Allogeneic tumor cell vaccine

Abstract

To provide an allogeneic tumor cell vaccine.SOLUTION: Provided is an allogeneic tumor cell vaccine, comprising: (1) a population of proliferation-incompetent, live, genetically engineered tumor cells expressing one or more tumor-specific antigens, the population comprising at least three stably expressed immunomodulatory molecules, at least three immunomodulatory molecules being OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L) including CD80, CD86, or both, to induce one or more subpopulations of PBMCs to proliferate in response to the expressed immunomodulatory molecules and subsequently enter an effector phase to kill tumor cells, and the subpopulations of PBMC cells comprising one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes; and (2) a pharmaceutically acceptable carrier.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application is a continuation-in-part of U.S. Provisional Application No. 15 / 821,105 (filed November 22, 2017), which claims the benefit of priority to U.S. Provisional Application No. 62 / 425,424 (filed November 22, 2016).

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on June 9, 2020, is named 128663-00119_SL.txt and is 263,710 bytes in size.

[0003] The invention described relates generally to immunological approaches to the treatment of cancer, and more particularly to cancer vaccines comprising modified tumor cells. [Background technology]

[0004] The human immune system is generally divided into two arms, termed "innate" and "adaptive." The innate arm of the immune system is primarily responsible for the early inflammatory response via many soluble factors, including the complement and chemokine / cytokine systems, and includes many specialized cell types, including mast cells, macrophages, dendritic cells (DCs), and natural killer cells. The adaptive arm includes delayed and sustained antibody responses, along with CD8+ and CD4+ T cell responses, which play an important role in immunological memory against antigens. A third arm of the immune system can be identified as including γδ T cells and T cells with limited T cell receptor repertoires, such as natural killer T (NKT) cells and mucosal-associated invariant T (MAIT) cells.

[0005] Cells of the immune system There are numerous cellular interactions that make up the immune system. These interactions occur through specific receptor-ligand pairs that transmit signals in both directions, so that each cell receives instructions based on the temporal and spatial distribution of these signals.

[0006] Mouse models have been extremely useful in discovering immunoregulatory pathways, but the clinical utility of these pathways does not necessarily translate from inbred mouse strains to outbred human populations, which may have individuals with varying degrees of dependency on individual immunoregulatory pathways.

[0007] Cells of the immune system include lymphocytes, monocytes / macrophages, dendritic cells, the closely related Langerhans cells, natural killer (NK) cells, mast cells, basophils, and other members of the myeloid lineage of cells. In addition, a series of specialized epithelial and stromal cells provide the anatomical environment in which immunity occurs, often by secreting key factors that regulate proliferation and / or gene activation in cells of the immune system, which also play a direct role in the inductive and effector phases of the response. (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0008] Cells of the immune system are found in peripheral, organized tissues such as the spleen, lymph nodes, intestinal Peyer's patches, and tonsils. Lymphocytes are also found in central lymphoid organs, the thymus, and bone marrow, where they undergo developmental stages that prepare them to mediate the myriad responses of the mature immune system. Lymphocytes and macrophages constitute a recirculating pool of cells found in the blood and lymph, delivering immunocompetent cells to sites where they are needed and enabling locally generated immunity to become systemic (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0009] The term "lymphocyte" refers to small white blood cells that form in lymphoid tissues throughout the body. In normal adults, they comprise approximately 22-28% of the total circulating white blood cell population and play a major role in defending the body against disease. Individual lymphocytes are specialized in that, through recombination of genetic material, they are determined to respond to a limited set of structurally related antigens (e.g., producing T cell and B cell receptors). This determination, which exists before the immune system's first contact with a given antigen, is represented by the presence of receptors specific for the antigen's determinants (epitopes) on the lymphocyte's surface membrane. Each lymphocyte contains a population of T cells, all of which have identical binding sites. One set of lymphocytes, or clones, differs from another clone in the structure of its receptor's binding region and therefore in the epitopes it can recognize. Lymphocytes differ from one another not only in the specificity of their receptors but also in their functions (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0010] Two broad classes of lymphocytes are recognized: B lymphocytes (B cells), which are the precursors of antibody-secreting cells, and T lymphocytes (T cells).

[0011] B lymphocytes B lymphocytes originate from hematopoietic cells in the bone marrow. Mature B cells can be activated by antigens that display epitopes recognized by their cell surface. The activation process can be direct, i.e., dependent on cross-linking of membrane Ig molecules by antigen (cross-linking-dependent B cell activation), or indirect, i.e., via interaction with helper T cells in a process called cognate help. In many physiological situations, receptor cross-linking stimulation and cognate help synergize to produce a more vigorous B cell response (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0012] Cross-linking-dependent B cell activation requires that an antigen present multiple copies of an epitope complementary to the binding site of a cell surface receptor, since each B cell expresses an Ig molecule with an identical variable region. This requirement is met by other antigens with repeated epitopes, such as microbial capsular polysaccharides and viral envelope proteins. Cross-linking-dependent B cell activation is the primary protective immune response mounted against these microorganisms (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0013] Cognate help allows B cells to mount responses to antigens for which they are unable to bridge their receptors, while at the same time providing costimulatory signals that rescue B cells from inactivation when they are stimulated by weaker bridge events. Cognate help depends on the binding of antigen by the B cell's membrane immunoglobulin (Ig), endocytosis of the antigen, and its fragmentation into peptides within the endosomal / lysosomal compartment of the cell. Some of the resulting peptides are loaded into the grooves of a specific set of cell surface proteins known as class II major histocompatibility complex (MHC) molecules. The resulting class II / peptide complexes are expressed on the cell surface and are then bound to CD4 + It acts as a ligand for the antigen-specific receptors of a set of T cells, designated as CD4. + T cells bear receptors on their surface specific for the class II / peptide complex of B cells. B cell activation does not depend solely on binding to a T cell via its T cell receptor (TCR); this interaction also allows the T cell's activating ligand (CD40 ligand) to bind to its receptor (CD40) on B cells, signaling B cell activation. Furthermore, T helper cells secrete several cytokines that regulate the proliferation and differentiation of stimulated B cells by binding to cytokine receptors on B cells (Paul, W. E., "Chapter 1: The immune system: an introduction," in Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0014] During cognate help to antibody production, CD40 ligand binds activated CD4 +Transiently expressed on T helper cells, it binds to CD40 on antigen-specific B cells, thereby transmitting a secondary costimulatory signal. The latter signal is important for B cell proliferation and differentiation and for the generation of memory B cells by preventing apoptosis of antigen-encountering germinal center B cells. Overexpression of CD40 ligand on both B and T cells has been implicated in the production of pathogenic autoantibodies in human SLE patients (Desai-Mehta, A. et al., "Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production," J. Clin. Invest. Vol. 97(9), 2063-2073, (1996)).

[0015] T lymphocytes T lymphocytes originate from precursors in hematopoietic tissues, differentiate in the thymus, and then disseminate to peripheral lymphoid tissues and the recirculating pool of lymphocytes. T lymphocytes, or T cells, mediate a wide range of immune functions. These include the ability to help B cells develop into antibody-producing cells, augment the bactericidal activity of monocytes / macrophages, inhibit certain immune responses, directly kill target cells, and mobilize inflammatory responses. These effects depend on T cell expression of specific cell surface molecules and secretion of cytokines (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0016] T cells differ from B cells in their mechanism of antigen recognition. Immunoglobulins, or B cell receptors, bind to distinct epitopes on the surface of soluble molecules or particles. B cell receptors recognize epitopes displayed on the surface of natural molecules. While antibodies and B cell receptors evolved to bind to and defend against microorganisms in extracellular fluids, T cells mediate their functions by recognizing antigens on the surface of other cells and interacting with and modifying these antigen-presenting cells (APCs). There are three major types of APCs in peripheral lymphoid organs that can activate T cells: dendritic cells, macrophages, and B cells. The most potent of these are dendritic cells, whose sole function is to present foreign antigens to T cells. Immature dendritic cells are present in tissues throughout the body, including the skin, intestine, and airways. When they encounter invading microorganisms at these sites, they endocytose the pathogens and their products and transport them through lymph nodes to regional lymph nodes or gut-associated lymphoid organs. Encounter with a pathogen induces the maturation of dendritic cells from antigen-capturing cells into APCs capable of activating T cells. APCs display three types of protein molecules on their surface that are responsible for activating T cells into effector cells: (1) MHC proteins, which present foreign antigens to the T cell receptor; (2) costimulatory proteins, which bind to complementary receptors on the T cell surface; and (3) intercellular adhesion molecules, which allow T cells to bind to the APC long enough to be activated ("Chapter 24: The adaptive immune system," Molecular Biology of the Cell, Alberts, B. et al., Garland Science, NY, (2002)).

[0017] T cells are subdivided into two distinct classes based on the cell surface receptors they express. Most T cells express a T cell receptor (TCR) consisting of an α chain and a β chain. A small group of T cells expresses a receptor made up of a γ chain and a δ chain. Some α / β T cells express the coreceptor molecule CD4 (CD4 + T cells) and those expressing CD8 (CD8 +There are two sublineages of immune cells (immune T cells) that differ in how they recognize antigens and in their effector and regulatory functions.

[0018] CD4 + T cells are the primary regulatory cells of the immune system. Their regulatory function depends on the expression of both their cell surface molecules, such as CD40 ligand, whose expression is induced upon T cell activation, and the wide range of cytokines that are secreted upon activation.

[0019] Like CD4+ helper T cells, CD8+ (cytotoxic) T cells are generated in the thymus and express the T cell receptor. However, rather than CD4 molecules, cytotoxic T cells express CD8, a dimeric coreceptor typically composed of one CD8α chain and one CD8β chain. CD8+ T cells recognize peptides presented by MHC class I molecules found on all nucleated cells. The CD8 heterodimer binds to a conserved portion of MHC class I (the α3 region) during T cell / antigen-presenting cell interactions. CD8+ T cells (often called cytotoxic T lymphocytes, or CTLs) are important for immune defense against intracellular pathogens, including viruses and bacteria, as well as tumor surveillance. Once CD8+ T cells recognize their antigens and are activated, they have three major mechanisms for killing infected or malignant cells. The first is the secretion of cytokines, primarily TNF-α and IFN-γ, which have antitumor and antiviral microbial effects. The second major function is the production and release of cytotoxic granules. These granules, also found on NK cells, contain two protein families: perforin and granzymes. Perforin, like the membrane attack complex of complement, forms pores in the target cell membrane. These pores allow granzymes, also found in cytotoxic granules, to enter infected or malignant cells. Granzymes are serine proteases that cleave intracellular proteins, halting viral protein production and ultimately triggering apoptosis of the target cell. CD8+ T cells can release the granules, kill the infected cell, and then migrate to a new target to kill it again. This process is often referred to as serial killing. The third major function of CD8+ T cell destruction of infected cells is through the Fas / FasL interaction. Activated CD8+ T cells express FasL on their cell surface and bind to its receptor, Fas, on the surface of target cells. This binding causes Fas molecules on the surface of target cells to trimerize and attract signaling molecules. These signaling molecules then lead to the activation of the caspase cascade, which also triggers apoptosis of the target cell.Because CD8+ T cells can express both molecules, Fas / FasL interaction is a mechanism by which CD8+ T cells kill each other, a process known as fratricide, and eliminate immune effector cells during the final contraction phase of the immune response.

[0020] T cells also mediate important effector functions, some of which are determined by the pattern of cytokines they secrete, which can be directly toxic to target cells or can mobilize powerful inflammatory mechanisms.

[0021] Furthermore, T cells, especially CD8 + T cells can become cytotoxic T lymphocytes (CTLs) that can effectively lyse target cells that express antigens recognized by CTLs (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0022] T cell receptors (TCRs) recognize complexes consisting of peptides derived from proteolytic cleavage of antigens bound to specialized grooves in class II or class I MHC proteins. CD4 + T cells recognize only peptide / class II complexes, whereas CD8 + T cells recognize peptide / class I complexes (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0023] Ligands for TCRs (i.e., peptide / MHC protein complexes) are generated within APCs. Typically, class II MHC molecules bind peptides derived from proteins ingested by APCs through the process of endocytosis. These peptide-loaded class II molecules are then expressed on the cell surface, where they are then transported to CD4 receptors with TCRs that can recognize the expressed cell surface complexes. + It can bind to T cells. + T cells are specialized to react with antigens derived from extracellular sources (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0024] In contrast, class I MHC molecules are loaded primarily with peptides derived from endogenously synthesized proteins, such as viral proteins. These peptides are generated from cytosolic proteins by proteolytic degradation in the proteosome and translocated to the rough endoplasmic reticulum. Such peptides, typically nine amino acids in length, bind to class I MHC molecules and are transported to the cell surface, where they are expressed by CD8 receptor-expressing cells. + It can be recognized by T cells, which allows the T cell system, especially CD8 +T cells can detect cells that express proteins that are different from, or produced in much greater abundance than, those of the rest of the organism's cells (e.g., viral antigens) or mutant antigens (e.g., active oncogene products), even if the intact form of those proteins is neither expressed on the cell surface nor secreted (Paul, W.E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W.E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0025] T cells can also be classified based on their function as helper T cells: T cells involved in the induction of cell-mediated immunity; suppressor T cells; and cytotoxic T cells.

[0026] Helper T cells Helper T cells are T cells that stimulate B cells to mount antibody responses to proteins and other T cell-dependent antigens. T cell-dependent antibodies are immunogens in which distinct epitopes appear only once or a limited number of times, rendering them unable or insufficiently able to crosslink B cell membrane immunoglobulins (Ig). B cells bind antigens via their membrane Ig, and the complexes undergo endocytosis. Within endosomal and lysosomal compartments, antigens are fragmented into peptides by proteolytic enzymes, and one or more of the resulting peptides are loaded onto class II MHC molecules, which then pass through this vesicular compartment. The resulting peptide / class II MHC complexes are then transported to the B cell surface membrane. T cells with receptors specific for the peptide / class II molecule complex recognize this complex on the surface of B cells (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).

[0027] B cell activation depends on both T cell TCR binding and the interaction between the T cell's CD40 ligand (CD40L) and CD40 on the B cell. T cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of interaction with antigen-binding cells (APCs) that express both CD80 or CD86 and the cognate antigen recognized by the T cell's TCR. CD80 / CD86 are generally expressed by activated, but not resting, B cells, and helper interactions involving activated B cells and T cells result in efficient antibody production. However, in many cases, the initial induction of CD40L on T cells depends on their recognition of antigens on the surface of APCs, such as dendritic cells, that constitutively express CD80 / 86. These activated helper T cells then efficiently interact with and help B cells. Cross-linking of membrane Ig on B cells, in concert with the CD40L / CD40 interaction, even if inefficient, leads to vigorous B cell activation. Subsequent events in the B cell response, including proliferation, Ig secretion, and class switching of the Ig class being expressed, depend on or are enhanced by the action of T cell-derived cytokines (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0028] CD4 + T cells are primarily cells that secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (T H 2 cells), or cells that primarily produce IL-2, IFNγ, and lymphotoxin (T H T H 2 cells are highly effective at helping B cells develop into antibody-producing cells, while T HT cells are effective inducers of cellular immune responses, including enhanced bactericidal activity of monocytes and macrophages and increased efficiency in lysing microorganisms in the resulting intracellular vesicular compartments. H CD4 with a 2-cell phenotype (i.e., IL-4, IL-5, IL-6, and IL-10) + T cells are efficient helper cells, but T H These cells also have the ability to act as helpers (Paul, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, WE, Lippicott-Raven Publishers, Philadelphia, (1999)).

[0029] Natural killer (NK) cells Natural killer (NK) cells are lymphocytes of the same family as T cells and B cells, derived from a common precursor cell. However, as cells of the innate immune system, NK cells are classified as group I innate lymphoid cells (ILCs) and rapidly respond to a variety of pathological challenges. NK cells defend against disease, for example, by killing virus-infected cells and detecting and controlling early signs of cancer. NK cells were first noted for their ability to kill tumor cells without priming or prior activation (in contrast to cytotoxic T cells, which require priming by antigen-presenting cells). They were named for this "natural" killing. Furthermore, NK cells secrete cytokines, such as IFNγ and TNFα, which act on other immune cells, such as macrophages and dendritic cells, to enhance the immune response.

[0030] While patrolling, NK cells are constantly in contact with other cells. Whether NK cells kill these cells depends on the balance of signals from activating and inhibitory receptors on the NK cell surface. Activating receptors recognize molecules expressed on the surface of cancer and infected cells and "switch on" the NK cell. Inhibitory receptors act as a check on NK cell killing. Most normal, healthy cells express MHC I receptors, which mark these cells as "self." Inhibitory receptors on the surface of NK cells recognize cognate MHC I, which "switches off" the NK cell and prevents killing. Cancer and infected cells often lose MHC I, making them susceptible to killing by NK cells. Once the decision to kill is made, NK cells release cytotoxic granules containing perforin and granzymes, causing lysis of the target cell.

[0031] Involvement of T cells in the induction of cellular immunity T cells can also act to enhance the ability of monocytes and macrophages to destroy intracellular microorganisms. In particular, interferon gamma (IFNγ) produced by helper T cells enhances several mechanisms by which mononuclear phagocytes destroy intracellular bacteria and parasites, including the production of nitric oxide and the induction of tumor necrosis factor (TNF) production. H1 T cells are effective in enhancing bactericidal activity because they produce IFNγ. H2 Two major cytokines produced by the cells, IL-4 and IL-10, block these activities (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

[0032] Regulatory T (Treg) cells Immune homeostasis is maintained by a controlled balance between the initiation and down-regulation of immune responses. Both mechanisms, apoptosis and T cell anergy (a tolerance mechanism in which T cells are essentially functionally inactivated after antigen encounter (Scwartz, RH, "T cell anergy", Annu. Rev. Immunol., Vol. 21: 305-334 (2003)), contribute to the down-regulation of immune responses. A third mechanism is the suppressor or regulatory CD4 + It is provided by the active suppression of activated T cells by T (Treg) cells (reviewed in Kronenberg, M. et al., "Regulation of immunity by self-reactive T cells", Nature, Vol. 435: 598-604 (2005)). CD4 + Treg (CD4 + CD25 + ) is a naturally occurring subset of anergic and suppressive T cells (Taams, LS et al., "Human anergic / suppressive CD4 + CD25 + CD4 + CD25 + Depletion of Tregs leads to systemic autoimmune disease in mice. Furthermore, transplantation of these Tregs prevents the development of autoimmune disease. + CD25 + Tregs, like their murine counterparts, are generated in the thymus and are characterized by their ability to suppress the proliferation of responder T cells through a cell-cell contact-dependent mechanism, their inability to produce IL-2, and an anergic phenotype in vitro. + CD25 + T cells are classified as suppressor (CD25 high ) cells and non-suppressor (CD25 low) cells. FOXP3, a member of the forkhead family of transcription factors, mediates the transcription of CD4+ cells in mice and humans. + CD25 + Expressed in Tregs and CD4 + CD25 + It has been shown that Rapamycin may be the master gene controlling Treg development (Battaglia, M. et al., "Rapamycin promotes expansion of functional CD4 + CD25 + Foxp3 + regulator T cells of both healthy subjects and type 1 diabetic patients”, J. Immunol., Vol. 177: 8338-8347, (2006)).

[0033] Cytotoxic T lymphocytes CD8 recognizes peptides derived from proteins produced within target cells + T cells have cytotoxic properties in that they direct the lysis of target cells. The mechanism of CTL-induced lysis involves the production by CTLs of perforin, a molecule that can insert into the membrane of target cells and promote their lysis. Perforin-mediated lysis is enhanced by granzymes, a series of enzymes produced by activated CTLs. Many activated CTLs also express large amounts of fas ligand on their surface. The interaction of fas ligand on the CTL surface with fas on the target cell surface initiates apoptosis of the target cells, resulting in their death. CTL-mediated lysis appears to be the primary mechanism for the destruction of virus-infected cells.

[0034] T memory cells After pathogen recognition and eradication by the adaptive immune response, the majority of T cells (90-95%) undergo apoptosis, and the remaining cells form a pool of memory T cells called central memory T cells (TCM), effector memory T cells (TEM), and resident memory T cells (TRM) (Clark, RA, “Resident memory T cells in human health and disease”, Sci. Transl. Med., 7, 269rv1, (2015)).

[0035] Compared to standard T cells, these memory T cells are long-lived and have distinct phenotypes, including expression of specific surface markers, rapid production of distinct cytokine profiles, the capacity for direct effector cell function, and unique homing distribution patterns. Memory T cells exhibit rapid responses upon re-exposure to their corresponding antigens to eliminate re-infection of the attacker and thereby rapidly restore balance to the immune system. Growing evidence demonstrates that autoimmune memory T cells hinder many attempts to treat or cure autoimmune diseases (Clark, RA, "Resident memory T cells in human health and disease," Sci. Transl. Med., Vol. 7, 269rv1, (2015)).

[0036] For an effective immune response to an antigen, antigen-presenting cells (APCs) must process and present antigen in the context of the appropriate major histocompatibility complex (MHC) to T cells, resulting in the stimulation of cytotoxic and helper T cells. Following antigen presentation, successful interaction of costimulatory molecules on both the APC and the T cell is required; otherwise, activation is aborted. GM-CSF and IL-12 function as effective pro-inflammatory molecules in many tumor models. For example, GM-CSF induces the proliferation and differentiation of myeloid precursor cells into dendritic cells (DCs), but additional signals are required to activate their maturation into effective antigen-presenting cells required for T cell activation. Barriers to effective immunotherapy include tolerance to target antigens, which may limit the induction of adequately sized and functional cytotoxic CD8 T cells, insufficient trafficking of generated T cells to the site of malignant cells, and poor persistence of induced T cell responses. DCs that phagocytose tumor cell debris process the material for MHC presentation, upregulate the expression of costimulatory molecules, and migrate to local lymph nodes to stimulate tumor-specific lymphocytes. This pathway leads to the proliferation and activation of CD4+ and CD8+ T cells that respond to tumor-associated antigens. Indeed, such cells can be frequently detected in patients' blood, lymphoid tissues, and malignant lesions.

[0037] Lymphocytes are a type of white blood cell involved in regulating the immune system. Lymphocytes are more common in the lymphatic system and include B cells, T cells, killer T cells, and natural killer (NK) cells. There are two broad categories of lymphocytes: T cells and B cells. T cells are responsible for cell-mediated immunity, while B cells are responsible for humoral immunity (related to antibodies). T cells are so named because these lymphocytes mature in the thymus. B cells mature in the bone marrow. B cells make antibodies that bind to pathogens and enable their destruction. CD4+ (helper) T cells coordinate the immune response. CD8+ (cytotoxic) T cells and natural killer (NK) cells can kill the body's own cells, for example, those infected with viruses or presenting antigenic sequences.

[0038] immune response Generally, the immune response is initiated by an individual's encounter with a foreign agent, e.g., an infectious microorganism. Infected individuals rapidly respond with both a humoral immune response, involving the production of antibody molecules specific to the antigenic determinants / epitopes of the immunogen, and a cell-mediated immune response, involving the expansion and differentiation of antigen-specific regulatory T cells and effector T lymphocytes, including both cytokine-producing cells and killer T cells capable of lysing infected cells. Primary immunization with a specific microorganism elicits antibodies and T cells specific to antigenic determinants / epitopes found on the microorganism, but typically fails to recognize or only poorly recognizes antigenic determinants expressed by unrelated microorganisms (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0039] As a result of this initial response, the immunized individual develops a state of immunological memory. If the same or closely related microorganism is encountered again, a secondary response ensues. This secondary response is generally more rapid and larger in magnitude, consisting of an antibody response composed of antibodies that bind to antigens with higher affinity and more effectively remove the microorganism from the body, as well as an enhanced and often more effective T cell response. However, the immune response to an infectious agent does not always result in elimination of the pathogen (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999), at p. 102).

[0040] Cancer immune tolerance Cancer is characterized by genetic instability in certain cells, but it has also been described as a disorder of the immune system, based on the fact that the immune system, at least in certain segments of the affected human population, is unable to respond optimally to cancerous cells with a clearly non-self phenotype that must be recognized as foreign. Several reasons have been proposed to explain this observation. For example, first, cancer cells are primarily composed of self-antigens, in stark contrast to the situation with infectious organisms. Some antigens classified as cancer antigens are actually normal antigens that are overexpressed or have mutations in only one or two amino acids in the polypeptide chain. Second, cancer cells downregulate MHC, thereby presenting fewer tumor-derived peptides via MHC. Third, cancer cells and associated tumor-associated macrophages express cytokines that dampen the immune response (see, e.g., Yu et al. (2007) Nature Rev. Immunol. 7:41-51). This dampening is caused, for example, by the secretion of interleukin-10 (IL-10) by cancer cells or associated macrophages. Fourth, unlike in infectious disease situations, cancer cells do not provide immune adjuvants. Pathogens express a variety of naturally occurring immune adjuvants in the form of Toll-like receptor (TLR) agonists and NOD agonists (see, e.g., Kleinnijenhuis et al. (2011) Clin. Dev. Immunol. 405310 (page 12)). Generally, optimal activation of dendritic cells requires contact of the immune adjuvant with one or more Toll-like receptors (TLRs) expressed by dendritic cells. Without dendritic cell activation, contact between dendritic cells and T cells (immune synapse) cannot result in optimal T cell activation.

