Cancer treatment with pre-existing microbial immunity

Abstract

Cancer treatments with pre-existing microbial immunization are provided. The present application relates to methods, compositions, and kits for redirecting a pre-existing immune response in an individual to reduce or stabilize cancer in an individual.

Description

[0001] This application is a divisional application of the Chinese patent application with the filing date of November 6, 2018, application number: 201880071646.5, and invention title "Cancer treatment by immunization with pre-existing microorganisms". Technical Field

[0002] The present invention relates to immunology and cancer therapy, including methods, compositions and kits for directing the existing immune response of a patient against cancer. Background Art

[0003] In healthy individuals, persistent asymptomatic viral infections are usually controlled by cell-mediated immunity and / or humoral immunity, but can reactivate in immunocompromised individuals. Cell-mediated immunity against some chronic viral infections increases with age and results in the induction of many fully functional virus-specific T cells. Cytomegalovirus (CMV) is a beta-herpesvirus that is highly prevalent globally (infecting 50 - 90% of the population) and is usually asymptomatic in healthy individuals. CMV establishes a lifelong persistent infection that requires long-term cellular immunity to prevent disease. Thus, CMV reactivation is a threat in immunosuppressed situations (e.g., in hematopoietic stem cell transplantation). In immunocompetent individuals, CD4 and CD8 T cell responses against CMV exhibit broad reactivity and high intensity against multiple CMV antigens, are highly prevalent in the general population, and increase with age (M. Bajwa et al., J Infect Dis 215, 1212 - 20 (2017)). Memory inflation, a hallmark of persistent cytomegalovirus infection, has been extensively studied in humans. CMV-specific CD8+ T cell responses can be divided into two types depending on whether they expand over time (inflationary) or remain quiescent after primary infection subsides (non-inflationary) (G. A. O'Hara, Trends Immunol 33:84 - 90 (2012)). During persistent CMV infection, the nature of the antigen and the pattern of antigen expression result in CD8+ T cells with a memory phenotype (non-inflationary) or an effector phenotype (inflationary). Mouse CMV infection also establishes a lifelong persistent infection by inducing an immune response that mimics the immune response against CMV in humans (Id).

[0004] The induction of anti-tumor T cell responses is crucial in the development of effective immunotherapies against cancer. Only a subset of cancer patients respond to current immunotherapies. Generation of T cell immunity against cancer antigens typically requires highly personalized approaches or relies on pre-existing anti-cancer T cells. In cancer patients, especially in the elderly, it is also difficult to generate robust de novo T cell immunity. Personalized approaches rely on vaccines against tumor-associated antigens, neoantigens (i.e., mutated self-antigens), or viral oncoproteins. Other methods are based on the adoptive transfer of chimeric antigen receptor-transduced T cells or the infusion of monoclonal antibodies, which require laborious identification of tumor-specific antigens and are applicable only to a subset of cancer types or subtypes. Finally, the adoptive transfer of ex vivo-expanded tumor-specific lymphocytes is a method aimed at harnessing naturally occurring anti-tumor responses. All of these methods are highly personalized and require the identification of tumor epitopes and / or ex vivo expansion of patient autologous cells.

[0005] In parallel, cytokine- or TLR ligand-based in situ tumor immunotherapies have been used, but they mainly target innate immune recognition mechanisms to alter the tumor immune microenvironment, thereby triggering immunogenic cancer cell death and promoting antigen epitope spreading.

[0006] Accordingly, there remains a need for a simple, widely applicable, antigen-agnostic immunotherapy approach to harness the role of the immune system in early and long-term cancer control by direct killing and promoting epitope spreading, respectively. SUMMARY OF THE INVENTION

[0007] The inventors have recognized that the complex adaptive cell-mediated immunity that has evolved over the years to strongly control chronic viral infections in aging individuals is the type of cell-mediated immunity that is effective in controlling tumor growth. To harness this type of anti-viral immunity to treat cancer, the inventors have developed a new method of in situ immunotherapy by targeting the tumor environment directly with highly functional pre-existing anti-viral T cells, either by using tumor-tropic papillomavirus pseudovirions or by in situ injection of minimal viral CD8 and CD4 T cell cytomegalovirus (CMV) epitopes. Presentation of viral epitopes in the tumor environment leads to in situ recruitment and activation of virus antigen-specific T cells, resulting in killing of otherwise virus-negative tumor cells and alterations in the tumor microenvironment. This method addresses the unmet need because it meets all the criteria for a successful immunotherapy by promoting and establishing both early and long-term cancer cell killing and epitope spreading.

[0008] Accordingly, the present disclosure provides methods of treating cancer in an individual by recruiting a pre-existing immune response to the site of the cancer to thereby treat the cancer. The pre-existing immune response can be an immune memory response present in an individual prior to being diagnosed with cancer. The pre-existing immune response can be a naturally occurring pre-existing immune response.

[0009] In these methods, recruiting the pre-existing immune response to cancer cells can include introducing an antigen not expressed by the cancer cells into the cancer prior to the start of treatment, wherein the antigen is recognized by one or more components of the pre-existing immune response.

[0010] These methods can include confirming that the individual has a pre-existing immune response to the antigen prior to introducing the antigen into the tumor. These methods can also include assessing the individual's pre-existing immune response to the antigen. In these methods, confirming the existence of the pre-existing immune response can include identifying a T cell response to the antigen in a sample from the individual.

[0011] In these methods, introducing the antigen can include injecting the antigen into the cancer. Additionally or alternatively, introducing the antigen can be accomplished by introducing a nucleic acid molecule encoding the antigen into the cancer. In these methods, the nucleic acid molecule can be DNA or RNA. For use of RNA, the RNA can be modified such that it is more resistant to degradation. The nucleic acid molecule can be introduced into cancer cells by injection. Additionally or alternatively, a viral vector or pseudovirion such as a papillomavirus pseudovirion can be used to introduce the nucleic acid molecule into the cancer.

[0012] In these methods, the antigen can be a viral antigen. For example, the antigen can be a polypeptide comprising at least one epitope from a cytomegalovirus (CMV) protein, which is recognized by one or more components of the pre-existing immune response. In these methods, the CMV protein can be selected from the group consisting of: pp50, pp65, pp150, IE-1, IE-2, gB, US2, US6, UL16, and UL18. The polypeptide can be a 9-15 mer MHC I-restricted peptide. Additionally or alternatively, the polypeptide can be at least a 15 mer MHC II-restricted peptide. Additionally or alternatively, the antigen comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 1-67. In these methods, one or more components of the immune response can be T cells.

[0013] In these methods, recruitment of the pre-existing immune response can alter the cancer microenvironment.

[0014] In these methods, the antigen can be administered in combination with a reagent that enhances the immune response. Exemplary reagents include reagents selected from the following: TLR agonists; IL-1R8 cytokine antagonists; intravenous immunoglobulin (IVIG); peptidoglycan isolated from Gram-positive bacteria; lipoteichoic acid isolated from Gram-positive bacteria; lipoproteins isolated from Gram-positive bacteria; lipoarabinomannan isolated from mycobacteria, zymosan isolated from yeast cell walls; polyadenylic acid - polyuridylic acid; poly(IC) (poly (IC)); lipopolysaccharide; monophosphoryl lipid A; flagellin; Gardiquimod; Imiquimod; R848; oligonucleotides containing CpG motifs, CD40 agonists, and 23S ribosomal RNA. In an exemplary method, the antigen can be administered in combination with poly-IC.

[0015] On the other hand, kits are provided for testing a patient and recruiting a pre-existing immune response to the site of cancer in the patient. These kits can include at least one CMV peptide antigen or nucleic acid encoding the peptide, a pharmaceutically acceptable carrier, a container, and a package insert or label indicating the administration of the CMV peptide, and the kits are for reducing at least one symptom of cancer in the patient.

[0016] This summary is neither intended nor should it be construed to represent the full extent and scope of the invention. Moreover, references herein to "the present disclosure" or aspects thereof should be understood to refer to certain embodiments of the invention and should not necessarily be construed to limit all embodiments to a particular description. The present disclosure is set forth in various levels of detail in this summary as well as in the drawings and the detailed description, and in this summary it is not intended to limit the scope of the present disclosure by including or excluding elements, components, etc. Other aspects of the invention will become apparent from the detailed description, particularly when used in conjunction with the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A Shows that murine cytomegalovirus (mCMV) infection induces a large-scale cytokine response against an mCMV peptide pool. Figure 1B Shows IFN-gamma production by splenic CD4+ and CD8+ T cells after peptide restimulation with the indicated MHC-I and MHC-II restricted mCMV peptides.

[0018] Figure 2A Shows an injection protocol for intratumoral transduction of solid tumors with an HPV Psv expressing mCMV antigen. Figure 2B and 2CThe tumor volumes were shown separately after intratumoral injection of HPV16 Psv expressing m122 and m45 or HPV Psv expressing red fluorescent protein (RFP).

