ras mutation(s) at 40 Y.U., or with placebo using three weekly doses starting between 21 and 35 days after resection (study visit Day 1, 8, 15), and all subjects received gemcitabine (GEM) 1000 mg/m² by intravenous infusion starting on study visit Day 24. Administration of GEM proceeded until either six monthly cycles were completed, GEM intolerance, study withdrawal, disease progression, or death occurred. Administration of study drug (GI-4000) proceeded until study withdrawal, disease recurrence, or death and could proceed beyond GEM cessation. Immune samples were collected from subjects on select study visits: Day 1 (baseline), 15, 24, 44, 52, 100, 108, 184 and then quarterly during on treatment and post study drug follow up phase (see Table 1). Fourteen subjects in the trial had glycine to arginine mutations at position 12 of Ras (G12R mutations).

Table 1. Dosing schedule and immune sampling schedule

[00160] Summary of Results for Subjects with Resection Margins Positive for Residual Tumor (Rl Subjects). In the GI-4000-02 phase 2 clinical trial, improvements in median recurrence free survival and median overall survival versus placebo were demonstrated in subjects having a favorable proteomic signature, as previously published (Richards D.A., et al. ESMO 14^th World Congress on Gastointestinal Cancer, Barcelona, Spain, June 27, 2012). In addition, in Rl subjects, a 2.6 month improvement in median overall survival (p=not significant) was demonstrated, with a 5 month improvement for immune responders (Richards et al, supra). In addition, generation of interferon-γ (IFN-γ) T cell responses (see methods described below) in Rl subjects receiving GI-4000 was demonstrated: 7/15 (46.7%) GI-4000 subjects vs. 1/12 (8.3%) placebo subjects (p=0.043) had an IFN-γ response to their G12 mutation (Richards et al., supra).

[00161] The following data presents results published subsequent to the present invention (Coeshott et al, AACR Ras Oncogenes: From Biology to Therapy (Abstract A28); 25 February 2014) and which show the surprising results related to the G12R mutations for GI-4000-02 as described herein.

[00162] Immune Analysis Methods. 102 subjects with no microscopic residual tumor in the resection margins (R0 subjects) and with adequate samples available were assayed for immune response. Twelve of these tested subjects had a G12R mutation (six in each treatment arm). Responses were measured by ELISpot assay for production of IFN- γ, a hallmark of antigen-specific CD4⁺ and CD8⁺ T cell activation. Peripheral blood mononuclear cells (PBMCs) were collected pre-treatment and at various times during treatment and were cryopreserved to enable longitudinal analysis. The PBMCs were stimulated ex vivo with Ras peptide pools containing the relevant Ras position 12 mutations (G12) that were present in the subject's tumor. Specifically, Ras peptides were provided as four pools of Ras peptides 10 or 15 amino acid residues in length that expressed the matched Ras mutation. In addition, a mismatched set of control peptides was also used for stimulation. For G12R subjects, the control/mismatch set of peptides was identical to the G12R peptides, except for position 12, at which there was a G12C mutation (See Table 2 for peptides). For G12C subjects, the control/mismatch set of peptides was the G12R set (Table 2). Similarly, peptides for G12D subjects (peptides having a G12D mutation at position 12) served as mismatched controls for G12V subjects and vice versa.

[00163] ELISpot Data Analysis. Data were expressed as numbers of IFN-y⁺ cells (or "spots") per million PBMC after subtraction of the appropriate mismatched peptide pool. On-treatment values were adjusted by subtraction of the baseline response for that peptide pool. Data were then analyzed by categorical assessments.

[00164] Categorical assessment: algorithms were pre-specified to evaluate the IFN-y⁺ T cell response. For a subject to be deemed a responder {i.e., an immune responder), the following criteria had to be met: 1. Baseline negative subjects: at least one G12 peptide pool or single peptide with an increase from baseline of >= 25 IFN-y⁺ cells/ 10⁶ PBMCs after subtraction of the control, mismatched peptide response. Additionally the raw score for the specific peptides before subtraction of mismatch control must increase on treatment. An increase is defined as a minimum mean increase of >= 25 IFN-Y⁺ cells/10⁶ PBMCs from the mean baseline value for that peptide pool or single peptide.