[0041] Insights into the mechanisms underlying immune evasion have served as the basis for the development of vaccines that induce effective antitumor immunity, along with combination treatment regimens that enhance the efficacy of therapeutic vaccination directly or indirectly through combination with immune checkpoint inhibitors or other therapies.

[0042] Immune surveillance and immunoediting Tumor immunoediting can be divided into three phases: elimination, equilibrium, and evasion. The elimination phase, also known as immune surveillance, is the process by which the immune system identifies cancerous or precancerous cells and eliminates them before they become uncontrollable. This phase can be completed when all cancerous or precancerous cells are eliminated. If some tumor cells are not eliminated, a temporary equilibrium between the immune system and tumor cell proliferation can be achieved. During this equilibrium phase, tumor cells can either remain dormant or continue to evolve by accumulating additional changes to their genomic DNA that can regulate the antigens they present. During this process, the immune system exerts selective pressure on evolving cells, giving tumor cells a survival advantage over those that are less likely to be recognized. Eventually, the immune response becomes unable to recognize tumor cells, resulting in the transition to the evasion phase, in which tumor cells gradually proliferate uncontrollably.

[0043] Tumor microenvironment Because tumors actively downregulate all stages of the antitumor immune response using a variety of strategies and mechanisms, the tumor microenvironment provides a consistently effective barrier to immune cell function. Many molecular mechanisms that cause immune cell dysfunction in the tumor microenvironment have been identified. These include mechanisms directly mediated by factors produced by the tumor and others resulting from alterations in normal tissue homeostasis in the presence of cancer. Most human tumors appear to be able to disrupt one or more stages of immune cell development, differentiation, migration, cytotoxicity, and other effector functions (T.L. Whiteside, The tumor microenvironment and its role in promoting tumor growth, Oncogene (2008) 27, 5904-5912).

[0044] One such mechanism is T reg (CD4 + CD25 bright Foxp3 + T cells) and bone marrow-derived cells (CD34 + CD33+ CD13 + CD11b + CD15 - ) in tumors, which is a common feature of human tumors and is associated with poor prognosis in cancer patients (TL Whiteside, The tumor microenvironment and its role in promoting tumor growth, Oncogene (2008) 27, 5904-5912). Under normal conditions, T reg T cells play an important role in preventing autoimmunity, but in cancer, Treg cells expand, migrate to tumors, downregulate the proliferation of autologous effector T cells, and use distinct molecular pathways to inhibit CD4 + CD25 - Cells and CD8 + CD25 - suppresses both the anti-tumor response of T cells and the T reg The cells express regulatory CD3 + CD4 + A heterogeneous population of T cells, including natural T reg These include antigen-specific Tr1 cells, and other less well-defined subsets of suppressor cells. Tr1 cells are induced in tumor microenvironments rich in IL-10, TGF-β, and prostaglandin E2 (PGE2), all of which have been shown to promote Tr1 generation (T.L. Whiteside, The tumor microenvironment and its role in promoting tumor growth, Oncogene (2008) 27, 5904-5912).

[0045] Myeloid suppressor cells (MSCs) also suppress T cell responses in the tumor microenvironment by secreting TGF-β or inducing TGF-β secretion. +Myeloid cell-derived MSCs have been identified in the peripheral blood of cancer patients. In tumor-bearing mice, MSCs accumulate in very large numbers in the spleen and peripheral circulation, exerting potent immunosuppression and promoting tumor growth. MSCs also control the availability of essential amino acids, such as L-arginine, and generate high levels of reactive oxygen species. MSCs found in tumors also constitutively express iNOS and arginase 1, an enzyme involved in L-arginine metabolism that has been shown to synergize with iNOS to increase superoxide and NO production and impede lymphocyte responses. GM-CSF, often secreted by tumor cells, recruits MSCs and induces dose-dependent in vivo immunosuppression and tumor promotion. At the same time, GM-CSF has been used as an immunoadjuvant in antitumor vaccines. GM-CSF was observed to increase a subset of TGF-β-producing MSCs in the circulation of metastatic melanoma patients. The simultaneous stimulatory and inhibitory roles suggest that GM-CSF and MSCs are involved in maintaining immune homeostasis in normal tissues, but in the tumor microenvironment, promoting tumor cell evasion (T.L. Whiteside, The tumor microenvironment and its role in promoting tumor growth, Oncogene (2008) 27, 5904-5912).

[0046] Tumor immunotherapy Cancer treatment is rapidly evolving as new molecular targets are discovered. Despite the emergence of biologics that target specific pathways (e.g., Herceptin®, Erbitux®) and small molecules designed against specific targets (e.g., Tamoxifen, Gleevec™), nonspecific modalities such as chemotherapy and radiation therapy remain the standard of care.

[0047] Anti-cancer immunotherapy has been a long-standing goal, and various approaches have been tested. One difficulty in developing this immunotherapy is that target antigens are often tissue-specific molecules found on both cancer and normal cells, which either do not elicit immune responses or exhibit nonspecific cell killing (Kaufman and Wolchok, eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). Furthermore, tumor cells have characteristics that make immune recognition difficult, such as the loss of expression of antigens that elicit immune responses, the absence of MHC class II, and down-regulated MHC class I expression. These characteristics can result in the non-recognition of tumor cells by both CD4+ and CD8+ T cells (Kaufman and Wolchok, eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). Tumors can also evade detection through active mechanisms such as the production of immunosuppressive cytokines (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).

[0048] DCs generated ex vivo by culturing hematopoietic progenitor cells or monocytes with a combination of cytokines have been tested as therapeutic vaccines for cancer patients for over a decade (Ueno H, et al., Immunol. Rev. (2010) 234: 199-212). For example, treatment of metastatic prostate cancer with sipuleucel-T (also known as APC 8015), a cell product based on enriched blood APCs cultured for a short period with a fusion protein of prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GM-CSF), resulted in a median survival extension of approximately 4 months in phase III trials (Higano CS, et al., Cancer (2009) 115: 3670-3679; Kantoff PW, et al., N. Engl. J. Med. (2010) 363: 411-422). This study concluded that DC-based vaccines are safe and can induce the expansion of circulating CD4+ and CD8+ T cells specific for tumor antigens. As a result of this and similar studies, sipuleucel-T was approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic prostate cancer, thereby paving the way for clinical development and regulation of next-generation cellular immunotherapy products (Palucka K and Banchereau J, Nature Reviews Cancer (April 2012) 12: 265-276).

[0049] Vaccination strategies involving DCs have been developed to induce tumor-specific effector T cells that can specifically reduce tumor burden and induce immunological memory to control tumor recurrence. For example, DCs can be cultured ex vivo with adjuvants and tumor-specific antigens, and then these cells can be injected back into patients to provide tumor-specific antigens to the DCs. Tumor cells obtained from resected tumors, needle biopsies, core biopsies, vacuum-assisted biopsies, or peritoneal lavage have been used to generate immunogenic compositions containing tumor-specific antigen-presenting dendritic cells.

[0050] Cancer Treatment Strategies Antibody therapies such as Herceptin™ and Erbitux™ are passive immunotherapies, but they have led to significant improvements in clinical outcomes, as measured by, for example, recurrence rates, progression-free survival, and overall survival. More recently, PD-1 and CTLA4 inhibitors have been reported to block distinct checkpoints in the active host immune response, sustaining endogenous anticancer immune responses. The term "immune checkpoint" refers to a series of inhibitory pathways necessary for maintaining self-tolerance and regulating the duration and magnitude of immune responses to minimize damage to normal tissues. Immune checkpoint molecules, such as PD-1, PD-L1, and CTLA-4, are cell surface signaling receptors that play a role in regulating T cell responses in the tumor microenvironment. Tumor cells have been shown to exploit and benefit from these checkpoints by upregulating their expression and activity. Due to the ability of tumor cells to direct several immune checkpoint pathways as a mechanism of immune resistance, it has been hypothesized that checkpoint inhibitors, which bind to and activate or inactivate immune cell molecules, may alleviate the inhibition of the immune response. Recent discoveries have identified immune checkpoints or targets, such as PD-1, PD-L1, PD-L2, CTLA-4, TIM3, LAG3, CCR4, OX40, OX40L, IDO, and A2AR, as proteins involved in immune evasion. Specific immune checkpoint inhibitors, including antibodies against CTLA-4, the PD-1 receptor, or its ligand, PD-L1, have produced impressive results in the clinic across a variety of cancers, leading to FDA approval of Yervoy™ (ipilimumab; a CTLA-4 antagonist), Opdivo™ (nivolumab; a PD-1 antagonist), and Keytruda™ (pembrolizumab; a PD-1 antagonist) for multiple oncology indications, with many more registrational trials underway.However, this therapy can only be successful if a patient has a pre-existing antitumor immune response (Pardoll, D., The blockade of immune checkpoints in cancer immunotherapy, Nature Reviews: Cancer, Vol. 12, April 2012, 253). Recent cell therapies, such as chimeric antigen receptor T-cell therapy (CAR-T), use synthetic biology to redirect T cells to specific cell-surface tumor antigens. Genetic modification of T cells is used to confer tumor antigen recognition through the transgenic expression of chimeric antigen receptors (CARs). CARs are engineered molecules that can be introduced into T cells to target tumor antigens (Frey, NV, Porter, DL, The Promise of Chimeric Antigen Receptor T-Cell Therapy, Oncology (2016); 30(1)) pii 219281). CAR T cells have shown some efficacy against hematological malignancies and, to a lesser extent, solid tumors. However, CAR T therapy has been shown to cause several types of toxicity, including cytokine release syndrome, neurotoxicity, non-tumor recognition, and anaphylaxis (Bonifant CL, et al., Toxicity and management in CAR T-cell therapy, Molecular Therapy - Oncolytics (2016) 3, 16011).

[0051] Therapeutic vaccination against cancer is an important modality that complements current standard treatments and has the potential to provide long-term cancer control. A prototypic example, GVAX™, is a GM-CSF gene-transduced tumor vaccine in either an autologous or allogeneic population of tumor cells. GM-CSF secretion from genetically modified tumor cells is thought to stimulate cytokine release at the vaccine site, activate antigen-presenting cells, and induce tumor-specific cellular immune responses (Eager, R. & Nemunaitis, J., GM-CSF Gene-Transduced Tumor Vaccines, Molecular Therapy, Vol. 12, No. 1, 18 (July 2005)). Although lethally irradiated tumor cell vaccines engineered to secrete GM-CSF (GVAX) have shown promising efficacy in various models of melanoma, renal cell, prostate, non-small cell lung, pancreatic, and head and neck squamous cell carcinoma, due to multiple immunological checkpoint blockades, GVAX as a monotherapy is unlikely to be clinically effective in advanced disease. There remains a need for improved compositions and methods for immunotherapy strategies to treat diseases such as cancer that can be refractory to traditional therapies.

[0052] Dendritic cell (DC)-tumor cell fusion has been developed to generate hybrid cells that express tumor-associated antigens derived from the parent tumor cells and are capable of processing and presenting such antigens to appropriate cells of the immune system. While DC-tumor cell fusion provides a greater variety of tumor antigens, it has met with limited success in human clinical trials, likely due to the required autologous components, product heterogeneity caused by DC cell maturation, and variability in antigen loading (Browning, M., Antigen-presenting cell / tumor cell fusion vaccines for cancer, Human Vaccines & Immunotherapeutics 9:7, 1545-1548; July 2013; Butterfield, L., Dendritic Cells in Cancer Immunotherapy Clinical Trials: Are We Making Progress?, Frontiers of Immunology, 2013, 4: 454).

[0053] Vaccine immunogenicity Vaccines against infectious pathogens are a prime example of specific receptor-ligand interactions used to shape immune responses with the therapeutic goal of preventing or mitigating infection (e.g., influenza vaccines). Generally, antigens are presented to the immune system in combination with adjuvants (e.g., synthetic small molecule immunomodulators).

[0054] The allogeneic tumor vaccines of the described invention differ from such vaccines in several important features. First, they are designed to treat existing tumors, but theoretically could also prevent tumor formation. Second, their effectiveness tends to be limited by the fact that while tumors express neoantigens (i.e., new non-self elements) that are foreign to the individual, they are also undoubtedly human tumor cells and therefore may not always be recognized as foreign (i.e., non-self) by the individual.

[0055] Despite the aforementioned difficulties, evidence is now emerging that 1) endogenous antitumor responses exist, 2) these immune responses can be modulated, and 3) this modulation can be measured in terms of overall survival in standard clinical trials.

[0056] According to some aspects of the described invention, a set of immunomodulatory factors that can be co-expressed either on tumor cell lines or tumor cell line variants derived from cancer patients or on allogeneic tumor cell lines or tumor cell line variants has been identified that, when used as tumor vaccines, can: 1) efficiently load endogenous antigen-presenting cells with a broad range of tumor antigens; 2) efficiently stimulate several cell types by enhancing normal signals received during an immune response; 3) interfere with mechanisms by which T regulatory cells suppress immune responses; 4) interfere with signals that are generally lost in immune responses; and 5) increase the overall survival rate of cancer patients vaccinated with such formulations. In certain embodiments, although the modified tumor cell lines or tumor cell line variants may be derived from the patient receiving the vaccine, an allogeneic tumor cell line or tumor cell line variant vaccine approach differs from a personalized therapeutic approach because the modified tumor cells are not necessarily derived from the individual who ultimately receives the vaccine. Instead, allogeneic tumor cell vaccines aim to focus the immune response on the many elements that individual tumors of the same tumor type share in common.

[0057] One strategy for utilizing the large number of potential tumor antigens for each individual type of cancer is to vaccinate with whole tumor cells to avoid accidentally excluding potentially relevant antigens. The invention described herein provides, among other things, vaccines comprising whole tumor cells carrying a range of tumor antigens and modified to express three or more immune modulators. Summary of the Invention

[0058] According to some embodiments, the described invention is an allogeneic tumor cell vaccine comprising: (1) a population of proliferation-incompetent, live, genetically engineered tumor cells expressing one or more tumor-specific antigens, the population comprising: at least three stably expressed immunomodulatory molecules, the at least three immunomodulatory molecules being OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, to induce one or more subpopulations of PBMCs to proliferate in response to the expressed immunomodulatory molecules and then enter an effector phase to kill the tumor cells, wherein the subpopulation of PBMC cells comprises one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes; (2) a pharmaceutically acceptable carrier; and The present invention provides an allogeneic tumor cell vaccine comprising:

[0059] According to some embodiments, the population of proliferation-incompetent, live, genetically engineered tumor cells expressing one or more tumor-specific antigens further comprises one or more additional stably expressed immunomodulatory molecules selected from R1-R44. According to some embodiments, the tumor cells are rendered proliferation-incompetent by irradiation. According to some embodiments, the induction of T lymphocytes comprises activation of a subpopulation of T lymphocytes, expansion of T lymphocytes, or both. According to some embodiments, the induction of NK cells comprises activation of a subpopulation of NK cells, expansion of a subpopulation of NK cells, or both. According to some embodiments, the induction of a subpopulation of DCs comprises activation of a subpopulation of DCs, expansion of a subpopulation of DCs, or both. According to some embodiments, the induction of a subpopulation of B lymphocytes comprises activation of a subpopulation of B lymphocytes, expansion of a subpopulation of B lymphocytes, or both. According to some embodiments, the subpopulation of NK cells comprises a subpopulation of memory-like NK cells. According to some embodiments, the subpopulation of T lymphocytes comprises a subpopulation of CD8+ cytotoxic T lymphocytes (CTLs). According to some embodiments, the subpopulation of T lymphocytes comprises a subpopulation of memory T cells. According to some embodiments, the subpopulation of T lymphocytes comprises a subpopulation of regulatory T cells. According to some embodiments, the subpopulation of T lymphocytes comprises a subpopulation of helper T cells. According to some embodiments, the subpopulation of B lymphocytes comprises a subpopulation of memory B cells.

[0060] According to some embodiments, the vaccine (1) enhances immune activation of cells effective to recognize and act against tumor cells containing the target tumor antigen in vivo without systemic inflammation, (2) reduces immune suppression in the tumor microenvironment of tumor cells containing the target tumor antigen, or (3) increases cell death of tumor cells expressing the target tumor antigen.

[0061] According to some embodiments, the tumor cells are derived from a cancer selected from the group consisting of melanoma, colorectal cancer, leukemia, chronic myeloid leukemia, prostate cancer, head and neck cancer, squamous cell carcinoma, tongue cancer, laryngeal cancer, tonsil cancer, hypopharyngeal cancer, nasopharyngeal cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, glioblastoma, and brain cancer. According to some embodiments, the melanoma tumor cells are characterized by expression of one or more of gp100, tyrosinase, Melan-A, tyrosinase-related protein (TRP-2-INT2), melanoma antigen-1 (MAGE-A1), NY-ESO-1, melanoma preferentially expressed antigen (PRAME), CDK4, and multiple myeloma oncogene 1 (MUM-1). According to some embodiments, the colorectal cancer tumor cells are characterized by expression of one or more of carcinoembryonic antigen (CEA), MAGE, HPV, human telomerase reverse transcriptase (hTERT), EPCAM, PD-1, PD-L1, p53, and cell surface-associated mucin 1 (MUC1).

[0062] According to some embodiments, the population of viable, growth-resistant tumor cells is derived from a biological sample from a subject. According to some embodiments, the population of viable, growth-resistant tumor cells is derived from a tumor cell line. According to some embodiments, the population of viable, growth-resistant tumor cells is effective at inducing immune activation without systemic inflammation. According to some embodiments, the vaccine induces an immune response that improves progression-free survival, overall survival, or both compared to a placebo control. According to some embodiments, the one or more additional stably expressed immunomodulatory molecules selected from R1-R44 are cytokines, TNF family members, secreted receptors, chaperones, IgG superfamily members, and / or chemokine receptors. According to some embodiments, the immunostimulatory molecules are displayed on the external surface of the genetically engineered tumor cells.

[0063] According to another aspect, the described invention provides a method of inducing an immune response against cancer in a subject, comprising parenterally or locally intratumorally administering to the subject an allogeneic tumor cell vaccine according to claim 1, wherein the allogeneic tumor cell vaccine matches the subject's cancer. According to some embodiments, the cancer is selected from melanoma or colorectal cancer. According to some embodiments, the subject has an infectious viral disease associated with the progression to cancer. According to some embodiments, the method further comprises administering to the subject a checkpoint inhibitor.

[0064] According to another aspect, the described invention is a method of treating cancer in a subject, comprising administering to the subject: (1) a population of proliferation-incompetent, live, genetically engineered tumor cells expressing one or more tumor-specific antigens, the population comprising: at least three stably expressed immunomodulatory molecules for inducing one or more subpopulations of PBMCs to proliferate in response to the expressed immunomodulatory molecules and then enter an effector phase to kill the tumor cells, wherein the at least three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and wherein the subpopulation of PBMC cells comprises one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes; (2) a pharmaceutically acceptable carrier; and In some embodiments, the effective amount improves clinical outcomes. In some embodiments, the effective amount improves progression-free survival, overall survival, or both, in a subject compared to a placebo control. In some embodiments, the cancer is melanoma or colorectal cancer.

[0065] According to another aspect, an allogeneic tumor cell vaccine comprising: (1) a population of proliferation-incompetent live genetically engineered tumor cells expressing one or more tumor-specific antigens, the population comprising: at least three stably expressed immunomodulatory molecules for inducing one or more subpopulations of PBMCs to proliferate in response to the expressed immunomodulatory molecules and then enter an effector phase to kill tumor cells, the at least three immunomodulatory molecules being OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and the subpopulation of PBMC cells comprising one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes; and (2) a pharmaceutically acceptable carrier, the vaccine comprises the steps of: providing an allogeneic parental tumor cell line comprising the population of live tumor cells; and introducing into the population of live tumor cells an exogenous nucleic acid encoding the stably expressed immunomodulatory molecules, wherein the immunomodulatory molecules induce one or more subpopulations of PBMCs to proliferate in response to the expressed immunomodulatory molecules and then enter an effector phase to kill tumor cells. introducing an exogenous nucleic acid encoding a stably expressed immunomodulatory molecule into a population of live tumor cells, wherein the immunomodulatory molecule is CD27 ligand (CD70); introducing an exogenous nucleic acid encoding a stably expressed immunomodulatory molecule into a population of live tumor cells, wherein the immunomodulatory molecule is CD28 ligand (CD28L) comprising CD80, CD86, or both, and wherein stable expression of OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L) comprising CD80, CD86, or both induces one or more subpopulations of PBMCs to proliferate in response to the expressed immunomodulatory molecule and then enter an effector phase to kill the tumor cells; generating tumor cell line variants by selecting tumor cell clones that stably express immunogenic amounts of the exogenous subset of immunomodulatory molecules;and selecting clonally derived cell line variants in a mixed lymphocyte-tumor cell reaction by one or more of the following parameters: cell proliferation, cell subset differentiation, cytokine release profile, and tumor cell lysis, wherein the selected clonally derived cell line variants are effective in stimulating activation of one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes;

[0066] According to some embodiments, the process for producing an allogeneic tumor cell vaccine further comprises introducing into the population of live tumor cells an exogenous nucleic acid encoding one or more stably expressed immunomodulatory molecules selected from R1-R44. According to some embodiments, the tumor cells are rendered proliferation incompetent by irradiation. According to some embodiments, the parent tumor cell line is derived from a tumor selected from the group consisting of melanoma and colorectal carcinoma. According to some embodiments, the exogenous nucleic acid comprises DNA or RNA. According to some embodiments, the introducing step comprises viral transduction. According to some embodiments, the introducing step comprises electroporation. According to some embodiments, the introducing step comprises utilizing one or more of liposome-mediated transfer, adenovirus, adeno-associated virus, herpes virus, retrovirus-based vectors, lipofection, and lentiviral vectors. According to some embodiments, the introducing step comprises introducing the exogenous nucleic acid by transfection with a lentiviral vector.