[0019] Figure 3A An injection protocol for intratumoral transduction of solid tumors with HPV Psv expressing mCMV antigen in combination with poly(I:C) (PIC) was depicted. Figure 3B - 3E This intratumoral transduction protocol was shown to slow down tumor growth. Figure 3F and 3G The infiltration of tumors by E7, m45, and m122-specific CD8+ T cells was shown by MHC-I tetramer staining and FACS analysis.

[0020] Figure 4A The effect on survival was shown, and Figure 4B The effect on tumor growth after intratumoral injection of MCMV MHC-I-restricted peptides in C57Bl / 6 mice infected with murine cytomegalovirus (mCMV) was shown.

[0021] Figure 5 The effect on tumor growth after intratumoral injection of different doses of mCMV MHC-I-restricted peptides in C57Bl / 6 mice infected with murine cytomegalovirus (mCMV) was shown.

[0022] Figure 6A and 6B The effect on tumor growth after intratumoral injection of a combination of mCMV MHC-I and MHC-II-restricted peptides in C57Bl / 6 mice infected with mCMV was shown. Figure 6C The E7, m45, m122-specific CD8+ T cell responses in the blood, as analyzed by FACS using MHC-I tetramers for each peptide, were shown, demonstrating that sequential intratumoral vaccination with mCMV CD4 followed by CD8 epitopes preferentially induces anti-tumor immunity.

[0023] Figure 7 The effect of complete clearance of primary tumors on long-term protection against secondary tumor challenges was shown.

[0024] Figure 8 mCMV infection was shown to induce an expansive CD8+ T cell response in C57Bl / 6 mice.

[0025] Figure 9A The production of IFN-γ by both expansive and non-expansive CD8+ T cells and the production of IFN-γ by CD4+ T cells were shown. Figure 9B The cytokine production by mCMV CD8+ T cells against an MHC-I-restricted peptide pool was shown.

[0026] Figure 10A Shows the experimental protocol timing for the murine TC1 tumor model with intratumoral administration of mCMV peptides. Figure 10B and 10C Shows the distribution of mCMV-specific CD8+ T cells in tumor-bearing mice. Expansionary (IE3; Figure 10B ) and non-expansionary (m45; Figure 10C ) specific CD8+ T cells were detected by FACS using MHC-I tetramer staining.

[0027] Figure 11A Shows the experimental protocol timing for the murine TC1 tumor model for gene expression analysis of the tumor microenvironment. Figure 11B - 11F Shows the tumor infiltration of CD45+ cells ( Figure 11B ), Th1 cells ( Figure 11C ), cytotoxic CD8 T cells ( Figure 11D ), NK cells ( Figure 11E ), or dendritic cells ( Figure 11F ) after intratumoral treatment.

[0028] Figure 12A and 12B Shows that intratumoral injection of mCMV CD8 epitopes delays tumor growth. Co-injection of poly(I:C) improves tumor control. Figure 12A Shows the effect of intratumoral injection of MHC-I-restricted mCMV peptides + / - poly(I:C) alone. Figure 12B Shows the effect of intratumoral injection of titrated MHC-I-restricted mCMV peptides.

[0029] Figure 13A and 13B Shows protection against TC1 tumor challenge by intratumoral injection of mCMV MHC-I and / or MHC-II peptides with poly(I:C). Sequential intratumoral inoculation with CD4, then CD8 MCMV epitopes inhibits tumor growth ( Figure 13A ) and promotes long-term survival ( Figure 13B ).

[0030] Figure 14 Shows the E7 tetramer-positive CD8+ T cell response in blood after 6 treatments with MHC-I-restricted selected m38, m45, and m122 peptides and / or MHC-II-restricted selected m139 peptide with or without poly(I:C) (30 ug) and saline or poly(I:C) alone as a control.

[0031] Figure 15 Shows that complete clearance of the primary tumor confers long-term protection against secondary tumor challenge.

[0032] Figure 16 Showing protection against MC38 tumor challenge by intratumoral injection of mCMV MHC-I and MHC-II peptides together with poly(I:C). Detailed Description

[0033] The present invention relates to novel methods for treating cancer. In particular, the present invention relates to methods for treating cancer in an individual by harnessing the individual's own immune system to attack cancer cells. The method exploits the fact that an individual has a pre-existing immune response that was initially not triggered in response to cancer, but rather by microorganisms in the environment. Since cancer cells typically do not express the microbial antigens that gave rise to the pre-existing immune response, it cannot be expected that such an immune response will attack the cancer. However, the inventors have found that such pre-existing immune responses can be recruited to attack cancer. One way to achieve this is by introducing one or more antigens recognized by the pre-existing immune response into the cancer, thereby causing the cells of the immune response to attack the cancer cells displaying the antigen. Thus, these methods do not target cancer cells that express the antigen prior to treating the cancer patient. For example, many glioblastoma cancer cells are found to express CMV antigens, and the methods of the present disclosure would not be used to treat such glioblastomas using the individual's pre-existing immunity against CMV. Additionally, the destruction of cancer cells can lead to the exposure of components of the pre-existing immune response to cancer cell antigens. This can lead to the initiation of an immune response against the cancer cell antigens. Thus, the general method of the present invention can be practiced by recruiting the pre-existing immune response in an individual to the site of the cancer such that the pre-existing immune response attacks the cancer. For example, recruitment can be achieved by introducing at least one antigen recognized by components of the individual's pre-existing immune response (such as T cells) into the cancer.

[0034] The present invention is not limited to the specific embodiments described herein, as they may vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0035] As used herein and in the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. Thus, the terms "a", "an", "one or more", and "at least one" may be used interchangeably. Similarly, the terms "comprising", "including", and "having" may be used interchangeably. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terms, such as "solely", "only", etc., or the use of "negative" limitations in the recitation of claim elements.

[0036] Certain features of the invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination for sake of brevity. All combinations of embodiments are expressly covered by the invention and are disclosed herein as if each combination was individually and expressly disclosed. Additionally, all sub-combinations are expressly covered by the invention and are disclosed herein as if each such sub-combination was individually and expressly disclosed herein.

[0037] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the provided publication dates may be different from the actual publication dates, which may need to be independently confirmed. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0039] In one aspect is a method of treating cancer in an individual, which comprises recruiting a pre-existing immune response to the cancer so as to treat the cancer.

[0040] As used herein, cancer refers to a disease in which abnormal cell division occurs without proper control of cell division and / or cell senescence. The term cancer is intended to cover solid tumors as well as hematogenous cancers. Generally, a tumor is an abnormal mass of tissue that typically does not contain cysts or areas of fluid. Solid tumors can be benign (not life-threatening) or malignant (life-threatening). Different types of solid tumors are named for the cell types that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. Hematologic cancers (also known as hematological cancers) are cancers that begin in cells in the blood-forming tissues (such as the bone marrow) or the immune system. Examples of hematologic cancers include leukemia, lymphoma, and multiple myeloma.

[0041] In some cancers, cells can invade tissues other than the tissue from which the original cancer cells originated. In some cancers, cancer cells can spread to other parts of the body through the blood and lymphatic systems. Thus, cancers are generally named for the organ or cell type in which they begin. For example, a cancer that originates in the colon is called colon cancer; a cancer that originates in the melanocytes of the skin is called melanoma, etc. As used herein, cancer can refer to carcinoma, sarcoma, adenocarcinoma, lymphoma, leukemia, etc., including solid and lymphoid cancers, gastric cancer, kidney cancer, breast cancer, lung cancer (including non-small cell and small cell lung cancer), bladder cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer (including hepatocarcinoma), lymphoma, including non-Hodgkin lymphoma (e.g., Burkitt's, small cell and large cell lymphoma) and Hodgkin lymphoma, leukemia, and multiple myeloma. In an exemplary embodiment, the cancer is lung cancer or adenocarcinoma.

[0042] As used herein, the terms individual, subject, patient, etc. are intended to encompass any mammal capable of developing cancer, where the preferred mammal is a human. The terms individual, subject, and patient do not, by themselves, denote a particular age, gender, race, etc. Thus, the present disclosure is intended to encompass individuals of any age, male or female. Similarly, the methods of the present invention can be applied to any human race, including, for example, Caucasians (whites), African Americans (blacks), Native Americans, Native Hawaiians, Hispanics, Latinos, Asians, and Europeans. Such characteristics may be important. In such cases, the important characteristics (e.g., age, gender, race, etc.) will be indicated. These terms also encompass both humans and non-human animals. Suitable non-human animals for testing or treating cancer include, but are not limited to, companion animals (i.e., pets), food animals, working animals, or zoo animals.

