Recombinant yeast lysates for inducing cellular immune responses

Abstract

The present invention relates to recombinant yeast lysates for inducing cellular immune responses. Methods of preparing immunogenic compositions comprising recombinant yeast lysates for inducing cellular immune responses and uses thereof are disclosed.

Description

[0001] This application is a divisional application. The application date of the original application is May 13, 2019, the application number is 201980032564.4 (PCT / US2019 / 031962), and the name of the invention is “Recombinant yeast lysate for inducing cellular immune response”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 62 / 671,587, filed on May 15, 2018. The entire disclosure of U.S. Provisional Patent Application No. 62 / 671,587 is incorporated herein by reference.

[0004] References to sequence listings

[0005] This application contains a sequence listing submitted as an electronic text file via EFS-Web. The text file named "3923-59-PCT_ST25" is 3000 bytes in size and was recorded on May 13, 2019. Pursuant to 37 CFR § 1.52(e)(5), the information contained in this text file is incorporated herein by reference. Technical Field

[0006] The present invention generally relates to immunotherapeutic compositions, particularly recombinant yeast lysates for inducing cellular immune responses, and uses thereof. Background Art

[0007] Vaccines are widely used to prevent diseases and to treat established diseases (therapeutic vaccines). However, there remains an urgent need to develop safe and effective vaccines and adjuvants for a variety of diseases, including those caused by infection with pathogenic agents, cancer, and other disorders that can be treated by inducing an immune response.

[0008] When not administered with an adjuvant, protein antigens (e.g., subunit vaccines, which can be developed by recombinant DNA technology) induce weak humoral (antibody) immunity, and therefore disappointingly show only limited immunogenicity so far. Another disadvantage of subunit vaccines and killed virus and recombinant live virus vaccines is that, although they appear to stimulate a strong humoral immune response when administered with an adjuvant, they fail to induce protective cellular immunity. Adjuvants are used experimentally to stimulate effective immune responses in mice, and are expected to be used in human vaccines, but few adjuvants are approved for use in humans. In fact, the only adjuvants approved for use in the United States are aluminum salts, aluminum hydroxide, and aluminum phosphate, none of which stimulate cell-mediated immunity. Aluminum salt formulations cannot be frozen or lyophilized, and such adjuvants are not effective for all antigens. In addition, most adjuvants do not lead to the induction of cytotoxic T lymphocytes (CTLs). CTLs are needed to kill cells that are synthesizing abnormal proteins (including viral proteins and mutated "self" proteins). Vaccines that stimulate CTLs are currently being extensively investigated against a variety of viruses (e.g., HIV, HCV, HPV, HSY, CMV, EBY), intracellular bacteria (e.g., tuberculosis), intracellular parasites (e.g., malaria, leishmaniasis, schistosomiasis, leprosy), and all cancers (e.g., melanoma, prostate cancer, ovarian cancer, etc.). Therefore, adjuvants that stimulate CTLs and cell-mediated immunity are generally needed.

[0009] Yeast has been used to produce subunit protein vaccines, including those tested in HIV vaccine trials and hepatitis B vaccines; however, in these cases, yeast is used to produce protein, but yeast cells or their subcellular fractions are not actually delivered to the patient. Yeast has also been fed to animals before immunization in an attempt to initiate an immune response in a nonspecific manner (i.e., stimulate phagocytosis and the production of complement and interferon). The results are unclear, and such regimens do not produce protective cellular immunity; see, for example, Fattal-German et al., 1992, Dev. Biol. Stand [Development of Biological Standardization]. 77, 115-120; Bizzini et al., 1990, FEMS Microbial. Immunol. [Federation of European Microbiological Societies Microbial Immunology] 2, 155-167.

[0010] U.S. Pat. No. 5,830,463 (Duke et al.), issued Nov. 3, 1998, describes the use of non-pathogenic yeast carrying at least one compound capable of modulating an immune response and demonstrates that such complexes are effective in stimulating both cell-mediated and humoral immunity. In particular, U.S. Pat. No. 5,830,463 demonstrates that yeast genetically engineered to express heterologous antigens can elicit both cell-mediated and humoral immune responses when administered to a mammal.

[0011] There is a need for improved vaccines that stimulate T cell (and particularly cytotoxic T lymphocyte (CTL)) mediated immunity against cell-associated or endogenous antigens. The targets of these vaccines include cells that infect viruses, intracellular bacteria and parasites, and cancer. The initiation of CTL-mediated immunity requires antigenic peptides to be presented in association with major histocompatibility (MHC) class 1 molecules on the surface of professional antigen presenting cells (APCs), particularly dendritic cells (DCs) (Ridge et al., Nature [Nature] 393: 474-8 1998). Dendritic cells are the main antigen presenting cells (APCs) used to initiate immune responses. Because DCs have unique abilities to activate naive CD4+ and CD8+ T cells, they play a vital role in initiating both MHC class II and class I restricted antigen-specific T cell responses (Ridge et al., Nature [Nature] 393: 474-8 (1998); Banchereau et al., Nature [Nature] 392: 245-52 (1998)). However, exogenously introduced antigens (e.g., antigens found in vaccines composed of antigenic proteins or killed pathogens) are primarily processed via the MHC class II pathway for presentation to CD4+ T cells (Moore et al., Cell [Cell] 54: 777-85 (1988)). These types of vaccines can stimulate effective humoral immunity, but are relatively ineffective in stimulating CD8+ CTLs. This shortcoming has led to research into vaccine strategies that specifically target DCs to present antigens via MHC class I in addition to MHC class II. It has been shown that DCs have unique pathways to process foreign antigens, particularly in microparticle form, for presentation via the MHC class I pathway (Rodriguez et al., Nat Cell Biol 1:362-368 (1999)). In this regard, various liposome-like microparticle formulations consisting of antigenic proteins or peptides with added adjuvants have shown promise in stimulating CTLs (Palo et al., Nat Med 1:649-53 (1995); O'Hagan, J Pharm Pharmacol 50:1-10 (1998)). The microparticle nature of these immunostimulatory complexes (ISCOMS) makes them susceptible to phagocytosis by DCs recruited to the vaccination site and partially activated by the adjuvant.

[0012] Cancer vaccines based on dendritic cells are also under intensive investigation. Dendritic cells are expanded ex vivo and "loaded" with peptides (derived from suspected tumor antigens) or mixed with the patient's cancer cells (DC engulf these cells and present tumor antigens). However, peptide loading efficiency is low and knowledge of the specific peptides used is required. Phagocytosis of dead tumor cells and debris results in class II MHC presentation, rather than class I MHC presentation, which is required for activation of CTLs. Therefore, there is a need in the art for improved vaccines, including improved DC vaccines.

[0013] The whole recombinant yeast-based therapeutic vaccine (i.e., Tarmogen) elicits CD4+, CD8+, and Th17 T cell responses that are capable of eliminating cancer cells and cells infected with microorganisms in humans. (TARgeted MOlecular immunoGEN, GlobeImmune, Inc., Louisville, CO) generally refers to a yeast vehicle that expresses one or more heterologous antigens extracellularly (on its surface), intracellularly (inside or in the cytoplasm), or both extracellularly and intracellularly. Tarmogens have been generally described in the art. See, e.g., U.S. Pat. No. 5,830,463. Responses to standalone / single-agent tarmogens have been shown to confer survival benefits in several different animal models. In humans, signs of such benefits have been observed in clinical trials in patients with pancreatic cancer and chronic hepatitis C, although, so far, statistical significance has not been achieved for primary endpoint readouts except in biomarker-selected patient subgroups. Other trials featuring these fully recombinant yeast-based therapeutic vaccines in combination with other immunomodulators are ongoing and may improve clinical efficacy. The increase in the magnitude of immune responses, particularly cytotoxic T cells (CTLs), elicited by these tarmogens may result in more effective target cell clearance, leading to efficacy in a greater percentage of patients.

[0014] In the 1990s, yeast-based immunotherapeutic compositions were introduced as novel compositions that induce immune responses through both the MHC class I-restricted pathway and the MHC class II-restricted pathway of antigen presenting cells (see U.S. Pat. No. 5,830,463). Although these compositions are initially exposed to the immune system as one or more exogenous antigens, yeast-based immunotherapeutic compositions are uniquely capable of triggering the induction of CD8+ cytotoxic T cell responses by cross-presenting antigens through the MHC class I-restricted pathway, and triggering the induction of CD4+ T cell responses by presenting antigens through the MHC class II-restricted pathway (see, e.g., U.S. Pat. Nos. 5,830,463 and 7,083,787, Stubbs et al., Nat. Med. 7:625-629 (2001) and Lu et al., Cancer Research 64:5084-5088 (2004)). Yeast-based immunotherapeutic compositions stimulate pattern recognition receptors (PRRs); upregulate adhesion molecules, co-stimulatory molecules, and MHC class I and class II molecules on antigen-presenting cells (including DCs); and induce the production of proinflammatory cytokines by antigen-presenting cells (e.g., TNF-α and IL-12) (see, e.g., Stubbs et al., supra; Brown et al., J Exp. Med. 197: 1119-1124 (2003)).

[0015] In the context of yeast-based immunotherapeutic compositions that can be engineered to express one or more antigens, the complexity of the mechanisms of action of yeast-based immunotherapies in terms of the immune system and therapeutic efficacy has not been fully determined. It is desirable to better understand how different individuals respond to immunization with yeast-based immunotherapeutic compositions so that immunotherapeutic strategies can be manipulated and personalized to more effectively elicit a desired immune response in an individual that is most appropriate for a given disease or condition. Summary of the invention

[0016] Various embodiments of the present invention are described below. However, the present invention is not limited to the embodiments described in the summary of the invention, because the invention described in the following description is also explicitly included therein.

[0017] One embodiment of the present invention relates to an immunogenic composition comprising (a) a yeast lysate prepared from yeast, wherein the lysate lacks yeast membrane and yeast cell wall; and (b) at least one antigen heterologous to the yeast.

[0018] Another embodiment of the present invention relates to an immunogenic formulation comprising: (a) a first immunogenic composition comprising (i) a yeast lysate prepared from yeast, wherein the lysate lacks yeast membranes and yeast cell walls; and (ii) at least one antigen heterologous to the yeast; and (b) a second immunogenic composition comprising (i) a yeast vehicle selected from the group consisting of whole yeast, yeast spheroplasts, yeast cytoplasts, yeast ghosts, and subcellular yeast particles; and (ii) at least one antigen heterologous to the yeast. In one aspect of this embodiment, the first immunogenic composition and the second immunogenic composition are combined in a ratio of about 90:10 to about 10:90.

[0019] Yet another embodiment of the present invention relates to a method for producing an immunogenic composition, the method comprising (a) lysing yeast cells; and (b) removing cell walls and cell membranes from the lysed cells, wherein the lysed cells contain antigens heterologous to the yeast. In one aspect of this embodiment, the lysing step is performed by a method selected from the group consisting of: glass bead crushing, high pressure homogenization, ultrasonic treatment, electrolysis, physical lysis, chemical lysis, and enzyme lysis. In yet another aspect of this embodiment, the removal step is performed by a method selected from the group consisting of: centrifugation, filtration, and affinity chromatography. In one aspect, the immunogenic composition and yeast vehicle are combined in a ratio of about 90:10 to about 10:90 yeast unit equivalents. In another aspect, before the step of removing cell walls and membranes, the lysed yeast cells are further treated by ultrasonic treatment to release antigens from the cell walls and cell membranes.

[0020] Another embodiment of the invention relates to a method of eliciting a cytotoxic T cell immune response in a subject, the method comprising administering to the subject an immunogenic composition comprising: (a) a yeast lysate prepared from yeast, wherein the lysate lacks yeast membrane and yeast cell wall; and (b) at least one antigen heterologous to the yeast.

[0021] In one aspect of this embodiment, the method further comprises combining the immunogenic composition with a composition comprising a whole recombinant killed yeast vehicle and at least one antigen heterologous to the yeast.

[0022] In one aspect of this embodiment, the immunogenic composition is administered to the subject by a route of administration selected from the group consisting of intravenous, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, inhalation, intranasal, oral, intraocular, intraarticular, intracranial, and intraspinal.

[0023] In one aspect of this embodiment, the immunogenic composition is formulated in a pharmaceutically acceptable excipient suitable for administration to a subject by injection.

[0024] In one aspect of this embodiment, the immunogenic composition is administered to the subject at a dose of about 0.1 YU to about 100 Y.U.

[0025] In one aspect of this embodiment, the immunogenic composition is administered to the subject at a dose of about 10 Y.U. to about 80 Y.U.

[0026] In one aspect of this embodiment, the immunogenic composition is administered once a week.

[0027] In one aspect of this embodiment, the immunogenic composition is administered every other week.

[0028] In one aspect of this embodiment, the immunogenic composition is administered monthly.

[0029] In one aspect of this embodiment, the composition comprises a plurality of antigens.

[0030] Still another embodiment of the invention relates to a method of eliciting an antigen-specific humoral immune response and an antigen-specific cell-mediated immune response in a subject, the method comprising administering to the subject an immunogenic composition comprising: (a) a yeast lysate prepared from yeast, wherein the lysate lacks yeast membrane and yeast cell wall; and (b) at least one antigen heterologous to the yeast.

[0031] In one aspect of this embodiment, the method further comprises administering to the subject a combination of the whole recombinant inactivated yeast and at least one antigen expressed by the yeast.

[0032] In one aspect of this embodiment, the immunogenic composition is administered to the subject by a route of administration selected from the group consisting of intravenous, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, inhalation, intranasal, oral, intraocular, intraarticular, intracranial, and intraspinal.

[0033] In one aspect of this embodiment, the immunogenic composition is formulated in a pharmaceutically acceptable excipient suitable for administration to a subject by injection.

[0034] In one aspect of this embodiment, the immunogenic composition is administered to the subject at a dose of about 0.1 YU to about 100 Y.U.

[0035] In one aspect of this embodiment, the immunogenic composition is administered to the subject at a dose of about 10 Y.U. to about 80 Y.U.

[0036] In one aspect of this embodiment, the immunogenic composition is administered once a week.

[0037] In one aspect of this embodiment, the immunogenic composition is administered every other week.

