JP2023536042A - Allogeneic tumor cell vaccine - Google Patents

Abstract

The described invention provides a core group of three immunomodulatory molecules for inducing one or more subpopulations of PBMCs to respond to expressed immunomodulatory molecules and enter an effector phase for killing tumor cells; and, optionally, an allogeneic tumor cell vaccine comprising a tumor cell line or tumor cell line variant genetically engineered to express an additional R immunomodulatory polypeptide. According to some embodiments, a tumor cell vaccine candidate is capable of inducing an immune response in a recipient cancer patient that cross-reacts with the patient's own (autologous) tumor cells, the effect of which is to induce anti-tumor immunity. is sufficient to enhance survival in a vaccinated patient cohort compared to a matched unvaccinated patient cohort. [Selection diagram] None

Description

RELATED APPLICATIONS This application claims priority benefit of U.S. Provisional Application No. 62/425,424 (filed November 22, 2016), U.S. Provisional Application No. 15/821,105 (filed November 22, 2017) ) is a continuation-in-part application.

SEQUENCE LISTING This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The above ASCII copy made on June 9, 2020 is 128663-00119_SL. txt and is 263,710 bytes in size.

The described invention relates generally to immunological approaches to cancer treatment, and more particularly to cancer vaccines comprising modified tumor cells.

The human immune system is generally divided into two groups called "natural immunity" and "adaptive immunity". The innate immune group of the immune system is primarily responsible for the early inflammatory response through many soluble factors, including the complement and chemokine/cytokine systems, mast cells, macrophages, dendritic cells (DC), and natural There are many specialized cell types, including killer cells. The group of adaptive immunity includes delayed and sustained antibody responses, along with CD8+ and CD4+ T cell responses that play an important role in immunological memory for antigens. A third group of the immune system has been identified as comprising γδ T cells and T cells with a limited T cell receptor repertoire such as natural killer T (NKT) cells and mucosa-associated invariant T (MAIT) cells. can be

Cells of the Immune System There are numerous cellular interactions that make up the immune system. Because these interactions signal in both directions, each cell occurs through specific receptor-ligand pairs that receive instructions based on the temporal and spatial distribution of these signals.

Mouse models have been very useful for discovering immunomodulatory pathways, but the clinical utility of these pathways does not necessarily translate from inbred mouse strains to outbred human populations. is not limited. This is because outbred human populations may have individuals who are dependent to varying degrees on individual immunoregulatory pathways.

Cells of the immune system include lymphocytes, monocytes/macrophages, dendritic cells, closely related Langerhans cells, natural killer (NK) cells, mast cells, basophils, and other members of the myeloid lineage of cells. include. Moreover, a set of specialized epithelial and stromal cells often provide the anatomical environment in which immunity occurs by secreting key factors that regulate proliferation and/or gene activation in cells of the immune system. , which also plays a direct role in the induction 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. ).

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 stages of development that prepare them to mediate the myriad responses of the mature immune system. Most lymphocytes and macrophages constitute the recirculating pool of cells found in the blood and lymph, deliver immunocompetent cells to the sites where they are needed, and render locally generated immunity systemic. (Paul, W. E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia Elphia, (1999) , at p.102).

The term "lymphocyte" is formed in lymphoid tissue throughout the body, constitutes about 22-28% of the total number of circulating white blood cells in normal adults, and plays a major role in defending the body against disease. refers to the small white blood cells that carry out Individual lymphocytes are specialized in that recombination of their genetic material has determined that they respond to a limited set of structurally related antigens (e.g., T-cell receptor and B-cell receptors). This determination, which exists before the first contact of the immune system with a given antigen, is expressed by the presence of receptors specific for the determinants (epitopes) of the antigen on the surface membrane of lymphocytes. Each lymphocyte has a population of T cells, all of which have identical binding sites. A set of lymphocytes, or clone, differs from another clone in the structure of its receptor binding regions and thus in the epitopes it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their function (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).

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

B Lymphocytes B lymphocytes are derived from hematopoietic cells of the bone marrow. Mature B cells can be activated by antigens that display epitopes recognized by their cell surface. The activation process is either direct, ie, dependent on the cross-linking of membrane Ig molecules by antigen (cross-linking-dependent B-cell activation), or indirect, ie, interaction with helper T cells in a process called cognate help. It can be through action. In many physiological situations, receptor bridging stimuli and cognate help synergize to produce more vigorous B-cell responses (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

Crosslink-dependent B cell activation requires that the antigen present multiple copies of the epitope complementary to the binding site of the cell surface receptor, as each B cell expresses an Ig molecule with identical variable regions. and Such requirements are met by other antigens with repetitive epitopes, such as microbial capsular polysaccharides and viral envelope proteins. Crosslink-dependent B-cell activation is the major protective immune response mounted against these organisms (Paul, W. E., "Chapter 1: The immune system: an introduction", Fundamental Immunology, 4th Edition , Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

Cognate help allows B cells to mount responses to antigens that are unable to cross-link their receptors, while simultaneously rescuing B cells from inactivation when stimulated by weak bridging events. Provides stimulus signals. Cognate help relies on antigen binding by B-cell membrane immunoglobulin (Ig), antigen endocytosis, and fragmentation into its 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 act as ligands for antigen-specific receptors on a set of T cells, designated as CD4 ⁺ T cells. CD4 ⁺ T cells carry receptors on their surface that are specific for B cell class II/peptide complexes. B-cell activation does not depend solely on its T-cell receptor (TCR)-mediated engagement with T-cells; binding to its receptor (CD40) and signals B-cell activation. In addition, 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, WE, "Chapter 1: The immune system: an introduction, "Fundamental Immunology, 4th Edition, Ed. Paul, W.E., Lippicott-Raven Publishers, Philadelphia, (1999)).

During cognate help to antibody production, CD40 ligand is transiently expressed on activated CD4 ⁺ T helper cells, which binds CD40 on antigen-specific B cells, thereby providing secondary co-stimulatory Transmits signals. The latter signal is important for B cell proliferation and differentiation, and for the generation of memory B cells by preventing apoptosis of germinal center B cells upon antigen encounter. Overexpression of the CD40 ligand on both B and T cells has been implicated in pathogenic autoantibody production in human SLE patients (Desai-Mehta, A. et al., “Hyperexpression of CD40 ligand by B and T cells”). Cells in human lupus and its role in pathogenic autoantibody production," J. Clin. Invest. Vol. 97(9), 2063-2073, (1996)).

T Lymphocytes T lymphocytes are derived from progenitors of hematopoietic tissue, differentiate in the thymus, and then seed peripheral lymphoid tissues and recirculating pools 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, the ability to increase the bactericidal action of monocytes/macrophages, inhibition of certain immune responses, direct killing of target cells, and recruitment of inflammatory responses. . These effects are dependent 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)).

T cells differ from B cells in their mechanism of antigen recognition. Immunoglobulins, or B-cell receptors, bind to discrete 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 have evolved to bind and defend against microorganisms in the extracellular fluid, T-cells recognize antigens on the surface of other cells and recruit these antigen-presenting cells ( It mediates T cell function by interacting with and modifying APC). 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 skin, intestines, and respiratory tract. When they encounter invading microorganisms at these sites, they endocytose the pathogens and their products and transport them through the lymph nodes to regional lymph nodes or gut-associated lymphoid organs. Pathogen encounter induces maturation of dendritic cells from antigen-capturing cells to APCs capable of activating T cells. APCs present three types of protein molecules on their surface that are responsible for activating T cells into effector cells: (1) MHC proteins that present foreign antigens to T cell receptors; (2) T cells. costimulatory proteins that bind to surface complementary receptors; and (3) intercellular adhesion molecules that allow T cells to bind to APCs long enough to be activated (see Chapter 24: The adaptive immune system). system, "Molecular Biology of the Cell, Alberts, B. et al., Garland Science, NY, (2002)).

T cells are subdivided into two distinct classes based on the cell surface receptors they express. Most T cells express the T cell receptor (TCR), which consists of α and β chains. A small group of T cells express receptors made from γ and δ chains. There are two sub-lines of α/β T cells, those that express the coreceptor molecule CD4 (CD4 ⁺ T cells) and those that express CD8 (CD8 ⁺ T cells). These cells differ in how they recognize antigens and in their effector and regulatory functions.

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 when T cells are activated, and a wide range of cytokines, which are secreted when activated. Dependent.

Similar to CD4+ helper T cells, CD8+ (cytotoxic) T cells are generated within the thymus and express T cell receptors. Cytotoxic T cells, however, do not express CD4 molecules, but CD8, a dimeric co-receptor normally composed of one CD8 alpha chain and one CD8 beta chain. CD8+ T cells recognize peptides presented by MHC class 1 molecules found on all nucleated cells. CD8 heterodimers bind to a conserved portion of MHC class 1 (α3 region) during T cell/antigen presenting cell interactions. CD8+ T cells (often called cytotoxic T lymphocytes, or CTLs) are important in immune defense against intracellular pathogens, including viruses and bacteria, and in tumor surveillance. Once CD8+ T cells recognize their antigen and become activated, they have three major mechanisms by which they kill infected or malignant cells. The first is the secretion of cytokines, mainly TNF-α and IFNγ, which have antitumor and antiviral microbial effects. The second major function is the production and release of cytotoxic granules. These granules, also found in NK cells, contain two protein families, perforins and granzymes. Perforin forms pores in the membrane of target cells, similar to the membrane attack complex of complement. This pore allows granzymes, also contained in cytotoxic granules, to enter infected or malignant cells. Granzymes are serine proteases that cleave intracellular proteins, halting the production of viral proteins and ultimately causing apoptosis of target cells. CD8+ T cells can release granules, kill infected cells, and then migrate to new targets to kill again. This is often called serial killing. A third major function of CD8+ T cell destruction of infected cells is through the Fas/FasL interaction. Activated CD8+ T cells express FasL on the cell surface and bind its receptor, Fas, on the surface of target cells. This binding trimerizes the Fas molecules on the surface of the target cell and draws the signaling molecules together. These signaling molecules lead to activation of the caspase cascade and also trigger apoptosis of target cells. Because CD8+ T cells can express both molecules, the Fas/FasL interaction is the mechanism by which CD8+ T cells, called fratricide, kill each other and eliminate immune effector cells in the final contractile phase of the immune response.

T cells also mediate important effector functions, some of which are determined by the pattern of cytokines they secrete. Cytokines can be directly toxic to target cells and can recruit powerful inflammatory mechanisms.

Furthermore, T cells, particularly CD8 ⁺ T cells, can become CTLs that can effectively lyse target cells expressing antigens recognized by cytotoxic T lymphocytes (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (199) 9)).

T-cell receptors (TCRs) recognize complexes composed of peptides derived from the proteolysis of antigens bound in specialized grooves of class II or class I MHC proteins. CD4 ⁺ T cells only recognize 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, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

TCR ligands (ie, peptide/MHC protein complexes) are made within the APC. In general, class II MHC molecules bind peptides derived from proteins ingested by APCs through endocytic processes. Class II molecules loaded with these peptides are then expressed on the surface of cells and can bind to CD4 ⁺ T cells with TCRs capable of recognizing the expressed cell surface complexes. CD4 ⁺ T cells are therefore specialized to react with antigens derived from extracellular sources (Paul, W.E., “Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

In contrast, Class I MHC molecules are predominantly loaded with peptides derived from internally synthesized proteins such as viral proteins. These peptides are generated from cytosolic proteins by proteolytic proteolysis and translocate to the rough endoplasmic reticulum. Such peptides, generally nine amino acids in length, bind to class I MHC molecules and are transported to the cell surface where they are recognized by CD8 ⁺ T cells expressing the appropriate receptor. can be This allows the T-cell system, particularly CD8 ⁺ T-cells, to produce proteins that are different from, or more likely than, those of cells in the rest of the organism (such as viral antigens) or variant antigens (such as active oncogene products). Cells expressing proteins that are produced in much greater amounts can be detected even if intact forms of those proteins are neither expressed nor secreted on the cell surface (Paul, WE, "Chapter 1: The immune system: an introduction, "Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

T cells can also be classified based on their function as helper T cells: T cells involved in inducing cell-mediated immunity; suppressor T cells; and cytotoxic T cells.

Helper T Cells Helper T cells are T cells that stimulate B cells to mount an antibody response against proteins and other T cell dependent antigens. T-cell dependent antibodies cause distinct epitopes to appear only once or a limited number of times, making them incapable or poorly able to cross-link membrane immunoglobulins (Ig) of B cells. is an immunogen. B cells bind antigen through their membrane Ig and the complex undergoes endocytosis. Within the 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 pass through this vesicular compartment. The resulting peptide/class II MHC complex is subsequently 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, WE, "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).

B cell activation depends on both T cell engagement through the TCR and the synergistic action between T cell CD40 ligand (CD40L) and CD40 on B cells. T cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of interaction with APCs expressing both the cognate antigen recognized by the TCR of T cells and CD80 or CD86. CD80/CD86 are generally expressed by activated but not resting B cells, and helper interactions involving activated B cells and T cells lead to 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 constitutively CD80/86-expressing APCs, such as dendritic cells. Such activated helper T cells then efficiently interact with and help B cells. Cross-linking of membrane Ig on B cells, even if inefficiently, cooperates with the CD40L/CD40 interaction to lead 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, are dependent on or enhanced by the actions of T cell-derived cytokines (Paul, W. et al. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999) ).

CD4 ⁺ T cells are cells that primarily secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (T _H 2 cells), or primarily produce IL-2, IFNγ, and lymphotoxin tend to differentiate into cells (T _H 1 cells). T _H 2 cells are highly effective in helping develop B cells into antibody-producing cells, while T _H 1 cells enhance the bactericidal action of monocytes and macrophages and the consequent intracellular It is an effective inducer of cellular immune responses, including increased efficiency in lysing microorganisms in the vesicular compartment. CD4 ⁺ T cells with a T _H 2 cell phenotype (ie, IL-4, IL-5, IL-6, and IL-10) are efficient helper cells, whereas T _H 1 cells have no helper (Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadel Phia, (1999) ).

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

During patrol, NK cells are in constant 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" NK cells. Inhibitory receptors serve as a check for NK cell killing. Most normal, healthy cells express MHC I receptors that mark these cells as "self." Inhibitory receptors on the surface of NK cells recognize the cognate MHC I, which "switches off" the NK cells to prevent killing. Cancer and infected cells often lose MHC I, making them susceptible to killing by NK cells. Once determined to kill, NK cells release cytotoxic granules containing perforin and granzymes, causing target cell lysis.

Involvement of T cells in inducing cell-mediated immunity T cells can also act to enhance the ability of monocytes and macrophages to destroy intracellular microbes. In particular, interferon gamma (IFNγ) produced by helper T cells is responsible for several of the ways mononuclear phagocytes destroy intracellular bacteria and parasites, including the production of nitric oxide and the induction of tumor necrosis factor (TNF) production. enhance the mechanism of T _H1 cells produce IFNγ and are therefore effective in enhancing bactericidal action. In contrast, IL-4 and IL-10, the two major cytokines produced by T _H2 cells, block their activity (Paul, WE, "Chapter 1: The immune system: an introduction, "Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia, (1999)).

Regulatory T (Treg) Cells Immune homeostasis is maintained by a regulated balance between the initiation and downregulation of immune responses. Apoptosis and T cell anergy, a tolerance mechanism whereby T cells are essentially functionally inactivated after antigen encounter (Scwartz, R. H., “T cell energy”, Annu. Rev. Immunol., Vol. 21 : 305-334 (2003)), both mechanisms contribute to the downregulation of the immune response.The third mechanism is the active suppression of activated T cells by suppressors or regulatory CD4 ⁺ T (Treg) cells. (reviewed in Kronenberg, M. et al., "Regulation of immunity by self-reactive T cells", Nature, Vol. 435: 598-604 (2005)) IL-2 receptor alpha ( CD4 ⁺ Tregs (CD4 ⁺ CD25 ⁺ ), which constitutively express the IL-2Rα) chain, are a subset of naturally occurring anergic and suppressive T cells (Taams, LS et al., "Human anergic/suppressive CD4 ⁺ CD25 ⁺ T cells: a highly differentiated and apoptosis-prone population", Eur. J. Immunol. Vol. ).Depletion of CD4 ⁺ CD25 ⁺ Tregs is associated with Moreover, transplantation of these Tregs prevents the development of autoimmune disease.Human CD4 ⁺ CD25 ⁺ Tregs, like their murine counterparts, are generated in the thymus and are cell-contact dependent. Characterized by an ability to suppress the proliferation of responder T cells via sexual mechanisms, an inability to produce IL-2, and an anergic phenotype in vitro, human CD4 ⁺ CD25 ⁺ T cells respond to the level of CD25 expression. can be divided into suppressive (CD25 ^high ) and non-suppressive (CD25 ^low ) cells.FOXP3, a member of the Forkhead family of transcription factors, is expressed on mouse and human CD4 ⁺ CD25 ⁺ Tregs, It has been shown to be the likely master gene controlling the development of CD4 ⁺ CD25 ⁺ Tregs (Battaglia, M. et al., "Rapamycin promotes expansion of functional CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulator T cells of both health"). subjects and type 1 diabetic patients", J. Immunol. , Vol. 177: 8338-8347, (2006)).

Cytotoxic T Lymphocytes CD8 ⁺ T cells that recognize peptides derived from proteins produced within target cells have cytotoxic properties in that they lead to 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 lysis of the cells. Perforin-mediated lysis is enhanced by granzymes, a series of enzymes produced by activated CTLs. Many active CTL also express a large number of fas ligands on their surface. Interaction of fas ligand on the surface of CTLs with fas on the surface of target cells initiates apoptosis of target cells, resulting in their death. CTL-mediated lysis appears to be the primary mechanism for destruction of virus-infected cells.

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

Compared to canonical T cells, these memory T cells display distinct phenotypes such as the expression of specific surface markers, the rapid production of different cytokine profiles, the capacity for direct effector cell function, and unique homing distribution patterns. It has a long life with mold. Memory T cells exhibit a rapid response upon re-exposure to their corresponding antigens to eliminate reinfection of the aggressor and thereby quickly restore balance to the immune system. Increasing evidence demonstrates that autoimmune memory T cells thwart 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)).

For an effective immune response to an antigen, antigen-presenting cells (APCs) must process and present antigens to T cells in the context of the appropriate major histocompatibility complex (MHC), resulting in cell Toxic T cells and T helper T cells are stimulated. Following antigen presentation, successful interaction of co-stimulatory molecules on both APCs and T cells is required or 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 progenitor cells into dendritic cells (DCs), but it is necessary to activate their maturation into effective antigen presenting cells required for T cell activation. signal is required. Barriers to effective immunotherapy include tolerance to the target antigen, which may limit induction of cytotoxic CD8 T cells of appropriate size and function, inadequate availability of generated T cells to sites of malignant cells. slow trafficking and decreased persistence of the induced T cell responses. DCs that phagocytose tumor cell debris process substances for MHC presentation, upregulate the expression of co-stimulatory molecules, and migrate to regional lymph nodes to stimulate tumor-specific lymphocytes. This pathway leads to proliferation and activation of CD4+ and CD8+ T cells that respond to tumor-associated antigens. Indeed, such cells can frequently be detected in patient blood, lymphoid tissue, and malignant lesions.

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 and B cells are responsible for humoral immunity (associated with 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 allow them to be destroyed. CD4+ (helper) T cells coordinate the immune response. CD8+ (cytotoxic) T cells and natural killer (NK) cells, for example, can kill cells of the body that are infected with viruses or that present antigenic sequences.

Immune Response Generally, an immune response is initiated by an individual's encounter with a foreign source material, such as an infectious microorganism. Infected individuals develop a humoral immune response with the production of antibody molecules specific to the antigenic determinants/epitopes of the immunogen and cells producing antigen-specific regulatory T cells and cytokines and lysing infected cells. It responds rapidly with both a cell-mediated immune response that involves expansion and differentiation of effector T lymphocytes, including both killer T cells that can kill cells. Primary immunization with a particular microorganism elicits antibodies and T cells that are specific for antigenic determinants/epitopes found in the microorganism, but are usually unable or unable to recognize 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). elphia, (1999) ), at p.102).

As a result of this initial response, immunized individuals develop a state of immunological memory. Secondary responses ensue when the same or closely related organisms are encountered again. This secondary response is generally more rapid and larger in magnitude, and is composed of antibodies that bind to the antigen with higher affinity and more effectively clear the organism from the body, as well as antibody responses. enhanced and often consist of a more effective T-cell response. However, an 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).

Immune Tolerance in Cancer Although cancer is characterized by genetic instability of certain cells, the immune system, at least in certain segments of the affected human population, develops an apparently non-self immune system that should be recognized as foreign. It has also been described as a disorder of the immune system based on the fact that it fails to respond optimally to phenotypically cancerous cells. Several reasons are offered to explain the basis for this observation. For example, first, cancer cells are composed primarily of self-antigens, in sharp contrast to the situation of infectious organisms. Some antigens classified as cancer antigens are actually normal antigens that are overexpressed or that have mutations in only one or two amino acids of the polypeptide chain. Second, cancer cells down-regulate MHC, so they present less tumor cell-derived peptides through MHC. Third, cancer cells and associated tumor-associated macrophages express cytokines that dampen the immune response (see, eg, Yu et al (2007) Nature Rev. Immunol. 7:41-51). This attenuation is caused, for example, by secretion of interleukin-10 (IL-10) by cancer cells or associated macrophages. Fourth, unlike in infectious disease situations, cancer cells do not provide an immune adjuvant. 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)). see). In general, optimal activation of dendritic cells requires contact of an 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 activation of the T cells.

Insight into the underlying mechanisms of immune evasion may be useful along with combination therapy regimens that enhance the efficacy of therapeutic vaccination, either directly or indirectly through combination with immune checkpoint inhibitors or other therapeutics, as well as effective antimicrobials. It has served as a basis for the development of vaccines that induce tumor immunity.

Immunosurveillance and immunoediting Tumor immunoediting can be divided into three stages: elimination, equilibrium, and evasion. The elimination step, also known as immune surveillance, is the process by which the immune system identifies cancerous or precancerous cells and eliminates them before they get out of control. This step can be completed when all cancerous or precancerous cells are eliminated. If some tumor cells are not eliminated, a temporary equilibrium can be reached between the immune system and tumor cell proliferation. In this equilibrium phase, tumor cells can either remain dormant or continue to evolve by accumulating further changes to genomic DNA that can regulate the antigens they present. During this process, the immune system exerts selective pressure on evolving cells, whereby less recognized tumor cells have a survival advantage. Eventually, the immune response fails to recognize the cells of the tumor, resulting in an escape phase in which tumor cells progressively proliferate uncontrollably.

Tumor microenvironment The tumor microenvironment provides a consistently effective barrier to immune cell function, as tumors actively downregulate all stages of the antitumor immune response using a variety of strategies and mechanisms. . A number of molecular mechanisms have been identified that cause immune cell dysfunction in the tumor microenvironment. This includes mechanisms directly mediated by factors produced by tumors and other mechanisms resulting from alterations in normal tissue homeostasis in the presence of cancer. Most human tumors appear to be capable of interfering with one or more stages of immune cell development, differentiation, migration, cytotoxicity, and other effector functions (TL Whiteside, The tumor microenvironment and its role in promoting tumor growth, Oncogene (2008) 27, 5904-5912).

One such mechanism involves tumor accumulation of T _reg (CD4 ⁺ CD25 ^bright Foxp3 ⁺ T cells) and myeloid-derived cells (CD34 ⁺ CD33 ⁺ CD13 ⁺ CD11b ⁺ CD15 ⁻ ), which are associated with human tumors. and is associated with poor prognosis in cancer patients (T L Whiteside, The tumor microenvironment and its role in promoting tumor growth, Oncogene (2008) 27, 5904-5912). Under normal conditions, T _reg cells play an important role in preventing autoimmunity, but in cancer, T reg cells expand, migrate into tumors, downregulate the proliferation of autologous effector T cells, and play different molecular roles. pathway is used to suppress the anti-tumor responses of both CD4 ⁺ CD25 ^- cells and CD8 ⁺ CD25 ^- T cells. T _reg cells within tumors are a heterogeneous population of regulatory CD3 ⁺ CD4 ⁺ T cells, comprising a subset of natural T _reg , antigen-specific Tr1 cells, and other less well-defined suppressor cells. include. Tr1 cells are induced in a tumor microenvironment rich in IL-10, TGF-β, and prostaglandin E ₂ (PGE ₂ ), 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).

Myeloid suppressor cells (MSCs) secrete TGF-β or also suppress T cell responses in the tumor microenvironment that induce TGF-β secretion. Immunosuppressive CD34 ⁺ cell-derived myeloid cells have been identified in the peripheral blood of cancer patients. In tumor-bearing mice, MSCs accumulate in very large amounts in the spleen and peripheral circulation, exert potent immunosuppression, and promote tumor growth. MSCs also regulate the availability of essential amino acids such as L-arginine and generate high levels of reactive oxygen species. MSCs found in tumors are also iNOS and arginase, an enzyme involved in the metabolism of L-arginine, which has been shown to synergize with iNOS to increase superoxide and NO production and interfere with lymphocyte responses. 1 and are constitutively expressed. GM-CSF, often secreted by tumor cells, recruits MSCs and induces dose-dependent in vivo immunosuppression and tumor promotion, while GM-CSF has been used as an immune adjuvant for anti-tumor vaccines. GM-CSF was observed to increase a subset of TGF-β-producing MSCs in the circulation of patients with metastatic melanoma. Simultaneous stimulatory and inhibitory roles suggest that GM-CSF and MSCs are involved in maintaining immune homeostasis in normal tissues, whereas in the tumor microenvironment they promote tumor cell escape (T L Whiteside, The tumor microenvironment and its role in promoting tumor growth, Oncogene (2008) 27, 5904-5912).

Tumor Immunotherapy Cancer treatments are evolving rapidly as new molecular targets are discovered. Despite the emergence of biologics targeting specific pathways (e.g. Herceptin®, Erbitux®) and small molecules designed against specific targets (Tamoxifen, Gleevec™) , nonspecific modalities such as chemotherapy and radiotherapy remain the standard of care.

Anti-cancer immunotherapy has been a long-standing goal and various approaches have been tested. One difficulty in developing this immunotherapy is that the target antigen is often a tissue-specific molecule found on both cancer and normal cells and either does not induce immunity or exhibits non-specificity with respect to cell killing. (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). In addition, tumor cells possess features that make immune recognition difficult, such as loss of expression of antigens that elicit immune responses, lack of MHC class II, and downregulation of MHC class I expression. These characteristics may lead to 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)).

DCs generated ex vivo by culturing hematopoietic progenitor cells or monocytes with a combination of cytokines have been tested as therapeutic vaccines in cancer patients for over a decade (Ueno H, et al., Immunol. Rev. (2010) 234: 199-212). For example, sipuleucel-T (also known as APC 8015), a concentrated blood APC-based cell product that has been briefly cultured with a fusion protein of prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GM-CSF). ) resulted in an increase in median survival of about 4 months in a phase III trial (Higano C S, et al., Cancer (2009) 115: 3670-3679; Kantoff P W, et al., N. Engl. J. Med. (2010) 363: 411-422). This study concluded that DC-based vaccines are safe and can induce 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 the next generation of cellular immunotherapy products. (Palucka K and Banchereau J, Nature Reviews Cancer (April 2012) 12: 265-276).

