Self-assembling vaccines and combination therapies for cancer treatment

Abstract

It provides a vaccine platform that can be tailored to target any tumor type and appropriately stimulates the expansion of specific anti-tumor immune cells. [Solution] Provided are self-assembling pharmaceutical compositions comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated component (e.g., a tumor cell, a tumor antigen, a virus, or a viral antigen). The self-assembling pharmaceutical compositions can further comprise an immunotherapy (e.g., an anti-PD-1 antibody). Also provided are methods of using these pharmaceutical compositions for preventing and / or treating cancer or inducing an immune response. Also provided are methods of using the self-assembling pharmaceutical compositions in combination with immunotherapy (e.g., an anti-PD-1 antibody).

Description

[Background technology]

[0001] While vaccines are commonly associated with infectious diseases, they also have a long history in cancer treatment. Like immunotherapy, cancer vaccines have had limited success and are not widely used. Traditionally, cancer vaccines have been based on the whole tumor, potentially including signals from both healthy and cancerous cells, resulting in poor specificity and low overall activity. However, with the widespread availability of DNA sequencing, it is now possible to identify mutations specifically associated with tumor cells but absent in healthy cells. Some tumor-specific mutations can be exploited as tumor-specific antigens, or "neoantigens." These are novel immune targets that can be used to train a patient's immune system to specifically target cancerous cells. Numerous companies are working to develop pipelines and algorithms for harvesting tumors from patients and identifying targetable mutations that can be incorporated into personalized vaccines. However, target identification is only the initial hurdle. The method for presenting the target to the immune system is just as important, if not more so, than the actual target. Even a vaccine that delivers a good target can potentially be less effective without proper immune stimulation. Summary of the Invention [Problem to be solved by the invention]

[0002] Therefore, there is a need for a vaccine platform that can be targeted to any tumor type and appropriately stimulates the expansion of specific anti-tumor immune cells. [Means for solving the problem]

[0003] The present application provides compositions and methods for preventing and / or treating cancer. In certain aspects, the present application provides pharmaceutical compositions comprising, consisting essentially of, or consisting of a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently linked to a biotinylated peptide, the peptide (1) binding to an MHC class I molecule and (2) having less than 100% homology to a native sequence and / or a native microbiome sequence. In some embodiments, the peptide is one or more peptides selected from Table 1 (page 33). In some embodiments, the pharmaceutical composition is a vaccine. In some embodiments, the peptide is 5-50 amino acids in length (e.g., 8-12 amino acids in length).

[0004] In certain aspects, the present application provides a method for preventing and / or treating ovarian cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently linked to a biotinylated peptide, the peptide (1) binding to an MHC class I molecule and (2) having less than 100% homology to a native self-sequence and / or native microbiome sequence. In certain embodiments, the peptide is one or more peptides selected from Table 1 (page 33). In certain embodiments, the peptide is 5-50 amino acids in length (e.g., 8-12 amino acids in length). In certain embodiments, the pharmaceutical composition used in the methods described herein is a vaccine. In certain embodiments, the method is a method for treating ovarian cancer (e.g., serous or epithelial papillary ovarian cancer). In certain embodiments, the pharmaceutical composition is administered to the subject as a non-covalent complex.

[0005] In certain aspects, the present application provides a pharmaceutical composition comprising, consisting essentially of, or consisting of: (1) a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated tumor cell or a biotinylated tumor antigen; and (2) an immunotherapy. In some embodiments, the tumor antigen binds to an MHC class I molecule. In some embodiments, the tumor antigen has less than 100% homology to a self-native sequence and / or a native microbiome sequence. In some embodiments, the tumor antigen is 5-50 amino acids in length (e.g., 8-12 amino acids in length).

[0006] In certain aspects, the present application provides a method for preventing and / or treating cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising: (1) a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to biotinylated tumor cells or biotinylated tumor antigen; and (2) immunotherapy. In some embodiments, the tumor antigen binds to an MHC class I molecule. In some embodiments, the tumor antigen has less than 100% homology to a self-native sequence and / or a native microbiome sequence. In some embodiments, the tumor antigen is 5 to 50 amino acids in length (e.g., 8 to 12 amino acids in length). In certain embodiments, the method is a method for treating cancer. In some embodiments, the biotinylated tumor cells or biotinylated tumor antigen in the pharmaceutical compositions described herein are derived from the same type of cancer as the cancer to be prevented and / or treated. For example, the cancer can be ovarian cancer (e.g., serous or epithelial papillary ovarian cancer) or human papillomavirus (HPV)-associated cancer (e.g., HPV-induced cervical cancer, HPV-induced anal cancer, or HPV-induced head and neck cancer). In some embodiments, the cancer is induced by infection with an oncogenic virus (e.g., human papillomavirus (HPV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), or herpesvirus). In some embodiments, the method further includes a cancer therapy selected from the group consisting of radiation, a radiosensitizer, chemotherapy, and a second immunotherapy. The immunotherapy or the second immunotherapy can independently be an immune checkpoint inhibitor or an immunomodulatory agent. In some embodiments, the immunomodulatory agent is a CXCR4 / CXCR7 antagonist (e.g., AMD3100), a Jak / stat inhibitor (e.g., ruxolitinib), or near-infrared laser immunomodulation of skin-associated immune cells.

[0007] In some embodiments, the present application provides a method for preventing and / or treating cancer in a subject, comprising administering to the subject an immunotherapy in combination with an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated tumor cell or a biotinylated tumor antigen. In some embodiments, the tumor antigen binds to an MHC class I molecule. In some embodiments, the tumor antigen has less than 100% homology to a self-native sequence and / or a native microbiome sequence. In some embodiments, the tumor antigen is 5 to 50 amino acids in length (e.g., 8 to 12 amino acids in length). In some embodiments, the method is a method for treating cancer. In some embodiments, the immunotherapy and the pharmaceutical composition are administered simultaneously or sequentially. In some embodiments, the pharmaceutical composition is administered before the immunotherapy. In some embodiments, the biotinylated tumor cell or biotinylated tumor antigen in the pharmaceutical composition is derived from the same type of cancer as the cancer to be prevented or treated. For example, the cancer can be ovarian cancer (e.g., serous or epithelial papillary ovarian cancer), human papillomavirus (HPV)-associated cancer (e.g., HPV-induced cervical cancer, HPV-induced anal cancer, HPV-induced oral cancer, HPV-induced vulvar cancer, HPV-induced vaginal cancer, HPV-induced penile cancer, or HPV-induced head and neck cancer). In some embodiments, the cancer is induced by infection with an oncogenic virus (e.g., HPV, HCV, EBV, HIV, or herpes virus). In some embodiments, the method further includes a cancer therapy selected from the group consisting of radiation, a radiosensitizer, and chemotherapy.

[0008] In some embodiments, the biotin-binding protein is non-covalently bound to biotinylated tumor cells, which express an antigen on their surface, and in some embodiments, the tumor cells have been rendered replication-incompetent, for example, by irradiation.In certain embodiments, the biotinylated tumor cells are biotinylated sarcoma cells or biotinylated carcinoma cells, e.g., the biotinylated tumor cells are biotinylated fibrosarcoma cells, biotinylated myxosarcoma cells, biotinylated liposarcoma cells, biotinylated chondrosarcoma cells, biotinylated osteogenic sarcoma cells, biotinylated chordoma cells, biotinylated angiosarcoma cells, biotinylated hemangiosarcoma cells, biotinylated lymphangiosarcoma cells, biotinylated lymphangiosarcoma cells, biotinylated synovioma cells, biotinylated mesothelioma cells, biotinylated Biotinylated Ewing's tumor cells, biotinylated leiomyosarcoma cells, biotinylated rhabdomyosarcoma cells, biotinylated colon carcinoma cells, biotinylated colorectal carcinoma cells, biotinylated pancreatic cancer cells, biotinylated breast cancer cells, biotinylated ovarian cancer cells, biotinylated prostate cancer cells, biotinylated squamous cell carcinoma cells, biotinylated basal cell carcinoma cells, biotinylated adenocarcinoma cells, biotinylated sweat gland carcinoma cells, biotinylated sebaceous gland carcinoma cells, biotinylated papillary carcinoma cells, biotinylated papillary adenocarcinoma cells, biotinylated cystadenocarcinoma cells, biotinylated medullary carcinoma cells, biotinylated bronchogenic carcinoma cells Cancer cells, biotinylated renal cell carcinoma cells, biotinylated hepatoma cells, biotinylated biliary tract carcinoma cells, biotinylated choriocarcinoma cells, biotinylated seminoma cells, biotinylated embryonal carcinoma cells, biotinylated Wilms' tumor cells, biotinylated cervical cancer cells, biotinylated testicular tumor cells, biotinylated lung cancer cells, biotinylated small cell lung cancer cells, biotinylated bladder cancer cells, biotinylated epithelial carcinoma cells, biotinylated glioma cells, biotinylated astrocytoma cells, biotinylated medulloblastoma cells, biotinylated craniopharyngioma cells, biotinylated ependymoma cells , biotinylated pinealoma cells, biotinylated hemangioblastoma cells, biotinylated acoustic neuroma cells, biotinylated oligodendroglioma cells, biotinylated meningioma cells, biotinylated melanoma cells, biotinylated neuroblastoma cells, biotinylated retinoblastoma cells, biotinylated leukemia cells, biotinylated polycythemia vera cells, biotinylated lymphoma cells, biotinylated multiple myeloma cells, biotinylated Waldenstrom's macroglobulinemia cells, biotinylated head and neck cancer cells, biotinylated anal cancer cells, or biotinylated heavy chain disease cells. In certain embodiments, the biotinylated tumor cells are biotinylated ovarian cancer cells (e.g., biotinylated serous or epithelial papillary ovarian cancer cells).In certain embodiments, the biotinylated tumor cells are biotinylated human papillomavirus (HPV)-associated cancer cells (e.g., biotinylated HPV-induced cervical cancer, biotinylated HPV-induced anal cancer, or biotinylated HPV-induced head and neck cancer).

[0009] In some embodiments, the biotin-binding protein is non-covalently bound to a biotinylated tumor antigen. The tumor antigen can be a protein overexpressed by tumor cells or an immunogenic fragment thereof. The tumor antigen can also be a protein specifically mutated in tumor cells or an immunogenic fragment thereof. In some embodiments, the tumor antigen comprises a full-length or partially inactivated tumor-producing virus. In other embodiments, the tumor antigen comprises a protein derived from an tumor-producing virus or an immunogenic fragment thereof. The tumor-producing virus can be, for example, HPV, HCV, EBV, HIV, or a herpes virus. In some embodiments, the tumor antigen is a tumor-derived phosphopeptide. In some embodiments, the tumor antigen is capable of eliciting an immune response. In some embodiments, the tumor antigen is derived from a sarcoma cell or a carcinoma cell, such as a fibrosarcoma cell, a myxosarcoma cell, a liposarcoma cell, a chondrosarcoma cell, an osteogenic sarcoma cell, a chordoma cell, angiosarcoma cell, angioendothelial sarcoma cell, lymphangiosarcoma cell, lymphangioendothelial sarcoma cell, synovial tumor cell, a mesothelioma cell, a Ewing's tumor cell, a leiomyosarcoma cell, a rhabdomyosarcoma cell, a colon cancer cell, a colorectal cancer cell, a pancreatic cancer cell, a breast cancer cell, an ovarian cancer cell, a prostate cancer cell, a squamous cell carcinoma cell, a basal cell carcinoma cell, an adenocarcinoma cell, a sweat gland carcinoma cell, a sebaceous gland carcinoma cell, a papillary carcinoma cell, a papillary adenocarcinoma cell, a cystadenocarcinoma cell, a medullary carcinoma cell, a bronchogenic carcinoma cell, a renal cell carcinoma cell, a thyroid ... The tumor antigen is derived from ovarian cancer cells (e.g., serous or epithelial papillary ovarian cancer cells). In a preferred embodiment, the tumor antigen is one or more peptides selected from Table 1 (page 33).In certain embodiments, the tumor antigen is derived from a human papillomavirus (HPV)-associated cancer cell (e.g., HPV-induced cervical cancer, HPV-induced anal cancer, HPV-induced oral cancer, HPV-induced vulvar cancer, HPV-induced vaginal cancer, HPV-induced penile cancer, or HPV-induced head and neck cancer).

[0010] In some embodiments, the immunotherapy inhibits an immune checkpoint. In certain embodiments, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, and A2aR. For example, the immune checkpoint can be PD1 or PD-L1. In a preferred embodiment, the immunotherapy is an anti-PD-1 antibody. In some embodiments, the immunotherapy is an immunomodulatory agent selected from the group consisting of a CXCR4 / CXCR7 antagonist (e.g., AMD3100), a Jak / stat inhibitor (e.g., ruxolitinib), and near-infrared laser immunomodulation of skin-associated immune cells.

[0011] In some embodiments, the present application provides a method for preventing and / or treating HPV-associated cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated HPV virus or a biotinylated HPV viral antigen. In some embodiments, the HPV viral antigen binds to an MHC class I molecule. In some embodiments, the HPV viral antigen has less than 100% homology to a self-native sequence and / or a native microbiome sequence. In some embodiments, the HPV viral antigen is 5-50 amino acids in length (e.g., 8-12 amino acids in length). In some embodiments, the pharmaceutical composition used in the methods described herein is a vaccine. In certain embodiments, the method is a method for treating HPV-associated cancer (e.g., head and neck cancer or anal cancer). In some embodiments, the pharmaceutical composition is administered to the subject as a non-covalent complex.

[0012] In certain aspects, the present application provides a pharmaceutical composition comprising, consisting essentially of, or consisting of: (1) a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated HPV virus or a biotinylated HPV viral antigen; and (2) an immunotherapy. In some embodiments, the HPV viral antigen binds to an MHC class I molecule. In some embodiments, the HPV viral antigen has less than 100% homology to a self-native sequence and / or a native microbiome sequence. In some embodiments, the HPV viral antigen is 5-50 amino acids in length (e.g., 8-12 amino acids in length).

[0013] In certain aspects, the present application provides a method for preventing and / or treating HPV-associated cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising: (1) a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated HPV virus or a biotinylated HPV viral antigen; and (2) immunotherapy. In some embodiments, the HPV viral antigen binds to an MHC class I molecule. In some embodiments, the HPV viral antigen has less than 100% homology to a self-native sequence and / or a native microbiome sequence. In some embodiments, the HPV viral antigen is 5-50 amino acids in length (e.g., 8-12 amino acids in length). In certain embodiments, the method is a method for treating HPV-associated cancer (e.g., head and neck cancer or anal cancer). In some embodiments, the method further comprises a cancer therapy selected from the group consisting of radiation, radiosensitizers, chemotherapy, and a second immunotherapy. The immunotherapy or the second immunotherapy can independently be an immune checkpoint inhibitor or an immunomodulatory agent. In some embodiments, the immunomodulatory agent is a CXCR4 / CXCR7 antagonist (e.g., AMD3100), a Jak / stat inhibitor (e.g., ruxolitinib), or near-infrared laser immunomodulation of skin-associated immune cells.

[0014] In some embodiments, the present application provides a method for preventing and / or treating HPV-associated cancer in a subject, comprising administering to the subject an immunotherapy in combination with an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated HPV virus or a biotinylated HPV viral antigen. In some embodiments, the HPV viral antigen binds to an MHC class I molecule. In some embodiments, the HPV viral antigen has less than 100% homology to a self-native sequence and / or a native microbiome sequence. In some embodiments, the HPV viral antigen is 5 to 50 amino acids in length (e.g., 8 to 12 amino acids in length). In some embodiments, the method is a method for treating HPV-associated cancer (e.g., head and neck cancer or anal cancer). In some embodiments, the immunotherapy and the pharmaceutical composition are administered simultaneously or sequentially. In some embodiments, the pharmaceutical composition is administered before the immunotherapy. In some embodiments, the method further comprises a cancer therapy selected from the group consisting of radiation, radiosensitizers, and chemotherapy.

[0015] In some embodiments, the biotin-binding protein is non-covalently bound to a biotinylated HPV virus, and the biotinylated HPV virus expresses an antigen. The HPV virus can be a full-length or partially inactivated HPV virus. In other embodiments, the biotin-binding protein is non-covalently bound to a biotinylated HPV viral antigen. The biotinylated HPV viral antigen can be a biotinylated E6 protein, a biotinylated E7 protein, or a biotinylated immunogenic fragment thereof. In specific embodiments, the biotinylated HPV viral antigen is selected from Table 3. In some embodiments, the pharmaceutical composition increases the survival rate of a subject suffering from an HPV-associated cancer (e.g., head and neck cancer or anal cancer).

[0016] Additionally, there are numerous embodiments that can be applied to any aspect of the present disclosure and / or can be combined with any other embodiment described herein. For example, in certain embodiments, the biotin-binding protein is selected from the group consisting of avidin, streptavidin, and neutravidin. In certain embodiments, the biotin-binding protein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to that of avidin or streptavidin.

[0017] In some embodiments, the heat shock protein is a mammalian heat shock protein or a bacterial heat shock protein. In certain embodiments, the heat shock protein is a member of the hsp70 family. In particular embodiments, the heat shock protein is or is derived from MTB-HSP70. For example, the heat shock protein can have an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the pharmaceutical composition is a vaccine. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition increases the survival rate of a subject suffering from cancer (e.g., ovarian cancer, such as serous or epithelial papillary ovarian cancer, or HPV-associated cancer). In some embodiments, the pharmaceutical composition enhances an immune response. In some embodiments, the pharmaceutical composition enhances immune cell proliferation.

[0018] In certain aspects, the present application provides a method for producing a pharmaceutical composition described herein, comprising contacting a heat shock protein fused to a biotin-binding protein with a biotinylated peptide sufficient to form a non-covalent complex between the heat shock protein and the biotinylated peptide, wherein the peptide (1) binds to an MHC class I molecule and (2) has less than 100% homology to a native sequence and / or a native microbiome sequence. In certain embodiments, the peptide is one or more peptides selected from Table 1 (page 33).

[0019] In certain aspects, the present application provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition described herein. [Brief explanation of the drawings]

[0020] [Figure 1] A schematic diagram of self-assembling vaccines (SAVs) is shown. MTB-HSP70 is the immunostimulatory basic unit common to all SAVs, regardless of target. Avidin is attached to MTB-HSP70 to bind to the variable unit, which provides the specific targeting peptide sequence (marked the variable unit). [Figure 2] The percent survival of mice receiving various treatments is shown. DETAILED DESCRIPTION OF THE INVENTION

[0021] overview The present invention is based, at least in part, on the discovery that non-covalent association of a heat shock protein fusion with a biotinylated component (e.g., a tumor cell or tumor antigen) results in a self-assembling vaccine (SAV) that increases survival in mice bearing cancer (e.g., ovarian cancer). Notably, combined treatment of SAV with immunotherapy (e.g., an anti-PD-1 antibody) exhibited synergistic effects in increasing survival in these mice. These effects are due, at least in part, to enhanced immune cell proliferation. Accordingly, compositions and methods are provided for preventing and / or treating cancer using SAV alone or in combination with immunotherapy.

[0022] A major challenge for all vaccines is the cost and time required for development. These constraints are particularly severe for emerging infectious diseases and cancer, where vaccines must be individually designed for a target and then rapidly and cost-effectively manufactured. To address this challenge, in certain embodiments, the present invention relates to a modular platform consisting of immunostimulatory building blocks that can be linked to targeting modules that elicit an immune response. While this platform was initially developed to target infectious diseases, it has now been demonstrated that the targeting modules can be similarly delivered to various cancer types. In certain embodiments, the building block of the platform is MTB-HSP70, a bacterial protein known to have immune-stimulating properties, modified to include avidin. The addition of avidin allows the building block to be linked to a specific targeting module, such as a protein subunit called a peptide (see Figure 1). In certain embodiments, peptides used to target tumors can be selected from proteins abnormally abundant in tumors, tumor-specific mutations, and protein modifications that are hallmarks of cancer cells. Computational tools can be used to identify target-specific peptides that are predicted to provide good targets for the immune system, determine the appropriate structure of the peptide chain, and incorporate the necessary mutations to synthesize custom peptides.

[0023] In certain embodiments, these custom peptides are biotinylated and conjugated with avidin, forming a highly stable bond between the immunostimulatory and cancer-targeting components. This approach has been termed "self-assembling vaccines" or "SAVs" because it can be produced without the need for special chemical reactions or further purification. Previous studies have shown that the SAV approach can promote specific immune responses against bacterial and viral targets (Leblanc et al. (2014) Human Vaccin. Immunother. 10:3022-3038). Other reports have also shown that MTB-HSP70 can improve the function of tumor-targeting antibodies. Therefore, SAV technology can be used to create tumor-specific vaccines, which can optionally incorporate broadly immunostimulatory adjuvants. In certain embodiments, the vaccine targets tumors in multiple ways to generate favorable anti-tumor immune responses.

[0024] Ovarian cancer is an area of ​​particularly urgent unmet need, as the majority of women diagnosed with this disease present at a later stage. Most women respond to surgery and chemotherapy initially; however, a high percentage of women ultimately experience a recurrence within five years. While immunotherapy has shown great promise in other cancer types, results in ovarian cancer have been lackluster. With the recent enthusiasm surrounding immunotherapy, there has been a renewed focus on the development of cancer vaccines. Effective cancer treatments appear to combine the traditional approach of surgery and chemotherapy with regimens individually designed for each patient, including targeted drugs, immunotherapies, and personalized vaccines.

[0025] In certain embodiments, the SAV comprises a peptide derived from a protein known to be overexpressed by tumors or specifically mutated in cancer cells, hi certain embodiments, the peptide alone elicits an immune response.