[0038] In one aspect of this embodiment, the immunogenic composition is administered monthly.

[0039] In one aspect of this embodiment, the composition comprises a plurality of antigens.

[0040] In one aspect of any of the above embodiments of the invention, the antigen is expressed by yeast.

[0041] In any of the embodiments described herein, in one aspect, the antigen is added to the yeast lysate.

[0042] In any of the embodiments described herein, in one aspect, the antigen is selected from the group consisting of a viral antigen, a mammalian cell surface molecule, a bacterial antigen, a fungal antigen, a protozoan antigen, a helminth antigen, an ectoparasite antigen, a cancer antigen, and a tumor neoantigen.

[0043] In any of the embodiments described herein, in one aspect, the yeast expresses the heterologous antigen prior to yeast lysis.

[0044] In any of the embodiments described herein, in one aspect, the heterologous antigen is added to the lysed yeast.

[0045] In any of the embodiments described herein, in one aspect, further comprising combining the immunogenic composition with at least one antigen expressed by a fully recombinant inactivated yeast vehicle.

[0046] In any of the embodiments described herein, in one aspect, the yeast vehicle is heat-killed yeast cells.

[0047] In any of the embodiments described herein, in one aspect, the yeast is from the genus Saccharomyces.

[0048] In any of the embodiments described herein, in one aspect, the yeast is from Saccharomyces cerevisiae. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a schematic diagram of the in vivo CTL assay.

[0050] Figure 2It is shown that the mixture of clarified Tarmogen lysate (first bar in each group) and complete Tarmogen (second bar in each group) is more effective in producing CTL-mediated killing in vivo than complete Tarmogen alone. TEF2: constitutive promoter of yeast translation elongation factor; CUP1: yeast copper-inducible promoter Kb-cyto: Tarmogen (or its lysate) expressing a polypeptide having multiple B16F10 cell-derived, H-2Kb-restricted new epitopes plus H-2Kb-restricted SIINFEKL (SEQ ID NO: 9) epitopes; IAb-cyto: Tarmogen (or its lysate) expressing a polypeptide having multiple B16F10 cell-derived IA(b) binding new epitopes plus H-2Kb-restricted SIINFEKL (SEQ ID NO: 9) epitopes; Yvec: empty yeast vector; Ovax: Tarmogen (or its clarified yeast lysate (YL)) expressing chicken ovalbumin.

[0051] Figure 3 Shown is the IA from the immunization b In vitro intracellular cytokine staining in spleen cells of mice treated with -cyto Tarmogen: intact Tarmogen vs. cleaved Tarmogen. b -cyto intact yeast or IA b -cyto clarified yeast lysate (YL) was used for two immunizations (homologous prime / boost). In vitro stimulators: medium only, cRPMI + 10% FBS; ESAT6, 5 μM mTb ESAT6 peptide N-MTEQQWNFAGIEAAASAIQG (SEQ ID NO: 1); Peptide group 4, class II peptide Nant23 (qpysgspanVavytalvep) (SEQ ID NO: 2), Nant24 (sgkhkyrqtAmftatmppa) (SEQ ID NO: 3); Nant25 (nrtsvmfnfpEqatvkkvv) (SEQ ID NO: 4), Nant26 (sfetashdgeagpsPevlq) (SEQ ID NO: 5); Nant27 (mfnqgrafaAvrlpfcghk) (SEQ ID NO: 6), Nant28 (lwvhslrrlLecfyvsvfs) (SEQ ID NO:7) and Nant29 (epmftfvypTifplretpm) (SEQ ID NO:8), wherein each capitalized residue in the peptide sequence is a new mutation of B16F10. CD3 + CD4 +Flow cytometry plots of intracellular IFNγ and TNFα expression.

[0052] Figure 4 YE-NEO-001-93 vaccination produces antigen-specific CD4 + and CD8 + Cytokine production. Female C57BL / 6 mice were immunized with: i) a mixture of 2YU YL + 2YU of empty vector (Yvec) Tarmogen + 25μg α-CD40 antibody (93-YL) from the YE-NEO-001-93 prototype; ii) 2.5YU of complete YE-NEO-001-93 + 25μg αCD40 antibody (93-INT); or iii) no immunization as a negative control (initial). After 7 days, spleen cells were collected and stimulated with peptides in vitro for 6 hours in the presence of Brefeldin A (BFA). Representative flow charts are shown in Figure 4 Figure 2 shows the production of dual IFNγ after stimulation with SIINFEKL (SEQ ID NO: 9) peptide or control (DMSO, medium without peptide and containing solvent (DMSO) concentration matching that used in peptide stimulation). + TNFα + CD8 + Frequency of T cell generation; SIINFEKL (SEQ ID NO: 9): ovalbumin peptide 257-264.

[0053] Figure 5 The effect of adding lysate to whole Tramogen is shown. cI IN: whole yeast expressing class I neo-epitope fusions; cII IN: whole yeast expressing class II neo-epitope fusions; cI IN+cI YL: 1.25 YU of cI IN mixed with 1.25 YU of cI YL; cII IN+cIICL: 1.25 YU of cII IN mixed with 1.25 YU of cII YL. Day: Days after tumor challenge. Day 8 is the first bar in the group (except for cI IN+Ci YL, whose data for day 8 are not shown); Day 10 is the middle bar in the group (except for cI IN+Ci YL, whose first bar shows day 10); Day 12 is the last bar. DETAILED DESCRIPTION

[0054] The present invention relates to methods of making and using yeast lysate ("yeast lysate" or "YL") free of yeast membranes and yeast cell walls, which is used alone or in combination with a complete yeast-based immunotherapeutic composition as a means of increasing the magnitude of disease antigen-specific CTL responses compared to the complete immunotherapeutic composition alone. Without being bound by theory, the rationale for increasing the response is that faster / more efficient acquisition of antigen presenting cells (APCs), and / or exposure of APCs to double-stranded RNA and DNA or other cytokines derived from yeast cytoplasm can improve antigen presentation by T cells via more efficient MHC loading and stronger APC activation (the latter can occur, for example, by activating Toll-like receptors (TLRs) 3 and 9, which cannot be effectively activated by the complete Tarmogen alone).

[0055] As provided in the Examples herein, YL was tested in murine in vivo CTL assays and compared to a complete yeast-based immunotherapeutic, i.e., Tarmogen (T) alone. The results showed that in two experiments against five different Tarmogen vaccine candidates, YL significantly improved the magnitude of antigen-specific in vivo cytolytic activity compared to T. In these experiments, T elicited 0 to about 20% killing, while YL consistently elicited up to about 80% to 95% killing.

[0056] In a mouse in vivo CTL model, using yeast-expressed ovalbumin antigens and certain yeast-expressed multi-epitope antigens as test cases, the present invention resulted in significantly improved CTL activity compared to the complete Tarmogen alone. The results are shown in Example 1 and Figure 1 and Figure 2 This improved CTL activity could translate into significant advantages against infectious diseases and cancer in human clinical trials.

[0057] In the third example presented herein, YL was compared to intact yeast in terms of its ability to generate antigen-specific CD4+ T cell responses using intracellular cytokine staining. b Mice were immunized intradermally with 24-week-old immunizations of 100% IgG4+ T-cell neoepitopes expressed in 100% IgG4+ T-cell neoepitopes. Nine days after the second immunization, the spleens were removed and stimulated with a pool of vaccine-matched class II MHC-binding peptides, and the magnitude of the “recalled” (reactivated) CD4+ T-cell responses was measured using ICS combined with flow cytometric readouts. The results showed that IA b-cyto YL immunization generated 6-fold higher levels of neo-epitope-specific CD4+ T cells producing IFNγ and TNFα than whole yeast (YL generated 0.059% TNFα+ / IFNγ+ CD4 T cells vs. 0.01% for whole yeast, Figure 3 ).

[0058] In the fourth example presented herein, intracellular cytokine staining was used to compare YL with intact yeast in terms of its ability to generate antigen-specific CD8+T cell responses. Mice were inoculated intravenously with intact contrast lysed yeast (these yeasts express multi-epitopes (multiples of class I T cell new epitopes) fused to the positive control epitope SIINFEKL (SEQ ID NO: 9)). The Tarmogen is called "93" (named for the size of the multi-epitope of 93kDa), and it contains new epitopes with different arrangements and quantities compared to the variants used in the above examples. Immunizations were performed on days 0 and 38, and nine days after the second immunization, the spleen was removed and spleen cell preparations were stimulated with antigen-free (DMSO peptide solvent only in the growth medium, = simulation) peptides or SIINFEKL (SEQ ID NO: 9) peptides (this is the main murine class I MHC binding epitope). SIINFEKL (SEQ ID NO: 9) specific recall CD8+T cell response magnitude was measured using ICS, and then analyzed by flow cytometry. The results showed that YL immunization produced 20-fold higher levels of neo-epitope-specific CD8+ T cells producing IFNγ and TNFα than whole yeast (YL immunization produced 0.45% TNFα+IFNγ+CD8 T cells, compared to 0.022% produced by the mock product, Figure 4 ).

[0059] In the fifth example presented herein, the multi-epitope yeast K b -cyto("cI") and IA b -cyto ("cII") YL was added in equal parts (1:1) to the whole tarmogen (expressing the same / matching multi-epitopes) to determine whether YL(CL) could improve the degree of tumor protection provided by the whole yeast. Briefly, mice were immunized three times (once a week) with whole yeast ("cI IN" or "cII IN", 5YU total), or 2.5YU of whole yeast mixed with 2.5YU of YL (e.g., cI IN+cI CL). The concept here is that vaccination should elicit a T cell response targeting the neo-epitope that can kill or otherwise inhibit the growth of subcutaneously implanted B16F10 tumors in vivo. Vaccinations were performed in 4 different groups of mice to test class I multi-epitope yeast and class II multi-epitope yeast ( Figure 5). One week after the third immunization, mice were challenged with an aggressive B16F10 tumor cell line expressing neo-epitopes matching those contained in the yeast vaccine, and tumor growth was measured every other day through day 12 post-challenge. The results showed that whole yeast mixed with YL slowed tumor growth by 2- to 5-fold compared to whole yeast alone (e.g., comparing the day 10 time point of "cI IN + cI CL" to the same time point of "cI In" alone). The total amount of yeast material used for immunization in these groups was the same, yet tumor growth inhibition was up to 5-fold greater than in the group containing CL ( Figure 5 )

[0060] YL is considered to work differently from any other known vaccine because it can not only trigger TLR 2, 4, and 6 (whole yeast), but also TLR 3 (activated by dsRNA in lysate) and TLR 9 (activated by dsDNA in lysate). Other receptors bound by the YL mixture include Dectin-1 and mannose receptor (Haller et al.) (bound by the whole Tarmogen component). This extensive activation can lead to effective and complete APC activation via multiple cellular pathways. In addition, the presence of the whole Tarmogen in the mixture ensures that APC will undergo phagocytosis, followed by cross-presentation of antigens with APC maturation. Heterologous antigens and other cellular components (including DNA and RNA / RNP) in the lysate will enter the phagosome with the whole yeast, thereby inducing multiple activation pathways in the process.

[0061] One embodiment of the present invention relates to an immunogenic composition comprising (a) a yeast lysate prepared from yeast, wherein the lysate lacks yeast membrane and yeast cell wall; and (b) at least one antigen heterologous to the yeast.

[0062] Another embodiment of the present invention relates to an immunogenic formulation comprising: (a) a first immunogenic composition comprising (i) a yeast lysate prepared from yeast, wherein the lysate lacks yeast membranes and yeast cell walls; and (ii) at least one antigen heterologous to the yeast; and (b) a second immunogenic composition comprising (i) a yeast vehicle selected from the group consisting of whole yeast, yeast spheroplasts, yeast cytoplasts, yeast ghosts, and subcellular yeast particles; and (ii) at least one antigen heterologous to the yeast. In one aspect of this embodiment, the first immunogenic composition and the second immunogenic composition are combined in a ratio of about 90:10, about 80:20, about 70:30, about 60:40, about 50:50, about 40:60, about 30:70, about 20:80, or about 10:90. In a preferred embodiment, the ratio is about 50:50. Alternative embodiments include first and second immunogenic compositions using any range of the above ratios as endpoints (e.g., 90:10 to 10:90 or 80:20 to 30:70). In still another aspect, the percentage of the first immunogenic composition in the combined composition is about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, 30%, about 20%, or about 10%, or any integer percentage between 10% and 99%. The percentages are based on yeast units.

[0063] Yet another embodiment of the present invention relates to a method for producing an immunogenic composition, the method comprising (a) lysing yeast cells; and (b) removing cell walls and cell membranes from lysed cells, wherein the lysed cells contain antigens heterologous to the yeast. In one aspect of this embodiment, the lysis step is performed by a method selected from the group consisting of glass beads, high pressure homogenization, ultrasonic treatment, electrolysis, physical lysis, chemical lysis, and enzyme lysis. In yet another aspect of this embodiment, the step of removing cell walls and membranes is performed by a method selected from the group consisting of centrifugation, filtration, and affinity chromatography. In one aspect, the immunogenic composition is combined with a fully recombinant inactivated yeast vehicle comprising at least one antigen heterologous to fully recombinant inactivated yeast. In this embodiment, the composition can be combined with a yeast vehicle in a ratio of about 90:10, about 80:20, about 70:30, about 60:40, about 50:50, about 40:60, about 30:70, about 20:80, or about 10:90 yeast unit equivalents.

[0064] Another embodiment of the present invention relates to a method of eliciting a cytotoxic T cell immune response in a subject, the method comprising administering to the subject an immunogenic composition comprising: (a) a yeast lysate prepared from yeast, wherein the lysate lacks yeast membranes and yeast cell walls; and (b) at least one antigen heterologous to the yeast. The method may further comprise administering to the subject a combination of whole recombinant inactivated yeast and at least one antigen expressed by the yeast.

[0065] Still another embodiment of the present invention relates to a method for eliciting an antigen-specific humoral immune response and an antigen-specific cell-mediated immune response in a subject, the method comprising administering to the subject an immunogenic composition comprising: (a) a yeast lysate prepared from yeast, wherein the lysate lacks yeast membranes and yeast cell walls; and (b) at least one antigen heterologous to the yeast. The method may further comprise administering to the subject a combination of whole recombinant inactivated yeast and at least one antigen expressed by the yeast.