A vaccination strategy involving DCs was developed to induce tumor-specific effector T cells that can specifically reduce tumor burden and induce immune memory to control tumor recurrence. For example, DCs can be provided with tumor-specific antigens by culturing them ex vivo with adjuvants and tumor-specific antigens and then injecting these cells back into the patient. Tumor cells obtained from resected tumors, needle biopsies, core biopsies, vacuum-assisted biopsies, or peritoneal lavages are used to generate immunogenic compositions comprising tumor-specific antigen-presenting dendritic cells. It's here.

Cancer Treatment Strategies Antibody therapies such as Herceptin(TM) and Erbitux(TM) are passive immunotherapies but have significant clinical outcomes as measured, for example, by recurrence rate, progression-free survival, and overall survival. brought improvement. More recently, PD-1 inhibitors and CTLA4 inhibitors have been reported to block distinct checkpoints of active host immune responses and sustain endogenous anti-cancer immune responses. The term "immune checkpoint" refers to a series of inhibitory pathways that are required to maintain self-tolerance and regulate the duration and extent 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 take advantage of 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, checkpoint inhibitors that bind to molecules of immune cells to activate or deactivate them reduce inhibition of the immune response. assumed to be possible. Recent discoveries have identified immune checkpoints or targets such as PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, CCR4, OX40, OX40L, IDO, and A2AR as proteins involved in immune evasion. ing. Specific immune checkpoint inhibitors, including antibodies directed against CTLA-4, the PD-1 receptor or its ligand PD-L1, have produced impressive results in a variety of cancers in the clinic and have been demonstrated in multiple tumor indications. and many more registration trials underway, Yervoy™ (ipilimumab; CTLA-4 antagonist), Opdivo™ (nivolumab; PD-1 antagonist) and Keytruda™ (pembrolizumab; PD-1 antagonist) ) resulted in FDA approval. However, this therapy can only be successful if a pre-existing anti-tumor immune response is present in the patient (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), attempt to 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 transgenic expression of chimeric antigen receptors (CARs). CARs are engineered molecules that can be introduced into T cells to target tumor antigens (Frey, N.V., Porter, D.L., The Promise of Chimeric Antigen Receptor T-Cell Therapy, Oncology (2016); 30(1)) pii 219281). CAR T cells have been shown to have some efficacy against hematologic 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).

Therapeutic vaccination against cancer is an important modality that complements the current standard of care and has the potential to provide long-term control of cancer. An example of a prototime, GVAX™, is a GM-CSF gene-transduced tumor vaccine in either autologous or allogeneic populations of tumor cells. GM-CSF secretion of genetically modified tumor cells is thought to stimulate cytokine release at the vaccine site to 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)). A lethally irradiated tumor cell vaccine designed to secrete GM-C SF (GVAX) is effective against melanoma, renal cell, prostate, non-small cell lung, pancreatic, head and neck squamous cell carcinomas. Although it has shown promising efficacy in various models, 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 immunization strategies to treat diseases such as cancer that can be refractory to traditional therapies.

Dendritic cell (DC)-tumor cell fusions produce hybrid cells that express relevant tumor-associated antigens derived from parental tumor cells and also have the ability to process and present such antigens to appropriate cells of the immune system. Developed to produce. Although DC-tumor cell fusion provides a greater variety of tumor antigens, human Clinical trials have met with limited success (Browning, M., Antigen presenting cell/tumor cell fusion vaccines for cancer, Human Vaccines & Immunotherapeutics 9:7, 1545-1548; July 2013; Terfield, L., Dendritic Cells in Cancer Immunotherapy Clinical Trials: Are We Making Progress?, Frontiers of Immunology, 2013 4: 454).

Immunogenicity of Vaccines Vaccines against infectious agents are representative examples of specific receptor-ligand interactions that are used to shape immune responses with the therapeutic goal of preventing or reducing infection (e.g. , influenza vaccine). Antigens are generally presented to the immune system in combination with an adjuvant, such as a synthetic small molecule immunomodulator.

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

Despite the aforementioned difficulties, there is evidence that 1) intrinsic anti-tumor 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. has now become clear.

According to some aspects of the described invention, either on a tumor cell line or tumor cell line variant derived from a cancer patient, or on an allogeneic tumor cell line or tumor cell line variant. The array of immunomodulators that can be expressed when used as tumor vaccines 1) efficiently loads endogenous antigen-presenting cells with a wide range of tumor antigens, and 2) enhances normal signals received during immune responses. 3) interfere with the mechanisms by which T regulatory cells suppress the immune response, 4) interfere with the signals by which the immune response is generally extinguished, and 5) such was identified as able to act to enhance overall survival in cancer patients vaccinated with a formulation of In certain embodiments, the modified tumor cell line or tumor cell line variant may be derived from the patient receiving the vaccine, whereas the allogeneic tumor cell line or tumor cell line variant vaccine approach is a personalized therapeutic approach. different from This is because the modified tumor cells may not come from the individual who will ultimately be vaccinated. Instead, allogeneic tumor cell vaccines aim to focus the immune response on many elements that individual tumors of the same tumor type have in common.

One strategy for exploiting the large number of potential tumor antigens for each individual type of cancer is to vaccinate whole tumor cells to avoid falsely excluding potentially relevant antigens. is. The invention described herein provides, inter alia, vaccines comprising whole tumor cells harboring a panel of tumor antigens and engineered to express three or more immunomodulatory factors.

According to some aspects, the described invention is an allogeneic tumor cell vaccine comprising: (1) a live, growth-incompetent genetically engineered tumor expressing one or more tumor-specific antigens; A population of cells, said population comprising: at least 3 stably expressed immunomodulatory molecules, wherein said at least 3 immunomodulatory molecules sensitize one or more subpopulations of PBMCs in response to said expressed immunomodulatory molecules OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand, including CD80, CD86, or both (CD28L), to induce tumor cells to proliferate and enter the effector phase to kill tumor cells. ), wherein the subpopulation of PBMC cells comprises one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC) or B lymphocytes;
(2) a pharmaceutically acceptable carrier;
An allogeneic tumor cell vaccine is provided comprising:

According to some embodiments, the population of live, non-proliferative, genetically engineered tumor cells expressing one or more tumor-specific antigens comprises one or more additional tumor cells selected from R1-R44. Further included are stably expressed immunomodulatory molecules. 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 subpopulations of T lymphocytes, expansion of T lymphocytes, or both. According to some embodiments, the induction of NK cells comprises activation of the NK cell subpopulation, expansion of the NK cell subpopulation, or both. According to some embodiments, the induction of the DC subpopulation comprises activation of the DC subpopulation, expansion of the DC subpopulation, or both. According to some embodiments, the induction of the B lymphocyte subpopulation comprises activation of the B lymphocyte subpopulation, expansion of the B lymphocyte subpopulation, 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 (CTL). 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.

According to some embodiments, the vaccine enhances cellular immune activation effective to (1) recognize and act against tumor cells containing the target tumor antigen in vivo without systemic inflammation; (2) reduce immunosuppression in the tumor microenvironment of tumor cells containing the target tumor antigen; or (3) increase cell death of tumor cells expressing the target tumor antigen.

According to some embodiments, the tumor cell is melanoma, colorectal cancer, leukemia, chronic myelogenous leukemia, prostate cancer, head and neck cancer, squamous cell carcinoma, tongue cancer, laryngeal cancer, tonsil cancer, hypopharyngeal cancer It is derived from a cancer selected from the group consisting of carcinoma, nasopharyngeal carcinoma, breast cancer, colon cancer, lung cancer, pancreatic cancer, glioblastoma, and brain cancer. According to some embodiments, the melanoma tumor cells are 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 carcinoembryonic antigen (CEA), MAGE, HPV, human telomerase reverse transcriptase (hTERT), EPCAM, PD-1, PD-L1, p53, and Characterized by the expression of one or more of the cell surface associated mucin 1 (MUC1).

According to some embodiments, the population of viable, growth-resistant tumor cells is derived from a subject-derived biological sample. 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 live, growth-resistant tumor cells is effective to induce immune activation without systemic inflammation. According to some embodiments, the vaccine elicits 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 a chemokine receptor. According to some embodiments, the immunostimulatory molecule is displayed on the external surface of the genetically engineered tumor cells.

According to another aspect, the described invention provides a method of inducing an immune response against cancer in a subject, comprising administering the allogeneic tumor cell vaccine of claim 1 to the subject parenterally or intratumorally. wherein the allogeneic tumor cell vaccine is matched to 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 with progression to cancer. According to some embodiments, the method further comprises administering a checkpoint inhibitor to the subject.

According to another aspect, the described invention provides a method of treating cancer in a subject, comprising: (1) live proliferation-incompetent genes expressing one or more tumor-specific antigens; A population of tumor cells engineered such that the population: one or more subpopulations of PBMCs proliferate in response to the expressed immunomodulatory molecules and then enter the effector phase to kill the tumor cells. at least 3 stably expressed immunomodulatory molecules, wherein the at least 3 immunomodulatory molecules include OX40 ligand (OX40L), CD27 ligand (CD70), and CD80, CD86, or both CD28 ligand (CD28L), the subpopulation of PBMC cells comprising one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC) or B lymphocytes;
(2) a pharmaceutically acceptable carrier;
administering an allogeneic tumor cell vaccine comprising in an amount that reduces tumor burden in a subject. According to some embodiments, the effective amount improves clinical outcome. According to some embodiments, the effective amount improves the subject's progression-free survival, overall survival, or both as compared to a placebo control. According to some embodiments, the cancer is melanoma or colorectal cancer.

According to another aspect, (1) a population of live, non-proliferative, genetically engineered tumor cells expressing one or more tumor-specific antigens, said population comprising: one or more of PBMCs comprising at least three stably expressed immunomodulatory molecules for inducing the subpopulation to proliferate in response to the expressed immunomodulatory molecules and then enter the effector phase to kill tumor cells; The three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), which includes CD80, CD86, or both, and subpopulations of PBMC cells are T lymphocytes, natural killer (NK) cells, dendritic cells (DC) or B lymphocytes; and (2) a pharmaceutically acceptable carrier. providing an allogeneic parental tumor cell line comprising a population of tumor cells that have been modified; and introducing an exogenous nucleic acid encoding a stably expressed immunomodulatory molecule into the population of living tumor cells, introducing the immunomodulatory molecule is OX40 ligand (OX40L); and introducing an exogenous nucleic acid encoding the immunomodulatory molecule stably expressed in a population of living tumor cells, is a CD27 ligand (CD70); and introducing an exogenous nucleic acid encoding an immunomodulatory molecule stably expressed in a population of living tumor cells, wherein the immunomodulatory molecule is CD80, CD28 ligand (CD28L), including CD86, or both, and stable expression of OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, is associated with PBMC inducing or introducing one or more subpopulations to proliferate in response to the expressed immunomodulatory molecules and then enter the effector phase to kill the tumor cells; generating tumor cell line variants by selecting tumor cell clones that stably express immunogenic amounts; and, in mixed lymphocytic tumor cell reactions, the following parameters: cell proliferation, cell subset differentiation, Selecting a clonal cell line variant by one or more selected from cytokine release profile and tumor cell lysis, wherein the selected clonal cell line variant is T lymphocytes, natural killer (NK ) cells, dendritic cells (DCs), or B-lymphocytes.

According to some embodiments, the process for producing an allogeneic tumor cell vaccine includes live exogenous nucleic acids encoding one or more stably expressed immunomodulatory molecules selected from R1-R44. further comprising introducing into the population of tumor cells. According to some embodiments, the tumor cells are rendered proliferation incompetent by irradiation. According to some embodiments, the parental tumor cell line is derived from a tumor selected from the group consisting of melanoma and colorectal cancer. According to some embodiments, 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 utilizes one or more of liposome-mediated transfer, adenovirus, adeno-associated virus, herpes virus, retrovirus-based vectors, lipofection, and lentiviral vectors. including. According to some embodiments, the introducing step comprises introducing the exogenous nucleic acid by transfection of a lentiviral vector.

These and other advantages of the present invention will become apparent to those skilled in the art upon reference to the following detailed description.

FIG. 11 shows one embodiment of heteroclitic cross-reactivity between peptides native to tumor cell lines and peptides native to tumor cells of patients undergoing immunotherapy. Schematic representation of scFv-anti-biotin-G3 hinge-mIgG1 vector 1 construction. Schematic representation of the construction of full-length anti-biotin-G3 hinge-mIgG1 vector 2 is shown. Schematic representation of the construction of sGM-CSF/ires/mFLT3L vector 3 is shown. Schematic representation of the construction of sFLT3L/ires/(FLT3 signal-GM-CSF-Tm) vector 4 is shown. FIG. 2 shows a schematic diagram of the construction of mCD40L vector 5. FIG. Schematic representation of the organization of mTNFa vector 6 is shown. Schematic representation of the construction of mRANKL/ires/FLT3 signal-V5-scFV anti-biotin-Tm vector 7 is shown. A schematic diagram of vector 44 is shown. A schematic representation of vector 97 is shown. A schematic diagram of vector 84 is shown. A schematic diagram of vector 29 is shown. A schematic diagram of vector 107 is shown. A schematic diagram of vector 116 is shown. A schematic diagram of vector 86 is shown. A schematic representation of vector 18 is shown. 17 shows a schematic representation of vector 17. FIG. A schematic diagram of the vector 98 is shown. A schematic diagram of a vector 30 is shown. A schematic diagram of vector 109 is shown. A schematic diagram of the vector 106 is shown. A schematic diagram of vector 16 is shown. A schematic diagram of vector 83 is shown. A schematic diagram of the vector 31 is shown. A schematic diagram of vector 12 is shown. 1 shows a schematic diagram of Vector 99. FIG. A schematic diagram of vector 121 is shown. A schematic diagram of the vector 105 is shown. A schematic diagram of the vector 32 is shown. A schematic diagram of vector 37 is shown. A schematic diagram of the vector 22 is shown. A schematic representation of vector 19 is shown. A schematic diagram of the vector 20 is shown. A schematic diagram of vector 89 is shown. A schematic diagram of vector 21 is shown. A schematic diagram of the vector 23 is shown. A schematic diagram of the vector 108 is shown. A schematic diagram of vector 15 is shown. A schematic diagram of vector 124 is shown. A schematic diagram of vector 65 is shown. A schematic diagram of a vector 64 is shown. A schematic diagram of vector 88 is shown. A schematic diagram of the vector 96 is shown. A schematic diagram of vector 14 is shown. A schematic diagram of vector 119 is shown. A schematic diagram of vector 120 is shown. A schematic diagram of vector 45 is shown. A schematic diagram of a vector 60 is shown. A schematic diagram of vector 59 is shown. 1 shows a schematic diagram of vector 8. FIG. A schematic diagram of vector 128 is shown. A schematic diagram of the vector 35 is shown. 1 is a schematic diagram showing a general experimental format; FIG. 2 is a panel of graphs showing the results of flow cytometry experiments. Forward scatter (FSC) and side scatter (SSC) plots of size versus granularity. SK strains are represented by number code; SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 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; 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, presumably due to the presence of TNF-a and CD40L receptors on cells of epithelial origin. 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 immunomodulatory agents. The SK cell lines are designated by the following codes; SK, unmodified parent; 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. Panel of graphs showing representative flow cytometry staining of the indicated engineered surface markers: GM-CSF, FLT3L, TNF-α, and CD40L. SK strains are represented by number code; SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 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; 6 is a combination of 3, 4, and 6. Figure 3 shows the results of CyTOF mass cytometry single-cell phenotypic analysis of hPBMC responses to SK melanoma cells with alterations due to expression of immunomodulators. A shows viSNE density contour plots of CyTOF staining data showing relative changes in abundance and phenotype of immune cell subsets. B shows single cell phenotype analysis. SK strains are represented by number code; SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 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; 6 is a combination of 3, 4, and 6. CyTOF monocyte cluster analysis of hPBMC showing changes in activation marker CD40 expression after 1 day of stimulation with genetically modified SK lines indicated at a 1:5 cell ratio. SK strains are represented by number code; SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 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; 6 is a combination of 3, 4, and 6. CyTOF monocyte cluster analysis of hPBMC showing changes in activation marker CD86 expression after 1 day of stimulation with genetically modified SK lines indicated at a 1:5 cell ratio. SK strains are represented by number code; SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 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; 6 is a combination of 3, 4, and 6. CyTOF monocyte cluster analysis of hPBMC showing changes in activation marker CD69 expression after 1 day of stimulation with genetically modified SK lines indicated at a 1:5 cell ratio. SK strains are represented by number code; SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 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; 6 is a combination of 3, 4, and 6. CyTOF monocyte cluster analysis of hPBMC showing changes in activation marker CD25 expression after 1 day of stimulation with genetically modified SK lines indicated at a 1:5 cell ratio. SK strains are represented by number code; SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 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; 6 is a combination of 3, 4, and 6. CyTOF monocyte cluster analysis of hPBMC showing relative median (MEI) expression levels of monocyte markers CD40 and CD86. CyTOF monocyte cluster analysis of hPBMC showing relative median expression index (MEI) of CD4 T cell markers CD69 and CD25. FIG. 10 is a graph showing the results of Luminex multiplex cytokine profiling of human PBMC responses to SK parental and genetically modified SK strains. Control cultures included SK cells only, hPBMCs only, and hPBMCs stimulated with a mixture of anti-CD3 and anti-CD28 antibodies (1 μg/ml final concentration). Symbols indicate cytokine levels in pg/ml extrapolated from standard curves using recombinant cytokines. Absence of symbols indicates that no cytokine was detected. SK strains are represented by number code; SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 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; 6 is a combination of 3, 4, and 6. Figure 2 shows the results of flow cytometry experiments demonstrating that CD8+ T cells can be activated by genetically modified SK lines expressing immunomodulatory molecules. Flow control after incubation of parental cell line SKMEL2 ((i) and genetically modified 14-18-30 expressing SK-MEL-2 tumor cells (Fig. (ii)) with PBMC in a mixed lymphocyte tumor response assay. Forward scatter (FSC) and side scatter (SSC) plots of cytometry size and granularity, dotted ellipses in (i) and (ii) indicate lymphocyte gates, (iii) and (iv) mixed CD8 population following incubation of PBMC with parental cell line (iii) and engineered 14-18-30 expressing SKMEL2 tumor cells (iv) in a lymphocyte tumor response assay, dotted line in lower panel of graph. circles indicate CD8 gates. In vitro CD8+ T from hPBMCs comparing day 9 cultures of the parental SKMEL2 cell line (A) and a genetically modified 14-18-30 cell line expressing combinations of immunomodulators shown in Table 2 (B). Cell expansion results in tumor cell killing. Genetically modified SK strains (i) APX/15; (ii) APX/19; (iii) APX/22; (iv) APX/23; (v) Dendritic cells (DC) in APX/29, natural killers (NK) cells, and flow cytometry results demonstrating stimulation of a subpopulation of B cells. Genetically modified SK strains expressing immunomodulatory molecules ((i) parental; (ii) APX/3; (iii) APX/3-4; (iv) APX/3-4-5; (v) APX/ CyTOF data demonstrating differentiation of different subsets of PBMCs after stimulation with 3-4-6)). SK strains are represented by number code; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; , the uncleaved form of TNF-α. Genetically modified SK strains expressing immunomodulatory molecules ((i) parental; (ii) APX/3; (iii) APX/3-4; (iv) APX/3-4-5; (v) APX/ Detailed CyTOF data showing DC activation after stimulation with 3-4-6)) are shown. SK strains are represented by number code; 3, secreted GM-CSF and membrane-expressed FLT-3L; 4, secreted FLT3L and membrane-expressed GM-CSF; 5, uncleaved form of CD40L; , the uncleaved form of TNF-α. 6 days in the CD8 expansion assay using the SK parental line (left panel) versus the genetically modified 14-18-30 expressing SK-MEL-2 tumor cell line expressing the combinations of immunomodulators shown in Table 2 (right panel). Flow cytometry results comparing day 8 and day 8 time points are shown. FIG. 4 is a plot showing the mean and standard deviation results of xenograft treatment studies using NGS mice. The ends of each box are the upper and lower quartiles. The median is marked by a vertical line inside the box, and the whiskers are the two lines outside the box, extending to the highest and lowest observations. Human tumor cells were implanted into the flanks of NGS (NOD scid gamma) mice. Tumors were grown to 150 mm ³ . Mice were divided into two groups, a control group and a treatment group, and 6 mice were used per group. On day 30 (t=0), mice in the control group were inoculated with vehicle alone and mice in the treatment group were activated with 14-18-30-expressing ENLIST™ cells (“SUPLEXA™ cells”). 3×10 ⁶ PBMC were inoculated. Tumor size was measured at intervals up to 36 days post-inoculation. Differences between the two groups appeared within 5 days. After 22 days, the difference became statistically significant (*P<0.05;**P<005).

DEFINITIONS The term "activation" or "lymphocyte activation" refers to the stimulation of lymphocytes by specific antigens, non-specific mitogens, or allogeneic cells leading to the synthesis of RNA, proteins, and DNA and the production of lymphokines. , which is followed by proliferation and differentiation of various effector and memory cells. For example, mature B cells can be activated by encountering an antigen that exhibits an epitope recognized by its cell surface immunoglobulin Ig. The activation process can be direct, depending on the cross-linking of membrane Ig molecules by antigen (crosslink-dependent B-cell activation), or indirect, which occurs most efficiently in conjunction with close interactions with helper T cells. ("kind help process"). T cell activation depends on the interaction of the TCR/CD3 complex with its cognate ligand, a peptide that binds to the groove of class I or class II MHC molecules. The molecular events driven by receptor binding are complex. Activation of tyrosine kinases, leading to tyrosine phosphorylation of a series of substrates that regulate several signaling pathways, appears to occur during the earliest steps. These include a series of adapter proteins that link the TCR to the ras pathway, whose tyrosine phosphorylation enhances its catalytic activity, participates in the inositol phospholipid metabolic pathway, increases intracellular free calcium levels and activates protein kinase C. phospholipase Cγ1, which regulates cell growth and differentiation, as well as a series of other enzymes that control cell proliferation and differentiation. Full responsiveness of T cells requires, in addition to receptor binding, co-stimulatory activity delivered by accessory cells, such as binding of CD28 on T cells by CD80 and/or CD86 on antigen presenting cells (APCs). and The soluble products of activated B lymphocytes are immunoglobulins (antibodies). Soluble products of activated T lymphocytes are lymphokines.

B-Cell Activation Mature B-cells can be activated by encounter with antigens that display epitopes recognized by their cell surface immunoglobulin Ig. The activation process can be direct, depending on the cross-linking of membrane Ig molecules by antigen (crosslink-dependent B-cell activation), or indirect, which occurs most efficiently in conjunction with close interaction with helper T cells. ("kind help process"). The soluble products of activated B lymphocytes are immunoglobulins (antibodies).

T cell activation depends on the interaction of the TCR/CD3 complex with its cognate ligand, a peptide that binds to the groove of class I or class II MHC molecules. The molecular events driven by receptor binding are complex. Activation of tyrosine kinases, leading to tyrosine phosphorylation of a series of substrates that regulate several signaling pathways, appears to occur during the earliest steps. These include a series of adapter proteins that link the TCR to the ras pathway, whose tyrosine phosphorylation enhances its catalytic activity, participates in the inositol phospholipid metabolic pathway, increases intracellular free calcium levels and activates protein kinase C. phospholipase Cγ1, which regulates cell growth and differentiation, as well as a series of other enzymes that control cell proliferation and differentiation. Full responsiveness of T cells requires, in addition to receptor binding, co-stimulatory activity delivered by accessory cells, such as binding of CD28 on T cells by CD80 and/or CD86 on antigen presenting cells (APCs). and Soluble products of activated T lymphocytes are lymphokines.

Dendritic Cell Activation Pathogen invasion induces a rapid inflammatory response initiated through 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 first wave of GET pro-inflammatory cytokines and chemokines limit pathogen spread, recruit and activate immune cells, and eradicate invaders. Dendritic cells (DCs) play a role in initiating the next phase of immunity governed by pathogen-specific T- and B-cell action. With regard to early proinflammatory responses, DC activation is induced by PRR signals, which convert resting DCs into potent antigen-presenting cells that can promote expansion and effector differentiation of naive pathogen-specific T cells. Convert. 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.

As used herein, the term "CD8+ T cell activation" or "CD8+ T cell activation" refers to CD8+ T cell (CTL) proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, Refers to a process (eg, signaling event) that causes or results in one or more cellular responses selected from toxic activity and expression of activation markers. As used herein, an "activated CD8+ T cell" refers to a CD8+ T cell that has received an activating signal, thus proliferating, differentiating, 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 described herein.

As used herein, the term "expanding CD8+ T cells" or "CD8+ T cell expansion" refers to the process by which a population of CD8+ T cells undergoes a series of cell divisions, thereby increasing cell number. 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.

As used herein, the term "activate NK cells" or "NK cell activation" causes NK cells capable of killing cells deficient in MHC class I expression, or Refers to a process that results (eg, a signaling event). As used herein, "activated NK cells" refer to NK cells that have received an activating signal and are therefore capable of killing cells deficient in MHC class I expression. Suitable assays for measuring NK cell activation are known in the art and described herein.

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 cell number. The term "expanded NK cells" relates to NK cells obtained by NK cell expansion. Suitable assays for measuring NK cell expansion are known in the art and described herein.

As used herein, the term "administration" and its various grammatical forms as applied to a mammal, cell, tissue, organ, or biological fluid include exogenous ligands, reagents, placebos, Refers to, but is not limited to, contacting a molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition with a subject, cell, tissue, organ, biological fluid, or the like. "Administration" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also includes in vitro and ex vivo treatment of cells, eg, with reagents, diagnostics, binding compositions, or another cell.

As used herein, the term "allogeneic" means that the donor and recipient (host) are of different genetic makeup but of the same species. As used herein, an "allogeneic cell" refers to a cell that is not derived from the recipient, meaning the individual to whom the cell is administered, ie, has a different genetic make-up than 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 human patients, such as cancer patients, as disclosed herein. As used herein, "allogeneic tumor cells" refer to tumor cells 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 capable of stimulating an immune response against the tumor in the individual to whom the cells are administered. As used herein, "allogeneic cancer cells," eg, lung cancer cells, refer to cancer cells not derived from the recipient individual to whom the allogeneic cells are administered.

The term "amino acid residue" or "amino acid" or "residue" includes, but is limited to, naturally occurring amino acids and known analogues of naturally occurring amino acids that can function similarly to naturally occurring amino acids. used interchangeably to refer to amino acids that are incorporated into proteins, polypeptides, or peptides that are not Amino acids may be L-amino acids or D-amino acids. Amino acids can be replaced by synthetic amino acids that are altered to increase the half-life of the peptide, increase the potency of the peptide, or increase the bioavailability of the peptide. The one-letter designation of amino acids is primarily used herein. Such one-letter designations are as follows: A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; is arginine; S is serine; T is threonine; V is valine; W is tryptophan; The following represent groups of amino acids that are conservative substitutions for each other: 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).

The term "apoptosis" or "programmed cell death" refers to the non-damaging effects of blebbing, changes in cell membranes, e.g. loss of membrane asymmetry and cohesion, cell shrinkage, nuclear fragmentation, chromatin It refers to a highly regulated active process that contributes to biological homeostasis, which consists of a series of biochemical events leading to various morphological changes, including condensation of chromosomal DNA and fragmentation of chromosomal DNA.

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 caspases. It is 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.

Caspases involved in apoptosis transmit apoptotic signals in the proteolytic cascade, 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 receptors with a death domain (DD) like Fas.