[0026] In certain embodiments, the present invention relates to pharmaceutical compositions for administration in combination with immunotherapeutic agents (e.g., antibodies targeting PD-1). Immune cells exhausted from fighting infections or tumors often have elevated levels of PD-1 on their surface. Anti-PD-1 antibodies can bind to the surface of these immune cells, which have become less functional, and revitalize them. However, this treatment does not increase the number of anti-tumor immune cells in the body. In certain embodiments, the use of tumor-targeted vaccines (to increase the number of anti-cancer immune cells) in combination with anti-PD-1 (to restore and maintain their function) can produce results superior to those seen to date with either approach alone.

[0027] definition For clarity, before describing the present invention in detail, certain terms employed in the specification, examples, and claims are defined below.

[0028] Unless otherwise clear from the context, reference to the singular includes reference to the plural.

[0029] As used herein, "isolated protein" refers to a protein that is substantially free of other proteins, cellular material, and isolation and culture media when the protein is isolated from a cell or produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide, or fusion protein is derived, or, when chemically synthesized, substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations in which compositions of the invention are separated from cellular components of the cells from which they are isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations that contain less than about 30%, less than 20%, less than 10%, or less than 5% (by dry weight) cellular material. When the antibody, polypeptide, peptide, or fusion protein, or fragment thereof (e.g., biologically active fragment thereof), is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation, more preferably less than about 10%, and most preferably less than about 5%.

[0030] The terms "about" and "approximately" generally refer to an acceptable degree of error in the measured quantity given the type or precision of the measurement. In general, a typical degree of error is within 20% of a stated value or range of values, with within 10% being preferred and within 5% being more preferred. Alternatively, particularly in biological systems, the terms "about" and "approximately" can refer to a value within a certain multiple of the stated value, with within 5-fold being preferred and within 2-fold being more preferred. Unless otherwise specified, quantities described herein are approximate, i.e., the term "about" or "approximately" can be inferred unless otherwise specified.

[0031] The term "administering" is intended to include any route of administration that allows a drug to perform its intended function. Examples of biotherapeutic administration routes that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection may be a bolus injection or a continuous infusion. Depending on the route of administration, the drug may be coated or placed inside a selected material to protect it from natural conditions that may adversely affect its ability to perform its intended function. The drug may be administered alone or with a pharmaceutically acceptable carrier. The drug may also be administered as a prodrug that is converted to its active form in vivo.

[0032] "Parenteral" means a mode of administration other than enteral or topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intralesional, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intrasternal, buccal, epidural, intranasal, and infusion.

[0033] As used herein, the terms "co-administration" or "administered in combination" or "co-administering" refer to any mode of administration in which two or more different drugs are administered such that a first drug is administered while another drug is still effective in the body (e.g., the two drugs may be effective in a subject simultaneously, resulting in a synergistic effect of the two drugs). For example, the different drugs can be administered in the same formulation or in separate formulations, either simultaneously or sequentially. Thus, a subject receiving such treatment can benefit from the combined effect of the different drugs.

[0034] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both amino and acid functional groups and can be included in polymers of natural amino acids. Exemplary amino acids include the natural amino acids; their analogs, derivatives, and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the above. In this application, the names of natural amino acids are abbreviated according to IUPAC-IUB recommendations.

[0035] The term "antibody" refers to an immunoglobulin or a derivative thereof that retains specific binding ability, as well as a protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. The term "antibody" is intended to include full-length antibodies or antigen-binding fragments thereof. These proteins may be derived from natural sources or may be partially or completely synthetically produced. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class from any species, including any of the human immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. In an exemplary embodiment, the antibody used in the methods and compositions described herein is a derivative of the IgG class. An antibody may be artificial or natural.

[0036] The term "antibody fragment" refers to any derivative of an antibody that is less than full-length. In typical embodiments, an antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fc fragments, scFv fragments, Fv fragments, dsFv diabody fragments, and Fd fragments. Antibody fragments can be produced by any means. For example, they can be produced by enzymatic or chemical fragmentation of an intact antibody, recombinantly produced from a gene encoding a partial antibody sequence, or synthetically produced in whole or in part. Antibody fragments can optionally be single-chain antibody fragments. Alternatively, they can be composed of multiple molecular chains interconnected, for example, by disulfide bonds. They can also optionally be multimolecular complexes. Functional antibody fragments will generally be at least about 50 amino acids in length, more usually at least about 200 amino acids in length.

[0037] Antibodies may be polyclonal or monoclonal, and may be xenogeneic, allogeneic, or syngeneic, or may be modified (e.g., humanized, chimeric, etc.). Antibodies may be fully human. Preferably, antibodies of the invention bind specifically or substantially specifically to a biomarker polypeptide or a fragment thereof. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one type of antigen-binding site capable of immunoreacting with a particular epitope of an antigen, and the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple types of antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition generally exhibits a single binding affinity for a particular antigen with which it immunoreacts.

[0038] The antibody may also be a "humanized" antibody, i.e., an antibody produced by a non-human cell but with variable and constant regions that have been modified to more closely resemble antibodies produced by a human cell. Such antibodies can be obtained, for example, by modifying the amino acid sequence of a non-human antibody to incorporate amino acids present in human germline immunoglobulin sequences. The humanized antibodies of the invention can include amino acid residues, for example, in the CDRs, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced randomly or by in vitro site-directed mutagenesis or by in vivo somatic mutation). As used herein, the term "humanized antibody" also includes antibodies in which CDR sequences derived from the germline of another mammalian species are grafted onto human framework sequences.

[0039] By "antigen" is meant the target of an immune response induced by the compositions described herein. Antigens can be protein antigens, and are understood to include full-length proteins as well as fragments of such proteins that are presented on the surface of viruses or infected, foreign, or tumor cells in a subject, and peptides that are presented by infected, foreign, or tumor cells as a result of processing and presentation of such proteins, e.g., via the typical MHC class I or II pathway. Examples of such foreign cells include bacteria, fungi, and protozoa.

[0040] In certain embodiments, the "antigen" binds to an MHC class I molecule (e.g., an HLA molecule). Methods for testing the binding ability of an antigen to HLA class II and class I alleles are well known in the art. For example, HLA binding can be predicted using known algorithms (e.g., the EpiMatrix algorithm) or tested using standard in vitro HLA binding assays (e.g., competitive assays) such as those described in Scholzen et al. (2019) Frontiers in Immunology 10:1-22. Binding affinity can be measured using a competitive assay to determine IC 50 For example, IC in an HLA class II binding assay 50Proteins or peptides with IC values ​​of 100 μM or less can be considered "binders" and are considered to be "binders" under the binding conditions tested. 50 Proteins or peptides whose values ​​are too high to measure accurately (>100 μM) or do not produce a dose-dependent response are considered non-binders. 50 Proteins or peptides with IC values ​​below 1000 μM can be considered "binders" and have IC values ​​under the binding conditions tested. 50 Proteins or peptides whose values ​​are too high to measure accurately (>1000 μM) or do not produce a dose-dependent response are considered non-binders.

[0041] In some embodiments, the "antigen" exhibits no or minimal autoreactivity and / or microbiome reactivity. "Autoreactivity" can be estimated based on sequence homology between the antigen and a native self sequence. In some embodiments, the antigen has a sequence that is less than 100%, less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% identical to a native self sequence. "Microbiome reactivity" can be estimated based on sequence homology between the antigen and a native microbiome sequence. In some embodiments, the antigen has a sequence that is less than 100%, less than 99%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% identical to a native microbiome sequence. In some embodiments, homology analysis can be performed using known algorithms (e.g., the Janus Matrix algorithm), such as those described in Scholzen et al. (2019) Frontiers in Immunology 10:1-22. In certain embodiments, the "antigen" is a peptide of about 5 to about 100 amino acids in length, e.g., about 5 to about 90 amino acids in length, about 5 to about 80 amino acids in length, about 5 to about 70 amino acids in length, about 5 to about 60 amino acids in length, about 5 to about 50 amino acids in length, about 5 to about 45 amino acids in length, about 5 to about 40 amino acids in length, about 5 to about 35 amino acids in length, about 5 to about 30 amino acids in length, about 5 to about 25 amino acids in length, about 5 to about 20 amino acids in length, about 5 to about 15 amino acids in length, or about 8 to about 12 amino acids in length.In certain embodiments, the "antigen" is a peptide 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. Examples of bacterial antigens include protein A (PrA), protein G (PrG), and protein L (PrL). Examples of tumor antigens include, but are not limited to, the peptides listed in Table 1 (page 33). Examples of viral antigens include, but are not limited to, the peptides listed in Table 3 (page 38).

[0042] The term "antigen-binding site" refers to the region of an antibody that specifically binds to an epitope on an antigen.

[0043] The term "biotin-binding protein" refers to a protein that non-covalently binds biotin. The biotin-binding protein can be a monomer, dimer, or tetramer, capable of forming monovalent, divalent, or tetravalent pharmaceutical compositions, respectively, as described herein. Non-limiting examples include anti-biotin antibodies, avidin, streptavidin, and neutravidin. The avidin can include mature avidin or a sequence at least 80%, 85%, 90%, 95%, or 99% identical to the sequence identified in NCBI Accession No. NP_990651. The streptavidin can include, for example, a sequence at least 80%, 85%, 90%, 95%, or 99% identical to the sequence identified in NCBI Accession No. AAU48617. The term "biotin-binding protein" includes wild-type avidin, streptavidin, and neutravidin, which may form monomers, dimers, or tetramers, and their derivatives. Examples of such derivatives are described below and also in Laitinen, OH (2007), "Brave New (Strept)avidins in Biotechnology," Trends in Biotechnology 25(6):269-277, and Nordlund, HR (2003), "Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding," FEBS Letters 555:449-454, the contents of both of which are expressly incorporated herein by reference.

[0044] The term "tumor cells" when used in the context of an antigen-containing biotinylated moiety is intended to include whole tumor cells or portions thereof, provided that said portions contain the antigen of interest on their surface accessible for recognition by the immune system when a pharmaceutical composition comprising the biotinylated "tumor cells" is administered to a subject.

[0045] The term "cancer" or "tumor" or "hyperproliferation" refers to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortalization, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological traits.

[0046] Cancer cells are often in the form of tumors, but they may exist alone in an animal or may be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" encompasses both pre-malignant and malignant cancers. Examples of cancers include, but are not limited to, B-cell cancers (e.g., multiple myeloma, Waldenstrom's macroglobulinemia), heavy chain diseases (e.g., alpha, gamma, and mu chain diseases), benign monoclonal gammopathy, immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colon cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, and hematopoietic tissue cancer. Other non-limiting examples of types of cancer that are amenable to the methods encompassed by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, hemangiosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, biliary tract carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, Brain tumors, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia (e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia)), chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.In some embodiments, the cancer is epithelial, including but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. The epithelial cancer may also be characterized by a variety of other classifications, including but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0047] As used herein, the term "HPV-associated cancer" or "HPV-associated cancer" refers to any type of cancer associated with or caused by human papillomavirus (HPV) infection. Persistent infection with certain HPV types (e.g., types 16, 18, 31, and 45) is associated with cancers such as oropharyngeal cancer, laryngeal cancer, vulvar cancer, vaginal cancer, cervical cancer, penile cancer, and anal cancer. In certain embodiments, HPV-associated cancers include, but are not limited to, cervical cancer, head and neck cancer, oral cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, lung cancer, and oropharyngeal cancer. In particular embodiments, the HPV-associated cancer is cervical cancer, head and neck cancer, or anal cancer.

[0048] The terms "comprise" and "comprising" are inclusive and open, meaning that other elements may be included.

[0049] As used herein, the term "costimulatory molecule" includes any molecule capable of enhancing the stimulatory effect of an antigen-specific primary T cell stimulator or increasing the activity of said stimulator above the threshold level required for cell activation, resulting in activation of naive T cells. Such costimulatory molecules can be membrane-localized receptor proteins.

[0050] The term "effective amount" refers to an amount of a pharmaceutical composition sufficient to produce a desired result. An effective amount of a pharmaceutical composition can be administered in one or more administrations.

[0051] As used herein, the terms "therapeutically effective amount" and "effective amount" refer to an amount of a drug effective to produce a desired therapeutic effect on at least a subpopulation of cells in a subject, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0052] The term "engineered antibody" refers to a recombinant molecule comprising at least an antibody fragment containing an antigen-binding site derived from the variable domains of the antibody heavy and / or light chains, and optionally can include all or part of the variable and / or constant domains of an antibody from any of the Ig classes (e.g., IgA, IgD, IgE, IgG, IgM, and IgY). Examples of engineered antibodies include highly functional single-chain monoclonal antibodies and highly functional monoclonal antibodies. Examples of engineered antibodies are described in detail in PCT / US2007 / 061554, the disclosure of which is incorporated herein by reference in its entirety.

[0053] The term "epitope" refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody preferentially binds to a single, specific epitope of a molecule that can be defined molecularly. In the present invention, multispecific antibodies can recognize multiple epitopes.

[0054] "Fusion protein" refers to a hybrid protein containing sequences from at least two different proteins. These sequences can be from proteins of the same or different organisms. In various embodiments, a fusion protein can include one or more amino acid sequences linked to a first protein. When two or more amino acid sequences are fused to a first protein, the fused sequences can be multiple copies of the same sequence or different amino acid sequences. The first protein can be fused to the N-terminus, C-terminus, or N- and C-terminus of the second protein.

[0055] The term "Fab fragment" refers to a fragment of an antibody that contains the antigen-binding site and is produced by cleaving an antibody with papain, an enzyme that cleaves the hinge region of the disulfide bond between the heavy chains at the N-terminus, generating two Fab fragments from one antibody molecule.

[0056] The term "F(ab')2 fragment" refers to a fragment of an antibody that contains two antigen-binding sites and is generated by cleaving an antibody molecule with pepsin, an enzyme that cleaves the hinge region of the disulfide bond between the H chains at the C-terminus.

[0057] The term "Fc fragment" refers to a fragment of an antibody containing the constant domain of its heavy chain.

[0058] The term "Fv fragment" refers to a fragment of an antibody comprising the variable domains of its heavy and light chains.

[0059] "Genetic construct" means a nucleic acid (e.g., a vector, a plasmid, a viral genome, etc.) that contains a "coding sequence" for a polypeptide or is transcribable into biologically active RNA (e.g., an antisense, decoy, ribozyme, etc.) that can be transfected into a cell (e.g., a mammalian cell in certain embodiments) and that can result in expression of the coding sequence in the cell transfected with the construct. A genetic construct can include one or more regulatory elements operably linked to the coding sequence, as well as intron sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0060] By "host cell" is meant a cell into which a particular transfer vector can be introduced. The cell is optionally selected from in vitro cells, such as cells derived from a cell culture, ex vivo cells, such as cells derived from an organism, and in vivo cells, such as cells present in an organism. It should be understood that such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because some variation in successive generations may occur due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still within the scope of the term as used herein.

[0061] The term "immunogenic" refers to the ability of a substance to induce an immune response. An "immunogenic composition" or "immunogen" is a composition or substance that induces an immune response. An "immune response" refers to a subject's reaction to the presence of an antigen and can include at least one of antibody production, immune development, antigen hypersensitivity reaction development, and immune tolerance development. In certain embodiments, "immune response" refers to an anti-tumor immune response.

[0062] The term "including" is used herein to mean "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0063] The term "linker" is well known in the art and refers to a molecule or group of molecules that connect two covalent components, such as a heat shock protein and a biotin-binding protein. A linker can consist of only a linking molecule, or it can contain a spacer molecule in addition to the linking molecule to provide a certain distance between the linking molecule and the component.

[0064] The term "multivalent antibody" refers to an antibody or artificial antibody that contains two or more antigen recognition sites. For example, a "bivalent" antibody has two antigen recognition sites, and a "tetravalent" antibody has four antigen recognition sites. "Monospecific," "bispecific," "trispecific," "tetraspecific," etc. refer to the number of different antigen recognition site specificities (not the number of antigen recognition sites) present in a multivalent antibody. For example, all of the antigen recognition sites of a "monospecific" antibody bind to the same epitope. A "bispecific" antibody has at least one antigen recognition site that binds to a first epitope and at least one antigen recognition site that binds to a second epitope that is different from the first epitope. A "multivalent monospecific" antibody has multiple antigen recognition sites that all bind to the same epitope. A "multivalent bispecific" antibody has multiple antigen recognition sites, a predetermined number of which bind to a first epitope and a predetermined number of which bind to a second epitope that is different from the first epitope.

[0065] The term "multivalent" when used in reference to the self-assembling pharmaceutical compositions described herein refers to a heat shock fusion protein non-covalently linked to two or more biotinylated components. The term "bivalent" when used in reference to the self-assembling pharmaceutical compositions described herein refers to a heat shock fusion protein non-covalently linked to two biotinylated components (e.g., tumor cells or tumor antigens). The term "tetravalent" when used in reference to the self-assembling pharmaceutical compositions described herein refers to a heat shock fusion protein non-covalently linked to four biotinylated components (e.g., tumor cells or tumor antigens). The biotinylated components (e.g., tumor cells or tumor antigens) of a multivalent pharmaceutical composition can be the same or different component types.

[0066] The term "nucleic acid" refers to a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides, or modifications of either nucleotides. The term also includes, as equivalents, RNA or DNA analogs formed from nucleotide analogs, and, where applicable to the described embodiments, should be understood to include single- and double-stranded polynucleotides (e.g., sense or antisense).

[0067] The terms "patient" or "subject" or "host" are used interchangeably and refer to either a human or non-human animal, respectively. This term includes mammals such as humans, primates, livestock (e.g., cows, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice, rabbits, and rats).

[0068] The term "pharmaceutically acceptable" is used herein to describe pharmaceutical compositions that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0069] As used herein, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or additive (e.g., a liquid or solid filler, diluent, excipient, or solvent encapsulating material) that is involved in carrying or transporting the pharmaceutical composition of the present application from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) glycols such as propylene glycol. (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic, compatible materials utilized in pharmaceutical formulations.

[0070] Unless otherwise clear from the context, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein when referring to a gene expression product (e.g., an amino acid sequence as encoded by a coding sequence). "Protein" can also refer to an assembly of one or more proteins, such as an antibody. "Protein" can also refer to a protein fragment. A protein can be a post-translationally modified protein, such as a glycosylated protein. "Gene expression product" refers to a molecule produced as a result of transcription of all or part of a gene. Gene products include RNA molecules transcribed from a gene and proteins translated from such transcripts. A protein can be a naturally occurring isolated protein, a recombinant product, or a chemically synthesized product. The term "protein fragment" refers to a protein that is missing amino acid residues compared to the reference protein itself, but whose remaining amino acid sequence typically corresponds to the amino acid sequence of the reference protein. Such deletions can be at the amino terminus, carboxy terminus, or both of the reference protein. Fragments are generally at least about 5, 6, 8, or 10 amino acids in length, at least about 14, 20, 30, 40, or 50 amino acids in length, at least about 75, or at least about 100, 150, 200, 300, 500, or more amino acids in length. Fragments can be obtained by fragmenting a larger protein using a proteinase, or by recombinant methods, such as expressing only a portion of a nucleotide sequence encoding a protein (alone or fused to a nucleic acid sequence encoding another protein). In various embodiments, fragments can contain enzymatic activity and / or an interaction site of the reference protein, for example, with a cellular receptor. In another embodiment, fragments can be immunogenic. Mutations can be introduced into specific genetic loci of the protein by various known techniques that can enhance, rather than impair, the use of the protein in the methods described herein. Fragments can retain one or more biological activities of the reference protein.

[0071] As used herein, the term "self-assembling" refers to the ability of a heat shock protein fused to a biotin-binding protein to form a non-covalent complex with a biotinylated component as described herein, such ability being conferred by the non-covalent association of biotin with the biotin-binding protein.

[0072] The term "single-chain variable fragment" or "scFv" refers to an Fv fragment in which the heavy and light chain domains are linked. One or more scFv fragments can be linked to other antibody fragments (e.g., heavy or light chain constant domains) to form an antibody construct with one or more antigen recognition sites.

[0073] "Treating" a disease in a subject or a subject with a disease means administering pharmaceutical treatment (e.g., administering a drug) to the subject to reduce or prevent the extent of the disease. Treatment includes (but is not limited to) administering a composition, such as a pharmaceutical composition, and can be administered after the initiation of a pathological event.

[0074] As used herein, a therapeutic agent that "prevents" a condition (e.g., cancer) refers to a composition that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the administered sample relative to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample.

[0075] The term "vaccine" refers to a pharmaceutical composition that induces an immune response against a target antigen. A vaccine can also confer protective immunity in a subject.

[0076] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. An example of a preferred vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. Preferred vectors are those capable of autonomously replicating and / or expressing a nucleic acid to which it has been linked. A vector capable of causing expression of a gene when operably linked to the gene is referred to herein as an "expression vector." While the vector form generally refers to a circular double-stranded DNA loop that is not bound to a chromosome, expression vectors useful in recombinant DNA technology are often in the form of a "plasmid." Because plasmids are the most widely used form of vector, the terms "plasmid" and "vector" are used interchangeably herein. However, those skilled in the art will recognize that the present invention also encompasses other equivalent forms of expression vectors that perform equivalent functions and that become known in the art.

[0077] The term "survival" includes all of the following: survival until death (also referred to as overall survival) (where death may be from causes unrelated to the tumor or may be tumor-related); "recurrence-free survival" (where recurrence includes both local and distant recurrence); metastasis-free survival; and disease-free survival (where disease includes cancer and its related diseases). The survival period can be calculated based on a predetermined starting point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for treatment efficacy can be expanded to include chemotherapy response, probability of survival, probability of metastasis within a predetermined period, and probability of tumor recurrence.

[0078] The term "synergistic effect" refers to the combined effect of two or more anti-cancer agents (e.g., a pharmaceutical composition described herein in combination with an immunotherapy) that can be greater than the sum of the separate effects of the anti-cancer agents / therapies alone.

[0079] The term "phosphopeptide" refers to a phosphorylated peptide capable of inducing an immune response. The peptide can be phosphorylated on a serine, threonine, or tyrosine residue. In some embodiments, the phosphopeptide is derived from a cancer cell and can induce an anti-tumor immune response.