[0067] These and other advantages of the present invention will become apparent to those skilled in the art upon review of the following detailed description. [Brief explanation of the drawings]

[0068] [Figure 1] 1 shows one embodiment of heteroclitic cross-reactivity between peptides native to a tumor cell line and peptides native to tumor cells of a patient undergoing immunotherapy. [Figure 2] 1 shows a schematic diagram of the construction of scFv-anti-biotin-G3 hinge-mIgG1 Vector 1. [Figure 3] Schematic diagram of the construction of full-length anti-biotin-G3 hinge-mIgG1 Vector 2. [Figure 4] A schematic diagram of the construction of sGM-CSF / ires / mFLT3L vector 3 is shown. [Figure 5] A schematic diagram of the construction of sFLT3L / ires / (FLT3 signal-GM-CSF-Tm) vector 4 is shown. [Figure 6] 1 shows a schematic diagram of the construction of mCD40L vector 5. [Figure 7] 1 shows a schematic diagram of the construction of mTNFα vector 6. [Figure 8] A schematic diagram of the construction of mRANKL / ires / FLT3 signal-V5-scFV anti-biotin-Tm vector 7 is shown. [Figure 9] A schematic diagram of Vector 44 is shown. [Figure 10] A schematic diagram of Vector 97 is shown. [Figure 11] A schematic diagram of vector 84 is shown. [Figure 12] A schematic diagram of vector 29 is shown. [Figure 13] A schematic diagram of vector 107 is shown. [Figure 14] A schematic diagram of the vector 116 is shown. [Figure 15] A schematic diagram of Vector 86 is shown. [Figure 16] A schematic diagram of Vector 18 is shown. [Figure 17] A schematic diagram of Vector 17 is shown. [Figure 18] A schematic diagram of vector 98 is shown. [Figure 19] A schematic diagram of vector 30 is shown. [Figure 20] A schematic diagram of vector 109 is shown. [Figure 21] A schematic diagram of vector 106 is shown. [Figure 22] A schematic diagram of Vector 16 is shown. [Figure 23] A schematic diagram of Vector 83 is shown. [Figure 24] A schematic diagram of Vector 31 is shown. [Figure 25] A schematic diagram of Vector 12 is shown. [Figure 26] A schematic diagram of vector 99 is shown. [Figure 27] A schematic diagram of Vector 121 is shown. [Figure 28] A schematic diagram of vector 105 is shown. [Figure 29] A schematic diagram of Vector 32 is shown. [Figure 30] A schematic diagram of Vector 37 is shown. [Figure 31] A schematic diagram of Vector 22 is shown. [Figure 32] A schematic diagram of Vector 19 is shown. [Figure 33] A schematic diagram of vector 20 is shown. [Figure 34] A schematic diagram of Vector 89 is shown. [Figure 35] A schematic diagram of Vector 21 is shown. [Figure 36] A schematic diagram of Vector 23 is shown. [Figure 37] A schematic diagram of vector 108 is shown. [Figure 38] A schematic diagram of Vector 15 is shown. [Figure 39] A schematic diagram of vector 124 is shown. [Figure 40] A schematic diagram of Vector 65 is shown. [Figure 41] A schematic diagram of Vector 64 is shown. [Figure 42] A schematic diagram of Vector 88 is shown. [Figure 43] A schematic diagram of Vector 96 is shown. [Figure 44] A schematic diagram of Vector 14 is shown. [Figure 45] A schematic diagram of vector 119 is shown. [Figure 46] A schematic diagram of a vector 120 is shown. [Figure 47]A schematic diagram of Vector 45 is shown. [Figure 48] A schematic diagram of vector 60 is shown. [Figure 49] A schematic diagram of vector 59 is shown. [Figure 50] A schematic diagram of Vector 8 is shown. [Figure 51] A schematic diagram of vector 128 is shown. [Figure 52] A schematic diagram of Vector 35 is shown. [Figure 53] FIG. 1 is a schematic diagram showing a general experimental format. [Figure 54] This is a panel of graphs showing the results of flow cytometry experiments. Forward scatter (FSC) and side scatter (SSC) plots of size and granularity. SK lines are represented by a number code: SK, unmodified parent line; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α; 3-4 is a combination of 3 and 4; 3-4-5 is a combination of 3, 4, and 5; and 3-4-6 is a combination of 3, 4, and 6. Cell lines 6, 3-4-5, and 3-4-6 exhibit a larger, more granular phenotype, likely due to the presence of receptors for TNF-α and CD40L on the epithelial-derived cells. [Figure 55] 1 is a panel of graphs showing representative flow cytometry staining of CD4+ cells in hPBMCs in response to the indicated engineered cell lines with the indicated immunomodulators. SK cell lines are represented by the following codes: SK, unmodified parent line; 2, membrane-expressed IgG1; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; and 6, uncleaved form of TNF. [Figure 56](A) is a panel of graphs showing representative flow cytometry staining for the indicated engineered surface markers: GM-CSF, FLT3L, TNF-α, and CD40L. SK lines are represented by number codes: SK, unmodified parental line; 3, secreted GM-CSF and membrane-expressed FLT3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α; 3-4 is a combination of 3 and 4; 3-4-5 is a combination of 3, 4, and 5; and 3-4-6 is a combination of 3, 4, and 6. [Figure 57A] CyTOF mass cytometry single-cell phenotyping analysis of hPBMC responses to SK melanoma cells modified by expression of immune modulators. (A) viSNE density contour plot of CyTOF staining data showing relative changes in immune cell subset abundance and phenotype. (B) Single-cell phenotyping analysis. SK strains are represented by number codes: SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α; 3-4 is a combination of 3 and 4; 3-4-5 is a combination of 3, 4, and 5; and 3-4-6 is a combination of 3, 4, and 6. [Figure 57B] CyTOF mass cytometry single-cell phenotyping analysis of hPBMC responses to SK melanoma cells modified by expression of immune modulators. (A) viSNE density contour plot of CyTOF staining data showing relative changes in immune cell subset abundance and phenotype. (B) Single-cell phenotyping analysis. SK strains are represented by number codes: SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α; 3-4 is a combination of 3 and 4; 3-4-5 is a combination of 3, 4, and 5; and 3-4-6 is a combination of 3, 4, and 6. [Figure 58A]CyTOF monocyte cluster analysis of hPBMCs showing changes in expression of the activation marker CD40 after 1 day of stimulation with the indicated genetically modified SK lines at a cell ratio of 1:5. SK lines are represented by number codes: SK, unmodified parent line; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α; 3-4 is a combination of 3 and 4; 3-4-5 is a combination of 3, 4, and 5; and 3-4-6 is a combination of 3, 4, and 6. [Figure 58B] CyTOF monocyte cluster analysis of hPBMCs showing changes in activation marker CD86 expression after 1 day of stimulation with the indicated genetically modified SK lines at a cell ratio of 1:5. SK lines are represented by number codes: SK, unmodified parent line; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α; 3-4 is a combination of 3 and 4; 3-4-5 is a combination of 3, 4, and 5; and 3-4-6 is a combination of 3, 4, and 6. [Figure 58C] CyTOF monocyte cluster analysis of hPBMCs showing changes in activation marker CD69 expression after 1 day of stimulation with the indicated genetically modified SK lines at a cell ratio of 1:5. SK lines are represented by number codes: SK, unmodified parent line; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α; 3-4 is a combination of 3 and 4; 3-4-5 is a combination of 3, 4, and 5; and 3-4-6 is a combination of 3, 4, and 6. [Figure 58D]CyTOF monocyte cluster analysis of hPBMCs showing changes in expression of the activation marker CD25 after 1 day of stimulation with the indicated genetically modified SK lines at a cell ratio of 1:5. SK lines are represented by number codes: SK, unmodified parent line; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α; 3-4 is a combination of 3 and 4; 3-4-5 is a combination of 3, 4, and 5; and 3-4-6 is a combination of 3, 4, and 6. [Figure 58E] CyTOF monocyte cluster analysis of hPBMCs showing relative median expression levels (MEI) of monocyte markers CD40 and CD86. [Figure 58F] CyTOF monocyte cluster analysis of hPBMCs showing the relative median expression index (MEI) of the CD4 T cell markers CD69 and CD25. [Figure 59] This graph shows the results of Luminex multiplex cytokine profiling of human PBMC responses to the parental SK strain and genetically modified SK strains. Control cultures included SK cells alone, hPBMCs alone, and hPBMCs stimulated with a mixture of anti-CD3 and anti-CD28 antibodies (final concentration 1 μg / ml). Symbols indicate cytokine levels in pg / ml estimated from a standard curve using recombinant cytokines. Absence of a symbol indicates no cytokine was detected. SK strains are represented by a number code: SK, unmodified parental strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α; 3-4 is a combination of 3 and 4; 3-4-5 is a combination of 3, 4, and 5; and 3-4-6 is a combination of 3, 4, and 6. [Figure 60]Figure 1 shows the results of a flow cytometry experiment demonstrating that CD8+ T cells can be activated by genetically modified SKMEL2 tumor cells expressing immunomodulatory molecules. Flow-controlled cytometry size and granularity forward scatter (FSC) and side scatter (SSC) plots after incubation of the parental cell line SKMEL2 ((i)) and genetically modified 14-18-30-expressing SK-MEL-2 tumor cells ((ii)) with PBMCs in a mixed lymphocyte tumor response assay. The dotted oval in (i) and (ii) indicates the lymphocyte gate. (iii) and (iv) show the CD8 population after incubation of PBMCs with the parental cell line (iii) and genetically modified 14-18-30-expressing SKMEL2 tumor cells (iv) in a mixed lymphocyte tumor response assay. The dotted circle in the bottom panel of the graph indicates the CD8 gate. [Figure 61A] Comparing day 9 cultures of the parental SKMEL2 cell line (A) and the genetically modified 14-18-30 cell line (B) expressing a combination of immune modulators shown in Table 2 shows that in vitro CD8+ T cell expansion from hPBMCs results in tumor cell killing. [Figure 61B] Comparing day 9 cultures of the parental SKMEL2 cell line (A) and the genetically modified 14-18-30 cell line (B) expressing a combination of immune modulators shown in Table 2 shows that in vitro CD8+ T cell expansion from hPBMCs results in tumor cell killing. [Figure 62] Flow cytometry results demonstrating stimulation of dendritic cells (DCs), natural killer (NK) cells, and subpopulations of B cells in genetically modified SK lines (i) APX / 15; (ii) APX / 19; (iii) APX / 22; (iv) APX / 23; and (v) APX / 29 are shown. [Figure 63]CyTOF data demonstrating differentiation of various subsets of PBMCs after stimulation with genetically engineered SK lines expressing immunomodulatory molecules ((i) parental; (ii) APX / 3; (iii) APX / 3-4; (iv) APX / 3-4-5; (v) APX / 3-4-6). SK lines are represented by number codes: 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α. [Figure 64] Detailed CyTOF data showing DC activation after stimulation with genetically engineered SK lines expressing immunomodulatory molecules ((i) parental; (ii) APX / 3; (iii) APX / 3-4; (iv) APX / 3-4-5; (v) APX / 3-4-6). SK lines are represented by number codes: 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; 6, uncleaved form of TNF-α. [Figure 65] Flow cytometry results comparing day 6 and day 8 time points in a CD8 expansion assay using the SK parental line (left panel) versus the genetically modified 14-18-30-expressing SK-MEL-2 tumor cell line (right panel) expressing the combinations of immune modulators shown in Table 2 are shown. [Figure 66]This plot shows the mean and standard deviation results of a xenograft treatment study using NGS mice. The edges of each box represent the upper and lower quartiles. The median is marked by a vertical line within the box, and the whiskers are the two lines outside the box extending to the highest and lowest observed values. Human tumor cells were implanted into the flanks of NGS (NOD scid gamma) mice. Tumors were allowed to grow to 150 mm3. Mice were divided into two groups: control and treatment, with six mice per group. On day 30 (t=0), control mice were inoculated with vehicle alone, and treatment mice were inoculated with 3 x 106 PBMCs activated with 14-18-30-expressing ENLIST™ cells ("SUPLEXA™ cells"). Tumor size was measured at intervals up to 36 days post-inoculation. Differences between the two groups emerged within five days. After 22 days, the differences became statistically significant (*P<0.05; **P<0.05). DETAILED DESCRIPTION OF THE INVENTION

[0069] definition The terms "activation" or "lymphocyte activation" refer to the stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells, resulting in the synthesis of RNA, protein, and DNA, and the production of lymphokines, followed by the proliferation and differentiation of various effector and memory cells. For example, mature B cells can be activated by encountering an antigen that displays an epitope recognized by its cell surface immunoglobulin Ig. The activation process can be direct, relying on cross-linking of membrane Ig molecules by antigen (cross-linking-dependent B cell activation), or indirect, occurring most efficiently in association with close interaction with helper T cells (the "cognate help process"). T cell activation depends on the interaction of the TCR / CD3 complex with its cognate ligand, i.e., a peptide that binds to the groove of class I or class II MHC molecules. The molecular events set in motion by receptor engagement are complex. The earliest step appears to be activation of tyrosine kinases, leading to the tyrosine phosphorylation of a series of substrates that control several signaling pathways. These include a series of adaptor proteins that link the TCR to the ras pathway; phospholipase Cγ1, whose tyrosine phosphorylation enhances its catalytic activity and is involved in the inositol phospholipid metabolic pathway, leading to an increase in intracellular free calcium concentration and activation of protein kinase C; and a series of other enzymes that control cell proliferation and differentiation. Full T cell responsiveness requires, in addition to receptor engagement, costimulatory activity delivered by accessory cells, such as the binding of CD28 on the T cell by CD80 and / or CD86 on antigen-presenting cells (APCs). Soluble products of activated B lymphocytes are immunoglobulins (antibodies). Soluble products of activated T lymphocytes are lymphokines.

[0070] B cell activation Mature B cells can be activated by encountering an antigen that displays an epitope recognized by its cell surface immunoglobulin Ig. The activation process can be direct, relying on cross-linking of membrane Ig molecules by the antigen (cross-linking-dependent B cell activation), or indirect, occurring most efficiently in association with close interaction with helper T cells (the "cognate help process"). The soluble products of activated B lymphocytes are immunoglobulins (antibodies).

[0071] T cell activation depends on the interaction of the TCR / CD3 complex with its cognate ligand, a peptide that binds to the groove of a class I or class II MHC molecule. The molecular events set in motion by receptor binding are complex. The earliest step appears to be activation of tyrosine kinases, which result in the tyrosine phosphorylation of a series of substrates that control several signaling pathways. These include a series of adaptor proteins that link the TCR to the ras pathway; phospholipase Cγ1, whose tyrosine phosphorylation enhances its catalytic activity and contributes to the inositol phospholipid metabolic pathway, leading to an increase in intracellular free calcium concentration and activation of protein kinase C; and a series of other enzymes that control cell proliferation and differentiation. In addition to receptor engagement, full T cell responsiveness requires costimulatory activity delivered by accessory cells, such as the ligation of CD28 on T cells by CD80 and / or CD86 on antigen-presenting cells (APCs). The soluble products of activated T lymphocytes are lymphokines.

[0072] dendritic cell activation Pathogen invasion induces a rapid inflammatory response initiated through the recognition of pathogen-derived molecules by pattern recognition receptors (PRRs) expressed on both immune and non-immune cells. Joffre, O., et al., Immunol. Rev. (2009) 277(1): 234-47. The initial wave of proinflammatory cytokines and chemokines limits pathogen spread and recruits and activates immune cells to eradicate the invader. Dendritic cells (DCs) are responsible for initiating the next phase of immunity, which is dominated by the action of pathogen-specific T cells and B cells. Regarding the initial proinflammatory response, DC activation is induced by PRR signaling, which converts resting DCs into potent antigen-presenting cells capable of promoting the expansion and effector differentiation of naive pathogen-specific T cells. Although DCs can be indirectly activated by inflammatory cytokines, these cells are unable to induce functional T cell responses and may function in tolerance induction.

[0073] As used herein, the term "CD8+ T cell activation" or "CD8+ T cell activation" refers to a process (e.g., a signaling event) that causes or results in one or more cellular responses selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers of CD8+ T cells (CTLs). As used herein, "activated CD8+ T cells" refer to CD8+ T cells that have received an activation signal and therefore exhibit one or more cellular responses selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring CD8+ T cell activation are known in the art and are described herein.

[0074] As used herein, the term "expanding CD8+ T cells" or "CD8+ T cell expansion" refers to a process in which a population of CD8+ T cells undergoes a series of cell divisions, thereby increasing the number of cells. The term "expanded CD8+ T cells" relates to CD8+ T cells obtained by CD8+ T cell expansion. Suitable assays for measuring T cell expansion are known in the art and described herein.

[0075] As used herein, the term "activating NK cells" or "NK cell activation" refers to a process (e.g., a signaling event) that causes or results in an NK cell that is capable of killing cells that lack MHC class I expression. As used herein, an "activated NK cell" refers to an NK cell that has received an activating signal and is therefore capable of killing cells that lack MHC class I expression. Suitable assays for measuring NK cell activation are known in the art and are described herein.

[0076] As used herein, the term "expanding NK cells" or "NK cell expansion" refers to the process by which a population of NK cells undergoes a series of cell divisions, thereby increasing the cell number. The term "expanded NK cells" refers to NK cells obtained by NK cell expansion. Suitable assays for measuring NK cell expansion are known in the art and are described herein.

[0077] As used herein, the term "administration" and its various grammatical forms as applied to a mammal, cell, tissue, organ, or biological fluid refers to, but is not limited to, the contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical, therapeutic, diagnostic, or composition with a subject, cell, tissue, organ, or biological fluid, etc. "Administration" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments of a cell, for example, with a reagent, diagnostic, binding composition, or another cell.

[0078] As used herein, the term "allogeneic" means that the donor and recipient (host) have different genetic makeups but are of the same species. As used herein, "allogeneic cells" refer to cells that are not derived from the recipient, meaning the individual to whom the cells are administered, i.e., have a different genetic makeup from the recipient individual. Allogeneic cells are generally obtained from the same species as the individual to whom the cells are administered. For example, allogeneic cells can be human cells for administration to a human patient, such as a cancer patient, as disclosed herein. As used herein, "allogeneic tumor cells" refer to tumor cells that are not derived from the recipient, meaning the individual to whom the allogeneic cells are administered. Generally, allogeneic tumor cells express one or more tumor antigens that can stimulate an immune response against the tumor in the individual to whom the cells are administered. As used herein, "allogeneic cancer cells," e.g., lung cancer cells, refer to cancer cells that are not derived from the recipient individual to whom the allogeneic cells are administered.

[0079] The terms "amino acid residue" or "amino acid" or "residue" are used interchangeably to refer to amino acids incorporated into proteins, polypeptides, or peptides, including, but not limited to, naturally occurring amino acids and known analogs of natural amino acids that can function similarly to naturally occurring amino acids. Amino acids may be L- or D-amino acids. Amino acids may be replaced by synthetic amino acids that are modified to increase the half-life of the peptide, increase the potency of the peptide, or increase the bioavailability of the peptide. The single-letter designations for amino acids are primarily used herein. Such single letter designations are as follows: A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; G is glycine; H is histidine; I is isoleucine; K is lysine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; R is arginine; S is serine; T is threonine; V is valine; W is tryptophan; and Y is tyrosine. The following represent groups of amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0080] The term "apoptosis" or "programmed cell death" refers to a highly regulated, active process contributing to biological homeostasis that consists of a series of biochemical events that result in a variety of morphological changes, including blebbing, changes in the cell membrane, e.g., membrane asymmetry and loss of adhesion, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation, without causing damage to the organism.

[0081] Apoptotic cell death is induced by many different factors and involves multiple signaling pathways, some dependent on caspase proteases (a class of cysteine ​​proteases) and others independent of caspase. It can be triggered by many different cellular stimuli, including cell surface receptors that lead to activation of apoptotic signaling pathways, mitochondrial responses to stress, and cytotoxic T cells.

[0082] Caspases involved in apoptosis transmit the apoptotic signal in a proteolytic cascade, where caspases cleave and activate other caspases, which then degrade other cellular targets, leading to cell death. Caspases at the top of the cascade include caspase-8 and caspase-9. Caspase-8 is the first caspase involved in responding to death domain (DD)-containing receptors, such as Fas.

[0083] Receptors in the TNF receptor family are involved in the induction of apoptosis and inflammatory signaling. The Fas receptor (CD95) mediates apoptotic signaling via Fas ligand expressed on the surface of other cells. Fas-FasL interaction plays an important role in the immune system, and deficiencies in this system lead to autoimmunity, suggesting that Fas-mediated apoptosis eliminates autoreactive lymphocytes. Fas signaling is also involved in immune surveillance to eliminate transformed and virus-infected cells. When Fas binds to oligomerized FasL on another cell, apoptotic signaling is activated through a cytoplasmic domain called the death domain (DD), which interacts with signaling adaptors including FAF, FADD, and DAX, activating the caspase proteolytic cascade. Caspase-8 and caspase-10 are activated first, which then cleave and activate downstream caspases and various cellular substrates, leading to cell death.

[0084] Mitochondria participate in the apoptosis signaling pathway through the release of mitochondrial proteins into the cytoplasm. Cytochrome c, a key protein in electron transport, is released from mitochondria in response to apoptotic signals and activates the mitochondrial-released protease Apaf-1. Activated Apaf-1 activates caspase-9 and the rest of the caspase pathway. Smac / DIABLO is released from mitochondria and inhibits IAP proteins, which normally interact with caspase-9 to inhibit apoptosis. Regulation of apoptosis by Bcl-2 family proteins occurs when family members form a complex that enters the mitochondrial membrane and regulates the release of cytochrome c and other proteins. TNF family receptors that trigger apoptosis directly activate the caspase cascade but can also activate Bid, a Bcl-2 family member that activates mitochondria-mediated apoptosis. Bax, another Bcl-2 family member, is activated by this pathway and localizes to the mitochondrial membrane, increasing its permeability and releasing cytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation and block apoptosis. Similar to cytochrome c, AIF (apoptosis-inducing factor) is a protein found in mitochondria and is released from mitochondria upon apoptotic stimuli. While cytochrome c is involved in caspase-dependent apoptosis signaling, AIF release stimulates caspase-independent apoptosis and translocates into the nucleus where it binds to DNA. DNA binding by AIF stimulates chromatin condensation and DNA fragmentation, likely through the recruitment of nucleases.

[0085] The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which interacts with Apaf-1, leading to its autocleavage and activation of caspase-9. Caspase-3, caspase-6, and caspase-7 are downstream caspases that are activated by upstream proteases and act to cleave cellular targets.

[0086] Granzyme B and perforin proteins released by cytotoxic T cells induce apoptosis in target cells, forming transmembrane pores and possibly through caspase cleavage, although a caspase-independent mechanism of granzyme B-mediated apoptosis has been suggested.

[0087] Fragmentation of the nuclear genome by multiple nucleases activated by apoptotic signaling pathways, resulting in the creation of nucleosome ladders, is a characteristic cellular response to apoptosis. One of the nucleases involved in apoptosis is the caspase-activated DNAse (CAD), DNA fragmentation factor (DFF). DFF / CAD is activated during apoptosis through cleavage of its associated inhibitor, ICAD, by caspase proteases. DFF / CAD interacts with chromatin components, such as topoisomerase II and histone H1, to condense chromatin structure and likely recruit CAD to chromatin. Another apoptosis-activating protease is endonuclease G (EndoG). EndoG is encoded by the nuclear genome but localizes to mitochondria in normal cells. EndoG may be involved in mitochondrial genome replication and apoptosis. Apoptotic signaling triggers the release of EndoG from mitochondria. The EndoG pathway and the DFF / CAD pathway are independent, as the EndoG pathway also occurs in cells lacking DFF.

[0088] Hypoxia and subsequent reoxygenation can lead to the release of cytochrome c and apoptosis. Glycogen synthase kinase-3 (GSK-3), a serine-threonine kinase ubiquitously expressed in most cell types, appears to mediate or enhance apoptosis through many stimuli that activate the mitochondrial cell death pathway. (Loberg, RD, et al., J. Biol. Chem. 277(44): 41667-673(2002)) It has been demonstrated to induce caspase-3 activation and activate the proapoptotic tumor suppressor gene p53. It has also been suggested that GSK-3 promotes the activation and translocation of Bax, a proapoptotic Bcl-2 family member, and that Bax induces cytochrome c release upon aggregation and mitochondrial localization. Akt is a key regulator of GSK-3, and its phosphorylation and inactivation may mediate some of Akt's antiapoptotic effects.

[0089] As used herein, the term "autologous" means derived from the same individual.

[0090] As used herein, the term "cancer" refers to a disease in which abnormal cells divide uncontrollably and can invade other tissues. There are over 100 different types of cancer. Most cancers are named after the organ or cell type in which they originate. For example, cancer that originates in the colon is called colon cancer; cancer that begins in the melanocytes of the skin is called melanoma. Cancer types can be divided into broader categories. The major categories of cancer are: carcinoma (meaning cancer that begins in the skin or tissues lining or covering internal organs, and their subtypes, including adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma); sarcoma (meaning cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue); leukemia (meaning cancer that begins in blood-forming tissues (e.g., bone marrow) and produces large amounts of abnormal blood cells that enter the bloodstream); lymphoma and myeloma (meaning cancer that begins in the cells of the immune system); and cancer of the central nervous system (meaning cancer that begins in the tissues of the brain and spinal cord). The term "myelodysplastic syndrome" refers to a type of cancer in which the bone marrow does not produce enough healthy blood cells (white blood cells, red blood cells, and platelets) and abnormal cells are present in the blood and / or bone marrow. Myelodysplastic syndrome can lead to acute myeloid leukemia (AML).

[0091] As used herein, the term "cell line" means a permanently established cell culture developed from a single cell and therefore consisting of cells with a uniform genetic make-up that will grow indefinitely.

[0092] As used herein, the term "chemotherapy" refers to treatment that uses drugs to stop the growth of cancer cells.

[0093] As used herein, the term "contacting" and its various grammatical forms refer to the state or condition of being in contact with or in immediate or local proximity. Bringing a composition into contact with a target destination can occur by any means of administration known to those of skill in the art.

[0094] As used herein, the term "costimulatory molecule" refers to one of two or more molecules that are presented on the cell surface and are responsible for activating T cells to become effector cells. For example, the MHC protein that presents foreign antigens to the T cell receptor also requires a costimulatory protein to bind to a complementary receptor on the T cell surface to activate the T cell.

[0095] As used herein, the term "cytokine" refers to small, soluble protein substances secreted by cells that have a variety of effects on other cells. Cytokines mediate many important physiological functions, including proliferation, development, wound healing, and immune responses. They act by binding to their cell-specific receptors located within the cell membrane, which initiate distinct signaling cascades within the cell, ultimately resulting in biochemical and phenotypic changes in the target cell. Cytokines can act both locally and remotely from the site of release. They include type I cytokines, which include many interleukins and several hematopoietic growth factors; type II cytokines, including interferons and interleukin-10; tumor necrosis factor ("TNF")-related molecules, including TNFα and lymphotoxin; immunoglobulin superfamily members, including interleukin-1 ("IL-1"); and chemokines, a family of molecules that play important roles in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on cells depending on their state. Cytokines often regulate the expression of other cytokines and trigger cascades of other cytokines. Non-limiting examples of cytokines include, for example, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), Fms-related tyrosine kinase 3 ligand (FLT3LG), Flt3, interleukin-1 (IL-1), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 / IL-23 P40, IL13, IL-15, IL-15 / IL15-RA, IL-17, IL-18, IL-21, IL-23, TGF-β, MCP-1, TNF-α, and interferon α (IFNα), IFNγ.