The TNF receptor family of receptors has been implicated in the induction of apoptosis and inflammatory signaling. The Fas receptor (CD95) mediates apoptotic signaling by Fas ligand expressed on the surface of other cells. The Fas-FasL interaction plays an important role in the immune system, and lack of this system leads to autoimmunity, indicating that Fas-mediated apoptosis eliminates autoreactive lymphocytes. Fas signaling is also involved in immune surveillance to eliminate transformed and virus-infected cells. Binding of Fas to oligomerized FasL on another cell activates apoptotic signaling through a cytoplasmic domain called the death domain (DD) that interacts with signaling adapters, including FAF, FADD, and DAX. , activates the caspase proteolytic cascade. Caspase-8 and caspase-10 are activated first, then cleave and activate downstream caspases and various cellular substrates leading to cell death.

Mitochondria are involved in apoptotic signaling pathways 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 Apaf-1, a mitochondrial released protease. Activated Apaf-1 activates caspase-9 and the rest of the caspase pathway. Smac/DIABLO is released from mitochondria and inhibits IAP proteins that normally interact with caspase-9 to inhibit apoptosis. Apoptosis regulation by Bcl-2 family proteins occurs when family members form complexes that enter the mitochondrial membrane and regulate the release of cytochrome c and other proteins. The TNF family receptors that cause 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. Cytochrome C is associated with caspase-dependent apoptotic signaling, whereas 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, presumably through recruitment of nucleases.

The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria and interacts with Apaf-1, causing self-cleavage 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.

Granzyme B and perforin proteins released by cytotoxic T cells induce apoptosis in target cells, forming transmembrane pores and possibly triggering apoptosis through the cleavage of caspases, although granzyme B-mediated apoptotic caspases Independent mechanisms have been suggested.

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

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

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

As used herein, the term "cancer" refers to a disease in which abnormal cells can divide uncontrolled and invade other tissues. There are over 100 types of cancer. Most cancers are named after the organ or cell type in which they arise. For example, cancer that originates in the colon is called colon cancer; cancer that arises from melanocytes in the skin is called melanoma. Cancer types can be grouped into broader categories. The main categories of cancer are: carcinomas (which arise in the skin or tissues that line or cover internal organs, including adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma) cancer, and subtypes thereof); sarcoma (meaning cancer that arises in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues); ), which refers to cancers that produce large numbers of abnormal blood cells and invade the blood); lymphomas and myeloma (which refer to cancers that arise in cells of the immune system); Cancer (meaning cancers that form 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 make 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 myelogenous leukemia (AML).

As used herein, the term "cell line" means a permanently established cell culture that has developed from a single cell and thus has a homogeneous genetic makeup that grows indefinitely. consists of cells with

As used herein, the term "chemotherapy" refers to treatment that uses drugs to stop cancer cells from growing.

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

As used herein, the term "co-stimulatory molecule" refers to one of two or more molecules that are presented on the cell surface and have a role in activating T cells into effector cells. . For example, MHC proteins that present foreign antigens to T cell receptors also require co-stimulatory proteins that bind to complementary receptors on the T cell surface to effect T cell activation.

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 allow them to initiate distinct signaling cascades within the cell that ultimately lead to biochemical and result in phenotypic changes. Cytokines can act both locally and remotely from the site of release. Type I cytokines, including many interleukins, and several hematopoietic growth factors; Type II cytokines, including interferon and interleukin-10; Tumor necrosis factor (“TNF”)-related molecules, including TNFα and lymphotoxin; It includes members of the immunoglobulin superfamily, 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 the state of the cell. Cytokines often modulate the expression of other cytokines and trigger cascades of other cytokines. Non-limiting examples of cytokines include 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.

The term "cytotoxic T lymphocytes" (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 through extrinsic and intrinsic pathways.

As used herein, the term "dendritic cell" or "DC" refers to a morphologically similar cell type found in various lymphoid and non-lymphoid tissues that presents foreign antigens to T cells. It represents a diverse population (see Steinman, Ann. Rev. Immunol. 9:271-296 (1991)).

As used herein, the term "derived from" includes any manner of receiving, obtaining, or modifying something from the original source.

As used herein, the term "derivative" or "variant" with respect to a peptide or DNA sequence (e.g., an immunomodulator peptide sequence) refers to a non-identical peptide or DNA sequence that has been altered from its original sequence. . Differences in sequence may be by design, the result of alterations in sequence or structure. Designed alterations may be those specifically designed and introduced into a sequence for a particular purpose. Certain such alterations can be made in vitro using a variety of 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 cells refers to cell lines that have been modified (e.g., modified to express a recombinant DNA sequence) from their original cell line. Point.

The term "detectable marker" encompasses both selectable markers and assay markers. The term "selectable marker" includes drug resistance markers, antigenic markers useful for fluorescence-activated cell sorting, adhesion markers such as receptors for adhesion ligands that allow selective adhesion of expression constructs, etc. refers to a variety of gene products that can be selected or screened for transformed cells.

The term "detectable response" refers to any signal or response that can be detected in an assay that 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, polarized light, fluorescence, phosphorescence, transmission, reflection, or resonance transfer. Detectable responses also include chromatographic mobility, turbidity, electrophoretic mobility, mass spectra, ultraviolet spectra, infrared spectra, nuclear magnetic resonance spectra, and X-ray diffraction. Alternatively, the detectable response can be the result of an assay to measure one or more properties of the biological material such as melting point, density, electrical conductivity, surface acoustic waves, 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. Detection reagents may include markers to facilitate detection.

As used herein, the term "differentiate" and its various grammatical forms refer to developmental processes involving an increase in the level of organization or complexity of cells or tissues that have more specialized functions. Point.

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

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

Engineered leukocyte stimulating cells (“ENLIST™ cells”) are genetically engineered to express a core group of three immunomodulatory molecules that are used to stimulate mononuclear cells for the treatment of cancer. means a population of tumor cells that are incapable of proliferation.

As used herein, the term "enriching" means increasing the proportion of a desired substance, e.g., increasing the relative frequency of a subtype of cell compared to its natural frequency in a cell population. point to 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. The specific markers used in the selection process are important because developmental stages and activation-specific responses can alter the antigenic profile of cells, regardless of the specific technique used for enrichment.

As used herein, the term "exogenous polypeptide" refers to a polypeptide that is not produced by wild-type cells of that type or is present at a lower level in wild-type cells than in cells containing the exogenous polypeptide. Refers to peptides. According to some embodiments, an exogenous polypeptide is a polypeptide encoded by a nucleic acid introduced into a cell, which nucleic acid is optionally not retained by the cell.

As used herein, the term "exogenous immunomodulatory molecule" refers to a cognate polypeptide (e.g., receptor) specific for immune cells, e.g., immune killer cells (e.g., NK cells or CD8+ T cells). comprises (e.g., intracellularly or on the cell surface) allogeneic cells (e.g., allogeneic cell lines) that bind functionally, thereby transmitting signals that mediate immune cell stimulation, such as immune cell proliferation, activation, and expansion. A provided polypeptide is included. 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.

As used herein, the term "express" or "expression" refers to mRNA biosynthesis, polypeptide biosynthesis, polypeptide activation by e.g. activation of expression by recruitment of Expression can be, for example, increasing the number of genes encoding the polypeptide, increasing transcription of the genes (such as by placing the genes under the control of a constitutive promoter), increasing translation of the genes, competing Augmentation can be achieved by a number of approaches, including genetic knockout, or a combination of these and/or other approaches.

The term "expression vector" refers to a DNA molecule that contains a gene for expression in a host cell. Gene expression is typically 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 element.

As used herein, the terms "first" and "second" with respect to exogenous immunomodulatory molecules are convenient to distinguish when more than one type of exogenous stimulatory polypeptide is present. used as The use of these terms is not intended to imply any particular order or orientation of the exogenous stimulatory polypeptides unless explicitly stated.

As used herein, the term "flow cytometry" refers to a tool for examining cell phenotypes and characteristics. It senses cells or particles as they move in a fluid stream through a laser (light amplification by stimulated emission of radiation)/light beam that passes through a sensing area. Relative light scattering and color-distinguished fluorescence of microparticles are measured. Analysis and identification of cell streams is based on size, granularity, and whether the cells carry fluorescent molecules in the form of either antibodies or dyes. As the cell passes 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. Proportional. Light can enter the cell and thus 90° light (right angle, side) scatter can be labeled with fluorochrome-conjugated antibodies or stained with fluorescent membrane, cytoplasmic, or nuclear dyes. Thus, discrimination of cell type, presence of membrane receptors and antigens, membrane potential, pH, enzymatic activity, and DNA content can be facilitated. Flow cytometers are multi-parameter, recording several measurements for each cell, thus making it possible to distinguish homogeneous subpopulations within heterogeneous populations (Marion G. Macey, Flow cytometry: principles and applications, Humana Press, 2007). Fluorescence-activated cell sorting (FACS) enables the separation of distinct cell populations that have too similar physical properties to separate by size or density, and uses fluorescent tags to detect differentially expressed surface proteins. , allowing fine discrimination between populations of physically homogeneous cells.

The terms "functional equivalent" or "functionally equivalent" are used interchangeably herein and refer to substances, molecules, polynucleotides, proteins, peptides or polypeptides that have a similar or identical effect or use. Refers to peptides.

As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid associated with the expression of a given RNA or protein. Thus, genes include the coding regions (typically including polypeptide coding sequences) for the expressed RNAs, and often include the regulatory sequences necessary 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 specifically designed to have desired parameters.

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 and cross-reacts with the original less immunogenic antigen.

The terms "immune response" and "immune-mediated" refer to any function 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. are used interchangeably herein to refer to expression.

As used herein, the term "immunosuppression" and other grammatical forms refer to the body's immune response and reduction in the ability of the immune system to fight infections and other diseases. For example, some immunosuppression may be drug-induced or due to disease.

The terms "immunomodulator," "immunomodulator," and "immunomodulatory" can directly or indirectly increase or decrease an immune response by chemokines, cytokines, and other mediators of the immune response. Used interchangeably herein to refer to a substance, agent, or cell that can.

As used herein, the term "immunostimulatory amount" of the disclosed compositions quantifies the blood population of antigen-specific CD4+ T cells by a measurable amount or Blood populations may be quantified to detect and quantify antigen-specific T cells, or suitable subjects (e.g., dendritic cells not loaded with tumor-specific cells, or peptides derived from tumor-specific cells). 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%, e.g., an ELISPOT assay ( an amount of an immunogenic composition effective to stimulate an immune response, as measured by cellular immune response), ICS (intracellular cytokine staining assay), and major histocompatibility complex (MHC) tetramer assay. Point.

As used herein, the term "genomically integrated" refers to a recombinant DNA sequence that is co-ligated with genomic DNA comprising the genome of the host cell.

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

As used herein, the term "labeling" refers to the process of distinguishing compounds, structures, proteins, peptides, antibodies, cells or cellular components by introducing traceable moieties. Common traceable moieties include fluorescent antibodies, fluorophores, dyes or fluorochromes, stains or fluorescent stains, markers, fluorescent markers, chemical stains, differential stains, differential labels, and radioisotopes, including: Not limited.

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

As used herein, the term "mediate" and its various grammatical forms mean to bring about a result.

As used herein, the term "minimal residual disease" refers to the very few cancer cells that remain in the body during or after treatment. Minimal residual disease can only be found by highly sensitive tests that can find one cancer cell out of one million normal cells.

The term "mixed lymphocyte tumor response" or "MLTR" is similar to the mixed lymphocyte reaction, but instead of using allogeneic lymphocytes to stimulate the response, allogeneic tumor cells are used instead. Used interchangeably herein to refer to the reaction used. The MLTR method involves contacting tumor cells being tested for immunogenicity with mixed lymphocytes from peripheral blood mononuclear cells, followed by lymphocyte cell proliferation, lymphocyte cell subset differentiation, and lymphocyte cell subset differentiation. measuring one or more of cytokine release profile and tumor cell death.

As used herein, the term "modify" and its various grammatical forms refer to changes in form or nature thereof.

As used herein, the term "modulate" and its various grammatical forms means to adjust, alter, adapt, or adjust to some measure or proportion. Such modulation can be any change, including undetectable changes.

As used herein in reference to an immune response to a tumor cell, the term "modified" or "modulated" refers to one or more refers to altering the shape or characteristics of the immune response to tumor cells through recombinant DNA technology.

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

As used herein, the term "natural killer (NK) cells" refers to lymphocytes of the same family as T and B cells, classified as Group I innate lymphocytes. They have the ability to kill tumor cells without priming or prior activation, in contrast to cytotoxic T cells, which require priming by antigen-presenting cells. NK cells secrete cytokines such as IFNγ and TNFα and 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 and infected cells and switch on NK cells. Inhibitory receptors serve as a check for NK cell killing. Most normal, healthy cells express MHCI receptors that mark them as "self." Inhibitory receptors on the surface of NK cells recognize the cognate MHCI, which switches off NK cells to prevent killing. Once determined to kill, NK cells release cytotoxic granules containing perforin and granzymes, causing target cell lysis. Natural killer reactivities, including cytokine secretion and cytotoxicity, are associated with several germline-encoded inhibitory receptors, such as killer immunoglobulin-like receptors (KIRs) and natural cytotoxic receptors (NCRs). Controlled by a balance of receptors and activated receptors. The presence of MHC class I molecules on target cells functions as one of the inhibitory ligands for killer cell immunoglobulin-like receptors (KIRs), MHC class I-specific receptors on NK cells. KIR receptor binding blocks NK activation and, paradoxically, retains the ability to respond to successive encounters by inducing an inactivating signal. Therefore, if the KIR is sufficiently capable of binding to MHC class I, this binding will abolish the killing signal and allow the target cell to survive. In contrast, target cell killing can proceed when NK cells are unable to bind sufficiently to MHC class I on the target cell. 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.

The term "nucleic acid" is used herein to refer to a polymer of deoxyribonucleotides or ribonucleotides in either single- or double-stranded form, unless otherwise limited to naturally occurring nucleotides. It includes known analogues that have the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a similar manner (eg, peptide nucleic acids). Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide or fragment thereof of the invention. Such nucleic acid molecules need not be 100% identical to the 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 invention include any nucleic acid molecule that encodes a polypeptide or fragment thereof of the invention. Such nucleic acid molecules need not be 100% identical to the 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 a pair that forms a double-stranded molecule with a complementary polynucleotide sequence (e.g., a gene as described herein) or portion thereof under conditions of varying stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507). Measuring the effects of base mismatches by quantifying the rate at which the two strands anneal can yield information about the base sequence similarity between the two annealed strands. A selectively hybridizing nucleic acid is detectable under stringent hybridization conditions (e.g., at least twice background) than its hybridization to non-target nucleic acid sequences and Substantial exclusion undergoes hybridization of a nucleic acid sequence to a specific nucleic acid target sequence.

"Substantially identical" refers to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein A polypeptide or nucleic acid molecule that exhibits at least 50% identity to one). For example, such sequences are at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at the amino acid or nucleic acid level, the sequences used for comparison. , 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.

Sequence identity is typically measured using sequence analysis software (e.g., BLAST, BESTFIT, GAP, sequence analysis software packages of University of Wisconsin Biotechnology Center, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). , or the PILEUP/PRETTYBOX program). 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; , tyrosine. An exemplary approach to determining the degree of identity can use the BLAST program, with probability scores between e-3 and e-100 indicating closely related sequences.

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. It begins with an initiation triplet (ATG) followed by a chain of triplets each encoding an amino acid and ends with a termination triplet (TAA, TAG or TGA).

The phrase "operably linked" means that (1) a first sequence(s) or domain is a second sequence(s) or domain or a region under the control of that second sequence or domain; (2) the promoter sequence is positioned sufficiently proximal to the second sequence(s) or domain so that it can affect the A functional linkage between a promoter and a second sequence that initiates and mediates transcription of a DNA sequence corresponding to a. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where required to join two protein coding regions, are in the same reading frame. According to some embodiments, the phrase "operably linked" means that two or more protein domains or polypeptides are in a state in which each protein domain or polypeptide of the resulting fusion protein performs its original function. refers to a ligation that is linked or joined through recombinant DNA techniques or chemical reactions to retain

As used herein, the term “overall survival” (OS) refers to the time a patient diagnosed with a disease, such as cancer, is still alive at the date of diagnosis of the disease, such as cancer, or the start of treatment. Refers to the length of the period from either.

As used herein, the term "parenteral" and other grammatical forms refer to administration of substances that occur within the body other than through the mouth or gastrointestinal tract. For example, as used herein, the term "parenteral" includes, for example, subcutaneous (ie, injection under the skin), intramuscular (ie, injection into the muscle), intravenous (ie, intravenous introduction into the body by injection (i.e., administration by injection), including intrathecal (i.e., injection into the spinal cord or into the subarachnoid space of the brain), intrasternal, or injection techniques point to

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.

As used herein, the term "pharmaceutical composition" means a composition that is used to prevent, reduce the intensity of, curatively or otherwise treat a target condition, syndrome, disorder, or disease. point to

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier conventional to the administration of pharmaceuticals with which the isolated polypeptides of the invention remain stable and bioavailable. refers to any substantially non-toxic carrier that can be used from Pharmaceutically acceptable carriers must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the mammal being treated. Additionally, there is a need to maintain the stability and bioavailability of the active agent. Pharmaceutically acceptable carriers may be liquid or solid, designed to provide the desired volume, concentration, etc. when combined with the active agent and other ingredients of a given composition. It can be selected in consideration of the administration method.

The term "pharmaceutically acceptable salt," as used herein, does not have undue toxicity, irritation, allergic response, etc. and is commensurate with a reasonable benefit/risk ratio Refers to salts that are suitable for use in contact with human and lower animal tissue within the scope of sound medical judgment. For use in medicine, salts should be pharmaceutically acceptable, but 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-toluene. Sulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. "Pharmaceutically acceptable salt" means a salt that does not have undue toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit/risk ratio, and within the scope of sound medical judgment. and salts suitable for use in contact with tissue of lower animals. Pharmaceutically acceptable salts are well known in the art. For example, P.I. H. 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 according to the invention or separately by reacting a free base functional group with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate. salt, digluconate, glycerophosphate, hemisulfinate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethione acid), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate, but It is not limited to these. Basic nitrogen-containing groups also include lower alkyl halides such as, for example, methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and diamyl sulfates; , long-chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides, such as benzyl and phenethyl bromides; can be graded. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric acid, as well as oxalic, maleic, succinic, and Organic acids such as citric acid are included. Basic addition salts combine a carboxylic acid-containing moiety with a suitable base such as a pharmaceutically acceptable hydroxide, carbonate or bicarbonate of a metal cation, or with ammonia or an organic primary, secondary or tertiary salt. It can be prepared in situ during the final isolation and purification of the compounds according to the invention by reacting with amines. Pharmaceutically acceptable salts include cations based on alkali metal or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts, as well as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethyl Non-toxic quaternary ammonia and amine cations including, but not limited to, amines, trimethylamine, triethylamine, diethylamine, ethylamine, and the like. Other representative organic amines useful in forming base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. Pharmaceutically acceptable salts are also made physiologically acceptable using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid. It can be obtained by providing an anion. Alkali metal (eg, sodium, potassium, or lithium) or alkaline earth metal (eg, calcium or magnesium) salts of carboxylic acids can also be made.

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

The terms "polypeptide," "peptide," and "protein" also refer to modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. including. As is well known, and as noted above, it will be appreciated that polypeptides may not be perfectly linear. For example, polypeptides may branch as a result of ubiquitination, and they generally undergo branching as a result of post-translational events, including natural processing events and those resulting from artificial manipulations that do not occur in nature. It may be cyclic with or without inclusion. Cyclic, branched and branched cyclic polypeptides can be synthesized by non-translational natural processes and also by wholly synthetic methods. According to some embodiments, peptides are of any length or size.

As used herein, the term “proliferate” and its various grammatical forms refer to a process that results in an increase in cell number, by a balance between cell division and cell death or cell loss through differentiation. Defined.

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 are generally composed of multiple domains that are connected to each other through flexible regions of the polypeptide chain.

The following terms are used herein to describe 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, eg, as a segment of a full-length cDNA or gene sequence, or as a complete cDNA or gene sequence. (b) The term "comparison window" refers to a contiguous specific segment of a polynucleotide sequence that can be compared to a reference sequence, the portion of the polynucleotide sequence within the comparison window being the optimal fit for the two sequences. may contain additions or deletions (ie, gaps) relative to a reference sequence (not containing additions or deletions) for a proper alignment. Generally, the comparison window is at least 20 contiguous nucleotides in length, optionally at least 30 contiguous nucleotides in length, at least 40 contiguous nucleotides in length, at least 50 contiguous nucleotides in length, , at least 100 contiguous nucleotides in length, or longer. Those skilled in the art understand that gap penalties are typically introduced and subtracted from the number of matches to avoid high similarity to a reference sequence due to the inclusion of gaps in the polynucleotide sequence. Methods of aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison is performed by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. Math. 2:482 (1981)) according to Needleman and Wunsch, J. Am. Mol. Biol. 48:443 (1970) by the homology alignment algorithm; Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); Intelligenetics, Mountain View, Calif. CLUSTAL in PC/Gene Programs by Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr. , Madison, Wis. , USA, GAP, BESTFIT, BLAST, FASTA, and TFASTA; The CLUSTAL program is described in 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 similarity searches of databases include: BLASTN for nucleotide query sequences against nucleotide database sequences, BLASTX for nucleotide query sequences against protein database sequences, 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. Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al. , Eds. , Greene Publishing and Wiley-Interscience, New York (1995). Unless otherwise stated, 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 analyzes is publicly available, eg, through the US National Center for Biotechnology Information. The algorithm identifies high-scoring sequence pairs (HSPs) by first identifying short-length words W. Obtain query sequences that match or satisfy a positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Expansion of word hits in each direction is stopped when: the cumulative alignment score drops by the amount X from its maximum achieved value; accumulation of one or more negative-scoring residue alignments leads to a cumulative score of less than or equal to 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 wordlength (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 wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915). In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, eg, Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873- 5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability of a match between two nucleotide or amino acid sequences occurring by chance. . BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequence that may be homopolymeric tracts, short periodic repeats, or regions rich in one or more amino acids. Such low-complexity regions can align between unrelated proteins even though other regions of the proteins are completely different. Several low-complexity filter programs can be used to reduce such low-complexity alignments. For example, the low-complexity filters of SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) are , can be used alone or in combination. The term "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to the residues of the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. used herein for - When using percentage sequence identities for proteins, residue positions that are not identical often differ by conservative amino acid substitutions, i.e., amino acid residues that have similar chemical properties (e.g., charge or hydrophobicity) It will be understood that other amino acid residues have been substituted with other amino acid residues that do not alter the functional properties of the molecule. Where amino acid sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to compensate for the conservative nature of the substitutions. 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 ordinary skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage 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. Scoring of conservative substitutions is performed as implemented in 1, the PC/GENE program (Intelligenetics, Mountain View, Calif., USA), for example, in Meyers and Miller, Computer Applic. Biol. Sci. , 4:11-17 (1988). (d) The term "percentage of sequence identity" as used herein means a value determined by comparing two optimally aligned sequences over a comparison window, wherein A portion of a polynucleotide sequence may contain additions or deletions (ie, gaps) as compared to a reference sequence (not containing additions or deletions) for optimal alignment of the two sequences. Percentage (%) is obtained by determining the number of positions where the same nucleobase or amino acid residue is present in both sequences to obtain the number of matching positions and the number of matching positions to the total number of positions within the comparison window. and multiplying the result by 100 to obtain the percentage sequence identity. (e) The term "substantial identity" of a polynucleotide sequence means a sequence identity of at least 70% when compared to a reference sequence using one of the alignment programs written using standard parameters , means a polynucleotide comprising a sequence having at least 80% sequence identity, at least 90% sequence identity, and at least 95% sequence identity. Those skilled in the art can adjust these values appropriately to take into account codon degeneracy, amino acid similarity, reading frame alignment, etc., to determine the corresponding identity of proteins encoded by two nucleotide sequences. would recognize Substantial identity of amino acid sequences for these purposes usually means at least 60%, or at least 70%, at least 80%, at least 90%, or at least 95% sequence identity. Another indication that nucleotide sequences are substantially identical is whether the two molecules will 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 a copy of a nucleic acid is made using the maximum codon degeneracy allowed 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. is. Mutations can also be made in the nucleotide sequence of the protein by reference to the genetic code, including taking into account codon degeneracy.

As used herein, the term "priming" (or "priming") refers to the process of increasing susceptibility to. It is used in an immunological sense to refer to the process by which specific antigens are presented to naive lymphocytes, causing them to differentiate. Priming involves several steps: antigen uptake, processing, cell surface expression bound to MHC molecules by antigen-presenting cells, recycling and antigen-specific capture of helper T cell precursors in lymphoid tissues, and Proliferation and Differentiation (Janeway, CA, Jr., "The priming of helper T cells, Semin. Immunol. 1(1): 13-20 (1989)).

As used herein, the term “progression-free survival” or “PFS” refers to the length of time during and after treatment for a disease, such as cancer, that a patient lives with the disease but does not get worse. point to In clinical trials, measuring progression-free survival is one way of determining how well a new treatment works.

As used herein with respect to cancer, the term "recurrence" usually refers to cancer that has recurred (come back) after a period during which no cancer was detected. Cancer can come back in the same place as the original (primary) tumor or in another place in the body.

As used herein, the term “recurrence-free survival (RFS)” refers to the period of time a patient lives without signs or symptoms of their cancer after primary treatment for cancer. Also called disease-free survival (DFS) and progression-free survival (PFS).

The term "release" or "release of cytokine effector molecules" refers to the complex and stringent processes in which soluble mediators of the immune response are delivered from a given immune cell type to the external environment following activation of signaling cascades in response to receptor stimulation. It is meant to refer to a process controlled by In the classical secretory pathway, cytokines with signal peptides are co-translationally 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 for constitutive delivery to the cell surface or other organelles in the trans-Golgi network (TGN). be. Specialized cell types provide additional modes of secretion by loading cytokines and other cargo into granules for storage and later release. 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 directly by immunoglobulin or complement receptor-mediated signaling, or by pathogens through a variety of cellular receptors, including pattern recognition receptors such as TLRs.

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

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

The term "reporter gene" ("reporter") or "assay marker" refers to a gene and/or peptide that can be detected or easily identified and measured. Reporter expression 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, nucleic acid sequence markers, and the like. Researchers can add the reporter gene to another gene of interest in cell culture, bacteria, animals, or plants. For example, some reporters are selectable markers or confer a property on the organism that expresses them, allowing the organism 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 in the same DNA construct and insert it into a cell or organism. For bacterial or eukaryotic cells in culture, this may be in the form of a plasmid. Commonly used reporter genes can include, but are not limited to, fluorescent proteins, luciferase, beta-galactosidase, and selectable markers such as chloramphenicol and kanomycin.

The term "secretion," as used herein when referring to cells, refers to the process by which molecules produced within the cell move into the extracellular space.

As used herein, the term "serial killer cell" refers to a population of cells that exhibit the ability to kill cells infected with multiple tumors or pathogens while exhibiting resistance to such killing. . There are multiple types of cells that exhibit this effector function, such as NK cells, NKT cells, LAK cells, CIK cells, MAIT cells, CD8+ CTL, CD4+ CTL, and the like. Serial killer effector function can be direct through cytolytic or cytotoxic activity, or indirect through immunoregulation of other cells and proteins that target pathogenic and cancerous cells. obtain.