[0080] Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclatures and techniques used in chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are those commonly used and well known in the art.

[0081] Biotinylated components As used herein, the term "biotinylated component" refers to a biotinylated protein, a biotinylated cell, or a biotinylated virus. Non-limiting examples of biotinylated components include biotinylated tumor antigens, biotinylated tumor cells, and biotinylated costimulatory molecules. The biotinylated component (e.g., tumor cells, tumor antigens, viruses, or viral antigens) is administered to a subject along with a heat shock protein fusion as described herein.

[0082] In one embodiment, the biotinylated tumor cells or tumor antigens are derived from a subject, which may be the same or a different subject to which the pharmaceutical composition is to be administered. For example, tumor cells or tumor antigens against which an immune response is desired can be isolated from the subject and, optionally, expanded or cloned in vitro. The tumor cells or tumor antigens can then be biotinylated in vitro using methods known in the art. The biotinylated tumor cells or tumor antigens can then be administered together with the heat shock protein fusions described herein to the same subject from which the tumor cells or tumor antigens were isolated, thereby developing a personalized vaccine. Alternatively, the biotinylated tumor cells or tumor antigens can be administered together with the heat shock protein fusions described herein to a subject other than the subject from which the tumor cells or tumor antigens were isolated. The latter approach allows for the development of a general population vaccine against cancer when administered to the general population.

[0083] Both approaches offer significant advantages over conventional techniques: they only require the identification of tumor cells or tumor antigens to the extent that they can be correlated with a specific cancer and isolated from a subject. This represents a novel approach for targeting antigens whose sequences are unknown or whose structures cannot even be identified. Thus, the present invention enables the production of pharmaceutical compositions for inducing immune responses against one or more antigens, whether known or unidentified and uncharacterized. Personalized vaccines also offer another advantage over conventional vaccines in that they eliminate the issue of HLA restriction, since the tumor cells or tumor antigens are derived from the same host as the subject to whom the biotinylated tumor cells or tumor antigens are to be administered.

[0084] In certain embodiments, the tumor cells or tumor antigens can be derived from a cancer cell line.

[0085] The tumor cells or tumor antigens can be derived from the same type of cancer as the cancer to be prevented and / or treated by the pharmaceutical compositions described herein. The tumor cells or tumor antigens can also be derived from a different type of cancer than the cancer to be prevented and / or treated by the pharmaceutical compositions described herein. The tumor cells or tumor antigens can also be derived from a cancer with the same genetic mutation as the cancer to be prevented and / or treated by the pharmaceutical compositions described herein. The tumor cells or tumor antigens can also be derived from a cancer with a different genetic mutation than the cancer to be prevented and / or treated by the pharmaceutical compositions described herein.

[0086] Any tumor cell or tumor antigen can be biotinylated and administered to a subject along with a heat shock protein fusion moiety described herein, such that when administered along with the heat shock fusion proteins described herein, the biotinylated tumor cell or tumor antigen will elicit an anti-tumor immune response.

[0087] Biotinylated tumor cells In certain embodiments, tumor cells are biotinylated and administered with the heat shock protein fusions described herein. Tumor cells can be isolated from a subject. A common method for obtaining purified tumor cells is to isolate and purify them from various tumor tissues, such as surgically removed tumor tissue, ascites, or malignant pleural effusions. Cancer cells can also be purified from fresh biopsy specimens from cancer patients or animal tumor models. Biopsy specimens often contain heterogeneous cell populations, including normal tissue, blood, and cancer cells. Purified cancer cell compositions preferably contain more than 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more total viable cancer cells, or any range or value therebetween. Numerous methods can be used to purify cancer cells from heterogeneous populations.

[0088] In one embodiment, cancer cells are isolated using laser microdissection. Targeted cancer cells can be carefully excised from thin tissue sections prepared for microscopic examination. In this method, a thin plastic film is placed over the tissue section, and a focused infrared laser beam pulse is directed at the area containing the selected cells. A small circle of the plastic film melts, binding the underlying cells. The trapped cells are then harvested for further analysis. This technique is suitable for isolating and analyzing cells from different parts of a tumor, allowing their similar distinguishing properties to be compared. Recently, this technique has been used to analyze pituitary cells from dispersed tissue and culture populations containing heterogeneous mixtures of pituitary, thyroid, and carcinoid tumor cells, as well as to analyze single cells present in various sarcomas.

[0089] In another embodiment, fluorescence-activated cell sorting (FACS) (also known as flow cytometry) is used to sort and analyze distinct cell populations. Cells bearing a cell marker or other specific marker of interest are tagged with an antibody, or generally a mixture of antibodies, that binds to the cell marker. Each antibody for a different marker is labeled with a detectable molecule, particularly a fluorescent dye that can be distinguished from other fluorescent dyes bound to other antibodies. The presence or absence of a specific labeled antibody is determined by passing the tagged or "stained" cell stream through a light source that excites the fluorescent dye and emission spectrum from the detected cells. Simultaneous detection of different fluorescent dyes (also known in the art as multicolor fluorescent cell sorting) allows cells displaying distinct sets of cell markers to be identified and isolated from other cells in the population. Other FACS parameters can also be used to select cells based on size and viability, including, but not limited to, side scatter (SSC), forward scatter (FSC), and vital dye staining (e.g., with propidium iodide). FACS sorting and analysis of HSCs and related lineage cells is well known in the art and is described, for example, in U.S. Patent Nos. 5,137,809, 5,750,397, 5,840,580, and 6,465,249; Manz et al. (202) Proc. Natl. Acad. Sci. USA 99:11872-11877; and Akashi et al. (200) Nature 404:193-197. General guidance regarding fluorescence-activated cell sorting is provided, for example, in Shapiro (2003) Practical Flow Cytometry, 4th Ed., Wiley-Liss (2003) and Ormerod (2000) Flow Cytometry: A Practical Approach, 3rd Ed., Oxford University Press.

[0090] Another method for isolating useful cell populations utilizes solid or insoluble supports immobilized with antibodies or ligands that interact with specific cell surface markers. In immunoadsorption techniques, cells are contacted with an antibody-loaded support (e.g., a bead column, flask, magnetic particles, etc.), and unbound cells are removed. Immunoadsorption techniques can be scaled up to directly process large numbers of cells in clinical specimens. Suitable supports include, but are not limited to, plastic, cellulose, dextran, polyacrylamide, agarose, and other materials known in the art (e.g., Pharmacia Sepharose 6MB macrobeads). When solid supports containing magnetic or paramagnetic beads are used, cells bound to the beads can be easily isolated using a magnetic separator (see, e.g., Kato and Radbruch (1993) Cytometry 14:384-92). Affinity chromatography cell separation typically involves flowing a cell suspension over a support with its selective ligand immobilized on its surface. The ligand interacts with its specific target molecule on the cells and is captured by the matrix. The bound cells are released by adding an eluent to the column running buffer, and the released cells are washed through the column and collected as a homogenous population. Those skilled in the art will appreciate that adsorption techniques are not limited to those utilizing specific antibodies; nonspecific adsorption can also be used. For example, silica adsorption is a simple method for removing phagocytes from cell preparations. One of the most common uses of this technique is the isolation of circulating tumor cells (CTCs) from the blood of patients with breast, non-small cell lung, prostate, and colon cancer using antibodies against EpCAM, a cell surface glycoprotein found to be highly expressed in epithelial cancers.

[0091] FACS and most batch immunoadsorption techniques can be applied to both positive and negative selection methods (see, e.g., U.S. Pat. No. 5,877,299). In positive selection, desired cells are labeled with antibodies and separated from the remaining unlabeled / unwanted cells. In negative selection, unwanted cells are labeled and separated. Another type of negative selection that can be used is the use of antibody / complement therapy or immunotoxins to separate unwanted cells.

[0092] In yet another embodiment, one of the latest advancements in microfluidics technology is used to isolate cancer cells. This method uses a microfluidic chip with spiral-shaped channels that can isolate circulating tumor cells (CTCs) from blood based on their size. When a blood sample is pumped into the device, inertial and centrifugal forces act as the cells move rapidly through the channels, causing small cells to flow along the outer wall and larger cells, including CTCs, to flow along the inner wall. Researchers have used this chip technology to isolate CTCs from the blood of patients with metastatic lung or breast cancer.

[0093] A recently published paper (Lin et al. Small (2015) 11:4394-4402) found that fluorescent nanodiamonds (FNDs) can be used to label and isolate slow-growing / dormant cancer stem cells. However, the authors noted that it has been difficult to isolate and track these cells over the long term using traditional fluorescent markers. They concluded that nanoparticles do not cause DNA damage or impair cell growth and are superior to EdU and CFSE fluorescent labels in terms of long-term tracking capabilities.

[0094] Of course, cell purification or isolation also includes combinations of the above methods. A typical combination may include an initial step effective to remove unwanted cells and bulk cellular material. A second step may involve isolating cells expressing markers common to one or more of the progenitor cell populations by immunoadsorption to antibodies immobilized on a support. Additional high-resolution steps of different cell types (e.g., FACS sorting using antibodies against a set of specific cell markers) may also be used to obtain a substantially pure population of desired cells.

[0095] In certain other embodiments, the cancer cells are derived from a cancer cell line.

[0096] In the present invention, tumor cells should be treated before being introduced or reintroduced into a subject so that they no longer replicate and are harmless to the subject. In some embodiments, the tumor cells are replication-incompetent. In certain embodiments, the tumor cells have been rendered replication-incompetent by irradiation (e.g., gamma and / or UV irradiation) and / or administration of a substance that disables cell replication (e.g., a compound that disrupts cell membranes, an inhibitor of DNA replication, an inhibitor of mitotic spindle formation during cell division, etc.). In some embodiments, sublethal doses of irradiation can be used. For example, the tumor cells can be irradiated with a sublethal dose before or after biotinylation to suppress cell proliferation before administration of the self-assembling vaccine and reduce the risk of new neoplastic lesions arising. Of course, irradiation is only one example of a method for rendering cells replication-incompetent, and other methods for rendering cancer cells unable to divide while still maintaining their ability to elicit anti-tumor immunity are encompassed by the present invention.

[0097] In some embodiments, the tumor cells express an antigen on their surface, which antigen may or may not be identified or characterized. When administered to a subject along with a heat shock protein fusion, the non-covalent complex induces an immune response against the tumor antigen on the tumor cells. In some embodiments, the immune response is a "cytotoxic T cell" response against the antigen expressing the tumor cells, thereby targeting the tumor cells for destruction.

[0098] The tumor cells may be cells of a cancer type to be treated or prevented by the method of the present invention. Examples of such cells include, but are not limited to, human sarcoma or carcinoma cells, such as fibrosarcoma cells, myxosarcoma cells, liposarcoma cells, chondrosarcoma cells, osteogenic sarcoma cells, chordoma cells, angiosarcoma cells, hemangioendothelial sarcoma cells, lymphangiosarcoma cells, lymphangioendothelial sarcoma cells, synovial tumor cells, mesothelioma cells, Ewing's tumor cells, leiomyosarcoma cells, rhabdomyosarcoma cells, colon cancer cells, colorectal cancer cells, pancreatic cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer cells, squamous cell carcinoma cells, basal cell carcinoma cells, adenocarcinoma cells, sweat gland carcinoma cells, sebaceous gland carcinoma cells, papillary carcinoma cells, papillary adenocarcinoma cells, cystadenocarcinoma cells, medullary carcinoma cells, bronchogenic carcinoma cells, renal cell carcinoma cells, hepatoma cells, biliary tract carcinoma cells, choriocarcinoma cells, seminoma cells, embryonal carcinoma cells, and Wilms' tumor. The cell is a cervical cancer cell, a testicular tumor cell, a lung cancer cell, a small cell lung cancer cell, a bladder cancer cell, an epithelial carcinoma cell, a glioma cell, an astrocytoma cell, a medulloblastoma cell, a craniopharyngioma cell, an ependymoma cell, a pinealoma cell, a hemangioblastoma cell, an acoustic neuroma cell, an oligodendroglioma cell, a meningioma cell, a melanoma cell, a neuroblastoma cell, a retinoblastoma cell, a leukemia (e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia)), a chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), a polycythemia vera cell, a lymphoma (Hodgkin's disease and non-Hodgkin's disease) cell, a multiple myeloma cell, a Waldenstrom's macroglobulinemia cell, or a heavy chain disease cell.

[0099] In some embodiments, the biotinylated tumor cells are biotinylated ovarian cancer cells (e.g., serous or epithelial papillary ovarian cancer cells). In some embodiments, the biotinylated tumor cells are biotinylated HPV-associated cancer cells (e.g., human papillomavirus (HPV)-induced cervical cancer cells, HPV-induced head and neck cancer cells, or HVP-induced anal cancer cells).

[0100] b. Biotinylated tumor antigen In some embodiments, tumor antigens are biotinylated and administered with the heat shock protein fusions described herein. "Antigen" refers to the target of the immune response induced by the compositions described herein. Antigens can be protein antigens, and are understood to include full-length proteins as well as fragments of such proteins that are presented on the surface of viruses or infected, foreign, or tumor cells in a subject, and peptides that are presented by infected, foreign, or tumor cells as a result of processing and presentation of such proteins, e.g., via the typical MHC class I or II pathway. Examples of such foreign cells include bacteria, fungi, and protozoa.

[0101] In certain embodiments, the term "tumor antigen" of the present invention encompasses tumor-associated proteins and any portion or peptide of the tumor-associated protein capable of eliciting an anti-tumor response in a subject. The tumor antigen can be a protein overexpressed by tumor cells or an immunogenic fragment thereof. The tumor antigen can also be a protein specifically mutated in tumor cells or an immunogenic fragment thereof. In certain embodiments, the tumor antigen is a tumor-derived phosphopeptide. The tumor antigen can be any tumor-associated protein, a fragment of the protein, a modified version of the protein (e.g., a phosphorylated protein or peptide), or a functionally equivalent variant of the protein capable of eliciting an immune response. "Functionally equivalent variants" include, but are not limited to, peptides with partial sequence homology, peptides with one or more specific conservative and / or non-conservative amino acid mutations, peptide conjugates, chimeric proteins, fusion proteins, and peptide nucleic acids.

[0102] The tumor antigen can be an antigen associated with the type of cancer to be treated or prevented by the methods of the invention. In some embodiments, the tumor antigen is associated with a sarcoma or carcinoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, hemangiosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal carcinoma, or the like. cell carcinoma, hepatoma, biliary tract cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, head and neck cancer, anal cancer, or heavy chain disease.

[0103] In certain embodiments, the tumor antigen is associated with ovarian cancer (e.g., serous or epithelial papillary ovarian cancer). In some embodiments, the tumor antigen is associated with HPV-associated cancer. In certain embodiments, the tumor antigen is associated with cervical cancer (e.g., human papillomavirus (HPV)-induced cervical cancer). In certain embodiments, the tumor antigen is associated with head and neck cancer (e.g., HPV-induced head and neck cancer). In certain embodiments, the tumor antigen is associated with anal cancer (e.g., HPV-induced anal cancer).

[0104] In some embodiments, the tumor antigen comprises a full-length or partially inactivated oncogenic virus. In some embodiments, the tumor antigen comprises a protein or immunogenic fragment thereof derived from an oncogenic virus. The oncogenic virus can be, for example, HPV, HCV, EBV, HIV, or a herpes virus.

[0105] In certain embodiments, the tumor antigen is a peptide derived from a tumor-associated protein. As used herein, the term "peptide" refers to natural peptides (degradation products or synthetically synthesized peptides) and also to peptide mimetics, such as peptoids and semipeptoids, which are peptide analogs and may be modified, for example, to enhance the peptide's stability in vivo or its immunogenicity. Such modifications include, but are not limited to, cyclization, N-terminal modifications, C-terminal modifications, peptide bond modifications (e.g., but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH, or CF=CH), backbone modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are specifically described in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin, Pergamon Press (1992), the entire disclosure of which is incorporated herein by reference.

[0106] As used herein, the term "protein-derived" refers to peptides derived from one or more specific proteins, and also to homologous peptides derived from equivalent regions of proteins homologous to the specific proteins of the same or other species, provided that these peptides are effective as anti-tumor vaccines. The term also refers to permissible amino acid modifications and peptidomimetics designed based on the amino acid sequence of the specific protein or its homologous proteins.

[0107] In certain embodiments, peptides used to target tumors can be selected from proteins abnormally abundant in tumors, tumor-specific mutations, and protein modifications that are hallmarks of cancer cells. The peptides can be identified through DNA sequencing or literature review. Computer tools can be used to identify target-specific peptides predicted to provide good targets for the immune system, determine the appropriate structure of their peptide chains, and / or incorporate the necessary mutations for custom peptide synthesis. For example, neoantigens and tumor-associated antigens identified by exome DNA sequencing of the tumor cells can be selected through algorithmic analysis. Using computer algorithms (e.g., the EpiMatrix or JanusMatrix algorithms), the immunogenic peptides can be top-selected for computer-predicted specific HLA binding and bottom-selected for autoreactivity, microbiome reactivity, and / or immunosuppressive activity. The HLA binding of the predicted peptides can also be tested in standard peptide-HLA binding in vitro assays, such as those described in Scholzen et al. (2019) Frontiers in Immunology 10:1-22, the entire disclosure of which is incorporated herein by reference.

[0108] All selected peptides can be tested for eliciting an immune response, an important criterion for enhancing the immune system's anti-tumor function. For example, the strength and specificity of the immune response to cancer-targeting peptides delivered via the SAV platform can be measured. These results can be compared to previous reports on other peptide-based approaches to identify underperforming peptides and guide further optimization. In certain embodiments, the self-assembling vaccines described herein are used to deliver a single tumor-associated peptide. In certain embodiments, the self-assembling vaccines are used to deliver multiple tumor-associated peptides. In certain embodiments, the multivalent self-assembling vaccines described herein are used to deliver multiple tumor-associated peptides. For example, the self-assembling vaccines described herein are used to deliver a complete repertoire of peptides, providing the immune system with a broad set of tumor targets. In certain embodiments, peptides derived from tumor-associated antigens, such as peptides derived from mesothelin or folate receptor alpha, can be used to generate the SAVs described herein. In certain embodiments, peptides derived from neoantigens, such as peptides derived from Ipo13, Rpl5, or Pkp4, can be used to generate the SAVs described herein. In certain embodiments, peptides from multiple species (e.g., two, three, four, etc.) can be linked together by linkers and used as a single peptide in the same SAV. The amino acid sequences of exemplary tumor antigens are shown in Table 1 below.

[0109] [Table 1]

[0110] Also included in Table 1 are polypeptide molecules comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity over its entire length to the amino acid sequence of any of the SEQ ID NOs listed in Table 1. Such polypeptides can have the function of the full-length polypeptide as detailed herein.

[0111] In certain embodiments, the tumor antigen has an amino acid sequence comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive amino acids of an amino acid sequence set forth in Table 1. In some embodiments, the consecutive amino acids correspond to an amino acid sequence set forth in Table 1.

[0112] In certain embodiments, the tumor antigen has an amino acid sequence consisting essentially of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids of an amino acid sequence set forth in Table 1. In some embodiments, the contiguous amino acids correspond to an amino acid sequence set forth in Table 1.

[0113] In certain embodiments, the tumor antigen has an amino acid sequence consisting of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive amino acids of the amino acid sequence. In some embodiments, the consecutive amino acids correspond to an amino acid sequence set forth in Table 1.

[0114] In some embodiments, the tumor antigen has an amino acid sequence comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in Table 1. In some embodiments, the contiguous amino acids match an amino acid sequence set forth in Table 1.

[0115] In some embodiments, the tumor antigen has an amino acid sequence consisting essentially of 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in Table 1. In some embodiments, the contiguous amino acids match an amino acid sequence set forth in Table 1.

[0116] In some embodiments, the tumor antigen has an amino acid sequence consisting of 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in Table 1. In some embodiments, the contiguous amino acids match an amino acid sequence set forth in Table 1.

[0117] As is well known to those skilled in the art, polypeptides with substantial sequence similarity can elicit the same or a similar immune response in a host animal. Thus, in certain embodiments, derivatives, equivalents, variants, fragments, or mutants of the tumor antigen proteins or fragments thereof are also suitable for use in the methods and compositions described herein.

[0118] In certain embodiments, the present application provides variants or derivatives of the tumor antigens. A modified polypeptide may have an altered amino acid sequence, e.g., by conservative substitution, but still elicit an immune response reactive with the unmodified protein antigen and is considered a functional equivalent. As used herein, the term "conservative substitution" refers to the replacement of an amino acid residue with another, biologically similar residue. It is well known in the art that amino acids within the same conserved residue group can generally be substituted for each other without substantially affecting protein function. In certain embodiments, a derivative, equivalent, variant, or mutant of the tumor antigen is a polypeptide that has at least 85% homology with the sequence of the tumor antigen protein or fragment thereof. In some embodiments, the homology is at least 90%, at least 95%, or at least 98%.

[0119] In certain embodiments, the tumor antigen can be produced by recombinant DNA technology. For example, a nucleic acid molecule encoding the tumor antigen can be cloned into an expression vector, and the expression vector can be introduced into host cells to express the tumor antigen in the host cells. The tumor antigen can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.

[0120] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One example of a vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments can be ligated. Another type of vector is a viral vector, into which other DNA segments can be ligated. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors are integrated into the genome of the host cell upon introduction into the host cell and are replicated along with the host genome (e.g., mammalian non-episomal vectors). Furthermore, some vectors are capable of driving the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because plasmids are the most widely used form of vector, the terms "plasmid" and "vector" can be used interchangeably herein. However, the invention is intended to include other equivalent forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0121] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because some variation in successive generations may occur due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein. Host cells can be any prokaryotic or eukaryotic cell. For example, the tumor antigen can be expressed in bacterial cells such as E. coli, insect cells, yeast, or mammalian cells (e.g., Fao hepatoma cells, primary hepatocytes, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those of skill in the art.