2. Baseline positive subjects: any G12 peptide pool or single peptide with a baseline response of >= 25 IFN-y⁺ cells/10⁶ PBMCs after subtraction of control, mismatched peptide response and a two fold or greater increase in the response to that specific pool/peptide on treatment after subtraction of control, mismatched peptide response, PLUS a second product related peptide response of >=25 IFN-y⁺ cells/ 10⁶ PBMCs.

[00165] G12R Immune Responses. Of 14 G12R R0 subjects enrolled in the study, 12 subjects were tested for IFN-γ responses, with one subject omitted from further analysis due to lack of a baseline sample. The immune responses of these G12R R0 subjects as a subset of patients were particularly notable. For the 1 1 analyzed G12R subjects, irrespective of treatment arm, seven (63.6%) had a treatment-emergent G12R- specific immune response. Three other G12R subjects (27.3%) had substantial baseline responses, but these responses did not increase further on treatment and these three subjects were thus deemed non-responders by categorical assessment based on the pre- specified criteria set forth above. Therefore, there was a high frequency of responders among the evaluable G12R subjects. In addition, the responses of five G12R subjects for at least one timepoint were of high amplitude (i.e. , with an average raw spots/well value > 100 after baseline and mismatch pool adjustment). The G12R-directed responses were also highly focused on a single peptide pool, denoted "pool 16", containing five 10 residue peptide sequences (see Table 2 for peptide sequences), suggesting a CD8⁺-dominated T cell response. There was no response to the 9 residue peptide (peptide 4), used in the assay as a single peptide not a pool (shown in Table 2). The response to pool 16 was highly specific for the G12R mutation since there was no response to a mismatched control set of peptides (denoted "pool 2," see Table 2) that were identical to the peptide sequences in pool 16 except for the presence of cysteine at position 12 instead of arginine.

[00166] Figs. 7A-7J show the IFN-γ ELISpot responses of the individual subjects described above (the seven G12R responders, also referred to as "categorical responders on treatment", and the three G12R subjects who were categorized as "non-responders" based on pre-defined criteria, but who had substantial pre-existing baseline responses, also referred to as "categorical baseline responders"). The treatment category for each subject is also indicated (black bars = G12R peptide pool 16; white bars = G12C peptide pool 2).

[00167] These responses were in contrast to those seen for subjects with the other G12 mutations in this study: G12V, G12C and G12D, for which there was no obvious focusing of the response to a single peptide pool (data not shown) and for which the frequency of categorical immune responders was lower (37.2% across both treatment arms).

Table 2. Peptide sequences

[00168] Discussion of Immune Responses. Previous studies in patients with Ras- mutation positive cancers, have reported T cell responses to G12V, G12C, G12D and G12R mutations (Khleif S. N. et al, J. Immunother. 22: 155-165, 1999; Gjertsen, M.K. et al, Int. J. Cancer. 72:784-790, 1997; Gjertsen, M.K. et al, Int. J. Cancer 92:441-50, 2001; Gjertsen, M.K. et al, J Mol Med. 81 :43-50, 2003; Kubuschok B., et al, Clin. Cancer Res 12: 1365-1372, 2006; Carbone, D.P. et al, J. Clin. Oncology 23:5099- 5107, 2005; Qin H., et al, Cancer Res. 55:2984-7, 1995; Abrams, S.I. et al, Cellular Immunology 182: 137-151, 1997). Gjertsen et al. (2003) defined a T cell response that was specific for a G12C peptide, K-Ras 8-16, and that was restricted to HLA-A*0302 (and to a lesser extent to HLA-A*0301) in a single pancreas cancer patient after repeated immunization with Ras peptides of 17 residues in length plus the immunostimulant GM- CSF (granulocyte/macrophage colony stimulating factor). This 9 residue peptide sequence spanning K-Ras 8-16 has also previously been identified as an HLA-A3 binding motif (Bertazzoli, C. et al. Tumori 83:847-855, 1997.