As used herein, the term "stably expressed exogenous immunomodulatory molecule" refers to stimulating T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or B lymphocytes. an exogenous immunomodulatory molecule expressed for a period of time sufficient to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or B lymphocytes to Point. According to some embodiments, the time period is from 1 hour to 72 hours, such as 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 exceeds 72 hours.

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

As used herein, the term "stimulating immune cells" or "stimulating immune cells" refers to activation of cellular responses, e.g., activation of immune cells, e.g., NK cells and/or CD8+ T cells. and/or refers to processes (including, for example, signaling events or stimuli) that cause or result in expansion. According to some embodiments, stimulating immune cells (e.g., NK cells and/or CD8+ T cells) provides stimuli or signals (e.g., stimulatory polypeptides) that lead to activation and/or expansion of immune cells. means to provide.

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

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

As used herein, the phrase "a subject in need thereof" refers, unless the context and usage of the phrase dictates otherwise, to (i) administering an immunogenic composition according to the described invention. (ii) receive an immunogenic composition according to the described invention; or (iii) a patient who has received an immunogenic composition according to the described invention.

The term "SUPLEXA™ cells" refers to autologous blood cells that have been stimulated in vitro by engineered leukocyte stimulating cells ("ENLIST™ cells").

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, ingredient, or component of the described composition of the invention responsible for the intended therapeutic effect. The terms "therapeutic agent" and "active agent" are used interchangeably herein. As used herein, the term "therapeutic ingredient" refers to a therapeutically effective dose (ie, dose and frequency of administration) that eliminates, reduces, or prevents the progression of a particular disease symptom in a percentage of the population. An example of a commonly used therapeutic moiety is the ED50, which represents the dose at a particular dose that is therapeutically effective against a particular disease manifestation in 50% of the population.

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. used for However, dosage levels will be based on a variety of factors including type of injury, age, weight, sex, medical condition of the patient, severity of the condition, route of administration, and the particular active agent used. Thus, dosage regimens may 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 prophylactic amounts of the described composition of the invention. In prophylactic or prophylactic applications of the described invention, the pharmaceutical composition or medicament is administered in an amount sufficient to eliminate or reduce the risk or biochemical, histological and and/or in an amount sufficient to delay the onset of the disease, disorder or condition, including behavioral symptoms, complications thereof, and intermediate pathological phenotypes that appear during the onset of the disease, disorder or condition. It is administered to a patient susceptible to or otherwise at risk for a disorder or condition. It is generally preferred to use the maximum dose, ie, the dose that is safest according to some medical judgment. The terms "dose" and "dosage" are used interchangeably herein.

As used herein, the term "therapeutic effect" means the result of treatment, which result is considered desirable and beneficial. A therapeutic effect may include, directly or indirectly, prevention, reduction, or elimination of symptoms of disease. A therapeutic effect can also include, directly or indirectly, arresting progression, alleviation, or elimination of symptoms of disease.

For any therapeutic agent described herein, a therapeutically effective amount can be initially determined from preliminary in vitro studies and/or animal models. A therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dosage to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the skilled artisan.

General principles for determining therapeutic efficacy 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. The principles are summarized below.

Pharmacokinetic principles provide the basis for modifying dosing regimens to achieve the desired degree of therapeutic effect while minimizing unacceptable side effects. Additional guidance on dose modification can be obtained in situations where the plasma concentration of the drug can be measured and related to the therapeutic window.

Pharmaceutical 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 pharmaceutical products are considered bioequivalent if the rate and extent of bioavailability of the active ingredients of the two products do not differ significantly under appropriate test conditions.

The term "therapeutic window" refers to the concentration range that provides therapeutic effect without unacceptable toxicity. After administration of a dose of drug, its effects usually show a characteristic temporal pattern. There is a lag period before the drug concentration exceeds the minimum effective concentration (“MEC”) for the desired effect. Following onset of response, the intensity of the effect increases as the drug continues to be absorbed and distributed. This reaches a peak after which drug removal leads to a decrease in the strength of the effect, which disappears when the drug concentration falls below the MEC. Therefore, the duration of action of a drug is determined by the period during which the concentration exceeds the MEC. The goal of therapy is to obtain and maintain concentrations within the therapeutic window for the desired response with minimal toxicity. A drug response below the MEC for a desired effect will be subtherapeutic, but for an adverse effect the probability of toxicity will 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 duration of action of the drug, but at the risk of increasing the potential for side effects. Therefore, increasing the dose is not a useful strategy to extend the duration of action of a drug unless the drug is non-toxic.

Alternatively, another dose of drug must be administered to maintain concentrations within the therapeutic window. In general, the lower end of a drug's therapeutic range appears to be approximately equal to the drug concentration that produces about half the maximum possible therapeutic effect, and the upper end of the therapeutic range is toxic to about 5% or less to about 10% of patients. It seems to work. These values are highly variable, and while some patients may benefit greatly from drug concentrations above the therapeutic range, other patients may suffer significant toxicity at much lower values. The goal of treatment is to maintain steady-state drug levels within a therapeutic window. For most drugs, the actual concentration associated with this desired range may or may not be known; It is sufficient to understand how frequency affects drug levels. For the few drugs with small (2- to 3-fold) differences between concentrations that produce efficacy and toxicity, plasma concentration ranges relevant to effective therapy have been defined.

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

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 drug relevant to the therapeutic window. To maintain a selected steady-state or target concentration (“maintenance dose”), the drug administration rate is adjusted so that the rate of input equals the rate of loss. Once the clinician has selected the desired concentration of drug in plasma and is aware of the drug's clearance and bioavailability in a particular patient, an appropriate dose and dosing interval can be calculated.

As used herein, the term "treating" means to negate, substantially inhibit, slow, or reverse the progression of a condition; Including ameliorating or substantially preventing the appearance of clinical symptoms of the condition. Treating further refers to achieving one or more of the following: (a) reducing the severity of the disorder; (b) preventing the development of symptoms characteristic of the disorder(s) being treated. (c) limiting exacerbation of symptoms characteristic of the disorder(s) being treated; (d) 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).

As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

As used herein, the terms "tumor burden" or "tumor burden" refer to the number of cancer cells, the size of a tumor, or the amount of cancer in the body.

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

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

As used herein, the term "vaccine" means a substance or substance that directs the immune system to respond to tumors or microorganisms or to help the body recognize and destroy cancer cells or microorganisms. Refers to a group of substances. The term vaccine also refers to man-made stimuli (eg, infectious agents, cancer cells) used to stimulate a strong immune response to their exposure.

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

As used herein, the term "variant" refers to a polypeptide that differs from the original protein by one or more amino acid substitutions, deletions, insertions, or other alterations. These modifications do not significantly alter the biological activity of the original protein. Variants often retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the biological activity of the original protein. Hold. A variant's biological activity may also be higher than that of the original protein. Variants may be naturally occurring, such as by allelic variation or polymorphism, or may be deliberately engineered.

The amino acid sequence of a variant is substantially identical to that of the original protein. In many embodiments, a variant has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more overall sequence identity or sequence identity with the original protein. share similarities. Sequence identity or similarity can be determined using various methods known in the art, such as Basic Local Alignment Tool (BLAST), dot matrix analysis, or dynamic programming methods. 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 a variant and the original protein may be substantially identical in one or more regions, but may differ in other regions.

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 natural populations. The terms "wild-type" and "naturally occurring" are used interchangeably.

II. Allogeneic Vaccines This disclosure features allogeneic tumor cell vaccines comprising tumor cells expressing exogenous immunomodulatory molecules, and methods of using allogeneic tumor cell vaccines to stimulate an immune response. Vaccine proteins are capable of inducing an immune response for use in the described invention, eg, in the treatment of cancer and infectious diseases. According to some embodiments, the allogeneic tumor cell vaccines described herein are effective to recognize and act against tumor cells containing target tumor antigens in vivo without systemic inflammation. It is effective in enhancing cellular immune activation, decreasing immunosuppression in the tumor microenvironment of tumor cells containing the target tumor antigen, or increasing cell death of tumor cells expressing the target tumor antigen. According to some embodiments, the allogeneic tumor cell vaccines described herein are capable of immune activation without systemic inflammation.

According to some aspects, the present disclosure provides (1) a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killers ( genetically engineered to stimulate one or more of NK) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC) or a population of tumor cells comprising a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes; and (2) a pharmaceutically acceptable carrier. It features a tumor cell vaccine.

Tumor-Specific Antigens According to some embodiments, the present disclosure provides populations of proliferation-incompetent tumor cells that express one or more tumor-specific antigens. According to some embodiments, tumor-specific antigens may be encoded by the primary open reading frames of gene products that are differentially expressed by tumors but not by normal tissues. According to some embodiments, tumor-specific antigens may be encoded by mutated genes, intronic sequences, or translated alternative open reading frames, pseudogenes, antisense strands, or products of gene translocation events. can represent According to some embodiments, tumor cells present a wide range of tumor-specific antigens, many of which are of unknown nature. According to some embodiments, the tumor antigen is a neoantigen.

According to some embodiments, the tumor-specific antigen is selected from one of the following groups: (a) unmutated shared antigen (e.g., melanoma-associated antigen (MAGE), B-melanoma) (b) differentiation antigens (e.g., prostate-specific membrane antigen [PSMA] and prostate-specific antigen ( PSA), 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 that provide novel epitopes for immune recognition (e.g., mutated ras, rearranged bcr/abl, mutated p53); (d) unique idiotypes. (e.g. immunoglobulin antigens of myeloma and B-cell myeloma, T-cell receptor (TCR) expressed in CTCL); (e) epitopes from oncoviruses (e.g. human papillomavirus encoded E6 and E7 proteins; Epstein-Barr virus-associated antigen present in primary cerebral lymphoma); , from antigens listed in the publicly available cancer antigen peptide database (caped.icp.ucl.ac.be/Peptide/list on the world wide web, hereby incorporated by reference in its entirety) Selected According to some embodiments, the tumor-specific antigen is selected from the antigens shown in Table 1, shown below.

According to some embodiments, the tumor cell is melanoma, colorectal cancer, leukemia, chronic myelogenous leukemia, prostate cancer, head and neck cancer, squamous cell carcinoma, tongue cancer, laryngeal cancer, tonsil cancer, hypopharyngeal cancer It is derived from a cancer selected from the group consisting of carcinoma, nasopharyngeal carcinoma, breast cancer, colon cancer, lung cancer, pancreatic cancer, glioblastoma, and brain cancer.

According to some embodiments, the melanoma tumor cells are gp100, tyrosinase, Melan-A, tyrosinase-related protein (TRP-2-INT2), melanoma antigen-1 (MAGE-A1), NY-ESO-1 , the preferentially expressed antigen of melanoma (PRAME), CDK4 and multiple myeloma oncogene 1 (MUM-1).

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

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

Immunological antigen specificity for a particular type of cancer cell can be derived from one or more specific fingerprints of multiple tumor antigens, where a particular antigen is expressed by a wide variety of tumor cells, whereas it is expressed by a small number of tumor types. From the fact that it has particular use in immunotherapy, from the fact that a specific collection of MHC class I-presentable and MHC class II-presentable epitopes are present on a particular polypeptide or polypeptide fragment, and to induce immune tolerance. can occur by omitting one or more peptides that may Those of ordinary skill in the art are familiar with, for example, the US government website, ncbi. nlm. Related nucleic acid and polypeptide sequences can be found at nih.

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

According to some embodiments, the tumor antigen specificity of the present invention can be determined by the parental tumor cell line or tumor cell line variant selected for modification by an immunomodulator.

Parental Cell Lines According to some embodiments, the tumor cell line or tumor cell line variant is from any public source (e.g., DCTD Tumor Repository operated by NIH, Charles River Laboratories Inc.) or commercial source. (eg, ATCC, Sigma Alrich, Thermo Fischer Scientific, Genescript, DSM2). According to some embodiments, new cell lines can be newly established from tumor cells derived from tumors of cancer patients.

According to some embodiments, cancer tissue, 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, the cancer cells include, but are not limited to, established non-small cell lung cancer (NSCLC), bladder cancer, melanoma, ovarian cancer, renal cell carcinoma, prostate cancer, sarcoma, breast cancer, It can be obtained from established tumor cell lines or tumor cell line variants, such as squamous cell carcinoma, head and neck, hepatocellular, pancreatic, or colon cancer cell lines.

According to some embodiments, the established cell line comprises the LNCaP clone FGC (ATCC CRL-1740) derived from metastatic prostate cancer that has itself migrated to the lymph nodes. According to some embodiments, the established cell line comprises the PC-3 (ATCC CRL-1435) cell line derived from metastatic prostate cancer that has itself migrated to bone. According to 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).

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

According to some embodiments, established cell lines are COO-G, DU4475, ELL-G, HIG-G, MCF/7, MDA-MB-436, MX-1, SW-613, and VAN - containing one or more of the breast cancer cell lines called G. According to some embodiments, the established cell lines comprise one or more of the alveolar soft part sarcoma cell lines designated ASPS and ASPS-1. According to some embodiments, the established cell lines are lung cell lines designated LX-1, COS-G, H-MESO-1, H-MESO-1A, NCI-H23, and NCI-H460. including one or more of According to some embodiments, the established cell lines are CX-5, GOB-G, HCC-2998, HCT-15, KLO-G, KM20L2, MRI-H-194, LOVO I, LOVO II, and one or more of the colon cancer cell lines called MRI-H-250. According to some embodiments, the established cell lines are NIS-G, TRI-G, WIL-G, MRI-H-121B, MRI-H-187, MRI-H-221, and MRI-H including one or more of the melanoma cell lines called -255. According to some embodiments, the established cell line is one of the cervical cancer cell lines designated MRI-H-177, MRI-H-186, MRI-H-196, and MRI-H-215. including one or more. According to some embodiments, the established cell lines include one or more of kidney cancer cell lines designated MRI-H-121 and MRI-H-166. According to some embodiments, the established cell lines comprise one or more of the endometrial cancer cell lines designated MRI-H-147 and MRI-H-220. According to some embodiments, the established cell lines are one or more of ovarian cancer cell lines designated MRI-H-258, MRI-H-273, MRI-H-1834, and SWA-G. including. According to some embodiments, the established cell lines include one or more of sarcoma cell lines designated HS-1, OGL-G, and DEL-G. According to some embodiments, the established cell line comprises an epidermoid cell line called DEAC-1. According to some embodiments, the established cell line comprises an epidermoid cell line called 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 called DAU. According to some embodiments, said established cell lines described herein are, for example, the American Type Culture Collection (ATCC), the European Cell Culture Collection (ECACC), or the Budapest Treaty Article 7 are commercially available from any depository listed as an International Depository (IDA) of

Exemplary established cell lines, according to some embodiments, include one or more of the following cell lines:

According to some embodiments, the choice of parental cell line from which the tumor cell line or tumor cell line variant can be derived influences the specificity of the allogeneic vaccine. For example, use of a tumor cell line or tumor cell line variant derived from metastatic prostate cancer that has migrated to the patient's bone results in an allogeneic vaccine that induces an immune response specific for metastatic prostate cancer in the patient's bone. obtain.

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

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

For example, according to some embodiments, the tumor cell line or tumor cell line variant is a solid tumor, including tumor cells including cancer stem cells, a metastatic cancer, including metastatic tumor cells including cancer stem cells, or It can be derived from non-metastatic cancer. According to some embodiments, the cancer is bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gingiva, head, kidney, liver, It may originate from the lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. According to some embodiments, the cancer is of histological type, e.g., cancer that arises in the tissue that lines or covers the skin or internal organs (carcinoma); cartilage, fat, muscle, blood vessels, fibrous tissue ( cancers that arise in the bones or soft tissues of the body, including sarcoma); cancers that arise in blood-forming tissues (leukemia); cancers that arise in cells of the immune system (lymphoma); cancers that arise in plasma cells (myeloma) ), or brain/spinal cord cancer.

Examples of carcinomas include giant cell carcinoma and spindle cell carcinoma, small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoid cell carcinoma; basal cell carcinoma; adenocarcinoma; gastrinoma, cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; cervical adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cyst Adenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; Adenocarcinoma with epithelial metaplasia; Sertoli cell carcinoma; Embryonal carcinoma; Choriocarcinoma.

Fibrosarcoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic Rhabdomyosarcoma; sarcoma; synovial sarcoma; angiosarcoma; Kaposi's sarcoma; lymphangiosarcoma; osteosarcoma; gingivosarcoma blasts; amelocytoma, malignant; myeloblastic fibrosarcoma; myeloid sarcoma; mast cell sarcoma.

Plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myelogenous leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; leukemia; hairy cell leukemia; but not limited to.

Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytes; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; malignant melanoma; apigmented melanoma; superficial melanoma; malignant melanoma in giant pigmented nevi; epithelioid melanoma; Not limited.

Examples of brain/spinal cord cancers include pineal tumor, malignant; chordoma; glioma, malignant; ependymoma; blastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectoderm; cerebellar sarcoma; Membraneoma, malignant; neurofibrosarcoma; schwannoma, malignant include, but are not limited to.

ovarian stromal tumor; capsule; granulosacytoma; androblastoma; Leydig cell tumor; lipid cell tumor; nevus blue, malignant; fibrous histiocytoma, malignant; mixed tumor, malignant; Müllerian mixed tumor; nephroblastoma; hepatoblastoma; dermatoma, malignant; dysgerminoma, teratoma, malignant; ovarian goiter, malignant; mesonephroma, malignant; hemangioendothelioma, malignant; malignant histiocytosis; immunoproliferative small bowel disease.

For any given tumor type, several tumor cell lines or tumor cell line variants may be commercially available. According to some embodiments, pools of some of these cell lines, either as whole cell mixtures or by making membrane preparations from whole cell mixtures, are used to form an array of cell surface tumor antigens for that tumor type. can provide

According to some embodiments, the tumor cell or tumor cell line or tumor cell line variant is rendered growth incapable by irradiation.

Exogenous Immunomodulatory Molecules According to some embodiments, exogenous immunomodulatory molecules of the invention are polypeptides that mediate stimulation of immune cells alone or in combination with other exogenous immunomodulatory molecules. According to some embodiments, the exogenous immunomodulatory molecules of the invention, alone or in combination with other exogenous immunomodulatory molecules, are T lymphocytes, natural killer (NK) cells, dendritic cells (DC ), or polypeptides that mediate stimulation of B lymphocytes. According to some embodiments, the NK cells are memory-like NK cells. According to some embodiments, the T lymphocytes are cytotoxic T lymphocytes (CTL) (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 invention that the population of tumor cells comprising exogenous immunomodulatory molecules 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, may be T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, dendritic cells (DC), or B lymphocytes. effective to stimulate one or more of

According to some embodiments, stimulating immune cells refers to expansion of immune cells. According to some embodiments, stimulating immune cells refers to activating immune cells. According to some embodiments, stimulating immune cells refers to increasing immune cell cytotoxicity. According to some embodiments, "stimulating immune cells" refers to a combination of one or more of expansion, activation, and/or increased cytotoxicity of immune cells. According to some embodiments, one or more exogenous immunomodulatory molecules expressed by the population of tumor cells are immune cells (e.g., T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, dendritic cells). It is effective in activating and/or expanding dysmorphic 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 immune killer cells (e.g., T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, effective in activating and/or expanding dendritic cells (DC) or B lymphocytes) in vivo. Described herein are assays for detecting whether exogenous immunostimulatory molecules are effective in stimulating immune killer cells. 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 T lymphocytes, Genetically engineered to stimulate one or more of natural killer (NK) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes (e.g., CD8+ T cells), natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, comprising a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes.

According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 3 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least four stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 5 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 5 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 6 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 7 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 8 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 9 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 10 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or comprising at least 11 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 12 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 13 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or comprising at least 14 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 15 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or comprising at least 16 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or comprising at least 17 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 18 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 19 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 20 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or comprising at least 21 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 22 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 23 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 24 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 25 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 26 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 27 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 28 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 29 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes. According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or at least 30 stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes.

According to some embodiments, the allogeneic vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK ) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or characterized by the expression of stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes.

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 are chimeric or fusion molecules, e.g., two or more each encoding at least one domain of a protein. are made by joining separate genes 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 the first tumor cell or population of tumor cells is a first A second tumor cell or population of tumor cells comprises a second immunomodulatory molecule. According to some embodiments, the allogeneic vaccines described herein comprise tumor cells comprising one or more exogenous immunomodulatory molecules, wherein the first tumor cell or population of tumor cells is a first It comprises one immunomodulatory molecule and a second immunomodulatory molecule, and the second tumor cell or population of tumor cells comprises a third immunomodulatory molecule. Thus, it is understood that the exogenous immunomodulatory molecules described herein can be present in the tumor cell population in cis (all on the same cell) or trans (each on different cells or a combination of each). According to some embodiments, exogenous immunostimulatory molecules are displayed on the surface of genetically engineered tumor cells.

According to some embodiments, the exogenous immunomodulatory molecule has a pre-existing or a combination of all three for their ability to reverse the immunosuppression of According to some embodiments, combinations of immunomodulatory molecules are evaluated and selected by human mixed lymphocytic tumor cell responses. According to some embodiments, the exogenous immunomodulatory molecule is selected from cytokines, TNF family members, secretory receptors, chaperones, IgG superfamily members, and chemokine receptors.

According to some embodiments, an allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes , natural killer (NK) cells, dendritic cells (DC) or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes, the exogenous immunomodulatory molecules comprising one or more cytokine proteins; Regulatory molecules include one or more TNF family member proteins; exogenous immunomodulatory molecules include one or more secreted receptor proteins; exogenous immunomodulatory molecules include one or more chaperone proteins; exogenous immunomodulatory The molecule comprises one or more IgG superfamily member proteins; and/or the exogenous immunomodulatory molecule comprises one or more chemokine receptor proteins.

According to some embodiments, an allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells comprise one or more genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC) or B lymphocytes of the population, the population of which is characterized by T lymphocytes, natural killer (NK) cells comprising a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate cells, dendritic cells (DCs), or B lymphocytes, wherein the exogenous immunomodulatory molecules are one or more cytokine family member proteins and one or more TNF family member proteins; the exogenous immunomodulatory molecule comprises one or more cytokine family member proteins and one or more secretory receptor proteins; the exogenous immunomodulatory molecule comprises one or more comprises a cytokine family member protein and one or more chaperone proteins; exogenous immunomodulatory molecule comprises one or more cytokine family member protein and one or more IgG superfamily member protein; exogenous immunomodulatory molecule comprises: Contains one or more cytokine family member proteins and one or more chemokine receptor proteins.

According to some embodiments, an allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells comprise one or more genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC) or B lymphocytes of the population, the population of which is characterized by T lymphocytes, natural killer (NK) cells comprising a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate cells, dendritic cells (DCs), or B lymphocytes, wherein the exogenous immunomodulatory molecules are one or more TNF family member proteins and one or more secreted receptor proteins; the exogenous immunomodulatory molecule comprises one or more TNF family member proteins and one or more chaperone proteins; the exogenous immunomodulatory molecule comprises one or more TNF family Includes member proteins and one or more IgG superfamily member proteins; exogenous immunomodulatory molecules include one or more TNF family member proteins and one or more chemokine receptor proteins.

According to some embodiments, an allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells comprise one or more genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC) or B lymphocytes of the population, the population of which is characterized by T lymphocytes, natural killer (NK) cells comprising a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate cells, dendritic cells (DCs), or B lymphocytes, wherein the exogenous immunomodulatory molecules are one or more secretory receptor proteins and one or more chaperone proteins; the exogenous immunomodulatory molecule comprises one or more secreted receptor proteins and one or more IgG superfamily member proteins; the exogenous immunomodulatory molecule comprises one or more secreted It includes a receptor protein and one or more chemokine receptor proteins.

According to some embodiments, an allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells comprise one or more genetically engineered to stimulate one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC) or B lymphocytes of the population, the population of which is characterized by T lymphocytes, natural killer (NK) cells comprising a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate cells, dendritic cells (DCs), or B lymphocytes, wherein the exogenous immunomodulatory molecules are one or more TNF family member proteins and one or more secreted receptor proteins; the exogenous immunomodulatory molecule comprises a chaperone protein and one or more IgG superfamily member proteins; the exogenous immunomodulatory molecule comprises one or more chaperone proteins and one including the above chemokine receptor proteins.

According to some embodiments, an allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes , natural killer (NK) cells, dendritic cells (DC), or B lymphocytes, the populations of which are T lymphocytes, natural killer (NK) cells, dendritic cells (DCs), or a plurality of stably expressed exogenous immunomodulatory molecules effective to stimulate B lymphocytes, wherein the exogenous immunomodulatory molecules are one or more IgG superfamily member proteins and one including the above chemokine receptor proteins.

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

According to some embodiments, the exogenous immunomodulatory molecule is selected from the group shown in Table 2. According to some embodiments, exogenous immunomodulatory molecules are derived from mice. According to some embodiments, the exogenous immunomodulatory molecule is of human origin.

According to some embodiments, the exogenous immunomodulatory molecule is selected from one or more of TNF family members, secretory receptors, chaperone proteins, IgG superfamily members, chemokine receptors. According to some embodiments, the TNF family member is selected from TNF family members listed in Table 2. According to some embodiments, the secretory receptor is selected from those listed in Table 2. According to some embodiments, the chaperone protein is selected from chaperone proteins listed in Table 2. According to some embodiments, the IgG superfamily member is selected from IgG superfamily members listed in Table 2. According to some embodiments, the chemokine receptor is selected from the chemokine receptors listed in Table 2.

According to some embodiments, the exogenous immunomodulatory molecules of Table 2 are membrane-bound (ie, contain membrane anchors). According to other embodiments, the exogenous immunomodulatory molecule is secreted. According to some embodiments, the membrane-bound form of the immunomodulator is 4-1BB ligand, BAFF, April, CD40 ligand, CD80, CD86, Flt3 ligand, GM-CSF, HSP90, ICOS ligand, IL-12, One or more selected from the group consisting of 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.

According to some embodiments, the exogenous immunomodulatory molecule of Table 2 is a molecule having a wild-type amino acid sequence. According to some embodiments, the exogenous immunomodulatory molecules of Table 2 are molecules with variant amino acid sequences.

According to some embodiments, the exogenous immunomodulatory molecule is 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, transmembrane region so as to remove Engineered FLT-3 ligand, GM-CSF, CD8 membrane anchor and GMCSF engineered to have 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 one or more exogenous immunomodulatory molecules comprises at least three essential immunomodulatory molecules, wherein the at least three essential immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70) , and CD28 ligand (CD28L), which includes CD80, CD86 or both. Additional immunomodulatory components identified as R may also be present, according to some embodiments.