[0066] The immunogenic composition of the present invention includes yeast lysate prepared from yeast, wherein the lysate lacks yeast membrane and yeast cell wall. This yeast lysate is prepared from yeast that has been lysed (i.e., yeast in which the cell wall and membrane have been destroyed, thereby exposing the yeast cell contents to the rest of the composition). Yeast lysate can be prepared from inactivated (such as heat-inactivated) yeast or from live yeast. Yeast can contain disease-associated antigens expressed from plasmids or integrated chromosomal alleles inside yeast. For example, yeast can be lysed by glass bead crushing, such as by mixing with PBS and 500 μL of acid-washed 0.2 μm glass beads with a total volume of 1.5 mL and shaking the mixture vigorously in a mechanical stirrer until the cells are broken (such as> 97% of the cells are broken) to lyse the yeast. Alternatively, yeast can be lysed by other methods, including high-pressure homogenization, ultrasonic treatment, and electrical, physical, chemical and enzyme techniques. In preferred embodiments, at least about 80% of the cells are disrupted, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%.

[0067] Preferred genera of yeast strains for producing yeast lysates include Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia, more preferably Saccharomyces, Candida, Hansenula, Pichia and Schizosaccharomyces, and particularly preferably Saccharomyces. Preferred species of yeast strains include Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica. lipolytica).

[0068] The lysed yeast cell composition is then further processed to remove yeast membranes and yeast cell walls by any suitable method, thereby producing a yeast lysate lacking yeast membranes and yeast cell walls. For example, yeast membranes and yeast cell walls can be removed from the lysed yeast by centrifugation to produce a lysate (supernatant) that does not contain cell walls and membranes, such as by centrifuging the lysed yeast at 16,000 rpm for 5 minutes at 25° C. Alternatively, the cell walls and membranous fragments of the lysate can be removed by means other than centrifugation after disruption. For example, filtering or treating cells with conA beads is an alternative method. It should be recognized that although the present invention describes the removal of yeast membranes and yeast cell walls, or references to lysates lacking yeast membranes and yeast cell walls, suitable methods for removing materials may not remove 100% of the yeast membranes and yeast cell walls from the lysate. In some embodiments, at least about 80% of the yeast membrane and / or yeast cell wall is removed, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%.

[0069] In addition to yeast lysates lacking yeast membranes and yeast cell walls, the immunogenic compositions of the present invention also include at least one antigen that is heterologous to the yeast (this term includes the immunogenic domains of the antigen). The heterologous antigen can be expressed by the yeast before lysis, or the antigen can be combined with the yeast before or after lysis and before or after the yeast membrane and yeast cell wall are removed from the lysate. In certain embodiments, the antigen is provided as a fusion protein (which may include two or more antigens). In one aspect, the fusion protein may include two or more immunogenic domains of one or more antigens, or two or more epitopes of one or more antigens.

[0070] The present invention includes at least one "yeast-based immunotherapeutic composition" (this phrase can be used interchangeably with "yeast-based immunotherapeutic product", "yeast-based immunotherapeutic composition", "yeast-based composition", "yeast-based immunotherapeutic agent" or "yeast-based vaccine", which can be a yeast lysate lacking yeast membrane and yeast cell wall) used alone or in combination with a complete yeast-based immunotherapeutic composition (such as Tarmogen). The yeast-based immunotherapeutic composition elicits an immune response sufficient to obtain at least one therapeutic benefit in a subject. More particularly, the yeast-based immunotherapeutic composition is a composition comprising a YL component (alone or in combination with a complete yeast-based immunotherapeutic composition) and is capable of eliciting or inducing an immune response, such as a cellular immune response, including but not limited to a T cell-mediated cellular immune response. In one aspect, the yeast-based immunotherapeutic composition useful in the present invention is capable of inducing a CD8+ and / or CD4+ T cell-mediated immune response, and in one aspect, induces a CD8+ and CD4+ T cell-mediated immune response. Optionally, the yeast-based immunotherapeutic composition is capable of eliciting a humoral immune response. Yeast-based immunotherapeutic compositions useful in the present invention can, for example, elicit an immune response in an individual, thereby treating a disease or disorder, or symptoms resulting from a disease or disorder, in the individual.

[0071] The yeast-based immunotherapeutic compositions of the present invention may be "prophylactic" or "therapeutic." When provided prophylactically, the immunotherapeutic compositions of the present invention are provided prior to any symptoms of a disease or condition. Prophylactic administration of the immunotherapeutic compositions is used to prevent or ameliorate or delay the onset of any subsequent disease. When provided therapeutically, the immunotherapeutic compositions are provided at or after the onset of symptoms of the disease. The term "disease" refers to any deviation from the normal health of an animal, and includes states when symptoms of the disease are present, as well as conditions where deviations have occurred (e.g., tumor growth, infection, etc.) but symptoms have not yet manifested.

[0072] In one embodiment, the yeast lysate of the present invention is prepared from a complete yeast-based immunotherapeutic composition. In addition, in the present invention, such a complete yeast-based immunotherapeutic composition can be combined with a yeast lysate-based composition. Such a complete yeast-based immunotherapeutic composition generally comprises a yeast vehicle and an antigen heterologous to the yeast.

[0073] Such whole yeast-based immunotherapeutic compositions and methods of making and generally using the compositions are also described in detail in, for example, U.S. Pat. No. 5,830,463, U.S. Pat. No. 7,083,787, U.S. Pat. No. 7,465,454, U.S. Patent Publication No. 2007-0224208, U.S. Patent Publication No. US2008-0003239, and Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research 64:5084-5088 (2004), and Bernstein et al., Vaccine 2008 Jan. 24; 26(4):509-21, each of which is incorporated herein by reference in its entirety. These yeast-based immunotherapeutic products have been shown to elicit immune responses, including cellular and humoral immune responses. Yeast-based immunotherapeutic products can kill target cells expressing a variety of antigens in a variety of animal species, and can kill the target cells via antigen-specific CD4+ and CD8+ mediated immune responses. Additional studies have shown that yeast is greedily engulfed by dendritic cells and directly activates dendritic cells, which then present yeast-related proteins to CD4+ and CD8+ T cells in an efficient manner. See, for example, Stubbs et al. Nature Med. 5:625-629 (2001) and U.S. Pat. No. 7,083,787.

[0074] In any yeast-based immunotherapeutic composition used in the present invention, the following aspects related to yeast vehicles are included in the present invention. According to the present invention, a yeast vehicle is any yeast cell (e.g., whole cell 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 the therapeutic composition of the present invention. Thus, a yeast vehicle can include, but is not limited to, a live whole yeast microorganism (i.e., a yeast cell with all its components (including a cell wall)), a killed (dead) or inactivated whole yeast microorganism or a derivative thereof, including: yeast spheroplasts (i.e., yeast cells lacking a cell wall), yeast cytoplasm (i.e., yeast cells lacking a cell wall and a nucleus), yeast slough (i.e., yeast cells lacking a cell wall, a nucleus, and a cytoplasm), a subcellular yeast membrane extract or a portion thereof (also referred to as a yeast membrane particle, formerly referred to as a subcellular yeast particle), any other yeast particle or yeast cell wall preparation.

[0075] Yeast spheroplasts are usually produced by enzymatic digestion of the yeast cell wall. This method is described, for example, in Franzusoff et al., 1991, Meth. Enzymol [Methods in Enzymology] 194, 662-674, which is incorporated herein by reference in its entirety.

[0076] Yeast cytoplasm is usually produced by enucleation of yeast cells. This method is described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55, which is incorporated herein by reference in its entirety.

[0077] Yeast exuviae are typically produced by resealing a permeabilized or lysed cell and may, but need not, contain at least some of the cell's organelles. Such methods are described, for example, in Franzusoff et al., 1983, J. Biol. Chem. [Journal of Biological Chemistry] 258, 3608-3614 and Bussey et al., 1979, Biochim. Biophys. Acta [Biological Chemistry and Biophysics] 553, 185-196, each of which is incorporated herein by reference in its entirety.

[0078] Yeast membrane particles (subcellular yeast membrane extracts or portions thereof) refer to yeast membranes lacking a native nucleus or cytoplasm. The particles can be of any size, including a range from the size of native yeast membranes to the size of microparticles produced by ultrasonic treatment or other membrane disruption methods known to those skilled in the art and then resealed. Methods for producing subcellular yeast membrane extracts are described, for example, in Franzusoff et al., 1991, Meth. Enzymol. [Methods in Enzymology] 194, 662-674. It is also possible to use portions of yeast membrane particles containing yeast membrane portions and (when the antigen is recombinantly expressed by yeast before preparing yeast membrane particles) antigens or other target proteins. Antigens or other target proteins can be carried inside the membrane, on any surface of the membrane, or a combination thereof (i.e., the protein is both inside and outside the membrane, and / or across the membrane of the yeast membrane particle). In one embodiment, the yeast membrane particle is a recombinant yeast membrane particle, which can be a complete, destroyed, or destroyed and resealed yeast membrane, which contains at least one desired antigen or other target protein on the membrane surface, or is at least partially embedded in the membrane.

[0079] One example of a yeast cell wall preparation is an isolated yeast cell wall carrying an antigen on its surface or at least partially embedded within the cell wall, such that the yeast cell wall preparation stimulates a desired immune response against a disease target when administered to an animal.

[0080] Any yeast strain can be used to produce a yeast vehicle (for producing a yeast lysate or for use in combination with a yeast lysate). Yeast is a unicellular microorganism belonging to one of the following three classes: Ascomycetes, Basidiomycetes and Imperfecta. A consideration for selecting the type of yeast to be used as an immunomodulator is the pathogenicity of the yeast. In one embodiment, the yeast is a non-pathogenic strain, such as Saccharomyces cerevisiae. Non-pathogenic yeast strains are selected to minimize any adverse reactions to the individual to whom the yeast vehicle is administered. However, if the pathogenicity of the yeast can be eliminated by any means known to those skilled in the art (e.g., mutant strains), pathogenic yeast can be used. According to one aspect of the invention, non-pathogenic yeast strains are used.

[0081] The genus that can be used for yeast strain of the present invention includes but is not limited to Saccharomyces, Candida (can be pathogenic), Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. On the one hand, the genus of Saccharomyces is selected from Saccharomyces cerevisiae, Candida, Hansenula, Pichia or Schizosaccharomyces, and in one aspect, Saccharomyces cerevisiae is used. The species that can be used for yeast strain of the present invention includes but is not limited to Saccharomyces cerevisiae, Karl's yeast, Candida albicans, Candida kefir, Candida tropicalis, Cryptococcus laurensis, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus milk variant, Pichia pastoris, Rhodotorula, Schizosaccharomyces pombe and Yarrowia lipolytica. It should be understood that many species in these species include various subspecies, types, subtypes, etc. that are intended to be included in the above-mentioned species. In one aspect, the yeast species used in the present invention include Saccharomyces cerevisiae, Candida albicans, Hansenula polymorpha, Pichia pastoris and Schizosaccharomyces pombe. Saccharomyces cerevisiae is useful because it is relatively easy to manipulate and is "generally recognized as safe" or "GRAS" (GRAS, FDA proposed rule 62FR18938, April 17, 1997) for use as a food additive. One embodiment of the present invention is a yeast strain (e.g., a Saccharomyces cerevisiae cir° strain) that is capable of replicating plasmids to a particularly high copy number. A Saccharomyces cerevisiae strain is one such strain that is capable of supporting expression vectors that allow one or more target antigens and / or antigen fusion proteins and / or other proteins to be expressed at high levels. In addition, any mutant yeast strain 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 enzymes that extend N-linked glycosylation.

[0082] In one embodiment, the yeast vehicle of the present invention can be fused with a cell type (such as a dendritic cell or macrophage) to which the yeast vehicle and antigen / agent delivery are received, thereby achieving particularly effective delivery of the yeast vehicle and (in many embodiments) one or more antigens or other agents to the cell type. As used herein, the fusion of the yeast vehicle with the target cell type refers to the ability of the yeast cell membrane or its particles to fuse with the membrane of the target cell type (such as a dendritic cell or macrophage) to cause syncytium formation. As used herein, syncytium is a multinuclear protoplasm produced by cell merging. Many viral surface proteins (including immunodeficiency viruses, such as those of HIV, influenza virus, poliovirus, and adenovirus) and other fusogens (such as those involving fusion between eggs and sperm) have been shown to be able to achieve fusion between two membranes (i.e., between viruses and mammalian cell membranes or between mammalian cell membranes). For example, a yeast vehicle that produces HIV gp120 / gp41 heterologous antigens on its surface can fuse with CD4+T lymphocytes. However, it should be noted that it is not necessary to incorporate the targeting moiety into the yeast vehicle, although this may be desirable in some cases. In the case where the yeast vehicle expresses the antigen extracellularly, this may be another advantage of the yeast vehicle of the present invention. In general, dendritic cells (and other cells, such as macrophages) readily take up yeast vehicles useful in the present invention.

[0083] The immunogenic composition of the present invention comprises at least one antigen that is heterologous to the yeast forming the composition. The antigen in YL or the complete immunotherapeutic composition can be expressed by yeast (for YL, before its cleavage), or can be added to the immunotherapeutic composition. The one or more antigens can be viral antigens, mammalian proteins, mammalian cell surface molecules, bacterial antigens, fungal antigens, protozoan antigens, helminth antigens, ectoparasite antigens and cancer antigens.

[0084] According to the present invention, the general term "antigen" herein refers to: any part of a protein (peptide, partial protein, full-length protein), wherein the protein is naturally occurring or synthetically derived; a cell composition (whole cell, cell lysate or broken cell); a microorganism or cell (whole microorganism, lysate or broken cell) or carbohydrate, or other molecule or part thereof. In some embodiments, an antigen can elicit an antigen-specific immune response (e.g., humoral and / or cell-mediated immune response) to the same or similar antigens encountered by elements of the immune system (e.g., T cells, antibodies). The term "cancer antigen" can be used interchangeably with the term "tumor-specific antigen", "tumor-associated antigen", "cancer-associated target" or "tumor-associated target" or "neoantigen" herein.