[0122] In another variant, protein production can be carried out using an in vitro translation system. In vitro translation systems are generally translation systems that are cell-free extracts containing at least the minimum components necessary for translating RNA molecules into proteins. In vitro translation systems generally contain at least ribosomes, tRNA, initiator methionine tRNAMet, and proteins or complexes involved in translation (e.g., eIF2, eIF3, cap-binding (CB) complex (eIF4F) containing cap-binding protein (CBP) and eukaryotic translation initiation factor 4F). Various in vitro translation systems are known in the art, and commercially available kits are also available. Examples of in vitro translation systems include eukaryotic cell lysates such as rabbit reticulocyte lysate, rabbit oocyte lysate, human cell lysate, insect cell lysate, and wheat germ extract. Lysates are commercially available from manufacturers such as Promega, Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO / BRL, Grand Island, NY. In vitro translation systems generally contain macromolecules such as enzymes, translation initiation factors, elongation factors, chemical reagents, and ribosomes. Additionally, in vitro transcription systems may be used. Such systems generally contain at least RNA polymerase holoenzyme, ribonucleotides, and the necessary transcription initiation factors, elongation factors, and termination factors. In vitro transcription and translation can be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs.

[0123] As an alternative to recombinant expression, tumor antigens can be chemically synthesized using standard peptide synthesis techniques. Chemical synthesis can be performed using a variety of methods well known in the art, including stepwise solid-phase synthesis, conformational religation of peptide fragments, enzymatic ligation of cloned or synthesized peptide segments, and semisynthetic methods using chemical ligation. Native chemical ligation utilizes the chemoselective reaction of two unprotected peptide segments to generate a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously rearranges to yield a full-length ligation product with a native peptide bond at the ligation site. The full-length ligation product is chemically identical to the protein produced by cell-free synthesis. Where permitted, the full-length ligation product can be refolded and / or oxidized to form a protein molecule containing native disulfide bonds.(From 6,184,344 to 6,174,530; see also TWMuir et al.,Curr.Opin.Biotech.(1993):vol.4,p420;M.Miller,et al.,Science(1989):vol.246,p1149;A.Wlodawer,et al.,Science(1989):vol.245,p616;LHHuang,et al.,Biochemistry(1991):vol.30,p7402;M.Sclmolzer,et al al.,Int.J.Pept.Prot.Res.(1992):vol.40,p180-193;K.Rajarathnam,et al.,Science(1994):vol.264,p90;REOfford,“Chemical Approaches to Protein Engineering”,in Protein Design and the Development of New Therapeutics and Vaccines,JBHook,G.Post,Eds.,(Plenum Press,New York,1990)pp.253-282;CJAWallace,et al.,J.Biol.Chem.(1992):vol.267,p3852;L.Abrahamsen,et al.,Biochemistry(1991):vol.30,p4151;TKChang,et al.,Proc.Natl.Acad.Sci.USA(1994)91:12544-12548;M.Schnlzer,et al.,Science(1992):vol.,3256,p221; al., Chem.Pharm.Bull.(Tokyo)(1985)33:184P.).

[0124] Furthermore, native tumor antigens can be isolated from cancer cells or tissues bearing the tumor antigen by an appropriate purification scheme using standard protein purification techniques, for example, using tumor antigen-specific antibodies. Cancer cells or tissues bearing the tumor antigen can be isolated from a subject. Exemplary methods for isolating and purifying tumor cells or tumor tissues are described above. In certain other embodiments, the tumor antigen can be isolated from a cancer cell line bearing the tumor antigen.

[0125] c. Biotinylated virus or viral antigen In some embodiments, a biotinylated virus or viral antigen can be administered to a subject together with a heat shock protein fusion as described herein. The subject can be suffering from a cancer induced by infection with an oncogenic virus (e.g., HPV, HCV, EBV, HIV, or herpes virus). To prevent and / or treat cancer, a biotinylated virus or viral antigen can be administered to a subject together with a heat shock protein fusion as described herein. In some embodiments, the cancer is induced by infection with an oncogenic virus (e.g., HPV, EBV, HIV, or herpes virus). In certain embodiments, the cancer is an HPV-associated cancer (e.g., cervical cancer, head and neck cancer, or anal cancer).

[0126] The biotinylated virus administered with the heat shock protein fusion as described herein can include a biotinylated oncogenic virus (e.g., HPV, HCV, EBV, HIV, or herpes virus). In certain embodiments, the biotinylated virus is a biotinylated full-length or partially inactivated oncogenic virus (e.g., HPV, HCV, EBV, HIV, or herpes virus). In a preferred embodiment, the biotinylated virus expresses an antigen capable of inducing an immune response (e.g., anti-tumor immunity).

[0127] Biotinylated viral antigens administered with heat shock protein fusions as described herein can include proteins or immunogenic fragments thereof derived from oncogenic viruses (e.g., HPV, HCV, EBV, HIV, or herpes viruses). Examples of immunogenic tumor antigens that can be biotinylated are described in Stevanovic, S. et al. (2017) Science 356:200-205, the entire disclosure of which is incorporated herein by reference. In certain embodiments, the biotinylated viral antigen is a biotinylated HPV viral antigen. The term "HPV viral antigen" refers to a protein, peptide, or functionally equivalent fragment derived from an HPV virus that is capable of eliciting an immune response (e.g., anti-tumor immunity). HPV viral antigens include, but are not limited to, the viral oncoproteins E6 and E7 and immunogenic fragments thereof. E6 and E7 are two major viral oncoproteins that induce cell immortalization and maintain a transformed phenotype during tumor progression, making them useful for the development of therapeutic vaccines. The amino acid sequence of E6 from HPV16, a high-risk HPV species, is publicly available in the GenBank database as NP_041325.1. The amino acid sequence of E7 from HPV16 is publicly available in the GenBank database as NP_041326.1. Exemplary biotinylated viral antigens for use in the compositions and methods of the invention are listed in Table 3 below and further illustrated in the Examples.

[0128] [Table 2]

[0129] Table 3 also includes polypeptide molecules comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity over its entire length to the amino acid sequence of any of the SEQ ID NOs listed in Table 3. Such polypeptides can have the function of the full-length polypeptide as detailed herein.

[0130] Heat shock protein fusions "Heat shock protein" refers to a gene encoded by a "heat shock gene" or stress gene, which is activated or otherwise detectably upregulated upon contact or exposure of an organism (containing said gene) to stressors such as heat shock, hypoxia, glucose depletion, heavy metal salts, inhibitors of energy metabolism and electron transport, and protein denaturants, or certain benzoquinone ansamycins. Nover, L., Heat Shock Response, CRC Press, Inc., Boca Raton, FL (1991). "Heat shock protein" also includes homologous proteins encoded by genes within known stress gene families, although such homologous genes are not themselves induced by the stressor.

[0131] "Heat shock protein fusion" refers to a heat shock protein linked to a biotin-binding protein. For example, a heat shock protein can be linked to the C-terminus or N-terminus of a biotin-binding protein to create a heat shock protein fusion. Administration of a heat shock protein fusion with a biotinylated component (e.g., a tumor cell or tumor antigen) described herein can stimulate or enhance humoral and / or cellular immune responses, including CD8 cytotoxic T cell (CTL) responses, against the antigen of interest.

[0132] For example, heat shock proteins that can be used in accordance with the present invention include, but are not limited to, BiP (also known as grp78), Hsp10, Hsp20-30, Hsp60, hsp70, hsc70, gp96 (grp94), hsp60, hsp40, Hsp100-200, Hsp100, Hsp90, and members of their families. Particularly preferred heat shock proteins are BiP, gp96, and hsp70, as exemplified below. Specific groups of heat shock proteins include Hsp90, Hsp70, Hsp60, and Hsp20-30, with Hsp70 and Hsp60 being more preferred. Members of the hsp70 family are most preferred.

[0133] Examples of Hsp10 include GroES and Cpn10. Hsp10 is generally found in Escherichia coli and in the mitochondria and chloroplasts of eukaryotic cells. Hsp10 forms a seven-membered ring and associates with Hsp60 oligomers. Hsp10 is also involved in protein folding.

[0134] An example of Hsp60 is Hsp65 from Mycobacterium spp. Bacterial Hsp60 is also commonly known as GroEL, such as GroEL from Escherichia coli. Hsp60 forms large homo-oligomeric complexes and appears to play a major role in protein folding. Hsp60 homologs exist in mitochondria and chloroplasts of eukaryotic cells.

[0135] Examples of Hsp70 include Hsp72 and Hsc73 from mammalian cells, DnaK from bacteria, particularly Mycobacteria such as Mycobacterium leprae, Mycobacterium tuberculosis (MTb), and Mycobacterium bovis (e.g., Bacille-Calmette-Guerin; herein referred to as Hsp71), DnaK from Escherichia coli, yeast, and other prokaryotes, as well as BiP and Grp78. Hsp70 specifically binds ATP and unfolded proteins and is involved in protein folding and unfolding and the assembly and dissociation of protein complexes. In a preferred embodiment, the heat shock protein is or is derived from MTb HSP70. The full-length protein sequences of M. tuberculosis HSP70 and M. bovis HSP70 are shown in Table 2 as SEQ ID NOs: 1 and 2, respectively. Heat shock protein fusions used in connection with the methods described herein can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:2.

[0136] [Table 3] TIFF2026041826000004.tif84168

[0137] Table 2 also includes polypeptide molecules comprising an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity over its entire length to the amino acid sequence of any of the SEQ ID NOs listed in Table 2. Such polypeptides can have the function of the full-length polypeptide as detailed herein.

[0138] Examples of Hsp90 include HtpG in Escherichia coli, Hsp83 and Hsc83 in yeast, and Hsp90α, Hsp90β, and Grp94 in humans. Hsp90 binds to a series of proteins, which are generally cellular regulatory molecules such as steroid hormone receptors (e.g., glucocorticoid, estrogen, progesterone, and testosterone receptors), transcription factors, and protein kinases that play a role in signal transduction mechanisms. Hsp90 proteins also participate in the formation of numerous large protein complexes that include other heat shock proteins.

[0139] Examples of Hsp100 include mammalian Hsp110, yeast Hsp104, ClpA, ClpB, ClpC, ClpX, and ClpY. Yeast Hsp104 and E. coli ClpA form hexameric particles, while E. coli ClpB forms tetrameric particles, and their assembly appears to require adenine nucleotide binding. Clp protease produces a 750 kDa hetero-oligomer composed of ClpP (a proteolytic subunit) and ClpA. ClpB-Y are structurally related to ClpA, but unlike ClpA, they do not appear to form complexes with ClpP.

[0140] An example of Hsp100-200 is Grp170 (glucose-regulated protein), which resides in the ER lumen in the pre-Golgi compartment and appears to play a role in immunoglobulin folding and assembly.

[0141] In accordance with the present invention, natural or recombinant mutants of heat shock proteins can be used. For example, but not limited to, the present invention provides for the use of heat shock proteins that have been mutated to facilitate secretion from the cell (e.g., those that have been mutated or deleted to facilitate endoplasmic reticulum anchoring, such as KDEL or its homologs; such mutants are described in PCT Application No. PCT / US96 / 13233 (WO97 / 06685), which is incorporated herein by reference).

[0142] In certain embodiments, the heat shock proteins of the present invention are obtained from Enterobacter, Mycobacterium (particularly Mycobacterium leprae, Mycobacterium tuberculosis, M. vaccae, M. smegmatis, and Mycobacterium bovis), Escherichia coli, yeast, Drosophila, vertebrates, birds, chickens, mammals, rats, mice, primates, or humans.

[0143] The pharmaceutical compositions provided herein may contain individual amino acid residues modified by oxidation or reduction. Furthermore, various substitutions, deletions, or additions may be made to the amino acid or nucleic acid sequence, with the net effect of maintaining or further enhancing the enhanced biological activity of the heat shock protein. Due to the degeneracy of the genetic code, for example, considerable variation may occur in nucleotide sequences encoding the same amino acid sequence. The term "heat shock protein" also includes fragments of heat shock proteins derived from heat shock proteins, provided that such fragments contain epitopes involved in enhancing the immune response to an antigen of interest. Heat shock protein fragments may be obtained by using proteinases or by recombinant methods, such as expressing only a portion of a nucleotide sequence encoding a stress protein (either alone or fused to a nucleic acid sequence encoding another protein). The heat shock protein may contain mutations introduced at specific genetic loci by various known techniques to enhance its effect on the immune system. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Drinkwater and Klinedinst Proc. Natl. Acad. Sci. USA 83:3402-3406 (1986); Liao and Wise, Gene 88:107-111 (1990); Horwitz et al., Genome 3:112-117 (1989).

[0144] In certain embodiments, for example, heat shock protein fusions comprising chemical conjugates of a heat shock protein and a biotin-binding protein, the heat shock proteins used in the present invention are isolated heat shock proteins, i.e., the heat shock proteins have been selected and isolated from the host cells in which they are produced. In certain embodiments in which a heat shock protein is recombinantly expressed as a fusion of a heat shock protein with a biotin-binding protein, the heat shock protein fusions used in the present invention are isolated heat shock protein fusions, i.e., the heat shock protein fusions have been selected and isolated from the host cells in which they are produced. Such isolation can be carried out as described herein using conventional protein isolation methods known in the art. Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182, Academic Press, Inc., San Diego, Calif. (1990). Exemplary methods for generating fusions of heat shock proteins with biotin-binding proteins are further described in PCT Publication No. WO2009 / 129502, the disclosure of which is incorporated herein by reference in its entirety.

[0145] Self-assembling vaccines As described in more detail below, multiple biotinylated components (e.g., tumor cells or tumor antigens) can be administered with a heat shock protein fusion. In this manner, multivalent pharmaceutical compositions can be created and administered to a subject. Creating multivalent pharmaceutical compositions allows for the production of "supercharged" or more potent vaccines and therapeutics.

[0146] When the pharmaceutical composition is multivalent, the administered biotinylated components (e.g., tumor cells or tumor antigens) can be any combination of the biotinylated components (e.g., tumor cells or tumor antigens) described herein. For example, biotinylated components (e.g., tumor cells or tumor antigens) of the same or different species can be administered together with a heat shock protein fusion as described herein, provided that the biotin-binding protein, and thus the heat shock protein fusion, is multivalent or capable of binding multiple biotinylated components (e.g., tumor cells or tumor antigens). For example, the wild-type biotin-binding protein avidin has four biotin-binding sites and is therefore capable of binding four biotinylated components (e.g., tumor cells or tumor antigens). In this example, the four sites are conjugated to four biotinylated moieties (e.g., tumor cell or tumor antigens), which can be mixed and matched in any possible permutation of one, two, three, or four identical biotinylated moieties (e.g., tumor cell or tumor antigens) as described herein based on moiety type. Four identical biotinylated moieties (e.g., tumor cell or tumor antigens) can be conjugated to the four biotin binding sites.

[0147] Thus, an effective amount of a first component species biotinylated tumor cells or tumor antigen is administered to a subject together with a heat shock protein fused to a biotin-binding protein sufficient to form a pharmaceutical composition comprising four parts of the first component species biotinylated tumor cells or tumor antigens and one part of the heat shock protein fused to a biotin-binding protein. Alternatively, an effective amount of first and second component species biotinylated tumor cells or tumor antigens may be administered to a subject together with a heat shock protein fused to a biotin-binding protein sufficient to form a pharmaceutical composition comprising three parts of the first component species biotinylated tumor cells or tumor antigens, one part of the second component species biotinylated tumor cells or tumor antigens, and one part of the heat shock protein fusion. In another embodiment, an effective amount of biotinylated tumor cells or tumor antigens of the first and second component species may be administered to a subject together with a heat shock protein fused to a biotin-binding protein sufficient to form a pharmaceutical composition comprising two parts biotinylated tumor cells or tumor antigens of the first component species, two parts biotinylated tumor cells or tumor antigens of the second component species, and one part heat shock protein fusion.

[0148] When the self-assembling pharmaceutical composition is bivalent, an effective amount of a first component species biotinylated tumor cell or tumor antigen can be administered to a subject together with a heat shock protein fused to a biotin-binding protein sufficient to form a pharmaceutical composition comprising two portions of the first component species biotinylated tumor cell or tumor antigen and one portion of a heat shock protein fusion. Alternatively, an effective amount of a first and second component species biotinylated tumor cell or tumor antigen can be administered to a subject together with a heat shock protein fused to a biotin-binding protein sufficient to form a pharmaceutical composition comprising one portion of the first component species biotinylated tumor cell or tumor antigen, one portion of the second component species biotinylated tumor cell or tumor antigen, and one portion of a heat shock protein fusion.

[0149] The multivalent pharmaceutical composition can include a costimulatory molecule or a blocking group (i.e., biotin alone or biotin conjugated to a nonfunctional molecule). Examples of costimulatory molecules that can be administered in connection with the present invention include B7 molecules, including B7-1 (CD80) and B7-2 (CD86), CD28, CD58, LFA-3, CD40, B7-H3, CD137 (4-1BB), and interleukins (e.g., IL-1, IL-2, or IL-12). As one example, one biotinylated component containing a costimulatory molecule can be administered together with i) three other biotinylated components containing a tumor cell or tumor antigen; and ii) one heat shock protein fused to a biotin-binding protein. As another example, two biotinylated components containing a costimulatory molecule can be administered together with i) two other biotinylated components containing a tumor cell or tumor antigen; and ii) one heat shock protein fused to a biotin-binding protein. In another example, three biotinylated components comprising a costimulatory molecule can be administered together with i) one other biotinylated component comprising a tumor cell or tumor antigen; and ii) one heat shock protein fused to a biotin-binding protein.

[0150] By controlling the noncovalent interaction of avidin, streptavidin, or neutravidin with biotin, pH-sensitive mutants of avidin, streptavidin, or neutravidin can be used to obtain heat shock protein fusions of desired stoichiometric compositions with various permutations and combinations of biotinylated tumor cells or tumor antigens, as described herein. Selection of wild-type or specific mutant forms of biotin-binding proteins (e.g., avidin) can be used to control the desired valency of pharmaceutical compositions (e.g., monomeric, dimeric, or tetrameric forms of avidin). Monovalent or bivalent vaccines can also be created by using heat shock fusion proteins containing other avidin, streptavidin, or neutravidin mutant proteins that bind biotin monovalently or bivalently. An example of an avidin mutant is described in the Examples section below. An example of a pH-sensitive point mutant of avidin that binds biotin in a pH-controllable manner is Y33H. Another mutant has Met96, Val115, and Ile117 substituted with histidine, and optionally Trp110 substituted with histidine. This approach to controlling biotin-streptavidin binding is described in Laitinen, OH (2007), "Brave New (Strept)avidins in Biotechnology," Trends in Biotechnology 25(6):269-277, and Nordlund, HR (2003), "Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding," FEBS Letters 555:449-454, the disclosures of both of which are incorporated herein by reference.

[0151] Methods for producing self-assembling vaccines In one embodiment of the present invention, a composition is comprised of two parts: a heat shock protein fused to a biotin-binding protein and a biotinylated component (e.g., a tumor cell or tumor antigen) that is the target of an immune response to the antigen against which an immune response is desired. Because the production of biotinylated antigens or antibodies is well-known, can be performed quickly, and can enhance vaccine production capacity, the present invention provides for the rapid and simple production of large quantities of pharmaceutical compositions (e.g., vaccines). As described herein, a single heat shock protein fusion can be administered in combination with any of a number of different biotinylated components (e.g., tumor cells or tumor antigens), eliminating the need to synthesize a new heat shock fusion protein de novo each time a new target antigen of interest is identified. Thus, once the heat shock protein fusion to be administered has been determined and produced, this production method is particularly rapid.

[0152] A heat shock protein fused to a biotin-binding protein can be produced as follows. Heat shock proteins can be produced from natural sources using standard techniques, as described, for example, in Flynn et al., Science 245:385-390 (1989), or they can be produced using recombinant techniques, such as expressing a genetic construct encoding the heat shock protein in a suitable host cell, such as a bacterial, yeast, or mammalian cell. Fusion proteins containing a heat shock protein and a biotin-binding protein can be produced by recombinant means. For example, a nucleic acid encoding the heat shock protein can be ligated to either end of a nucleic acid sequence encoding the biotin-binding protein so that the coding sequences for the biotin-binding protein and the heat shock protein are in a common translational reading frame and can be expressed as a fusion protein containing both proteins. The ligated sequence is inserted into an appropriate vector selected based on the desired expression characteristics and host cell type. In the following example, the nucleic acid sequence is assembled in a vector suitable for protein expression in the bacterium Escherichia coli. After expression in a selected host cell, the fusion protein can be purified by conventional biochemical separation techniques or by immunoaffinity methods using antibodies against either portion of the fusion protein. Alternatively, the fusion protein sequence can be tagged with a tag (e.g., an oligohistidine tag, as described in the Examples below) in a selected vector, allowing the expression of a tagged fusion protein, which can then be purified by affinity methods using an antibody or other material with a reasonably high affinity for the tag. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182, Academic Press, Inc., San Diego, CA (1990).When a vector tag suitable for expression in mammalian cells (e.g., one of the vectors described below) is used, the heat shock protein fusion can be expressed and purified from mammalian cells. Alternatively, a mammalian expression vector (containing a sequence encoding the fusion protein) can be administered to a subject, resulting in expression of the heat shock protein fusion protein in the subject's cells. Nucleic acids encoding the heat shock protein can also be produced chemically and then inserted into a vector suitable for production and purification of the fusion protein or administration to a subject. Additionally, fusion proteins can be produced chemically.

[0153] Techniques for creating fusion genes are well known in the art. Briefly, various DNA fragments encoding different polypeptide sequences can be ligated according to conventional techniques, utilizing blunt or overhanging ends for ligation, restriction enzyme digestion to provide suitable ends, filling in overhanging ends as needed, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that generate complementary overhangs between two adjacent gene fragments, followed by annealing to create a chimeric gene sequence (see, e.g., *Current Protocols in Molecular Biology*, eds. Ausubel et al., John Wiley & Sons, 1992). Thus, an isolated nucleic acid is provided that comprises a fusion gene in which a gene encoding a heat shock protein is fused to a gene encoding a biotin-binding protein.