[0096] The term "cytotoxic T lymphocyte" (CTL) refers to effector CD8+ T cells. Cytotoxic T cells kill by inducing apoptosis in their targets. They induce target cells to undergo programmed cell death via extrinsic and intrinsic pathways.

[0097] As used herein, the term "dendritic cell" or "DC" refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues that present foreign antigens to T cells (see Steinman, Ann. Rev. Immunol. 9:271-296 (1991)).

[0098] As used herein, the term "derived" encompasses any method for receiving, obtaining, or modifying something from a source of origin.

[0099] As used herein, the term "derivative" or "variant" with respect to a peptide or DNA sequence (e.g., an immunomodulatory factor peptide sequence) refers to a non-identical peptide or DNA sequence that has been modified from its original sequence. Differences in sequence may be the result of changes in sequence or structure by design. Engineered changes may be those that are specifically designed and introduced into a sequence for a particular purpose. Such specific changes can be made in vitro using various mutagenesis techniques. Such specifically generated sequence variants can be referred to as "mutants" or "derivatives" of the original sequence. As used herein, the term "derivative" or "variant" with respect to a cell refers to a cell line that has been modified from its original cell line (e.g., modified to express a recombinant DNA sequence).

[0100] The term "detectable marker" encompasses both selectable markers and assay markers. The term "selectable marker" refers to a variety of gene products by which cells transformed with an expression construct can be selected or screened, including drug resistance markers, antigen markers useful for fluorescence activated cell sorting, adhesion markers such as receptors for adhesion ligands that allow selective adhesion, etc.

[0101] The term "detectable response" refers to any signal or response that can be detected in an assay, which can be performed with or without a detection reagent. Detectable responses include, but are not limited to, radioactive decay and energy (e.g., fluorescence, ultraviolet, infrared, visible) emission, absorption, polarization, fluorescence, phosphorescence, transmission, reflection, or resonance transfer. Detectable responses also include chromatographic mobility, turbidity, electrophoretic mobility, mass spectrometry, ultraviolet spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and X-ray diffraction. Alternatively, a detectable response can be the result of an assay for measuring one or more properties of a biological material, such as melting point, density, conductivity, surface acoustic wave, catalytic activity, or elemental composition. A "detection reagent" is any molecule that produces a detectable response indicating the presence or absence of a substance of interest. Detection reagents include any of a variety of molecules, such as antibodies, nucleic acid sequences, and enzymes. To facilitate detection, a detection reagent may contain a marker.

[0102] As used herein, the term "differentiate" and its various grammatical forms refer to a developmental process involving an increase in the level of organization or complexity of cells or tissues with more specialized functions.

[0103] As used herein, the term "dose" refers to the amount of a therapeutic substance prescribed to be taken at one time.

[0104] As used herein, the term "effector cell" refers to a cell that carries out the ultimate response or function. For example, the primary effector cells of the immune system are activated lymphocytes and phagocytes.

[0105] Engineered leukocyte stimulator cells ("ENLIST™ cells") refer to a population of proliferation-incompetent tumor cells that have been genetically engineered to express a core group of three immune-modulating molecules used to stimulate mononuclear cells for the treatment of cancer.

[0106] As used herein, the term "enrich" refers to increasing the proportion of a desired substance, e.g., increasing the relative frequency of a cell subtype compared to its natural frequency in a cell population. Positive selection, negative selection, or both are generally considered necessary for any enrichment scheme. Selection methods include, but are not limited to, magnetic separation and FACS. Regardless of the specific technique used for enrichment, the specific markers used in the selection process are important because developmental stage and activation-specific responses can alter the antigenic profile of cells.

[0107] As used herein, the term "exogenous polypeptide" refers to a polypeptide that is not produced by wild-type cells of that type or that is present at lower levels in wild-type cells than in cells that contain the exogenous polypeptide. According to some embodiments, an exogenous polypeptide is a polypeptide encoded by a nucleic acid that has been introduced into a cell, where the nucleic acid is optionally not retained by the cell.

[0108] As used herein, the term "exogenous immunomodulatory molecule" includes polypeptides that comprise allogeneic cells (e.g., allogeneic cell lines) that specifically bind (e.g., intracellularly or on the cell surface) to a cognate polypeptide (e.g., a receptor) on an immune cell, e.g., an immune killer cell (e.g., an NK cell or a CD8+ T cell), thereby providing a signal that mediates immune cell stimulation, such as immune cell proliferation, activation, or expansion. According to one embodiment, the one or more exogenous immunomodulatory polypeptides are sufficient to stimulate immune killer cells ex vivo or in vivo. Exemplary exogenous immunomodulatory polypeptides are described in detail below.

[0109] As used herein, the term "express" or "expression" encompasses mRNA biogenesis, polypeptide biosynthesis, polypeptide activation, e.g., by post-translational modification, or activation of expression by a change in subcellular location or recruitment to chromatin. Expression can be increased by many approaches, including, for example, increasing the number of genes encoding polypeptides, increasing transcription of genes (such as by placing genes under the control of a constitutive promoter), increasing translation of genes, knocking out competing genes, or a combination of these and / or other approaches.

[0110] The term "expression vector" refers to a DNA molecule containing a gene that is expressed in a host cell. Typically, gene expression is placed under the control of specific regulatory elements, including, but not limited to, promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operably linked" to the regulatory elements.

[0111] As used herein, the terms "first" and "second" with respect to exogenous immunomodulatory molecules are used for convenience to distinguish when more than one type of exogenous stimulatory polypeptide is present. The use of these terms is not intended to confer a particular order or orientation of the exogenous stimulatory polypeptides unless explicitly stated.

[0112] As used herein, the term "flow cytometry" refers to a tool for examining cellular phenotypes and characteristics. It senses cells or particles as they move through a fluid stream through a laser (light amplification by stimulated emission of radiation) / light beam passing through a sensing region. It measures the relative light scattering and color-coded fluorescence of microparticles. Analysis and identification of flowing cells is based on their size, granularity, and whether the cells carry fluorescent molecules, either in the form of antibodies or dyes. As cells pass through the laser beam, light is scattered in all directions, and light scattered forward at small angles (0.5-10°) from the axis is proportional to the square of the radius of the sphere, i.e., the size of the cell or particle. Light can enter cells at 90° (right angle, side) scattering, which can then be labeled with fluorescent dye-conjugated antibodies or stained with fluorescent membrane, cytoplasm, or nuclear dyes. This facilitates identification of cell type, the presence of membrane receptors and antigens, membrane potential, pH, enzymatic activity, and DNA content. Flow cytometers are multiparameter, recording several measurements for each cell and are therefore able to distinguish homogeneous subpopulations within a heterogeneous population (Marion G. Macey, Flow cytometry: principles and applications, Humana Press, 2007). Fluorescence-activated cell sorting (FACS) allows the separation of distinct cell populations that have physical properties too similar to separate by size or density, and uses fluorescent tags to detect differentially expressed surface proteins, allowing fine distinctions to be made between physically homogeneous populations of cells.

[0113] The terms "functional equivalent" or "functionally equivalent" are used interchangeably herein and refer to a substance, molecule, polynucleotide, protein, peptide, or polypeptide that has a similar or identical effect or use.

[0114] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid involved in the expression of a given RNA or protein. Thus, a gene includes a region that encodes the expressed RNA (typically including a polypeptide-coding sequence) and often includes the regulatory sequences required for their expression. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and can include sequences designed to have specifically desired parameters.

[0115] The term "heteroclitic" is used herein to refer to a peptide that has greater biological potency than the original peptide. A "heteroclitic immunogen" is an immunogen that elicits an immune response that cross-reacts with the original, less immunogenic antigen.

[0116] The terms "immune response" and "immune-mediated" are used interchangeably herein to refer to any functional manifestation of a subject's immune system against either foreign or self-antigens, regardless of whether the outcome of these responses is beneficial or harmful to the subject.

[0117] As used herein, the term "immunosuppression" and other grammatical forms refer to a decrease in the body's immune response and the ability of the immune system to fight infections and other diseases. For example, some immunosuppression can be drug-induced or can result from disease.

[0118] The terms "immunomodulation," "immunomodulator," and "immunomodulator" are used interchangeably herein to refer to substances, agents, or cells that can directly or indirectly increase or decrease an immune response through chemokines, cytokines, and other mediators of the immune response.

[0119] As used herein, the term "immunostimulatory amount" of the disclosed compositions refers to an amount of an immunogenic composition effective to stimulate an immune response as measured by, for example, ELISPOT assay (cellular immune response), ICS (intracellular cytokine staining assay), and major histocompatibility complex (MHC) tetramer assay to detect and quantify antigen-specific T cells by measurable amounts, quantifying blood populations of antigen-specific CD4+ T cells, or quantifying blood populations of antigen-specific CD8+ T cells, or detecting and quantifying an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, when compared to a suitable control (e.g., a control composition in which dendritic cells are not loaded with tumor-specific cells or peptides derived from tumor-specific cells), or at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% when compared to a suitable control.

[0120] As used herein, the term "genomically integrated" refers to a recombinant DNA sequence that is co-linked into genomic DNA comprising the genome of the host cell.

[0121] As used herein, the term "Kaplan-Meier plot" or "Kaplan-Meier survival curve" refers to a plot of the probability that a clinical study subject will survive for a given period of time, taking into account many small intervals of time. Kaplan-Meier plots assume the following: (i) censored (i.e., lost) subjects always have the same chance of survival as subjects who continue to be followed; (ii) the survival probability is the same for subjects recruited early and late in the study; and (iii) an event (e.g., death) occurs at a specified time. The probability of an event occurring is calculated at a specific time point, and the successive probabilities are multiplied by the previously calculated probability to obtain a final estimate. The survival probability at a specific time point is calculated by dividing the number of surviving subjects by the number of subjects at risk. Subjects who die, drop out, or are discontinued from the study are not considered to be at risk.

[0122] As used herein, the term "labeling" refers to the process of distinguishing a compound, structure, protein, peptide, antibody, cell, or cellular component by introducing a traceable moiety. Common traceable moieties include, but are not limited to, fluorescent antibodies, fluorophores, dyes or fluorescent pigments, stains or fluorescent dyes, markers, fluorescent markers, chemical stains, differential stains, differential labels, and radioisotopes.

[0123] The terms "marker" or "cell surface marker" are used interchangeably herein and refer to antigenic determinants or epitopes found on the surface of a particular type of cell. Cell surface markers can facilitate the characterization of a cell type, its identification, and ultimately its isolation. Cell sorting techniques are based on cellular biomarker(s) in which the cell surface marker(s) can be used for either positive or negative selection, i.e., inclusion or exclusion, from a cell population.

[0124] As used herein, the term "mediate" and its various grammatical forms means to bring about a result.

[0125] As used herein, the term "minimal residual disease" refers to a very small number of cancer cells remaining in the body during or after treatment. Minimal residual disease can only be detected by highly sensitive tests that can find one cancer cell out of one million normal cells.

[0126] The terms "mixed lymphocyte tumor reaction" or "MLTR" are used interchangeably herein to refer to a reaction similar to the mixed lymphocyte reaction, but in which allogeneic lymphocytes are not used to stimulate the response, but instead allogeneic tumor cells are used. The MLTR method involves contacting tumor cells being tested for immunogenicity with mixed lymphocytes from peripheral blood mononuclear cells, and then measuring one or more of lymphocyte cell proliferation, lymphocyte cell subset differentiation, lymphocyte cytokine release profile, and tumor cell death.

[0127] As used herein, the term "modify" and its various grammatical forms refers to a change in form or nature.

[0128] As used herein, the term "modulate" and its various grammatical forms means to adjust, alter, adapt, or adjust a particular measure or proportion. Such adjustment may be any change, including an undetectable change.

[0129] As used herein with respect to an immune response to tumor cells, the terms "modified" or "modulated" refer to changing the form or characteristics of the immune response to tumor cells through one or more recombinant DNA techniques, so that immune cells are able to recognize and kill tumor cells.

[0130] As used herein, the terms "myeloid suppressor cells" or "myeloid-derived suppressor cells" refer to a heterogeneous population of cells characterized by their myeloid origin, immature state, and ability to potently suppress T cell responses. These cells regulate immune responses and tissue repair in healthy individuals and rapidly expand during inflammation.

[0131] As used herein, the term "natural killer (NK) cells" refers to lymphocytes classified as group I innate lymphocytes, belonging to the same family as T cells and B cells. In contrast to cytotoxic T cells, which require priming by antigen-presenting cells, they have the ability to kill tumor cells without priming or prior activation. NK cells secrete cytokines such as IFNγ and TNFα, which act on other immune cells, such as macrophages and dendritic cells, to enhance the immune response. Activating receptors on the surface of NK cells recognize molecules expressed on the surface of cancer cells and infected cells, turning on the NK cells. Inhibitory receptors act as a check on NK cell killing. Most normal, healthy cells express MHC1 receptors, which mark them as "self." Inhibitory receptors on the surface of NK cells recognize cognate MHC1s, which switch off the NK cells and prevent them from killing. Once the decision to kill is made, NK cells release cytotoxic granules containing perforin and granzymes, causing lysis of the target cell. Natural killer reactivity, including cytokine secretion and cytotoxicity, is controlled by the balance of several germline-encoded inhibitory and activating receptors, including killer immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs). The presence of MHC class I molecules on target cells acts as an inhibitory ligand for the killer immunoglobulin-like receptors (KIRs), the MHC class I-specific receptors on NK cells. KIR receptor binding blocks NK activation and, paradoxically, induces inactivating signals, thereby preserving the ability to respond to subsequent encounters. Thus, if KIRs can sufficiently bind to MHC class I, this binding neutralizes the killing signal, allowing the target cell to survive. In contrast, if NK cells cannot sufficiently bind to MHC class I on target cells, target cell killing can proceed. As a result, tumors that express low MHC class I and are thought to be able to evade T cell-mediated attack may instead be susceptible to NK cell-mediated immune responses.

[0132] The term "nucleic acid" is used herein to refer to a polymer of deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and, unless otherwise limited, encompasses known analogs that possess the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. The term "hybridize" refers to pairing to form a double-stranded molecule with a complementary polynucleotide sequence (e.g., a gene described herein) or portion thereof under various stringency conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507). Measuring the effect of base mismatches by quantifying the rate at which the two strands anneal can provide information about the base sequence similarity between the two annealed strands. Selectively hybridizing nucleic acids undergo hybridization to a specific nucleic acid target sequence under stringent hybridization conditions to a detectable extent (e.g., at least twice the background) over its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids.

[0133] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences set forth herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences set forth herein). For example, such a sequence may be at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0134] Sequence identity is typically measured using sequence analysis software (e.g., the BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs, which are sequence analysis software packages from the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, with a probability score between e-3 and e-100 indicating closely related sequences.

[0135] As used herein, the term "open reading frame" refers to a sequence of nucleotides in a DNA molecule that may encode a peptide or protein, beginning with an initiation triplet (ATG), followed by a chain of triplets each encoding an amino acid, and ending with a termination triplet (TAA, TAG, or TGA).

[0136] The phrase "operably linked" refers to (1) a first sequence(s) or domain(s) positioned sufficiently proximal to a second sequence(s) or domain(s) such that the first sequence(s) or domain(s) can affect the second sequence(s) or domain(s) or a region under the control of the second sequence(s) or domain(s), and (2) a functional linkage between a promoter and a second sequence such that the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. Generally, operably linked means that the linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. According to some embodiments, the phrase "operably linked" refers to a linkage in which two or more protein domains or polypeptides are joined or joined via recombinant DNA techniques or chemical reactions such that each protein domain or polypeptide of the resulting fusion protein retains its original function.

[0137] As used herein, the term "overall survival" (OS) refers to the length of time from either the date of diagnosis of a disease, such as cancer, or the start of treatment, that a patient diagnosed with a disease, such as cancer, is still alive.

[0138] As used herein, the term "parenteral" and other grammatical forms refer to the administration of a substance that occurs in the body other than by mouth or the digestive tract. For example, as used herein, the term "parenteral" refers to introduction into the body by injection (i.e., administration by injection), including, for example, subcutaneous (i.e., injection under the skin), intramuscular (i.e., injection into a muscle), intravenous (i.e., injection into a vein), intrathecal (i.e., injection around the spinal cord or into the subarachnoid space of the brain), intrasternal, or infusion techniques.

[0139] The terms "peripheral blood mononuclear cells" or "PBMCs" are used interchangeably herein to refer to blood cells with a single round nucleus, such as lymphocytes or monocytes.

[0140] As used herein, the term "pharmaceutical composition" refers to a composition used to prevent, reduce the intensity of, cure or otherwise treat a targeted condition, syndrome, disorder or disease.

[0141] As used herein, the term "pharmaceutically acceptable carrier" refers to any substantially non-toxic carrier conventionally usable for administering pharmaceuticals in which the isolated polypeptide of the present invention remains stable and bioavailable. A pharmaceutically acceptable carrier must be of sufficiently high purity and sufficiently low toxicity to make it suitable for administration to the mammal being treated. Furthermore, it must maintain the stability and bioavailability of the active agent. A pharmaceutically acceptable carrier may be liquid or solid and can be selected taking into account the planned method of administration to provide a desired volume, concentration, etc. when combined with the active agent and other components of a given composition.

[0142] The term "pharmaceutically acceptable salts," as used herein, refers to salts that are suitable for use in contact with the tissues of humans and lower animals, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio. When used in medicine, salts should be pharmaceutically acceptable, although non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromide, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts can also be prepared as alkali metal or alkaline earth salts, such as sodium, potassium, or calcium salts of the carboxylic acid group. "Pharmaceutically acceptable salt" means a salt that is suitable for use in contact with the tissues of humans and lower animals within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, PH Stahl, et al., describe pharmaceutically acceptable salts in detail in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley VCH, Zurich, Switzerland: 2002). Salts can be prepared in situ during the final isolation and purification of the compounds described in the present invention, or separately by reacting the free base functional group with a suitable organic acid.Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfinate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate salts. In addition, basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates; long-chain halides, e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides, e.g., benzyl and phenethyl bromides, and others. This results in water- or oil-soluble or dispersible products. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. The base addition salts can be prepared in situ during the final isolation and purification of the compounds according to the invention by reacting the carboxylic acid-containing moiety with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, or with ammonia or an organic primary, secondary, or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals, such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts, as well as non-toxic quaternary ammonia and amine cations, including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, and the like. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. Pharmaceutically acceptable salts can also be obtained, using standard procedures well known in the art, by reacting a sufficiently basic compound, such as an amine, with a suitable acid to provide a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium or magnesium) salts of carboxylic acids can also be made.

[0143] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The essential property of such analogues of naturally occurring amino acids, when incorporated into a protein, is that the protein will react specifically with antibodies elicited against a protein composed entirely of the same but naturally occurring amino acids.

[0144] The terms "polypeptide," "peptide," and "protein" also include modifications, including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. As is well known and discussed above, it is understood that polypeptides may not be entirely linear. For example, polypeptides may be branched as a result of ubiquitination, and they may generally be cyclic, with or without branching, as a result of post-translational events, including natural processing events and events brought about by artificial manipulation that do not occur in nature. Cyclic, branched, and branched cyclic polypeptides can also be synthesized by non-translational natural processes and by entirely synthetic methods. According to some embodiments, peptides are of any length or size.

[0145] As used herein, the term "proliferate" and its various grammatical forms refer to a process that results in an increase in cell number and is defined by a balance between cell division and cell loss through cell death or differentiation.

[0146] The terms "protein domain" and "domain" are used interchangeably to refer to a portion of a protein that has its own tertiary structure. Large proteins generally consist of multiple domains that are connected to each other through flexible regions of the polypeptide chain.

[0147] The following terms are used herein to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence," (b) "comparison window," (c) "sequence identity," (d) "percentage of sequence identity," and (e) "substantial identity." (a) The term "reference sequence" refers to a sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence, for example, as a segment of a full-length cDNA or gene sequence, or as the complete cDNA or gene sequence. (b) The term "comparison window" refers to a specific contiguous segment of a polynucleotide sequence, where a polynucleotide sequence can be compared to a reference sequence, and the portion of the polynucleotide sequence within the comparison window can contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Generally, the length of comparison window is at least 20 consecutive nucleotides, and can optionally be at least 30 consecutive nucleotides, at least 40 consecutive nucleotides, at least 50 consecutive nucleotides, at least 100 consecutive nucleotides or longer.Those skilled in the art will understand that in order to avoid the high similarity between polynucleotide sequence and reference sequence due to the gap that polynucleotide sequence contains, gap penalty is typically introduced and subtracted from the number of matching.The method of aligning sequences for comparison is well known in the art.Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. Math. 2:482 (1981)), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); by computerized implementations of these algorithms, including, but not limited to, CLUSTAL in the PC / Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA. The CLUSTAL program is described in detail by Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS 5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90 (1988); Huang, et al., Computer Applications in the Biosciences, 8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology, 24:307-331 (1994).The BLAST family of programs that can be used for database similarity searches include: BLASTN for nucleotide query sequences against nucleotide database sequences, BLASTX for nucleotide query sequences against protein database sequences, BLASTP for protein query sequences against protein database sequences, TBLASTN for protein query sequences against nucleotide database sequences, and TBLASTX for nucleotide query sequences against nucleotide database sequences. See Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Unless otherwise specified, the sequence identity / similarity values ​​provided herein refer to values ​​obtained using the BLAST 2.0 suite of programs using default parameters. Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analysis is publicly available, for example, from the National Center for Biotechnology Information. This algorithm identifies high-scoring sequence pairs (HSPs) by first identifying short words W. The query sequence is obtained if it matches or meets a positive threshold score T when aligned with words of the same length in database sequences. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as possible to increase the cumulative alignment score. The cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction is stopped if:The cumulative alignment score is reduced by a quantity X from its maximum achieved value; the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below zero; or 3) the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequence, which may be homopolymer tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions can align between unrelated proteins even if other regions of the proteins are completely different. To reduce such low-complexity alignments, several low-complexity filter programs can be used. For example, the low complexity filters SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) can be used alone or in combination.The terms "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences are used herein to refer to the residues of the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When using percentage sequence identity with respect to proteins, it is understood that non-identical residue positions often differ by conservative amino acid substitutions, i.e., amino acid residues are replaced with other amino acid residues that have similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the molecule. When amino acid sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0 and 1. Conservative substitution scoring is calculated according to, for example, the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988), as implemented in the PC / GENE program (Intelligenetics, Mountain View, Calif., USA). (d) The term "percentage of sequence identity" as used herein refers to a value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) with respect to the optimal alignment of the two sequences.The percentage (%) is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. (e) The term "substantial identity" of polynucleotide sequences means: "Substantial identity" refers to a polynucleotide containing a sequence that has at least 70%, at least 80%, at least 90%, and at least 95% sequence identity compared to a reference sequence using one of the alignment programs described with standard parameters. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to account for codon degeneracy, amino acid similarity, reading frame alignment, and the like, to determine the corresponding identity of proteins encoded by two nucleotide sequences. For these purposes, substantial identity of amino acid sequences typically means at least 60%, or at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. Another indicator that nucleotide sequences are substantially identical is whether two molecules hybridize to each other under stringent conditions. However, nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This can occur, for example, when copies of nucleic acids are created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid. Variations in the nucleotide sequence of the protein can also be made by reference to the genetic code, including taking into account codon degeneracy.

[0148] As used herein, the term "prime" (or "priming") refers to the process of increasing susceptibility to antigens. When used in an immunological sense, it refers to the process by which a specific antigen is presented to naive lymphocytes, causing them to differentiate. Priming involves several steps: antigen uptake, processing, cell surface expression in association with MHC molecules by antigen-presenting cells, recirculation and antigen-specific capture of helper T cell precursors in lymphoid tissues, and T cell proliferation and differentiation (Janeway, CA, Jr., "The priming of helper T cells," Semin. Immunol. 1(1): 13-20 (1989)).

[0149] As used herein, the term "progression-free survival" or "PFS" refers to the length of time a patient lives with a disease, such as cancer, during and after treatment, without the disease worsening. In clinical trials, measuring progression-free survival is one way to determine how well a new treatment works.

[0150] As used herein with respect to cancer, the term "recurrence" refers to cancer that has recurred (come back), usually after a period of time in which the cancer was not detected. The cancer may recur in the same place as the original (primary) tumor or in another location in the body.