[0043] As used herein, an immune or immunological response refers to the presence of a humoral and / or cellular response in an individual against one or more antigens. For the purposes of the present disclosure, a "humoral response" refers to an immune response mediated by B cells and antibody molecules (including secretory (IgA) or IgG molecules), while a "cellular response" is an immune response mediated by T lymphocytes and / or other white blood cells. An important aspect of cellular immunity involves the antigen-specific response of cytotoxic T cells (CTLs). CTLs are specific for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) on the cell surface. CTLs help induce and promote the destruction of intracellular microorganisms, or the lysis of cells infected by such microorganisms. Another aspect of cellular immunity involves the antigen-specific response of helper T cells. Helper T cells function to help stimulate the function of nonspecific effector cells against cells that display peptide antigens associated with MHC molecules on their surface and to focus the activity of such nonspecific effector cells. Cellular immune responses also refer to the production of cytokines, chemokines, and other such molecules by activated T cells and / or other white blood cells (including those derived from CD4+ and CD8+ T cells).

[0044] Accordingly, an immunological response can be a response that stimulates the production or activation of CTLs, and / or helper T cells. It can also stimulate the production of chemokines and / or cytokines. An immune response can also comprise an antibody-mediated immune response. Thus, an immunological response can include one or more of the following actions: production of antibodies (such as IgA or IgG) by B cells; and / or activation of inhibitory, cytotoxic, or helper T cells and / or T cells specific for an antigen. Such responses can be determined using standard immunoassays and neutralization assays known in the art.

[0045] As used herein, a pre-existing immune response is an immune response that exists in an individual prior to the commencement of cancer treatment. Thus, prior to the commencement of treatment of cancer using an antigen, an individual with a pre-existing immune response has an immune response against the antigen. A pre-existing immune response can be a naturally occurring immune response, or it can be an induced immune response. As used herein, a naturally occurring pre-existing immune response is an immune response in an individual that is elicited in response to an antigen (such as a bacterial or viral antigen) to which the individual has been inadvertently exposed. That is, an individual with a pre-existing immune response has not been deliberately exposed to the antigen to generate an immune response against the antigen. An induced pre-existing immune response is an immune response that is generated as a result of deliberate exposure to an antigen (such as when receiving a vaccine). A pre-existing immune response can be a naturally occurring immune response, or a pre-existing immune response can be an induced immune response.

[0046] As used herein, the phrase "recruit an immune response" refers to the process in which an antigen is administered to an individual such that the components of a pre-existing immune response travel through the body to the location where the antigen was administered, resulting in the attack of cells presenting the antigen by components of the immune system. As used herein, "components of the immune response" refers to cells that can bind an antigen and initiate an immune response against the antigen. Antigens useful in practicing the present invention are any molecules that can be recognized by cells of a pre-existing immune system, particularly T cells. An example of such a compound is a protein, such as a bacterial or viral protein.

[0047] As used herein, the phrase "treating cancer" refers to various outcomes regarding cancer. Treating cancer includes reducing the rate of increase in the number of cancer cells in a treated individual. Such a reduction in the rate of increase can be due to a slowing of cancer cell replication. Alternatively, the rate of cancer cell replication can be unaffected, and an increased number of cancer cells can be killed by a pre-existing immune response. In some aspects, treating cancer refers to a situation in which the number of cancer cells stops increasing but remains at a constant level. Such a situation can be caused by inhibition of cancer cell replication by recruiting a pre-existing immune response, or it can be due to a rate of cancer cell killing by a recruited pre-existing immune response balancing the rate of production of new cancer cells. Treating cancer is stabilizing the cancer such that the growth of the cancer is reduced or stopped, or reducing the number of cancer cells in a treated individual and / or in an individual without cancer (i.e., no detectable cancer cells).

[0048] In an embodiment, the step of recruiting a pre-existing immune response comprises introducing an antigen recognized by one or more components of the pre-existing immune response into the cancer. In a preferred embodiment, the antigen is not present in the cancer prior to treatment. Thus, one embodiment is a method of treating cancer in an individual, comprising recruiting a pre-existing immune response to the cancer by introducing an antigen recognized by one or more components of the pre-existing immune response into the cancer, wherein the antigen is not present in the cancer prior to treating the cancer. Thus, as described above, the pre-existing immune response can be a naturally occurring immune response or an induced immune response. Methods known in the art can be used to introduce the antigen into the cancer, and can vary depending on the type of cancer being treated. For example, one type of cancer is a solid tumor. In such cancers, the cancer cells replicate and remain adjacent to their parental cancer cells, resulting in the formation of a mass of tissue formed by the adjacent cancer cells. Since such cancers are cell masses, the antigen can be delivered directly to the mass or into the mass. One embodiment is a method of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a pre-existing immune response to the solid tumor by introducing an antigen recognized by one or more components of the pre-existing immune response into the solid tumor, wherein the antigen is not present in the solid tumor prior to treatment. In one embodiment, the pre-existing immune response is a naturally occurring immune response. In one embodiment, the pre-existing immune response is an induced immune response. In one embodiment, the antigen is delivered to the cancer (e.g., solid tumor) by injecting the antigen into the cancer (e.g., solid tumor). In such embodiments, the antigen is delivered directly into the cancer, either by direct binding to such molecules or by uptake and processing of the antigen by the cancer cells, thereby allowing the antigen to be displayed on the MHC I molecules of the cells. In these methods, the antigen can be combined with other molecules or compounds that enhance the uptake of the antigen and / or the presentation of the antigen to the immune system.

[0049] As described above, in these methods, the antigen can be a protein. As described above, these protein antigens can be directly injected into the cancer (e.g., a tumor). Thus, one embodiment is a method of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a pre-existing immune response to the solid tumor by injecting the solid tumor with an antigenic protein, wherein the antigenic protein is recognized by one or more components of the pre-existing immune response, and wherein the antigenic protein is not present in the solid tumor prior to treatment. Alternatively, the protein antigen can be introduced into the cancer by introducing a nucleic acid molecule encoding the protein into the cancer. Thus, one embodiment is a method of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a pre-existing immune response to the solid tumor by introducing a nucleic acid molecule encoding an antigenic protein into the solid tumor, wherein the antigenic protein is recognized by one or more components of the pre-existing immune response, and wherein the antigenic protein is not present in the solid tumor prior to treatment. Any suitable method known in the art can be used to introduce the nucleic acid molecule encoding the antigen into the cancer. One embodiment is a method of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a pre-existing immune response to the solid tumor by injecting a nucleic acid molecule encoding an antigenic protein into the solid tumor, wherein the antigenic protein is recognized by one or more components of the pre-existing immune response, and wherein the antigenic protein is not present in the solid tumor prior to treatment. In these methods, the nucleic acid molecule encoding the antigen can be injected as a naked nucleic acid molecule (i.e., a nucleic acid molecule not complexed with other molecules for delivery that are designed to enhance the stability of the nucleic acid molecule) or the injected nucleic acid molecule encoding the antigen can be complexed with one or more compounds designed to enhance the delivery, stability, or lifespan of the nucleic acid molecule. Examples of such compounds include lipids, proteins, carbohydrates, and polymers, including synthetic polymers.

[0050] Delivery vehicles such as recombinant viruses or pseudoviruses (pseudoparticles) can also be used to introduce nucleic acid molecules encoding more than one antigen into cancer. Examples of viruses that can be used to practice the methods of the present invention include, but are not limited to, adenoviruses, adeno-associated viruses, herpesviruses, and papillomaviruses. The use of such viruses for delivering nucleic acid molecules is known to those of skill in the art and is also disclosed in U.S. Patent No. 8,394,411, which is incorporated herein by reference. Examples of pseudoviruses that can be used to practice the methods of the present invention include, but are not limited to, hepatitis pseudoviruses, influenza pseudoviruses, and papillomavirus pseudoviruses. As used herein, a pseudovirus refers to a particle comprising viral capsid proteins assembled into virus-like particles (VLPs) that are capable of binding to and entering cancer cells. Such pseudoparticles may, but preferably do not, package subgenomic amounts of viral nucleic acid molecules. Methods for producing and using pseudoparticles are known in the art and are also described in U.S. Patent Nos. 6,599,739; 7,205,126; and 6,416,945, which are incorporated herein by reference in their entirety. Accordingly, the present disclosure provides methods of treating cancer in an individual, wherein the cancer is a solid tumor, comprising recruiting a pre-existing immune response to the solid tumor by introducing a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigenic protein into the tumor, wherein the antigenic protein is recognized by one or more components of the pre-existing immune response, and wherein the antigenic protein is not present in the solid tumor prior to treatment. Entry of a pseudovirus carrying the nucleic acid molecule of the present disclosure into a cell results in the cell expressing the encoded antigenic protein and subsequently presenting the antigen to the immune system. In these methods, the pseudovirus is a papillomavirus pseudovirus.