[0154] The nucleotide sequence encoding the heat shock protein fusion can be carried in a vector operably linked to at least one regulatory sequence. Of course, the design of the expression vector will depend on factors such as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. The vector's copy number, the ability to control that copy number, and the expression of other proteins encoded by the vector (e.g., antibiotic markers) should also be considered. Such vectors can be administered in any biologically effective medium (e.g., any formulation or composition capable of effectively transfecting cells ex vivo or in vivo with genetic material encoding the chimeric polypeptide). Approaches include viral vectors, such as recombinant retroviruses, adenoviruses, adeno-associated viruses, human immunodeficiency viruses, and herpes simplex virus type 1, or by inserting the nucleic acid into a recombinant bacterial or eukaryotic plasmid. Viral vectors can be used to directly transfect cells, or plasmid DNA can be delivered alone using, for example, cationic liposomes (lipofectin), derivatized (e.g., antibody-bound) polylysine conjugates, gramicidin S, artificial viral envelopes, or other similar intracellular vehicles. Nucleic acids can also be directly injected. Alternatively, calcium phosphate precipitation can be performed to facilitate the introduction of nucleic acids into cells.

[0155] The nucleic acids can be used to express and overexpress heat shock protein fusion proteins in cells grown in culture, eg, to produce the fusion proteins.

[0156] Also provided are host cells transfected with recombinant genes to express heat shock protein fusions. The host cells can be any prokaryotic or eukaryotic cell. For example, heat shock protein fusions can be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, insect, plant, or mammalian cells. If the host cell is a human cell, it may or may not be present in a living subject. Other suitable host cells are known to those of skill in the art. Additionally, tRNA molecules not typically present in the host may be added to the host cell to optimize expression of the polypeptide. Those of skill in the art will recognize other methods suitable for maximizing expression of the fusion polypeptide.

[0157] The cell culture comprises host cells, medium, and other by-products. Suitable media for cell culture are well known in the art. The fusion polypeptide can be secreted and isolated from the mixture of medium and cells containing the fusion polypeptide. Alternatively, the fusion polypeptide can be retained in the cytoplasm, and the cells harvested, lysed, and the protein isolated. Protein purification techniques known in the art can be used to isolate the fusion polypeptide from the cell culture medium, the host cells, or both, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification using antibodies specific for a particular epitope of the fusion.

[0158] Thus, nucleotide sequences encoding all or part of a heat shock protein fusion can be used for recombinant production of the protein by microbial or eukaryotic processes. Ligation of the sequences into a polynucleotide construct, such as an expression vector, and transformation or transfection into a eukaryotic (yeast, avian, insect, or mammalian) or prokaryotic (bacterial) host is standard practice. Similar techniques, or variations thereof, may be used to produce recombinant fusion polypeptides by microbiological means or tissue culture techniques in accordance with the present invention.

[0159] Expression vehicles for the production of recombinant proteins include plasmids and other vectors. For example, vectors suitable for the expression of fusion polypeptides include the following types of plasmids for expression in prokaryotic cells such as E. coli: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.

[0160] In another embodiment, the nucleic acid encoding the heat shock protein fusion polypeptide is operably linked to a bacterial promoter, e.g., the anaerobic Escherichia coli NirB promoter, e.g., the E. coli lipoprotein llp promoter described in Inouye et al. (1985) Nucl. Acids Res. 13:3101, the Salmonella pagC promoter (Miller et al., supra), the Shigella ent promoter (Schmitt and Payne, J. Bacteriol. 173:816 (1991)), the tet promoter on Tn10 (Miller et al., supra), or the Vibrio cholera ctx promoter. Any other promoter may also be used. The bacterial promoter may be constitutive or inducible. Exemplary inducible promoters are promoters induced by iron or under iron-limiting conditions. Indeed, it is believed that some bacteria (e.g., intracellular microorganisms) are subject to iron-limiting conditions in the host cytoplasm.Examples of iron-regulated promoters for FepA and TonB are known in the art and are described, for example, in the following references: Headley, V. et al. (1997) Infection & Immunity 65:818; Ochsner, UA et al. (1995) Journal of Bacteriology 177:7194; Hunt, MD et al. (1994) Journal of Bacteriology 176:3944; Svinarich, DM and S. Palchaudhuri. (1992) Journal of Diarrheal Diseases Research 10:139; Prince, RW et al. (1991) Molecular Microbiology 5:2823; Goldberg, MB et al. (1990) Journal of Bacteriology 172:6863; de Lorenzo, V. et al. (1987) Journal of Bacteriology 169:2624; and Hantke, K. (1981) Molecular & General Genetics 182:288.

[0161] The plasmid preferably contains sequences necessary for proper transcription of the nucleic acid in bacteria, such as a transcription termination signal. The vector may further contain sequences encoding factors that allow for the selection of bacteria containing the nucleic acid of interest (e.g., a gene encoding a protein that confers antibiotic resistance), and sequences necessary for amplification of the nucleic acid (e.g., a bacterial origin of replication).

[0162] In another embodiment, a signal peptide sequence is added to the construct to allow secretion of the fusion polypeptide from the cell. Such signal peptides are well known in the art.

[0163] In one embodiment, the strong phage T5 promoter, recognized by E. coli RNA polymerase, is used in conjunction with a lac operator repression module to generate tightly regulated, high-level expression of recombinant proteins in E. coli. In this system, protein expression is prevented in the presence of high concentrations of the lac repressor.

[0164] In one embodiment, the DNA is operably linked to a first promoter, and the bacterium further comprises second DNA encoding a first polymerase capable of mediating transcription from the first promoter, the DNA encoding the first polymerase being operably linked to a second promoter. In a preferred embodiment, the second promoter is a bacterial promoter such as those listed above. In a further preferred embodiment, each of the polymerases is a bacteriophage polymerase (e.g., SP6, T3, or T7 polymerase), and the first promoter is a bacteriophage promoter (e.g., SP6, T3, or T7 promoter, respectively). Plasmids containing bacteriophage promoters and encoding bacteriophage polymerases are commercially available, for example, from Promega (Madison, Wis.) and InVitrogen (San Diego, Calif.), or can be obtained directly from bacteriophage using standard recombinant DNA techniques (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, 1989). Bacteriophage polymerases and promoters are described in detail in, for example, Sagawa, H. et al. (1996) Gene 168:37; Cheng, X. et al. (1994) PNAS USA 91:4034; Dubendorff, J. W. and F. W. Studier (1991) Journal of Molecular Biology 219:45; Bujarski, J. J. and P. Kaesberg (1987) Nucleic Acids Research 15:1337; and Studier, F. W. et al. (1990) Methods in Enzymology 185:60. Such plasmids can be further modified depending on the particular embodiment of the heat shock protein fusion to be expressed.

[0165] In another embodiment, the bacterium further comprises DNA encoding a second polymerase capable of mediating transcription from the second promoter, wherein the DNA encoding the second polymerase is operably linked to a third promoter. The third promoter can be a bacterial promoter. Alternatively, to achieve high levels of transcription, three or more different polymerases and promoters can be introduced into the bacterium. Using one or more polymerases to mediate transcription in bacteria can significantly increase the amount of polypeptide in the bacterium compared to placing DNA directly under the control of a bacterial promoter in the bacterium. The choice of system to employ will depend on the particular application, for example, the desired amount of protein production.

[0166] Generally, a nucleic acid encoding a fusion protein is introduced into a host cell, such as by transfection, and the host cell is cultured under conditions that allow expression of the fusion protein. Methods for introducing nucleic acids into prokaryotic and eukaryotic cells are well known in the art. Suitable media for mammalian and prokaryotic host cell culture are well known in the art. Generally, the nucleic acid is placed under the control of an inducible promoter, and such a promoter is induced after a certain number of divisions of the host cell containing the nucleic acid encoding the fusion protein of interest. For example, if the nucleic acid is placed under the control of a β-galactose operator and repressor, the bacterial host cell will produce OD 600 Once the culture reaches a density of approximately 0.45-0.60, isopropyl-β-D-thiogalactopyranoside (IPTG) is added. The culture is then allowed to grow for a further period, giving the host cells time to synthesize protein. The culture is then typically frozen and can be stored frozen for a period of time prior to protein isolation and purification.

[0167] When prokaryotic host cells are used, the host cells can contain a plasmid expressing an internal T7 lysozyme, for example, expressed from the plasmid pLysSL (see Examples). Upon lysis of such host cells, the lysozyme is liberated, which then degrades the bacterial membrane.

[0168] Other sequences that can be included in vectors for expression in bacteria or other prokaryotic cells include a synthetic ribosome binding site, a strong transcription terminator to prevent transcription readthrough and ensure stability of the expressed protein (e.g., t0 from phage λ and t4 from the rrnB operon in E. coli), an origin of replication (e.g., ColE1), and a β-lactamase gene that confers ampicillin resistance.

[0169] Other host cells include prokaryotic host cells, and more preferred host cells are bacteria (e.g., E. coli). Other bacteria that can be used include Shigella spp., Salmonella spp., Listeria spp., Rickettsia spp., Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Francisella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp. Examples of suitable bacteria include Haemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., and Erysipelothrix spp. Most of these bacteria can be obtained from the American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, VA 20110-2209).

[0170] Many vectors are available for expressing recombinant proteins in yeast. For example, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are useful cloning and expression vehicles for introducing gene constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83). These vectors can replicate in E. coli due to the presence of the pBR322 onori and in S. cerevisiae due to the replication determinant of the yeast 2μ plasmid. In addition, drug resistance markers such as ampicillin may be used.

[0171] In certain embodiments, mammalian expression vectors contain prokaryotic sequences to facilitate amplification of the vector in bacteria and one or more eukaryotic transcription units for expression in eukaryotic cells. Examples of mammalian expression vectors suitable for transfection of eukaryotic cells include pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors. Some of these vectors have been modified with sequences derived from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses, such as bovine papillomavirus (BPV-1) and Epstein-Barr virus (pHEBo, pREP-derived, and p205), can be used for transient protein expression in eukaryotic cells. Methods utilized for constructing plasmids and transforming host organisms are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells and general recombinant procedures, see Chapters 16 and 17 of "Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989). In some cases, it may be desirable to express recombinant proteins using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., pBlueBac III, which contains β-gal).

[0172] In another variant, protein production can be carried out using an in vitro translation system. In vitro translation systems are generally translation systems that are cell-free extracts containing at least the minimum components necessary for translating RNA molecules into proteins. In vitro translation systems generally contain at least ribosomes, tRNA, initiator methionine tRNAMet, and proteins or complexes involved in translation (e.g., eIF2, eIF3, cap-binding (CB) complex (eIF4F) containing cap-binding protein (CBP) and eukaryotic translation initiation factor 4F). Various in vitro translation systems are known in the art, and commercially available kits are also available. Examples of in vitro translation systems include eukaryotic cell lysates such as rabbit reticulocyte lysate, rabbit oocyte lysate, human cell lysate, insect cell lysate, and wheat germ extract. Lysates are commercially available from manufacturers such as Promega, Madison, Wis.; Stratagene, La Jolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO / BRL, Grand Island, NY. In vitro translation systems generally contain macromolecules such as enzymes, translation initiation factors, elongation factors, chemical reagents, and ribosomes. Additionally, in vitro transcription systems may be used. Such systems generally contain at least RNA polymerase holoenzyme, ribonucleotides, and the necessary transcription initiation factors, elongation factors, and termination factors. RNA nucleotides for in vitro translation can be prepared using methods known in the art. In vitro transcription and translation can be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs.

[0173] If expression of a carboxy-terminal fragment of a protein, i.e., a truncated mutant, is desired, it may be necessary to add an initiation codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well known in the art that methionine at the N-terminal position can be enzymatically cleaved using the enzyme methionine aminopeptidase (MAP). MAP has been cloned from Escherichia coli (Ben-Bassat et al., (1987) J. Bacteriol. 169:751-757) and Salmonella typhimurium, and its in vitro activity has been demonstrated with recombinant proteins (Miller et al., (1987) PNAS USA 84:2718-1722). Thus, if removal of the N-terminal methionine is desired, it can be accomplished in vivo by expressing such recombinant protein in a host that produces MAP (e.g., E. coli or CM89 or Saccharomyces cerevisiae), or in vitro by using purified MAP (e.g., the method of Miller et al.).

[0174] When using plant expression vectors, expression of heat shock protein fusions can be driven by any of a number of promoters, including viral promoters such as the 35S and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature, 310:511-514) and the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J., 6:307-311), as well as plant promoters such as the small subunit promoter of RUBISCO (Coruzzi et al., 1994, EMBO J., 3:1671-1680; Broglie et al., 1984, Science, 224:838-843), and heat shock promoters (e.g., soybean Hsp17.5-E or Hsp17.3-B) (Gurley et al., 1986, Mol. Cell. Biol., 6:559-565). These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, etc. For details of such techniques, see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, New York, Section VIII, pp. 421-463, and Grierson & Corey, 1988, Plant Molecular Biology, 2nd Ed., Blackie, London, Ch. 7-9.

[0175] Another expression system that can be used to express protein tags or fusion proteins containing protein tags is an insect system. One example of such a system uses Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector for expressing foreign genes. This virus grows in Spodoptera frugiperda cells. PGHS-2 sequences can be cloned into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter). Successful insertion of the coding sequence will inactivate the polyhedrin gene, resulting in the production of non-protected recombinant virus (i.e., virus lacking the protein coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells, allowing the inserted gene to be expressed (see, e.g., Smith et al., 1983, J. Virol., 46:584; Smith, U.S. Patent No. 4,215,051).

[0176] In one specific embodiment of the insect system, DNA encoding the heat shock protein fusion protein is cloned downstream of the polyhedrin promoter into the pBlueBacIII recombinant transfer vector (Invitrogen, San Diego, Calif.) and transfected into Sf9 insect cells (derived from Spodoptera frugiperda ovary cells, commercially available from Invitrogen, San Diego, Calif.) to generate recombinant virus. After plaque purification of the recombinant virus, high-titer virus stocks are prepared and used to infect Sf9 or High Five™ (BTI-TN-5B1-4 cells, derived from Trichoplusia ni ovary homogenate, commercially available from Invitrogen, San Diego, Calif.) insect cells to produce large amounts of the appropriately post-translationally modified protein of interest.

[0177] In other embodiments, the heat shock protein fusion and the biotin-binding protein are produced separately and then linked together (e.g., non-covalently linked). For example, the heat shock protein fusion and the biotin-binding protein are produced separately in vitro, purified, and then mixed under conditions that allow the tag to be linked to the protein of interest. For example, the heat shock protein and / or the biotin-binding protein can be obtained (isolated) from a source known to contain the protein, produced or harvested from cell culture, produced by cloning and expressing a gene encoding the desired heat shock protein fusion, or chemically synthesized. Additionally, nucleic acid sequences encoding the desired heat shock protein fusions can be chemically synthesized. Such mixtures of protein conjugates can have properties that differ from those of the individual fusion proteins.

[0178] A linker (also called a "linker molecule" or "crosslinker") may be used to link the heat shock protein and the biotin-binding protein. Linkers include chemicals that can react with specific chemical groups in several molecules, usually two, to link these molecules. Most known crosslinkers react with amine, carboxyl, and sulfhydryl groups. The selection of the target chemical group is important because it can affect the biological activity of the protein being linked. For example, maleimides react with sulfhydryl groups and can inactivate Cys-containing proteins that require Cys to be linked to the target. Linkers can be homofunctional (containing the same type of reactive group), heterofunctional (containing different reactive groups), or photoreactive (containing groups that become reactive upon irradiation).

[0179] Linker molecules can affect various properties of the resulting composite. The length of the linker should be considered in light of molecular flexibility during the conjugation process and the availability of the conjugated molecule to its target (e.g., a cell surface molecule). It is believed that longer linkers improve the biological activity and ease of manufacture of the compositions of the present invention. The geometric configuration of the linker can be used to orient the molecule for optimal interaction with the target. Linkers with flexible geometric configurations can be used to bind to other proteins, thereby adapting the conformation of the crosslinked protein. The type of linker can be varied to achieve various other goals. For example, the aryl structure of MBuS has been shown to be less immunogenic than the aromatic spacer of MBS. Furthermore, the hydrophobicity and functionality of the linker molecule can be controlled by the physical properties of the component molecules. For example, the hydrophobicity of a polymer linker can be controlled by the order of the monomer units comprising the polymer (e.g., block polymers with alternating hydrophobic and hydrophilic monomer blocks).

[0180] Chemical techniques for producing and utilizing a wide variety of molecular linkers are well known in the art, and many pre-made linkers used to link molecules are commercially available from vendors such as Pierce Chemical Co., Roche Molecular Biochemicals, and United States Biological.

[0181] The produced and / or isolated heat shock protein is fused to a biotin-binding protein and administered to a subject with a desired biotinylated component sufficient to form a non-covalent association between the biotin moiety and the biotin-binding protein. The heat shock protein fusion and one or more biotinylated components (e.g., tumor cells or tumor antigens) can be administered simultaneously or sequentially. If administered simultaneously, the heat shock protein fusion and one or more biotinylated components (e.g., tumor cells or tumor antigens) can be administered as a mixture or as a non-covalent complex. If administered as a non-covalent complex, the heat shock protein fused to the biotin-binding protein can be non-covalently bound to the desired biotinylated component (e.g., tumor cells or tumor antigens) in vitro or in vivo after production and / or isolation.

[0182] The non-covalent complex can be formed by contacting a heat shock protein fused to a biotin-binding protein with a biotinylated component (e.g., a tumor cell or tumor antigen) under conditions sufficient to promote binding of the biotin-binding protein to biotin, such conditions being known in the art.

[0183] The genes for various heat shock proteins have been cloned and sequenced, any of which may be used to generate heat shock protein fusions, including, but not limited to, gp96 (human: Genebank accession number X15187; Maki et al., Proc. Natl. Acad. Sci. USA 87:5658-5562 (1990); mouse: Genebank accession number M16370; Srivastava et al., Proc. Natl. Acad. Sci. USA 84:3807-3811 (1987)), BiP (mouse: Genebank accession number U16277; Haas et al., Proc. Natl. Acad. Sci. USA 85:2250-2254 (1988); human: Genebank accession number M19645; Ting et al., DNA 7:275-286 (1988)), hsp70 (mouse: Genebank accession number M35021; Hunt et al., Gene 87:199-204 (1990); human: Genebank accession number M24743; Hunt et al., Proc. Natl. Acad. Sci. USA 82:6455-6489 (1995)), and hsp40 (human: Genebank accession number D49547; Ohtsuka K., Biochem. Biophys. Res. Commun. 197:235-240 (1993)).

[0184] A heat shock protein fused to a biotin-binding protein can be non-covalently bound to a biotinylated component (eg, a tumor cell or tumor antigen).

[0185] Tumor cells or tumor antigens administered with heat shock proteins can be conjugated with biotin by means known in the art. Prior to conjugation with biotin, tumor cells or tumor antigens can be produced and / or isolated using methods known in the art. Recombinant techniques can be used in much the same way as described herein for heat shock protein fusions. After production and / or isolation of tumor cells or tumor antigens, one or more biotin molecules can be directly conjugated to the tumor cells or tumor antigens. Biotin can also be indirectly conjugated to tumor cells or tumor antigens via a linker. Biotin should be conjugated to a region that sterically allows interaction between biotin and the biotin-binding protein. Biotinylation kits and reagents are available commercially from Pierce (Rockford, IL) and can be used to produce the biotinylated components described herein.

[0186] The sequences of many different antigens have been cloned and characterized by DNA sequence analysis and can be included in the compositions described herein. Bacterial vectors containing complete or partial cellular or viral genomes or antigens can be obtained from various resource banks, such as the American Tissue Culture Collection (ATCC). Other antigens that can be used can also be isolated and typed by methods well established for this purpose and known in the art.

[0187] immunotherapy In certain embodiments, the self-assembling vaccines described herein can be administered in conjunction with immunotherapy.

[0188] The term "immunotherapy" refers to any treatment that uses specific parts of a subject's immune system to fight diseases, such as cancer. The subject's own immune system is stimulated (or suppressed) with or without the administration of one or more drugs for this purpose. Immunotherapies designed to induce or amplify an immune response are referred to as "immunostimulatory therapy." Immunotherapies designed to decrease or suppress an immune response are referred to as "immunosuppressive therapy." Drugs suspected of having an immune system effect on genetically modified, transplanted cancer cells can be assayed to determine whether they are immunotherapeutic agents and to examine the effect of a given genetic modification on modulating the immune response. In some embodiments, immunotherapy is cancer cell-specific. In some embodiments, immunotherapy can be "non-targeted" therapy, which refers to the administration of drugs that do not selectively interact with immune system cells but instead modulate immune system function. Representative examples of non-targeted therapies include, but are not limited to, chemotherapy, gene therapy, and radiation therapy.

[0189] Immunotherapy is a form of targeted therapy and can involve, for example, the use of cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are potentially useful in cancer treatment because they can infect and lyse cancer cells without harming normal cells. Oncolytic virus replication promotes tumor cell destruction while simultaneously providing dose amplification at the tumor site. Oncolytic viruses can also function as vectors for anti-cancer genes, specifically delivering anti-cancer genes to tumor sites. Immunotherapy can be a form of passive immunization for short-term host protection, achieved by administering preformed antibodies against cancer or disease antigens (e.g., monoclonal antibodies, optionally linked to chemotherapeutic agents or toxins, to tumor antigens). For example, anti-VEGF and mTOR inhibitors have been shown to be effective in treating renal cell carcinoma. Immunotherapy can also focus on the use of epitopes of cancer cell lines that are recognized by cytotoxic lymphocytes. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, and the like can be used to selectively regulate biomolecules associated with tumor or cancer development, progression and / or pathology.