According to some embodiments, the allogeneic vaccine comprises one or more tumor-specific antigens and three stably expressed essential exogenous immunomodulatory molecules effective to stimulate the MNC population; It contains a population of proliferation incompetent tumor cells expressing OX40L, CD70 and CD28L. According to some embodiments, the ENLST™ cell population comprises one or more tumor-specific antigens and three stably expressed requisite exogenous antigens effective to stimulate synergistic expansion of CTLs. and CD28L, including CD80, CD86, or both. According to some embodiments, the allogeneic vaccine is 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 , 21, 22, 23, 24, or 25 immunomodulators. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stably expressed exogenous immunomodulatory It is engineered to stably express the molecules OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and one R subset, which includes 3-25 global immune regulators. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stably expressed exogenous immunomodulatory It is engineered to stably express the molecules OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and two R-subsets, which include 3-25 global immune regulators. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stably expressed exogenous immunomodulatory It is engineered to stably express the molecules OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and three R subsets, which include 3-25 global immune regulators. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three requisite stably expressed exogenous Engineered to stably express immunomodulatory molecules, CD28L, including OX40L, CD70, and CD80, CD86, or both, and four R-subsets, including 3-25 global immunomodulators. be. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three requisite stably expressed exogenous Engineered to stably express the immunomodulatory molecules CD28L, which includes OX40L, CD70, and CD80, CD86, or both, and the 5 R subsets, which include 3-25 global immunomodulators. be. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three requisite stably expressed exogenous Engineered to stably express the immunomodulatory molecules CD28L, including OX40L, CD70, and CD80, CD86, or both, and 6 R subsets, including 3-25 global immunomodulators. be. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three requisite stably expressed exogenous Engineered to stably express the immunomodulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and the 7 R subsets, which include 3-25 global immunomodulators. be. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three requisite stably expressed exogenous It is engineered to express the immunomodulatory molecules, OX40L, CD70, and CD28L, which includes CD80, CD86, or both, and the 8 R subsets, which include 3-25 global immunomodulators. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three requisite stably expressed exogenous Engineered to stably express the immunomodulatory molecules CD28L, including OX40L, CD70, and CD80, CD86, or both, and 9 R subsets, including 3-25 global immunomodulators. be. According to some embodiments, the allogeneic vaccine comprises a population of tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three requisite stably expressed exogenous Genetically engineered to stably express immunomodulatory molecules, OX40L, CD70, and CD28L, including CD80, CD86, or both, and 10 R-subsets, including 3-25 global immunomodulators be done.

According to some embodiments, the exogenous immunomodulatory molecule ^R1 is APRIL. According to some embodiments, the exogenous immunomodulatory molecule ^R2 is BAFF. According to some embodiments, exogenous immunomodulatory molecule R ³ is 4-IBB ligand. According to some embodiments, the exogenous immunomodulatory molecule ^R4 is CD30L. According to some embodiments, exogenous immunomodulatory molecule ^R5 is CD40 ligand. According to some embodiments, the exogenous immunomodulatory molecule ^R6 is CD80. According to some embodiments, the exogenous immunomodulatory molecule ^R7 is CD86. According to some embodiments, exogenous immunomodulatory molecule ^R8 is FLT-3 ligand. According to some embodiments, the exogenous immunomodulatory molecule ^R9 is HSP-70. According to some embodiments, the exogenous immunomodulatory molecule R ¹⁰ is HSP-90. According to some embodiments, exogenous immunomodulatory molecule R ¹¹ is an ICOS ligand. According to some embodiments, the exogenous immunomodulatory molecule ^R12 is IL-10R. According to some embodiments, the exogenous immunomodulatory molecule R ¹³ is IL-12. According to some embodiments, exogenous immunomodulatory molecule R ¹⁴ is IL-15. According to some embodiments, exogenous immunomodulatory molecule R ¹⁵ is IL-18. According to some embodiments, exogenous immunomodulatory molecule R ¹⁶ is IL-2. According to some embodiments, the exogenous immunomodulatory molecule R ¹⁷ is IL-21. According to some embodiments, exogenous immunomodulatory molecule R ¹⁸ is IL-23. According to some embodiments, the exogenous immunomodulatory molecule R ¹⁹ is IL-7. According to some embodiments, the exogenous immunomodulatory molecule ^R20 is LIGHT. According to some embodiments, the exogenous immunomodulatory molecule ^R21 is a RANK ligand. According to some embodiments, the exogenous immunomodulatory molecule ^R22 is the TGF-b receptor. According to some embodiments, the exogenous immunomodulatory molecule ^R23 is TNF. According to some embodiments, the exogenous immunomodulatory molecule R ²⁴ is GM-CSF.

According to some embodiments, the exogenous immunomodulatory molecule R is from 1 to 30 comprehensive, 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 APRIL, BAFF, 4-IBB ligands; CD30 ligand, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove the transmembrane region, GM-CSF, CD8 membrane anchor and engineered to have an IRES compatible signal sequence GMCSF, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, CD8 with membrane anchor an exogenous immunomodulatory molecule selected from the group consisting of IL-7, LIGHT, OX-40 ligand, RANK ligand, TGF-b receptor, and TNF that has been engineered. According to some embodiments, the exogenous immunomodulatory molecule is between 1 and 30 global, ie 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, and at least 3 The immunomodulatory molecule is OX40 ligand (OX40L), CD27 ligand, and CD28 ligand, including CD80, CD86, or both, and additional immunomodulatory components are identified as R ¹ -R ²⁴ , APRIL, BAFF , 4-IBBL ligand (4-IBBL), CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove the transmembrane region, CD8 membrane anchor and IRES compatible signal sequence. GMCSF engineered to have 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.

According to some embodiments, the exogenous immunomodulatory molecule R is between 1 and 20 comprehensive, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, removes 12, 13, 14, 15, 16, 17, 18, 19, or 20 APRIL, BAFF, 4-1BB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, transmembrane domains GM-CSF, GMCSF engineered to have a CD8 membrane anchor and 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 an exogenous immunomodulatory molecule selected from the group consisting of TNF. According to some embodiments, the exogenous immunomodulatory molecule R is between 1 and 20 comprehensive, 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 3 immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand , additional immunomodulatory components identified to be R ¹ -R ²⁴ , APRIL, BAFF, 4-IBB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, transmembrane domain to remove Engineered FLT-3 ligand, GMCSF engineered to have CD8 membrane anchor and IRES compatible signal sequence, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL- 18, selected from the group consisting of 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 be done.

According to some embodiments, the exogenous immunomodulatory molecule R is 1-10 global, ie 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 transmembrane region, GM-CSF, CD8 membrane anchor and IRES compatible GMCSF, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, engineered to have a signal sequence, An exogenous immunomodulatory molecule selected from the group consisting of 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 molecule R is 1-10 global, ie 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 including exogenous immunomodulatory molecules, at least three of which are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand; and additional immunomodulatory components identified as R ¹ -R ²⁴ , APRIL , BAFF, 4-IBB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, FLT-3 ligand engineered to remove transmembrane region, CD8 membrane anchor and IRES compatible signal sequence Engineered GMCSF, HSP-70, HSP-90, ICOS ligand, IL-10R, IL-12, IL-15, IL-18, IL-2, IL-21, IL-23, IL-7, CD8 membranes selected from the group consisting of IL-7, LIGHT, RANK ligand, TGF-b receptor, and TNF engineered to have an anchor.

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

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

According to some embodiments, exogenous immunomodulatory molecule R removes 14 APRIL, BAFF, 4-1BB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, transmembrane domain GM-CSF, GM-CSF engineered to have a CD8 membrane anchor and 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 an exogenous immunomodulatory molecule selected from the group consisting of TNF. According to some embodiments, the exogenous immunomodulatory molecules comprise 14 exogenous immunomodulatory molecules, wherein at least 3 immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand, and CD28 ligand; Additional immunomodulatory factors are recognized to be R ¹ -R ²⁴ , engineered to remove APRIL, BAFF, 4-IBB ligand, CD30L, CD40 ligand, CD80, CD86, FLT-3 ligand, transmembrane domain engineered FLT-3 ligand, CD8 membrane anchor and GM-CSF engineered to have 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 selected.

According to some embodiments, the exogenous immunomodulatory molecule 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 Each of the receptor and TNF are wild-type molecules. According to some embodiments, the exogenous immunomodulatory molecule 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 Each of the receptor and TNF is a mutant or variant sequence.

According to some embodiments, the exogenous immunomodulatory molecule ^R24 is a CD86 variant engineered to have an IRES-matched signal sequence. According to some embodiments, the exogenous immunomodulatory molecule ^R25 is a FLT3L variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule ^R26 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 ^R27 is an HSP70 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule ^R28 is an HSP-90B1 (GRP94/96) variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule ^R29 is an HSP90 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule ^R30 is an ICOSL variant engineered to have an IRES-compatible signal sequence. According to some embodiments, the exogenous immunomodulatory molecule ^R31 is an IL10R variant engineered to remove the transmembrane region. According to some embodiments, exogenous immunomodulatory molecule R ³² is an IL-Rα variant (VSV-GM-CSF tag) engineered to remove the transmembrane domain. According to some embodiments, the exogenous immunomodulatory molecule ^R33 is an IL12 variant engineered to be single chain containing a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule ^R34 is an IL15 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule ^R35 is an IL18 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule ^R36 is an IL2 variant engineered to have a CD8 membrane anchor and an IRES matching sequence. According to some embodiments, the exogenous immunomodulatory molecule ^R37 is an IL21 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule ^R38 is an IL23 variant engineered to be single chain containing a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule ^R39 is an IL17 variant engineered to have a CD8 membrane anchor. According to some embodiments, the exogenous immunomodulatory molecule ^R40 is a TGFb-R variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory ^R41 molecule is a TGFb receptor III variant engineered to remove the transmembrane region. According to some embodiments, the exogenous immunomodulatory molecule ^R42 is a mIFNα variant engineered to be membrane bound. According to some embodiments, the exogenous immunomodulatory molecule ^R43 is a mIFNαγ variant engineered to be membrane bound. According to some embodiments, the exogenous immunomodulatory molecule ^R44 is a CD40L variant that is cleavage resistant.

Table 3 below shows the R groups, R ¹ -R ⁴⁴ .

According to some embodiments, the at least 12 vectors comprise 14 immunomodulatory factors, and the at least 3 immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD80, CD86, or Both are CD28 ligands (CD28L), and the remaining 11 immunomodulators are selected from R ¹ -R ⁴⁴ in Table 3. According to some embodiments, the at least 11 vectors comprise 14 immunomodulatory factors, and the at least 3 immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD80, CD86, or Both are CD28 ligands (CD28L), and the remaining 11 immunomodulators are selected from R ¹ -R ⁴⁴ in Table 3. According to some embodiments, at least 10 vectors comprise 14 immunomodulatory factors, and at least 3 immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD80, CD86, or Both are CD28 ligands (CD28L), and the remaining 11 immunomodulators are selected from R ¹ -R ⁴⁴ in Table 3. According to some embodiments, the 14 immunomodulatory agents are selected from Table 2, and the at least 3 immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD80, CD86, or both and the remaining 11 immunomodulators are selected from R ¹ -R ⁴⁴ in Table 3, 14 immunomodulators contained in 12 vectors. According to some embodiments, the 14 immunomodulatory agents are selected from Table 2, and the at least 3 immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD80, CD86, or both and the remaining 11 immunomodulators are selected from R ¹ -R ⁴⁴ in Table 3, 14 immunomodulators contained in 11 vectors. According to some embodiments, the 14 immunomodulatory agents are selected from Table 2, and the at least 3 immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD80, CD86, or both and the remaining 11 immunomodulators are selected from R ¹ -R ⁴⁴ in Table 3, 14 immunomodulators contained in 10 vectors. A vector may further include a tag.

According to some embodiments, the immunomodulatory factor is codon optimized. "Codon-optimized" means that the codons of a polynucleotide encoding a protein are used in certain organisms prior to others so that the encoded protein can be more efficiently expressed in that particular organism. It means to modify at a codon. The genetic code is degenerate because most amino acids are described by several codons called "synonyms" or "synonymous codons." However, codon usage by a particular organism is not random and is biased toward a particular codon triplet. Such codon usage biases may be associated 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 in an organism's genome. and could be even higher.

Cytokines According to one embodiment, the present disclosure includes a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK) cells, Genetically engineered to stimulate one or more of dendritic cells (DCs), or B lymphocytes, the population includes one or more cytokines, including allogeneic tumor cell vaccines. Accordingly, the present disclosure encompasses cytokines, including full-length cytokines, fragments, homologues, variants, or mutants. Cytokines include proteins that can affect the biological function of another cell. Biological functions affected by cytokines can 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 binding immune cells (e.g., T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, dendritic cells (DC), or B lymphocytes).

According to some embodiments, the cytokine is 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, selected from IL-21, IL-23, TGF-β, MCP-1, TNF-α and interferon alpha (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).

According to some embodiments, cytokines are secreted. According to some embodiments, the cytokine is membrane bound.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)
Granulocyte-macrophage colony-stimulating factor (GM-CSF; colony-stimulating factor 2; CSF2) is found on monocytes/macrophages and activated T cells and acts as a growth factor to stimulate and recruit 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 that contains a 17 amino acid signal peptide that can be cleaved to produce the mature 127 amino acid protein. The biological activity of GM-CSF occurs through 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 (eg, 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).

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

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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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.

According to some embodiments, a tumor cell line or tumor cell line variant may contain one or more proteins comprising a fusion between GM-CSF and HLA-I to allow membrane expression. According to some embodiments, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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.

Fms-like tyrosine kinase-3 ligand (Flt-3L)
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 dendritic cell production and development. Mice lacking Flt3L have low levels of dendritic cells, and administration of Flt3L to mice or humans results in very high levels of dendritic cells (Shortman et al., Steady-state and inflammatory dendritic-cell development, Nature Reviews Immunology, Vol.7.19-30 (2007)).

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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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.

According to some embodiments, the tumor cell line or tumor cell line variant comprises a soluble form of Flt3L. According to some embodiments, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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.

One of ordinary skill in the art, after armed with the teachings provided herein, will understand that the present invention encompasses any cytokine, whether now well known to those of ordinary skill in the art or discovered in the future. would do.

According to some embodiments, allogeneic tumor cell vaccines comprising populations of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens are used in one or more (e.g., 2, 3, 4 , 5, or more) cytokines, or variants or fragments thereof.

TNF Family Members According to one embodiment, the present disclosure includes a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK) including allogeneic tumor cell vaccines genetically engineered to stimulate one or more of cells, dendritic cells (DC), or B lymphocytes, the population comprising one or more TNF family members . Accordingly, the present disclosure encompasses TNF family member proteins, including full length, fragments, homologues, variants, or mutants of TNF family proteins. According to some embodiments, the TNF superfamily member is 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, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18, TNFβ, TNFSF1B, TNFγ, ectodysplasin Select from one or more of A (EDA) be done. According to some embodiments, the TNF superfamily member is TNFα. According to some embodiments, the TNF superfamily member is CD40L.

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

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 extracellular proteolytic cleavage and can function as cytokines. These proteins are expressed primarily by immune cells and regulate diverse cellular functions such as proliferation, differentiation, apoptosis, and embryogenesis, as well as regulation of immune responses and inflammation. The superfamily contains 19 members that bind to 29 members of the TNF receptor superfamily.

OX40L (TNFSF4, bTNF superfamily member 4)
OX40 ligand (OX40L) (CD252, TNFSF4), originally called glycoprotein 34 kDa (GP34), belongs to the TNF superfamily. It is mainly expressed on the surface of antigen-presenting cells (APC), including activated dendritic cells (DC), 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. Am. Immunol (2002) 168: 661-670. doi: 10.4049/jimmunol. 168.2.661; Ohshima, Y.; et al. , Blood (1998) 92:3338-3345].

OX40 (ACT35, CD134, TNFRSF4) is constitutively expressed on the cell surface of activated CD4+ T cells [Id., citing Ogawa R, et al. , Cytokine Growth Factor Rev. (2008) 19:253-262. doi: 10.1016/j. cytog fr. 2008.04.003, Paterson DJ, et al. Mol Immunol. (1987) 24:1281-1290. doi: 10.1016/0161-5890(87)90122-2]. It specifically binds OX40L and initiates a series of responses that contribute to promoting the proliferation and survival of CD4+ T cells and the secretion of cytokines [Id., 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 costimulatory signals as potent as CD28 costimulation ( Ditto). OX-40R binding enhances effector and memory effector T cell function by upregulating IL-2 production and extending effector T cell lifespan (Id.).

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

According to some embodiments of the disclosed invention, a tumor cell line or tumor cell line variant can be engineered to express a membrane-bound form of OX40L of SEQ ID NO: 108 on the membrane of the tumor cell. According to some embodiments, a 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:108. According to some embodiments, a 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:108. According to some embodiments, a 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:108. According to some embodiments, a 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:108. According to some embodiments, a 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:108. According to some embodiments, a 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, a 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, a 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, a 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.

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 as well as NK cells. The CD27 ligand and its receptor CD27 modulate immune responses by promoting T cell expansion and differentiation and NK enhancement. CD27 signals prevent apoptosis of antigen-specific CD8+ T cells late in the primary CD8+ T cell response. Lack of CD27 signal diminishes the quality of memory CD8+ T cell responses. However, memory CD8+ T cells, which, like naive cells, express surface CD27, do not require CD27 costimulation during secondary responses. Thus, in vivo, CD27 modulates primary antigen-specific CD8+ T cell responses by acting indirectly to prevent apoptosis of CD8+ T cells late in the primary response and is required for optimal quality of memory cells, although normally is not required for primed secondary CD8+ T cell responses (Dolfi, DV, et a., J. Immunol. (2008) 180(5):2912-2921). Full-length CD27 ligand (CD70) is a 193 amino acid protein, composed 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.

According to some embodiments of the disclosed invention, a tumor cell line or tumor cell line variant can be engineered to express a membrane-bound form of CD70 on the membrane of the tumor cell.

According to some embodiments of the disclosed invention, a tumor cell line or tumor cell line variant can be engineered to express a soluble form of CD70.

According to some embodiments of the disclosed invention, the tumor cell line or tumor cell line variant can be engineered to express the membrane-bound form of CD70 of SEQ ID NO: 109 on the membrane of the tumor cell. According to some embodiments, a 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:109. According to some embodiments, a 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:109. According to some embodiments, a 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:109. According to some embodiments, a 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:109. According to some embodiments, a 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:109. According to some embodiments, a 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, a 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, a 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, a 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

Naive CD8 T cells require more co-stimulatory activity to drive them to become activated effector cells than naive CD4 T cells. This requirement can be met in two ways. The simplest is priming by activated DCs with high intrinsic co-stimulatory activity. In some viral infections, dendritic cells are sufficiently activated to induce the IL-2 required for direct induction of CD8 T cells and their differentiation into cytotoxic effector cells, without the help of CD4 T cells. produces This property of DCs has been exploited to generate cytotoxic T cell responses against tumors. However, in most viral infections, activation of CD8 T cells 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 CDT cells by further activating APCs. B7 expressed by DCs primarily activates CD4 T cells to express IL-2 and CD40L. CD40L binds to CD40 on DCs and delivers additional signals that increase B7 and 4-IBBL expression by dendritic cells. This provides additional co-stimulation to naive CD8 T cells. IL-2 produced by activated CD4 T cells also acts to promote differentiation of effector CD8 T cells (Murphy, Kenneth. Janeway's Immunobiology: 8th ed. Chapter 15: Garland Science. (2012). ), at 372).

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

According to some embodiments of the disclosed invention, the R subset of immunomodulators can include membrane-bound forms of 4-IBBL. According to some embodiments of the disclosed invention, the R subset of immunomodulators can include soluble forms of 4-IBBL.

CD40L
The ligand for CD40, known as CD154 or CD40L, is a type II transmembrane protein that varies in molecular weight between 32 and 39 kDa due to post-translational modifications (Elgueta R et al., Molecular mechanism and function of CD40/CD40L 229(1):10.1111/j.1600-065X.2009.00782.x.doi:10.1111/j.1600-065X.2009.00 782.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 similar activity to the transmembrane form (Id., 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).

In nature, CD40L is a member of the TNF superfamily and is characterized by a sandwich extracellular structure composed of a β-sheet, an α-helical loop, and a β-sheet that allows trimerization of CD40L (Id., see Citation, 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 (Id., citing Carbone E et al., J Exp Med. 1997 Jun 16; 185 (12):2053-60). The widespread expression of the co-stimulatory pair of CD40L and CD40 indicates the pivotal role they play in various cell-mediated immune processes.

CD40L has three binding partners: CD40, α5β1 integrin, and αIIbβ3 integrin. CD40L acts as a co-stimulatory molecule and is particularly important on a subset of T cells called T follicular helper cells (TFH cells). CD40L binds CD40 on the surface of B cells and promotes cell-to-cell communication, thereby promoting B cell maturation and function. Defects in the CD40L gene are unable to undergo immunoglobulin class switching and are associated with hyper-IgM syndrome. Without CD40L, germinal center formation also ceases, preventing affinity maturation of antibodies, a key process of the adaptive immune system.

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

CD40 is also found in several types of cancer, including solid tumors and hematologic malignancies. CD40-mediated signaling in hematological cancers may mediate growth or regression, whereas CD40 signaling in solid tumors is only tumoricidal. These features are also seen in the SCID mouse model and are therefore likely due to TNF death domain signaling. There is also evidence of immunomodulation. For example, blocking the CD40/CD40L pathway reduces the protective efficacy of GM-CSF-secreting melanoma vaccines (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).

Tumor cell vaccines expressing CD40L have proven useful in cancer models. For example, conjugation 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 anti-tumor activity in preclinical mouse models (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)).

According to some embodiments, the R subset of immunomodulators can include CD40 ligand (CD40L). According to some embodiments of the disclosed invention, a tumor cell line or tumor cell line variant can be engineered to express an uncleaved form of the CD40L peptide of SEQ ID NO:6. According to some embodiments, a 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:6. According to some embodiments, a 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:6. According to some embodiments, a 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:6. According to some embodiments, a 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:6. According to some embodiments, a 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:6. According to some embodiments, a 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, a 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, a 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, a 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.

According to some embodiments, a tumor cell line or tumor cell line variant can be engineered to express an uncleaved form of the membrane-bound CD40L peptide of SEQ ID NO:7 on the membrane surface of the tumor cell. According to some embodiments, a 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:7. According to some embodiments, a 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:7. According to some embodiments, a 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:7. According to some embodiments, a 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:7. According to some embodiments, a 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:7. According to some embodiments, a 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, a 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, a 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, a 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.

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 the immunogenic response. TNF can be produced by other cell types such as, for example, CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons.

In its primary role as a regulator of immune cells, TNF inhibits fever, apoptotic cell death, cachexia, inflammation, and tumor formation; inhibits viral replication; sepsis vial IL-1 and IL-6 producing cells; can trigger the initiation of a response to Dysregulated TNF production is associated with a variety of human diseases, including Alzheimer's disease, major depression, psoriasis, and inflammatory bowel disease (IBD). TNF can be produced ectopically in the setting of malignancies and parallels parathyroid hormone both in causes of secondary hypercalcemia and in cancers where overproduction is associated.

TNF consists of a 26 kDa membrane-bound form and a 17 kDa soluble cytokine form. The soluble form of TNF is derived from 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 metalloprotease that recognizes the cleavage site of the extracellular domain of full-length TNF (Rieger, R., Chimeric form of tumor necrosis factor-alpha has enhanced surface expression and antibody activity, Cancer Gene Therapy, 2009, 16, 53-64). Deletion of the TNF cleavage site improves the membrane stability of TNF (Id.).

TNF is known to have antiproliferative and cytotoxic effects on cells, reduce tumor blood flow and tumor vascular damage, and stimulate the immune response by stimulating the activity of macrophages and NK cells. can be adjusted. However, the use of TNF as a therapeutic agent itself has been limited by dose-dependent hypotension and capillary leakage that can lead to sepsis-like syndromes. Therefore, it needs to be presented in a way that limits systemic effects. TNF has been added to standard chemotherapeutic agents to improve response rates. Other approaches for administering TNF include injection of adenoviruses modified to express TNF in gastrointestinal malignancies. TNF compounds have also been developed that target tumor blood vessels (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 in treating inflammatory diseases such as psoriasis and rheumatoid arthritis. Recognizing this role, molecules such as antibodies have been designed to interfere with TNF activity. However, such therapy carries 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.

According to some embodiments, the subset of R members may include TNF. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a membrane-bound form of TNF on the membrane of the tumor cell. For example, according to some embodiments, the cell line variant comprises the peptide of SEQ ID NO:8. According to some embodiments, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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, a 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.

According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express an uncleaved, membrane-bound form of TNF. For example, according to some embodiments, a tumor cell line or tumor cell line variant may comprise the TNF protein of SEQ ID NO: 8 with one or more of the amino acids VRSSSRTPSDKP (SEQ ID NO: 104) deleted (e.g., See SEQ ID NO:26).

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

According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express an uncleaved membrane-bound chimeric form of CD40L and TNF. For example, according to some embodiments, the ligand binding portion of the TNF molecule is fused with the transmembrane domain and proximal extracellular domain of CD40L such that TNF lacks a defined TNF alpha cleaving enzyme (TACE) site. can be According to some embodiments, intracellular, transmembrane, and partial extracellular portions of CD40L may be fused with the extracellular region of TNF distal to the TACE cleavage site. According to some embodiments, a CD40L/TNF chimeric form may 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 connecting peptide between 1-30 amino acids in length. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically 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, a tumor cell line or tumor cell line variant can be genetically engineered to contain 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, a tumor cell line or tumor cell line variant may be genetically engineered to contain 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, a tumor cell line or tumor cell line variant can be genetically engineered to contain 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, a tumor cell line or tumor cell line variant may be genetically engineered to contain 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, a tumor cell line or tumor cell line variant may be genetically engineered to contain 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, a tumor cell line or tumor cell line variant may be genetically engineered to contain 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, a tumor cell line or tumor cell line variant can be genetically engineered to contain 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, a tumor cell line or tumor cell line variant can be genetically engineered to contain a fusion protein having at least 99% sequence identity to the proteins of SEQ ID NO:9 and SEQ ID NO:10. .

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

According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express an uncleaved membrane-bound chimeric form of CD40L and TNF. For example, according to some embodiments, a ligand portion of a TNF molecule can be fused with an extracellular portion of CD40L, wherein CD40L comprises an extracellular portion that is uncleaved, and TNF is associated with a defined TACE site ( for example, the cleavage site between amino acids 76 and 77). According to some embodiments, some or all of the CD40L peptide sequences are fused to the extracellular region of the TNF peptide sequences distal to the TACE cleavage site. According to some embodiments, a chimeric form of CD40L/TNF may comprise the sequence of SEQ ID NO:31. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain a fusion protein having at least 60% sequence identity to the protein of SEQ ID NO:31. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain a fusion protein having at least 70% sequence identity to the protein of SEQ ID NO:31. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain a fusion protein having at least 80% sequence identity to the protein of SEQ ID NO:31. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain a fusion protein having at least 90% sequence identity to the protein of SEQ ID NO:31. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain a fusion protein having at least 95% sequence identity to the protein of SEQ ID NO:31. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain a fusion protein having at least 96% sequence identity to the protein of SEQ ID NO:31. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain a fusion protein having at least 97% sequence identity to the protein of SEQ ID NO:31. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain a fusion protein having at least 98% sequence identity to the protein of SEQ ID NO:31. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain a fusion protein having at least 99% sequence identity to the protein of SEQ ID NO:31.

According to some embodiments, allogeneic tumor cell vaccines comprising populations of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens are used in one or more (e.g., 2, 3, 4 , 5, or more), or variants or fragments thereof.

Secretory Receptors According to one embodiment, the present disclosure includes a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK) Genetically engineered to stimulate one or more of cells, dendritic cells (DCs), or B lymphocytes, the population comprising one or more secretory receptors, including allogeneic tumor cell vaccines .