[0051] Any suitable method known in the art can be used to effect the introduction of a virus or pseudovirus comprising a nucleic acid molecule encoding an antigen into cancer. For example, a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigen can be injected near the cancer or directly into the cancer. Alternatively, a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigen can be administered to an individual by a route that results in delivery of the recombinant virus or pseudovirus to the cancer. Examples of such routes include, but are not limited to, intravenous (IV) injection, intramuscular (IM) injection, intraperitoneal (IP) injection, subcutaneous (SC) injection, and oral delivery. Accordingly, one embodiment is a method of treating cancer in an individual, comprising administering to the individual a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigenic protein, wherein the cancer is a solid tumor, wherein the antigenic protein is recognized by one or more components of the pre-existing immune response, and wherein the antigenic protein is not present in the solid tumor prior to treatment. In these methods, the recombinant virus or pseudovirus can be injected directly into the solid tumor, or the recombinant virus or pseudovirus can be delivered using a method selected from IV injection, IM injection, IP injection, SC injection, and oral delivery.

[0052] The methods of the present disclosure can be used to treat hematological cancers. Hematological cancers, blood cancers, hematological malignancies, etc. begin in cells of blood-forming tissues (such as bone marrow) or the immune system. Examples of blood cancers include leukemia, lymphoma, and multiple myeloma. Such cancers begin when cells of the blood-forming tissues or cells of the immune system lose control of cell replication and begin to replicate in an uncontrolled manner. Once formed, hematological cancer cells can enter the blood or lymphatic system, resulting in a significant increase in the number of cancer cells in the blood and / or lymphatic system. For example, leukemia is a cancer found in the blood and bone marrow. Leukemia is caused by the uncontrolled replication of white blood cells, resulting in a large increase in the number of abnormal white blood cells in the blood and lymphoid tissues. These abnormal white blood cells cannot function properly, and thus an individual with leukemia cannot fight infections. Accordingly, the present disclosure provides methods of treating hematological cancers in an individual, including recruiting a pre-existing immune response to hematological cancer cells in an individual by introducing an antigen recognized by one or more components of the pre-existing immune response into the hematological cancer cells, wherein the antigen is not present in the hematological cancer cells or on the hematological cancer cells prior to treatment. In these methods, the pre-existing immune response can be a naturally occurring immune response or an induced immune response. Any suitable method can be used to introduce the antigen into the hematological cancer cells. In these methods, the antigen can be introduced into the hematological cancer cells by administering the antigen to the individual in a form that results in delivery of the antigen to the hematological cancer cells. For example, methods selected from IV injection, IM injection, IP injection, SC injection, and oral administration can be used to administer the antigen to the individual. In these methods, the antigen can be targeted to the hematological cancer cells, for example, by conjugating the antigen to a protein that binds to a molecule on the hematological cancer cells.

[0053] The antigen can also be introduced into the hematological cancer cells by introducing a nucleic acid molecule encoding an antigenic protein into the hematological cancer cells in an individual. Accordingly, the present disclosure provides methods of treating hematological cancers in an individual, which include recruiting a pre-existing immune response against hematological cancer by administering a nucleic acid molecule encoding an antigenic protein to the individual, wherein the antigenic protein is recognized by one or more components of the pre-existing immune response, and wherein the antigenic protein is not present in the hematological cancer cells or on the hematological cancer cells prior to treatment. Any suitable method known in the art can be used to administer the nucleic acid molecule encoding the antigen to the individual. For example, the nucleic acid molecule encoding the antigen can be injected as a naked nucleic acid molecule. Alternatively or additionally, the nucleic acid molecule encoding the antigen can be complexed with one or more compounds designed to enhance the delivery, stability, or lifespan of the nucleic acid molecule. Examples of such compounds include lipids, proteins, carbohydrates, and polymers, including synthetic polymers.

[0054] It is also possible to use a delivery vehicle such as a recombinant virus or pseudovirus to introduce a nucleic acid molecule encoding more than one antigen into a hematological cancer cell. Examples of such delivery vehicles have been described previously herein. Examples of viruses that can be used to practice the methods of the present invention include, but are not limited to, adenovirus, adeno-associated virus, herpesvirus, and papillomavirus. Examples of pseudoviruses that can be used to practice the methods of the present invention include, but are not limited to, hepatitis pseudovirus, influenza pseudovirus, and papillomavirus pseudovirus. Accordingly, the present disclosure provides a method of treating a hematological cancer in an individual, comprising recruiting a pre-existing immune response against a solid tumor by introducing a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigenic protein into the tumor, wherein the antigenic protein is recognized by one or more components of the pre-existing immune response, and wherein the antigenic protein was not present in the hematological cancer cell or on the hematological cancer cell prior to treatment.

[0055] Any suitable method known in the art can be used to effect the introduction of a virus or pseudovirus comprising a nucleic acid molecule encoding an antigen into the cancer. For example, a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigen can be administered to an individual by a route that results in delivery of the recombinant virus or pseudovirus to the cancer. Examples of such routes include, but are not limited to, intravenous (IV) injection, intramuscular (IM) injection, intraperitoneal (IP) injection, subcutaneous (SC) injection, and oral administration. Accordingly, the present disclosure provides a method of treating a hematological cancer in an individual, comprising administering to the individual a recombinant virus or pseudovirus comprising a nucleic acid molecule encoding an antigenic protein, wherein the antigenic protein is recognized by one or more components of the pre-existing immune response, and wherein the antigenic protein was not present in the hematological cancer cell or on the hematological cancer cell prior to treatment. The recombinant virus or pseudovirus can be delivered using a method selected from the group consisting of: IV injection, IM injection, IP injection, SC injection, and oral administration.

[0056] The methods disclosed herein use one or more antigens to recruit pre - existing immune responses to cancer. Any antigen can be used, provided that the antigen is recognized by one or more components of a pre - existing immune response and that the antigen is not present in or on cancer cells prior to treatment. Examples of useful antigens include, but are not limited to, viral and bacterial antigens. An example of a viral antigen that can be used in the methods of the present invention is an antigen comprising at least one epitope from a cytomegalovirus protein. As used herein, an epitope is a cluster of amino acid residues that is recognized by the immune system, thereby triggering an immune response. Such epitopes can consist of contiguous amino acid residues (i.e., amino acid residues adjacent to each other in a protein), or they can consist of non - contiguous (i.e., amino acid residues not adjacent to each other in a protein), but spatially very close amino acid residues in the final folded protein. Those skilled in the art generally understand that an epitope requires a minimum of six amino acid residues to be recognized by the immune system. Thus, the methods of the present invention can include using an antigen comprising at least one epitope from a cytomegalovirus protein. Any suitable CMV protein can be used to generate an antigen that can be used to practice the methods of the present invention, provided that the antigen recruits a pre - existing immune response to cancer. Examples of CMV proteins suitable for the methods disclosed herein include, but are not limited to, CMV pp50, CMV pp65, CMV pp150, CMV IE - 1, CMV IE - 2, CMV gB, CMV US2, CMV UL16, and CMV UL18. Examples of such proteins and their useful fragments are disclosed in U.S. Patent Publication Nos. 2005 / 00193344 and 2010 / 0183647, both of which are incorporated herein by reference in their entirety. Useful fragments can also include any one or combination of peptides comprising the amino acid sequences of SEQ ID NOs: 1 - 67.

[0057] One or more antigens can also be used to practice the disclosed methods, each antigen independently comprising an amino acid sequence that is a variant of at least 8 contiguous amino acid sequences from a CMV protein. As used herein, a variant refers to a protein or nucleic acid molecule whose sequence is similar but not identical to a reference sequence, where the activity (e.g., immunogenicity) of the variant protein (or the protein encoded by the variant nucleic acid molecule) is not significantly altered. These sequence variations can be naturally occurring variations or they can be engineered using genetic engineering techniques known to those of skill in the art. Examples of such techniques can be found in Sambrook J, Fritsch E F, Maniatis T et al., in Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, pp. 9.31 - 9.57 or Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 - 6.3.6.

[0058] With respect to variants, any type of change in the amino acid sequence is permitted, so long as the resulting variant protein retains the ability to elicit an immune response. Examples of such variations include, but are not limited to, deletions, insertions, substitutions, and combinations thereof. For example, with respect to proteins, it is well known to those of skill in the art that one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids can typically be removed from the amino and / or carboxyl termini of a protein without significantly affecting the activity of the protein. Similarly, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids can typically be inserted into a protein without significantly affecting the activity of the protein.

[0059] As noted, a variant protein can contain amino acid substitutions relative to a reference protein (e.g., a wild - type protein). Any amino acid substitution is permitted so long as it does not significantly affect the activity of the protein. In this regard, it is understood in the art that amino acids can be classified based on their physical properties. Examples of such groups include, but are not limited to, charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids. Preferred variants containing substitutions are those in which an amino acid is replaced with an amino acid from the same group. Such substitutions are referred to as conservative substitutions.