[0190] Immunotherapy can be passive immunization for short-term protection of the host, achieved by administering preformed antibodies against cancer or disease antigens (e.g., monoclonal antibodies, optionally linked to chemotherapeutic agents or toxins, administered to tumor antigens). Immunotherapy can also focus on the use of epitopes in cancer cell lines recognized by cytotoxic lymphocytes. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, and the like can be used to selectively modulate biomolecules associated with tumor or cancer development, progression, and / or pathology.

[0191] In certain embodiments, the immunotherapy described herein includes at least one immunogenic chemotherapy. The term "immunogenic chemotherapy" refers to any chemotherapy that has been demonstrated to induce immunogenic cell death, detectable by the release of one or more damage-associated molecular pattern (DAMP) molecules, including, but not limited to, calreticulin, ATP, and HMGB1 (Kroemer et al. (2013), Annu. Rev. Immunol., 31:51-72). Specific representative examples of consensus immunogenic chemotherapy include, among others, 5'-fluorouracil, anthracyclines (e.g., doxorubicin), and platinum-based agents (e.g., oxaliplatin).

[0192] In certain embodiments, the immunotherapy comprises inhibitors of one or more immune checkpoints. The term "immune checkpoint" refers to a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by downregulating or suppressing the anti-tumor immune response. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, IDO, CD39, CD73, and A2aR (see, e.g., WO 2012 / 177624). The term further encompasses biologically active protein fragments and nucleic acids encoding full-length immune checkpoint proteins and biologically active protein fragments thereof. In certain embodiments, the term further includes any fragment that conforms to the homology descriptions provided herein. In one embodiment, the immune checkpoint is PD-1.

[0193] Immune checkpoints and their sequences are well known in the art, and representative embodiments are described below. For example, the term "PD-1" refers to a member of the immunoglobulin gene superfamily that functions as a co-inhibitory receptor, with its ligands PD-L1 and PD-L2. PD-1 was previously identified using a subtraction cloning approach to select for genes upregulated during TCR-induced activated T cell death. PD-1 is a member of the CD28 / CTLA-4 family of molecules because of its ability to bind to PD-L1. Like CTLA-4, PD-1 is rapidly induced on the surface of T cells in response to anti-CD3 (Agata et al. (1996) Int. Immunol. 8:765). However, unlike CTLA-4, PD-1 is also induced on the surface of B cells (in response to anti-IgM). PD-1 is also expressed on thymocytes and a subset of myeloid cells (Agata et al. (1996) supra; Nishimura et al. (1996) Int. Immunol. 8:773).

[0194] The nucleic acid and amino acid sequences of representative human PD-1 biomarkers are publicly available in the GenBank database as NM_005018.2 and NP_005009.2 (see also Ishida et al. (1992) 20 EMBO J 11:3887; Shinohara et al. (1994) Genomics 23:704; U.S. Patent No. 5,698,520). PD-1 has an extracellular region containing an immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al. (1994) Genomics 23:704; and U.S. Patent No. 5,698,520) and immunoreceptor compatibility tyrosine-based motifs (ITSMs). These characteristics also define a larger family of polypeptides called immunoinhibitory receptors, which also includes gp49B, PIR-B, and killer inhibitory receptors (KIRs) (Vivier and Daeron (1997) Immunol. Today 18:286). The tyrosine-phosphorylated ITIM and ITSM motifs of these receptors are often thought to interact with SH2-domain containing phosphatases, leading to inhibitory signaling. A subset of these immunoinhibitory receptors binds to MHC polypeptides; for example, KIRs and CTLA4 bind to B7-1 and B7-2. A phylogenetic relationship between MHC and B7 genes has been suggested (Henry et al. (1999) Immunol. Today 20(6):285-8). The nucleic acid and polypeptide sequences of PD-1 orthologues in organisms other than humans are known, including, for example, mouse PD-1 (NM_008798.2 and NP_032824.1), rat PD-1 (NM_001106927.1 and NP_001100397.1), canine PD-1 (XM_543338.3 and XP_543338.3), bovine PD-1 (NM_001083506.1 and NP_001076975.1), and chicken PD-1 (XM_422723.3 and XP_422723.2).

[0195] PD-1 polypeptides are inhibitory receptors that can suppress immune cell effector function by transmitting inhibitory signals to immune cells, or, when present in a soluble monomeric form, can promote costimulation of immune cells (e.g., by competitive inhibition). Preferred PD-1 family members are identical in sequence to PD-1 and bind to one or more B7 family members (e.g., B7-1, B7-2), PD-1 ligands, and / or other polypeptides on antigen-presenting cells.

[0196] The term "PD-1 activity" includes the ability of a PD-1 polypeptide to modulate inhibitory signals in activated immune cells, for example, by binding to a natural PD-1 ligand on an antigen-presenting cell. Modulation of inhibitory signals in immune cells modulates immune cell proliferation and / or cytokine secretion by immune cells. Thus, the term "PD-1 activity" includes the ability of a PD-1 polypeptide to bind to its natural ligand, to modulate immune cell costimulatory or inhibitory signals, and to modulate an immune response.

[0197] The term "PD-1 ligand" refers to the binding partner of the PD-1 receptor and includes both PD-L1 (Freeman et al. (2000) J. Exp. Med. 192:1027-1034) and PD-L2 (Latchman et al. (2001) Nat. Immunol. 2:261). At least two human PD-1 ligand polypeptides exist. PD-1 ligand proteins contain a signal sequence, as well as IgV and IgC domains, a transmembrane domain, and a short cytoplasmic tail. PD-L1 (for sequence data, see Freeman et al. (2000)) and PD-L2 (for sequence data, see Latchman et al. (2001) Nat. Immunol. 2:261) are both members of the B7 family of polypeptides. PD-L1 and PD-L2 are both expressed in the placenta, spleen, lymph nodes, thymus, and heart. PD-L2 is also expressed in the pancreas, lung, and liver, whereas PD-L1 is expressed in fetal liver. Both PD-1 ligands are upregulated on activated monocytes and dendritic cells, but PD-L1 expression is more widespread. For example, PD-L1 is known to be constitutively expressed and highly upregulated on murine hematopoietic cells (e.g., T cells, B cells, macrophages, dendritic cells (DCs), and bone marrow-derived mast cells) and non-hematopoietic cells (e.g., endothelial cells, epithelial cells, and muscle cells), whereas PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast cells (Butte et al. (2007) Immunity 27:111).

[0198] PD-1 ligands constitute a family of polypeptides that conserve certain structural and functional characteristics. The term "family," as used with respect to proteins or nucleic acid molecules, refers to two or more proteins or nucleic acid molecules that share a common structural domain or motif and sufficient amino acid or nucleotide sequence homology, as defined herein. Such family members can be natural or non-natural, and can be from the same or different species. For example, a family can include a first protein of human origin and another, different protein of human origin, or it can include a homolog of a non-human origin. Family members can also share common functional characteristics. PD-1 ligands are members of the B7 polypeptide family. As used herein, the terms "B7 family" or "B7 polypeptide" include costimulatory polypeptides that share sequence homology with B7 polypeptides (e.g., B7-1, B7-2, B7h) (Swallow et al. (1999) Immunity 11:423) and / or PD-1 ligands (e.g., PD-L1 or PD-L2). For example, human B7-1 and B7-2 share approximately 26% amino acid sequence identity when compared using the NCBI BLAST program with default parameters (Blosum62 matrix with gap penalties set to existence 11 and extension 1) (see the NCBI website). The term B7 family also includes variants of these polypeptides that can regulate immune cell function. B7 family molecules are conserved in many regions, including the signaling domain, IgV domain, and IgC domain. The IgV domain and IgC domain are Ig superfamily member domains well known in the art. These domains correspond to a structural unit with a distinct folding pattern called the Ig fold. The Ig fold is a sandwich structure of two β-sheets, each composed of antiparallel β-strands of 5-10 amino acids, stacked together in a single layer. The disulfide bond between the two sheets is conserved in most, if not all, domains.The IgC domains of Ig, TCR, and MHC molecules share the same sequence pattern, known as the C1 set within the Ig superfamily. Other IgC domains belong to other sets. IgV domains also share a common sequence pattern, known as the V set domains. IgV domains are longer than IgC domains and have one extra pair of beta strands.

[0199] Preferred B7 polypeptides can promote or suppress immune cell responses by providing costimulatory or inhibitory signals to immune cells. For example, B7 family members that bind to costimulatory receptors enhance T cell activation and proliferation, while B7 family members that bind to inhibitory receptors suppress costimulation. Furthermore, the same B7 family member can increase or decrease T cell costimulation. For example, PD-1 ligand can induce immune cell costimulation when it binds to a costimulatory receptor, and, for example, when present in a soluble form, can suppress immune cell costimulation. PD-1 ligand polypeptides can transmit inhibitory signals to immune cells when it binds to an inhibitory receptor. Preferred B7 family members include B7-1, B7-2, B7h, PD-L1, or PD-L2, and soluble fragments or derivatives thereof. In one embodiment, B7 family members bind to one or more receptors on immune cells (e.g., CTLA4, CD28, ICOS, PD-1 and / or other receptors) and, depending on the receptor, can deliver inhibitory or costimulatory signals to immune cells, preferably T cells.

[0200] Modulation of costimulatory signals results in modulation of immune cell effector functions. Thus, the term "PD-1 ligand activity" includes the ability of a PD-1 ligand polypeptide to bind to its natural receptor (e.g., PD-1 or B7-1), to modulate immune cell costimulatory or inhibitory signals, and to modulate an immune response.

[0201] The term "PD-L1" refers to a specific PD-1 ligand. Two forms of the human PD-L1 molecule have been identified. One form is a native PD-L1 soluble polypeptide, i.e., it has a short hydrophilic domain and no transmembrane domain, and is referred to herein as PD-L1S. The second form is a cell-associated polypeptide, i.e., it has a transmembrane domain and a cytoplasmic domain, and is referred to herein as PD-L1M. The nucleic acid and amino acid sequences of representative human PD-L1 biomarkers for PD-L1M are publicly available in the GenBank database as NM_014143.3 and NP_054862.1. The PD-L1 protein contains an IgV domain and an IgC domain in addition to a signal sequence. In addition, the nucleic acid and polypeptide sequences of PD-L1 orthologs in organisms other than humans are also known, including mouse PD-L1 (NM_021893.3 and NP_068693.1), rat PD-L1 (NM_001191954.1 and NP_001178883.1), canine PD-L1 (XM_541302.3 and XP_541302.3), bovine PD-L1 (NM_001163412.1 and NP_001156884.1), and chicken PD-L1 (XM_424811.3 and XP_424811.3).

[0202] The term "PD-L2" refers to another specific PD-1 ligand. PD-L2 is a B7 family member expressed on various APCs, including dendritic cells, macrophages, and bone marrow-derived mast cells (Zhong et al. (2007) Eur. J. Immunol. 37:2405). PD-L2 expressed on APCs can suppress T cell activation through PD-1 ligation and costimulate T cell activation through a PD-1-independent mechanism (Shin et al. (2005) J. Exp. Med. 201:1531). Furthermore, ligation of PD-L2 expressed on dendritic cells results in enhanced dendritic cell cytokine expression and survival (Radhakrishnan et al. (2003) J. Immunol. 37:1827; Nguyen et al. (2002) J. Exp. Med. 196:1393). The nucleic acid and amino acid sequences of representative human PD-L2 biomarkers are known in the art and are also publicly available in the GenBank database as NM_025239.3 and NP_079515.2. PD-L2 proteins are characterized by common structural elements. In some embodiments, PD-L2 proteins contain at least one of a signal peptide domain, a transmembrane domain, an IgV domain, an IgC domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. As used herein, a "signal sequence" or "signal peptide" refers to a peptide of about 15 or more amino acids at the N-terminus of a secreted or membrane-bound polypeptide that functions to target a polypeptide containing such a sequence to the lipid bilayer and is cleaved in the secreted or membrane-bound polypeptide. The signal sequence contains a number of hydrophobic amino acid residues. For example, the signal sequence contains at least about 10 to 30 amino acid residues, preferably about 15 to 25 amino acid residues, more preferably about 18 to 20 amino acid residues, and even more preferably about 19 amino acid residues, of which at least about 35 to 65% are hydrophobic amino acid residues (e.g., valine, leucine, isoleucine, or phenylalanine), preferably about 38 to 50%, and more preferably about 40 to 45%.In another embodiment, amino acid residues 220-243 of the native human PD-L2 polypeptide and amino acid residues 201-243 of the mature polypeptide comprise a transmembrane domain. As used herein, the term "transmembrane domain" includes an amino acid sequence of approximately 15 amino acid residues in length that spans a cell membrane. More preferably, the transmembrane domain comprises at least about 20, 25, 30, 35, 40, or 45 amino acid residues and spans the cell membrane. Transmembrane domains are rich in hydrophobic residues and generally adopt an α-helical structure. In preferred embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or more of the amino acids in the transmembrane domain are hydrophobic, such as leucine, isoleucine, tyrosine, or tryptophan. Transmembrane domains are described, for example, in Zagotta, W.N. et al. (1996) Annu. Rev. Neurosci. 19:235-263. In yet another embodiment, amino acid residues 20-120 of native human PD-L2 polypeptide and amino acid residues 1-101 of the mature polypeptide comprise an IgV domain. Amino acid residues 121-219 of native human PD-L2 polypeptide and amino acid residues 102-200 of the mature polypeptide comprise an IgC domain. As used herein, the terms IgV domain and IgC domain are recognized in the art as Ig superfamily member domains. These domains correspond to structural units with a distinct folding pattern called the Ig fold. The Ig fold is a sandwich structure consisting of two β-sheets, each composed of antiparallel β-strands of 5-10 amino acids, with the disulfide bond between the two sheets conserved in most, if not all, domains. The IgC domains of Ig, TCR, and MHC molecules share a common sequence pattern, referred to as the C1 set within the Ig superfamily. Other IgC domains belong to other sets. IgV domains also share a common sequence pattern, referred to as the V-set domain. The IgV domain is longer than the IgC domain and has one extra pair of strands.In yet another embodiment, amino acid residues 1-219 of native human PD-L2 polypeptide and amino acid residues 1-200 of the mature polypeptide comprise the extracellular domain. As used herein, the term "extracellular domain" refers to the N-terminal amino acids that extend as a tail from the surface of a cell. The extracellular domain of the present invention comprises an IgV domain and an IgC domain, and optionally a signal peptide domain. In yet another embodiment, amino acid residues 244-273 of native human PD-L2 polypeptide and amino acid residues 225-273 of the mature polypeptide comprise the cytoplasmic domain. As used herein, the term "cytoplasmic domain" refers to the C-terminal amino acids that extend as a tail into the cytoplasm of a cell. In addition, the nucleic acid and polypeptide sequences of PD-L2 orthologs in organisms other than humans are also known, including mouse PD-L2 (NM_021396.2 and NP_067371.1), rat PD-L2 (NM_001107582.2 and NP_001101052.2), canine PD-L2 (XM_847012.2 and XP_852105.2), bovine PD-L2 (XM_586846.5 and XP_586846.3), and chimpanzee PD-L2 (XM_001140776.2 and XP_001140776.1).

[0203] The terms "PD-L2 activity," "PD-L2 biological activity," or "PD-L2 functional activity" refer to activity of a PD-L2 protein, polypeptide, or nucleic acid molecule on a PD-L2-responsive cell or tissue, or on a PD-L2 polypeptide binding partner, as measured in vivo or in vitro according to standard techniques. In one embodiment, PD-L2 activity is a direct activity, such as association with a PD-L2 binding partner. As used herein, a "target molecule" or "binding partner" refers to a molecule to which a PD-L2 polypeptide naturally binds or interacts, such that a PD-L2-mediated function is achieved. In an exemplary embodiment, the PD-L2 target molecule is the receptor RGMb. Alternatively, PD-L2 activity is an indirect activity, such as cell signaling activity mediated by the interaction of a PD-L2 polypeptide with its natural binding partner (i.e., a physiologically important interacting macromolecule involved in immune or other biologically important functions) (e.g., RGMb). The biological activities of PD-L2 are as described herein. For example, the PD-L2 polypeptides of the invention may have one or more of the following activities: 1) binding to and / or modulating the activity of the receptor RGMb, PD-1, or other natural binding partners of PD-L2; 2) modulating intracellular or intercellular signaling; 3) modulating the activation of immune cells (e.g., T lymphocytes); and 4) modulating the immune response of an organism (e.g., a mouse or a human).

[0204] "Immune checkpoint blockade therapy" refers to the use of agents that inhibit immune checkpoint nucleic acids and / or proteins. Inhibition of one or more immune checkpoints can prevent or neutralize inhibitory signaling and upregulate immune responses to more effectively treat cancer. Exemplary agents useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands that can bind to and / or interact with immune checkpoint proteins or fragments thereof, as well as RNA interference, antisense, nucleic acid aptamers, and the like that can downregulate the expression and / or activity of immune checkpoint nucleic acids or fragments thereof. Exemplary agents for upregulating immune responses include antibodies against one or more immune checkpoint proteins that block the interaction of the proteins with their native receptors; inactivated forms of one or more immune checkpoint proteins (e.g., dominant-negative polypeptides); small molecules or peptides that block the interaction of one or more immune checkpoint proteins with their native receptors; fusion proteins that bind to their native receptors (e.g., the extracellular portion of an immune checkpoint inhibitory protein fused to the Fc portion of an antibody or immunoglobulin); and nucleic acid molecules that block the transcription or translation of immune checkpoint nucleic acids. Such agents can directly block the interaction of one or more immune checkpoint proteins with their native receptors (e.g., antibodies), preventing inhibitory signaling and upregulating the immune response. Alternatively, some agents can indirectly block the interaction of one or more immune checkpoint proteins with their native receptors, preventing inhibitory signaling and upregulating the immune response. For example, soluble forms of immune checkpoint protein ligands, such as stabilized extracellular domains, can bind to their receptors, indirectly reducing the effective concentration of the receptors that can bind to appropriate ligands. In one embodiment, anti-PD-1 antibodies, anti-PD-L1 antibodies, and / or anti-PD-L2 antibodies, alone or in combination, are used to inhibit immune checkpoints.These embodiments are also applicable to specific therapies directed against specific immune checkpoints, such as the PD-1 pathway (e.g., PD-1 pathway blockade therapy, also known as PD-1 pathway inhibitor therapy).

[0205] In a preferred embodiment, the immunotherapy used in the compositions and methods of the present invention is an agent that inhibits PD1 or PD-L1. Such agents include, but are not limited to, small molecule inhibitors, CRISPR guide RNA (gRNA), RNA interference agents, antisense oligonucleotides, peptide or peptidomimetic inhibitors, aptamers, antibodies, or intrabodies. In a specific embodiment, the agent that inhibits PD1 or PD-L1 is a PD1 or PD-L1 blocking antibody. Exemplary anti-PD-1 antibodies that can be used in the present invention include, but are not limited to, Keytruda (Merck, Inc.).

[0206] In some embodiments, the immunotherapy used in the compositions and methods of the present invention is an immunomodulatory agent. Such agents include, but are not limited to, CXCR4 / CXCR7 antagonists (e.g., AMD3100), Jak / stat inhibitors (e.g., ruxolitinib), and near-infrared laser immunomodulation of skin-associated immune cells. Examples of near-infrared laser immunomodulation of skin-associated immune cells are described in Kimizuka, Y. et al. J. Immun. 2018, 201(12) 3587-3603 and Gelfand, J. et al. FASEB J. 2019, 33(2), 3074-3081, the entire disclosures of which are incorporated herein by reference.

[0207] Pharmaceutical Compositions and Administration In some embodiments, the pharmaceutical compositions provided herein comprise a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated moiety (e.g., a tumor cell or tumor antigen). In some embodiments, the pharmaceutical composition further comprises an immunotherapy (e.g., an anti-PD-1 antibody). In particular embodiments, the tumor antigen that is biotinylated and non-covalently bound to the heat shock protein fusion is a peptide selected from Table 1 or Table 3. The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier.

[0208] The heat shock protein fusion and biotinylated component (e.g., tumor cell or tumor antigen) produced as described above can be purified to a purity suitable for use as a pharmaceutical composition. Generally, a purified composition will have a single component species that represents greater than about 85% of all component species present in the composition, greater than about 85%, 90%, 95%, 99%, or more of all component species present. The component species of interest can be purified to near homogeneity (i.e., contaminating component species cannot be detected in the composition by conventional detection methods), and the composition will consist essentially of a single component species. Given the teachings herein, one of skill in the art will be able to purify the heat shock protein fusion and biotinylated component (e.g., tumor cell or tumor antigen), or noncovalent complexes thereof, using standard purification techniques, such as immunoaffinity chromatography and size exclusion chromatography. Protein purity can be measured by numerous methods known to those of skill in the art, including, for example, terminal amino acid sequence analysis, gel electrophoresis, and mass spectrometry.

[0209] Thus, provided are pharmaceutical compositions comprising the heat shock protein fusion and a biotinylated component (e.g., a tumor cell or tumor antigen), or a noncovalent complex thereof. In one aspect, provided are pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the pharmaceutical compositions described herein combined with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. In another aspect, in certain embodiments, the pharmaceutical compositions can be administered neat or in admixture with a pharmaceutically acceptable carrier, and may also be administered in combination with other agents. Thus, combination therapy includes sequential, simultaneous, and separate administration, i.e., coadministration, in which the therapeutic effect of a previously administered agent is not completely lost upon administration of a subsequent agent.

[0210] The heat shock protein fusion and biotinylated component (e.g., tumor cell or tumor antigen), or noncovalent complex thereof, as described herein, can be administered to a subject in a variety of ways. Administration routes include systemic, peripheral, parenteral, enteral, topical, and transdermal (e.g., slow-release polymers). Any other suitable route of administration can also be used, including infusion or bolus injection, or absorption through epithelial or mucocutaneous linings. Additionally, the compositions described herein can contain and be administered in the presence or absence of other pharmacologically acceptable components, such as biologically active substances (e.g., adjuvants such as alum), surfactants (e.g., glycerides), excipients (e.g., lactose), vehicles, diluents, and additives. Furthermore, the compositions can be used ex vivo as a means of stimulating leukocytes obtained from a subject for in vitro induction, expansion, and proliferation of antigen-specific immune cells, followed by reintroduction into the subject.