[00169] A 10 residue peptide in pool 16 used in the GI-4000-02 ELISpot studies (against which the majority of responses were seen) incorporates the sequence K-Ras 8-16, suggesting that a single peptide in pool 16 is binding to HLA-A3 (A*0301 or A*0302), or to other alleles which have the same peptide anchoring requirements as A* 0301 or A* 0302. Five G12R subjects in the present example have an HLA-A3 allele (all A*0301) and all had an IFN-γ ELISpot response either pre-existing at baseline or emerging on treatment.

[00170] The 9 residue motif described by Bertazzoli et al. (1997, supra) is defined by having a lysine (K) residue at the C-terminus and a valine (V) residue in anchor position 2. The peptide in pool 16 has the sequence VVVGARGVGK (SEQ ID NO:22). The 10 residue sequence could potentially be trimmed at the N-terminus by proteases in order to bind HLA-A3 as a 9 residue or could bind as a 10 residue using the valine (V) at position 2 or 3 as the anchor residue. Bertazzoli et al. (1997, supra) showed that WT and G12 mutations G12A, G12D and G12V also bind to HLA-A3; however, all had improved binding over G12R 9 residue peptides. G12C, the mismatch mutation used as the control in ELISpot testing of the G12R subjects (see Table 2; pool 2), was not tested for binding by Bertazzoli et al.

[00171] Ras is an endogenous protein and therefore antigen presentation would presumably be handled through the MHC Class I pathway. Evidence for endogenous processing of mutant Ras has been provided by data in which T cells from patients with Ras mutations killed target tumor cells expressing the same mutation but not a WT Ras tumor cell in an MHC Class I-restricted fashion (Khleif et al. 1999, supra; Fossum, B. et al, Cancer Immunol. Immunother. 40:165-72, 1995). Thus, the IFN-γ response to Ras could be dominated by CD8⁺ T cells. In the current study, as discussed above, there is evidence that immune responders with a G12R mutation are recognizing an HLA-A3 restricted peptide and therefore the response being measured is a CD8⁺ T cell response.

[00172] Clinical Outcomes for G12R Subjects. For all G12R R0 subjects, regardless of treatment arm, there was a pronounced improvement in median overall survival (335 days) compared to all other mutations (see Table 4, and Figs. 2 and 3 (1167 vs. 832 days). Median overall survival was also greatly improved (568 days) in GI-4000 treated G12R R0 subjects compared to placebo-treated G12R R0 subjects (compare Table 5 vs. Table 6, and see Table 7, compare Fig. 4 vs. Fig.5 and see Fig. 6). Median overall survival was also greatly increased (1517 vs. 744 days; improvement of 773 days) for G12R RO subjects receiving GI-4000 versus all other RO subjects receiving GI-4000 (see Table 5, Fig. 4). For placebo subjects, there was only a 94 day improvement in median overall survival for G12R RO versus non-G12R RO subjects (see Table 6; Fig. 5). These data indicate that the presence of the Ras G12R mutation affords a survival advantage in resected pancreas cancer patients, and particularly RO pancreas cancer patients, and that treatment with immunotherapy targeting Ras G12R, such as the GI-4000 product described in this trial (GI-4020), can further improve survival, presumably by generating a G12R-specific tumor-directed immune response. While the results in the present invention were discovered through analysis of pancreas cancer RO subjects (see above), without being bound by theory, the present inventors believe that the discoveries of the present invention can be extended to all Ras mutation-positive pancreas cancer, whether RO or Rl , and additionally, to other Ras mutation-positive cancers.

[00173] In addition, for three RO subjects with Ras Q61H mutations (i.e., WT glutamine (Q) at position 61 of Ras is replaced by histidine (H)) in the GI -4000-02 trial, who received the same yeast immunotherapy product as the G12R subjects (the product referred to as "GI-4020" which contains both the G12R and Q61H mutations in the Ras antigen construct), but who did not have tumors with G12R mutations (i.e., the patient tumor had only Ras Q61H mutations), the overall survival for the two Q61H subjects in the GI-4000 arm was higher than for the one subject in the placebo arm (528 and 663 days in the GI-4000 arm vs. 112 days in the placebo arm; see Figs. 2 and 4). This improvement in outcome, which was only discovered as a result of the G12R observations described above, taken together with the data presented above, suggests that the GI-4020 containing a G12R antigen is more immunogenic than other GI-4000 products that do not contain the G12R antigen. These Q61H subjects were not tested for immune response.