[0060] Naturally occurring residues can be classified based on common side chain properties: 1) hydrophobic: Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr; 3) acidic: Asp, Glu; 4) basic: Asn, Gln, His, Lys, Arg; 5) residues affecting strand orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.

[0061] For example, a non-conservative substitution can involve swapping a member of one of these categories for a member from another category.

[0062] When making amino acid changes, the hydrophilicity index of the amino acids can be considered. Each amino acid is assigned a hydrophilicity index based on its hydrophobicity and charge characteristics. The hydrophilicity indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is generally understood in the art the importance of the hydrophilic amino acid index in conferring interactive biological functions to proteins (Kyte et al., 1982, J. Mol. Biol. 157:105-31). It is known that certain amino acids can be substituted for other amino acids having similar hydrophilicity indices or scores and still retain similar biological activity. When making changes based on the hydrophilicity index, substitutions of amino acids with hydrophilicity indices within ±2 are preferred, substitutions of amino acids within ±1 are particularly preferred, and substitutions of amino acids within ±0.5 are even more particularly preferred.

[0063] It should also be understood in the art that substitution of similar amino acids can be effectively carried out based on hydrophilicity, especially in cases where the resulting biologically functionally equivalent proteins or peptides are intended for use in immunological inventions, as in the present case. The maximum local average hydrophilicity of a protein (controlled by the hydrophilicity of its neighboring amino acids) is related to its immunogenicity and antigenicity, i.e., to the biological properties of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When making changes based on similar hydrophilicity values, substitution of amino acids with hydrophilicity values within ±2 is preferred, substitution of amino acids within ±1 is particularly preferred, and substitution of amino acids within ±0.5 is even more particularly preferred. Epitopes can also be identified from the primary amino acid sequence based on hydrophilicity.

[0064] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art when such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of a protein, or to increase or decrease the immunogenicity, solubility or stability of a protein. Exemplary amino acid substitutions are shown in the following table:

[0065]

[0066] As used herein, the phrase "significantly affects protein activity" means that the protein activity is reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. With respect to the present invention, such activity can be measured, for example, as the ability of the protein to induce neutralizing antibodies or to induce a T cell response. Methods for determining such activity are known to those skilled in the art.

[0067] The methods of the present disclosure can use one or more antigens, each independently comprising at least 6 contiguous amino acids, at least 10 contiguous amino acids, at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, or at least 100 contiguous amino acids from a CMV protein. The methods of the present disclosure can use one or more antigens, each independently comprising an amino acid sequence that is at least 85% identical, at least 95% identical, at least 97% identical, or at least 99% identical to at least 10 contiguous amino acids, at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, or at least 100 contiguous amino acids from a CMV protein. The methods of the present disclosure can use one or more antigens, each independently comprising at least 6 contiguous amino acids, at least 10 contiguous amino acids, at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, or at least 100 contiguous amino acids from a CMV protein. The methods of the present disclosure can use one or more antigens, each independently comprising an amino acid sequence that is at least 95% identical, at least 97% identical, or at least 99% identical to 9 to 15 contiguous amino acid residues from a CMV protein, wherein the antigen is an MHC I-restricted antigen. The methods of the present disclosure can use one or more antigens, each independently comprising 9 to 15 contiguous amino acid residues from a CMV protein, wherein the antigen is an MHC I-restricted antigen. The methods of the present disclosure can use one or more antigens, each independently comprising an amino acid sequence that is at least 95% identical, at least 97% identical, or at least 99% identical to at least 15 contiguous amino acid residues from a CMV protein, wherein the antigen is an MHC II-restricted antigen. The methods of the present disclosure can use one or more antigens, each independently comprising at least 15 contiguous amino acid residues from a CMV protein, wherein the antigen is an MHC II-restricted antigen. The methods of the present disclosure can use one or more antigens, each independently comprising an amino acid sequence that is at least 95% identical, at least 97% identical, or at least 99% identical to a peptide consisting of a sequence selected from the group consisting of peptides comprising the amino acid sequences of SEQ ID NOs: 1-67 or any combination thereof. The methods of the present disclosure can use one or more antigens, each independently consisting of an amino acid sequence that is at least 95% identical, at least 97% identical, or at least 99% identical to a sequence selected from the group consisting of peptides comprising the amino acid sequences of SEQ ID NOs: 1-67 or any combination thereof. The methods of the present disclosure can use one or more antigens, each independently consisting of a sequence selected from the group consisting of peptides comprising the amino acid sequences of SEQ ID NOs: 1-67 or any combination thereof.

[0068]

[0069]

[0070] The methods of the present invention include treating an individual for cancer by recruiting a pre-existing immune response to the cancer. In these methods, it may be known that the individual has a pre-existing immune response to an antigen prior to the initiation of cancer treatment. Prior to initiating cancer treatment, the individual may be tested to confirm the presence of the pre-existing immune response. Thus, these methods can include treating cancer in an individual by confirming that the individual has a pre-existing immune response to an antigen, wherein the antigen is not present in or on the cancer. The antigen is then administered to the individual confirmed to have the pre-existing immunity such that the antigen is introduced into the cancer, thereby treating the cancer.

[0071] Such methods can be used to treat any cancer described herein, including any solid tumor and / or hematological cancer.

[0072] Any method for confirming that an individual to be treated has a pre-existing immune response to an antigen can be used to practice the methods of the present invention. Examples of such methods include identifying B cells that recognize a specific antigen, antibodies that recognize a specific antigen, T cells that recognize a specific antigen, or T cell activity initiated in response to a specific antigen in a sample from the individual. Any suitable sample from the individual can be used to identify the pre-existing immune response. Examples of suitable samples include, but are not limited to, whole blood, serum, plasma, and tissue samples. As used herein, a B cell, T cell, or antibody recognizing a specific antigen refers to the ability of such B cell, T cell, or antibody to specifically bind the antigen. Specific binding of a B cell, T cell, or antibody to an antigen refers to the B cell, T cell, or antibody binding to the specific antigen with an affinity greater than the binding affinity of the same B cell, T cell, or antibody for a molecule unrelated to the antigen. For example, a B cell, T cell, or antibody that recognizes an antigen from the CMV pp50 protein or is specific for an antigen from the CMV pp50 protein binds the CMV pp50 antigen with an affinity significantly greater than the binding affinity of the same B cell, T cell, or antibody for a protein unrelated to the CMV pp50 protein, such as human albumin. Specific binding between two entities can be scientifically represented by its dissociation constant, which is typically less than about 10 -6 less than about 10 -7 or less than about 10 -8The concepts of specific binding between molecules and between cells and molecules, and methods for measuring such binding, are well known to those of ordinary skill in the art, including but not limited to enzyme immunoassays (e.g., ELISA), immunoprecipitation, immunoblot assays, and other immunoassays, as described, for example, in Sambrook et al., supra, and Harlow et al., Antibodies, a Laboratory Manual (Cold Spring Harbor Labs Press, 1988). Such methods are also described in U.S. Patent No. 7,172,873, which is incorporated herein by reference. Methods for measuring T cell activation in a sample from an individual are also known to those skilled in the art. Examples of such methods are disclosed in U.S. Patent Publication No. 2003 / 003485 and U.S. Patent No. 5,750,356, both of which are incorporated herein by reference.

[0073] Such methods generally involve contacting a sample containing T cells from an individual with an antigen and measuring T cell activation of the sample. Methods for measuring T cell activation are also well known in the art and are further disclosed in Walker, S., et al., Transplant Infectious Disease, 2007:9:165-70; and Kotton, C.N. et al. (2013) Transplantation 96, 333.

[0074] The commercially available CMV test (QuantiFERON™-CMV, QIAGEN Sciences Inc., Germantown, MD) can be used as an in vitro diagnostic test that uses a peptide mixture mimicking human cytomegalovirus protein (CMV) to stimulate cells in heparinized whole blood. Individuals exposed to a disease / infection have specific T cell lymphocytes in their blood that maintain an immune memory against the antigens (immunoreactive molecules) of the disease / infection in question. Addition of an antigen to blood collected from a primed individual results in rapid restimulation of antigen-specific effector T cells, leading to the release of cytokines (e.g., IFN-γ). Effector T cells are capable of rapid response upon exposure to the priming antigen. Thus, the production of IFN-γ in response to antigen exposure is a specific marker of the cellular immune response to that antigen. This IFN-γ response can be used to quantify the immune response. Detection of interferon-gamma (IFN-γ) by enzyme-linked immunosorbent assay (ELISA) is used to identify the in vitro response to peptide antigens associated with CMV infection. The intended use of QuantiFERON™-CMV is to monitor the anti-CMV immune level in humans.