[0211] For pharmaceutical compositions containing biotinylated tumor cells, 0.1 × 10 per kg of subject body weight 6 pieces, 0.2×10 6 pieces, 0.3×10 6 pieces, 0.4×10 6 pieces, 0.5×10 6 pieces, 0.6×10 6 pieces, 0.7×10 6pieces, 0.8×10 6 pieces, 0.9×10 6 pieces, 1.0×10 6 pieces, 5.0×10 6 pieces, 1.0×10 7 pieces, 5.0×10 7 pieces, 1.0×10 8 pieces, 5.0×10 8 In some cases, tumor cells can be administered in amounts of 1 x 10 per kg of body weight, or more, or any range or number therebetween. The number of cells implanted can be adjusted based on the level of engraftment desired within a given time period. Generally, 1 x 10 per kg of body weight is administered as needed. 5 ~Approx. 1×10 9 Approximately 1 x 10 per kg of body weight 6 ~Approx. 1×10 8 or approximately 1 x 10 per kg of body weight 7 In certain embodiments, a total of at least about 0.1 x 10 cells can be transplanted for an average sized mouse. 6 pieces, 0.5×10 6 pieces, 1.0×10 6 pieces, 2.0×10 6 pieces, 3.0×10 6 pieces, 4.0×10 6 pieces, or 5.0 x 10 6 Transplantation of individual cells is effective.

[0212] Administration can be carried out using methods generally known in the art. The pharmaceutical composition comprising the cells can be introduced to the desired site by direct injection or any other means used in the art, including but not limited to intravascular, intracerebral, parenteral, intraperitoneal, intravenous, epidural, intraspinal, intrasternal, intraarticular, intrasynovial, intrathecal, intraarterial, intracardiac, or intramuscular administration.

[0213] For example, transplanted cells can be engrafted into a target subject via a variety of routes. These routes include, but are not limited to, intravenous administration, subcutaneous administration, administration to a specific tissue (e.g., local implantation), injection into the femoral medullary cavity, injection into the spleen, and administration under the renal capsule of a fetal liver. In certain embodiments, the cancer vaccines of the present invention are injected intratumorally or subcutaneously into a subject. Cells may be administered in a single infusion or by continuous infusion over a predetermined period sufficient to produce the desired effect. Exemplary methods for transplantation, engraftment assessment, and marker phenotyping of transplanted cells are well known in the art (see, e.g., Pearson et al. (2008) Curr. Protoc. Immunol. 81:15.21.1-15.21.21; Ito et al. (2002) Blood 100:3175-3182; Traggiai et al. (2004) Science 304:104-107; Ishikawa et al. Blood (2005) 106:1565-1573; Shultz et al. (2005) J. Immunol. 174:6477-6489; and Holyoake et al. (1999) Exp. Hematol. 27:1418-1427).

[0214] Furthermore, the pharmaceutical compositions of the present invention can be administered to a subject or applied externally to a subject in a biocompatible form suitable for pharmaceutical administration. A "biocompatible form suitable for in vivo administration" refers to an administration form in which the therapeutic effects outweigh any toxic effects. Administration of pharmaceutical compositions as described herein can be in any pharmacological form containing a therapeutically active amount of a drug, alone or in combination with a pharmaceutically acceptable carrier. As used herein, the term "therapeutically effective amount" refers to an amount of a drug effective to produce some desired therapeutic effect (e.g., cancer treatment) at a reasonable benefit / risk ratio.

[0215] Administration of a therapeutically active amount of a pharmaceutical composition of the present invention is defined as an amount effective to achieve the desired result at the dosage and for the time required. For example, a therapeutically active amount of a drug may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the peptide to induce a desired response in the individual. Dosage regimens can be adjusted to obtain the optimal therapeutic response. For example, divided doses may be administered daily, or the dose may be proportionally reduced as required by the therapeutic situation.

[0216] The combined formulation or co-administration of the individual agents results in an effective amount of each desired modulator being present in the patient at one time.

[0217] The pharmaceutical compositions described herein can be administered in any suitable manner, such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active agent can be coated with a material to protect the active agent from the action of enzymes, acids, and other natural conditions that may inactivate the active agent. For example, for administration of pharmaceutical compositions by routes other than parenteral administration, it may be desirable to coat the pharmaceutical composition with, or administer in combination with, a material to prevent the inactivation of the pharmaceutical composition.

[0218] The pharmaceutical composition may be administered to an individual in a suitable vehicle, diluent, or adjuvant, or may be administered in combination with an enzyme inhibitor or encapsulated in a suitable vehicle such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. The term "adjuvant" is used in its broadest sense and includes any immunostimulant, such as interferon. Adjuvants contemplated herein include resorcinol and nonionic surfactants (e.g., polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether). Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEEP), and trasylol. Liposomes include water-in-oil-in-water (W / O / W) emulsions and conventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

[0219] The pharmaceutical composition can be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, or in oils. Under normal storage and use conditions, preservatives can be added to these preparations to prevent the growth of microorganisms.

[0220] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the compositions are preferably sterile and must be fluid to the extent that easy puncture is possible. They are also preferably stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferable to add isotonic agents (e.g., sugars, polyalcohols such as mannitol and sorbitol, sodium chloride) to the compositions. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0221] Sterile injection solution can be prepared by adding the pharmaceutical composition of the present invention in the required amount to a suitable solvent together with one or a combination of the above-mentioned components as needed, and then sterilizing by filtration.Generally, dispersion is prepared by adding an active agent to a sterile solvent containing a basic dispersion medium and other necessary components selected from the above-mentioned components.For the preparation of sterile powder for sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying, and powder is obtained from a solution of the active agent and other desired components that has been previously sterilized by filtration.

[0222] Compositions for administration include solutions in which the pharmaceutical composition is dissolved in a pharmaceutically acceptable base, such as an aqueous base. Various aqueous bases can be used, including, for example, buffered saline solutions. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances (e.g., pH adjusting / buffering agents, toxicity adjusting agents, etc.) necessary to approximate physiological conditions (e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.). The concentration of the biomarker-specific substance in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., depending on the particular administration method selected and the needs of the subject.

[0223] The pharmaceutical compositions are provided as sterile solutions of known concentrations, but may also be provided in lyophilized form and rehydrated with sterile water prior to administration. Actual methods for preparing administrable compositions will be recognized or understood by those skilled in the art and are detailed in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).

[0224] When the pharmaceutical composition is suitably protected as described above, it can be orally administered, for example, with an inert diluent or an assimilable edible base. As used herein, "pharmaceutically acceptable base" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and additives for pharmaceutically active substances is well known in the art. Except insofar as it is incompatible with the active agent, use of any conventional media or additive in pharmaceutical compositions is contemplated. Supplementary active agents can also be added to the compositions.

[0225] It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, "dosage unit form" means physically discrete units suitable as unitary doses for the mammalian subject to be treated, each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect together with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by and directly depend on (a) the inherent characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the constraints inherent in the art of formulating such active agents with respect to treating hypersensitivity in individuals.

[0226] Furthermore, heat shock protein fusion proteins can be administered by in vivo expression of nucleic acids encoding such protein sequences in human subjects. Expression of such nucleic acids and contact with biotinylated components (e.g., tumor cells or tumor antigens) can also be performed ex vivo as a means of stimulating leukocytes obtained from a subject for reintroduction into the subject after in vitro induction, expansion, and proliferation of antigen-specific immune cells. Expression vectors suitable for driving expression of heat shock protein fusion proteins can be selected from a wide variety of vectors currently used in the art. Vectors capable of producing high levels of expression and effective for introducing genes of interest are preferred. For example, recombinant adenovirus vector pJM17 (All et al., Gene Therapy 1:367-84 (1994); Berkner KL, Biotechniques 6:616-24 (1988)), second-generation adenovirus vector DE1 / DE4 (Wang and Finer, Nature Medicine 2:714-6 (1996)), or adeno-associated virus vector AAV / Neo (Muro-Cacho et al., J. Immunotherapy 11:231-7 (1992)) can be used. Furthermore, recombinant retrovirus vectors MFG (Jaffee et al., Cancer Res. 53:2221-6 (1993)) or LN, LNSX, LNCX, LXSN (Miller and Rosman, Biotechniques 7:980-9 (1989)) can be used. Alternatively, herpes simplex virus vectors such as pHSV1 (Geller et al., Proc. Nat'l Acad. Sci. 87:8950-4 (1990)) or vaccinia virus vectors such as MVA (Sutter and Moss, Proc. Nat'l Acad. Sci. 89:10847-51 (1992)) can be used.

[0227] Commonly used specific expression units containing a promoter and 3' sequence include those incorporated in the plasmids CDNA3 (Invitrogen), AH5, pRC / CMV (Invitrogen), pCMU II (Paabo et al., EMBO J. 5:1921-1927 (1986)), pZip-Neo SV (Cepko et al., Cell 37:1053-1062 (1984)), and pSRa (DNAX, Palo Alto, CA). Introduction of genes into expression units and / or vectors can be carried out using genetic engineering techniques, such as those described in manuals such as Molecular Cloning and Current Protocols in Molecular Biology (Sambrook, J., et al., Molecular Cloning, Cold Spring Harbor Press (1989); Ausubel, F. M., et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience (1989)). The resulting expressible nucleic acid can be introduced into the cells of a human subject by any method that allows the nucleic acid to be introduced into cells in an expressible form, such as as part of a viral vector as described above, as naked plasmid or other DNA, or encapsulated in targeted liposomes or erythrocyte ghosts (Friedman, T., Science, 244:1275-1281 (1989); Rabinovich, N. R. et al., Science. 265:1401-1404 (1994)).Methods of introduction include direct injection into tissues and tumors, liposome transfection (Fraley et al., Nature 370:111-117 (1980)), receptor-mediated endocytosis (Zatloukal et al., Ann. NY Acad. Sci. 660:136-153 (1992)), and particle gun gene transfer (Eisenbraun et al., DNA & Cell. Biol. 12:791-797 (1993)).

[0228] The amount of heat shock protein fusion and biotinylated component (e.g., tumor cell or tumor antigen) or noncovalent complex thereof in the compositions of the present invention is an amount that generates an effective immunostimulatory response in a subject. An effective amount is an amount that generates an immune response when administered. Furthermore, the amount of heat shock protein fusion and biotinylated component or noncovalent complex thereof administered to a subject will vary depending on various factors, including the heat shock protein fusion and biotinylated component used, the subject's size, age, weight, general health, sex, and diet, as well as their general immune responsiveness. Adjustment and manipulation of the established dose range is within the skill of those in the art. For example, the amount of heat shock protein fusion, biotinylated component, or noncovalent complex thereof can be about 1 microgram to about 1 gram, preferably about 100 micrograms to about 1 gram, or about 1 milligram to about 1 gram. An effective amount of a composition containing an expression vector is an amount that, when administered, induces an immune response against the antigen targeted by the pharmaceutical composition. Furthermore, the amount of expression vector administered to a subject will vary depending on various factors, including the heat shock protein fusion being expressed, the subject's size, age, weight, general health, sex, and diet, as well as their general immune responsiveness. Other factors that need to be considered are the route of administration and the type of vector used. For example, when administering prophylactic or therapeutic treatment using a viral vector containing a nucleic acid encoding a heat shock protein fusion, an effective amount would be 10 mg of helper-free replication-deficient virus per kg of body weight. 4 ~10 12 The range is 10 per kg of body weight. 5 ~1011 10 per kg of body weight is more preferable. 6 ~10 10 would be most preferable.

[0229] Determination of an effective amount of fusion protein and biotinylated component, or non-covalent complex thereof, effective to induce an immune response in a subject is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0230] Effective doses can be estimated initially from in vitro assays. For example, doses can be formulated in animal models to induce an immune response using techniques well known in the art. Those skilled in the art can readily optimize human administration based on animal data. Dosage and administration intervals can be adjusted individually. For example, when used as a vaccine, the protein and / or cell line of the present invention can be administered approximately one to three times over a period of one to 36 weeks. Three doses, spaced approximately three to four months apart, may be administered, followed by periodic booster vaccinations. Alternative protocols suitable for individual patients are possible. An appropriate dose is the amount of protein or cell line, when administered as described above, capable of generating an immune response in an immunized patient sufficient to protect the patient from disease or infection for at least one to two years.

[0231] The composition may further comprise an adjuvant to enhance the immune response. The protein may also be suspended in an oil emulsion to delay the release of such proteins in vivo after injection. The optimal ratio of each component in the formulation can be determined by techniques well known to those skilled in the art.

[0232] Any of a variety of adjuvants can be used in the vaccines of the present invention to enhance the immune response. Most adjuvants contain substances to protect the antigen from rapid catabolism (e.g., aluminum hydroxide or mineral oil) and specific or nonspecific stimulators of the immune response (e.g., lipid A or Bordetella pertussis). Suitable adjuvants are commercially available, including, for example, incomplete Freund's adjuvant and complete Freund's adjuvant (Difco Laboratories) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A, quil A, SBAS1c, SBAS2 (Ling et al., 1997, Vaccine 15:1562-1567), SBAS7, Al(OH)3, and CpG oligonucleotides (WO96 / 02555).

[0233] In the vaccines of the present invention, the adjuvant can induce a Th1-type immune response. Suitable adjuvant systems include, for example, monophosphoryl lipid A, preferably a combination of 3-O-deacylated monophosphoryl lipid A (3D-MPL) and an aluminum salt. Enhanced systems include a combination of monophosphoryl lipid A and a saponin derivative, particularly a combination of 3D-MLP and the saponin QS21, as disclosed in WO 94 / 00153, or a composition in which QS21 has been detoxified with cholesterol to reduce reactive symptoms, as disclosed in WO 96 / 33739. Previous experiments have demonstrated a clear synergistic effect of the combination of 3D-MLP and QS21 in inducing both humoral and Th1-type cellular immune responses. WO95 / 17210 describes that QS21, 3D-MLP and tocopherol suspended in an O / W (oil-in-water) emulsion form a particularly potent adjuvant, which can be added to the formulation.

[0234] Treatment method The present invention provides prophylactic and therapeutic methods for treating subjects at risk (or susceptible to) or currently suffering from cancer. The cancer can be a solid cancer or a hematopoietic cancer. The cancer can be a sarcoma or carcinoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, hemangiosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, leiomyosarc ... Examples of cancers that may be present include bladder cancer, epithelial carcinoma, glioma, biliary tract cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, head and neck cancer, anal cancer, and heavy chain disease.

[0235] In certain embodiments, the cancer is ovarian cancer, such as serous or epithelial papillary ovarian cancer. In some embodiments, the cancer is induced by infection with an oncogenic virus (e.g., HPV, HCV, EBV, HIV, or herpes virus). In certain embodiments, the cancer can be an HPV-associated cancer (e.g., human papillomavirus (HPV)-induced cervical cancer, HPV-induced head and neck cancer, or HPV-induced anal cancer).

[0236] In one embodiment, the cancer is of the same cancer type or has the same genetic mutation as the biotinylated tumor cells or tumor antigen, hi another embodiment, the cancer is of a different cancer type or has a different genetic mutation than the biotinylated tumor cells or tumor antigen.

[0237] The heat shock protein fusions and biotinylated components (e.g., tumor cells or tumor antigens) described herein can be administered to a subject to induce or enhance an anti-tumor immune response in the subject. The heat shock protein fusions can simply enhance the immune response (i.e., function as immunogenic compositions) or can confer protective immunity (i.e., function as vaccines). Accordingly, the present application also provides methods for inducing an immune response using the pharmaceutical compositions described herein.

[0238] a. Preventive methods In one aspect, the present invention provides a method of preventing ovarian cancer in a subject by administering to the subject an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently linked to a biotinylated peptide, and the peptide is selected from Table 1 or Table 3.

[0239] In another aspect, the present invention provides a method for preventing cancer (e.g., ovarian cancer, head and neck cancer, anal cancer, or cervical cancer) in a subject by administering to the subject an effective amount of a pharmaceutical composition comprising: (1) a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated tumor cell or a biotinylated tumor antigen (e.g., one or more peptides selected from Table 1 or Table 3); and (2) an immunotherapy (e.g., an anti-PD-1 antibody).

[0240] In yet another aspect, the present invention provides a method for preventing cancer (e.g., ovarian cancer, head and neck cancer, anal cancer, or cervical cancer) in a subject, comprising administering to the subject an immunotherapy (e.g., an anti-PD-1 antibody) in combination with an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated tumor cell or a biotinylated tumor antigen (e.g., one or more peptides selected from Table 1 or Table 3).

[0241] In yet another aspect, the invention provides a method for preventing an HPV-associated cancer (e.g., head and neck cancer or anal cancer) in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated HPV virus or a biotinylated HPV viral antigen. In certain embodiments, the subject has or has been exposed to HPV.

[0242] Administration of a prophylactic agent (e.g., a pharmaceutical composition described herein) can occur before the manifestation of symptoms characteristic of cancer, such that cancer is prevented or delayed in its progression. In certain embodiments, administration of a prophylactic agent (e.g., a pharmaceutical composition described herein) protects the subject from recurrent cancer.

[0243] b. Therapeutic methods Another aspect of the present invention relates to a method of treating a subject suffering from cancer. For example, in one aspect, the present invention provides a method of treating ovarian cancer in a subject by administering to the subject an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently linked to a biotinylated peptide, and the peptide is selected from Table 1 or Table 3.

[0244] In another aspect, the present invention provides a method of treating cancer (e.g., ovarian cancer, head and neck cancer, anal cancer, or cervical cancer) in a subject by administering to the subject an effective amount of a pharmaceutical composition comprising: (1) a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated tumor cell or a biotinylated tumor antigen (e.g., one or more peptides selected from Table 1 or Table 3); and (2) an immunotherapy (e.g., an anti-PD-1 antibody).

[0245] In yet another aspect, the present invention provides a method of treating cancer (e.g., ovarian cancer, head and neck cancer, anal cancer, or cervical cancer) in a subject, comprising administering to the subject an immunotherapy (e.g., an anti-PD-1 antibody) in combination with an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated tumor cell or a biotinylated tumor antigen (e.g., one or more peptides selected from Table 1 or Table 3).

[0246] In yet another aspect, the present invention provides a method for treating an HPV-associated cancer (e.g., head and neck cancer or anal cancer) in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated HPV virus or a biotinylated HPV viral antigen.

[0247] c. Combination therapy In certain embodiments, the self-assembling vaccines described herein can be administered in conjunction with immunotherapy. The immunotherapy and the self-assembling vaccine can be administered simultaneously or sequentially. For example, the self-assembling vaccine can be administered before, simultaneously with, or after the immunotherapy.

[0248] The pharmaceutical compositions described herein can also be administered in combination with non-targeted therapies (e.g., chemotherapeutic agents, hormones, anti-angiogenic agents, radiolabeled compounds, surgery, cryotherapy, and / or radiation therapy). The term "non-targeted therapy" refers to the administration of a substance that does not selectively interact with a selected biomolecule but still treats cancer. Representative examples of non-targeted therapies include, but are not limited to, chemotherapy, gene therapy, and radiation therapy.

[0249] In one embodiment, chemotherapy is used. Chemotherapy involves the administration of chemotherapeutic agents, including, but not limited to, those selected from the following classes of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, antimitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogs, plant alkaloids and toxins, and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents such as cisplatin, treosulfan, and trofosfamide; plant alkaloids such as vinblastine, paclitaxel, and docetaxel; DNA topoisomerase inhibitors such as teniposide, crisnatol, and mitomycin; folate antagonists such as methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs such as 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs such as mercaptopurine and thioguanine; DNA antimetabolites such as 2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and mitotic inhibitors such as halichondrin, colchicine, and rhizoxin. Compositions containing one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG includes fludarabine, cytosine arabinoside (Ara-C), and G-CSF. CHOP includes cyclophosphamide, vincristine, doxorubicin, and prednisone. The above examples of chemotherapeutic agents are illustrative and not limiting. For example, the pharmaceutical compositions described herein can be administered in combination with a therapeutically effective dose of a chemotherapeutic agent. In another embodiment, the pharmaceutical composition is administered in combination with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The Physicians' Desk Reference (PDR) discloses dosages of chemotherapeutic agents used to treat various cancers. Therapeutically effective dosing regimens and dosages of these chemotherapeutic agents vary depending on the particular cancer being treated, the extent of the disease, and other factors known to physicians in the art, and can be determined by a physician.

[0250] In another embodiment, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external beam radiation therapy, interstitial implantation of radioisotopes (I-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, P32 intraperitoneal radiation therapy, and / or total abdominal and pelvic radiation therapy. For a general discussion of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J.B. Lippencott Company, Philadelphia. Radiation therapy can be performed as external beam radiation therapy or teletherapy, in which radiation is delivered from a distant source. Radiation therapy can also be performed as internal beam radiation therapy or brachytherapy, in which a radioactive source is placed inside the body near the cancer cells or tumor mass. Also contemplated is the use of photodynamic therapy, which involves the administration of photosensitizers such as hematoporphyrin and its derivatives, verteporfin (BPD-MA), phthalocyanines, photosensitizer Pc4, demethoxyhypocrelin A, and 2BA-2-DMHA.