According to some embodiments, the R immunomodulator can comprise one or more (eg, 2, 3, 4, 5, or more) secretory receptor proteins, or variants or fragments thereof. According to some embodiments, the secretory receptor is IL10R, TGFβR3, or both.

Interleukin-10 (IL-10) is an important immunosuppressive cytokine produced by a variety of 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 signals emanating from the cell surface. IL-10R is a heterotetramer composed of two subunits of IL-10Rα and two subunits of IL-10Rβ (Id., citing Moore, KW, et al., Annu. Rev. Immunol (2001) 19: 683-765). The IL-10Rα subunit is unique to IL-10 signaling, whereas the IL-10Rβ subunit is shared with other cytokine receptors such as IL-22, IL-26 and interferon-λ (Id.). IL-10 downstream signaling through IL-10R inhibits the induction of proinflammatory cytokines by blocking NF-κB-dependent signals (Id., citing Saraiva, M., and O'Garra , A. Nat. Rev. Immunol. (2010) 10:180-181).

Transforming growth factor-beta 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. Expression levels of TβRIII are 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 abundantly expressed TGF-β superfamily receptor and TGF-β superfamily member TGF-β1, TGF-β2, or TGF-β3, inhibin, BMP-2, BMP-4, BMP -7, and GDF-5, thereby functioning as TGF-β superfamily co-receptors and presenting these ligands to their respective signaling receptors, TGF-β1 to Smad transcription factors, BMP , or activates or represses (in the case of inhibins) activin signaling. For example, in the case of TGF-β1, 2, or 3, TβRIII presents a 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 causing phosphorylation of Smad2/3. Phosphorylation of Smad2 and Smad3 results in 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 Smad signaling through its receptors, TβRIII also mediates ligand-dependent and independent p38 pathway signaling. TβRIII can also undergo ectodomain shedding to generate soluble TβRIII (sTβRIII). It binds and sequesters members of the TGF-β superfamily and inhibits their signaling. Although sTβRIII expression has been demonstrated to correlate with cell surface expression of TβRIII, little is known about the regulation of sTβRIII production. Shedding of TβRIII is partially mediated by the membrane-type matrix metalloproteinases (MT-MMPs) MT1-MMP and/or MT3-MMP, and plasmin, a serine proteinase that has been shown to cleave the extracellular domain of TβRIII. can be mediated. In addition, shedding of TβRIII is regulated by the tyrosine phosphatase inhibitor pervanadate. In support of this, MT-MMP and the 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 GAIP-interacting protein C-terminus (GIPC), a PDZ domain-containing protein. 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 ongoing invasion of breast cancer. The cytoplasmic domain of TβRIII is phosphorylated by TβRII, resulting in TβRIII binding to the scaffold protein β-arrestin2. TβRIII/β-arrestin2 interaction results in co-internalization of β-arrestin2/TβRIII/TβRII and downregulation of TGF-β signaling. The interaction between TβRIII and β-arrestin2 modulates BMP and TGF-beta signaling. TβRIII forms a complex with the BMPI-type receptor ALK6 in a beta-arrestin2-dependent manner and mediates ALK6 internalization and stimulation of ALK6-specific BMP signaling events. TβRIII negatively regulates NFκ-B signaling in the context of breast cancer through interaction with β-arrestin 2, resulting in epithelial cell adhesion to fibronectin, fibril formation, and α5β1 internalization Regulates focal adhesion formation through modulation of trafficking to focal adhesions, activates Cdc42, alters the actin cytoskeleton, and suppresses migration of normal and cancerous ovarian epithelial cells. During development, TβRIII plays an important role in the formation of the atrioventricular eminence of the heart. Consistent with the critical role of TβRIII during development, TGFβR3 null mice are embryonic lethal due to heart and liver defects. TGFβR3 was recently identified as a tumor suppressor of multiple types of human cancer, including breast, lung, ovarian, pancreatic and prostate cancer. Loss of TGFβR3 in these cancer types correlates with disease progression, leading to increased motility and invasion in vitro and invasion and metastasis in vivo (http://atlasgeneticsoncology.org /Genes/TGFBR3ID42541ch1p33.html, visited Aug. 26, 2019).

Chaperones According to some embodiments, the subset of R immunomodulators may comprise one or more chaperone proteins. According to one embodiment, the present disclosure includes a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK) cells, dendritic Genetically engineered to stimulate one or more of DCs, or B lymphocytes, the population includes allogeneic tumor cell vaccines containing one or more chaperone proteins. Accordingly, the present disclosure encompasses chaperone proteins, including full-length, fragments, homologues, variants, or mutants of chaperone proteins. Chaperones are a group of functionally related proteins that assist in protein folding within cells under physiological and stress conditions. According to some embodiments, the chaperone protein is GRP78/BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, protein disulfide isomerase (PDI), peptidyl prolyl cis-trans-isomerase (PPI) , Erp57, Hsp60, Hsp70, Hsp90, Hsp100.

According to some embodiments, the chaperone protein is membrane bound.

According to some embodiments, allogeneic tumor cell vaccines comprising populations of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens are used in one or more (e.g., 2, 3, 4 , 5, or more), or variants or fragments thereof.

Immunoglobulin superfamily (IgSF)
According to some embodiments, the subset of R immunomodulators may comprise one or more IgSF proteins. According to one embodiment, the present disclosure includes a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK) cells, dendritic Genetically engineered to stimulate one or more of DCs, or B lymphocytes, the population includes allogeneic tumor cell vaccines containing one or more IgS family proteins. Accordingly, the present disclosure encompasses members of the IgSF superfamily, including full-length, fragments, homologues, variants, or mutants of IgSF superfamily members. The immunoglobulin superfamily (IgSF) is a class of proteins involved in cell adhesion, binding, and recognition processes. Molecules are classified as members of this superfamily based on common structural features with immunoglobulins. They all have domains known as immunoglobulin domains or folds. Members of the IgSF include cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors and intracellular muscle proteins. be 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); co-receptors (e.g. CD4, CD8); costimulatory molecules or inhibitory molecules (e.g. CD28, Cd80, CD86); receptors on natural killer cells (e.g. killer cell immunoglobulin-like receptors (KIR)); receptors on leukocytes (e.g. leukocyte immunoglobulin-like receptors) IGSF CAMs (eg NCAM, ICAM-1); cytokine receptors; growth factor receptors; receptor tyrosine kinases/phosphatases; IgG binding receptors.

According to some embodiments, the IgSF family member is membrane bound.

Poliovirus receptor (PVR/CD155) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. PVR/CD155 mediates NK cell adhesion and induces NK cell effector functions. PVR/CD155 binds to two different NK cell receptors, CD96 and CD226. These interactions accumulate at cell-cell contact sites and lead to the formation of mature immune synapses between NK cells and target cells. This causes adhesion and secretion of lytic granules and IFN-γ (IFNγ), activates cytotoxicity of activated NK cells, and promotes NK cell-target cell modular exchange and PVR transfer to NK cells. can.

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.

The CD48 antigen (surface antigen class 48), also called B-lymphocyte activation marker (BLAST-1) or signaling lymphocyte activation molecule 2 (SLAMF2), is the protein encoded by the CD48 gene in humans. CD48 is a member of the CD2 subfamily of IgSFs, 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, endothelial cells and is involved in the activation and differentiation pathways of these cells.

NK-TB antigen (NTBA) is a surface molecule expressed on NK cells, T cells and B cells. On human NK cells, NTBA has been shown to act primarily as a co-receptor, as it can only induce cytolytic activity in cells expressing high surface densities of natural cytotoxic receptors (NCRs). Molecular cloning has revealed that NTBA is a member of the Ig superfamily characterized by structural features that allow it to be assigned to the CD2 family.

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 the NK-TB antigen.

Immunoglobulins (Igs) are glycoproteins produced by immune cells. Antibodies are serum proteins whose molecules have small regions on their surface that are complementary to small chemical clusters on their target. There are at least two of these complementary regions (called complementary determining regions (CDRs), or antibody-binding sites, or antigen-binding sites) per antibody molecule, with some types of antibody molecules having 10, 8, or In some species, as many as a dozen may react with corresponding complementary regions (antigenic determinants or epitopes) on the antigen to bind together several molecules of a multivalent antigen to form a lattice. Immunoglobulins play an important role in the immune response by binding to specific antigens such as those displayed by bacteria and viruses. According to some embodiments, the binding of immunoglobulins to antigens can target them for destruction by the subject's immune cells.

The basic structural units of an entire antibody molecule are four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each containing about 440 amino acids). including). The two heavy chains and the two light chains are bound together by a combination of non-covalent and covalent (disulfide) bonds. The molecule is composed of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of the light chain and the N-terminal region of the heavy chain. Both light and heavy chains normally cooperate to form the antigen-binding surface.

There are five classes of antibodies in mammals: IgA, IgD, IgE, IgG, and IgM, each with its own class of heavy chains—α (for IgA), δ (for IgD), ε ( for IgE), γ (for IgG), and μ (for IgM). In addition, IgG immunoglobulins have four subclasses (IgG1, IgG2, IgG3, IgG4), each having γ1, γ2, γ3, and γ4 heavy chains. Secreted IgM is a pentamer composed of five four-chain units, providing a total of ten antigen-binding sites. Each pentamer contains one copy of the J chain covalently inserted between two adjacent tail regions.

A diverse library 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 a single polypeptide chain in which the heavy and light chain variable domains are joined by a polypeptide spacer are generated by randomly combining the heavy and light chain V genes using PCR. be able to.

According to some embodiments, a tumor cell line or tumor cell line variant may be engineered to express an IgG1 heavy chain constant region. In nature, the Ig gamma-1 (IgG-1) chain C region is the protein encoded by the human IGHG1 gene. According to some embodiments, the tumor cell line or tumor cell line variant may express the membrane-bound IgG-1 chain C protein of SEQ ID NO:1. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a secretory form of IgG-1 chain C of SEQ ID NO:2. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a secretory form of IgG-1 chain C of SEQ ID NO:3. According to some embodiments, the tumor cell line or tumor cell line variant has at least 60% It can be engineered to contain a fusion protein with sequence identity. According to some embodiments, the tumor cell line or tumor cell line variant has at least 70% It can be engineered to contain a fusion protein with sequence identity. According to some embodiments, the tumor cell line or tumor cell line variant has at least 80% It can be engineered to contain a fusion protein with sequence identity. According to some embodiments, the tumor cell line or tumor cell line variant has at least 90% It can be engineered to contain a fusion protein with sequence identity. According to some embodiments, the tumor cell line or tumor cell line variant has at least 95% It can be engineered to contain a fusion protein with sequence identity. According to some embodiments, the tumor cell line or tumor cell line variant has at least 96% It can be engineered to contain a fusion protein with sequence identity. According to some embodiments, the tumor cell line or tumor cell line variant has at least 97% It can be engineered to contain a fusion protein with sequence identity. According to some embodiments, the tumor cell line or tumor cell line variant has at least 98% It can be engineered to contain a fusion protein with sequence identity. According to some embodiments, the tumor cell line or tumor cell line variant has at least 99% It can be engineered to contain a fusion protein with sequence identity.

According to some embodiments, the tumor cell line or tumor cell line variant comprises , SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, and SEQ ID NO:46. According to some embodiments, the tumor cell line or tumor cell line variant comprises , SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, and SEQ ID NO:46. According to some embodiments, the tumor cell line or tumor cell line variant comprises , SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, and SEQ ID NO:46. According to some embodiments, the tumor cell line or tumor cell line variant comprises , SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, and SEQ ID NO:46. According to some embodiments, the tumor cell line or tumor cell line variant comprises , SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, and SEQ ID NO:46. According to some embodiments, the tumor cell line or tumor cell line variant comprises , SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, and SEQ ID NO:46. According to some embodiments, the tumor cell line or tumor cell line variant comprises , SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, and SEQ ID NO:46. According to some embodiments, the tumor cell line or tumor cell line variant comprises , SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:45, and SEQ ID NO:46. According to some embodiments, the tumor cell line or tumor cell line variant comprises , 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 can be engineered to express an IgG protein capable of binding to a tumor cell specific antigen. For example, a tumor cell line or tumor cell line variant can be engineered to express an IgG protein capable of binding to the extracellular region of a prostate cancer-specific antigen, such as the prostate-specific membrane antigen (PSMA) (Chang, S., Overview of Prostate-Specific Membrane Antigen, Reviews in Urology, Vol.6 Suppl.10, S13 (2004)). According to some embodiments, a tumor cell line or tumor cell line variant can be engineered to express an IgG protein capable of binding an immune cell specific antigen. For example, a tumor cell line or tumor cell line variant can be engineered to express an IgG protein capable of binding a T cell marker such as CD3, CD4, or CD8. According to another example, a tumor cell line or tumor cell line variant can be engineered to express an IgG protein capable of binding a dendritic cell marker, such as CD11c or CD123.

According to some embodiments, a tumor cell line or tumor cell line variant may be engineered to express an IgG3 heavy chain constant region. In nature, the IgG3 heavy chain constant region contains the CH1-hinge-CH2-CH3 domains and in humans is encoded by the IGHG3 gene. The IGHG3 gene contains structural polymorphisms involving different hinge lengths. According to some embodiments, the tumor cell line or tumor cell line variant can be engineered to express the IgG3 heavy chain constant region of SEQ ID NO:4. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 lacking amino acids 1-76. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 lacking amino acids 1-76. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically 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, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:16) comprising the E213Q variant. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:17) comprising the P221L variant. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:18) comprising the E224Q variant. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:19) comprising the Y226F variant. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:20) comprising the D242N variant. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:21) comprising the N245D variant. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:22) comprising the T269A variant. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:23) comprising the S314N variant. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:24) comprising the S314 variant. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to express a derivative of SEQ ID NO:4 (SEQ ID NO:25) comprising the F366Y variant.

According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO:4. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO:4. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO:4. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO:4. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO:4. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO:4. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO:4. According to some embodiments, a tumor cell line or tumor cell line variant can be genetically engineered to contain one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO:4. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO:4.

According to some embodiments, a tumor cell line or tumor cell line variant may be engineered to express one or more IgG3 heavy chain variable regions. According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express lambda/kappa light chain constant regions and/or light chain variable regions. According to some embodiments, the IgG hinge region binds to FcyR receptors on immune cells. According to some embodiments, IgG is effective in activating FcyRs and enhancing presentation of antigens (eg, PSA associated with prostate cancer cells).

According to some embodiments, tumor cell lines or tumor cell line variants may be engineered to express intact monoclonal or polyclonal antibodies on the cell surface of tumor cells. According to some embodiments, intact monoclonal or polyclonal antibodies can be engineered to deliver molecules that elicit an immunogenic response. For example, according to some embodiments, intact monoclonal antibodies can be engineered to bind DNA and deliver CpG motifs to immune cells.

の３０－ｍｅｒのＣｐＧである。ＣｐＧモチーフに関して本明細書で使用される場合、「Ｅ」はＧ－ホスホロチオエートであり、この連結はヌクレオチドの３’末端を指す（すなわち、ホスホロチオエート結合は、ヌクレオチド骨格の非架橋酸素を硫黄原子に置き換える）。いくつかの実施形態によれば、配列

のビオチン化された３０－ｍｅｒである。いくつかの実施形態によれば、配列

の３０－ｍｅｒのＣｐＧである。いくつかの実施形態によれば、配列

のビオチン化された３０－ｍｅｒのＣｐＧである。 According to some embodiments, the immunostimulatory activity of bacterial DNA can be mimicked by engineering immunomodulators to deliver unmethylated CpG motifs to immune cells. For example, according to some embodiments, IgG can be engineered to bind biotin and then deliver the biotinylated CpG to cells of the immune system. According to some embodiments, the CpG motifs can bind directly or indirectly to the surface of tumor cells of tumor cell vaccines to prevent systemic effects. According to some embodiments, the CpG motif may be conjugated to one or more antigens presented on the surface of tumor cells from a tumor cell line or tumor cell line variant. According to some embodiments, the CpG is a class A CpG. According to some embodiments, the CpG is a class B CpG. According to some embodiments, the CpG is a Class C CpG. According to some embodiments, the array

30-mer CpG. As used herein for CpG motifs, "E" is G-phosphorothioate and the linkage refers to the 3' end of the nucleotide (i.e., the phosphorothioate linkage replaces the non-bridging oxygen of the nucleotide backbone with a sulfur atom). ). According to some embodiments, the array

is a biotinylated 30-mer of According to some embodiments, the array

30-mer CpG. According to some embodiments, the array

is a biotinylated 30-mer CpG of

According to some embodiments, IgG can be engineered as a hybrid of one or more IgG subclasses. For example, according to some embodiments, IgG includes sequences from IgG1 and IgG3. According to some embodiments, IgG can be engineered to have an affinity for biotin. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO:45. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO:45. According to some embodiments, a tumor cell line or tumor cell line variant can be genetically engineered to contain one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO:45. According to some embodiments, a tumor cell line or tumor cell line variant can be genetically engineered to contain one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO:45. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO:45. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO:45. According to some embodiments, a tumor cell line or tumor cell line variant can be genetically engineered to contain one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO:45. According to some embodiments, a tumor cell line or tumor cell line variant may be genetically engineered to contain one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO:45. According to some embodiments, a tumor cell line or tumor cell line variant can be genetically engineered to contain one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO:45.

According to some embodiments, the IgG comprises one or more mutations compared to wild-type IgG that enhance the affinity of the IgG for an Fc receptor (FcyR). According to some embodiments, a tumor cell line or tumor cell line variant can be genetically engineered to contain one or more proteins of SEQ ID NO:45 with one or more of the mutations T323A and E325A. According to some embodiments, the tumor cell line or tumor cell line variant has at least 60% sequence identity to one or more proteins of SEQ ID NO:41, SEQ ID NO:30, and SEQ ID NO:43. can be genetically engineered to contain one or more proteins with According to some embodiments, the tumor cell line or tumor cell line variant has at least 70% sequence identity to one or more proteins of SEQ ID NO:41, SEQ ID NO:30, and SEQ ID NO:43. can be genetically engineered to contain one or more proteins with According to some embodiments, the tumor cell line or tumor cell line variant has at least 80% sequence identity to one or more proteins of SEQ ID NO:41, SEQ ID NO:30, and SEQ ID NO:43. can be genetically engineered to contain one or more proteins with According to some embodiments, the tumor cell line or tumor cell line variant has at least 90% sequence identity to one or more proteins of SEQ ID NO:41, SEQ ID NO:30, and SEQ ID NO:43. can be genetically engineered to contain one or more proteins with According to some embodiments, the tumor cell line or tumor cell line variant has at least 95% sequence identity to one or more proteins of SEQ ID NO:41, SEQ ID NO:30, and SEQ ID NO:43. can be genetically engineered to contain one or more proteins with According to some embodiments, the tumor cell line or tumor cell line variant has at least 96% sequence identity to one or more proteins of SEQ ID NO:41, SEQ ID NO:30, and SEQ ID NO:43. can be genetically engineered to contain one or more proteins with According to some embodiments, the tumor cell line or tumor cell line variant has at least 97% sequence identity to one or more proteins of SEQ ID NO:41, SEQ ID NO:30, and SEQ ID NO:43. can be genetically engineered to contain one or more proteins with According to some embodiments, the tumor cell line or tumor cell line variant has at least 98% sequence identity to one or more proteins of SEQ ID NO:41, SEQ ID NO:30, and SEQ ID NO:43. can be genetically engineered to contain one or more proteins with According to some embodiments, the tumor cell line or tumor cell line variant has at least 99% sequence identity to one or more proteins of SEQ ID NO:41, SEQ ID NO:30, and SEQ ID NO:43. can be genetically engineered to contain one or more proteins with

According to some embodiments, an allogeneic tumor cell vaccine comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are one or more (e.g., two , 3, 4, 5, or more) IgSF proteins, or variants or fragments thereof.

Chemokine Receptors According to some embodiments, the subset of R members may comprise one or more chemokine receptors. Chemokine receptors are defined as modulators that activate cellular responses upon binding of chemokines. Twenty-three subtypes of human chemokine receptors have been identified, all of which are members of the seven transmembrane (7TM) domain superfamily of receptors. They can be divided into two major groups: G protein-coupled chemotactic chemokine receptors (n=19) and atypical chemokine receptors (n=4). The chemokine binding, membrane anchoring, and signaling domains of both groups of receptors are derived from a single polypeptide chain. There is structural and biochemical evidence that these receptors form homodimers and heterodimers.

According to some embodiments, the chemokine receptor is membrane bound.

According to one embodiment, the present disclosure includes a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, wherein the tumor cells are T lymphocytes, natural killer (NK) cells, dendritic Genetically engineered to stimulate one or more of DCs, or B lymphocytes, the population includes allogeneic tumor cell vaccines containing one or more chemokine receptors. Accordingly, the present disclosure encompasses chemokine receptors, including full-length, fragments, homologues, variants, or mutants of chemokine receptors. Cytokines include proteins that are effective in influencing the biological function of another cell. Biological functions affected by cytokines can include, but are not limited to, cell proliferation, cell differentiation, or cell death. For example, the chemokine receptors of the present disclosure can stimulate immune cells such as T lymphocytes (e.g., CD8+ T cells), natural killer (NK) cells, dendritic cells (DC), or B lymphocytes. .

According to some embodiments, the chemokine receptor is CXCR1, CXCR2, CXCR3, CXCR5, CXCR6, CXCR8, CCR8, CCR1, CCR2, CCR3, CCR5, CCR4, CCR6, CCR7, CCR9, CCR10, CXCR1, and CXCR3 is selected from one or more of

CD28 ligand (CD28L)
Ligation of the CD28 receptor on T cells provides an important second signal alongside T cell receptor (TCR) ligation for naive T cell activation (Esenstein, JH et al, Immunity (2016) 44(5): 973-988). CD28 promotes key intracellular biochemical events such as unique phosphorylation and transcriptional signaling, metabolism, production of key cytokines, chemokines, and survival signals essential for long-term expansion and differentiation of T cells. (Id., citing Bluestone, JA et al., Immunity. (2006) 24: 233-238; Bour-Jordan, H. et al., Immunol Rev. (2011) 241: 180-205; Martin, PJ Weiss, A. et al., J Immunol.(1986) 137:819-825).

CD28 is an early member of a subfamily of costimulatory molecules characterized by extracellular variable immunoglobulin-like domains. Other members of the subfamily include ICOS, CTLA4, PD1, PD1H, and BTLA (Id., citing Chen, L. and Flies, D.B., Nat Rev Immunol. 2013; 13:227 -242). CD28 is constitutively expressed on mouse T cells, whereas expression of other family members, ICOS and CTLA4, is induced in response to T cell receptor stimulation and cytokines such as interleukin-2 (IL-2). . CD28 is expressed on approximately 80% of human CD4+ T cells and 50% of CD8+ T cells. The percentage of CD28-positive T cells in humans declines with age. Expression of CD28 has also been identified on other cell lineages, including bone marrow stromal cells, plasma cells, neutrophils, and eosinophils, although the functional importance of CD28 in these cells is not fully understood. (Id., citing Gray Parkin, K., et al., J Immunol. (2002) 169:2292-2302; Rozanski, CH et al., J Exp Med. (2011) 208:1435-1446 Venuprasad, K., et al., Eur J Immunol.(2001) 31:1536-1543; Woerly, G. et al., Clin Exp Allergy.(2004) 34:1379-1387).

The CD28 ligands CD80 and CD86 differ in their expression patterns, multimeric state, and functionality, adding another layer of complexity to the regulation of CD28 signaling. CD80 exists primarily in a dimeric form on the cell surface, whereas CD86 is a monomer (Id., citing Bhatia, S. et al., Proc Natl Acad Sci USA. (2005) 102: 15569-155742005). CD86 is constitutively expressed on antigen-presenting cells (APCs) and is rapidly upregulated upon spontaneous stimulation of APCs (Id., citing Lenschow, DJ et al., J Immunol. (1994) 153:1990). -1997). On the other hand, the other CD28 ligand, CD80, is upregulated at later time points (Id., cited in Sharpe, AJ and Freeman, GJ, Nat Rev Immunol. (2002) 2:116-126). Therefore, CD86 may be more important in initiating the immune response. CD80 and CD86 are induced by different stimuli of different cell types and are not functionally interchangeable.

CD28 and CTLA4 have opposite effects on T cell stimulation. CD28 provides an activating signal and CTLA4 an inhibitory signal. This is now considered a classic immune checkpoint (Id., citing Krummel, MF and Allison, JP, J Exp Med. 1995; 182:459-465; Walunas, TL et al., Immunity (1994) 1:405-413). ICOS, which also contributes to activation, binds to its ligand B7H2 (ICOSL). It also functions as a ligand for human CD28 and CTLA4 (Id., citing Chen, L. and Flies, DB, Nat Rev Immunol. (2013) 13:227-242; Yao, S. et al., Immunity (2011) 34:729-740). Thus, this family of receptors and ligands is rather complex in both binding patterns and biological effects. Overall, the opposing roles of CD28 and ICOS relative to CTLA4 allow this family of receptors and ligands to function as rheostats of the immune response through competing pro- and anti-inflammatory effects ( Ditto).

It has been suggested that CD80 and CD86 may themselves also function as signaling receptors, as ligation with CTLA4Ig has been shown to regulate tryptophan metabolism of APCs (Grohmann, U et al., Nat Immunol. (2002) 3:1097-1101). In addition to T cells, plasma cells also express CD28. Although the CD28 signal can regulate antibody production by plasma cells or survival of plasma cells, the precise role played by CD28 in plasma cell biology is still unclear (Id., citing Njau, NM and Jacob, J. ., Adv Exp Med Biol. (2013) 785:67-75).

The CD28 gene is composed of four exons that encode a 220 amino acid protein that is expressed as a 44 kDa glycosylated disulfide-linked homodimer on the cell surface. Members of the CD28 family share many common functions. These receptors are composed of paired V-set immunoglobulin superfamily (IgSF) domains bounded by a single transmembrane domain and a cytoplasmic domain that contains key signaling motifs (Id., citing Carreno, BM and Collins, M, Annu Rev Immunol. (2002) 20: 29-53). The CD28 and CTLA4 ligands, CD80 and CD86, are composed of a single V-set and a C1-set of IgSF domains. The interaction of these co-stimulatory receptors with ligands is mediated through the MYPPPY motif (SEQ ID NO: 105) within the receptor V-set domain (Evans, EJ et al., Nat Immunol. (2005) 6: 271-279; Metzler, WJ et al., Nat Struct Biol. (1997) 4: 527-531).

Binding of CD28 by its ligand initiates signaling events that depend on the specific binding of the protein to the cytoplasmic tail of CD28. Despite the lack of intrinsic enzymatic activity, the 41-amino acid cytoplasmic tail of human CD28 contains a highly conserved protein that is phosphorylated in response to TCR or CD28 stimulation and binds SH2 domains and targets in a phosphotyrosine-dependent manner. It contains a tyrosine-based signaling motif. A proline-rich sequence within the cytoplasmic tail also binds SH3 domain-containing proteins. In particular, the membrane-proximal YMNM motif (SEQ ID NO: 106) and distal PYAP motif (SEQ ID NO: 107) form complexes with several kinases and adapter proteins, some of which are associated with SH2 and/or SH3 It has been shown that it can bind to either or both motifs through domain interactions (Id., cited in Boomer, JS and Green, JM, Cold Spring Harb Perspect Biol. (2010) 2:a002436). These motifs are important for IL-2 gene expression mediated by CD28-dependent activation of NFAT, AP-1, and NF-κB family transcription factors (Id., citing Fraser, JD et al. June, CH et al., Mol Cell Biol.(1987) 7:4472-4481;Thompson, CB et al., Proc Natl Acad Sci U S A.(1989). 86:1333-1337).