[0085] Antigens can be as small as a single epitope or larger, and can contain multiple epitopes. Therefore, the size of an antigen can be as small as about 5-16 amino acids (e.g., small peptides), and as large as: a domain of a protein, a partial protein (peptide or polypeptide), a full-length protein (including multimers and fusion proteins), a chimeric protein, or an agonist protein or peptide. In addition, an antigen can include a carbohydrate.

[0086] When referring to stimulating an immune response, the term "immunogen" is a subset of the term "antigen" and, therefore, in some cases, it can be used interchangeably with the term "antigen." As used herein, an immunogen describes an antigen that elicits a humoral and / or cell-mediated immune response (i.e., is immunogenic), such that administration of the immunogen to an individual under appropriate circumstances (e.g., as part of a yeast-based immunotherapy composition) will elicit or induce an antigen-specific immune response to the same or similar antigen encountered by the individual's immune system.

[0087] An "immunogenic domain" of a given antigen can be any portion, fragment or epitope of the antigen (e.g., a peptide fragment or subunit or an antibody epitope or other conformational epitope) that contains at least one epitope that acts as an immunogen when administered to an animal. For example, a single protein can contain multiple different immunogenic domains. An immunogenic domain need not be a linear sequence within a protein, as in the case of a humoral immune response.

[0088] An epitope is defined herein as a single immunogenic site within a given antigen that is sufficient to elicit an immune response. Those skilled in the art will recognize that T cell epitopes differ from B cell epitopes in size and composition, and that epitopes presented via the class I MHC pathway differ from epitopes presented via the class II MHC pathway. An epitope may be a linear sequence or a conformational epitope (conserved binding region).

[0089] Antigens contemplated for the present invention include any antigens that are expected to trigger an immune response, and in particular, any antigens that will be beneficial to an individual for a therapeutic immune response to the antigen. For example, antigens may include but are not limited to any antigens relevant to pathogens, including viral antigens, fungal antigens, bacterial antigens, helminth antigens, parasite antigens, ectoparasite antigens, protozoan antigens or antigens from any other infective agent. Antigens may also include any antigen relevant to a particular disease or condition, whether from a pathogenic source or a cell source, including but not limited to cancer antigens, antigens (such as diabetes antigens) relevant to autoimmune diseases, allergic reaction antigens (allergens), mammalian cell molecules carrying one or more mutant amino acids, proteins expressed before or during birth by mammalian cells, proteins induced to express by insertion of epidemiological factors (such as viruses), proteins induced to express by gene translocations, and proteins induced to express by the mutation of an adjusting sequence. These antigens may be natural antigens or genetically engineered antigens modified in some way (such as, changing sequence or producing fusion protein). It should be understood that in some embodiments (ie, when the antigen is expressed from a recombinant nucleic acid molecule by a yeast vehicle), the antigen can be a protein or any epitope or immunogenic domain thereof, a fusion protein or a chimeric protein, rather than a whole cell or microorganism.

[0090] Other antigens useful in the yeast-based immunotherapeutic compositions of the invention include antigens involved in suppressing unwanted or harmful immune responses, such as those caused by allergens, autoimmune antigens, inflammatory factors, antigens involved in GVHD, certain cancers, septic shock antigens, and antigens involved in transplant rejection.

[0091] In one aspect of the invention, antigens that can be used in one or more immunotherapeutic compositions of the invention include any antigen associated with cancer or a tumor. In one aspect, antigens include antigens associated with a pre-tumor state or a proliferative state. Antigens can also be associated with cancer or cause cancer. Such antigens can be tumor-specific antigens, tumor-associated antigens (TAA) or tissue-specific antigens, their epitopes and epitope agonists thereof. Cancer antigens include, but are not limited to, antigens from any tumor or cancer, including, but not limited to, melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, hemangioendothelioma, mastocytoma, leukemia, lymphoma, primary liver cancer, lung cancer, pancreatic cancer, gastrointestinal cancer (including colorectal cancer), renal cell carcinoma, hematopoietic tumors and metastatic cancers thereof.

[0092] Suitable cancer antigens include, but are not limited to, carcinoembryonic antigen (CEA) and epitopes thereof, such as CAP-1, CAP-1-6D (GenBank Accession No. M29540), MART-1 (Kawakami et al., J. Exp. Med. [Journal of Experimental Medicine] 180:347-352, 1994), MAGE-1 (U.S. Pat. No. 5,750,395), MAGE-3, GAGE ​​(U.S. Pat. No. 5,648,226), GP-100 (Kawakami et al., Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 91:6458-6462, 1992), MUC-1, MUC-2, point mutant Ras oncoprotein, normal and point mutant p53 oncoprotein (Hollstein et al., Nucleic Acids Res. [Nucleic Acids Research] 22:3551-3555, 1994), PSMA (Israeli et al. Cancer Res. [Cancer Research] 53:227-230, 1993), tyrosinase (Kwon et al. PNAS [Proceedings of the National Academy of Sciences of the United States of America] 84:7473-7477, 1987), TRP-1 (gp75) (Cohen et al. Nucleic Acids Res. [Nucleic Acids Research] 18:2807-2808, 1990; U.S. Patent No. 5,840,839), NY-ESO-1 (Chen et al. PAS 94:1914-1918, 1997), TRP-2 (Jackson et al., EMBO J, 11:527-535, 1992), TAG72, KSA, CA-125, PSA, HER-2 / neu / c-erb / B2 (U.S. Pat. No. 5,550,214), EGFR, hTERT, p73, B-RAF, adenomatous polyposis coli (APC), Myc, Hippel-Lindau (von Hippel-Lindau) protein (VHL), Rb-1, Rb-2, androgen receptor (AR), Smad4, MDR1, Flt-3, BRCA-1, BRCA-2, Bcr-Abl, pax3-fkhr, ews-fli-1, Brachyury, HERV-H, HERV-K, TWIST, mesothelin, NGEP, modifications of such antigens and tissue-specific antigens, splice variants of such antigens, and / or epitope agonists of such antigens. Other cancer antigens are known in the art. Other cancer antigens can also be identified, isolated and cloned by methods known in the art (e.g., those disclosed in U.S. Patent No. 4,514,506). Cancer antigens can also include one or more growth factors and splice variants of each.

[0093] Cancer antigens can also be non-self neoantigens generated by genetic mutations of normal cell proteins within the tumor. These new mutations can generate novel T cell neoepitopes and therefore represent a large number of potential new targets for yeast-based immunotherapy.

[0094] In one aspect of the invention, antigens useful in one or more immunotherapeutic compositions of the invention include any antigen associated with a pathogen or a disease or condition caused by or associated with a pathogen. Such antigens include, but are not limited to, any antigen associated with a pathogen, including viral antigens, fungal antigens, bacterial antigens, helminth antigens, parasite antigens, ectoparasite antigens, protozoan antigens, or antigens from any other infectious agent.

[0095] In one aspect, antigens are from viruses, including but not limited to adenovirus, arenavirus, bunyavirus, coronavirus, coxsackievirus, cytomegalovirus, Epstein-Barr virus, arbovirus, hepadnavirus, hepatitis virus, herpes virus, influenza virus, slow virus, measles virus, mumps virus, myxovirus, orthomyxovirus, papillomavirus, papovavirus, parainfluenza virus, paramyxovirus, parvovirus, microRNA virus, poxvirus, rabies virus, respiratory syncytial virus, reovirus, rhabdovirus, rubella virus, togavirus and varicella virus. Other viruses include T lymphocyte trophic virus, such as human T cell lymphotrophic virus (HTLV, such as HTLV-I and HTLV-II), bovine leukemia virus (BLVS) and feline leukemia virus (FLV). Slow virus includes but is not limited to human (HIV, including HIV-1 or HIV-2), simian (SIV), cat (FIV) and canine (CIV) immunodeficiency virus. In one embodiment, viral antigens include those from non-oncogenic viruses.

[0096] In another aspect, the antigen is from an infectious agent of the genus Aspergillus, Bordatella, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Escherichia, Francisella, Gonococcus, Histoplasma, Leishmania, Mycobacterium, In one aspect, the infectious agent is selected from Plasmodium falciparum or Plasmodium vivax.

[0097] In one aspect, the antigen is from a bacterium selected from the following section: Enterobacteriaceae, Micrococcaceae, Vibrionaceae, Pasteurellaceae, Mycoplasmataceae, and Rickettsiaceae. In one aspect, the bacterium is selected from the following genus: Pseudomonas, Bordetella, Mycobacterium, Vibrio, Bacillus, Salmonella, Francisella, Staphylococcus, Streptococcus, Escherichia, Enterococcus, Pasteurella, and Yersinia. In one aspect, the bacterium is from a species selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas mallei, Pseudomonas pseudomallei, Bordetella pertussis, Mycobacterium tuberculosis, Mycobacterium leprae, Francisella tularensis, Vibrio cholerae, Bacillus anthracis, Salmonella enteric, Yersinia pestis, Escherichia coli, and Bordetella bronchiseptica.

[0098] In one aspect, antigen is from fungi, and this fungi includes but is not limited to the fungi from Saccharomyces cerevisiae species, Aspergillus species, Cryptococcus species, Coccidioides species, Neurospora species, Histoplasma species or Blastomyces species. In one aspect, fungi is from selected from following species: Aspergillus fumigatus (Aspergillusfumigatus), Aspergillus flavus (A.flavus), Aspergillus niger (A.niger), Aspergillus terreus (A.terreus), Aspergillus nidulans (A.nidulans), Coccidioides immitis (Coccidioides immitis), Posadas Coccidioides posadasii (Coccidioides posadasii) or Cryptococcus neoformans (Cryptococcus neoformans). The most common species of Aspergillus causing invasive disease include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus and Aspergillus nidulans, and for example can be found in patients with immunosuppression or T cell or phagocytic dysfunction. Aspergillus fumigatus is relevant with asthma, pulmonary aspergilloma and invasive aspergillosis. Coccidioidomycosis, also known as San Joaquin Valley Fever, is a fungal disease caused by Coccidioides immitis and can cause acute respiratory infections and chronic lung conditions or spread to the meninges, bones, and joints. The methods of the present invention also target cryptococcosis-related conditions, such as in non-immunosuppressed or immunosuppressed subjects (such as subjects infected with HIV).

[0099] In some embodiments, the antigen is a fusion protein. In one aspect of the invention, the fusion protein can include two or more antigens. In one aspect, the fusion protein can include two or more immunogenic domains, or two or more epitopes of one or more antigens. Immunotherapeutic compositions containing such antigens can provide antigen-specific immunization for a wide range of patients. For example, a multi-domain fusion protein that can be used for the present invention can have multiple domains, wherein each domain is composed of a peptide from a specific protein, and the peptide is composed of at least 4 amino acid residues located on both sides of the protein and including a mutant amino acid found in the protein, wherein the mutation is associated with a specific disease or symptom.

[0100] In one embodiment, constructs particularly useful for the expression of heterologous antigens in yeast are used to produce fusion proteins used as components of yeast-based immunotherapeutic compositions used in the present invention. Typically, one or more desired antigenic proteins or peptides are fused at their amino termini to: (a) specific synthetic peptides that stabilize the expression of the fusion protein in a yeast vehicle or prevent post-translational modification of the expressed fusion protein (such peptides are described in detail, for example, in U.S. Patent Publication No. 2004-0156858A1, published on August 12, 2004, which is incorporated herein by reference in its entirety); (b) at least a portion of an endogenous yeast protein, wherein any fusion partner can significantly enhance the stability of protein expression in yeast and / or prevent yeast cells from post-translational modification of the protein (such proteins are also described in detail, for example, in U.S. Patent Publication No. 2004-0156858A1, supra); and / or (c) at least a portion of a yeast protein that allows the fusion protein to be expressed on the surface of yeast (e.g., Aga protein, described in more detail herein). In addition, the present invention includes the use of peptides fused to the C-terminal of the construct encoding the antigen, particularly for the selection and identification of proteins. Such peptides include but are not limited to any synthetic or natural peptides, such as peptide tags (e.g., 6X His) or any other short epitope tags. The peptide attached to the C-terminal of the antigen according to the present invention can be used with or without the addition of the above-mentioned N-terminal peptide.

[0101] In one embodiment, a synthetic peptide that can be used for a fusion protein is linked to the N-terminus of an antigen, the peptide consisting of at least two amino acid residues that are heterologous to the antigen, wherein the peptide stabilizes the expression of the fusion protein in a yeast vehicle or prevents post-translational modification of the expressed fusion protein. The synthetic peptide and the N-terminal portion of the antigen together form a fusion protein that has the following requirements: (1) the first amino acid residue of the fusion protein is methionine (i.e., the first amino acid in the synthetic peptide is methionine); (2) the second amino acid residue of the fusion protein is not glycine or proline (i.e., the second amino acid in the synthetic peptide is not glycine or proline); (3) the amino acid residues at positions 2-6 of the fusion protein are not methionine (i.e., whether it is part of a synthetic peptide or part of a protein (if the synthetic peptide is less than 6 amino acids), the amino acids at positions 2-6 do not include methionine); and (4) the amino acids at positions 2-6 of the fusion protein are not lysine or arginine (i.e., whether it is part of a synthetic peptide or part of a protein (if the synthetic peptide is less than 5 amino acids), the amino acids at positions 2-6 do not include lysine or arginine). Synthetic peptides can be as short as two amino acids, but in one aspect are at least 2-6 amino acids (including 3, 4, 5 amino acids), and can be greater than 6 amino acids (whole integers), up to about 200 amino acids, 300 amino acids, 400 amino acids, 500 amino acids or more.

[0102] In one embodiment, the fusion protein comprises an amino acid sequence of M-X2-X3-X4-X5-X6, wherein M is methionine; wherein X2 is any amino acid 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 proline. Exemplary synthetic sequences that enhance the stability of antigen expression in yeast cells and / or prevent post-translational modification of proteins in yeast include the sequence MADEAP (SEQ ID NO: 10). In addition to enhancing the stability of the expression product, the fusion partner does not appear to have a negative impact on the immune response to the vaccination antigen in the construct. In addition, synthetic fusion peptides can be designed to provide an epitope that can be recognized by a selection agent (such as an antibody).