Table 4. Analysis of the Duration of Overall Survival from Randomization

(G12R Compared to All Other Mutations)

(Intent-to-Treat Population with RO Resection)

G12R All Other

Subjects R0 Subjects

(N=14) (N=123)

Subject Status

Alive 5 (35.7%) 33 (26.8%)

Death 9 (64.3%) 90 (73.2%)

Hazard ratio³ (G12R Subjects / All Other RO Subjects) 0.601

95% Confidence Interval for Hazard Ratio³ [0.302, 1.194]

p-value³ 0.146

Time from Date of Randomization to Death

Median (days)^b 1167.0 832.0

95% Confidence Interval for Median^b [842.0, .] [663.0, 970.0]

Based on Cox proportional hazards model with G12R status as the fixed effect.

Based on Kaplan-Meier methodology.

Note: Percentages are based on the number of subjects in each group.

Table 5: Analysis of the Duration of Overall Survival from Randomization

(G12R Compared to All Other Mutations)

(Intent-to-Treat Population with R0 Resection, GI-4000 Group Only)

G12R All Other Subjects R0 Subjects (N=7) (N=62)

Subject Status

Alive 3 (42.9%) 18 (29.0%) Death 4 (57.1%) 44 (71.0%)

Hazard ratio³ (G12R Subjects / All Other R0 Subjects) 0.465

95% Confidence Interval for Hazard Ratio³ [0.167, 1.297]

p-value³ 0.144

Time from Date of Randomization to Death

Median (days)^b 1517.0 744.0

95% Confidence Interval for Median^b [853.0, .] [551.0, 1034.0]

³ Based on Cox proportional hazards model with G12R status as the fixed effect.

b Based on Kaplan-Meier methodology.

Note: Percentages are based on the number of subjects in each group. Table 6: Analysis of the Duration of Overall Survival from Randomization

(G12R Compared to All Other Mutations)

(Intent-to-Treat Population with R0 Resection, Placebo Group Only)

G12R All Other Subjects R0 Subjects (N=7) (N=61)

Subject Status

Alive 2 ( 28.6%) 15 ( 24.6%) Death 5 ( 71.4%) 46 ( 75.4%)

Hazard ratio³ (G12R Subjects / All Other R0 Subjects) 0.777

95% Confidence Interval for Hazard Ratio³ [0.307, 1.962]

p-value³ 0.593

Time from Date of Randomization to Death

Median (days)^b 949.0 854.5

95% Confidence Interval for Median^b [735.0, .] [633.0, 1096.0]

b Based on Kaplan-Meier methodology.

Note: Percentages are based on the number of subjects in each group.

Table 7: Analysis of the Duration of Overall Survival from Date of Randomization by

Treatment Group

(Intent-to-Treat Population with R0 Resection and G12R Status)

G12R G12R Gemcitabine Gemcitabine + GI-4000 + Placebo (N=7) (N=7)

Subject Status

Alive 3 ( 42.9%) 2 (28.6%) Death 4 ( 57.1%) 5 (71.4%)

Hazard ratio³ (Gemcitabine + GI-4000 / Gemcitabine + Placebo) 0.502

95% Confidence Interval for Hazard Ratio³ [0.133, 1.890]

p-value³ 0.308

Time from Date of Randomization to Death

Median (days)^b 1517.0 949.0

95% Confidence Interval for Median^b [853.0, .] [735.0, .]

³ Based on Cox proportional hazards model with treatment group as the fixed effect.

b Based on Kaplan-Meier methodology.

Note: Percentages are based on the number of subjects in each group.

[00174] These data indicate that the G12R mutation affords a survival advantage that may be linked to its ability to induce a more robust T cell immune response than other Ras mutations. [00175] In contrast, in one Japanese study, G12R and G12D mutations were found to be negative prognostic indicators of survival in unresectable pancreas cancer patients (Ogura, T. et al, J. Gastroenterol. 48:640-646, 2013). Without being bound by theory, the present inventors believe that this may indicate a difference in the immune response capacity of resectable vs. unresectable patients, or potentially a population difference, since Ogura et al. only reviewed one clinical site. Moreover, administration of a Ras G12R immunotherapy composition to such patients may, according to the present invention, reverse the prognosis for such patients.