The membrane-proximal YXXM motif is shared between CD28, CTLA4, and ICOS and is the consensus site for the p85 subunit of the lipid kinase phosphatidylinositol 3-kinase (PI3K) (Id., citing August, A. et al.). and Dupont, B. Int Immunol.(1994) 6:769-774;Pages, F., et al., Nature.(1994) 369: 327-329; A. (1994) 91: 2834-2838; Rudd, CE and Schneider, H., Nat Rev Immunol.(2003) 3: 544-556). In addition to the +3 methionine of the CD28 sequence that confers PI3K specificity, YMNM (SEQ ID NO: 106), the +2 asparagine confers specificity for the adapter proteins GRB2 and GADS on CD28 (Id., citing Cai, YC Kim, HH et al., J Biol Chem.(1998) 273: 296-301; Okkenhaug, K. and Rottapel, R., 1998; Raab, M et al., Proc Natl Acad Sci USA (1995) 92: 8891-8895; Stein, PH et al., Mol Cell Biol. (1994) 14: 3392-3402). Both ICOS and CTLA4 can bind to PI3K but are incapable of binding to GRB2. This may explain some of the functional and signaling differences between these co-stimulatory receptors (Id., cited in Rudd, CE and Schneider, H Nat Rev Immunol. (2003) 3: 544- 556). The importance of the YMNM motif (SEQ ID NO: 106) in mediating proliferation and IL-2 secretion is controversial. Signaling events downstream of the C-terminal PYAP motif (SEQ ID NO: 107) include phosphorylation and activation of the kinases PDK1 and PKCθ, and subsequent inactivation of GSK3β, ultimately leading to IL-2-mediated NFAT-dependent Encouraging gene transcription is included. Binding and activation of Src kinase Lck via SH3 (Id., citing Holdorf, AD et al., J Exp Med. (1999) 190: 375-384; King, PD et al., J Immunol. ( 1997) 158: 580-590) have been proposed as potential regulators of this pathway. The adapter proteins GRB2 and GADS bind CD28 either through the SH3 domain to the distal PYAP motif (SEQ ID NO: 107) or through the SH2 domain to the membrane-proximal YMNM motif (SEQ ID NO: 106). can be combined. However, it is the C-terminal PYAP motif (SEQ ID NO: 107) that is thought to play a greater role in NF-κB activation, and other important NF-κB activation factors such as Lck discussed above. It has been suggested that the signaling molecule binds to the C-terminal PYAP motif (SEQ ID NO: 107) (Id., citing Holdorf, AD et al., J Exp Med. (1999) 190: 375-384). Watanabe, R. et al., J Immunol. (2006) 177:1085-1091).

CD28 ligation is important in promoting conventional T cell proliferation and effector function, but also promotes the anti-inflammatory function of regulatory T (Treg) cells. CD28 thus plays both pro- and anti-inflammatory roles, depending on the cell type and the context in which it is expressed. CD28 signaling is important for enabling effector T cells to overcome Treg cell-mediated immunosuppression (Id., citing Lyddane, C et al., J Immunol. (2006) 176: 3306- 3310), but CD28 in another context prevents spontaneous autoimmunity by promoting Treg function (Id., citing Salomon B. et al., Immunity. 2000; 12:431-440).

CD28 supports T cell homeostasis and functions in a variety of ways. CD28 signals support expression of miR17-92 family members that are critical for maximal IL-10 production by Treg cells (de Kouchkovsky, D et al., J Immunol. (2013) 191: 1594-1605). Thymocytes require simultaneous TCR and CD28 signals to upregulate Foxp3 and differentiate into Treg cells. CD28 is also required for the generation of peripherally induced Treg cells. CD4+CD25- T cells required CD28 ligation to differentiate into functional Foxp3+ Treg cells when activated with TGF-β.

According to some embodiments of the disclosed invention, populations of proliferation-incompetent tumor cells can be engineered to express the membrane-bound form of CD80 of SEQ ID NO: 110 on ENLIST™ membranes. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO:110. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO:110. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO:110. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO:110. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO:110. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO:110. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO:110. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO:110. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO:110.

According to some embodiments of the disclosed invention, the population of proliferation-incompetent tumor cells expresses a membrane-bound form of CD86 of SEQ ID NO: 111 on the membrane of the population of proliferation-incompetent tumor cells. can be manipulated. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 60% sequence identity to the protein of SEQ ID NO:111. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 70% sequence identity to the protein of SEQ ID NO:110. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 80% sequence identity to the protein of SEQ ID NO:111. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 90% sequence identity to the protein of SEQ ID NO:111. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 95% sequence identity to the protein of SEQ ID NO:111. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 96% sequence identity to the protein of SEQ ID NO:111. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 97% sequence identity to the protein of SEQ ID NO:111. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 98% sequence identity to the protein of SEQ ID NO:111. According to some embodiments, a population of proliferation-incompetent tumor cells can be engineered to contain one or more proteins having at least 99% sequence identity to the protein of SEQ ID NO:111.

According to some embodiments, the allogeneic vaccine uses human peripheral blood mononuclear cells from healthy subjects and cancer patients to examine inter-individual variability as well as the standard for patient differences. It is adapted for rapid in vitro evaluation, thus avoiding animal experiments.

According to some embodiments, the allogeneic vaccine is adapted to provide clinical benefit in the short term by induction of a strong anti-allogeneic vaccine response and in the long term endogenous, unmodified adapted to provide long-lived cross-reactive responses to host tumors. According to some embodiments, the immune response to an allogeneic tumor cell vaccine is a combination of peptides specific to the tumor cell line or tumor cell line variant and peptides specific to the tumor cells of the patient receiving the vaccine. Including heteroclitic cross-reactivity (see, eg, FIG. 1). According to some embodiments, heteroclitic cross-reactivity is immunogenic through enhanced binding of T cell receptors to tumor cell peptide-MHC complexes that normally provide a non-immunogenic surface. to enhance According to some embodiments, the allogeneic tumor cell vaccine comprises modified peptides compared to tumor cells of a subject with cancer, wherein the modified peptides are tumor cells from the patient with cancer. provides an immunogenic surface that provides heteroclitic cross-reactivity to non-immunogenic peptides. According to some embodiments, heteroclitic and alloreactive antigen recognition of tumor cell vaccines provides a broad spectrum of antigens useful for inducing immune responses against tumor cells in vaccinated patients. . According to some embodiments, allogeneic vaccines are adapted to provide clinical benefit in the form of, for example, progression-free survival, recurrence-free survival, or overall survival. According to some embodiments, allogeneic vaccines are effective in providing heteroclitic immunity-induced tumor immunity (Dyall R., et al., Heteroclitic Immunization Induces Tumor Immunity, J. Exp. Med., Vol. 188, No. 9, November 2, 1998, incorporated herein by reference in its entirety).

According to some embodiments, the allogeneic vaccine is derived from a tumor cell line or tumor cell line variant that has been genetically modified to contain a recombinant immunomodulatory signal that is expressed in therapeutic amounts. According to some embodiments, allogeneic vaccines are derived from a homogenous starting material, such as a tumor cell line or tumor cell line variant, and multiple distinct biologics, either soluble or membrane-bound. It is expressed in the starting material. According to some embodiments, the expression and activity of the soluble and membrane-bound forms are confirmed in vitro using peripheral blood mononuclear cells by flow cytometry and mixed lymphocyte tumor assays, respectively. According to some embodiments, the expression and activity of the soluble and membrane-bound forms enhances peripheral blood mononuclear cells of vaccinated cancer patients against allogeneic tumor cells used for immunization in vitro. Used and confirmed by flow cytometry and mixed lymphocyte tumor assays.

According to some embodiments, the allogeneic vaccine comprises exogenous immunomodulatory molecules, each encoding a membrane-bound or secreted signaling molecule. According to some embodiments, each membrane-bound immunomodulatory molecule is active in a spatially and temporally restricted manner primarily to provide signaling at the time and place of antigen presentation. The following doses are adapted to deliver therapeutic doses. According to some embodiments, membrane-bound immunomodulatory molecules are adapted to reduce the potential for systemic side effects. According to some embodiments, the secreted immunomodulatory molecule is adapted to deliver a local rather than a systemic signal.

According to some aspects, an allogeneic vaccine comprises genetic material effective to genetically introduce one or more immunomodulatory molecules into a tumor cell line or tumor cell line variant. According to some embodiments, the genetic material can be introduced by viral transduction techniques and isolated by positive selection of genetically introduced immunomodulators. For example, according to some embodiments, positive selection of genetically introduced immunomodulator molecules comprises selection using antibodies. According to some embodiments, the immunomodulatory molecule is diverse with respect to its effect on important immune cell subsets such as dendritic cells and lymphocyte subpopulations (e.g., T cells, natural killer cells, and T regulatory cells). and complementary. According to some embodiments, the allogeneic vaccine comprises different immunomodulatory molecules directed to different immunoregulatory pathways on different immune cell subsets, all pathways affecting immunity in individual cancer patients. They do not contribute equally to the primary response. According to some embodiments, the immunomodulatory molecule genetically introduced into the tumor cell line or tumor cell line variant is stably expressed.

According to some embodiments, an allogeneic tumor cell vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the allogeneic tumor cells comprising: It contains a core of three stably expressed essential exogenous immunomodulatory molecules, OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), which includes CD80, CD86, or both. According to some embodiments, the allogeneic tumor cell vaccine further comprises one or more additional subsets of stably expressed exogenous immunomodulatory molecules designated as group R (of the core chemical structure ), each subset contains 3-25 immunomodulators. According to some embodiments, the subset R is , 22, 23, 24, or 25 immunomodulators. According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and 3-25 immunomodulators are genetically engineered to stably express two R subsets; According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. are genetically engineered to stably express exogenous immunomodulatory molecules, 4-IBBL, GM-CSF, OX40L, and two R-subsets containing 3-25 immunomodulators. According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. exogenous immunomodulatory molecules, OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and 3 to 25 immunomodulators expressed in are genetically engineered to stably express two R subsets; According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and 3-25 immunomodulators are genetically engineered to stably express the four R subsets, including According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and 3-25 immunomodulators are genetically engineered to stably express the five R subsets, including According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and 3-25 immunomodulators It is genetically engineered to stably express the six R subsets, including According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and 3-25 immunomodulators are genetically engineered to stably express 7 R subsets, including According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and 3-25 immunomodulators is engineered to stably express 8 R subsets, including According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and 3-25 immunomodulators are genetically engineered to stably express the nine R subsets, including According to some embodiments, an allogeneic vaccine of the present disclosure comprises a population of proliferation-incompetent tumor cells expressing one or more tumor-specific antigens, the tumor cells comprising at least three stable tumor cells. OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, and 3-25 immunomodulators It is genetically engineered to stably express 10 R subsets, including

Evaluation of Immunogenicity Mixed Lymphocytic Tumor Cell Reactivity According to some embodiments, the transgenic immunomodulators are tested for their immunogenic potential by the mixed lymphocytic tumor cell reaction (MLTR). can be evaluated. The MLTR assay allows tumor cells of a tumor cell line or tumor cell line variant to elicit an immune response from mixed lymphocytes in vitro by incubating mixed lymphocytes with a tumor cell line or tumor cell line variant (or control) for several days. including making sure This method is a rapid in vitro method for assessing mixed lymphocyte responses to tumor cells or lysates, including lymphocyte cell proliferation, lymphocyte cell subset differentiation, lymphocyte cytokine release profiles, and tumor cell death. can be provided. This approach allows comprehensive monitoring of cellular, humoral, or both immune responses against phenotypically altered transfected tumor cells using human peripheral blood mononuclear cells. MLTR can also provide an alternative for mouse tumor survival studies and can lead to the selection of optimal tumor cell lines or tumor cell line variants for anti-tumor responses. A similar assay is described by Hunter TB et al. , (2007) Scandanavian J.; Immunology 65, 479-486, which is incorporated herein by reference in its entirety.

MLTR was first described by Stjernsward J, et al. in 1970 (Clin Exp Immunol 6: 963-668, incorporated herein by reference in its entirety) and was based on the mixed lymphocyte response (MLR) method. Uchida A, et al. describe the generation of specific and non-specific killer T cells using MLTR (Int J. Canc (1988) 41: 651-656, incorporated herein by reference in its entirety). MLTR is described, for example, in Eini et al., which uses the MLTR assay to assess immune response modulation (eg, elevation of pro-inflammatory cytokine release). (Biochemical Pharmacology Volume 98, Issue 1, 1 November 2015, Pages 110-118, incorporated herein by reference in its entirety); et al. , (Eur J Immunol. 1995 May;25(5):1163-7, incorporated herein by reference in its entirety); and superantigen staphylococcal enterotoxin A (SEA)-coated using the MLTR assay. Xiu et al. to determine the effect of tumor cells on tumor-specific T cell responses. (J Mol Med (Berl). 2007 May;85(5):511-21. Epub 2007 Jan 12, incorporated herein by reference in its entirety). model.

According to some embodiments, the tumor cell line or tumor cell line variant is obtained by contacting the transfected tumor cells with mixed lymphocytes from peripheral blood mononuclear cells, followed by cell proliferation, differentiation of cell subsets, Immunogenic potential can be tested by measuring at least one of cytokine release profiles and tumor cell lysates.

According to some embodiments, mixed lymphocytes can be obtained from peripheral blood mononuclear cells isolated by a Ficoll-Paque gradient. Briefly, anticoagulant-treated blood can be diluted with PBS/EDTA ranging from 1:2 to 1:4 to reduce red blood cell aggregation. The diluted blood can then be layered on top of the Ficoll-Paque solution in a centrifuge tube without mixing. The layered blood/Ficoll-Paque was centrifuged at 400 xg for 40 minutes at 18°C-20°C without centrifugal brake, and from top to bottom, the first fraction containing plasma, mononuclear Blood fractions can be formed comprising a second fraction containing spheres, a third fraction containing Ficoll-Paque medium, and a fourth fraction containing granulocytes and red blood cells.

Fractions can be further processed to isolate specific fraction components. For example, for further processing of the mononuclear cells, a second fraction containing the mononuclear cells can be carefully removed from the Ficoll-Paque gradient using a Pasteur pipette. Alternatively, the second fraction can be removed directly by puncturing the tube with a needle and withdrawing the second fraction directly. The second fraction is then washed and centrifuged with PBS/EDTA three times at 300 xg at 18°C and 20°C, discarding the supernatant after each round.

According to some embodiments, the tumor cell line or tumor cell line variant is co-cultured with PBMC containing lymphocytes for 7 days in the presence of an immunomodulatory factor derived from the tumor cell line or tumor cell line variant. direct assessment of the activation of anti-tumor responses.

According to some embodiments, one parameter used to measure lymphocyte activation may be cell proliferation. According to some embodiments, proliferation can be detected by ³ H-thymidine incorporation. Briefly, about 5×10 ³ cells of a tumor cell line or tumor cell line variant can be co-cultured with about 1×10 ⁶ mixed lymphocytes in a round bottom 96-well plate. After 3 days of culture, cells may be pulsed with 1 μCi of ³ H-thymidine for 18 hours. Cells can then be harvested onto filtermats and ³ H-thymidine incorporation measured using a scintillation counter. Proliferation of a tumor cell line or tumor cell line variant relative to a non-transfected tumor cell control can be measured. Increased, decreased or no change in proliferation compared to controls are possible outcomes.

According to some embodiments, another parameter for measuring lymphocyte activation may be the cytokine release profile. For example, the number of responsive T cells in mixed lymphocyte populations can be quantified by enzyme-linked immunospot (ELISpot) analysis of IFN-γ and/or IL-2 production by PBMC. Briefly, PBMC containing mixed lymphocytes and tumor cell lines or tumor cell line variants can be co-cultured for 3-7 days. Co-cultured cells can then be harvested and incubated on ELISpot plates precoated with anti-IFN-γ and/or anti-IL-2 antibodies. After 20 hours, cells can be removed by washing twice with distilled water and twice with wash buffer. The ELISpot plate can then be contacted with biotinylated anti-IFN-γ and/or anti-IL-2 antibody and streptavidin alkaline phosphatase in blocking buffer for 1-2 hours. After washing, the plate is contacted with alkaline phosphatase substrate until dark spots appear. The plates are then washed with tap water and air dried. The spots are then quantified manually or by a plate reader and compared to controls of untransfected tumor cell lines or tumor cell line variants.

According to some embodiments, another parameter for measuring lymphocyte activation may be by quantifying differentiation of cell subsets. For example, differentiation of CD45+/CD3+ T lymphocytes into CD45+/CD3+/CD4+ helper T lymphocytes, CD45+/CD3+/CD8+ cytotoxic T lymphocytes, and CD45+/CD3+/CD25+ activated T lymphocytes was analyzed by flow cytometric analysis. can be quantified by

According to some embodiments, another parameter for measuring lymphocyte activation may be by quantifying tumor cell cytotoxicity. Tumor cell cytotoxicity can be measured by a number of established methods. For example, according to some embodiments, the LDH-Cytotoxicity Colorimetric Assay Kit (BioVision Catalog No. K311-400) is used to detect lactate dehydrogenase (LDH) released from injured cells into the growth medium. Tumor cell cytotoxicity can be determined by testing. Briefly, 100 μl of medium from each of the control group (containing untransfected tumor cells), the experimental group (containing tumor cells transfected with an immunomodulatory factor), and medium alone were added to 96 wells. It can be pipetted into the wells of the plate. 100 μl of the LDH reaction mixture containing dye solution and catalyst solution can then be added to the wells of the 96-well plate and incubated for 30 minutes at room temperature. Samples can then be measured for absorbance at 490-500 nm using a microtiter plate reader.

Stimulation of Immune Cells As described herein, the present disclosure includes populations of proliferation-incompetent tumor cells that express one or more tumor-specific antigens, wherein the tumor cells are isolated from one or more immune cells. Allogeneic tumor cell vaccines that are genetically engineered to stimulate cells (e.g., one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or B lymphocytes) provide. Stimulation of immune cells can enhance normal cell function or can initiate normal cell function in abnormal cells.

According to some embodiments, stimulating T lymphocytes comprises activating and/or expanding T lymphocytes. According to some embodiments, stimulating NK cells comprises activating and/or expanding NK cells. According to some embodiments, stimulating DCs comprises activating and/or expanding DCs. According to some embodiments, stimulating B lymphocytes comprises activating and/or expanding B lymphocytes.

According to some embodiments, the present disclosure provides allogeneic tumor cells, including populations of proliferation-incompetent tumor cells, expressing one or more tumor-specific antigens effective to stimulate immune killer cells. provide vaccines. Immune killer cells that can be stimulated include, for example, cytolytic T cells (CD8+ cells), memory CD8+ T cells, and NK cells. According to one embodiment, the killer immune cells are natural killer (NK) cells. According to one embodiment, the NK cells are memory-like NK cells. According to one embodiment, killer immune cells are CD8+ T cells. According to one embodiment, the CD8+ T cells are memory T cells. Accordingly, the invention also provides cell populations resulting from stimulation with allogeneic tumor cell vaccines described herein.

According to some embodiments, the allogeneic tumor cell vaccine simultaneously stimulates more than one type of immune killer cell, e.g., cytolytic T cells (CD8+ cells), memory CD8+ T cells, and NK cells. It is a feature of the present invention that it is effective in stimulating. According to some embodiments, the allogeneic tumor cell vaccine is effective in stimulating both CD8+ T cells and NK cells.

According to some embodiments, the allogeneic tumor cell vaccine stimulates CD8+ T cells and expression of certain exogenous immunomodulatory molecules is more effective than others compared to other exogenous stimulatory molecules. Stimulate killing CD8+ T cells.

According to some embodiments, the allogeneic tumor cell vaccine stimulates NK cells and expression of certain exogenous immunomodulatory molecules is more effective than others compared to other exogenous stimulatory molecules. Stimulates killing NK cells.

According to some embodiments, "stimulating immune killer cells" refers to expansion of immune killer cells. According to some embodiments, "stimulating immune killer cells" refers to activation of immune killer cells. According to some embodiments, "stimulating immune killer cells" refers to increasing the cytotoxicity of immune killer cells.

According to some embodiments, the allogeneic tumor cell vaccines described herein are sufficient to stimulate a population of immune killer cells ex vivo. In other embodiments, the allogeneic tumor cell vaccines described herein are sufficient to stimulate a population of immune killer cells in vivo.

Activation and Expansion of NK Cells According to one embodiment, the allogeneic tumor cell vaccines described herein are effective in activating NK cells.

According to one embodiment, the allogeneic tumor cell vaccines described herein are effective in expanding NK cells.

Degranulation/Cytotoxicity Distinct functional characteristics of NK cells retain their inherent ability to "natural kill" cellular targets without prior sensitization.

According to one embodiment, the tumor cell vaccines described herein are effective in activating and expanding NK cells such that the allogeneic tumor cell vaccines described herein activate and expanded NK cells show higher degranulation activity compared to control NK cells. For example, degranulation activity can be estimated through determination of CD107a expression, eg, by flow cytometry. Surface expression of CD107a is closely correlated with degranulation and release of cytotoxic granules. Degranulation, as measured by CD107a expression, correlates with cytotoxic activity of effector cells such as NK cells. Methods of determining degranulation activity through determination of CD107a expression are well known to those of skill in the art (e.g. Alter G, Malenfant J M, Altfeld M. CD107a as a functional marker for the identification of natural killer cell activity. J Imm Unol Methods 2004;294:15-22, the entire contents of which are incorporated herein by reference).

According to one embodiment, the expanded and activated NK cells obtained after ex vivo or in vivo stimulation with an allogeneic tumor cell vaccine of the invention are expanded, e.g., as measured by degranulation activity. increased cytotoxicity by at least about 50%, about 60%, about 70%, about 80%, or about 90% compared to untreated NK cells. According to one embodiment, the expanded and activated NK cells contain at least about 100% increased cytotoxicity compared to non-expanded NK cells. According to one embodiment, the expanded and activated NK cells contain at least about 200% increased cytotoxicity as compared to non-expanded NK cells. According to one embodiment, the expanded and activated NK cells comprise at least about 300% increased cytotoxicity compared to non-expanded NK cells ex vivo. According to one embodiment, the expanded and activated NK cells comprise at least about 400% increased cytotoxicity compared to non-expanded NK cells ex vivo.

According to one embodiment, the expanded and activated NK cells obtained after ex vivo or in vivo stimulation with an allogeneic tumor cell vaccine of the invention are at least about 50% as compared to non-expanded NK cells. , comprising about 60%, about 70%, about 80% or about 90% increased degranulation activity. According to one embodiment, the expanded and activated NK cells comprise at least about 100% increased degranulation activity compared to non-expanded NK cells. According to one embodiment, the expanded and activated NK cells comprise at least about 200% increased degranulation activity compared to non-expanded NK cells. According to one embodiment, the expanded and activated NK cells comprise at least about 300% increased degranulation activity compared to NK cells that have not been expanded ex vivo. According to one embodiment, the expanded and activated NK cells comprise at least about 400% increased degranulation activity compared to NK cells that have not been expanded ex vivo.

Markers of NK Cell Maturation and Activation Human NK cells are phenotypically characterized by expression of CD56 and absence of CD3 and can be further subdivided into CD56 ^bright and CD56 ^dim populations. The CD56 ^bright population has interferon-γ (IFNγ), tumor necrosis factor-β (TNF-B), tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GMCSF), IL-10, and IL It produces immunomodulatory cytokines, including -13(4). The CD56 ^dim subset is the terminally differentiated progeny of the CD56 ^bright population and is primarily responsible for exerting cytolytic functions. However, CD56 ^dim NK cells, after induction of the cells via the NKp30-activating receptor NKp46, or after stimulation with a combination of IL-2, IL-12, IL-15, release cytokines, specifically IFNγ. can be produced.

According to one embodiment, various markers of NK cell maturation and/or activation can be detected using, for example, flow cytometry methods. For example, a classical marker for NK cells is the activating receptor FcγRIII, also called CD16.

NK cell activation results in the release of cytotoxic granules, including perforin and various granzymes, and cytokine production, most notably interferon-γ (IFNγ). In addition, cell surface expression of death-inducing ligands belonging to the tumor necrosis factor (TNF) family, such as Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL), also affects death receptors (DR) on target cells, i.e. Induces activation of the caspase enzyme cascade through binding to Fas, DR4 (TRAIL-RI), and DR5 (TRAIL-RII).

According to one embodiment, the allogeneic tumor cell vaccine described herein comprises at least about 20% of at least one NK cell activating receptor (e.g., activating receptors listed in Table 3), upregulate by about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, or more . According to one embodiment, the allogeneic tumor cell vaccines described herein comprise at least one NK cell activating receptor at least about 75%, i.e. at least about 76%, at least about 77%, at least about 78% %, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88 %, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% %, at least about 99%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180 %, at least about 190%, at least about 200% upregulation. According to one embodiment, an allogeneic tumor cell vaccine described herein upregulates at least one NK cell activating receptor by at least about 100%. According to one embodiment, an allogeneic tumor cell vaccine described herein upregulates at least one NK cell activating receptor by at least about 200%.

According to another embodiment, an allogeneic tumor cell vaccine described herein downregulates expression of at least one NK cell receptor, such as an inhibitory receptor or chemokine receptor (eg, CCR7). For example, a particular NK cell inhibitory receptor is called KIR (killing inhibitory receptor or CD158). Non-limiting examples of inhibitory receptors are inhibitory killer immunoglobulin-like receptor (KIR), GL183, KIR2DL1, Lir-1, NKB1, and NKG2A.

According to one embodiment, the allogeneic tumor cell vaccines described herein comprise at least one NK cell inhibitory receptor (e.g., inhibitory receptors listed in Table 4 below) at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, 120% , at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 220%, at least about 230%, Downregulate by at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, or more. According to one embodiment, an allogeneic tumor cell vaccine described herein downregulates at least one NK cell inhibitory receptor by at least about 75%. According to one embodiment, an allogeneic tumor cell vaccine described herein downregulates at least one NK cell inhibitory receptor by at least about 100%. According to one embodiment, an allogeneic tumor cell vaccine described herein downregulates at least one NK cell inhibitory receptor by at least about 200%.

Changes in receptor expression can be calculated by the following mean fluorescence intensity (MFI) ratios:
MFI _dayX /MFI _day0
where x is the number of days of NK cell expansion.

If the MFI of the day X sample is higher than day 0, the MFI ratio will be higher than one. This indicates the relative degree of upregulation of that receptor. Thus, for example, an MFI ratio of 1.5 means 50% upregulation of a particular receptor. Calculation of MFI ratio is well known to those skilled in the art.

Various NK cell activating or inhibitory receptors are shown in Table 4 below. Bold indicates family.

Activation and Expansion of CD8+ T Cells According to one embodiment, the allogeneic tumor cell vaccines described herein are effective in activating CD8+ T cells. According to one embodiment, the allogeneic tumor cell vaccines described herein are effective in expanding CD8+ T cells. According to one embodiment, the allogeneic tumor cell vaccines described herein are effective in activating and expanding CD8+ T cells.

T cell activation and expansion can be measured by various assays as described herein. For example, T cell activities that can be measured include induction of T cell proliferation, induction of signaling in T cells, induction of expression of activation markers in T cells, induction of cytokine secretion by T cells, and Includes toxic activity. For example, in certain embodiments CD8+ T cell activation is measured by a proliferation assay.