[0075] Thus, in any method of the present disclosure for treating cancer in an individual, it can be first confirmed that the individual has a pre-existing immune response against an antigen that is not present in or on the cancer. Such a pre-existing immune response can be confirmed by identifying the following in a sample from the individual:

[0076] i) B cells that recognize a specific antigen;

[0077] ii) antibodies that recognize a specific antigen;

[0078] iii) T cells that recognize a specific antigen; and,

[0079] iv) T cell activity initiated in response to a specific antigen.

[0080] Then the specific antigen can be administered to an individual confirmed to have a pre-existing immune response such that the antigen is introduced into the cancer, thereby treating the cancer.

[0081] In any method provided in the present disclosure, other reagents can be used in combination with (i.e., administered with) CMV antigens to enhance immune regulation or recruitment within the practice of the present invention. Such other reagents include TLR agonists; intravenous immunoglobulin (IVIG); peptidoglycan isolated from Gram-positive bacteria; lipoteichoic acid isolated from Gram-positive bacteria; lipoproteins isolated from Gram-positive bacteria; lipoarabinomannan isolated from mycobacteria, zymosan isolated from yeast cell walls; polyadenylic acid - polyuridylic acid; poly(I:C); lipopolysaccharide; monophosphoryl lipid A; flagellin; Gardiquimod; Imiquimod; R848; oligonucleotides containing CpG motifs, CD40 agonists, and 23S ribosomal RNA. In a preferred aspect of these methods, the TLR agonist is poly(I:C).

[0082] Another aspect of the present disclosure is a kit for testing an individual and recruiting a pre-existing immune response to cancer in an individual. The kit can comprise at least one CMV peptide antigen or nucleic acid encoding the peptide, a pharmaceutically acceptable carrier, a container, and a package insert or label indicating the administration of the CMV peptide to reduce at least one symptom of cancer in a patient. These kits can further include means for testing the patient's antigenic response to the CMV antigen. For example, the kit can include sterilized plastic articles for obtaining and testing whole blood samples and for in vitro testing of the response to CMV peptide antigens and / or detecting interferon-gamma (IFN-γ) by enzyme-linked immunosorbent assay (ELISA) to identify the in vitro response to these peptide antigens.

[0083] Examples

[0084] Chronic viral infections that are normally well controlled by the host, such as human cytomegalovirus (hCMV), typically lead to the induction of increasing numbers of fully functional virus-specific T cells with age. Using a murine mCMV model that mimics key aspects of the human immune response to hCMV, the inventors have developed methods and reagents for attracting these antiviral T cells to tumors, subsequently killing tumor cells and inducing a potent epitope spreading to tumor neoantigens, which results in an adaptive immune response that confers long-term control of tumor growth and provides protection against re-challenge with homologous tumor cells.

[0085] Example 1

[0086] Murine cytomegalovirus infection induces cytokine responses against an mCMV peptide library

[0087] C57Bl / 6 mice were infected with 1x10^4 pfu murine cytomegalovirus (mCMV). Blood samples were collected on day 12 post-infection. Blood leukocytes were re-stimulated with a library of selected immunogenic peptides from m38, m45, m57, m122, 1m39, m141, and m164 mCMV proteins. IFN-gamma, TNF-alpha, and IL-2 cytokine production by CD8+ T cells was evaluated by intracellular cytokine staining and analyzed by fluorescence-activated cell sorting (FACS) ( Figure 1A ). Blood samples were collected two months post-infection. Expanded (m122)- and non-expanded (m45)-specific CD8+ T cells were detected by FACS using MHC-I tetramer staining. Memory CD8+ T cell responses were mapped against mCMV. Spleens were collected six months post-infection. IFN-gamma production by CD8+ and CD4+ T cells was evaluated by intracellular cytokine staining after in vitro stimulation with m38, m45, m122 MHC-I-restricted and m139 560-574 MHC-II-restricted mCMV peptides ( Figure 1B ).

[0088] Example 2

[0089] Intratumoral transduction of solid tumors with HPV Psv expressing mCMV antigens

[0090] C57Bl / 6 mice were infected with 1x10^4 pfu murine cytomegalovirus (mCMV). Six months post-infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins (injection protocol, Figure 2A ). Tumor growth was measured using electronic calipers. On days 13 and 15 post-tumor injection, HPV16 Psv expressing m122 and m45 was injected intratumorally ( Figure 2B) or HPV Psv expressing red fluorescent protein (RFP) Figure 2C ) (10^8 infectious units per PsV).

[0091] Example 3

[0092] Intratumoral transduction of solid tumors with mCMV antigens in combination with poly(I:C)

[0093] C57Bl / 6 mice were infected with 1x10^4 pfu murine cytomegalovirus (mCMV). Four months after infection, the mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins Figure 3A ). In the presence or absence of poly(I:C) (30 µg) (PIC), the tumors were injected intratumorally on days 11 and 13 with HPV16 expressing m122, m38, and m45 or control RFP, on days 16 and 18 with HPV45 expressing m122, m38, and m45 or control RFP, and on days 21 and 23 with HPV58 expressing m122, m38, and m45 or control RFP (10^8 infectious units per PsV). Tumor growth was measured using electronic calipers Figure 3B - 3E ). These tumor volume / growth data showed that intratumoral transduction of solid tumors with HPV Psv expressing mCMV antigens slowed tumor growth, and co-administration with poly(I:C) further slowed tumor growth (compare Figure 3B and 3D ; and compare Figure 3C and 3E ). Infiltration of the tumors by E7 Figure 3F ), m45, and m122 Figure 3G )-specific CD8+ T cells was analyzed by MHC-I tetramer staining and FACS. These data showed that tumor infiltration by CD8+ T cells was significantly enhanced when these CMV antigens were co-administered with poly(I:C).

[0094] Example 4

[0095] Intratumoral injection of mCMV MHC-I-restricted peptides confers increased survival

[0096] C57Bl / 6 mice were infected with 1x10^4 pfu murine cytomegalovirus (mCMV). Four months after infection, the mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins Figure 3A)。On days 11, 13, 16, 18, 21, and 23, the selected m38, m45, and m122 peptides (1 μg each), with or without poly(I:C) (30 μg), and saline as a control or poly(I:C) alone were used for intratumoral injection of the tumors. Animal deaths were recorded ( Figure 4A ), and tumor growth was measured using electronic calipers ( Figure 4B ). These data indicate that intratumoral injection of mCMV MHC-I-restricted peptides delayed tumor growth and conferred increased survival.

[0097] Example 5

[0098] Intratumoral injection of mCMV MHC-I-restricted peptides delays tumor growth

[0099] C57Bl / 6 mice were infected with 1x10^4 pfu murine cytomegalovirus (mCMV). Four months after infection, the mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. On days 11, 13, 16, 18, 21, and 23, the selected m38, m45, and m122 peptides at decreasing doses (1 μg, 0.1 μg, and 0.01 μg), with or without poly(I:C) (30 μg), and saline as a control or poly(I:C) alone were used for intratumoral injection of the tumors. Tumor growth was measured using electronic calipers ( Figure 5 ). These data indicate that intratumoral injection of mCMV MHC-I-restricted peptides delayed tumor growth.

[0100] Example 6

[0101] Combination of mCMV MHC-I and MHC-II-restricted peptides delays tumor growth

[0102] C57Bl / 6 mice were infected with 2.5x10^5 mCMV. Four months after infection, the mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. From day 12 to day 28, the tumors were injected intratumorally 6 times with MHC-I-restricted selected m38, m45, and m122 peptides and / or MHC-II-restricted m139 selected peptides or saline. All peptides were injected together with poly(I:C) (30 μg). The groups were injected 6 times with MHC-I, or 6 times with MHC-II peptides, or 6 times with MHC-I and MHC-II peptides together, or 3 times with MHC-I peptides followed by 3 times with MHC-II peptides, or 3 times with MHC-II peptides followed by 3 times with MHC-I peptides. Tumor growth was measured using electronic calipers ( Figure 6A and 6B)。These data indicate that intratumoral injection of a combination of mCMV MHC-I and MHC-II restricted peptides delays tumor growth. For each peptide, E7, m45, m122-specific CD8+ T cell responses in the blood were also analyzed by FACS using MHC-I tetramers ( Figure 6C )。These data indicate that sequential intratumoral inoculation with mCMV CD4 followed by CD8 epitopes preferentially induces anti-tumor immunity.

[0103] Example 7

[0104] Complete clearance of primary tumors confers long-term tumor protection

[0105] Protected C57Bl / 6 mice that survived primary tumor challenge as described in Example 6 were s.c. injected with 2 x 10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins on the opposite side of the body from the primary challenge. As a control for tumor take, young (12-week-old) and age-matched (10-month-old) mice were challenged with TC-1 tumor cells. Tumor growth was measured using electronic calipers ( Figure 7 )。These data indicate that complete clearance of primary tumors confers long-term protection against secondary tumor challenge.