[00176] Clinical Outcomes: HLA-A3 subjects. For R0 subjects in the clinical trial GI-4000-02, HLA-A3 was a negative predictor of survival, regardless of treatment arm and Ras mutation (see Table 8). A significant difference in median overall survival occurred between HLA-A3 subjects (n=30, includes HLA-A*0301, -A*0302 and - A*03XKS subjects) and subjects without the HLA-A3 allele (n=105) (569 vs. 934 days; hazards ratio (HR) =1.615, p=0.039). However, for the small number of subjects with both G12R mutation and HLA-A3 (n=5, all A* 0301), regardless of treatment arm, improved survival occurred and there was no difference between the median overall survival or these subjects and all other R0 subjects (see Table 9; 949 vs. 853 days; HR=0.940, p=0.903) suggesting that these subjects were rescued by an immune response to the G12R mutation, either a naturally derived response and/or by stimulation with the yeast-based immunotherapy product containing the optimally immunogenic G12R sequence (GI-4020). As mentioned above, all five subjects had an IFN-γ ELISpot response either pre-existing at baseline on emerging on treatment.

Table 8: Analysis of the Duration of Overall Survival by HLA-A3 Type Status (Intent-to-Treat Population with R0 Resection)

Non-

HLA-A3 HLA-A3 (N=30) (N=105)

Subject Status

Alive 5 (16.7%) 32 (30.5%) Death 25 (83.3%) 73 (69.5%)

Hazard ratio^b (HLA-A3 / Non-HLA-A3) 1.615

95% Confidence Interval for Hazard Ratio^b [1.024, 2.546]

p-value^b 0.039

Time from Date of Randomization to Death

Median (days)⁰ 569.5 934.0

95% Confidence Interval for Median⁰ [403.0, 949.0] [735.0, 1118.0] ^a Includes HLA-A*0301, A*0302 and A*03XKS subjects.

b Based on Cox proportional hazards model with HLA-A3 status as the fixed effect.

0 Based on Kaplan-Meier methodology.

Note: Percentages are based on the number of subjects in each group.

Table 9: Analysis of the Duration of Overall Survival by HLA-A3 Type and G12R

Status

(Intent-to-Treat Population with R0 Resection)

G12R and All Other HLA-A3^a R0 Subjects (N=5) (N=130)

Subject Status

Alive 1 (20.0%) 36 (27.7%) Death 4 (80.0%) 94 (72.3%)

Hazard ratio" (G12R and HLA-A3 / All Other R0 Subjects) 0.940

95% Confidence Interval for Hazard Ratio^b [0.345, 2.562]

p-value^b 0.903

Time from Date of Randomization to Death

Median (days)⁰ 949.0 853.0

95% Confidence Interval for Median⁰ [814.0, .] [695.0, 1031.0] ^a HLA-A*0301

b Based on Cox proportional hazards model with the combination of G12R and HLA-A3 status as the fixed effect.

⁰ Based on Kaplan-Meier methodology.

Note: Percentages are based on the number of subjects in each group.

Example 2

[00177] The following example describes experiments showing how G12R-containing immunotherapy compositions can act as agonists of T cells that are specific for Ras antigens containing non-G12R antigens. [00178] Briefly, tetramers or pentamers of HLA-A3-G12R peptides are produced in order to isolate G12R- specific T cells and create G12R-specific T cell lines to be used as positive controls in the following experiments. T cell lines are then also created from cells of subjects from the GI -4000-02 clinical trial with Ras G12V or G12D mutations that were deemed to be immune responders by IFN-γ ELISpot. These lines are preferentially stimulated by G12R-containing peptides and compared to stimulation with peptides bearing the patient's mutation (G12V or G12D). PBMCs from G12V and G12D subjects in GI-4000-02 are restimulated with both their "cognate" peptides (G12V or G12D respectively) and with G12R peptides.