[0151] As used herein, the term "recurrence-free survival (RFS)" refers to the length of time a patient lives without signs or symptoms of cancer after primary treatment for that cancer. Also referred to as disease-free survival (DFS) and progression-free survival (PFS).

[0152] The terms "release" or "release of cytokine effector molecules" refer to the complex and tightly regulated process by which soluble mediators of the immune response are delivered from a given immune cell type to the external environment after activation of a signaling cascade in response to receptor stimulation. In the classical secretory pathway, cytokines bearing a signal peptide are cotranslationally inserted into the endoplasmic reticulum (ER) for synthesis as soluble or transmembrane precursors. They are then transported in vesicles to the Golgi complex for further processing and loaded into vesicles or carriers at the trans-Golgi network (TGN) for constitutive delivery to the cell surface or other organelles. In specialized cell types, additional modes of secretion are provided by loading cytokines and other cargo into granules for storage and later release. See Lacy, P. and Stow, JL, "Cytokine release from innate immune cells: association with diverse membrane trafficking pathways," Blood (2011) 118: 9-18. Cytokine release can be triggered by signaling through immunoglobulin or complement receptors, or directly by pathogens through a variety of cellular receptors, including pattern recognition receptors such as TLRs.

[0153] As used herein, the term "response rate" refers to the percentage of patients whose cancer shrinks or disappears after treatment.

[0154] As used herein, the term "resistant cancer" refers to a cancer that does not respond to treatment at the start of such treatment or at some point during such treatment.

[0155] The term "reporter gene" ("reporter") or "assay marker" refers to a gene and / or peptide that can be detected or easily identified and measured. Expression of a reporter can be measured at either the RNA or protein level. Gene products that can be detected in experimental assay protocols include, but are not limited to, marker enzymes, antigens, amino acid sequence markers, cell phenotype markers, and nucleic acid sequence markers. Researchers can add a reporter gene to another gene of interest in cell culture, bacteria, animals, or plants. For example, some reporters are selectable markers, or confer characteristics to organisms that express them, allowing them to be easily identified and assayed. To introduce a reporter gene into an organism, researchers can place the reporter gene and the gene of interest on the same DNA construct and insert it into the cell or organism. For bacteria or eukaryotic cells in culture, this can be in the form of a plasmid. Commonly used reporter genes include, but are not limited to, fluorescent proteins, luciferase, beta-galactosidase, and selectable markers such as chloramphenicol and kanomycin.

[0156] The term "secretion," as used herein in reference to a cell, refers to the process by which molecules produced within the cell move into the extracellular space.

[0157] As used herein, the term "serial killer cells" refers to a population of cells that exhibit the ability to kill multiple tumor- or pathogen-infected cells while also exhibiting resistance to such killing. There are several types of cells that exhibit this effector function, including NK cells, NKT cells, LAK cells, CIK cells, MAIT cells, CD8+ CTLs, and CD4+ CTLs. Serial killer effector function can be direct, via cytolytic or cytotoxic activity, or indirect, via immunomodulation of other cells and proteins that target pathogenic and cancerous cells.

[0158] As used herein, the term "stably expressed exogenous immunomodulatory molecule" refers to an exogenous immunomodulatory molecule that is expressed for a period sufficient to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, the period is between 1 hour and 72 hours, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 71, or 72 hours. According to some embodiments, the period is greater than 72 hours.

[0159] As used herein, the term "stimulate" in any of its various grammatical forms refers to inducing activation or increasing activity.

[0160] As used herein, the terms "stimulate an immune cell" or "stimulating an immune cell" refer to a process (e.g., including a signaling event or stimulus) that causes or results in a cellular response, e.g., activation and / or expansion, of an immune cell, e.g., an NK cell and / or a CD8+ T cell. According to some embodiments, stimulating an immune cell (e.g., an NK cell and / or a CD8+ T cell) refers to providing a stimulus or signal (e.g., a stimulatory polypeptide) that results in the activation and / or expansion of the immune cell.

[0161] As used herein, the term "sufficient to stimulate an immune cell" refers to an amount or level of a signaling event or stimulus, e.g., an exogenous immunomodulatory polypeptide, that promotes a cellular response in an immune cell.

[0162] As used herein, the terms "subject" or "individual" or "patient" are used interchangeably to refer to members of an animal species of mammalian origin, including humans.

[0163] As used herein, the phrase "subject in need thereof" refers to a patient who (i) is being administered an immunogenic composition according to the described invention, (ii) is receiving an immunogenic composition according to the described invention, or (iii) has received an immunogenic composition according to the described invention, unless the context and usage of the phrase dictates otherwise.

[0164] The term "SUPLEXA™ cells" refers to autologous blood cells that have been stimulated in vitro with engineered leukocyte stimulator cells ("ENLIST™ cells").

[0165] As used herein, the term "therapeutic agent" refers to a drug, molecule, nucleic acid, protein, metabolite, composition, or other substance that provides a therapeutic effect. As used herein, the term "active" refers to the active ingredient, component, or constituent of the described compositions of the present invention that is responsible for the intended therapeutic effect. The terms "therapeutic agent" and "active agent" are used interchangeably herein. As used herein, the term "therapeutic agent" refers to a therapeutically effective dose (i.e., dose and frequency of administration) that eliminates, reduces, or prevents the progression of a specific disease manifestation in a certain proportion of the population. An example of a commonly used therapeutic agent is the ED50, which represents the dose at a particular dose that is therapeutically effective against a specific disease manifestation in 50% of the population.

[0166] The terms "therapeutic amount," "therapeutically effective amount," "effective amount," or "pharmaceutically effective amount" of an active agent are used interchangeably to refer to an amount sufficient to provide the intended benefit of treatment. However, dosage levels are based on a variety of factors, including the type of injury, age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent used. Thus, dosage regimens can vary widely but can be routinely determined by a physician using standard methods. Furthermore, the terms "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" include prophylactic or preventative amounts of the compositions of the described invention. In prophylactic or preventative applications of the described invention, a pharmaceutical composition or medicament is administered to a patient susceptible to or otherwise at risk of a disease, disorder, or condition in an amount sufficient to eliminate or reduce the risk of, or delay the onset of, the disease, disorder, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes manifested during the development of the disease, disorder, or condition. In general, it is preferred to use the maximum dose, i.e., the safest dose according to some medical judgment. The terms "dose" and "administration" are used interchangeably herein.

[0167] As used herein, the term "therapeutic effect" refers to a result of treatment, which is determined to be desirable and beneficial. Therapeutic effect may include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. Therapeutic effect may also include, directly or indirectly, the prevention, reduction, or elimination of the progression of disease symptoms.

[0168] For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from preliminary in vitro studies and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. Adjusting the dose to achieve maximum efficacy based on the above-mentioned methods and other well-known methods is within the ability of one skilled in the art.

[0169] The following summarizes general principles for determining therapeutic efficacy, which can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference.

[0170] Pharmacokinetic principles provide the basis for modifying dosing regimens to achieve the desired degree of therapeutic effect while minimizing unacceptable side effects. In situations where plasma concentrations of a drug can be measured and related to a therapeutic window, additional guidance on dosage modifications can be obtained.

[0171] Drug products are considered pharmaceutically equivalent if they contain the same active ingredient and are identical in strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent drug products are considered bioequivalent if, under appropriate test conditions, the rate and extent of bioavailability of the active ingredient in the two products do not differ significantly.

[0172] The term "therapeutic window" refers to the concentration range that produces a therapeutic effect without unacceptable toxicity. After administration of a given dose of a drug, its effect usually exhibits a characteristic temporal pattern. There is a lag period before the drug concentration exceeds the minimum effective concentration ("MEC") for the desired effect. Following the onset of the response, the intensity of the effect increases as the drug continues to be absorbed and distributed. This peaks, after which drug elimination results in a decrease in the intensity of the effect, which disappears when the drug concentration falls below the MEC. Therefore, the duration of a drug's action is determined by the period during which the concentration exceeds the MEC. The goal of treatment is to achieve and maintain a concentration within the therapeutic window for the desired response with minimal toxicity. A drug response below the MEC for the desired effect would be subtherapeutic, while for adverse effects, the probability of toxicity would increase above the MEC. Increasing or decreasing the drug dose shifts the response curve up or down the intensity scale, which is used to modulate the drug's effect. Increasing the dose also prolongs the drug's duration of action, but at the risk of increasing the potential for side effects. Therefore, unless the drug is nontoxic, increasing the dose is not a useful strategy for extending the duration of a drug's action.

[0173] Alternatively, another dose of drug must be administered to maintain concentrations within the therapeutic window. Generally, the lower limit of a drug's therapeutic range is considered to be approximately equal to the drug concentration that produces about half of the maximum possible therapeutic effect, and the upper limit of the therapeutic range is that at which toxic effects occur in about 5% or less to about 10% of patients. These values ​​are highly variable, and while some patients may benefit greatly from drug concentrations above the therapeutic range, others may suffer significant toxicity at much lower values. The therapeutic goal is to maintain steady-state drug levels within the therapeutic window. For most drugs, the actual concentrations associated with this desired range are not, or need not be, known; it is sufficient to understand that efficacy and toxicity are generally concentration-dependent and that drug dosage and frequency of administration affect drug levels. For the few drugs for which there is a small (2- to 3-fold) difference between the concentrations that produce efficacy and toxicity, plasma concentration ranges associated with effective therapy have been defined.

[0174] In this case, a target level strategy is reasonable, in which a desired target steady-state concentration of the drug (usually in plasma) associated with efficacy and minimal toxicity is selected and the dose expected to achieve this value is calculated. Drug concentrations are then measured, and the dose adjusted, if necessary, to more closely approximate the target.

[0175] In most clinical situations, drugs are administered in a series of repeated doses or as a continuous infusion to maintain a steady-state concentration of the drug relative to the therapeutic window. To maintain a selected steady-state or target concentration (the "maintenance dose"), the drug administration rate is adjusted so that the input rate equals the output rate. Once the clinician selects the desired concentration of the drug in plasma and knows the clearance and bioavailability of that drug in a particular patient, the appropriate dose and administration interval can be calculated.

[0176] As used herein, the term "treating" includes negating, substantially inhibiting, slowing, or reversing the progression of a condition, substantially ameliorating the clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition. Treating further refers to achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting the onset of symptoms characteristic of the disorder(s) being treated; (c) limiting the worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting the recurrence of the disorder(s) in patients who have previously experienced the disorder(s); and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder(s).

[0177] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0178] As used herein, the term "tumor burden" or "tumor burden" refers to the number of cancer cells, the size of the tumor, or the amount of cancer in the body.

[0179] As used herein, the term "vaccinated" refers to being treated with a vaccine.

[0180] As used herein, the term "vaccination" refers to treatment with a vaccine.

[0181] As used herein, the term "vaccine" refers to a substance or group of substances intended to induce the immune system to respond to a tumor or microorganism, or to help the body recognize and destroy cancer cells or microorganisms. The term vaccine also refers to an artificial stimulus (e.g., an infectious agent, cancer cells) used to stimulate a strong immune response to that exposure.

[0182] As used herein, the term "vaccine therapy" refers to a type of treatment that uses a substance or group of substances to stimulate the immune system to destroy tumors or infectious organisms.

[0183] As used herein, the term "variant" refers to a polypeptide that differs from an original protein by one or more amino acid substitutions, deletions, insertions, or other modifications. These modifications do not significantly alter the biological activity of the original protein. Often, a variant retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the biological activity of the original protein. The biological activity of a variant may also be greater than the biological activity of the original protein. Variants may exist naturally, such as through allelic variation or polymorphism, or may be intentionally engineered.

[0184] The amino acid sequence of the variant is substantially identical to that of the original protein. In many embodiments, the variant shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more overall sequence identity or sequence similarity with the original protein. Sequence identity or sequence similarity can be determined using various methods known in the art, such as Basic Local Alignment Tool (BLAST), dot matrix analysis, or dynamic programming. In one example, sequence identity or sequence similarity is determined using the Genetics Computer Group (GCG) program GAP (Needleman-Wunsch algorithm). The amino acid sequences of the variant and the original protein may be substantially identical in one or more regions, but may differ in other regions.

[0185] As used herein, the term "wild-type" refers to the typical form of an organism, strain, gene, protein, nucleic acid, or characteristic as it exists in nature. Wild-type refers to the most common phenotype in a natural population. The terms "wild-type" and "naturally occurring" are used interchangeably.

[0186] II. Allogeneic Vaccines The present disclosure features allogeneic tumor cell vaccines comprising tumor cells expressing exogenous immunomodulatory molecules, and methods of using allogeneic tumor cell vaccines to stimulate immune responses. The vaccine proteins can be used in the described inventions, for example, to induce immune responses in the treatment of cancer and infectious diseases. According to some embodiments, the allogeneic tumor cell vaccines described herein are effective in enhancing immune activation of cells effective in recognizing and acting against tumor cells containing target tumor antigens in vivo without systemic inflammation, reducing immune suppression in the tumor microenvironment of tumor cells containing target tumor antigens, or increasing cell death of tumor cells expressing target tumor antigens. According to some embodiments, the allogeneic tumor cell vaccines described herein are capable of immune activation without systemic inflammation.

[0187] According to some aspects, the disclosure features an allogeneic tumor cell vaccine that includes: (1) a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population includes a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes; and (2) a pharmaceutically acceptable carrier.

[0188] tumor-specific antigens According to some embodiments, the present disclosure provides a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens. According to some embodiments, the tumor-specific antigen may be encoded by the primary open reading frame of a gene product that is differentially expressed by tumors but not by normal tissues. According to some embodiments, the tumor-specific antigen may be encoded by a mutated gene, an intron sequence, or a translated alternative open reading frame, a pseudogene, an antisense strand, or may represent the product of a genetic translocation event. According to some embodiments, tumor cells provide a wide range of tumor-specific antigens, many of which are of unknown nature. According to some embodiments, the tumor antigen is a neoantigen.

[0189] According to some embodiments, the tumor-specific antigen is selected from one of the following groups: (a) non-mutated shared antigens (e.g., melanoma-associated antigen (MAGE), B-melanoma antigen (BAGE), kidney tumor antigen (RAGE), and cancer-testis antigens (e.g., NY-ESO); (b) differentiation antigens (e.g., prostate-specific membrane antigen [PSMA] and prostate-specific antigen (PSA) in prostate cancer, Mart1 / MelanA and tyrosinase present in many melanomas, and carcinoembryonic antigen (CEA) present in the majority of colon cancers, which are tissue-restricted and present on lineage-specific tumor cells; (c) mutated oncogenes and tumor suppressor genes (e.g., mutated ras, rearranged bcr / abl, mutated p53) that provide novel epitopes for immune recognition; (d) unique idiotypes (e.g., For example, immunoglobulin antigens of myeloma and B-cell myeloma, T-cell receptors (TCRs) expressed in CTCL; (e) epitopes derived from oncoviruses (e.g., E6 and E7 proteins encoded by human papillomavirus, Epstein-Barr virus-associated antigens present in primary cerebral lymphoma); (f) non-mutated oncofetal proteins such as CEA, alpha-fetoprotein, and survivin. According to some embodiments, the tumor-specific antigen is selected from antigens listed in the publicly available Cancer Antigen Peptide Database (on the World Wide Web at caped.icp.ucl.ac.be / Peptide / list, incorporated herein by reference in its entirety). According to some embodiments, the tumor-specific antigen is selected from antigens listed in Table 1, set forth below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0190] According to some embodiments, the tumor cells are from a cancer selected from the group consisting of melanoma, colorectal cancer, leukemia, chronic myeloid leukemia, prostate cancer, head and neck cancer, squamous cell carcinoma, tongue cancer, laryngeal cancer, tonsil cancer, hypopharyngeal cancer, nasopharyngeal cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, glioblastoma, and brain cancer.

[0191] According to some embodiments, the melanoma tumor cells are characterized by expression of one or more of gp100, tyrosinase, Melan-A, tyrosinase-related protein (TRP-2-INT2), melanoma antigen-1 (MAGE-A1), NY-ESO-1, preferentially expressed antigen in melanoma (PRAME), CDK4, and multiple myeloma oncogene 1 (MUM-1).

[0192] According to some embodiments, the colorectal cancer tumor cells are characterized by expression of one or more of carcinoembryonic antigen (CEA), MAGE, HPV, human telomerase reverse transcriptase (hTERT), EPCAM, PD-1, PD-L1, p53, cell surface-associated mucin 1 (MUC1).

[0193] Immunological antigen specificity can arise from one or more amino acid sequences of the antigen, from the degree of expression of that antigen by tumor cells, from post-translational modifications of the antigen, and the like.

[0194] Immunological antigen specificity for a particular type of cancer cell can arise from one or more of the specific fingerprints of multiple tumor antigens, from the fact that a particular antigen is expressed by a wide variety of tumor cells but has particular use in immunotherapy for a few tumor types, from the fact that a particular collection of MHC class I-presentable and MHC class II-presentable epitopes is present on a particular polypeptide or polypeptide fragment, and by omitting one or more peptides that may induce immune tolerance. Those skilled in the art can find relevant nucleic acid and polypeptide sequences, for example, at the U.S. government website, ncbi.nlm.nih.

[0195] According to some embodiments, the tumor cells are derived from a sample from a subject. According to some embodiments, the tumor cells are derived from a tumor cell line or tumor cell line variant.

[0196] According to some embodiments, the tumor antigen specificity of the present invention may be determined by the parent tumor cell line or tumor cell line variant selected for modification with the immunomodulator.

[0197] Parent cell line According to some embodiments, tumor cell lines or tumor cell line variants may be derived from established cell lines from any public source (e.g., NIH, DCTD Tumor Repository operated by Charles River Laboratories Inc.) or commercial source (e.g., ATCC, Sigma Alrich, Thermo Fischer Scientific, Genescript, DSM2). According to some embodiments, new cell lines may be established de novo from tumor cells derived from tumors of cancer patients.

[0198] According to some embodiments, cancer tissues, cancer cells, cells infected with cancer-causing agents, other pre-neoplastic cells, and cell lines of human origin can be used as sources. According to some embodiments, cancer cells can be obtained from established tumor cell lines or tumor cell line variants, such as, but not limited to, established non-small cell lung cancer (NSCLC), bladder cancer, melanoma, ovarian cancer, renal cell carcinoma, prostate cancer, sarcoma, breast cancer, squamous cell carcinoma, head and neck cancer, hepatocellular carcinoma, pancreatic cancer, or colon cancer cell lines.

[0199] In some embodiments, the established cell line comprises the LNCaP clone FGC (ATCC CRL-1740) derived from metastatic prostate cancer that has migrated to lymph nodes. In some embodiments, the established cell line comprises the PC-3 (ATCC CRL-1435) cell line derived from metastatic prostate cancer that has migrated to bone. In some embodiments, the tumor cell line or tumor cell line variant is derived from one or more of the following ATCC cell lines: VCaP (ATCC CRL-2876); MDA PCa 2b (ATCC CRL-2422); or DU 145 (ATCC HTB-81).

[0200] According to some embodiments, the established cell line comprises the SK-MEL-2 clone (ATCC HTB-68), itself derived from a thigh skin metastasis.

[0201] According to some embodiments, the established cell lines include one or more of the breast cancer cell lines designated COO-G, DU4475, ELL-G, HIG-G, MCF / 7, MDA-MB-436, MX-1, SW-613, and VAN-G. According to some embodiments, the established cell lines include one or more of the alveolar soft part sarcoma cell lines designated ASPS and ASPS-1. According to some embodiments, the established cell lines include one or more of the lung cell lines designated LX-1, COS-G, H-MESO-1, H-MESO-1A, NCI-H23, and NCI-H460. According to some embodiments, the established cell lines include one or more of the colon cancer cell lines designated CX-5, GOB-G, HCC-2998, HCT-15, KLO-G, KM20L2, MRI-H-194, LOVO I, LOVO II, and MRI-H-250. According to some embodiments, the established cell lines include one or more of the melanoma cell lines designated NIS-G, TRI-G, WIL-G, MRI-H-121B, MRI-H-187, MRI-H-221, and MRI-H-255. According to some embodiments, the established cell lines include one or more of the cervical cancer cell lines designated MRI-H-177, MRI-H-186, MRI-H-196, and MRI-H-215. According to some embodiments, the established cell lines include one or more of the renal cancer cell lines designated MRI-H-121 and MRI-H-166. According to some embodiments, the established cell lines include one or more of the endometrial cancer cell lines designated MRI-H-147 and MRI-H-220. According to some embodiments, the established cell line comprises one or more of the ovarian cancer cell lines designated MRI-H-258, MRI-H-273, MRI-H-1834, and SWA-G. According to some embodiments, the established cell line comprises one or more of the sarcoma cell lines designated HS-1, OGL-G, and DEL-G. According to some embodiments, the established cell line comprises an epidermoid cell line designated DEAC-1. According to some embodiments, the established cell line comprises an epidermoid cell line designated DEAC-1.According to some embodiments, the established cell line comprises a glioblastoma cell line designated SF295. According to some embodiments, the established cell line comprises a prostate cancer cell line designated CWR-22. According to some embodiments, the established cell line comprises a Burkitt's lymphoma cell line designated DAU. According to some embodiments, the aforementioned established cell lines described herein are commercially available, for example, from the American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC), or any of the depositories listed as International Depositary Authorities (IDAs) under Article 7 of the Budapest Treaty.

[0202] According to some embodiments, exemplary established cell lines include one or more of the following cell lines: [Table 2-1] [Table 2-2]

[0203] According to some embodiments, the choice of parent cell line from which the tumor cell line or tumor cell line variant may be derived influences the specificity of the allogeneic vaccine. For example, the use of a tumor cell line or tumor cell line variant derived from metastatic prostate cancer that has migrated to the bone of a patient may result in an allogeneic vaccine that elicits an immune response specific to the metastatic prostate cancer in the patient's bone.

[0204] According to some embodiments, tumor cell lines or tumor cell line variants can be derived from parent cells that contain universal cancer-specific antigens. For example, the use of parent tumor cell lines or tumor cell line variants derived from metastatic prostate cancer that has migrated to a patient's bone can result in an allogeneic vaccine that elicits an immune response against all prostate cancer cells.

[0205] According to some embodiments, tumor cell lines or tumor cell line variants are derived from patient-derived cells from various cancers. According to some embodiments, fresh tissue surgically removed from the tumor is enzymatically digested with type IV collagenase, followed by collection of disaggregated cells. According to some embodiments, the disaggregated cells can then be grown in vitro in a growth medium containing 10% fetal bovine serum on an extracellular matrix substrate, such as collagen or fibronectin, to promote adhesion. Then, according to some embodiments, the adherent cells can be passaged until the immortal cancer cells outgrow the non-cancerous fibroblasts.

[0206] For example, in some embodiments, tumor cell lines or tumor cell line variants may be derived from solid tumors containing tumor cells containing cancer stem cells, metastatic cancers containing metastatic tumor cells containing cancer stem cells, or non-metastatic cancers. In some embodiments, cancers may originate from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, testes, tongue, or uterus. In some embodiments, cancers may be of various histological types, such as cancers originating in tissues lining or covering the skin or internal organs (carcinoma); cancers originating in bone or soft tissues of the body, including cartilage, fat, muscle, blood vessels, and fibrous tissue (sarcoma); cancers originating from hematopoietic tissues (leukemia); cancers originating from cells of the immune system (lymphoma); cancers originating in plasma cells (myeloma), or brain / spinal cord cancer.

[0207] Examples of carcinomas include giant cell and spindle cell carcinoma, small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; ciliary body carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid tumors; carcinoid tumors; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma ;granular cell carcinoma;follicular adenocarcinoma;non-encapsulated sclerosing carcinoma;adrenal cortical carcinoma;endometrial carcinoma;cutaneous adnexal carcinoma;apocrine gland carcinoma;sebaceous gland carcinoma;cervical adenocarcinoma;mucoepidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease, acinar cell carcinoma of the breast;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia;Sertoli cell carcinoma;embryonal carcinoma;choriocarcinoma.

[0208] Examples of sarcomas include, but are not limited to, glomangiosarcoma; sarcoma; fibrosarcoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; carcinosarcoma; synovial sarcoma; angiosarcoma; Kaposi's sarcoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; myeloblastic gingival sarcoma; ameloblastoma, malignant; myeloblastic fibrosarcoma; myeloid sarcoma; and mast cell sarcoma.

[0209] Examples of leukemias include, but are not limited to, leukemia, lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; and hairy cell leukemia.

[0210] Examples of lymphomas and myelomas include, but are not limited to, malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranulomatous; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other certain non-Hodgkin's lymphomas; malignant melanoma; amelanotic melanoma; superficial melanoma; malignant melanoma in giant pigmented nevi; epithelioid cell melanoma; and multiple myeloma.

[0211] Examples of brain / spinal cord cancers include, but are not limited to, pineal tumor, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant.