[0251] In another embodiment, hormone therapy is used. Hormone therapy treatment can include, for example, hormone agonists, hormone antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogens, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)), vitamin D3 analogs, antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0252] Another embodiment uses hyperthermia, a method of exposing body tissue to high temperatures (up to 106°F). Heat helps shrink tumors by damaging cells and stripping them of substances they need to survive. Hyperthermia can be local, regional, or whole-body hyperthermia using external and internal heating devices. Hyperthermia is almost always used in conjunction with other therapies (e.g., radiation therapy, chemotherapy, and biological therapy) to enhance their effectiveness. Local hyperthermia refers to the heating of a very small area, such as a tumor. This area can be heated externally using radiofrequency waves applied to the tumor from an external device. To achieve internal heating, one of several sterile probes can be used, including thin heated tubes or hollow tubes filled with warm water; piercing microwave antennas; and radiofrequency electrodes. Regional hyperthermia involves heating an organ or limb. A magnet and a high-energy device are placed over the area to be heated. Another approach, called perfusion, involves removing a portion of the patient's blood, heating it, and then pumping it into the area to be heated. Whole-body hyperthermia is used to treat metastatic cancer that has spread throughout the body. This method can be performed using a warm water blanket, heated wax, induction coils (similar to those used in electric blankets), or a heating chamber (similar to a large incubator). Hyperthermia does not cause a significant increase in radiation-related side effects or complications. However, because it directly heats the skin, it can cause discomfort or significant local pain in approximately half of treated patients. Blisters may occur, but they generally heal quickly.

[0253] In yet another embodiment, photodynamic therapy (PDT, also known as photoradiotherapy, phototherapy, or photochemotherapy) is used to treat some types of cancer. This method is based on the discovery that certain chemicals called photosensitizers can kill certain types of single-celled organisms when exposed to light. PDT destroys cancer cells by combining a certain frequency of laser light with the photosensitizer. In PDT, the photosensitizer is injected into the bloodstream and absorbed by cells throughout the body. The photosensitizer remains in cancer cells longer than normal cells. When treated cancer cells are exposed to laser light, the photosensitizer absorbs the light, producing reactive oxygen species, which destroy the treated cancer cells. Light exposure must be carefully timed so that most of the photosensitizer has been excreted from healthy cells but is still present in cancer cells. The laser light used in PDT can be transmitted through optical fibers (very thin glass threads). The optical fibers are positioned near the cancer to deliver the appropriate amount of light. To treat lung cancer, the fiber optic can be guided into the lungs through a bronchoscope, and to treat esophageal cancer, it can be guided into the esophagus through an endoscope. An advantage of PDT is that it minimizes damage to healthy tissue. However, because currently used laser light cannot penetrate tissue thicker than approximately 3 centimeters (slightly larger than 1.125 inches), PDT is primarily used to treat tumors on or just below the surface of the skin or the lining of internal organs. Photodynamic therapy can cause skin and eye sensitivity to light for at least six weeks after treatment. Patients are advised to avoid direct sunlight and strong indoor light for at least six weeks. If patients must go outdoors, they should wear protective clothing, including sunglasses. Other temporary side effects of PDT are related to specific treatment sites and include coughing, swallowing, abdominal pain, and painful breathing or shortness of breath. In December 1995, the U.S. Food and Drug Administration (FDA) approved a photosensitizer called porfimer sodium, or Photofrin®, for the relief of symptoms from obstructing esophageal cancer and for esophageal cancer that has not responded satisfactorily to laser treatment alone. In January 1998, the FDA approved porfimer sodium for the treatment of early-stage non-small cell lung cancer in patients for whom conventional lung cancer treatments are inadequate.The National Cancer Institute and other organizations are sponsoring clinical trials (research studies) to evaluate the use of photodynamic therapy in several types of cancer, including bladder cancer, brain cancer, laryngeal cancer, and oral cancer.

[0254] In yet another embodiment, laser therapy is used to destroy cancer cells with high-intensity light. This technique is often used to relieve cancer symptoms, such as bleeding and blockage, especially when other treatments fail to cure the cancer. It can also be used to treat cancer by shrinking or destroying tumors. The term "laser" refers to the amplification of light by stimulated emission of radiation. Ordinary light, such as light from a light bulb, has many wavelengths and spreads in all directions. Laser light, on the other hand, has a specific wavelength and is focused into a narrow beam. This type of high-intensity light contains a large amount of energy. Lasers are very powerful and can be used to cut steel or shape diamonds. Lasers can also be used to repair damaged retinas inside the eye (in place of scalpels) or for very precise surgical procedures, such as cutting tissue. There are several different types of lasers, but only three are widely used in medical applications. First, there is the carbon dioxide (CO2) laser, which can remove a thin layer of skin from the surface without penetrating deep into the skin. This technique is particularly useful for treating tumors that have not spread deep into the skin and certain precancerous conditions. As an alternative to traditional scalpel surgery, CO2 lasers can also cut the skin. For this reason, they are used to remove skin cancer. Next, there is the neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. Light from this laser can penetrate deeper into tissue than light from other types of lasers and rapidly coagulate blood. It can also be delivered via fiber optics to difficult-to-reach areas of the body. This type of laser is sometimes used to treat laryngeal and pharyngeal cancer. Finally, there is the argon laser. This laser penetrates only the surface layers of tissue, making it useful in dermatological and ophthalmic surgery. It is also used with photosensitive dyes to treat tumors in a procedure called photodynamic therapy (PDT). Lasers offer several advantages over standard surgical tools: They are more precise than scalpels. They rarely come into contact with surrounding skin or other tissue, protecting tissue near the incision. The heat generated by the laser sterilizes the surgical site, reducing the risk of infection.The precision of lasers allows for smaller incisions, shortening the required surgical time. Healing times are often faster because laser heat seals blood vessels, reducing bleeding, swelling, and scarring. Laser surgery can be less cumbersome. For example, fiber optics can be used to guide laser light to internal body parts without large incisions. More procedures can be performed in outpatient settings. Lasers can be used to treat cancer in two ways: by shrinking or destroying tumors with heat, or by activating chemicals called photosensitizers that destroy cancer cells. In PDT, photosensitizers accumulate in cancer cells and, when stimulated with light, produce a reaction that kills the cancer cells. CO2 and Nd:YAG lasers have been used to shrink or destroy tumors. These lasers can be used with endoscopes, which are tubes that doctors use to examine certain internal body parts, such as the bladder. Some lasers can transmit light through flexible endoscopes equipped with fiber optics. This allows doctors to aim the laser beam with great precision, allowing them to view and treat internal parts that are otherwise inaccessible. Lasers can also be used in conjunction with a low-power microscope, allowing physicians to clearly visualize the treatment area. When used with other devices, laser systems can create incisions as small as 200 microns in diameter, smaller than the width of a microfine thread. Lasers are used to treat many types of cancer. Laser surgery is the standard treatment for certain stages of glottic (vocal cord), cervical, skin, lung, vaginal, vulvar, and penile cancer. In addition to its use to destroy cancer, laser surgery is also used to help relieve symptoms caused by cancer (palliative care). For example, lasers can be used to shrink or destroy tumors blocking a patient's trachea (throat), making breathing easier. Lasers are also sometimes used for palliation in colon and anal cancer. Laser interstitial thermotherapy (LITT) is one of the latest developments in laser therapy. LITT uses the same concept as a cancer treatment called hyperthermia: heat can help shrink tumors by damaging cells or depriving them of substances they need to survive.In this treatment, a laser is directed into the interstitial space (the area between organs) of the body, where the laser light increases the temperature of the tumor and damages or destroys cancer cells.

[0255] The above-described therapeutic methods and / or pharmaceutical compositions can be used in combination with other forms of conventional therapy (e.g., standard cancer treatments known to those of skill in the art), and can be administered sequentially before or after the conventional therapy. The administration duration and / or dose of the cancer vaccine can vary depending on the particular self-assembling vaccine or the particular combination therapy. Those of skill in the art will understand the appropriate administration duration for a particular cancer therapeutic agent. The present invention contemplates the continuous evaluation of the optimal administration schedule for each cancer therapeutic agent, with the subject's cancer phenotype, as determined by the methods of the present invention, being a factor in determining the optimal administration dose and schedule.

[0256] kit The present invention provides kits for expressing or administering a heat shock protein fused to a biotin-binding protein. Such kits can be comprised of a nucleic acid encoding the heat shock protein fused to a biotin-binding protein. The nucleic acid can be carried on a plasmid or vector (e.g., a bacterial plasmid or a viral vector). Other kits include a heat shock protein fused to a biotin-binding protein. In some embodiments, the kit can further include an immunotherapy, such as an anti-PD-1 antibody. Additionally, the present invention provides kits for producing and / or purifying a heat shock protein fused to a biotin-binding protein. The kits described herein can optionally include biotinylated tumor cells and / or tumor antigens. In certain embodiments, such kits include tumor cells and / or tumor antigens as described herein and a biotinylation agent.

[0257] The present invention provides kits for preventing and / or treating cancer in a patient. For example, the kits can include one or more of the pharmaceutical compositions described above, and optionally instructions for use thereof. In yet another embodiment, the present invention provides kits including one or more pharmaceutical compositions and one or more devices for administering such compositions.

[0258] The kit components can be packaged for manual, partially automated, or fully automated performance of the methods. In other embodiments of the kit, instructions for its use can be provided.

[0259] The following examples describe other embodiments of the present invention. The following examples further illustrate the present invention but should not be construed as limiting the invention to these examples. The contents and figures of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference. [Example]

[0260] [Example 1] Treatment of ovarian cancer Study design Ovarian cancer patients often present at a later stage of disease progression, resulting in tumors with fewer mutations that are not easily detected by the immune system. This study used an immunocompetent mouse model of ovarian cancer. Similar to the human disease, the ID8 model exhibits fewer mutations, as seen in later-stage patients, and recurrent disseminated tumors. Previously, anti-PD-1 therapy has shown limited efficacy in the ID8 model. To maximize the potential of SAV targeting ovarian cancer, we combined an ID8-specific SAV with anti-PD-1 antibody therapy. Given the advantages and challenges of ovarian cancer therapy and the ID8 model system, we selected this approach as a "high-bar" test of the SAV cancer approach, aiming to demonstrate improved survival in mice administered the vaccine alone and potentially enhance the efficacy of anti-PD-1 antibody therapy.

[0261] b. Treatment and results In our SAV cancer vaccine trial for ovarian cancer, we tested mutant proteins (neoantigens) and peptides derived from proteins over-abundant in tumors as a basis for providing the immune system with a broad set of tumor targets (see Table 1). Mice were first injected with ovarian cancer cells and then vaccinated 10 days later with SAV containing tumor-targeting peptides. Administration of anti-PD-1 antibodies began 3 days after vaccination and continued every 3 days until day 60 of the experiment. Mice were monitored daily, with tumor growth measured weekly for the first 4 weeks.

[0262] The results demonstrated that administration of anti-PD-1 antibodies, SAV cancer vaccines, or a combination of both extended the survival of tumor-bearing mice compared with control mice treated with either peptides or MAV proteins alone. Notably, administration of the SAV cancer vaccine alone or in combination with an anti-PD-1 antibody improved survival compared with mice treated with the anti-PD-1 antibody alone. The most significant improvement in survival was observed in mice treated with the SAV cancer vaccine in combination with an anti-PD-1 antibody, with three of seven mice surviving for more than 100 days. In contrast, although initial survival was improved in mice treated with an anti-PD-1 antibody, overall survival in this group was not improved compared with control mice. Taken together, the survival data demonstrate that the combination of SAV and an anti-PD-1 antibody can improve outcomes for ovarian cancer patients. Immunological studies of tumor-infiltrating lymphocytes revealed that the SAV / anti-PD-1 antibody combination resulted in the highest levels of immune cell proliferation of all treatment groups, which was at least partly responsible for the improved survival of this group of mice compared to mice receiving other treatments (see Figure 2).

[0263] c. Conclusion This study confirmed the presence of targetable proteins and mutations in the ID8 model of ovarian cancer. Next, candidate sequences derived from the selected tumor targets were identified, and peptides were designed and synthesized to construct SAVs targeted to ID8 tumors. The results demonstrated that the SAV cancer vaccine was effective alone and demonstrated synergistic effects when combined with immunotherapy (e.g., anti-PD-1 antibody therapy). Larger mouse groups were used to provide sufficient statistical power and to provide a clear measure of the efficacy of the SAV platform in preclinical cancer settings.

[0264] Example 2: Hypothetical Example - Treatment of Ovarian Cancer in a Preclinical Model The promising results obtained in the challenging model system described in Example 1 suggest further refinement and investigation of the SAV cancer vaccine. The SAV cancer platform can be improved in several ways. First, we will use variants of MAVs developed in other anti-cancer trials to improve immune system stimulation. Second, we will measure the strength and specificity of the immune response to cancer-targeting peptides delivered by the SAV platform. Next, we will compare these results with previous reports of other peptide-based approaches to identify underperforming peptides and guide further optimization. This evaluation of immune stimulation will include a second class of tumor-derived peptides, called phosphopeptides. Although not included in the above studies, these peptides can also be recognized by the immune system and increase the number of targets within the tumor. The ID8 model can use a range of cancer cells at an early dose. Reducing the tumor cell dose slows tumor growth. Given the challenging nature of the ID8 model, we will evaluate mice injected with reduced amounts of cancerous cells. Previous experiments have shown that reducing cell numbers slows tumor growth and improves overall survival. The advantage of this approach is that the vaccine will fight tumors for longer and reduce the overall burden of fighting them. Finally, we will conduct large-scale trials of the modified SAV cancer vaccine that are sufficiently powered to provide robust statistics on survival and assays to generate clear data on the performance of the SAV platform.

[0265] After refinements are made, large-scale studies will be conducted with sufficient statistical power to provide robust descriptions of changes in immune function and tumor biology in response to treatment, as well as survival groups. All studies will include detailed descriptions of the immune system using CyTOF, providing a broader and more detailed assessment than traditional flow cytometry can provide. Tumors will be archived for later retrieval using RNAseq, providing information on the behavior of both cancer and immune cells within the tumor. Archived tumors can also be used for layered imaging analysis to map the distribution of drugs and immune cells in the tumor.

[0266] Example 3: Hypothetical Example - Treating HPV-Associated Cancer Murine model systems that are amenable to immunotherapy and vaccination will also be explored. Three model systems will be evaluated. The first system is a model of human papillomavirus (HPV)-induced cervical cancer, the second most common gynecological cancer, and provides a number of well-validated HPV viral antigens. The efficacy of SAV for the prevention and / or treatment of HPV-associated head and neck and anal cancers will also be evaluated.

[0267] HPV16 is used to generate the TC-1 model. The vaccine is designed to target the E6 and / or E7 epitopes. Because C57 and Balb / c mice have different MHC alleles, the peptides for C57 and Balb / c mice are different (see Table 4). C57BL / 6 female mice are used. Epitopes derived from HPV E6 and E7 proteins are selected from consensus sequences in the literature (see Table 5). 21 days for production. st No optimization by Century is required.

[0268] [Table 4]

[0269] The peptides used in this study are as follows: E6 QLLRREVYDFAFRDLC (SEQ ID NO: 3) E7 GQAEPDRAHYNIVTFCCKCD (SEQ ID NO: 4)

[0270] [Table 5]

[0271] This list is a literature citation and is not intended to represent an exhaustive search. They correspond to predictions obtained using publicly available MHC epitope selection tools. They represent consensus sequences used in numerous studies of HPV-driven tumor models.

[0272] The sequences used overlap with sequences applicable to human tumors driven by HPV-16. These sequences share approximately 70% homology with HPV types 31, 33, 45, 58, and 73. A different peptide set can be used for human testing. For example, the human HLA-DR11 allele binds to the peptide HPV16E6 amino acid (AA) 52-62, but HPV16E6AA43-57 was used.

[0273] Literature reference All publications, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including definitions therein, will control.

[0274] All polynucleotide and polypeptide sequences that cite accession numbers correlating to entries in public databases, such as those maintained on the World Wide Web by The Institute for Genomic Research (TIGR) at tigr.org and / or those maintained on the World Wide Web by the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov, are also incorporated herein by reference in their entirety.

[0275] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A pharmaceutical composition comprising an immune checkpoint inhibitor and a heat shock protein fused with a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to biotinylated tumor cells, biotinylated tumor antigens, biotinylated HPV virus, or biotinylated HPV virus antigens.

2. The pharmaceutical composition according to claim 1, wherein the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1.

3. The pharmaceutical composition according to claim 2, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

4. The biotinylated tumor cells are biotinylated fibrosarcoma cells, biotinylated myxosarcoma cells, biotinylated liposarcoma cells, biotinylated chondrosarcoma cells, biotinylated ossogenic sarcoma cells, biotinylated chordoma cells, biotinylated angiosarcoma cells, biotinylated intravascular sarcoma cells, biotinylated lymphangiosarcoma cells, biotinylated intralymphatic sarcoma cells, biotinylated synovial tumor cells, biotinylated mesothelioma cells, biotinylated Ewing's tumor cells, biotinylated leiomyosarcoma cells, biotinylated rhabdomyosarcoma cells, and biotinylated colon cancer cells. Cells, biotinylated colorectal cancer cells, biotinylated pancreatic cancer cells, biotinylated breast cancer cells, biotinylated ovarian cancer cells, biotinylated prostate cancer cells, biotinylated squamous cell carcinoma cells, biotinylated basal cell carcinoma cells, biotinylated adenocarcinoma cells, biotinylated sweat gland carcinoma cells, biotinylated sebaceous gland carcinoma cells, biotinylated papillary carcinoma cells, biotinylated papillary adenocarcinoma cells, biotinylated cystadenocarcinoma cells, biotinylated medullary carcinoma cells, biotinylated bronchogenic carcinoma cells, biotinylated renal cell carcinoma cells, biotinylated hepatoma cells, biotinylated biliary tract carcinoma Cells, biotinylated choriocarcinoma cells, biotinylated seminoma cells, biotinylated embryonic carcinoma cells, biotinylated Wilms' tumor cells, biotinylated cervical cancer cells, biotinylated testicular tumor cells, biotinylated lung cancer cells, biotinylated small cell lung cancer cells, biotinylated bladder cancer cells, biotinylated epithelial carcinoma cells, biotinylated glioma cells, biotinylated astrocytoma cells, biotinylated medulloblastoma cells, biotinylated craniopharyngioma cells, biotinylated ependymoma cells, biotinylated pineal glandoma cells, biotinylated angioblastoma cells, biotinylated angioblastoma cells A pharmaceutical composition according to any one of claims 1 to 3, wherein the cells are neurotumor cells, biotinylated oligodendroglioma cells, biotinylated meningioma cells, biotinylated melanoma cells, biotinylated neuroblastoma cells, biotinylated retinoblastoma cells, biotinylated leukemia cells, biotinylated polycythemia vera cells, biotinylated lymphoma cells, biotinylated multiple myeloma cells, biotinylated Waldenström macroglobulinemia cells, biotinylated head and neck cancer cells, biotinylated anal cancer cells, or biotinylated heavy chain disease cells.

5. The pharmaceutical composition according to claim 4, wherein the biotinylated tumor cells are biotinylated ovarian cancer cells.

6. The pharmaceutical composition according to claim 5, wherein the biotinylated ovarian cancer cells are biotinylated serous or epithelial papillary ovarian cancer cells.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the biotinylated tumor cells are biotinylated HPV-related cancer cells.

8. The pharmaceutical composition according to claim 7, wherein the biotinylated HPV-related cancer cells are biotinylated HPV-induced head and neck cancer cells, biotinylated HPV-induced cervical cancer cells, or biotinylated HPV-induced anal cancer cells.

9. The pharmaceutical composition according to any one of claims 1 to 3, wherein the tumor antigen comprises a full-length or partially inactivated tumor-producing virus, or a protein derived from a tumor-producing virus or an immunogenic fragment thereof.

10. The pharmaceutical composition according to claim 9, wherein the tumor-producing virus is human papillomavirus (HPV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), or herpesvirus.

11. The pharmaceutical composition according to any one of claims 1 to 3, wherein the tumor antigen is derived from ovarian cancer cells.

12. The pharmaceutical composition according to any one of claims 1 to 3, wherein the tumor antigen is one or more peptides selected from Table 1.

13. The pharmaceutical composition according to any one of claims 1 to 3, wherein the tumor antigen is derived from HPV-related cancer cells.

14. The pharmaceutical composition according to claim 13, wherein the HPV-related cancer cells are HPV-induced head and neck cancer cells, HPV-induced cervical cancer cells, or HPV-induced anal cancer cells.

15. The pharmaceutical composition according to any one of claims 1 to 3, wherein the HPV virus is a full-length or partially inactivated HPV virus.

16. The pharmaceutical composition according to any one of claims 1 to 3, wherein the biotinylated HPV virus antigen is selected from Table 3.

17. A pharmaceutical composition according to any one of claims 1 to 16 for inducing an immune response in a target.

18. Use of the pharmaceutical composition according to any one of claims 1 to 16 for the manufacture of a pharmaceutical for inducing an immune response in a target.

19. A pharmaceutical composition according to any one of claims 1 to 16 for preventing and / or treating a target cancer.

20. The pharmaceutical composition according to claim 19, wherein the cancer is ovarian cancer or HPV-related cancer.

21. Use of the pharmaceutical composition according to any one of claims 1 to 16 for the manufacture of a pharmaceutical for preventing and / or treating cancer in a subject.

22. The use according to claim 21, wherein the cancer is ovarian cancer or HPV-related cancer.

23. A pharmaceutical composition comprising a heat shock protein fused with a biotin-binding protein for preventing and / or treating cancer in a subject, wherein the pharmaceutical composition is administered in combination with immunotherapy, and the biotin-binding protein is non-covalently bound to biotinylated tumor cells, biotinylated tumor antigens, biotinylated HPV virus, or biotinylated HPV virus antigens.

24. The pharmaceutical composition according to claim 23, wherein the cancer is ovarian cancer or HPV-related cancer.

25. Use of a pharmaceutical composition comprising a heat shock protein fused with a biotin-binding protein for the manufacture of a pharmacopoeia for the prevention and / or treatment of cancer in a subject, wherein the pharmaceutical composition is administered in combination with immunotherapy, and the biotin-binding protein is non-covalently bound to biotinylated tumor cells, biotinylated tumor antigens, biotinylated HPV virus, or biotinylated HPV virus antigens.

26. The use according to claim 25, wherein the cancer is ovarian cancer or HPV-related cancer.

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