[0103] The present invention includes delivering (administering, immunizing) the composition of the present invention to a subject. The administration process can be performed in vitro or in vivo, but is usually performed in vivo. Ex vivo administration refers to performing a partial delivery step outside the patient's body, such as administering the composition of the present invention to a cell colony (dendritic cell) taken out of the patient's body under the condition that a yeast vehicle, one or more antigens and any other medicament or composition are loaded into the cell, and the cells are returned to the patient. This can include YL or YL mixed with tarmogen. The therapeutic composition of the present invention can be returned to the patient or administered to the patient (by any suitable mode of administration).

[0104] The composition can be systemic, mucosal and / or near the position of the target site (for example, near a tumor). According to the type of disease to be prevented or treated, the antigen used, and / or the target cell colony or tissue, those skilled in the art will clearly see the suitable route of administration. Various acceptable methods of administration include but are not limited to intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (for example, entering the carotid artery), subcutaneous administration, retroorbital administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, suction (for example, aerosol), intracranial, intraspinal, intraocular, intraauricular, intranasal, oral, pulmonary administration, immersion of catheter and direct injection into tissue. In one aspect, route of administration includes: intravenous, intraperitoneal, subcutaneous, intracutaneous, intranodular, intramuscular, transdermal, suction, intranasal, oral, intraocular, intraarticular, intracranial and intraspinal. Parenteral delivery can include intradermal, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheter and venous catheter approach. Delivery in the ear may include ear drops, delivery in the nose may include nose drops or intranasal injection, and delivery in the eye may include eye drops. Aerosol (inhalation) delivery may also be carried out using methods standard in the art (see, e.g., Stribling et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 189: 11277-11281, 1992, which is incorporated herein by reference in its entirety). Other routes of administration for regulating mucosal immunity may be used to treat viral infections. Such routes include bronchial, intradermal, intramuscular, intranasal, other inhalation, rectal, subcutaneous, topical, transdermal, vaginal and urethral routes. In one aspect, the immunotherapeutic composition of the present invention is administered subcutaneously.

[0105] The immunogenic compositions of the present invention may be formulated into a pharmaceutically acceptable excipient suitable for administration to a subject by injection.

[0106] With respect to the yeast-based immunotherapeutic compositions of the present invention, generally, a suitable single dose refers to a dose that, when administered one or more times over an appropriate period of time, is effective to provide the composition of the present invention to a given cell type, tissue, or region of a patient's body in an amount effective to elicit an antigen-specific immune response. For example, in one embodiment, a single dose of the composition of the present invention is about 1 x 10 5 About 5x10 7 In one aspect, a single dose of a yeast vehicle of the invention is about 0.1 YU (1 x 10 6 cells) to about 200 Y.U. (2x 10 9 cells), including any intermediate doses, at 0.1 x 10 6cells (i.e. 1.1x10 6 , 1.2x 10 6 , 1.3x 10 6 ...) increments. In one embodiment, the dosage includes a dosage between 1 Y.U. and 80 Y.U., and in one aspect, a dosage between 10 Y.U. and 40 Y.U. In one embodiment, the dosage is administered at different sites of the individual but during the same administration period. For example, a 40 Y.U. dose can be administered by injection at four different sites of the individual at a dosage of 10 Y.U. during one administration period. In one embodiment, a dosage of 1 Y.U. to about 200 Y.U. includes both YL and Tarmogen.

[0107] For example, "boosters" or "boosts" of the therapeutic composition are administered when the immune response to an antigen is reduced or as needed to provide an immune response to one or more specific antigens or to induce a memory response. After the initial administration, the intervals for administration of booster doses can be about every 1, 2, 3, 4, 5, 6, 7 or 8 weeks, every month, every two months, every quarter, every year, every few years. In one embodiment, the administration regimen is about 1 x 10 per kg of the organism's body weight. 5 About 5x 10 7 The composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more over a period of time ranging from weeks to months to years.

[0108] The immunogenic compositions of the present invention can be administered to a subject weekly, every other week, and / or monthly. The composition can be administered once every 40 days. In still another aspect, the composition can be administered once every 60 days. In still another aspect, the composition can be administered once every 38 to 40 days.

[0109] In one aspect, the lysate is inhaled to treat lung disease. This can occur whether or not mixed with the intact Tarmogen, and can be achieved via pressurized metered dose inhalers (pMDIs), nebulizers, and dry powder inhalers (DPIs).

[0110] The present invention contemplates methods of producing yeast vehicles and expressing yeast vehicles, combining and / or associating yeast vehicles with antigens and / or other proteins and / or agents of interest to produce yeast-based immunotherapeutic compositions.

[0111] According to the present invention, the terms "yeast-lysate-antigen complex", "yeast vehicle-antigen complex" or "yeast-antigen complex" are generally used to describe any association of a yeast lysate or yeast vehicle with an antigen, and can be used interchangeably with "yeast-based immunotherapy composition" when such a composition is used to elicit an immune response as described above. Such associations include expression of the antigen by yeast (recombinant yeast), introduction of the antigen into the yeast, physical attachment of the antigen to the yeast, and mixing the yeast and antigen together (such as in a buffer or other solution or formulation). These types of complexes are described in detail below. It will be appreciated that the yeast lysate-antigen complex can be formed from the yeast vehicle-antigen complex by the methods described herein.

[0112] In one embodiment, yeast cells used to prepare yeast vehicles are transfected with heterologous nucleic acid molecules encoding proteins (e.g., antigens or agents) so that the proteins are expressed by the yeast cells. Such yeasts are also referred to herein as recombinant yeast or recombinant yeast vehicles. The yeast cells can then be loaded into dendritic cells as complete cells, or the yeast cells can be killed, or they can be derivatized (e.g., by forming yeast spheroplasts, cytoplasts, bacterial exuviae, or subcellular particles, any of which will then load the derivatives into dendritic cells). Yeast spheroplasts can also be directly transfected with recombinant nucleic acid molecules (e.g., spheroplasts are produced by whole yeast and then transfected) to produce recombinant spheroplasts expressing antigens or other proteins.

[0113] In one aspect, yeast cells or yeast spheroplasts used to prepare yeast vehicles are transfected with recombinant nucleic acid molecules encoding one or more antigens or other proteins, so that the antigens or other proteins are recombinantly expressed by the yeast cells or yeast spheroplasts. In this aspect, yeast cells or yeast spheroplasts that recombinantly express one or more antigens or other proteins are used to produce yeast vehicles comprising yeast cytoplasts, yeast exuviae, or yeast membrane particles or yeast cell wall particles or fractions thereof.

[0114] In general, yeast vehicles and one or more antigens or other agents may be associated by any of the techniques described herein. In one aspect, the yeast vehicle is loaded with one or more antigens and / or one or more agents intracellularly. In another aspect, one or more antigens and / or one or more agents are attached to the yeast vehicle in a covalent or non-covalent form. In yet another aspect, the yeast vehicle is associated with one or more antigens and / or one or more agents by mixing. In another aspect, and in one embodiment, one or more antigens and / or one or more agents are recombinantly expressed by the yeast vehicle or by yeast cells or yeast spheroplasts from which the yeast vehicle is derived.

[0115] The number of antigens and / or other proteins produced by the yeast vehicles of the invention is any number of antigens and / or other proteins that can reasonably be produced by a yeast vehicle, and generally ranges from at least one to at least about 6 or more (including about 2 to about 6) heterologous antigens and / or other proteins.

[0116] The expression of antigen or other protein in yeast vehicle of the present invention is completed using technology known to those skilled in the art. In short, the nucleic acid molecule encoding at least one desired antigen or other protein is inserted into the expression vector in a manner that the nucleic acid molecule is operably connected to the transcription control sequence, thereby being able to affect the constitutive or regulated expression of the nucleic acid molecule when converted into a host yeast cell. The nucleic acid molecule encoding one or more antigens and / or other proteins can be on one or more expression vectors operably connected to one or more expression control sequences. Particularly important expression control sequences are those sequences (e.g., promoters and upstream activation sequences) that control transcription initiation. 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 expressed in Saccharomyces cerevisiae include, but are not limited to, promoters of genes encoding the following yeast proteins: CUP1, alcohol dehydrogenase I (ADH1) or II (ADH2), phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), translation elongation factor EF-1α (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as TDH3 for triosephosphate dehydrogenase), galactokinase (GAL1), galactose-1-phosphate uracil transferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome c1 (CYC1), Sec7 protein (SEC7), and acid phosphatase (PHO5), including hybrid promoters such as the ADH2 / GAPDH and CYC1 / GAL10 promoters, and including the ADH2 / GAPDH promoter (induced when the glucose concentration in the cell is low (e.g., about 0.1% to about 0.2%)), as well as the CUP1 promoter and the TEF2 promoter. Likewise, many upstream activating sequences (UAS) (also referred to as enhancers) are known. Upstream activating sequences for expression in Saccharomyces cerevisiae include, but are not limited to, UASs of genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7, and GAL10, as well as other UASs activated by the GAL4 gene product, wherein in one aspect the ADH2 UAS is used. Since the ADH2 UAS is activated by the ADR1 gene product, when a heterologous gene is operably linked to the ADH2 UAS, it may be more preferred to overexpress the ADR1 gene. Transcription termination sequences for expression in Saccharomyces cerevisiae include termination sequences of α-factor, GAPDH, and CYC1 genes.

[0117] Transcription control sequences for expressing genes in methylotrophic yeast include transcription control regions of genes encoding alcohol oxidase and formate dehydrogenase.

[0118] According to the present invention, nucleic acid molecules can be transfected into yeast cells by any method, by which nucleic acid molecules are applied to cells, and include but are not limited to diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption and protoplast fusion. Transfected nucleic acid molecules can be integrated into yeast chromosomes 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, complete yeast microorganisms or yeast spheroplasts can also be transfected with desired nucleic acid molecules, antigens are produced therein, and then these microorganisms or spheroplasts are further processed using techniques known to those skilled in the art to produce cytoplasm, capsids or subcellular yeast membrane extracts or fractions thereof containing desired antigens or other proteins, and yeast cytoplasm, yeast capsids and yeast membrane particles or cell wall preparations are recombinantly produced.

[0119] Effective conditions for producing recombinant yeast vehicles and expressing antigens and / or other proteins (e.g., medicaments as described herein) by yeast vehicles include effective culture media in which yeast strains can be cultured. Effective culture media are typically aqueous culture media containing absorbable carbohydrates, nitrogen and phosphate sources, as well as suitable salts, minerals, metals and other nutrients (e.g., vitamins and growth factors). Culture media may contain complex nutrients or may be limited minimal culture media. Yeast strains of the present invention may be cultured in various containers (including but not limited to bioreactors, conical flasks, test tubes, microtiter dishes and culture dishes). Culture is carried out at a temperature, pH and oxygen content suitable for yeast strains. Such culture conditions are completely within the professional knowledge of those of ordinary skill in the art (see, e.g., Guthrie et al. (editor), 1991, Methods in Enzymology [enzymology method], Vol. 194, Academic Press [academic press], San Diego).

[0120] In some aspects of the invention, yeast is grown under neutral pH conditions, and in particular in a medium where the pH level is maintained at at least 5.5 (i.e., the pH of the medium is not allowed to drop below pH 5.5). In other aspects, yeast is grown at a pH level of about 5.5. In other aspects, yeast is grown at a pH level of about 5.6, 5.7, 5.8 or 5.9. In another aspect, yeast is grown at a pH level of about 6. In another aspect, yeast is grown at a pH level of about 6.5. In other aspects, yeast is grown at a pH level of about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In other aspects, yeast is grown at a pH level of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. pH levels are important for yeast culture. Those skilled in the art will recognize that the culturing process includes not only the initiation of the yeast culture, but also the maintenance of the culture. Since yeast cultures are known to become acidic (i.e., the pH decreases) over time, care must be taken to monitor the pH level during the culturing process. It is still contemplated within the scope of the present invention to carry out yeast cell culturing when the pH level of the culture medium drops below 6, provided that the pH of the culture medium is raised to at least 5.5 at some point during the culturing process. Therefore, the longer the yeast is grown in a culture medium of at least pH 5.5 or higher, the better the results in obtaining yeast with the desired characteristics.

[0121] In one aspect, the yeast is cultured so that the pH level of the culture medium is not less than pH 5.5. In some cases, the pH drops below 5.5 for no more than 5 minutes. In other cases, the pH drops below 5.5 for no more than 10 minutes. In other cases, the pH drops below 5.5 for no more than 1 hour. In another aspect, the yeast is cultured so that the pH level of the culture medium is not less than 5.0. In some cases, the pH drops below 5.0 for no more than 5 minutes. In other cases, the pH drops below 5.0 for no more than 10 minutes, preferably no more than 20, 30, 40, 50 or 60 minutes. In other cases, the pH drops below 5.0 for no more than 1 hour. Therefore, the longer the yeast is grown in a culture medium of at least pH 5.5 or higher, the better the results in obtaining yeast with desired characteristics as described below.

[0122] In one aspect, culturing yeast cells using a neutral pH method means that the yeast cells are grown at a neutral pH for at least 50% of the yeast culture time. More preferably, the yeast is grown at a neutral pH for at least 60% of its culture time, more preferably for at least 70% of its culture time, more preferably for at least 80% of its culture time, and most preferably for at least 90% of its culture time.

[0123] In another aspect, growing yeast at neutral pH comprises culturing the yeast cells at neutral pH for at least five minutes, preferably at least 15 minutes at neutral pH, more preferably at least one hour at neutral pH, more preferably at least two hours, even more preferably at least three hours or more.