[0212] Other examples of cancer include, but are not limited to, thymoma; ovarian stromal tumor; theca; granulosa cell tumor; androblastoma; Leydig cell tumor; lipid cell tumor; paraganglioma; extramammary paraganglioma; pheochromocytoma; blue nevus, malignant; fibrous histiocytoma, malignant; mixed tumor, malignant; Mullerian mixed tumor; nephroblastoma; hepatoblastoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; mesothelioma, malignant; dysgerminoma; teratoma, malignant; ovarian goiter, malignant; mesonephroma, malignant; hemangioendothelioma, malignant; hemangiopericytoma, malignant; chondroblastoma, malignant; granular cell tumor, malignant; malignant histiocytosis; and immunoproliferative small intestinal disease.

[0213] For any given tumor type, several tumor cell lines or tumor cell line variants may be commercially available. According to some embodiments, pooling several of these cell lines, either as a mixture of whole cells or by making membrane preparations from the mixture of whole cells, can provide an array of cell surface tumor antigens for that tumor type.

[0214] According to some embodiments, the tumor cells or tumor cell lines or tumor cell line variants are proliferation incompetent by irradiation.

[0215] Exogenous immunomodulatory molecules According to some embodiments, the exogenous immunomodulatory molecule of the present invention is a polypeptide that mediates stimulation of immune cells, alone or in combination with other exogenous immunomodulatory molecules. According to some embodiments, the exogenous immunomodulatory molecule of the present invention is a polypeptide that mediates stimulation of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, alone or in combination with other exogenous immunomodulatory molecules. According to some embodiments, the NK cells are memory-like NK cells. According to some embodiments, the T lymphocytes are cytotoxic T lymphocytes (CTLs) (CD8+ T cells). According to some embodiments, the T lymphocytes are memory T cells. According to some embodiments, the T lymphocytes are regulatory T cells. According to some embodiments, the T lymphocytes are helper T cells. According to some embodiments, the B lymphocytes are memory B cells. According to some embodiments, it is a feature of the present invention that a population of tumor cells comprising an exogenous immunomodulatory molecule is effective in stimulating more than one type of immune cell. For example, allogeneic tumor cells, including populations of proliferation-incompetent tumor cells of the present disclosure, are effective in stimulating one or more of T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.

[0216] According to some embodiments, stimulating immune cells refers to the expansion of immune cells. According to some embodiments, stimulating immune cells refers to the activation of immune cells. According to some embodiments, stimulating immune cells refers to an increase in the cytotoxicity of immune cells. According to some embodiments, "stimulating immune cells" refers to a combination of one or more of the expansion, activation, and / or increase in cytotoxicity of immune cells. According to some embodiments, one or more exogenous immunomodulatory molecules expressed by the population of tumor cells are effective to activate and / or expand immune cells (e.g., T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes) ex vivo. According to some embodiments, one or more exogenous immunomodulatory molecules expressed by the population of tumor cells are effective to activate and / or expand immune killer cells (e.g., T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes) in vivo. Assays for detecting whether an exogenous immune stimulatory molecule is effective in stimulating immune killer cells are described herein. According to one aspect, the present disclosure provides an allogeneic tumor cell vaccine comprising a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immune modulatory molecules effective in stimulating T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.

[0217] According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least three stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least four stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least five stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least five stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least six stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least seven stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, wherein the tumor cells are: and a population of tumor cells that are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, wherein the population comprises at least eight stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, wherein the population comprises at least nine stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 10 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 11 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprises at least 12 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 13 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 14 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 15 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 16 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least 17 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least 18 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 19 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 20 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 21 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 22 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least 23 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least 24 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 25 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 26 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least 27 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprising at least 28 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 29 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes. According to some embodiments, an allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and wherein the population comprises at least 30 stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.

[0218] According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population is characterized by expression of a stably expressed exogenous immunomodulatory molecule effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes.

[0219] According to some embodiments, the population of tumor cells comprises a first exogenous immunomodulatory molecule and a second exogenous immunomodulatory molecule. According to some embodiments, the population of tumor cells comprises a first exogenous immunomodulatory molecule, a second exogenous immunomodulatory molecule, and a third exogenous stimulatory molecule. According to some embodiments, the first exogenous immunomodulatory molecule and the second exogenous immunomodulatory molecule comprise chimeric or fusion molecules, e.g., molecules created by joining two or more separate genes, each encoding at least one domain of a protein, such that the genes are transcribed and translated as a single unit to produce a single polypeptide. According to some embodiments, the allogeneic vaccines described herein comprise tumor cells comprising one or more exogenous immunomodulatory molecules, wherein a first tumor cell or population of tumor cells comprises the first immunomodulatory molecule and a second tumor cell or population of tumor cells comprises the second immunomodulatory molecule. According to some embodiments, the allogeneic vaccines described herein comprise tumor cells comprising one or more exogenous immunomodulatory molecules, wherein a first tumor cell or population of tumor cells comprises a first immunomodulatory molecule and a second immunomodulatory molecule, and a second tumor cell or population of tumor cells comprises a third immunomodulatory molecule. It is therefore understood that the exogenous immunomodulatory molecules described herein can be present in a tumor cell population in cis (all on the same cell) or trans (each or a combination of each on different cells). According to some embodiments, the exogenous immunostimulatory molecules are displayed on the surface of genetically engineered tumor cells.

[0220] According to some embodiments, the exogenous immunomodulatory molecules are specifically selected from the group for their ability to initiate and / or maintain an anti-tumor immune response, and / or for their ability to override pre-existing immunosuppression characteristically present in cancer patients, or a combination of all three. According to some embodiments, the combination of immunomodulatory molecules is evaluated and selected by human mixed lymphocyte-tumor cell reaction. According to some embodiments, the exogenous immunomodulatory molecule is selected from a cytokine, a TNF family member, a secreted receptor, a chaperone, an IgG superfamily member, and a chemokine receptor.

[0221] According to some embodiments, the allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, wherein the exogenous immunomodulatory molecules comprise one or more cytokine proteins; the exogenous immunomodulatory molecules comprise one or more TNF family member proteins; the exogenous immunomodulatory molecules comprise one or more secreted receptor proteins; the exogenous immunomodulatory molecules comprise one or more chaperone proteins; the exogenous immunomodulatory molecules comprise one or more IgG superfamily member proteins; and / or the exogenous immunomodulatory molecules comprise one or more chemokine receptor proteins.

[0222] According to some embodiments, the allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, wherein the exogenous immunomodulatory molecules comprise one or more cytokine family member proteins and one or more TNF family member proteins; the exogenous immunomodulatory molecules comprise one or more cytokine family member proteins and one or more secreted receptor proteins; the exogenous immunomodulatory molecules comprise one or more cytokine family member proteins and one or more chaperone proteins; the exogenous immunomodulatory molecules comprise one or more cytokine family member proteins and one or more IgG superfamily member proteins; and the exogenous immunomodulatory molecules comprise one or more cytokine family member proteins and one or more chemokine receptor proteins.

[0223] According to some embodiments, the allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, wherein the exogenous immunomodulatory molecules comprise one or more TNF family member proteins and one or more secreted receptor proteins; the exogenous immunomodulatory molecules comprise one or more TNF family member proteins and one or more chaperone proteins; the exogenous immunomodulatory molecules comprise one or more TNF family member proteins and one or more IgG superfamily member proteins; or the exogenous immunomodulatory molecules comprise one or more TNF family member proteins and one or more chemokine receptor proteins.

[0224] According to some embodiments, the allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the population comprising a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the exogenous immunomodulatory molecules comprising one or more secreted receptor proteins and one or more chaperone proteins; the exogenous immunomodulatory molecules comprising one or more secreted receptor proteins and one or more IgG superfamily member proteins; and the exogenous immunomodulatory molecules comprising one or more secreted receptor proteins and one or more chemokine receptor proteins.

[0225] According to some embodiments, the allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the population comprising a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the exogenous immunomodulatory molecules comprising one or more TNF family member proteins and one or more secreted receptor proteins; the exogenous immunomodulatory molecules comprising one or more chaperone proteins and one or more IgG superfamily member proteins; and the exogenous immunomodulatory molecules comprising one or more chaperone proteins and one or more chemokine receptor proteins.

[0226] According to some embodiments, the allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, and the population comprises a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the exogenous immunomodulatory molecules comprising one or more IgG superfamily member proteins and one or more chemokine receptor proteins.

[0227] According to some embodiments, the exogenous immunomodulatory molecule is an immunostimulatory molecule.

[0228] According to some embodiments, the exogenous immunomodulatory molecule is selected from the group shown in Table 2. According to some embodiments, the exogenous immunomodulatory molecule is derived from a mouse. According to some embodiments, the exogenous immunomodulatory molecule is derived from a human. [Table 3-1] [Table 3-2]

[0229] According to some embodiments, the exogenous immunomodulatory molecule is selected from one or more of a TNF family member, a secreted receptor, a chaperone protein, an IgG superfamily member, and a chemokine receptor. According to some embodiments, the TNF family member is selected from the TNF family members listed in Table 2. According to some embodiments, the secreted receptor is selected from the secreted receptors listed in Table 2. According to some embodiments, the chaperone protein is selected from the chaperone proteins listed in Table 2. According to some embodiments, the IgG superfamily member is selected from the IgG superfamily members listed in Table 2. According to some embodiments, the chemokine receptor is selected from the chemokine receptors listed in Table 2.

[0230] According to some embodiments, the exogenous immunomodulatory molecule of Table 2 is membrane-bound (i.e., comprises a membrane anchor). According to other embodiments, the exogenous immunomodulatory molecule is secreted. According to some embodiments, the membrane-bound form of the immunomodulatory factor is one or more selected from the group consisting of 4-1BB ligand, BAFF, April, CD40 ligand, CD80, CD86, Flt3 ligand, GM-CSF, HSP90, ICOS ligand, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL7, LIGHT, OX40 ligand, RANK ligand, and TNF. According to some embodiments, the secreted form of the immunomodulatory factor is one or more selected from the group consisting of Flt3 ligand, GM-CSF, IL10R, IL7, and TGFβ receptor.

[0231] According to some embodiments, the exogenous immunomodulatory molecules of Table 2 are molecules having a wild-type amino acid sequence. According to some embodiments, the exogenous immunomodulatory molecules of Table 2 are molecules having a variant amino acid sequence.

[0232] According to some embodiments, the exogenous immunomodulatory molecule is one or more selected from the group consisting of 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane domain, GM-CSF, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, OX-40 ligand, RANK ligand, TGF-b receptor, and TNF.

[0233] According to some embodiments, the one or more exogenous immunomodulatory molecules include at least three essential immunomodulatory molecules, the at least three essential immunomodulatory molecules being OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both. According to some embodiments, an additional immunomodulatory component, identified as R, may also be present.

[0234] According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens and three stably expressed essential exogenous immunoregulatory molecules, OX40L, CD70, and CD28L, effective to stimulate an MNC population. According to some embodiments, the ENLST™ cell population comprises a population of tumor cells expressing one or more tumor-specific antigens and three stably expressed essential exogenous immunoregulatory molecules, OX40L, CD70, and CD28L, including CD80, CD86, or both, effective to stimulate the synergistic expansion of CTLs. According to some embodiments, the allogeneic vaccine further comprises one or more subsets of R immunoregulators comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 immunoregulators. According to some embodiments, an allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express at least three stably expressed exogenous immunomodulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and one R subset comprising 3-25 global immunomodulators. According to some embodiments, an allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express at least three stably expressed exogenous immunomodulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and two R subsets comprising 3-25 global immunomodulators. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express at least three stably expressed exogenous immunoregulatory molecules: OX40L, CD70, and CD28L, including CD80, CD86, or both, and three R subsets, including 3-25 global immunoregulatory factors.According to some embodiments, an allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express at least three essential stably expressed exogenous immunoregulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and four R subsets, which include 3-25 global immunoregulatory factors. According to some embodiments, an allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express at least three essential stably expressed exogenous immunoregulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and five R subsets, which include 3-25 global immunoregulatory factors. According to some embodiments, an allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express at least three essential stably expressed exogenous immunoregulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and six R subsets, which include 3-25 global immunoregulators. According to some embodiments, an allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express at least three essential stably expressed exogenous immunoregulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and seven R subsets, which include 3-25 global immunoregulators. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to express at least three essential stably expressed exogenous immunoregulatory molecules, OX40L, CD70, and CD28L, including CD80, CD86, or both, and eight R subsets, including 3-25 global immunoregulatory factors.According to some embodiments, an allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express at least three essential stably expressed exogenous immunomodulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and nine R subsets, which include 3-25 global immunomodulators. According to some embodiments, an allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are genetically engineered to stably express at least three essential stably expressed exogenous immunomodulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and ten R subsets, which include 3-25 global immunomodulators.

[0235] According to some embodiments, the exogenous immunomodulatory molecule R 1 According to some embodiments, the exogenous immunomodulatory molecule R 2 According to some embodiments, the exogenous immunomodulatory molecule R 3 is a 4-IBB ligand. According to some embodiments, the exogenous immunomodulatory molecule R 4 According to some embodiments, the exogenous immunomodulatory molecule R 5 is a CD40 ligand. According to some embodiments, the exogenous immunomodulatory molecule R 6 According to some embodiments, the exogenous immunomodulatory molecule R 7 According to some embodiments, the exogenous immunomodulatory molecule R 8 is an FLT-3 ligand. According to some embodiments, the exogenous immunomodulatory molecule R 9 According to some embodiments, the exogenous immunomodulatory molecule R 10 is HSP-90. According to some embodiments, the exogenous immunomodulatory molecule R 11 is an ICOS ligand. According to some embodiments, the exogenous immunomodulatory molecule R 12According to some embodiments, the exogenous immunomodulatory molecule R 13 According to some embodiments, the exogenous immunomodulatory molecule R 14 According to some embodiments, the exogenous immunomodulatory molecule R 15 According to some embodiments, the exogenous immunomodulatory molecule R 16 According to some embodiments, the exogenous immunomodulatory molecule R 17 According to some embodiments, the exogenous immunomodulatory molecule R 18 According to some embodiments, the exogenous immunomodulatory molecule R 19 According to some embodiments, the exogenous immunomodulatory molecule R 20 According to some embodiments, the exogenous immunomodulatory molecule R 21 is a RANK ligand. According to some embodiments, the exogenous immunomodulatory molecule R 22 is a TGF-β receptor. According to some embodiments, the exogenous immunomodulatory molecule R 23 According to some embodiments, the exogenous immunomodulatory molecule R 24 is GM-CSF.

[0236] According to some embodiments, the exogenous immunomodulatory molecule R is selected from 1 to 30, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, of: APRIL, BAFF, 4-IBB ligand, CD30 ligand, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 engineered to remove its transmembrane domain, The exogenous immunomodulatory molecule includes an exogenous immunomodulatory molecule selected from the group consisting of GM-CSF, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor, and TNF. According to some embodiments, the exogenous immunomodulatory molecules comprise 1 to 30, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 exogenous immunomodulatory molecules, wherein at least three of the immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand, including CD80, CD86, or both, and the additional immunomodulatory component is R. 1 -R 24 and is selected from the group consisting of APRIL, BAFF, 4-IBBL ligand (4-IBBL), CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane domain, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF.

[0237] According to some embodiments, the exogenous immunomodulatory molecule R is selected from 1 to 20, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, of APRIL, BAFF, 4-1BB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove the transmembrane domain, GM-CSF, CD8 membrane antigen receptor agonist (CAMP), and the like. The exogenous immunomodulatory molecule R comprises an exogenous immunomodulatory molecule selected from the group consisting of GMCSF, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor, and TNF. According to some embodiments, the exogenous immunomodulatory molecule R comprises 1 to 20 exogenous immunomodulatory molecules, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 exogenous immunomodulatory molecules, wherein at least three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand, and the additional immunomodulatory component is R. 1 -R 24 and is selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane domain, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, RANK ligand, TGF-b receptor, and TNF.

[0238] According to some embodiments, the exogenous immunomodulatory molecule R comprises 1 to 10 exogenous immunomodulatory molecules selected from the group consisting of, inclusively, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, APRIL, BAFF, 4-1BB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane domain, GM-CSF, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor, and TNF. According to some embodiments, the exogenous immunomodulatory molecule R comprises 1 to 10 exogenous immunomodulatory molecules, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 exogenous immunomodulatory molecules, wherein at least three of the immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand, and the additional immunomodulatory component is R. 1 -R 24 and is selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane domain, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, RANK ligand, TGF-b receptor, and TNF.

[0239] According to some embodiments, the exogenous immunomodulatory molecule R comprises 5 to 20 exogenous immunomodulatory molecules selected from the group consisting of APRIL, BAFF, 4-1BB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane domain, GM-CSF, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor, and TNF, inclusive. According to some embodiments, the exogenous immunomodulatory molecule R comprises 5 to 20 exogenous immunomodulatory molecules, inclusive, i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 exogenous immunomodulatory molecules, wherein at least three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand, and the additional immunomodulatory component is R. 1 -R 24 and is selected from the group consisting of APRIL, BAFF, CD27 ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane region, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF.

[0240] According to some embodiments, the exogenous immunomodulatory molecule R comprises 10 to 15 exogenous immunomodulatory molecules selected from the group consisting of APRIL, BAFF, 4-1BB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane domain, GM-CSF, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, OX-40 ligand, RANK ligand, TGF-β receptor, and TNF, inclusive. According to some embodiments, the exogenous immunomodulatory molecule R comprises 10 to 15 exogenous immunomodulatory molecules, inclusive, i.e., 10, 11, 12, 13, 14, or 15 exogenous immunomodulatory molecules, wherein at least three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand, and the additional immunomodulatory component is R 1 -R 24 and is selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane domain, GMCSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, RANK ligand, TGF-b receptor, and TNF.

[0241] According to some embodiments, the exogenous immunomodulatory molecule R comprises an exogenous immunomodulatory molecule selected from the group consisting of 14 exogenous immunomodulatory molecules: APRIL, BAFF, 4-1BB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane region, GM-CSF, GM-CSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, OX-40 ligand, RANK ligand, TGF-b receptor, and TNF. According to some embodiments, the exogenous immunomodulatory molecules include 14 exogenous immunomodulatory molecules, at least three of which are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand, and the additional immunomodulatory factor is R 1 -R 24 and is selected from the group consisting of APRIL, BAFF, 4-IBB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove its transmembrane domain, GM-CSF engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, IL-7 engineered to have a CD8 membrane anchor, LIGHT, RANK ligand, TGF-β receptor, and TNF.

[0242] According to some embodiments, each of the exogenous immunomodulatory molecules 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, GM-CSF, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, LIGHT, OX-40 ligand, RANK ligand, TGF-b receptor, and TNF is a wild-type molecule. According to some embodiments, each of the exogenous immunomodulatory molecules 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, GM-CSF, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, LIGHT, OX-40 ligand, RANK ligand, TGF-b receptor, and TNF is a mutant or variant sequence.

[0243] According to some embodiments, the exogenous immunomodulatory molecule R 24 is a CD86 variant engineered to have an IRES-compatible signal sequence. According to some embodiments, the exogenous immunomodulatory molecule R 25 is a FLT3L variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule R 26 is a GM-CSF variant engineered to have a CD8 membrane anchor and an IRES-compatible signal sequence. According to some embodiments, the exogenous immunomodulatory molecule R 27 is an HSP70 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 28 is an HSP-90B1 (GRP94 / 96) variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 29 is an HSP90 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 30is an ICOSL variant engineered to have an IRES-compatible signal sequence. According to some embodiments, the exogenous immunomodulatory molecule R 31 is an IL10R variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule R 32 is an IL-Rα variant (VSV-GM-CSF tag) engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule R 33 is a single-chain engineered IL12 variant containing a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 34 is an IL15 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 35 is an IL18 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 36 is an IL2 variant engineered to have a CD8 membrane anchor and an IRES compatible sequence. According to some embodiments, the exogenous immunomodulatory molecule R 37 is an IL21 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 38 is a single-chain engineered IL23 variant containing a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 39 is an IL17 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule R 40 is a TGFb-R variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory R 41 The molecule is a TGFb receptor III variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule R 42 is a mIFNα variant engineered to be membrane-bound. According to some embodiments, the exogenous immunomodulatory molecule R 43is a mIFNαγ variant engineered to be membrane-bound. According to some embodiments, the exogenous immunomodulatory molecule R 44 is a CD40L variant that is resistant to cleavage.

[0244] Table 3 below shows the R group, R 1 -R 44 This shows: [Table 4-1] [Table 4-2]

[0245] According to some embodiments, at least 12 vectors comprise 14 immunomodulatory factors, at least three of which are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and the remaining 11 immunomodulatory factors are selected from the R 1 -R 44 According to some embodiments, at least 11 vectors comprise 14 immunomodulatory factors, at least three of which are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and the remaining 11 immunomodulatory factors are selected from R in Table 3. 1 -R 44 According to some embodiments, at least 10 vectors comprise 14 immunomodulatory factors, at least three of which are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and the remaining 11 immunomodulatory factors are selected from the R 1 -R 44According to some embodiments, the 14 immunomodulatory factors are selected from Table 2, at least three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and the remaining 11 immunomodulatory factors are selected from Table 3. 1 -R 44 In some embodiments, the 14 immunomodulatory factors are selected from Table 2, and at least three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and the remaining 11 immunomodulatory factors are selected from Table 3. 1 -R 44 In some embodiments, the 14 immunomodulatory factors are selected from Table 2, and at least three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and the remaining 11 immunomodulatory factors are selected from Table 3. 1 -R 44 The 14 immunomodulatory factors are selected from the following:

[0246] In some embodiments, the immunomodulatory factor is codon-optimized. "Codon optimization" refers to modifying the codons of a polynucleotide encoding a protein with codons that are used more frequently in a particular organism than in other organisms, so that the encoded protein can be expressed more efficiently in that organism. The genetic code is degenerate because most amino acids are described by several codons called "synonyms" or "synonymous codons." However, the codon usage frequency of a particular organism is not random, but is biased toward certain codon triplets. Such codon usage bias may be even higher in association with specific genes, genes of common function or ancestral origin, proteins expressed at high levels relative to low-copy-number proteins, or group protein-coding regions of an organism's genome.

[0247] cytokines According to one embodiment, the present disclosure encompasses an allogeneic tumor cell vaccine comprising a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the population comprising one or more cytokines. Accordingly, the present disclosure encompasses cytokines, including full-length, fragments, homologs, variants, or mutants of cytokines. Cytokines include proteins that can affect the biological function of another cell. Biological functions affected by cytokines may include, but are not limited to, cell proliferation, cell differentiation, or cell death. According to some embodiments, cytokines of the present disclosure can bind to specific receptors on the surface of cells, thereby stimulating immune cells (e.g., T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes).

[0248] According to some embodiments, the cytokine is selected from granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), Fms-related tyrosine kinase 3 ligand (FLT3LG), interleukin-1 (IL-1), IL-1a, IL-1b, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12p40, IL-12p70, IL-12 / IL-23 P40, IL13, IL-15, IL-15 / IL15-RA, IL-17, IL-17A, IL-18, IL-21, IL-23, TGF-β, MCP-1, TNF-α, and interferon α (IFNα), IFNγ, MIP1b, Rantes, Tweak, and TREM-1. According to some embodiments, the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF). According to some embodiments, the cytokine is Fms-related tyrosine kinase 3 ligand (FLT3LG).

[0249] According to some embodiments, the cytokine is secreted. According to some embodiments, the cytokine is membrane bound.

[0250] Granulocyte-macrophage colony-stimulating factor (GM-CSF) Granulocyte-macrophage colony-stimulating factor (GM-CSF; colony-stimulating factor 2; CSF2) is found in monocytes / macrophages and activated T cells and acts as a growth factor that stimulates and recruits dendritic cells. GM-CSF is a monomeric glycoprotein secreted by cells of the immune system, as well as endothelial cells and fibroblasts. Human GM-CSF is a 144-amino acid protein containing a 17-amino acid signal peptide that can be cleaved to generate the mature 127-amino acid protein. GM-CSF's biological activity is mediated by binding to heteromeric cell surface receptors expressed on monocytes, macrophages, granulocytes, lymphocytes, endothelial cells, and alveolar epithelial cells. The GM-CSF receptor (GM-CSFR) typically has low expression (e.g., 20–200 / cell) but high affinity (Shi Y et al., Granulocyte-macrophage colony-stimulating factor (GM-CSF) and T-cell responses: what we do and don't know, Cell Research (2006) 16: 126–133).