[00179] It is expected that G12R acting as an agonist will produce a marked shift in the peptide dose response and increases in the levels of IFN-γ produced compared to the G12V or G12D peptides, which is an accepted method to identify agonist activity in other peptides (Salazar E., et al, Int. J. Cancer 15:829-838, 2000). PBMCs that are expected to be particularly relevant in this experiment are from G12V or G12D subjects who had demonstrated high "non-specific" responses to the mismatch peptide pools used as controls in the GI-4000-02 ELISpot assays.

Example 3

[00180] The following example describes immunization with a G12R-containing immunotherapy composition to reverse or slow the poor survival associated with Ras non- G12R mutations and with Ras-mutated cancers in general.

[00181] The following example describes a phase 2 clinical trial using yeast-Ras G12R immunotherapeutic compositions.

[00182] A randomized phase 2 clinical trial in patients with pancreas cancer, colorectal cancer, or NSCLC is run using a yeast-Ras G12R immunotherapeutic composition as described in Example 1 {e.g., GI-4020), as the active treatment. At least 100 or more subjects with Ras mutation-positive cancer are enrolled. Subject inclusion criteria include the detection in the subject's tumor of a Ras mutation, including any subjects with a non- G12R mutation (any other mutation at position G12 or a mutation at a different position, such as Q61). Subject inclusion criteria include HL A- A3 -positive subjects (subjects with at least one allele of HLA-A*0301 or HLA-A*0302, or another rare allele with similar peptide anchoring properties), which can be stratified or treated as a separate arm, or alternatively, PBMCs from HL A- A3 -positive subjects are tested for an in vitro immune response to Ras G12R peptides, and if positive, subjects are enrolled in the trial. Subjects with G12R mutations may be excluded or the trial may be stratified for subjects with G12R mutations. The trial is run as a double-blind or open- label, placebo-controlled, multi-center trial. All patients receive standard of care therapy (e.g., gemcitabine), with treatment arm patients receiving several serial injections of yeast-Ras G12R immunotherapeutic composition during treatment, alone or together with a yeast-Ras immunotherapeutic composition targeting the subject's actual Ras mutation for non-G12R subjects. The primary endpoint is time to progression or overall survival. Additional secondary endpoints can include antigen-specific T cell responses (e.g., Ras-specific CD8⁺ T cells emerging or expanding on treatment), maintenance of lymph node negativity, or progression to metastases.

[00183] The yeast-Ras G12R immunotherapeutic composition is expected to be safe and well-tolerated with no significant toxicities. In addition, the yeast-Ras G12R immunotherapeutic composition is expected to produce treatment-emergent Ras-specific T cell responses and/or an improvement in pre-existing Ras-specific baseline T cell responses in at least some or a majority of patients, including those that are HLA-A3- positive and those with non-G12R mutations. Some or a majority of patients are also expected to have stabilized disease, delayed time to progression, increased rate of overall survival, reduced rate of tumor growth, or reduced tumor burden.

[00184] While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

What is Claimed is:

1. A method to treat a Ras mutation-positive cancer, comprising administering to a subject who has a Ras mutation-positive cancer that has been selected, prior to the step of administering, to be negative for a Ras G12R mutation, an immunotherapeutic composition comprising:

a) a yeast vehicle; and

b) a Ras G12R antigen.

2. The method of Claim 1, wherein the subject is HLA-A3-positive.

3. A method to treat a Ras mutation-positive cancer, comprising administering to a subject who has a Ras mutation-positive cancer and who has been pre-selected as being HLA- A3 -positive, an immunotherapeutic composition comprising:

a) a yeast vehicle; and

b) a Ras G12R antigen.

4. The method of any one of Claims 1 to 3, wherein the subject has also been pre-selected as an immune responder in vitro to a Ras G12R antigen.

5. A method to treat a Ras mutation-positive cancer, comprising:

a) testing cancer cells from subjects with a Ras mutation-positive cancer in vitro to identify the specific Ras mutation or mutations in the cancer of the subjects; and

b) selecting subjects with a Ras mutation-positive cancer that is identified in (a) as being Ras G12R mutation-negative; and

c) administering to the subjects selected in (b) an immunotherapeutic composition comprising:

i) a yeast vehicle; and

ii) a Ras G12R antigen.