[0124] As used herein, the general term "neutral pH" refers to a pH range between about pH 5.5 and about pH 8, and in one aspect, between about pH 6 and about 8. It will be appreciated by those skilled in the art that when measured with a pH meter, there will be slight fluctuations (e.g., one tenth or one hundredth). Therefore, using a neutral pH to cultivate yeast cells means that yeast cells grow at a neutral pH for most of the time they are cultivated. Using a neutral pH in culturing yeast promotes several biological effects, which are desirable features for using yeast as an immunomodulatory medium. In one aspect, culturing yeast at a neutral pH allows yeast to grow well, and does not have any negative impact on cell generation time (e.g., slowing down doubling time). Yeast can continue to grow to high density without losing the flexibility of its cell wall. In another aspect, using a neutral pH allows the production of yeast with a flexible cell wall and / or yeast sensitive to cell wall digestion enzymes (e.g., glucanase) at all collection densities. This trait is ideal because yeast with flexible cell walls can induce abnormal immune responses, such as by promoting the secretion of cytokines (e.g., interferon-γ (IFN-γ)) in cells of the host yeast. In addition, greater accessibility to antigens located in the cell wall can be provided by such culture methods. In another aspect, the use of neutral pH for some antigens allows the release of disulfide-bound antigens by dithiothreitol (DTT) treatment, which is impossible when such antigen-expressing yeast is cultured in a culture medium at a lower pH (e.g., pH 5). Finally, in another aspect, yeast cultured using a neutral pH method triggers an increase in the production of at least TH1 type cytokines (including but not limited to IFN-γ, interleukin 12 (IL-12) and IL-2), and may also trigger an increase in the production of other cytokines (e.g., proinflammatory cytokines (e.g., IL-6)).

[0125] In one embodiment, controlling the amount of yeast glycosylation is used to control the antigen expression of yeast (especially on the surface). The amount of yeast glycosylation can affect the immunogenicity and antigenicity of surface expressed antigens because the sugar moieties are often large. Therefore, in the regulation of yeast according to the present invention, the presence of sugar moieties on the yeast surface and their impact on the three-dimensional space around one or more target antigens should be considered. Any method can be used to reduce (or increase, if desired) the amount of glycosylation of yeast. For example, yeast mutant strains that have been selected to have low glycosylation (e.g., mnn1, och1, and mnn9 mutants) can be used, or glycosylation receptor sequences on the target antigen can be eliminated by mutation. Alternatively, yeast with shortened glycosylation patterns, such as Pichia pastoris, can be used. Yeast can also be treated using methods that reduce or change glycosylation.

[0126] In one embodiment of the invention, as an alternative to recombinantly expressing antigens or other proteins in yeast vehicles, yeast vehicles are loaded with proteins or peptides in cells, or loaded with carbohydrates or other molecules used as antigens and / or as immunomodulators or bioreaction modifiers according to the present invention. Subsequently, yeast vehicles currently containing antigens and / or other proteins in cells can be applied to patients or loaded into carriers (such as dendritic cells). Peptides and proteins can be directly inserted 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 water bath ultrasound). Yeast vehicles that can directly load peptides, proteins, carbohydrates or other molecules include complete yeast, as well as spheroplasts, bacterial sloughs or cytoplasmic bodies, which can be loaded with antigens and other medicaments after production. Alternatively, complete yeast can be loaded with antigens and / or medicaments, and spheroplasts, bacterial sloughs, cytoplasmic bodies or subcellular particles can then be prepared therefrom. In this embodiment, any number of antigens and / or other agents can be loaded into the yeast vehicle, for example by loading microorganisms, by loading mammalian tumor cells or parts thereof to provide from at least 1, 2, 3, 4 or any integer to hundreds or thousands of antigens and / or other agents.

[0127] In another embodiment of the present invention, antigen and / or other agents are physically attached to yeast vehicles. The physical attachment of antigen and / or other agents to yeast vehicles can be completed by any method suitable in the art, including covalent and non-covalent association methods, including but not limited to chemical cross-linking of antigen and / or other agents to the outer surface of yeast vehicles or biological connection of antigen and / or other agents to the outer surface of yeast vehicles (such as by using antibodies or other binding partners). For example, chemical cross-linking can be achieved by the following methods, including glutaraldehyde bonding, photoaffinity labeling, carbodiimide treatment, treatment with chemicals capable of connecting disulfide bonds, and standard treatment with other cross-linked chemicals in the art. Alternatively, chemicals can be contacted with yeast vehicles that change the charge of the yeast membrane lipid bilayer or the composition of the cell wall, so that the outer surface of the yeast is more easily fused or combined with antigens and / or other agents with specific charge characteristics. Targeting agents (such as antibodies, binding peptides, soluble receptors and other ligands) can also be incorporated into antigens as fusion proteins or otherwise associated with antigens to combine the antigen with yeast vehicles.

[0128] When antigens or other proteins are expressed on the yeast surface or physically attached to the yeast surface, the spacer arms can be carefully selected in one aspect to optimize the expression or content of the antigens or other proteins on the surface. The size of the one or more spacer arms will affect the amount of antigens or other proteins exposed to bind to the yeast surface. Therefore, depending on the one or more antigens or one or more other proteins used, those skilled in the art will select spacer arms that make the antigens or other proteins on the yeast surface have appropriate spacing. In one embodiment, the spacer arm is a yeast protein of at least 450 amino acids. The spacer arm has been discussed in detail above.

[0129] Another consideration for optimizing antigen surface expression is whether the antigen and spacer arm combination should be expressed as a monomer or dimer or trimer, or even more units linked together. The use of monomers, dimers, trimers, etc. allows for proper spacing or folding of the antigen so that some portion (if not all) of the antigen is displayed on the surface of the yeast vehicle in a manner that makes it more immunogenic.

[0130] In yet another embodiment, the yeast vehicle and antigen or other protein are associated with each other through a more passive, non-specific or non-covalent binding mechanism (such as by gently mixing the yeast vehicle and antigen or other protein in a buffer or other suitable formulation (e.g., a mixing agent)).

[0131] In one embodiment of the invention, both the yeast vehicle and the antigen or other protein are loaded intracellularly into a carrier (such as a dendritic cell or macrophage) to form a therapeutic composition or vaccine of the invention. Alternatively, the antigen or other protein can be loaded into dendritic cells in the absence of a yeast vehicle.

[0132] In one embodiment of the invention, the yeast vehicle used in the present invention includes a yeast vehicle that has been killed or inactivated. Killing or inactivating yeast can be accomplished by any of various suitable methods known in the art. For example, heat inactivation of yeast is a standard method for yeast inactivation, and those skilled in the art can (if desired, by standard methods known in the art) monitor the structural changes of the target antigen. As described herein, yeast lysates are made in another way of inactivating yeast. Alternatively, other methods of inactivating yeast can also be used, such as chemical methods, electrical methods, radiation methods or UV methods. See, for example, methods disclosed in standard yeast culture textbooks, such as Methods of Enzymology [enzymology methods], Volume 194, Cold Spring Harbor Publishing [Cold Spring Harbor Publishing] (1990). Any inactivation strategy used should take into account the secondary, tertiary or quaternary structure of the target antigen, and maintain this structure to optimize its immunogenicity.

[0133] Yeast lysate and yeast vehicle can be formulated into yeast-based immunotherapy compositions or products of the present invention using a variety of techniques known to those skilled in the art, including preparations for administration to subjects that are directly or first loaded into carriers (such as dendritic cells). For example, yeast vehicles can be dried by lyophilization. Preparations containing yeast vehicles can also be prepared by packaging yeast in cakes or tablets, such as yeast for baking or brewing operations. In one aspect, yeast lysate and yeast vehicles can be frozen. In addition, yeast vehicles can be mixed with pharmaceutically acceptable excipients (such as isotonic buffers tolerated by hosts or host cells). Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and aqueous solutions of other physiologically balanced salts. Non-aqueous vehicles can also be used, such as fixed oils, sesame oil, ethyl oleate, or triglycerides. Other useful preparations include suspensions containing viscosity enhancers (such as sodium carboxymethylcellulose, sorbitol, glycerol, or dextran). Excipients can also contain a small amount of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m-cresol or o-cresol, formalin and benzyl alcohol. Standard formulations can be liquid injections or solids, which can be absorbed in a suitable liquid as a suspension or solution for injection. Thus, in non-liquid formulations, excipients may include, for example, dextrose, human serum albumin and / or preservatives, to which sterile water or saline may be added before administration.

[0134] In one embodiment of the invention, the composition may include a biological response modifier compound, or the ability to produce such a modifier (ie, by transfecting a yeast vehicle with a nucleic acid molecule encoding such a modifier). Biological response modifiers have been described above.

[0135] The compositions of the present invention may further include any other compound useful for protecting a subject from a particular disease or condition, including an infectious disease or cancer, or any compound that treats or ameliorates any symptom of such an infection.

[0136] The present invention also includes a test kit comprising any of the compositions described herein or any individual component of the compositions described herein. The reagent can exist in a free form, or can be fixed on a substrate (such as a plastic dish, a microarray plate, a test tube, a test stick, etc.). The test kit can also include suitable reagents for detecting reagents and / or for marking positive or negative controls, washing solutions, dilution buffers, etc. The test kit can also include a set of written instructions for using the test kit and explaining the results. In one embodiment, the test kit is configured as a high-throughput assay kit. The test kit can be prepared and used for any clinical, research or diagnostic method of the present invention.

[0137] With regard to the medicaments useful in the present invention, in one aspect, the protein or antibody is administered in an amount between about 50 U / kg and about 15,000 U / kg of the subject's body weight. In another embodiment, the protein or antibody is administered in an amount of about 0.01 μg to about 10 mg / kg of the patient's body weight, and more preferably in an amount of about 0.1 μg to about 100 μg of the patient's body weight. When the compound to be delivered is a nucleic acid molecule, an appropriate single dose can result in at least about 1 pg of protein expressed per mg of total tissue protein per μg of nucleic acid delivered. Small molecules are delivered according to the preferred dose specified for a given small molecule, and can be determined by those skilled in the art.

[0138] In one aspect of the invention, the medicament is administered simultaneously with the yeast-based immunotherapy composition. In one aspect of the invention, the medicament is administered sequentially with the yeast-based immunotherapy composition. In another embodiment, the medicament is administered before the yeast-based immunotherapy composition is administered. In another embodiment, the medicament is administered after the yeast-based immunotherapy composition is administered. In one embodiment, the medicament is administered with alternating doses with the yeast-based immunotherapy composition, or in one embodiment, the yeast-based composition is administered between one or more consecutive doses of the medicament or with one or more doses of the medicament at a specified interval, or vice versa. In one embodiment, the yeast-based immunotherapy composition is administered with one or more doses within a period of time before the start of the administration of the medicament. In other words, the yeast-based immunotherapy composition is administered as a monotherapy for a period of time, and then the medicament is added (administered simultaneously with a new dose of yeast-based immunotherapy, or administered in an alternating manner with yeast-based immunotherapy). Alternatively, a medicament may be administered for a period of time before the start of the administration of the yeast-based immunotherapy composition. In one aspect, the yeast is engineered to express or carry the medicament, or different yeasts are engineered or produced to express or carry the medicament.

[0139] Virus-based immunotherapeutic compositions generally include a viral vector comprising a viral genome or portion thereof (e.g., a recombinant virus) and a nucleic acid sequence encoding at least one antigen from a pathogenic agent or disease state (e.g., one or more cancer antigens, one or more infectious disease antigens, and / or at least one immunogenic domain thereof). In some embodiments, the virus-based immunotherapeutic composition further includes at least one viral vector comprising one or more nucleic acid sequences encoding one or more immunostimulatory molecules. In some embodiments, genes encoding immunostimulatory molecules and antigens are inserted into the same viral vector (same recombinant virus).

[0140] As used herein, with respect to the administration of a composition, the term "simultaneously" means that each composition, and in particular the first dose of such a composition, is administered substantially at the same time or within the same dosing cycle, or within a time period (e.g., no more than 1-2 days) during which the immune system is activated by an immunotherapeutic composition. For clarity, simultaneous administration does not require that all compositions be administered at the exact same time, but rather that the administration of all compositions should be performed within a predetermined dose administration of the patient in order to activate the immune system and simultaneously achieve the effects of the medicament (e.g., one composition may be administered first, followed by an immediate or immediate administration of a second composition, and so on). In some cases, such as when the compositions are administered to the same site, the compositions may be provided in the form of a mixture, although each composition may be administered sequentially within the same dosing time period even if administered to the same site. In one aspect, the compositions are administered within the same 1-2 days, and in another aspect on the same day, and in another aspect within the same 12 hour period, and in another aspect within the same 8 hour period, and in another aspect within the same 4 hour period, and in another aspect within the same 1, 2, or 3 hour period, and in another aspect within the same 1, 2, 3, 4, 6, 7, 8, 9, or 10 minutes.

[0141] In one embodiment of the invention, a yeast-based immunotherapeutic composition and one or more agents are administered simultaneously, but to different physical sites of the patient. For example, one composition or agent may be administered to one or more sites of the individual's body, while another composition or agent may be administered to one or more different sites of the individual's body, for example, on different sides of the body or near different draining lymph nodes. In another embodiment, the immunotherapeutic composition and agent are administered to the patient simultaneously and at the same or substantially adjacent sites. A substantially adjacent site refers to a site that is not exactly the same as the injection site to which the first composition or agent is administered, but is very close (adjacent) to the first injection site. In one embodiment, the immunotherapeutic composition and agent are administered as a mixture. Some embodiments may include a combination of administration methods.

[0142] In the methods of the present invention, the compositions and therapeutic compositions can be applied to animals (including any vertebrates), and in particular to any member of the vertebrate class, mammals, including but not limited to primates, rodents, livestock, and domestic pets. Livestock include mammals to be consumed or to produce useful products (e.g., sheep for wool production). Mammals to be protected include humans, dogs, cats, mice, rats, goats, sheep, cattle, horses, and pigs.

[0143] An "individual" is a vertebrate, such as a mammal, including but not limited to humans. Mammals include but are not limited to livestock, sport animals, pets, primates, mice, and rats. The term "individual" can be used interchangeably with the terms "animal," "subject," or "patient."

[0144] 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. 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 (1998); and Methods in yeast genetics: a laboratory course manual. Rose et al., Cold Spring Harbor Laboratory Press (1990). Press [Cold Spring Harbor Laboratory Press] (1993); The Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics [Yeast: Genome Dynamics, Protein Synthesis, and Energetics], Broach et al., eds., Spring Harbor Laboratory Press [Cold Spring Harbor Laboratory Press] (1992); Molecular Cloning: A Laboratory Manual, second edition [Molecular Cloning: Laboratory Manual, second edition] (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition [Molecular Cloning: Laboratory Manual, third edition] (Sambrook and Russell, 2001), (hereinafter collectively referred to as "Sambrook");Current Protocols in Molecular Biology (FM 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, Spring Harbor Laboratory Press, Cold Spring Harbor, New York (hereinafter collectively referred to 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 [Cazaret and Duer's Toxicology: Basic Science of Toxicology], edited by C. Klaassen, 6th edition (2001); and Vaccines [Vaccines], edited by S. Plotkin and W. Orenstein, 3rd edition (1999).