[0251] In some mouse models, vaccination with syngeneic murine melanoma cells secreting GM-CSF stimulates stronger and longer-lasting antitumor immunity than vaccines produced with other cytokines. Melanoma patients treated with soluble GM-CSF as adjuvant therapy have shown increased disease-free survival compared with controls. GM-CSF has been used as an immune adjuvant in a variety of ways, including systemic and local application of soluble GM-CSF, GM-CSF fusion proteins, transfection of tumor cells with GM-CSF, and injection of GM-CSF DNA. Recombinant GM-CSF has been used as an adjuvant for a variety of peptide, protein, and viral vaccines and has been shown to be an effective adjuvant in patients with melanoma, breast cancer, and ovarian cancer. GM-CSF-containing fusion proteins have also been shown to enhance the immunogenicity of antigens. GM-CSF is being tested for use in gene therapy approaches using allogeneic or autologous GM-CSF-expressing cells as a vaccine (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). Such vaccines have had varying degrees of efficacy across several different types of cancer.

[0252] According to some embodiments, the tumor cell line or tumor cell line variant may express the GM-CSF peptide of SEQ ID NO: 13. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO: 13. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO: 13.

[0253] According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins comprising a fusion between GM-CSF and HLA-I to enable membrane expression. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO: 42 or SEQ ID NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO: 42 or SEQ ID NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO: 42 or SEQ ID NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO: 42 or SEQ ID NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO: 42 or SEQ ID NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO: 42 or SEQ ID NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO: 42 or SEQ ID NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO: 42 or SEQ ID NO: 5. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO: 42 or SEQ ID NO: 5.

[0254] Fms-like tyrosine kinase-3 ligand (Flt-3L) The human Flt3L protein is a membrane-bound hematopoietic four-helix bundle cytokine encoded by the FLT3LG gene. Flt3L functions as a growth factor that stimulates the proliferation and differentiation of various blood cell progenitor cells and is essential for the production and development of dendritic cells. Mice lacking Flt3L have low levels of dendritic cells, and administration of Flt3L to mice or humans results in significantly higher levels of dendritic cells (Shortman et al., Steady-state and inflammatory dendritic-cell development, Nature Reviews Immunology, Vol. 7, pp. 19-30 (2007)).

[0255] According to some embodiments, the tumor cell line or tumor cell line variant may express the Flt3L peptide of SEQ ID NO: 14. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO: 14. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO: 14. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO: 14. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO: 14. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO: 14. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO: 14. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO: 14. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO: 14. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO: 14.

[0256] According to some embodiments, the tumor cell line or tumor cell line variant comprises a soluble form of Flt3L. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO: 44. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO: 44. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO: 44. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO: 44. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO: 44. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO: 44. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO: 44. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO: 44. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO: 44.

[0257] Those skilled in the art, after being armed with the teachings provided herein, will understand that the present invention encompasses any cytokine, whether currently known to those skilled in the art or discovered in the future.

[0258] According to some embodiments, the allogeneic tumor cell vaccine, which comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, comprises one or more (e.g., 2, 3, 4, 5, or more) cytokines, or variants or fragments thereof.

[0259] TNF family members According to one embodiment, the present disclosure encompasses an allogeneic tumor cell vaccine comprising a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the population comprising one or more TNF family members. Thus, the present disclosure encompasses TNF family member proteins, including full-length, fragments, homologs, variants, or mutants of TNF family proteins. According to some embodiments, the TNF superfamily member is selected from one or more of tumor necrosis factor alpha (TNFα), CD40 ligand (CD40L), OX40 ligand (OX40L), FAS ligand (FASL), CD27 ligand (CD27L), CD30 ligand (CD30L), CD137 ligand (CD137L), TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFS12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18, TNFβ, TNFSF1B, TNFγ, and ectodysplasin A (EDA). According to some embodiments, the TNF superfamily member is TNFα. According to some embodiments, the TNF superfamily member is CD40L.

[0260] According to some embodiments, the TNF family member is membrane bound.

[0261] The tumor necrosis factor (TNF) superfamily is a protein superfamily of type II transmembrane proteins that contain TNF homology domains and form trimers. Members of this superfamily are released from the cell membrane by exoproteolytic cleavage and can function as cytokines. These proteins are primarily expressed by immune cells and regulate diverse cellular functions, including proliferation, differentiation, apoptosis, and embryogenesis, as well as regulating immune responses and inflammation. The superfamily contains 19 members that bind to 29 members of the TNF receptor superfamily.

[0262] OX40L (TNFSF4, bTNF superfamily member 4) OX40 ligand (OX40L) (CD252, TNFSF4), originally called glycoprotein 34 kDa (GP34), belongs to the TNF superfamily. It is primarily expressed on the surface of antigen-presenting cells (APCs), including activated dendritic cells (DCs), B cells, macrophages, T cells, and endothelial cells [Huang, L. et al., J. Trans. Med. (2018) 16: 74; doi:10.1186 / s12967-018-1436-4, citing DeSmedt, T et al., J. Immunol (2002) 168: 661-670. doi: 10.4049 / jimmunol.168.2.661; Ohshima, Y. et al., Blood (1998) 92:3338-3345].

[0263] OX40 (ACT35, CD134, TNFRSF4) is constitutively expressed on the cell surface of activated CD4+ T cells [Ibid., citing Ogawa R, et al., Cytokine Growth Factor Rev. (2008) 19:253-262. doi:10.1016 / j.cytogfr.2008.04.003, Paterson DJ, et al. Mol Immunol. (1987) 24:1281-1290. doi: 10.1016 / 0161-5890(87)90122-2]. It specifically binds to OX40L and initiates a series of responses that contribute to promoting CD4+ T cell proliferation and survival and cytokine secretion [Ibid., citing Kaur D, Brightling C. Chest. (2012) 141:494-499. doi: 10.1378 / chest.11-1730]. The OX-40 receptor (OX-40R) is a transmembrane protein found on the surface of activated CD4(+) T cells (Weinberg, AD, et al., "OX-40: life beyond the effector T cell stage," Semin. Immunol. (1998) 10(6): 471-80). Upon binding of agonists such as anti-OX-40 antibodies or OX-40 ligand (OX-40L) during antigen presentation to T cell lines, OX-40R generates a costimulatory signal as potent as CD28 costimulation (Ibid.). OX-40R ligation enhances effector and memory effector T cell function by upregulating IL-2 production and extending effector T cell lifespan. (Id.)

[0264] CD25-Foxp3- naive CD4 T cells acquire Foxp3, driven by TGF-βR and IL-2R signaling, leading to differentiation into inducible Tregs (iTregs) (So, T et al., Cytokine Growth Factor Rev. (2008) 19(3-4): 253-62). Costimulatory signals from OX40 have been shown to antagonize Foxp3 induction in antigen-responsive naive CD4 T cells and suppress the development of large numbers of CD25+Foxp3+ iTregs (ibid., citing Vu MD, et al. Blood. (2007) 110:2501-10; So T, Croft M. J Immunol. (2007) 179:1427-30).

[0265] According to some embodiments of the disclosed invention, tumor cell lines or tumor cell line variants may be engineered to express a membrane-bound form of OX40L of SEQ ID NO: 108 on the membrane of tumor cells. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO: 108. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO: 108. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO: 108. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO: 108. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO: 108. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO: 108. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO: 108. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO: 108. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO: 108.

[0266] CD27 ligand (CD70) CD27 ligand (CD70), a type II transmembrane protein, is a member of the TNF superfamily. It is expressed on activated T and B lymphocytes and NK cells. CD27 ligand and its receptor, CD27, regulate immune responses by promoting T cell expansion and differentiation and NK cell recruitment. CD27 signaling prevents apoptosis of antigen-specific CD8+ T cells late in the primary CD8+ T cell response. Lack of CD27 signaling reduces the quality of memory CD8+ T cell responses. However, memory CD8+ T cells, which express surface CD27 like naive cells, do not require CD27 costimulation during secondary responses. Thus, in vivo, CD27 indirectly regulates primary antigen-specific CD8+ T cell responses by preventing apoptosis of CD8+ T cells late in the primary response and is required for optimal memory cell quality, but is not normally required for primed secondary CD8+ T cell responses (Dolfi, DV, et al., J. Immunol. (2008) 180(5):2912-2921). Full-length CD27 ligand (CD70) is a 193-amino acid protein consisting of a 17-amino acid cytoplasmic domain, a 21-amino acid transmembrane domain, and a 155-amino acid extracellular domain. Human soluble CD70 corresponds to the 155-amino acid extracellular domain of the full-length CD70 protein.

[0267] According to some embodiments of the disclosed invention, tumor cell lines or tumor cell line variants may be engineered to express a membrane-bound form of CD70 on the membrane of the tumor cells.

[0268] According to some embodiments of the disclosed invention, tumor cell lines or tumor cell line variants may be engineered to express a soluble form of CD70.

[0269] According to some embodiments of the disclosed invention, tumor cell lines or tumor cell line variants may be engineered to express a membrane-bound form of CD70 of SEQ ID NO: 109 on the membrane of tumor cells. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO: 109. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO: 109. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO: 109. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO: 109. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO: 109. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO: 109. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO: 109. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO: 109. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO: 109. 4-IBBL

[0270] Naive CD8 T cells require more costimulatory activity to become activated effector cells than naive CD4 T cells. This requirement can be met in two ways. The simplest is priming with activated DCs, which have high intrinsic costimulatory activity. In some viral infections, dendritic cells are sufficiently activated to directly induce CD8 T cells to produce IL-2, which is necessary for their differentiation into cytotoxic effector cells, without the help of CD4 T cells. This property of DCs has been exploited to generate cytotoxic T cell responses against tumors. However, in the majority of viral infections, CD8 T cell activation requires additional help provided by CD4 effector T cells. CD4 T cells that recognize relevant antigens presented by APCs can amplify the activation of naive CD4 T cells by further activating the APCs. B7 expressed by DCs initially activates CD4 T cells to express IL-2 and CD40L. CD40L binds to CD40 on DCs, providing an additional signal that increases the expression of B7 and 4-IBBL by DCs. This provides additional costimulation to naive CD8 T cells. IL-2 produced by activated CD4 T cells also acts to promote the differentiation of effector CD8 T cells (Murphy, Kenneth. Janeway's Immunobiology: 8th ed. Chapter 15: Garland Science. (2012), at 372).

[0271] 4-IBB has an expression pattern that follows primary T cell activation and is restricted to activated CD4+ and CD8+ T cells (Guinn, B, et al., J. Immuno. (1999) 162: 5003-5010). Engagement of the 4-IBB receptor has been shown to relay potent costimulatory signals in activated T cells, enhancing their proliferation and cytokine secretion (ibid.). Such signaling prevents activation-induced cell death after TCR crosslinking in the absence of other accessory signals (ibid.). 4-IBBL, the high-affinity ligand for 4-IBB expressed on the surface of activated APCs, is a type II membrane protein that shows homology to members of the TNF receptor family. T cells purified from CD28- / - mice have been shown to secrete cytokines and proliferate in response to 4-IBBL-expressing lymphoma. This response can be inhibited by a soluble 4-IBB receptor fusion protein (ibid.). In the absence of CD28 signals, 4-IBBL:4-IBB interactions have been shown to play a role in generating Th2 responses in mixed lymphocyte reactions (Id.).

[0272] According to some embodiments of the disclosed invention, the R subset of immunomodulators may comprise a membrane-bound form of 4-IBBL. According to some embodiments of the disclosed invention, the R subset of immunomodulators may comprise a soluble form of 4-IBBL.

[0273] CD40L The ligand for CD40, known as CD154 or CD40L, is a type II transmembrane protein whose molecular weight varies between 32 and 39 kDa due to post-translational modifications (Elgueta R et al., Molecular mechanism and function of CD40 / CD40L engagement in the immune system. Immunological reviews. 2009;229(1):10.1111 / j.1600-065X.2009.00782.x. doi:10.1111 / j.1600-065X.2009.00782.x, citing van Kooten C et al., J. Leukoc Biol. 2000 Jan;67(1):2-17.). The soluble form of CD40L has been reported to have activity similar to that of the transmembrane form (Ibid., citing Graf D et al., Eur J Immunol. 1995 Jun;25(6):1749-54; Mazzei GJ et al., J Biol Chem. 1995 Mar 31;270(13):7025-8).

[0274] In nature, CD40L is a member of the TNF superfamily and is characterized by a sandwich extracellular structure composed of a beta sheet, an alpha helix loop, and a beta sheet, which allows CD40L trimerization (ibid., citing Karpusas M et al., Structure. 1995 Oct 15;3(10):1031-9). CD40L is expressed primarily by activated T cells, as well as activated B cells and platelets. Under inflammatory conditions, it is also induced in monocytes, natural killer cells, mast cells, and basophils (ibid., citing Carbone E et al., J Exp Med. 1997 Jun 16;185(12):2053-60). The widespread expression of the CD40L and CD40 costimulatory pair indicates their crucial role in various cellular immune processes.

[0275] CD40L has three binding partners: CD40, α5β1 integrin, and αIIbβ3 integrin. CD40L acts as a costimulatory molecule and is particularly important for a subset of T cells called T follicular helper cells (TFH cells). CD40L binds to CD40 on the B cell surface, facilitating cell-to-cell communication and promoting B cell maturation and function. Defects in the CD40L gene result in an inability to undergo immunoglobulin class switching and are associated with hyper-IgM syndrome. Absence of CD40L also halts germinal center formation and prevents antibody affinity maturation, a critical process in the adaptive immune system.

[0276] CD40 has been found to be expressed on APCs, while its ligand, CD40L, is found on activated T cells. CD40 has been found to play an important role in humoral immune responses, enabling APCs to activate T cells. Several pathologies, including lupus and atherosclerosis, have been linked to the CD40 / CD40L pathway, but anti-CD40L antibodies have only limited clinical application in the treatment of thrombotic complications due to CD40 expression on activated platelets (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).

[0277] CD40 is also found in several types of cancer, including solid tumors and hematologic malignancies. While CD40-mediated signaling in hematologic cancers can mediate proliferation or regression, CD40 signaling in solid tumors is exclusively tumoricidal. These features are also observed in SCID mouse models and are thought to be due to TNF death domain signaling. There is also evidence of immunomodulation. For example, blockade of the CD40 / CD40L pathway reduces the protective effect of GM-CSF-secreting melanoma vaccines (Kaufman and Wolchok, eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).

[0278] CD40L-expressing tumor cell vaccines have proven useful in cancer models. For example, ligation of CD40 with CD40L or anti-CD40 antibodies has shown synergistic effects with GM-CSF, IFN-γ, IL-2, and CTLA-4 blockade. Furthermore, anti-CD40 antibodies have been reported to have antitumor activity in preclinical mouse models (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).

[0279] According to some embodiments, the R subset of immune modulators may comprise CD40 ligand (CD40L). According to some embodiments of the disclosed invention, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved form of the CD40L peptide of SEQ ID NO: 6. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO: 6. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO: 6. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO: 6. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO: 6. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO: 6. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO: 6. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO: 6. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO: 6. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO: 6.

[0280] According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved form of the membrane-bound CD40L peptide of SEQ ID NO: 7 on the membrane surface of tumor cells. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO: 7. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO: 7. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO: 7. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO: 7. According to some embodiments, tumor cell lines or tumor cell line variants may comprise one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO: 7. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO: 7. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO: 7. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO: 7. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO: 7.

[0281] Tumor necrosis factor alpha (TNFα) Tumor necrosis factor (TNF; tumor necrosis factor alpha (TNFα); cachexin, cachectin) is a cytokine produced primarily by activated macrophages and lymphocytes and involved in systemic inflammation. It is also one of the cytokines involved in the acute phase of immunogenic responses. TNF can also be produced by other cell types, such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons.

[0282] In its primary role as a regulator of immune cells, TNF can induce fever, apoptotic cell death, cachexia, inflammation, and tumorigenesis; inhibit viral replication; and initiate responses to sepsis via IL-1 and IL-6-producing cells. Dysregulated TNF production has been linked to a variety of human diseases, including Alzheimer's disease, major depression, psoriasis, and inflammatory bowel disease (IBD). TNF can be ectopically produced in the setting of malignancies, paralleling parathyroid hormone in both the cause of secondary hypercalcemia and the cancers in which its overproduction is associated.

[0283] TNF consists of a 26 kDa membrane-bound form and a 17 kDa soluble cytokine form. The soluble form of TNF is derived from the proteolytic cleavage of the membrane-bound form by TNF-α-converting enzyme (TACE) (Grell M. et al., The Transmembrane Form of Tumor Necrosis Factor Is the Prime Activating Ligand of the 80 kDa Tumor Necrosis Factor Receptor, Cell, Vol. 83, 793-802). TACE is a matrix metalloproteinase that recognizes a cleavage site in the extracellular domain of full-length TNF (Rieger, R., Chimeric form of tumor necrosis factor-alpha has enhanced surface expression and antitumor activity, Cancer Gene Therapy, 2009, 16, 53-64). Deletion of the cleavage site of TNF improves membrane stability of TNF (ibid.).

[0284] TNF has antiproliferative and cytotoxic effects on cells, is known to reduce tumor blood flow and vascular damage, and can modulate immune responses by stimulating macrophage and NK cell activity. However, its use as a therapeutic agent has been limited by dose-dependent hypotension and capillary leakage, which can lead to sepsis-like syndromes. Therefore, it must be delivered in a manner that limits systemic effects. TNF has been added to standard chemotherapy agents to improve response rates. Other approaches for administering TNF include the injection of adenoviruses engineered to express TNF in gastrointestinal malignancies. TNF compounds that target tumor vasculature have also been developed (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). Recombinant TNF has been used as an immunostimulant under the name tasonermin, while Humira® is an antibody against TNF and is useful for treating inflammatory diseases (e.g., psoriasis and rheumatoid arthritis). Recognizing this role, molecules such as antibodies have been designed to block TNF activity. However, such treatments pose the risk of initiating a cytokine storm caused by inappropriate systemic release of cytokines, resulting in a potentially fatal positive feedback loop of leukocyte activation / cytokine release.

[0285] According to some embodiments, a subset of R members may include TNF. According to some embodiments, tumor cell lines or tumor cell line variants may be genetically engineered to express a membrane-bound form of TNF on the membrane of tumor cells. For example, according to some embodiments, the cell line variant comprises the peptide of SEQ ID NO: 8. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO: 8. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO: 8. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO: 8. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO: 8. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO: 8. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO: 8. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO: 8. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO: 8. According to some embodiments, the tumor cell line or tumor cell line variant may comprise one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO: 8.

[0286] According to some embodiments, tumor cell lines or tumor cell line variants can be genetically engineered to express uncleaved membrane-bound forms of TNF. For example, according to some embodiments, tumor cell lines or tumor cell line variants can comprise the TNF protein of SEQ ID NO: 8, in which one or more of the amino acids VRSSSRTPSDKP (SEQ ID NO: 104) are deleted (see, for example, SEQ ID NO: 26).

[0287] According to some embodiments, tumor cell lines or tumor cell line variants may be genetically engineered to express a soluble form of TNF. According to some embodiments, tumor cell lines or tumor cell line variants may express the TNF protein of SEQ ID NO: 8 in which part or all of the transmembrane region has been deleted. For example, according to some embodiments, tumor cell lines or tumor cell line variants may comprise a derivative TNF protein of SEQ ID NO: 8 in which one or more of the amino acids F, S, F, L, I, V, A, G, A, T, T, L, F, C, L, L, H, F, G, V, I have been deleted (see, e.g., SEQ ID NO: 27).

[0288] According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to express a non-cleavable membrane-bound chimeric form of CD40L and TNF. For example, according to some embodiments, the ligand-binding portion of a TNF molecule can be fused to the transmembrane and proximal extracellular domains of CD40L such that TNF lacks a defined TNF alpha cleaving enzyme (TACE) site. According to some embodiments, the intracellular, transmembrane, and partial extracellular portions of CD40L can be fused to the extracellular region of TNF distal to the TACE cleavage site. According to some embodiments, a CD40L / TNF chimeric form can comprise the CD40L sequence of SEQ ID NO: 9 and the TNF sequence of SEQ ID NO: 10. According to some embodiments, the CD40L / TNF sequences are operably linked via a linking peptide between 1 and 30 amino acids in length. According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to contain a fusion protein having at least 60% sequence identity to the proteins of SEQ ID NO: 9 and SEQ ID NO: 10. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 70% sequence identity to the proteins of SEQ ID NO:9 and SEQ ID NO:10. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 80% sequence identity to the proteins of SEQ ID NO:9 and SEQ ID NO:10. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 90% sequence identity to the proteins of SEQ ID NO:9 and SEQ ID NO:10. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 95% sequence identity to the proteins of SEQ ID NO:9 and SEQ ID NO:10. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 96% sequence identity to the proteins of SEQ ID NO:9 and SEQ ID NO:10.According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 97% sequence identity to the proteins of SEQ ID NO:9 and SEQ ID NO:10. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 98% sequence identity to the proteins of SEQ ID NO:9 and SEQ ID NO:10. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 99% sequence identity to the proteins of SEQ ID NO:9 and SEQ ID NO:10.

[0289] According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved membrane-bound form of TNF having at least 60% sequence identity to the protein of SEQ ID NO: 11. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved membrane-bound form of TNF having at least 70% sequence identity to the protein of SEQ ID NO: 11. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved membrane-bound form of TNF having at least 80% sequence identity to the protein of SEQ ID NO: 11. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved membrane-bound form of TNF having at least 90% sequence identity to the protein of SEQ ID NO: 11. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved membrane-bound form of TNF having at least 95% sequence identity to the protein of SEQ ID NO: 11. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved membrane-bound form of TNF having at least 96% sequence identity to the protein of SEQ ID NO: 11. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved membrane-bound form of TNF having at least 97% sequence identity to the protein of SEQ ID NO: 11. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved membrane-bound form of TNF having at least 98% sequence identity to the protein of SEQ ID NO: 11. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an uncleaved membrane-bound form of TNF having at least 99% sequence identity to the protein of SEQ ID NO: 11.

[0290] According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to express a non-cleavable membrane-bound chimeric form of CD40L and TNF. For example, according to some embodiments, the ligand portion of the TNF molecule can be fused to the extracellular portion of CD40L, where CD40L comprises an extracellular portion that is non-cleavable, and TNF lacks a defined TACE site (e.g., the cleavage site between amino acids 76 and 77). According to some embodiments, some or all of the CD40L peptide sequence is fused to the extracellular region of the TNF peptide sequence distal to the TACE cleavage site. According to some embodiments, the CD40L / TNF chimeric form can comprise the sequence of SEQ ID NO: 31. According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to contain a fusion protein having at least 60% sequence identity to the protein of SEQ ID NO: 31. According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to contain a fusion protein having at least 70% sequence identity to the protein of SEQ ID NO: 31. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to contain a fusion protein having at least 80% sequence identity to the protein of SEQ ID NO: 31. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to contain a fusion protein having at least 90% sequence identity to the protein of SEQ ID NO: 31. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to contain a fusion protein having at least 95% sequence identity to the protein of SEQ ID NO: 31. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to contain a fusion protein having at least 96% sequence identity to the protein of SEQ ID NO: 31. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to contain a fusion protein having at least 97% sequence identity to the protein of SEQ ID NO: 31.According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to contain a fusion protein having at least 98% sequence identity to the protein of SEQ ID NO: 31. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to contain a fusion protein having at least 99% sequence identity to the protein of SEQ ID NO: 31.

[0291] According to some embodiments, the allogeneic tumor cell vaccine, which comprises a population of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, comprises one or more (e.g., 2, 3, 4, 5, or more) TNF family member proteins, or variants or fragments thereof.

[0292] Secretory receptors According to one embodiment, the present disclosure encompasses an allogeneic tumor cell vaccine comprising a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the population comprising one or more secreted receptors.

[0293] According to some embodiments, the R immunomodulator may comprise one or more (e.g., 2, 3, 4, 5, or more) secreted receptor proteins, or variants or fragments thereof. According to some embodiments, the secreted receptor is IL10R, TGFβR3, or both.

[0294] Interleukin-10 (IL-10) is an important immunosuppressive cytokine produced by various leukocytes and non-hematopoietic cells (Shouval, DS., et al., Immunity (2014) 40:706-719). IL-10 mediates its anti-inflammatory effects through IL-10 receptor (IL-10R)-dependent signaling emanating from the cell surface. IL-10R is a heterotetramer composed of two subunits, IL-10Rα and IL-10Rβ (ibid., citing Moore, KW, et al., Annu. Rev. Immunol. (2001) 19:683-765). While the IL-10Rα subunit is unique to IL-10 signaling, the IL-10Rβ subunit is shared with other cytokine receptors, such as IL-22, IL-26, and interferon-λ (ibid.). IL-10 downstream signaling via IL-10R inhibits the induction of pro-inflammatory cytokines by blocking NF-κB-dependent signaling (Ibid., citing Saraiva, M., and O'Garra, A. Nat. Rev. Immunol. (2010) 10:180-181).