6. The method of Claim 5, wherein the subject has been further selected as being HLA-A3-positive.

7. A method to treat a Ras mutation-positive cancer, comprising:

a) testing a biological sample from a subject with a Ras-mutation positive cancer to identify the HLA type of the subject; and

b) administering to subjects who are HLA- A3 -positive an immunotherapeutic composition comprising:

i) a yeast vehicle; and

ii) a Ras G12R antigen.

8. The method of any one of Claims 1 to 7, wherein the yeast vehicle is a whole yeast.

9. The method of Claim 8, wherein the Ras G12R antigen has been expressed by the whole yeast.

10. The method of Claim 8 or Claim 9, wherein the whole yeast has been heat- inactivated.

11. The method of any one of Claims 1 to 10, wherein the yeast is from Saccharomyces.

12. A method to treat Ras mutation-positive cancer, comprising administering an immunotherapeutic composition comprising a Ras G12R antigen to a pre-selected subject, wherein the subject has been pre-selected as having a Ras mutation-positive cancer that does not express a Ras G12R mutation.

13. A method to treat a Ras mutation-positive cancer, comprising administering an immunotherapeutic composition comprising a Ras G12R antigen to a pre-selected subject, wherein the subject has been pre-selected as having a Ras mutation-positive cancer and as being HLA-A3-positive.

14. The method of any one of Claims 1 to 13, wherein the Ras G12R antigen comprises at least 9 amino acids of any one of SEQ ID NOs:3, 5, 7, 9, 11, or 13, wherein the Ras G12R antigen includes the amino acid at position 12 of SEQ ID NO:3, 5, 7, 9, 11 or 13, except that the glycine at that position in SEQ ID NO:3, 5, 7, 9, 11 or 13 has been substituted for an arginine.

15. The method of any one of Claims 1 to 14, wherein the Ras G12R antigen comprises the amino acid sequence of SEQ ID NO: 15.

16. The method of any one of Claims 1 to 14, further comprising administering to the subject a mutated Ras antigen that has the same mutation or mutations as the Ras mutation in the subject's cancer.

17. The method of Claim 16, wherein the mutated Ras antigen is contained within the same immunogenic composition as the Ras G12R antigen.

18. The method of Claim 16 or Claim 17, wherein the mutated Ras antigen is part of a fusion protein comprising the mutated Ras antigen and the Ras G12R antigen.

19. The method of any one of Claims 16 to 18, wherein the mutated Ras antigen comprises a G12 mutation that is not G12R.

20. The method of Claim 19, wherein the G12 mutation is selected from G12V, G12D, G12C, G12S, or G12A.

21. The method of any one of Claims 16 to 20, wherein the mutated Ras antigen comprises a Q61 mutation.

22. The method of Claim 21, wherein the Q61 mutation is selected from Q61L, Q61R or Q61H.

23. The method of any one of Claims 1 to 22, wherein the individual is being treated or has been treated with another therapy for cancer.

24. The method of Claim 23, wherein the therapy is radiation therapy, tumor resection, or chemotherapy.

25. The method of Claim 24, wherein the therapy is administration of one or more additional immunotherapeutic compositions.

26. The method of Claim 25, wherein the additional immunotherapeutic composition is a yeast vehicle and a cancer antigen that is not a Ras G12R antigen.

27. The method of Claim 25, wherein the additional immunotherapeutic composition is an immune checkpoint inhibitor.

28. An immunotherapeutic composition comprising:

a) a yeast vehicle; and

b) a mutated Ras antigen, wherein the mutated Ras antigen comprises a G12R mutation and at least one additional Ras G12 mutation that is not a G12R mutation.

29. The immunotherapeutic composition of Claim 28, wherein the mutated Ras antigen comprises at least 9 amino acids of any one of SEQ ID NOs:3, 5, 7, 9, 11, or 13, wherein the antigen includes the amino acid at position 12 of SEQ ID NO:3, 5, 7, 9, 11 or 13, except that the glycine at that position in SEQ ID NO:3, 5, 7, 9, 11 or 13 has been substituted for an arginine.