[0145] General Definition

[0146] (Globe Immune, Inc., Louisville, Colorado) generally refers to a yeast vehicle that expresses one or more heterologous antigens extracellularly (on its surface), intracellularly (inside or in the cytoplasm), or both extracellularly and intracellularly. Certain yeast-based immunotherapeutic compositions and methods of making and generally using them are also described in detail in, for example, U.S. Pat. No. 5,830,463, U.S. Pat. No. 7,083,787, U.S. Pat. No. 7,736,642, Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research 64:5084-5088 (2004), and Bernstein et al., Vaccine 2008 Jan. 24; 26(4):509-21, each of which is incorporated herein by reference in its entirety.

[0147] As used herein, the term "analog" refers to a compound that is structurally similar to another compound but slightly different in composition (such as the replacement of one atom by an atom of a different element, or the presence of a specific functional group, or the replacement of one functional group by another). Therefore, an analog is a compound that is similar or equivalent in function and appearance to a reference compound, but has a different structure or origin.

[0148] The terms "substituted," "substituted derivative," and "derivative," when used to describe a compound, mean that at least one of the hydrogens bonded to the unsubstituted compound is replaced with a different atom or chemical moiety.

[0149] A "cell-mediated" immune response (used interchangeably with the term "cellular" immune response anywhere herein) generally refers to a response to an antigen by immune cells including T lymphocytes (including cytotoxic T lymphocytes (CTLs)), dendritic cells, macrophages, and natural killer cells, as well as all processes associated with such a response, including but not limited to the activation and proliferation of these cells, CTL effector function, the production of cytokines that affect the function of other cells involved in adaptive and innate immune responses, the generation of memory T cells, and stem cell-like memory cells.

[0150] "Vaccination" or "immunization" refers to the induction of an immune response to an antigen or an immunogenic portion thereof as a result of administration alone or with an adjuvant. Vaccination can result in a protective or therapeutic effect, wherein subsequent exposure to the antigen (or source of the antigen) induces an immune response to the antigen (or source) to reduce or prevent a disease or condition in the animal. The concept of vaccination is well known in the art. The immune response elicited by administration of an immunotherapeutic composition (vaccine) can be any detectable change in any aspect of the immune response (e.g., a cell-mediated response, a humoral response, cytokine production) compared to when the composition was not administered.

[0151] According to the present invention, "heterologous amino acids" refers to a sequence of amino acids that are not naturally found flanking a particular amino acid sequence (i.e., not found naturally in vivo), or are not relevant to the function of a particular amino acid sequence, or that, when present in a gene, are not encoded by nucleotides flanking a naturally occurring nucleic acid sequence encoding a particular amino acid sequence, if the standard codon usage of the organism from which the given amino acid sequence was derived is used to translate such nucleotides in the naturally occurring sequence. Thus, the at least two amino acid residues that are heterologous to an antigen are any two amino acid residues that are not naturally found flanking the antigen.

[0152] According to the present invention, reference to a "heterologous" protein or "heterologous" antigen (including heterologous fusion proteins) associated with the yeast vehicles of the present invention means that the protein or antigen is not a protein or antigen naturally expressed by yeast, although the fusion protein may include yeast sequences, or a protein or portion thereof naturally expressed by yeast (e.g., the Aga protein described herein). For example, for the purposes of the present invention, a fusion protein of an influenza hemagglutinin protein and a yeast Aga protein is considered a heterologous protein relative to the yeast vehicle because such a fusion protein is not naturally expressed by yeast.

[0153] According to the present invention, the phrase "selectively binds to" refers to the ability of the antibody, antigen binding fragment or binding partner of the present invention to preferentially bind to a specific protein. More particularly, the phrase "selectively binds to" refers to the specific binding of one protein to another protein (e.g., an antibody, a fragment thereof or a binding partner to an antigen), wherein the binding level measured as any standard assay (e.g., an immunoassay) is statistically significantly higher than the background control of the assay. For example, when performing an immunoassay, the control typically includes a reaction well / tube containing only an antibody or antigen binding fragment (i.e., in the absence of an antigen), wherein the amount of reactivity of the antibody or its antigen binding fragment in the absence of an antigen (e.g., non-specific binding to a well) is considered as background. Binding can be measured using a variety of methods standard in the art, including enzyme immunoassays (e.g., ELISA), immunoblotting assays, etc.).

[0154] The protein or polypeptide mentioned in the present invention include any fragment, domain, conformational epitope or homologue of full-length protein, fusion protein or such protein. More particularly, according to the present invention, the protein of separation is the protein (including polypeptide or peptide) taken out from its natural environment (that is, artificial manipulation has been carried out), and can include purified protein, partially purified protein, recombinantly produced protein and for example synthetically produced protein. Therefore, "separated" does not reflect the degree of purification of protein. In one aspect of the present invention, the protein of separation of the present invention is recombinantly produced. According to the present invention, the terms "modification" and "mutation" can be used interchangeably, particularly about the modification / mutation of the amino acid sequence of protein or its part.

[0155] As used herein, the term "homolog" is used to refer to a protein or peptide that differs from a naturally occurring protein or peptide by minor modifications to the naturally occurring protein or peptide (i.e., a "prototype" or "wild-type" protein) but retains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to: changes in one or several amino acid side chains; changes in one or several amino acids, including deletions (e.g., truncated forms of proteins or peptides), insertions and / or substitutions; changes in the stereochemistry of one or several atoms; and / or minor derivatization, including, but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and / or addition of glycosylphosphatidylinositol. Homologs may have enhanced, reduced, or substantially similar properties compared to naturally occurring proteins or peptides. Homologs may include agonists of proteins or antagonists of proteins. Homologs can be produced using techniques known in the art for producing proteins, including but not limited to direct modification of an isolated, naturally occurring protein, direct protein synthesis, or modification of a nucleic acid sequence encoding the protein using, for example, classical or recombinant DNA techniques to produce random or targeted mutations.

[0156] A homolog 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 90%, or at least about 95% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least about 99% identical (or any percentage identity between 45% and 99% in whole number increments) to the amino acid sequence of the reference protein. In one embodiment, a homolog comprises, consists essentially of, or consists of an amino acid sequence having less than 100% identity, less than about 99% identity, less than about 98% identity, less than about 97% identity, less than about 96% identity, less than about 95% identity, and so on in increments of 1%, to less than about 70% identity to the naturally occurring amino acid sequence of the reference protein.

[0157] As used herein, unless otherwise specified, references to percent (%) identity refer to homology estimates performed using: (1) BLAST 2.0 Basic BLAST homology searches with standard default parameters using blastp for amino acid searches and blastn for nucleic acid searches, with the query sequence being filtered for low complexity regions by default (described in Altschul, SF, Madden, TL, AA, Zhang, J., Zhang, Z., Miller, W., and Lipman, DJ (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402, which is incorporated herein by reference in its entirety); (2) BLAST 2 alignments (using the parameters described below); (3) and / or PSI-BLAST with standard default parameters (position-specific iterative BLAST). It should be noted that due to some differences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences can be identified as having significant homology using the BLAST 2 program, while a search performed in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify a top match to the second sequence. In addition, PSI-BLAST provides an automated, easy-to-use "spectrum" search version, which is a sensitive method for finding sequence homologues. The program first performs a gap BLAST database search. The PSI-BLAST program uses any important comparison information returned to build a position-specific scoring matrix that replaces the query sequence in the next round of database searches. Therefore, it should be understood that percentage identity can be determined by using any one of these programs.

[0158] Two specific sequences can be compared to each other using BLAST 2 sequences (as described in Tatusova and Madden, (1999), "Blast 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250, which is incorporated herein by reference in its entirety). BLAST 2 sequence comparisons are performed in blastp or blastn, wherein a gap BLAST search (BLAST 2.0) is performed between the two sequences using the BLAST 2.0 algorithm, thereby allowing for the introduction of gaps (deletions and insertions) in the resulting comparison. For purposes of clarity, BLAST 2 sequence comparisons are performed using the following standard default parameters.

[0159] For blastn, use the 0BLOSUM62 matrix:

[0160] Matching Reward = 1

[0161] Mismatch Penalty = -2

[0162] Opening (5) and Extending (2) Penalties

[0163] gap x_dropoff(50)expect(10)word size(11)filter(on)

[0164] For blastp, use the 0BLOSUM62 matrix:

[0165] Opening gap (11) and extending gap (1) penalties

[0166] gap x_dropoff (50) expect (10) word size (3) filter (on).

[0167] An isolated nucleic acid molecule refers to a nucleic acid molecule that has been removed from its natural environment (i.e., artificially manipulated), which is the genome or chromosome in which the nucleic acid molecule is found in nature. Thus, "isolated" does not necessarily reflect the degree to which a nucleic acid molecule has been purified, but indicates that the molecule does not include the entire genome or entire chromosome in which the nucleic acid molecule is found in nature. An isolated nucleic acid molecule may include a gene. An isolated nucleic acid molecule that includes a gene is not a chromosomal fragment that includes the gene, but rather a chromosomal fragment that includes the coding region and regulatory region associated with the gene but does not include other genes found naturally on the same chromosome. An isolated nucleic acid molecule may also include a specific nucleic acid sequence that is flanked (i.e., at the 5' and / or 3' end of the sequence) by other nucleic acids that are not normally flanking the specific nucleic acid sequence in nature (i.e., heterologous sequences). An isolated nucleic acid molecule may include DNA, RNA (e.g., mRNA), or a derivative of 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 a nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to nucleic acid molecules or nucleic acid sequences that can encode proteins or protein domains.

[0168] A recombinant nucleic acid molecule is a molecule that may include at least one of the nucleic acid sequences encoding any one or more of the proteins described herein, operably linked to at least one of any transcription control sequences that are effective to regulate the expression of one or more nucleic acid molecules in the cells to be transfected. Although the phrase "nucleic acid molecule" refers primarily to the physical nucleic acid molecule, and the phrase "nucleic acid sequence" refers primarily to the nucleotide sequence on the nucleic acid molecule, the two phrases may be used interchangeably, particularly with respect to nucleic acid molecules or nucleic acid sequences that are capable of encoding a protein. In addition, the phrase "recombinant molecule" refers primarily to a nucleic acid molecule that is operably linked to a transcription control sequence, but may be used interchangeably with the phrase "nucleic acid molecule" administered to an animal.

[0169] Recombinant nucleic acid molecules include recombinant vectors, which are any nucleic acid sequences operably connected to the isolated nucleic acid molecules of the present invention encoding fusion proteins, typically heterologous sequences, which can be recombinant to produce fusion proteins, and can be delivered to host cells according to the present invention. This vector can contain non-naturally found nucleic acid sequences near the isolated nucleic acid molecules to be inserted into the vector. The vector can be RNA or DNA, or it can be prokaryotic or eukaryotic, and in one aspect of the present invention, it is a virus or plasmid. Recombinant vectors can be used for cloning, sequencing and / or other operations of nucleic acid molecules, and can be used for delivering such molecules (for example, in DNA vaccines or vaccines based on viral vectors). Recombinant vectors can be used for the expression of nucleic acid molecules, and can also be referred to as expression vectors. Some recombinant vectors can be expressed in transfected host cells.

[0170] In the recombinant molecules of the present invention, the nucleic acid molecule is operably linked to an expression vector containing regulatory sequences (e.g., transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences of the present invention that are compatible with the host cell and control the expression of the nucleic acid molecule). In particular, the recombinant molecules of the present invention include nucleic acid molecules that are operably linked to one or more expression control sequences. The phrase "operably linked" refers to linking the nucleic acid molecule to the expression control sequence in a manner that allows the nucleic acid molecule to be expressed when transfected (i.e., transformed, transduced, or transfected) into a host cell.

[0171] According to the present invention, the term "transfection" is used to refer to any method capable of inserting an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) into a cell. When the term "conversion" is used to refer to the introduction of nucleic acid molecules into microbial cells (such as algae, bacteria and yeast), the term can be used interchangeably with the term "transfection". In microbial systems, the term "conversion" is used to describe the genetic changes caused by the acquisition of exogenous nucleic acids by microorganisms, and is essentially synonymous with the term "transfection". Therefore, transfection techniques include, but are not limited to, conversion, chemical treatment of cells, particle bombardment, electroporation, microinjection, liposome infection, adsorption, infection and protoplast fusion.

[0172] The following experimental results are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0173] Examples

[0174] Example 1

[0175] The following example shows that Clarified lysate and intact Mixing ratio separate complete The results showed that several different The heterologous antigen for which expression in yeast is directed contains the SIINFEKL (SEQ ID NO: 9) epitope of chicken ovalbumin.

[0176] method:

[0177] 1. Each Lysate preparation is as follows: in the logarithmic phase of growth, antigen expression is induced by adding copper (e.g., 375 μM for 3 h) from a copper-inducible promoter. The cells are washed and heat-killed at 56 ° C for 1 h. The heat-killed Tarmogen is mixed with PBS and 500 μL of acid-washed 0.2 μm glass beads in a total volume of 1.5 mL, and the mixture is vigorously shaken in a mechanical stirrer ("Fastprep" or "Bead beater") until >97% of the cells are broken. The mixture is centrifuged at 16,000 rpm and 25 ° C for 5 minutes, and the lysate (supernatant) without cell wall, membrane and glass beads is collected.

[0178] 2. Mix the lysate with the intact (2 YU each) plus 25 μg of anti-CD40 antibody was mixed, and the mixture was injected into C57BL / 6 mice via the retro-orbital route.