[0295] Transforming growth factor-β receptor 3 (TβRIII or TβR3) is an 853-amino acid transmembrane proteoglycan containing a short 41-amino acid cytoplasmic domain. It is ubiquitously expressed in almost all cell types. The expression level of TβRIII is cell type-specific. It is a member of the TGF-β superfamily signaling pathway and plays an essential role in mediating cell proliferation, apoptosis, differentiation, and migration in most human tissues. TTβRIII is the most abundant TGF-β superfamily receptor and functions as a TGF-β superfamily coreceptor by binding to TGF-β superfamily members TGF-β1, TGF-β2, or TGF-β3, inhibin, BMP-2, BMP-4, BMP-7, and GDF-5, presenting these ligands to their respective signaling receptors to activate or inhibit (in the case of inhibin) TGF-β1, BMP, or activin signaling to Smad transcription factors. For example, in the case of TGF-β1, 2, or 3, TβRIII presents the ligand to the TGF-β type II receptor (TβRII). Upon ligand binding, TbetaRII recruits and transphosphorylates the TGF-β type I receptor (TbetaRI), activating its kinase function and leading to phosphorylation of Smad2 / 3. Phosphorylation of Smad2 and Smad3 leads to the formation of a complex with Smad4, which accumulates in the nucleus. This complex, together with coactivators and corepressors, regulates the transcription of genes involved in proliferation, angiogenesis, apoptosis, and differentiation. In addition to regulating receptor-mediated Smad signaling, TβRIII also mediates ligand-dependent and -independent p38 pathway signaling. TβRIII can also undergo ectodomain shedding to generate soluble TβRIII (sTβRIII), which binds and sequester members of the TGF-β superfamily, inhibiting their signaling. Although it has been demonstrated that sTβRIII expression correlates with cell surface expression of TβRIII, little is known about the regulation of sTβRIII production.TβRIII shedding may be mediated in part by the membrane-type matrix metalloproteinases (MT-MMPs) MT1-MMP and / or MT3-MMP, as well as plasmin, a serine proteinase that has been shown to cleave the extracellular domain of TβRIII. Furthermore, TβRIII shedding is regulated by the tyrosine phosphatase inhibitor pervanadate. Supporting this, the MT-MMP and ADAM protease inhibitor TAPI-2 have been shown to inhibit TβRIII shedding. Modulation of TβRIII expression is sufficient to alter TGF-β signaling. The cytoplasmic domain of TβRIII interacts with the PDZ domain-containing protein GAIP-interacting protein C-terminal (GIPC). This stabilizes TβRIII cell surface expression and increases TGF-β signaling. The interaction between TβRIII and GIPC also plays an important role in TβRIII-mediated inhibition of TGF-β signaling, cell migration, and invasion during breast cancer progression. The cytoplasmic domain of TβRIII is phosphorylated by TβRII, resulting in binding of TβRIII to the scaffolding protein β-arrestin2. The TβRIII / β-arrestin2 interaction leads to co-internalization of β-arrestin2 / TβRIII / TβRII and downregulation of TGF-β signaling. The interaction between TβRIII and β-arrestin2 regulates BMP signaling and TGF-β signaling. TβRIII forms a complex with the BMP1 type receptor ALK6 in a β-arrestin2-dependent manner, mediating ALK6 internalization and stimulation of ALK6-specific BMP signaling events. TβRIII negatively regulates NFκB signaling in the context of breast cancer through its interaction with β-arrestin 2; it regulates epithelial cell adhesion to fibronectin, fibrillogenesis, and focal adhesion formation through regulating the internalization and trafficking of α5β1 to developing focal adhesions; it activates Cdc42, alters the actin cytoskeleton, and inhibits migration of normal and cancerous ovarian epithelial cells. During development, TβRIII plays an important role in the formation of the atrioventricular prominence of the heart. Consistent with a critical role for TβRIII during development, TGFβR3 null mice are embryonic lethal due to cardiac and liver defects.TGFβR3 has recently been identified as a tumor suppressor in multiple types of human cancer, including breast, lung, ovarian, pancreatic, and prostate cancer. Loss of TGFβR3 in these cancer types correlates with disease progression, resulting in increased motility and invasion in vitro and increased invasion and metastasis in vivo (http: / / atlasgeneticsoncology.org / Genes / TGFBR3ID42541ch1p33.html, accessed August 26, 2019).

[0296] chaperone According to some embodiments, the subset of R immune modulators may include one or more chaperone proteins. According to one embodiment, the present disclosure encompasses an allogeneic tumor cell vaccine comprising a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the population comprising one or more chaperone proteins. Thus, the present disclosure encompasses chaperone proteins, including full-length, fragments, homologs, variants, or mutants of chaperone proteins. Chaperones are a group of functionally related proteins that assist in the folding of proteins within cells under physiological and stress conditions. According to some embodiments, the chaperone protein is selected from one or more of GRP78 / BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, protein disulfide isomerase (PDI), peptidyl prolyl cis-trans-isomerase (PPI), Erp57, Hsp60, Hsp70, Hsp90, Hsp100.

[0297] According to some embodiments, the chaperone protein is membrane-bound.

[0298] According to some embodiments, the allogeneic tumor cell vaccine, comprising a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, comprises one or more (e.g., 2, 3, 4, 5, or more) chaperone proteins, or variants or fragments thereof.

[0299] Immunoglobulin superfamily (IgSF) According to some embodiments, the subset of R immune modulators may include one or more IgSF proteins. According to one embodiment, the present disclosure encompasses an allogeneic tumor cell vaccine comprising a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells being genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes, the population comprising one or more IgS family proteins. Accordingly, the present disclosure encompasses members of the IgSF superfamily, including full-length, fragments, homologs, variants, or mutants of IgSF superfamily members. The immunoglobulin superfamily (IgSF) is a class of proteins involved in cellular adhesion, binding, and recognition processes. Molecules are classified as members of this superfamily based on structural features shared with immunoglobulins; all possess domains known as immunoglobulin domains or folds. Members of the IgSF include cell surface antigen receptors, coreceptors and costimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors, and intracellular muscle proteins. Members of the IgSF can be classified as follows: antigen receptors (e.g., antibodies or immunoglobulins: IgA, IgD, IgE, IgG, IgM); antigen-presenting molecules (e.g., MHC class I, MHC class II); coreceptors (e.g., CD4, CD8); costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86); receptors on natural killer cells (e.g., killer cell immunoglobulin-like receptors (KIRs)); receptors on leukocytes (e.g., leukocyte immunoglobulin-like receptors (LILRs)); IGSF CAMs (e.g., NCAM, ICAM-1); cytokine receptors; growth factor receptors; receptor tyrosine kinase / phosphatases; and IgG-binding receptors.

[0300] According to some embodiments, the IgSF family member is membrane-bound.

[0301] The poliovirus receptor (PVR / CD155) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. PVR / CD155 mediates NK cell adhesion and triggers NK cell effector functions. PVR / CD155 binds to two distinct NK cell receptors, CD96 and CD226. These interactions result in accumulation at cell-cell contact sites, leading to the formation of a mature immune synapse between NK cells and target cells. This leads to adhesion and the secretion of lytic granules and IFN-γ (IFNγ), which activates the cytotoxicity of activated NK cells and may promote NK cell-target cell modular exchange and PVR transfer to NK cells.

[0302] Poliovirus receptor-related 2 (PVRL2), also known as nectin-2, is a single-pass type I membrane glycoprotein with two Ig-like C2-type domains and an Ig-like V-type domain. This protein is one of the plasma membrane components of adherens junctions.

[0303] The CD48 antigen (cluster of differentiation 48), also known as B lymphocyte activation marker (BLAST-1) or signaling lymphocyte activation molecule 2 (SLAMF2), is a protein encoded by the CD48 gene in humans. CD48 is a member of the CD2 subfamily of IgSF, which includes SLAM (signaling lymphocyte activation molecule) proteins such as CD84, CD150, CD229, and CD244. CD48 is found on the surface of lymphocytes and other immune cells, dendritic cells, and endothelial cells, and is involved in the activation and differentiation pathways of these cells.

[0304] NK-TB antigens (NTBAs) are surface molecules expressed on NK cells, T cells, and B cells. In human NK cells, NTBAs have been shown to act primarily as coreceptors, as they can trigger cytolytic activity only on cells expressing a high surface density of natural cytotoxicity receptors (NCRs). Molecular cloning has revealed that NTBAs are members of the Ig superfamily, characterized by structural features that allow their assignment to the CD2 family.

[0305] According to one embodiment, the IgSF protein is IgG. According to one embodiment, the IgSF protein is PVR / CD155. According to one embodiment, the IgSF protein is CD48. According to one embodiment, the IgSF protein is Nectin2. According to one embodiment, the IgSF protein is an NK-TB antigen.

[0306] Immunoglobulins (Ig) are glycoproteins produced by immune cells. Antibodies are serum proteins whose molecules possess small regions on their surface that are complementary to small chemical groups on their targets. These complementary regions (called complementarity-determining regions (CDRs), or antibody-binding sites, or antigen-binding sites), of which there are at least two per antibody molecule—some types of antibody molecules have as many as 10, 8, or even 12 in some species—can react with corresponding complementary regions (antigenic determinants or epitopes) on antigens, binding several molecules of multivalent antigen together to form a lattice. Immunoglobulins play an important role in the immune response by binding to specific antigens, such as those displayed by bacteria or viruses. According to some embodiments, immunoglobulin binding to antigens can target them for destruction by the subject's immune cells.

[0307] The basic structural unit of a whole antibody molecule consists of four polypeptide chains: two identical light (L) chains (each containing approximately 220 amino acids) and two identical heavy (H) chains (each containing approximately 440 amino acids). The two heavy chains and two light chains are bound together by a combination of non-covalent and covalent (disulfide) bonds. The molecule consists of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of a light chain and the N-terminal region of a heavy chain. Normally, both the light and heavy chains cooperate to form the antigen-binding surface.

[0308] Mammals have five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each with its own class of heavy chain—α (for IgA), δ (for IgD), ε (for IgE), γ (for IgG), and μ (for IgM). Furthermore, IgG immunoglobulins have four subclasses (IgG1, IgG2, IgG3, and IgG4), each with γ1, γ2, γ3, and γ4 heavy chains, respectively. Secretory IgM is a pentamer composed of five four-chain units, providing a total of 10 antigen-binding sites. Each pentamer contains one copy of the J chain covalently inserted between two adjacent tail regions.

[0309] Diverse libraries of immunoglobulin heavy (VH) and light (Vκ and Vλ) chain variable genes from peripheral blood lymphocytes can also be amplified by polymerase chain reaction (PCR) amplification. Genes encoding single polypeptide chains in which the heavy and light chain variable domains are linked by a polypeptide spacer can be generated by randomly combining heavy and light chain V genes using PCR.

[0310] According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express an IgG1 heavy chain constant region. In nature, the Ig gamma-1 (IgG-1) chain C region is a protein encoded by the human IGHG1 gene. According to some embodiments, tumor cell lines or tumor cell line variants may express the membrane-bound IgG-1 chain C protein of SEQ ID NO: 1. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a secreted form of IgG-1 chain C of SEQ ID NO: 2. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a secreted form of IgG-1 chain C of SEQ ID NO: 3. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to contain a fusion protein having at least 60% sequence identity to one or more of the proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 70% sequence identity to one or more of the proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 80% sequence identity to one or more of the proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 90% sequence identity to one or more of the proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 95% sequence identity to one or more of the proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 96% sequence identity to one or more of the proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 97% sequence identity to one or more of the proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 98% sequence identity to one or more of the proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise a fusion protein having at least 99% sequence identity to one or more of the proteins having the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0311] According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise fusion proteins having at least 60% sequence identity to one or more proteins having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise fusion proteins having at least 70% sequence identity to one or more proteins having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise fusion proteins having at least 80% sequence identity to one or more proteins having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise fusion proteins having at least 90% sequence identity to one or more proteins having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46. According to some embodiments, a tumor cell line or tumor cell line variant may be engineered to contain a fusion protein having at least 95% sequence identity to one or more proteins having the amino acid sequence of SEQ ID NO:12, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, and SEQ ID NO:46.According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise fusion proteins having at least 96% sequence identity to one or more proteins having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise fusion proteins having at least 97% sequence identity to one or more proteins having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise fusion proteins having at least 98% sequence identity to one or more proteins having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to comprise fusion proteins having at least 99% sequence identity to one or more proteins having the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, and SEQ ID NO: 46.

[0312] According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to express an IgG protein capable of binding to a tumor cell-specific antigen. For example, tumor cell lines or tumor cell line variants can be engineered to express an IgG protein capable of binding to the extracellular domain of a prostate cancer-specific antigen, such as prostate-specific membrane antigen (PSMA) (see Chang, S., Overview of Prostate-Specific Membrane Antigen, Reviews in Urology, Vol. 6 Suppl. 10, S13 (2004)). According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to express an IgG protein capable of binding to an immune cell-specific antigen. For example, tumor cell lines or tumor cell line variants can be engineered to express an IgG protein capable of binding to a T cell marker, such as CD3, CD4, or CD8. According to another example, tumor cell lines or tumor cell line variants can be engineered to express an IgG protein capable of binding to a dendritic cell marker, such as CD11c or CD123.

[0313] According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to express an IgG3 heavy chain constant region. In nature, the IgG3 heavy chain constant region comprises a CH1-hinge-CH2-CH3 domain and is encoded by the IGHG3 gene in humans. The IGHG3 gene contains structural polymorphisms, including different hinge lengths. According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to express an IgG3 heavy chain constant region of SEQ ID NO: 4. According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to express a derivative of SEQ ID NO: 4 in which amino acids 1-76 are missing. According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to express a derivative of SEQ ID NO: 4 in which amino acids 1-76 are missing. According to some embodiments, tumor cell lines or tumor cell line variants can be engineered to express a derivative of SEQ ID NO: 4 in which amino acids 77-98 are replaced with amino acids QMQGVNCTVSS (SEQ ID NO: 101). According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a derivative of SEQ ID NO: 4 comprising the E213Q variant (SEQ ID NO: 16). According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a derivative of SEQ ID NO: 4 comprising the P221L variant (SEQ ID NO: 17). According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a derivative of SEQ ID NO: 4 comprising the E224Q variant (SEQ ID NO: 18). According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a derivative of SEQ ID NO: 4 comprising the Y226F variant (SEQ ID NO: 19). According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a derivative of SEQ ID NO: 4 comprising the D242N variant (SEQ ID NO: 20). According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a derivative of SEQ ID NO: 4 comprising the N245D variant (SEQ ID NO: 21).According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a derivative of SEQ ID NO: 4 comprising the T269A variant (SEQ ID NO: 22). According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a derivative of SEQ ID NO: 4 comprising the S314N variant (SEQ ID NO: 23). According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express a derivative of SEQ ID NO: 4 comprising the S314 variant (SEQ ID NO: 24). According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to ex...

Claims

1. 1. An allogeneic tumor cell vaccine, comprising: (1) A population of proliferation-incompetent, live, genetically engineered tumor cells expressing one or more tumor-specific antigens, said population comprising at least three stably expressed immunomodulatory molecules, said at least three immunomodulatory molecules being OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, for inducing one or more subpopulations of PBMCs to proliferate in response to said expressed immunomodulatory molecules and then enter an effector phase to kill tumor cells; the subpopulation of PBMC cells comprising one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes; (2) a pharmaceutically acceptable carrier; and The allogeneic tumor cell vaccine comprising:

2. (a) a population of proliferation-incompetent, viable, genetically engineered tumor cells expressing one or more tumor-specific antigens is cultured in a culture medium containing a lysate of the tumor; 1 ~R 44 or (b) the tumor cells are rendered proliferation incompetent by irradiation; or (c) the tumor cells are derived from a cancer selected from the group consisting of melanoma, colorectal cancer, leukemia, chronic myeloid leukemia, prostate cancer, head and neck cancer, squamous cell carcinoma, tongue cancer, laryngeal cancer, tonsil cancer, hypopharyngeal cancer, nasopharyngeal cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, glioblastoma, and brain cancer; or (d) the population of viable growth-resistant tumor cells is derived from a biological sample derived from the subject; or (e) the population of viable, growth-resistant tumor cells is derived from a tumor cell line; or (f) the immune stimulatory molecule is displayed on the exterior surface of the engineered tumor cell; or (g) the induction of T lymphocytes includes activation of a subpopulation of T lymphocytes, expansion of T lymphocytes, or both; or (h) the induction of said NK cells comprises activation of a subpopulation of said NK cells, expansion of said subpopulation of said NK cells, or both; or (i) inducing the subpopulation of DCs comprises activating the subpopulation of DCs, expanding the subpopulation of DCs, or both; or (j) inducing the B lymphocyte subpopulation comprises activating the B lymphocyte subpopulation, expanding the B lymphocyte subpopulation, or both; The allogeneic tumor cell vaccine of claim 1 .

3. (a) the subpopulation of T lymphocytes comprises a subpopulation of CD8+ cytotoxic T lymphocytes (CTLs); or (b) the subpopulation of T lymphocytes comprises a subpopulation of memory T cells; or (c) the subpopulation of T lymphocytes comprises a subpopulation of regulatory T cells; (d) the subpopulation of T lymphocytes comprises a subpopulation of helper T cells; (e) the subpopulation of B lymphocytes comprises a subpopulation of memory B cells; The allogeneic tumor cell vaccine of claim 1 .

4. (1) the vaccine enhances immune activation of cells effective in recognizing and acting against tumor cells containing the target tumor antigen in vivo without systemic inflammation; or (2) the vaccine reduces immunosuppression in the tumor microenvironment of tumor cells containing the target tumor antigen; or (3) the vaccine increases cell death of tumor cells expressing the target tumor antigen; or (4) the population of viable, growth-resistant tumor cells induces immune activation without systemic inflammation; or (5) the vaccine induces an immune response that improves progression-free survival, overall survival, or both compared to a placebo control; The allogeneic tumor cell vaccine of claim 1 .

5. 3. The allogeneic tumor cell vaccine of claim 2, wherein the melanoma tumor cells are characterized by expression of one or more of gp100, tyrosinase, Melan-A, tyrosinase-related protein (TRP-2-INT2), melanoma antigen-1 (MAGE-A1), NY-ESO-1, preferentially expressed antigen of melanoma (PRAME), CDK4, and multiple myeloma oncogene 1 (MUM-1).

6. 3. The allogeneic tumor cell vaccine of claim 2, wherein the colorectal cancer tumor cells are characterized by expression of one or more of carcinoembryonic antigen (CEA), MAGE, HPV, human telomerase reverse transcriptase (hTERT), EPCAM, PD-1, PD-L1, p53, and cell surface-associated mucin 1 (MUC1).

7. The R 1 ~R 44 wherein the one or more additional stably expressed immunomodulatory molecules are selected from a cytokine, a TNF family member, a secreted receptor, a chaperone, an IgG superfamily member, and / or a chemokine receptor; The allogeneic tumor cell vaccine of claim 2 .

8. providing an allogeneic parental tumor cell line comprising a population of viable tumor cells; introducing into the population of viable tumor cells an exogenous nucleic acid encoding a stably expressed immunomodulatory molecule, wherein the immunomodulatory molecule is OX40 ligand (OX40L); introducing into said population of viable tumor cells an exogenous nucleic acid encoding a stably expressed immunomodulatory molecule, wherein said immunomodulatory molecule is CD27 ligand (CD70); introducing into the population of live tumor cells an exogenous nucleic acid encoding a stably expressed immunomodulatory molecule, wherein the immunomodulatory molecule is CD28 ligand (CD28L) comprising CD80, CD86, or both, and wherein stable expression of OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L) comprising CD80, CD86, or both induces one or more subpopulations of PBMCs to proliferate in response to the expressed immunomodulatory molecule and then enter an effector phase to kill tumor cells; generating tumor cell line variants by selecting tumor cell clones that stably express immunogenic amounts of the exogenous subset of immunomodulatory molecules; selecting clonally derived cell line variants in a mixed lymphocyte tumor cell reaction by one or more parameters selected from cell proliferation, cell subset differentiation, cytokine release profile, and tumor cell lysis, wherein the selected clonally derived cell line variants are effective in stimulating activation of one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes; 2. The allogeneic tumor cell vaccine of claim 1, produced by a process comprising:

9. R 1 ~R 44 10. The allogeneic tumor cell vaccine produced by the process of claim 8, further comprising introducing into the population of live tumor cells an exogenous nucleic acid encoding one or more stably expressed immunomodulatory molecules selected from:

10. (a) the tumor cells are rendered proliferation incompetent by irradiation, or (b) the parental tumor cell line is derived from melanoma or colorectal carcinoma; or (c) the exogenous nucleic acid comprises DNA or RNA; or (d) the introducing step comprises viral transduction; or (e) the introducing step comprises electroporation; or (f) the introducing step comprises utilizing one or more of liposome-mediated transfer, adenovirus, adeno-associated virus, herpes virus, retrovirus-based vectors, lipofection, and lentiviral vectors; or (g) the introducing step comprises introducing the exogenous nucleic acid by transfection of a lentiviral vector; 9. An allogeneic tumor cell vaccine produced by the process of claim 8.

11. 1. A method of inducing an immune response against cancer in a subject, comprising: (a) administering to the subject an allogeneic tumor cell vaccine parenterally or locally intratumorally, the allogeneic tumor cell vaccine comprising: (1) a population of proliferation-incompetent, live, genetically engineered tumor cells that express one or more tumor-specific antigens, the population comprising at least three stably expressed immunomodulatory molecules, the at least three immunomodulatory molecules being OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both; (2) a pharmaceutically acceptable carrier; and administering the allogeneic tumor cell vaccine to the subject parenterally or locally intratumorally; (b) inducing one or more subpopulations of peripheral blood mononuclear cells (PBMCs) to proliferate in response to the expressed immunomodulatory molecules and then enter an effector phase to kill tumor cells; Including, the subpopulation of PBMC cells comprises one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes; the allogeneic tumor cell vaccine is type-matched to the subject's cancer; The method.

12. 12. The method of claim 11, wherein the cancer is selected from melanoma or colorectal cancer.

13. 12. The method of claim 11, wherein the subject has an infectious viral disease associated with progression to cancer.

14. 12. The method of claim 11, further comprising administering a checkpoint inhibitor to the subject.

15. The population of proliferation-incompetent, viable, genetically engineered tumor cells expressing one or more tumor-specific antigens is 1 ~R 44 12. The method of claim 11, further comprising one or more additional stably expressed immunomodulatory molecules selected from:

16. (a) the population of viable, growth-resistant tumor cells is derived from a biological sample derived from a subject; or (b) the population of viable, growth-resistant tumor cells is derived from a tumor cell line; The method of claim 11.

17. 1. A method of treating cancer in a subject, comprising: wherein the cancer is melanoma or colorectal cancer, and the method comprises: (a)(1) a population of proliferation-incompetent, viable, genetically engineered tumor cells that express one or more tumor-specific antigens; (2) a pharmaceutically acceptable carrier; and administering to the subject an allogeneic tumor cell vaccine comprising: the population comprises at least three stably expressed immunomodulatory molecules, the at least three immunomodulatory molecules being OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both; administering the allogeneic tumor cell vaccine to the subject; (b) inducing one or more subpopulations of peripheral blood mononuclear cells (PBMCs) to proliferate in response to the expressed immunomodulatory molecules and then enter an effector phase to kill tumor cells; Including, the subpopulation of PBMC cells comprises one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or B lymphocytes in an amount effective to reduce tumor burden in the subject and improve progression-free survival, overall survival, or both, in the subject compared to a placebo control; The method.

18. The population of proliferation-incompetent, viable, genetically engineered tumor cells expressing one or more tumor-specific antigens is 1 ~R 44 18. The method of claim 17, further comprising one or more additional stably expressed immunomodulatory molecules selected from:

19. 18. The method of claim 17, further comprising rendering the population of viable, proliferation-incompetent, genetically engineered tumor cells proliferation-incompetent by irradiation.

20. (a) the population of viable, growth-resistant tumor cells is derived from a biological sample derived from a subject; or (b) the population of viable, growth-resistant tumor cells is derived from a tumor cell line; 18. The method of claim 17.

21. 18. The method of claim 17, wherein the immune stimulatory molecule is displayed on the exterior surface of the genetically engineered tumor cell.

22. (a) inducing the T lymphocytes comprises activating a subpopulation of the T lymphocytes, expanding the T lymphocytes, or both; or (b) inducing said NK cells comprises activating a subpopulation of said NK cells, expanding said subpopulation of NK cells, or both; or (c) inducing the subpopulation of DCs comprises activating the subpopulation of DCs, expanding the subpopulation of DCs, or both; or (d) inducing the subpopulation of B lymphocytes comprises activating the subpopulation of B lymphocytes, expanding the subpopulation of B lymphocytes, or both; 18. The method of claim 17.

Images