[0106] Example 8

[0107] Intratumoral injection of MCMV alters the tumor immune microenvironment

[0108] Two days after the end of the last intratumoral treatment, the effects of intratumoral injection of mCMV MHC-I and MHC-II restricted peptides with or without poly IC on the tumor immune microenvironment were analyzed in RNA samples for immune gene expression using the Nanostring Cancer immunology gene set (nCounter). Results were summarized as the change in score for each analyzed gene set. An overall score for differential expression of gene sets was performed relative to the saline-treated group (n = 4 per group). Microenvironmental features evaluated included: B cell function, interleukins, TNF superfamily, antigen processing, MHC, adaptability, transporter function, adhesion, NK cell function, T cell function, CD molecules, leukocyte function, complement pathway, microglial function, humoral, TLR, inflammation, dendritic cell function, interferon, innate, macrophage function, chemokines and receptors, senescence, apoptosis, cytokines and receptors, cancer progression, basic cell function, cell cycle, and pathogen response.

[0109] Example 9

[0110] mCMV infection induces expansive CD8 + T cell responses

[0111] C57Bl / 6 mice were infected with 5x10^3 pfu murine cytomegalovirus (mCMV). Blood samples were collected 1 or 5 months after infection. Expansive (IE3) and non-expansive (m45) specific CD8+ T cells were detected by FACS using MHC-I tetramer staining. As Figure 8 shown, mCMV infection induced a significant effector and memory CD8+ T cell response.

[0112] Example 10

[0113] mCMV infection induces robust CD8 + and CD4 + T cell responses

[0114] C57Bl / 6 mice were infected with 5x10^3 pfu murine cytomegalovirus (mCMV). Blood samples were collected on day 12 after infection. Spleen cells were restimulated with the indicated peptides and blood cells were restimulated with a selected library of immunogenic peptides from m38, m45, m57, m122, m139, m141 and m164 mCMV proteins. IFN-gamma, TNF-alpha and IL-2 cytokine production of CD4+ and CD8+ T cells was evaluated by intracellular cytokine staining and analyzed by FACS ( Figure 9A , 9B ). These results indicate that murine cytomegalovirus infection induces a large cytokine response.

[0115] Example 11

[0116] Tissue distribution of mCMV-specific CD8+ T cells

[0117] The distribution of mCMV-specific CD8+ T cells in tumor-bearing mice was studied. C57Bl / 6 mice were infected with 5x10^3 mCMV. The experimental schedule is shown in Figure 10A . Four months after infection, mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. Lymph nodes, spleen, salivary glands and tumor tissues were collected and expansive (IE3; Figure 10B ) and non-expansive (m45; Figure 10C ) specific CD8+ T cells were detected by FACS using MHC-I tetramer staining. Expression of resident memory T cell markers was evaluated using CD69 and CD103 antibodies. These results indicate that the TC1 tumor is infiltrated by mCMV-specific CD8+ T cells.

[0118] Example 12

[0119] Gene expression analysis of the tumor microenvironment

[0120] The expression of genes in tumor cells in a mouse model was studied after intratumoral treatment with the following (4 animals per group): saline; poly I:C (PIC) (50 μg); mCMV m139 peptide (MHC-II restricted / CD4) (CD4) (3 μg); mCMV m38, m122, m45 peptides (MHC-I restricted / CD8) (CD8) (1 μg each); mCMV m139 + poly I:C (PIC CD4) (3 μg each); mCMV m38, m122, m45 peptides (MHC-I restricted / CD8) + poly I:C (PIC CD8) (1 μg each). The tumors were treated 3 times at weeks 11, 13, and 16 after subcutaneous placement of TC1 tumor cells. The experimental protocol schedule is shown in Figure 11A . After treatment and harvesting of the tumors, tumor RNA was extracted using the QIACube. Tumor cell gene expression was analyzed using the NanoString Cancer Immunology Gene Set (NS_MM_CANCERIMM_C3400) in the form of gene transcripts that measure 770 genes in the Tumor PanCancer Immune Profiling Panel. Briefly, the normalized data was represented as a heatmap of gene set expression in specific biological processes (adaptive immunity, antigen processing, T cell function, dendritic cell function, NK cell function, interferon, TNF superfamily genes); a volcano plot of the gene expression changes relative to saline treatment was constructed (the plot represents the changes (expressed as fold increase or decrease) in the treatment groups relative to the control treatment (saline) that are statistically significant); a cell infiltration quantification algorithm (CD45, cytotoxic CD8, CD4 Th1, NK cells, and dendritic cells) was applied. The results showed that the greatest change in global significance scores occurred in animals treated with MHC-I restricted / CD8 and MHC-I restricted / CD8 + poly(I:C).

[0121] After intratumoral treatment, profiling of immune genes in whole tumor RNA showed significant upregulation of immune genes in three groups:

[0122] 1) mCMV m139 peptide: MHC-II restricted / CD4 - 3 mg (230 genes upregulated while 4 genes were downregulated);

[0123] 2) mCMV m38, IE3, m45 peptides: MHC-I restricted / CD8 - 1 mg (359 genes upregulated while 43 genes were downregulated);

[0124] 3) mCMV m38, IE3, m45 peptides: MHC-I restricted / CD8+ poly(I:C) (309 genes upregulated and 49 genes downregulated).

[0125] After intratumoral treatment, infiltration of leukocytes into the tumor was also analyzed. Figure 11B - 11F Tumor infiltration of different leukocytes was shown. These data indicate that intratumoral injection of CD8 mCMV epitopes (with or without poly(I:C)) induces recruitment of T cells and non-T cells (NK) into the tumor; and intratumoral injection of CD4 mCMV epitopes with poly(I:C) induces recruitment of T cells and non-T cells (NK) into the tumor; and intratumoral injection of poly(I:C) with CD8 or CD4 epitopes induces recruitment of dendritic cells into the tumor.

[0126] Example 13

[0127] Intratumoral injection of mCMV CD8 epitopes delays tumor growth

[0128] C57Bl / 6 mice were infected with 5x10^3 pfu murine cytomegalovirus (mCMV). Four months after infection, the mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. Tumor growth was measured using an electronic caliper. At days 11, 13, 16, 18, 21, and 23, the tumors were injected intratumorally with selected MHC-I restricted m38, m45, and m122 peptides (0.01, 0.1, or 1 μg each) with or without poly(I:C) (30 μg), and with saline or poly(I:C) alone as a control. Figure 12A and 12B It was shown that intratumoral injection of mCMV MHC-I restricted peptides delayed tumor growth, and co-injection with poly(I:C) improved tumor control.

[0129] Example 14

[0130] Protection against TC1 and MC38 tumor challenges was provided by intratumoral injection of mCMV MHC-I and / or MHC-II peptides with poly(I:C)

[0131] C57Bl / 6 mice were infected with 5x10^3 mCMV. Four months after infection, the mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. Tumor growth and survival were monitored. From day 12 to day 28, the tumors were injected intratumorally 6 times with MHC-I restricted selected m38, m45, and m122 peptides and / or MHC-II restricted m139 selected peptide, with or without poly(I:C) (30 μg), and with saline or poly(I:C) alone as a control. The groups were injected 6 times with MHC-I, or 6 times with MHC-II peptides, or 6 times with both MHC-I and MHC-II peptides together, or 3 times with MHC-I peptides followed by 3 times with MHC-II peptides in sequence, or 3 times with MHC-II peptides followed by 3 times with MHC-I peptides. Figure 13A showed that the combination of intratumoral injection of mCMV MHC-I and MHC-II restricted peptides delayed tumor growth, and Figure 13B showed that sequential intratumoral inoculation with CD4 (MHC-II), then CD8 (MHC-I) mCMV epitopes promoted long-term survival.

[0132] Example 15

[0133] E7 tetramer-positive CD8 + T cell responses in the blood after treatment

[0134] C57Bl / 6 mice were infected with 5x10^3 mCMV. Four months after infection, the mice were injected s.c. with 2x10^5 TC-1 tumor cells expressing E6 and E7 oncoproteins. Tumor size was measured using electronic calipers. From day 12 to day 28, the tumors were injected intratumorally 6 times with MHC-I restricted selected m38, m45, and m122 peptides and / or MHC-II restricted m139 selected peptide, with or without poly(I:C) (30 μg), and with saline or poly(I:C) alone as a control. All peptides were injected together with poly(I:C) (30 μg). The groups were injected 6 times with MHC-I, or 6 times with MHC-II peptides, or 6 times with both MHC-I and MHC-II peptides together, or 3 times with MHC-I peptides followed by 3 times with MHC-II peptides in sequence, or 3 times with MHC-II peptides followed by 3 times with MHC-I peptides. The E7, m45, m122-specific CD8+ T cell responses in the blood were analyzed by FACS using MHC-I tetramers for each peptide. Figure 14 showed that sequential intratumoral inoculation with mCMV CD4, then CD8 epitopes preferentially induced anti-tumor immunity.