30. The immunotherapeutic composition of Claim 28, wherein the at least one additional Ras G12 mutation is a mutation that has been detected in a subject who has a mutated Ras-positive cancer.

31. The immunotherapeutic composition of any one of Claims 28 to 30, wherein the mutated Ras antigen is a fusion protein comprising the Ras G12R mutation and the at least one additional Ras G12 mutation.

32. The immunotherapeutic composition of any one of Claims 28 to 31, wherein the at least one additional Ras G12 mutation is selected from G12V, G12D, G12C, G12S, or G12A.

33. The immunotherapeutic composition of any one of Claims 28 to 32, wherein the mutated Ras antigen further comprises at least one additional Ras mutation that is not a G12 mutation.

34. The immunotherapeutic composition of Claim 33, wherein the at least one additional Ras mutation that is not a G12 mutation is a Q61 mutation.

35. The immunotherapeutic composition of Claim 34, wherein the Q61 mutation is selected from Q61L, Q61R or Q61H.

36. The immunotherapeutic composition of any one of Claims 28 to 35, wherein the yeast vehicle is a whole yeast.

37. The immunotherapeutic composition of Claim 36, wherein the whole yeast has been heat-inactivated.

38. The immunotherapeutic composition of any one of Claims 28 to 37, wherein the yeast is from Saccharomyces.

39. A combination of immunotherapeutic compositions, wherein the combination comprises a mixture of:

a) a first yeast expressing a fusion protein having the amino acid sequence of SEQ ID NO: 15; and

b) a second yeast expressing a fusion protein having an amino acid sequence encoding a mutated Ras antigen, wherein the mutated Ras antigen does not include a G12R mutation.

40. The combination of immunotherapeutic compositions of Claim 39, wherein the second yeast expresses a fusion protein having an amino acid sequence selected from: SEQ ID NO : 17, SEQ ID NO : 19 or SEQ ID NO :21.

41. An immunotherapeutic composition comprising a yeast expressing a fusion protein having a first amino acid sequence selected from SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:21, wherein the fusion protein additionally includes a second amino acid sequence from a Ras protein comprising a G12R mutation.

42. The immunotherapeutic composition of Claim 41, wherein the second amino acid sequence is appended to the N- or C-terminus of the first amino acid sequence.

43. The immunotherapeutic composition of Claim 41 or 42, wherein the second amino acid sequence comprises at least 9 amino acids of any one of SEQ ID NOs:3, 5, 7, 9, 11, or 13, wherein the antigen includes the amino acid at position 12 of SEQ ID NO:3, 5, 7, 9, 11 or 13, except that the glycine at that position in SEQ ID NO:3, 5, 7, 9, 11 or 13 has been substituted for an arginine.

44. The immunotherapeutic composition of any one of Claims 28 to 43, for use to treat a Ras mutation-positive cancer, wherein the Ras mutation-positive cancer has been pre-selected as being Ras G12R-negative.

45. The immunotherapeutic composition of any one of Claims 28 to 43, for use to treat a Ras mutation-positive cancer in an HLA-A3 -positive subject.

46. Use of an immunotherapeutic composition of any one of Claims 28 to 43 in the preparation of a medicament for treating a Ras mutation-positive cancer, wherein the Ras mutation-positive cancer has been pre-selected as being Ras G12R-negative.

47. Use of an immunotherapeutic composition of any one of Claims 28 to 43 in the preparation of a medicament for treating a Ras mutation-positive cancer in an HLA- A3 -positive subject.

48. A method to pre-select a subject with a Ras mutation-positive cancer for treatment with an immunotherapy composition comprising a G12R antigen, comprising:

a) testing a biological sample from the subject to identify the HLA allele or alleles expressed by the subject;

b) testing a T cell-containing biological sample from the subject for an immune response to a Ras G12R antigen in vitro; and

c) pre-selecting subjects with at least one HLA- A3 allele and whose T cells respond to the Ras G12R antigen as subjects to be treated with an immunotherapy composition comprising a Ras G12R antigen.

49. The method of Claim 48, wherein the T-cell containing biological sample is a sample of peripheral blood mononuclear cells isolated from the subject.