[0179] 3.1 week later, the vaccinated mice were retro-orbitally injected with primary syngeneic splenocytes that had been "pulsed" with 100 μg / mL of SIINFEKL (SEQ ID NO: 9) peptide solution, washed to remove unbound peptide, and labeled with CFSE green dye for detection. Simultaneously injected was an unpulsed population that was labeled with a low concentration of CFSE to distinguish it from the peptide-pulsed population (internal control). Figure 1 A schematic diagram of the assay is shown. Injected splenocytes home to the spleen. If vaccination generates SIINFEKL (SEQ ID NO: 9)-specific CTLs, the peptide-pulsed cells will be killed. The extent of killing correlates with the efficacy of the vaccine.

[0180] 4. Remove spleens from vaccinated mice injected with target cells. Disperse spleens into single cell suspensions, remove red blood cells, and measure CTL-mediated killing by flow cytometry.

[0181] Example 2

[0182] This example shows that ovalbumin Mixing of (ovax) clarified lysate with intact Ovax resulted in almost complete (95%) killing of targets pulsed with SIINFEKL (SEQ ID NO:9), whereas 0%-20% killing was observed in mice vaccinated with intact Ovax. Figure 1 The difference is that the vaccine is injected into the tail vein instead of the retro-orbital vein (Table 1).

[0183] Table 1: In vivo CTL results in mice vaccinated with Tarmogen lysate plus whole Tarmogen

[0184] Vaccinations: #Mouse Average kill % Complete Ovax(2YU) 3 0％-20％ Ovax(2YU)+Ovax clarified lysate(2YU) 3 95％ none 3 0％

[0185] Example 3:

[0186] Figure 3 Shown is the IA from the immunization b In vitro intracellular cytokine staining in spleen cells of mice treated with -cyto Tarmogen: intact Tarmogen vs. cleaved Tarmogen. b -cyto-complete Tarmogen or IA b YL immunized mice intradermally with 2.5 YU / side (5 YU / mouse in total). On day 38, mice received a homologous booster vaccination, still 2.5 YU / side (5 YU / mouse in total). Nine days after the booster vaccination (day 47), spleens were collected and total splenocytes were stimulated with peptide pools (5 μM / peptide) or control peptides at 37°C for 2 hours. Brefeldin A was added and the cultures were returned to 37°C for another 4 hours and then stored at 4°C overnight. Cells were stained with fluorescent dye-labeled antibodies as described below (" Figure 4 Flow cytometry was performed using a Beckman Coulter CyAn instrument, collecting 1.5-2 x 10 5 CD3 + cells. CD3 + CD4 + Flow cytometry plots of intracellular IFNγ and TNFα expression.

[0187] Example 4

[0188] This example shows that YE-NEO-001-93 vaccination elicits antigen-specific CD4 + and CD8 + Cytokine production.

[0189] Immunization: Female C57BL / 6 mice were immunized retro-orbitally with 2YU of YE-NEO-001-93YL + 25μg α-CD40 antibody + 2YU-Yvec or with 2.5YU of complete YE-NEO-001-93 + 25μg α-CD40 antibody (n = 2 mice / group). All mice were injected with 150μL. Due to the small size of the spleen, three gender- and age-matched, initially unimmunized mice were used as negative controls.

[0190] Spleen cell collection and in vitro stimulation: On day 7 after immunization, spleens were collected from naive or vaccinated mice and all mice in each condition were pooled (naive mice: n=3, experimental vaccination: n=2). Single cell suspensions were prepared and red blood cells were lysed using ammonium chloride-potassium (ACK) lysis buffer. A total of 2 x 10 cells were stimulated with 5 μM peptide (SIINFEKL) (SEQ ID NO: 9) (total volume 200 uL) in 96-well U-bottom tissue culture treated plates. 6 cells / well. The amount of DMSO in the DMSO negative stimulation control was the same as that in the wells treated with SIINFEKL (SEQ ID NO: 9). The cells were incubated at 37°C, 5% CO2 for a total of 6 hours. BFA was added at time zero. After 6 hours, the cells were stored at 4°C for 12-18 hours.

[0191] Intracellular cytokine staining and flow cytometry analysis: The next day, ICS was performed using the eBioscience FoxP3 / transcription factor staining buffer set as follows. The cells were washed and then stained with CD8 ef450, CD4 APC-CY7, CD3APC and FC block (24G2) 1:200 in FACS buffer (PBS + 2% FBS + 2mM EDTA) on ice for 30 min. The cells were then washed and fixed / permeabilized. Intracellular staining was performed at RT for 30 min using IFNγPE and TNFαPE-CY7 at a ratio of 1:200 in FACS buffer in the presence of FC block and rat serum (2uL / 25uL). After washing, three wells for each condition were combined into a single sample in FACS buffer and stained using a BD FACSVerse TM Two replicates were analyzed under each condition, and 2-3x10 5 CD3 +Cells were analyzed. Data were analyzed using FlowJo v10. Figure 4 As shown, the lysate produced a 20-fold stronger antigen-specific signal than intact yeast.

[0192] Example 5

[0193] This example shows that Tarmogen clarified lysate enhances anti-tumor activity compared to intact Tarmogen alone.

[0194] Mice were vaccinated with whole Tarmogen alone or Tarmogen plus YL (once a week for a total of 3 times, subcutaneously (sc), on both sides). One week after the third immunization, mice were challenged with 30,000 B16F10 cells sc on the right flank, behind the immunization site. These B16F10 cells contain putative neo-epitopes that match those carried by the Tarmogen vaccine. Tumor growth was monitored with a digital caliper every other day until day 12. cI IN: whole yeast expressing class I neo-epitope fusions; cII IN: whole yeast expressing class II neo-epitope fusions; cI IN+cI YL: 1.25 YU of cI IN mixed with 1.25 YU of cI YL; cII IN+cIICL: 1.25 YU of cII IN mixed with 1.25 YU of cII YL. Days: days after tumor challenge.

[0195] The results showed that CL enhanced the antitumor activity up to approximately 2 to 5 times when added to the complete Tarmogen in an equal amount (1:1) ratio compared to the complete Tarmogen alone.

[0196] Although various embodiments of the present invention have been described in detail, it is apparent that those skilled in the art may make modifications and adaptations to those embodiments. However, it should be expressly understood that such modifications and adaptations are within the scope of the present invention as set forth in the following claims.

Claims

1. An immunogenic preparation, comprising: i. a yeast lysate prepared from Saccharomyces cerevisiae cells, wherein the lysate lacks yeast membranes and yeast cell walls, and the Saccharomyces cerevisiae cells contain Mycobacterium tuberculosis antigens; and ii. fully recombinant inactivated Saccharomyces cerevisiae and at least one Mycobacterium tuberculosis antigen, The yeast lysate and the fully recombinant inactivated Saccharomyces cerevisiae are combined in a ratio of 90:10 to 10:

90.

2. The formulation of claim 1, further comprising a second antigen selected from the group consisting of a viral antigen, a mammalian cell surface molecule, a bacterial antigen, and a fungal antigen.

3. The preparation of claim 1, wherein the preparation comprises a plurality of Mycobacterium tuberculosis antigens.

4. A method for producing an immunogenic composition, the method comprising: a. Lysis of Saccharomyces cerevisiae cells; b. removing cell walls and cell membranes from lysed cells, wherein the lysed cells contain Mycobacterium tuberculosis antigens; and c. combining the product of step b with a fully recombinant inactivated Saccharomyces cerevisiae vehicle and at least one Mycobacterium tuberculosis antigen, The product of step b and the yeast vehicle are combined in a ratio of 90:10 to 10:90 yeast unit equivalents.

5. The method of claim 4, wherein prior to the step of removing the cell walls and cell membranes, the lysed yeast cells are further treated by sonication.

6. The method of claim 4, wherein the yeast vehicle is heat-killed yeast cells.

7. The method of claim 4, wherein the lysis step is performed by a method selected from the group consisting of glass bead crushing, high pressure homogenization, sonication, and electrolysis.

8. The method of claim 4, wherein the cleavage step is performed by a method selected from the group consisting of physical cleavage, chemical cleavage, and enzymatic cleavage.

9. The method of claim 4, wherein the removing step is performed by a method selected from the group consisting of: centrifugation, filtration and affinity chromatography.

10. Use of an immunogenic composition for the preparation of a medicament for eliciting a cytotoxic T cell immune response to Mycobacterium tuberculosis infected cells in a subject, the immunogenic composition comprising: (i) a yeast lysate prepared from Saccharomyces cerevisiae cells, wherein the lysate lacks yeast membrane and yeast cell wall, and the Saccharomyces cerevisiae cells contain a Mycobacterium tuberculosis antigen; and (ii) a fully recombinant inactivated Saccharomyces cerevisiae vehicle and at least one Mycobacterium tuberculosis antigen, The yeast lysate and the fully recombinant inactivated Saccharomyces cerevisiae vector are combined in a ratio of 90:10 to 10:

90.

11. Use of an immunogenic composition for the preparation of a medicament for eliciting both a Mycobacterium tuberculosis-specific humoral immune response and a cell-mediated immune response in a subject, the immunogenic composition comprising: (i) a yeast lysate prepared from Saccharomyces cerevisiae cells, wherein the lysate lacks yeast membrane and yeast cell wall, and the Saccharomyces cerevisiae cells contain a Mycobacterium tuberculosis antigen; and (ii) a fully recombinant inactivated Saccharomyces cerevisiae vehicle and at least one Mycobacterium tuberculosis antigen, The yeast lysate and the fully recombinant inactivated Saccharomyces cerevisiae vector are combined in a ratio of 90:10 to 10:

90.

12. The method of claim 10 or 11, wherein the immunogenic composition is administered to the subject by a route of administration selected from the group consisting of intravenous, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, intranasal, oral, intraocular, intraarticular, intracranial, and intraspinal.

13. The use of claim 10 or 11, wherein the immunogenic composition is formulated in a pharmaceutically acceptable excipient suitable for administration to a subject by injection.

14. The use of claim 10 or 11, wherein the immunogenic composition is administered to the subject at a dose of 0.1 YU to 100 Y.U.

15. The use of claim 10 or 11, wherein the immunogenic composition is administered to the subject at a dose of 10 Y.U. to 80 Y.U.

16. The use of claim 10 or 11, wherein the immunogenic composition is administered once a week.

17. The use of claim 10 or 11, wherein the immunogenic composition is administered once every other week.

18. The use of claim 10 or 11, wherein the immunogenic composition is administered once a month.

19. The use according to claim 10 or 11, wherein the composition comprises a plurality of Mycobacterium tuberculosis antigens.

20. An immunogenic preparation, comprising: i. a yeast lysate prepared from Saccharomyces cerevisiae cells, wherein the lysate lacks yeast membranes and yeast cell walls; and ii. fully recombinant inactivated Saccharomyces cerevisiae; and iii. at least one Mycobacterium tuberculosis antigen, The yeast lysate and the fully recombinant inactivated Saccharomyces cerevisiae are combined in a ratio of 90:10 to 10:

90.

21. The formulation of claim 20, further comprising a second antigen selected from the group consisting of a viral antigen, a mammalian cell surface molecule, a bacterial antigen, and a fungal antigen.

22. The formulation of claim 20, wherein the formulation comprises a plurality of Mycobacterium tuberculosis antigens.

23. A method of producing an immunogenic composition, the method comprising: a. Lysis of Saccharomyces cerevisiae cells; b. removing cell walls and cell membranes from lysed cells; and c. combining the product of step b with a fully recombinant inactivated Saccharomyces cerevisiae vehicle and at least one Mycobacterium tuberculosis antigen, The product of step b and the yeast vehicle are combined in a ratio of 90:10 to 10:90 in terms of yeast unit equivalents.

24. The method of claim 23, wherein prior to the step of removing the cell walls and cell membranes, the lysed yeast cells are further treated by sonication.

25. The method of claim 23, wherein the yeast vehicle is heat-killed yeast cells.

26. The method of claim 23, wherein the lysing step is performed by a method selected from the group consisting of glass bead crushing, high pressure homogenization, sonication, and electrolysis.

27. The method of claim 23, wherein the cleavage step is performed by a method selected from the group consisting of physical cleavage, chemical cleavage, and enzymatic cleavage.

28. The method of claim 23, wherein the removing step is performed by a method selected from the group consisting of: centrifugation, filtration, and affinity chromatography.

29. Use of an immunogenic composition for the preparation of a medicament for eliciting a cytotoxic T cell immune response to Mycobacterium tuberculosis infected cells in a subject, the immunogenic composition comprising: (i) a yeast lysate prepared from Saccharomyces cerevisiae cells, wherein the lysate lacks yeast membranes and yeast cell walls; (ii) a fully recombinant inactivated Saccharomyces cerevisiae vector; and (iii) at least one Mycobacterium tuberculosis antigen, The yeast lysate and the fully recombinant inactivated Saccharomyces cerevisiae vector are combined in a ratio of 90:10 to 10:

90.

30. Use of an immunogenic composition for the preparation of a medicament for eliciting both a Mycobacterium tuberculosis-specific humoral immune response and a cell-mediated immune response in a subject, the immunogenic composition comprising: (i) a yeast lysate prepared from Saccharomyces cerevisiae cells, wherein the lysate lacks yeast membranes and yeast cell walls; (ii) a fully recombinant inactivated Saccharomyces cerevisiae vector; and (iii) at least one Mycobacterium tuberculosis antigen, The yeast lysate and the fully recombinant inactivated Saccharomyces cerevisiae vector are combined in a ratio of 90:10 to 10:

90.

31. The use of claim 29 or 30, wherein the immunogenic composition is administered to the subject by a route of administration selected from the group consisting of: intravenous, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, intranasal, oral, intraocular, intraarticular, intracranial, and intraspinal.

32. The use of claim 29 or 30, wherein the immunogenic composition is formulated in a pharmaceutically acceptable excipient suitable for administration to a subject by injection.

33. The use of claim 29 or 30, wherein the immunogenic composition is administered to the subject at a dose of 0.1 YU to 100 Y.U.

34. The use of claim 29 or 30, wherein the immunogenic composition is administered to the subject at a dose of 10 Y.U. to 80 Y.U.

35. The use of claim 29 or 30, wherein the immunogenic composition is administered once a week.

36. The use of claim 29 or 30, wherein the immunogenic composition is administered once every other week.

37. The use of claim 29 or 30, wherein the immunogenic composition is administered once a month.

38. The use of claim 29 or 30, wherein the composition comprises a plurality of Mycobacterium tuberculosis antigens.

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