[0135] Example 16

[0136] Long-term protection against secondary tumor challenge

[0137] Two million TC-1 tumor cells expressing E6 and E7 oncoproteins were s.c. injected into protected C57Bl / 6 mice that had survived primary tumor challenge on the opposite side of the primary challenge as described above. Tumor growth was measured using electronic calipers. As a control for tumor take, young (12-week-old) and age-matched (10-month-old) mice were challenged with TC-1 tumor cells. Figure 15 Complete clearance of the primary tumor was shown to confer long-term protection against secondary tumor challenge.

[0138] Example 17

[0139] Protection against MC38 tumor challenge was provided by intratumoral injection of mCMV MHC-I and MHC-II peptides with poly(I:C)

[0140] C57Bl / 6 mice were infected with 5x10^3 mCMV. Four months after infection, the mice were s.c. injected with 5x10^5 MC38 tumor cells from murine colon adenocarcinoma that exhibited hypermutation and microsatellite instability. Tumor growth was monitored. From day 12 to day 28, the tumors were injected intratumorally 6 times with MHC-I-restricted selected m38, m45, and m122 peptides and MHC-II-restricted m139 selected peptide in the presence of poly(I:C) (30 μg), or with MHC-II-restricted m139 selected peptide alone in the presence of poly(I:C) (30 μg) and with saline alone as a control. Figure 16 Complete clearance of the primary tumor was shown to confer long-term protection against secondary tumor challenge. Figure 16 Intratumoral injection of a combination of mCMV MHC-I and MHC-II-restricted peptides was shown to delay tumor growth and result in tumor clearance.

[0141] The studies described in Examples 1-17 showed that during potential mCMV infection, both non-expanding and expanding mCMV-specific T cells infiltrated tumors and redirected established antiviral T cells into solid tumors resulting in tumor regression and a dramatic alteration of the tumor immune microenvironment. The data also showed that redirecting established antiviral CD4+ T cells into solid tumors promoted epitope spreading to tumor-associated antigens and complete tumor clearance. Thus, these methods provide a widely applicable "antigen agnostic" tumor therapy based on pre-existing antiviral T cells. HPV L1 and L2 particles exhibit strong tropism for numerous tumor cells but do not bind to or infect intact epithelium. Thus, HPV PsV or VLP can be used to genetically or directly as a vector to direct anti-tumor agents to tumor cells.

[0142] Although the present invention has been described with reference to specific embodiments, those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. Additionally, many modifications can be made to adapt a particular situation, material, composition of matter, process, one or more process steps to the purpose, spirit, and scope of the present invention. All such modifications are intended to fall within the scope of the claims. The present invention provides the following embodiments.

[0143] 1. A method of treating cancer in an individual, comprising recruiting a pre-existing immune response to the site of the cancer so as to treat the cancer.

[0144] 2. The method of item 1, wherein the pre-existing immune response is a naturally occurring pre-existing immune response.

[0145] 3. The method of item 1 or 2, wherein recruiting the pre-existing immune response to cancer cells comprises introducing an antigen not expressed by the cancer cells into the cancer before the start of treatment, wherein the antigen is recognized by one or more components of the pre-existing immune response.

[0146] 4. The method of any one of the preceding items, wherein before introducing the antigen into the tumor, it is confirmed that the individual has a pre-existing immune response against the antigen.

[0147] 5. The method of item 4, wherein the step of confirming the existence of the pre-existing immune response comprises identifying a T cell response against the antigen in a sample from the individual.

[0148] 6. The method of any one of the preceding items, wherein the step of introducing the antigen comprises injecting the antigen into the cancer.

[0149] 7. The method of any one of the preceding items, wherein the step of introducing the antigen comprises introducing a nucleic acid molecule encoding the antigen into the cancer.

[0150] 8. The method of item 7, wherein the nucleic acid molecule is DNA.

[0151] 9. The method of item 7, wherein the nucleic acid molecule is RNA.

[0152] 10. The method of item 9, wherein the RNA is modified to be more resistant to degradation.

[0153] 11. The method of item 7, wherein the nucleic acid molecule is introduced into the cancer by injection.

[0154] 12. The method of item 7, wherein the nucleic acid molecule is introduced into the cancer using a viral vector.

[0155] 13. The method of item 12, wherein a pseudovirion is used to introduce the viral vector into the cancer.

[0156] 14. The method of item 13, wherein the pseudovirion is a papillomavirus pseudovirion.

[0157] 15. The method of any one of items 3 - 14, wherein the antigen is a viral antigen.

[0158] 16. The method of any one of items 3 - 14, wherein the antigen is a polypeptide comprising at least one epitope from a cytomegalovirus (CMV) protein, and wherein the at least one epitope is recognized by one or more components of the pre - existing immune response.

[0159] 17. The method of item 16, wherein the one or more components are T cells.

[0160] 18. The method of item 16, wherein the CMV protein is selected from the group consisting of: pp50, pp65, pp150, IE - 1, IE - 2, gB, US2, US6, UL16, and UL18.

[0161] 19. The method of item 16, wherein the polypeptide is an MHC I - restricted peptide of 9 - 15 amino acids.

[0162] 20. The method of item 16, wherein the polypeptide is an MHC II - restricted peptide of at least 15 amino acids.

[0163] 21. The method of item 16, wherein the antigen comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of: SEQ ID NO: 1 - 67.

[0164] 22. The method of item 16, wherein the antigen comprises a sequence selected from SEQ ID NO: 1 - 67.

[0165] 23. The method of any one of items 3 - 22, wherein the recruitment of the pre - existing immune response alters the cancer microenvironment, which is selected from B - cell function, interleukin, TNF superfamily, antigen processing, MHC, adaptive, transporter function, adhesion, NK - cell function, T - cell function, CD molecules, leukocyte function, complement pathway, microglial function, humoral, TLR, inflammation, dendritic - cell function, interferon, innate, macrophage function, chemokines and receptors, senescence, apoptosis, cytokines and receptors, cancer progression, basic cell function, cell cycle, and pathogen response.

[0166] 24. The method of any one of items 3-23, wherein the antigen is administered in combination with a reagent that enhances the immune response, and the reagent is selected from TLR agonists; IL-1R8 cytokine antagonists; intravenous immunoglobulin (IVIG); peptidoglycan isolated from Gram-positive bacteria; lipoteichoic acid isolated from Gram-positive bacteria; lipoproteins isolated from Gram-positive bacteria; lipoarabinomannan isolated from mycobacteria, zymosan isolated from yeast cell walls; polyadenylic acid - polyuridylic acid; poly(I:C); lipopolysaccharide; monophosphoryl lipid A; flagellin; Gardiquimod; Imiquimod; R848; oligonucleotides containing CpG motifs, CD40 agonists, and 23S ribosomal RNA.

[0167] 25. The method of any one of items 3-23, wherein the antigen is administered in combination with poly(I:C).

[0168] 26. The method of any one of the preceding items, wherein the cancer is a solid tumor.

[0169] 27. The method of any one of the preceding items, wherein the cancer is a hematological cancer.

[0170] 28. A kit for recruiting a pre-existing immune response to cancer in an individual, comprising at least one CMV peptide antigen or nucleic acid encoding said peptide, a pharmaceutically acceptable carrier, a container, and a package insert or label describing the administration of the CMV peptide to reduce cancer in a patient.

[0171] 29. A kit for testing a patient and recruiting a pre-existing immune response to a cancer site in the patient.

Claims

1. Use of at least one immunogenic antigen from a cytomegalovirus (CMV) protein and poly(I:C) in the preparation of a medicament for treating solid tumors in an individual, wherein each antigen independently comprises a sequence of one of SEQ ID NOs: 1-33, wherein the antigen recruits a pre-existing immune response against CMV to the solid tumor, wherein the antigen is not expressed by any cancer cells of the solid tumor in the individual, wherein the antigen is recognized by one or more components of the pre-existing immune response, and wherein the medicament is for injection into the solid tumor.

2. The use of claim 1, wherein the medicament comprises the antigen, and the antigen comprises SEQ ID NOs: 1-33.

3. The use of any one of claims 1-2, wherein prior to introducing the antigen into the tumor, it is confirmed that the individual has a pre-existing immune response against the antigen.

4. The use of claim 3, wherein the step of confirming the existence of the pre-existing immune response comprises identifying a T cell response against the antigen in a sample from the individual.

5. The use of any one of claims 1-4, further comprising the use of at least one CMV peptide, the CMV peptide comprising a sequence shown in one of SEQ ID NOs: 34-67.

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