Cytokine Secretion Activation of CD8+ T cells by the allogeneic tumor cell vaccines of the present invention is characterized by gamma interferon (IFNγ), tumor necrosis factor alpha (TNFa), interleukin-12 (IL-12), interleukin 2 (IL-2) ) can be assessed or measured by determining the secretion of cytokines such as In some embodiments, the ELISA measures cytokine secretion, e.g., gamma interferon (IFNγ), tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12) or interleukin-2 (IL-2) secretion. used to determine Using ELISPOT (enzyme-linked immunospot) technology, T cells that secrete a given cytokine (e.g., gamma interferon (IFNγ)) in response to stimulation by engineered tumor cells described herein. can be detected. T cells are cultured with engineered tumor cells in wells coated with anti-IFNγ antibodies. Secreted IFNγ is captured by the coated antibody and revealed with a secondary antibody conjugated to a chromogenic substrate. Thus, locally secreted cytokine molecules form spots, each spot corresponding to one IFNγ-secreting cell. The number of spots allows determination of the frequency of IFNγ-secreting cells in the analyzed sample. ELISPOT assays are assays for tumor necrosis factor alpha, interleukin-4 (IL-4), IL-5, IL-6, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor, and granzyme B-secreting lymphocytes. Detection has also been described (Klinman D, Nutman T. Current protocols in immunology. New York, N.Y.: John Wiley & Sons, Inc.; 1994. pp. 6, incorporated herein by reference in its entirety). .19.1-6.19.8).

Flow cytometric analysis of intracellular cytokines can be used to measure cytokine content in culture supernatants, but does not provide information on the number of T cells that actually secrete cytokines. When T cells are treated with secretory inhibitors such as monensin or brefeldin A, they accumulate cytokines in their cytoplasm upon activation (eg, by engineered red blood cells of the invention). After fixation and permeabilization of lymphocytes, intracellular cytokines can be quantified by cytometry. This technique makes it possible to determine the cytokines produced, the types of cells producing these cytokines, and the amount of cytokines produced per cell.

Cytotoxicity Activation of CD8+ T cells by the allogeneic tumor cell vaccines of the invention can be assessed by assaying CD8+ T cell cytotoxic activity.

T cell cytotoxic activity can be assessed by any suitable technique known to those of skill in the art. For example, samples containing T cells exposed to engineered red blood cells according to the present invention can be assayed for cytotoxic activity after a suitable period of time in standard cytotoxicity assays. Such assays can include, but are not limited to, chromium release CTL assays and Alamar Blue™ fluorescence assays known in the art.

Expansion/Expansion The ability of the allogeneic tumor cell vaccines of the invention to expand T cells can be assessed using CFSE staining. To compare the initial rate of cell expansion, cells are subjected to CFSE staining to determine the extent to which the allogeneic tumor cell vaccine induced T cell proliferation. CFSE staining provides a much more quantitative endpoint and allows simultaneous phenotyping of expanded cells. Each day after stimulation, an aliquot of cells is removed from each culture and analyzed by flow cytometry. CFSE staining makes the cells highly fluorescent. When cells divide, the fluorescence halves, and the more times the cells divide, the weaker the fluorescence. The ability of allogeneic tumor cell vaccines to induce T-cell proliferation is quantified by measuring the number of cells that divide 1, 2, 3, etc. times. Allogeneic tumor cell vaccines that induce the maximum number of cell divisions at a given time point are considered the most potent expansion factors.

To determine how well these allogeneic tumor cell vaccines promote long-term proliferation of T cells, cell growth curves can be generated. These experiments are set up like the CFSE experiments described above, but without CFSE. Every 2-3 days of culture, T cells are removed from each culture and counted using a Coulter Counter, which measures the number of cells present and the average volume of cells. Mean corpuscular volume is the best predictor of when to restimulate cells. In general, when T cells are properly stimulated, they triple in volume. If this volume is reduced by more than about half of the initial blast, it may be necessary to restimulate the T cells to maintain log-linear expansion (Levine et al., 1996, Science 272:1939-1943; Levine et al. ., 1997, J. Immunol. 159:5921-5930). Calculate the time it takes for each engineered red blood cell to induce 20 population doublings. The relative difference between each allogeneic tumor cell vaccine that induces this level of T cell expansion is one criterion by which a particular allogeneic tumor cell vaccine is evaluated.

In addition, the phenotype of cells expanded by each allogeneic tumor cell vaccine can be characterized to determine whether certain subsets are preferentially expanded. Prior to each restimulation, a phenotypic analysis of the expanding T cell population is performed, as suggested by Appay et al. (2002, Nature Med. 8, 379-385, incorporated herein by reference in its entirety). Expanded T cell differentiation using the definitions of CD27 and CD28 proposed by the authors and the definition of CCR7 proposed by Sallusto et al. Define a state. Intracellular staining for perforin and granzyme B can be used to perform macroscopic measurements to estimate cytolytic capacity.

Apoptotic Markers According to certain embodiments of the present invention, stimulation, activation and expansion of T cells with the allogeneic tumor cell vaccines described herein, in vivo or in vitro, protect against apoptosis, It enhances the expression of certain key molecules in T cells that otherwise prolong survival. Apoptosis is usually due to the induction of specific signals in T cells. Thus, the engineered tumor cells of the invention may provide protection to T cells from cell death resulting from stimulation of T cells. Thus, the present invention includes protection from premature death, or lack or depletion of recognized T cell proliferation markers such as Bcl-xL, growth factors, cytokines, or lymphokines normally required for T cell survival; Also included is enhanced T-cell proliferation by protection from cross-linking of Fas or tumor necrosis factor receptor (TNFR), or by exposure to certain hormones or stress.

III. Methods of Making Various methods of making allogeneic tumor cell vaccines are contemplated by the present disclosure.

According to some embodiments, the present disclosure provides an allogeneic parental tumor cell line; introducing into the tumor cell an exogenous nucleic acid encoding at least one exogenous immunomodulatory molecule; generating tumor cell line variants by selecting tumor cell clones that stably express immunogenic amounts of sexual immunomodulatory molecules; selecting a clonally derived cell line variant by one or more of parameters selected from differentiation, cytokine release profile, and tumor cell lysis. stably expressed at least three variants wherein the variant is effective to stimulate activation of one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or B lymphocytes An allogeneic tumor cell vaccine comprising a population of proliferation-incompetent tumor cells containing an exogenous immunomodulatory molecule is characterized. According to one embodiment, the tumor cells are rendered proliferation incompetent by irradiation. According to one embodiment, exogenous nucleic acid comprises DNA or RNA. According to one embodiment, the step of introducing comprises viral transduction. According to one embodiment, the introducing step comprises electroporation. According to one embodiment, 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 one embodiment, the introducing step comprises introducing the exogenous nucleic acid by transfection of a lentiviral vector.

Lentiviral Vectors The described invention provides nucleic acid constructs encoding two or more immunomodulatory factors that can be expressed in prokaryotic and eukaryotic cells. For example, the described invention provides expression vectors (eg, DNA or RNA-based vectors) that include nucleotide sequences encoding two or more immunomodulators. Additionally, the described invention provides methods for making the vectors described herein, as well as methods for introducing the vectors into suitable host cells for expression of the encoded polypeptides. In general, the methods provided herein involve constructing nucleic acid sequences encoding two or more immune modulators and cloning the sequences into an expression vector. Expression vectors can be introduced into host cells or incorporated into viral particles, either of which can be administered to a subject, eg, to treat cancer.

cDNAs or DNA sequences encoding two or more immune modulators can be obtained (and modified, if desired) using conventional DNA cloning and mutagenesis, DNA amplification, and/or synthetic methods. can. Generally, sequences encoding more than one immunomodulatory factor can be inserted into a cloning vector for purposes of genetic modification and replication prior to expression. Each coding sequence can be operably linked to regulatory elements, such as promoters, for expression of the encoded protein in suitable host cells in vitro and in vivo.

Expression vectors can be introduced into host cells to produce secreted immunomodulators. There are various techniques available for introducing nucleic acids into living cells. Techniques suitable for introducing nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, calcium phosphate precipitation, and others. mentioned. A number of techniques and reagents can also be used for in vivo gene transfer, including liposomes and natural polymer-based delivery vehicles such as chitosan and gelatin. Viral vectors are also suitable for in vivo transduction. In some situations it is desirable to provide targeting agents such as antibodies or ligands specific for cell surface membrane proteins. When liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis are, for example, capsid proteins or fragments thereof directed to specific cell types, antibodies to proteins that undergo internalization during circulation, and can be used to target proteins that target intracellular localization and enhance intracellular half-life and/or to facilitate their uptake. Techniques for receptor-mediated endocytosis are described, for example, in Wu et al. , J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al. , Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).

Gene delivery agents such as, for example, integration sequences can also be used if desired. Numerous integration sequences are known in the art (eg, Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. (see ). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), FIp (Broach, et al., Cell , 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (eg, Groth et al., J. Mol. Biol. 335:667- 678, 2004), sleeping beauty, mariner family transposases (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and retroviruses or lentiviruses. Antiviruses with components that provide for viral integration, such as LTR sequences, as well as AAV ITR sequences (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003).

Cells can be cultured in vitro or genetically engineered, for example. Host cells may be obtained from normal or diseased subjects, including healthy humans, cancer patients, private laboratory deposits, public culture collections such as the United States Type Culture Collection, or from commercial suppliers. can be done.

Cells that can be used in vivo to produce and secrete two or more immunomodulatory factors include epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocyte T lymphocytes, B lymphocytes, monocytes. , blood cells such as macrophages, neutrophils, eosinophils, megakaryocytes, or granulocytes; various stem or progenitor cells such as hematopoietic stem or progenitor cells (e.g., obtained from bone marrow); cord blood; peripheral blood; Examples include, but are not limited to, fetal liver and the like, and tumor cells (eg, human tumor cells). The choice of cell type depends on the type of tumor or infection being treated or prevented and can be determined by one skilled in the art.

Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. A host cell can be chosen that modifies and processes the expressed gene product in a specific way similar to the way the recipient processes its heat shock proteins (hsps).

According to some embodiments, expression constructs as provided herein can be introduced into antigenic cells. As used herein, an antigenic cell is a pre-neoplastic cell that has been infected with an infectious agent that causes cancer, such as a virus, but is not yet neoplastic; Antigenic cells that have been exposed to mutagens or cancer-causing agents can be included. Other cells that can be used are preneoplastic cells that have transitioned from normal to neoplastic morphology, as characterized by morphology or physiological or biochemical function.

Typically, the cancer cells and preneoplastic cells used in the methods provided herein are of mammalian origin. According to some embodiments, cancer cells (eg, human tumor cells) can be used in the methods described herein. Cell lines derived from preneoplastic lesions, cancerous tissue, or cancer cells can also be used. Cancer tissues, cancer cells, cells infected with cancer-causing agents, other preneoplastic cells, human-derived cell lines, and the like can be used. According to some embodiments, the cancer cells include, but are not limited to, established non-small cell lung cancer (NSCLC), bladder cancer, melanoma, ovarian cancer, renal cell carcinoma, prostate cancer, sarcoma, breast cancer, It can be obtained from established tumor cell lines or tumor cell line variants, such as squamous cell carcinoma, head and neck, hepatocellular, pancreatic, or colon cancer cell lines.

Parental cell lines are described supra.

Further, according to some embodiments, allogeneic tumor cell vaccines, when used in the various methods described herein, activate the patient's immune system against the disease of interest. provide an adjuvant effect that further allows for

Both prokaryotic and eukaryotic vectors can be used to express two or more immunomodulators in the methods provided herein. Prokaryotic vectors include E. E. coli sequence-based constructs (see, eg, Makrides, Microbiol Rev 1996, 60:512-538). E. Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, 1pp, phoA, recA, tac, T3, T7, and lambda P _L . Non-limiting examples of prokaryotic expression vectors include . lamda. Agt vector series such as gt11 (Huynh et al., in "DNA Cloning Techniques, Vol. I: A Practical Approach," 1984, (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185:60-89).

A variety of regulatory regions can be used for expression of allogeneic tumor vaccines in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include promoters associated with the metallothionein II gene, the glucocorticoid-responsive long terminal repeat of mouse mammary cancer virus (MMTV-LTR), the n-interferon gene, and the hsp70 gene. but not limited to (see Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock or stress promoters may also be advantageous for promoting expression of the fusion protein in recombinant host cells.

The following animal regulatory regions that exhibit tissue specificity and have been utilized in transgenic animals can also be used in tumor cells of certain tissue types: the elastase I gene regulatory region, which is active in pancreatic acinar cells (Swift et al. Ornitz et al., Cold Spring Harbor Symp Quant Biol 1986, 50:399-409; and MacDonald, Hepatology 1987, 7:425-515); gene control regions (Hanahan, Nature 1985, 315:115-122), immunoglobulin gene control regions that are active in lymphoid cells (Grosschedl et al., Cell 1984, 38:647-658; Adames et al., Nature 1985) , 318:533-538; and Alexander et al., Mol Cell Biol 1987, 7:1436-1444), a mouse mammary tumor virus regulatory region active in testis, breast, lymphocytes and mast cells (Leder et al. , Cell 1986, 45:485-495), the albumin gene regulatory region active in the liver (Pinkert et al., Genes Devel, 1987, 1:268-276), the alpha-fetoprotein gene regulatory region active in the liver ( Krumlauf et al., Mol Cell Biol 1985, 5:1639-1648; and Hammer et al., Science 1987, 235:53-58); Genes Devel 1987, 1:161-171), β-globin gene control regions active in myeloid cells (Mogram et al., Nature 1985, 315:338-340; and Kollias et al., Cell 1986, 46:89- 94); myelin basic protein gene regulatory region active in oligodendrocyte cells in the brain (Readhead et al., Cell 1987, 48:703-712); myosin light chain 2 gene regulatory region active in skeletal muscle. (Sani, Nature 1985, 314:283-286) and the gonadotropin-releasing hormone gene control region that is active in the hypothalamus (Mason et al. , Science 1986, 234:1372-1378).

Expression vectors can also contain transcription enhancer factors such as those found in SV40 virus, hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, and β-actin (Bittner et al., Meth Enzymol 1987). , 153:516-544; and Gorman, Curr Op Biotechnol 1990, 1:36-47). In addition, expression vectors can contain sequences that enable the vector to be maintained and replicated in more than one host cell, or to integrate into the host chromosome. Such sequences include, but are not limited to, origins of replication, autonomously replicating sequences (ARS), centromere DNA, and telomeric DNA.

In addition, the expression vector may carry one or more selectable or screenable marker genes for initially isolating, identifying, or tracking host cells containing DNA encoding the immunogenic proteins described herein. can contain. For long-term, high-yield production of gp96-Ig and T cell co-stimulatory fusion proteins, stable expression in mammalian cells may be useful. Many selection systems are available for mammalian cells. For example, herpes simplex virus thymidine kinase (Wigler et al., Cell 1977, 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski and Szybalski, Proc Natl Acad Sci USA 1962, 48:2026), and adenine phosphoribosyltransferase. (Lowy et al., Cell 1980, 22:817) can be used in tk ^- cells, hgprf ^- cells or aprf ^- cells, respectively. In addition, antimetabolite resistance has been linked to dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 1980, 77:3567; O'Hare et al., Proc Natl Acad Sci USA 1981). gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc Natl Acad Sci USA 1981, 78:2072); neomycin phosphotransferase (neo), which confers resistance to the aminoglycoside G-418 (Colberre- Garapin et al., J Mol Biol 1981, 150:1); and hygromycin phosphotransferase (hyg), which confers resistance to hygromycin (Santerre et al., Gene 1984, 30:147). can. Other selectable markers such as histidinol and Zeocin™ can also be used.

Some viral-based expression systems can also be used in mammalian cells to generate allogeneic tumor cell vaccines. Vectors that use DNA virus backbones include simian virus 40 (SV40) (Hamer et al., Cell 1979, 17:725), adenovirus (Van Doren et al., Mol Cell Biol 1984, 4:1653), adeno-associated Virus (McLaughlin et al., J Virol 1988, 62:1963) and bovine papilloma virus (Zinn et al., Proc Natl Acad Sci USA 1982, 79:4897). When adenovirus is used as an expression vector, the donor DNA sequence can be ligated to an adenovirus transcription/translation control complex, eg, the late promoter and tripartite leader sequence. This fusion gene can then be inserted into the adenoviral genome by in vitro or in vivo recombination. Insertions into non-essential regions of the viral genome (e.g., regions E1 or E3) can result in recombinant viruses that are viable in infected hosts and capable of expressing heterologous products (e.g., Logan and Shenk, Proc Natl Acad. Sci USA 1984, 81:3655-3659).

Bovine papillomavirus (BPV) can infect many higher vertebrates, including humans, and its DNA replicates episomally. A number of shuttle vectors have been developed for recombinant gene expression and exist as stable multicopy (20-300 copies/cell) extrachromosomal elements in mammalian cells. Typically, these vectors contain a segment of BPV DNA (entire genome or 69% transforming fragment), a broad host range promoter, a polyadenylation signal, a splice signal, a selectable marker, and a vector containing E . It contains "non-toxic" plasmid sequences that allow it to grow in E. coli. Following assembly and amplification in bacteria, the expression gene construct is transfected into cultured mammalian cells by, for example, calcium phosphate co-precipitation. For transformed non-phenotypical host cells, selection of transformants is accomplished by using dominant selectable markers such as histidinol and G418 resistance.

Alternatively, the vaccinia 7.5K promoter can be used (eg, Mackett et al., Proc Natl Acad Sci USA 1982, 79:7415-7419; Mackett et al., J Virol 1984, 49:857-864 and Panicali et al., Proc Natl Acad Sci USA 1982, 79:4927-4931). When using human host cells, vectors based on the Epstein-Barr virus (EBV) origin (OriP) and EBV nuclear antigen 1 (EBNA-1; trans-acting replication factor) can be used. Such vectors are, for example, EBO-pCD (Spickofsky et al., DNA Prot Eng Tech 1990, 2:14-18); pDR2 and . lamda. It can be used with a wide range of human host cells such as DR2 (available from Clontech Laboratories).

Allogeneic tumor cell vaccines can also be made in retroviral-based expression systems. Retroviruses, such as Moloney murine leukemia virus, can be used because most viral gene sequences can be removed and replaced with exogenous coding sequences, supplying the missing viral functions in trans. In contrast to transfection, retroviruses can efficiently infect and introduce genes into a wide range of cell types, including, for example, primary hematopoietic cells. In addition, the host range of infection by retroviral vectors can be manipulated through the selection of the envelope used to package the vector.

For example, retroviral vectors can include a 5' long terminal repeat (LTR), a 3' LTR, a packaging signal, a bacterial origin of replication, and a selectable marker. For example, a gp96-Ig fusion protein coding sequence can be inserted at a position between the 5'LTR and 3'LTR such that transcription from the 5'LTR promoter transcribes the cloned DNA. The 5'LTR contains a promoter (eg, LTR promoter), an R region, a U5 region, and a primer binding site, in that order. The nucleotide sequences of these LTR elements are well known in the art. Heterologous promoters as well as multiple drug selectable markers can also be included in the expression vector to facilitate selection of infected cells. McLauchlin et al. , Prog Nucleic Acid Res Mol Biol 1990, 38:91-135; Morgenstern et al. , Nucleic Acid Res 1990, 18:3587-3596; Choulika et al. , J Virol 1996, 70:1792-1798; Boesen et al. Salmons and Gunzberg, Human Gene Ther 1993, 4:129-141; and Grossman and Wilson, Curr Opin Genet Devel 1993, 3:110-114. .

Any of the cloning and expression vectors described herein can be synthesized and assembled from known DNA sequences using techniques known in the art. These exogenous regulatory regions and enhancer elements can be of a variety of origins, both natural and synthetic. Several vectors and host cells are commercially available. Non-limiting examples of useful vectors are described in Current Protocols in Molecular Biology, 1988, ed. Ausubel et al. , Greene Publish. Assoc. & Wiley Interscience, Supplement 5, and Clontech Laboratories, Stratagene Inc. , and Invitrogen, Inc. listed in commercial supplier catalogs such as

Recombinant Immunomodulatory Agents According to some embodiments, two or more immunomodulatory agents are transfected into cells of a tumor cell line or tumor cell line variant (e.g., lipids, calcium phosphate, cationic polymers, DEAE dextran , activated dendrimers, magnetic beads, electroporation, biolistic techniques, microinjection, laserfection/optoinjection) or for transduction (e.g. retroviruses, lentiviruses, adenoviruses, adeno-associated via virus) can be cloned into two or more plasmid constructs. According to some embodiments, recombinant DNAs encoding respective immunomodulator proteins can be cloned into lentiviral vector plasmids for integration into the genome of cells of a tumor cell line or tumor cell line variant. According to some embodiments, recombinant DNA encoding an immunomodulator protein can be cloned into a plasmid DNA construct encoding a selectable trait such as an antibiotic resistance gene. According to some embodiments, recombinant DNAs encoding immunomodulator proteins are cloned into plasmid constructs adapted to stably express the respective recombinant protein in cells of a tumor cell line or tumor cell line variant. can be made According to some embodiments, the transfected or transduced tumor cells are clonally expanded to recombinantly encode the respective immunomodulator proteins into the genome of cells of the tumor cell line or tumor cell line variant. Cell line variants with uniform DNA integration sites can be achieved.

Lentiviral Constructs According to some embodiments, DNA sequences encoding exogenous immunomodulatory molecules can be cloned into lentiviral vectors for transduction into mammalian cells. According to some embodiments, the lentiviral system comprises a lentiviral transfer plasmid encoding two or more immune modulator sequences, a packaging plasmid encoding GAG, POL, TAT, and REV sequences, and an ENV sequence. It may contain an encoding envelope plasmid. According to some embodiments, lentiviral transfer plasmids use the viral LTR promoter for gene expression. According to some embodiments, lentiviral transfer plasmids use hybrid promoters or other specialized promoters. According to some embodiments, the promoter of the lentiviral transfer plasmid is selected to express two or more immunoregulatory factor sequences at desired levels relative to other immunoregulatory sequences. According to some embodiments, the relative level is measured at the level of transcription as mRNA transcripts. According to some embodiments, the relative level is measured at the level of translation as protein expression.

Multicistronic Plasmid Constructs According to some embodiments, one or more immunomodulator sequences are multicistronic for co-expression of one immunomodulator with a second immunomodulator or other recombinant sequences. can be cloned into a sexual vector. According to some embodiments, immunomodulator sequences may be cloned into plasmids containing IRES elements to facilitate translation of two or more proteins from a single transcript. According to some embodiments, one or more immunomodulator sequences are cloned into a multicistronic vector containing sequences of self-cleaving 2A peptides to generate two or more exogenous immunomodulatory sequences from a single transcript. Produce regulatory molecules.

Genetic Introduction of Exogenous Immunomodulatory Molecules According to some embodiments, plasmid constructs containing recombinant immunomodulator sequences can be transfected or transduced into tumor cell lines or tumor cell line variants.

According to some embodiments, up to 25 immunomodulators (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 2, 22, 23, 24, or 25) can be cloned into 10 separate vectors for transduction into mammalian cells. According to some embodiments, up to 25 immunomodulators (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, or 25) can be cloned into 11 separate vectors for transduction into mammalian cells. According to some embodiments, up to 25 immunomodulators (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, or 25) can be cloned into 12 separate vectors for transduction into mammalian cells. According to some embodiments, 14 or more immunomodulators (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or above) can be cloned into 10 separate vectors for transduction into mammalian cells. According to some embodiments, 14 or more immunomodulators (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or above) can be cloned into 11 separate vectors for transduction into mammalian cells. According to some embodiments, 14 or more immunomodulators (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or above) can be cloned into 12 separate vectors for transduction into mammalian cells. According to some embodiments, 14 or more immunomodulators (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or above) can be cloned into 13 separate vectors for transduction into mammalian cells. According to some embodiments, 14 or more immunomodulators (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or above) can be cloned into 14 separate vectors for transduction into mammalian cells. According to some embodiments, 14 or more immunomodulators (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or above) can be cloned into 14 or more separate vectors for transduction into mammalian cells.

According to some embodiments, the vector construct further comprises one or more tags, as described herein.

Lentiviral Systems According to some embodiments, a lentiviral system can be used in which a transfer vector, an envelope vector, and a packaging vector having immunomodulator sequences are each transduced into a host cell for virus production. be affected. According to some embodiments, lentiviral vectors can be transfected into 293T cells either by calcium phosphate precipitation transfection, lipid-based transfection, or electroporation and incubated overnight. For embodiments in which immunomodulator sequences may be associated with fluorescent reporters, examination of 293T cells for fluorescence can be checked after overnight incubation. 293T cell medium containing viral particles can be harvested 2 or 3 times every 8-12 hours and centrifuged to sediment detached cells and cell debris. The medium can then be used directly, frozen or concentrated as desired.

Tumor cell lines or tumor cell line variants can be grown to about 70% confluency under standard tissue culture conditions. Cells can then be treated in fresh medium with hexadimethrine bromide (to enhance cell transduction) and lentiviral particles containing the recombinant construct and incubated for 18-20 hours before changing the medium. can.

Lipid-Based Transfection According to some embodiments, cells of a tumor cell line or tumor cell line variant can be transfected with immunomodulator sequences using lipid-based transfection methods. According to some embodiments, established lipid-based transfection reagents such as Lipofectamine can be used. Tumor cell lines or tumor cell line variants can be grown to approximately 70-90% confluence in tissue culture vessels. Plasmid constructs containing appropriate amounts of Lipofectamine® and immunomodulator sequences can be diluted separately in tissue culture medium and incubated briefly at room temperature. Lipofectamine® diluted in medium and the plasmid construct can be mixed together and incubated briefly at room temperature. The plasmid and lipofectamine mixture can then be added to the cells of the tumor cell line or tumor cell line variant in the tissue culture vessel and incubated for 1-3 days under standard tissue culture conditions.

Selection of Expressing Clones According to some embodiments, tumor cells of a tumor cell line or tumor cell line variant transfected with an immunomodulator sequence may be selected for varying levels of expression.

According to some embodiments, the immunomodulator sequence can be accompanied by an antibiotic resistance gene, which is used to select clones with stable integration of recombinant DNA encoding the immunomodulator sequence. can do. According to some embodiments, immunomodulator sequences can be cloned into plasmid constructs containing antibiotic resistance, such as neomycin/kanamycin resistance genes. Transfected cells are treated with antibiotics for 1-2 more weeks with daily medium changes according to the manufacturer's protocol. At some point during antibiotic treatment, there is massive tumor cell death of all cells that have not stably integrated the antibiotic resistance gene, leaving small colonies of stably expressing clones. Each stably expressing clone can be picked, cultured in separate tissue culture vessels, and tested for immunomodulator expression levels by established methods such as Western blot, flow cytometry, and fluorescence microscopy. .

According to some embodiments, transfected tumor cells can be selected for high expression of immunomodulators by fluorescence activated cell sorting (FACS). According to some embodiments, an immunomodulator sequence can be associated with one or more fluorescent proteins (eg, GFP) that can be used to quantify the expression of the immunomodulator. For example, a bicistronic plasmid containing an immunomodulator sequence connected to a GFP sequence via an IRES sequence will result in both immunomodulator and GFP proteins translated from the same transcript. Thus, GFP expression levels serve as a surrogate for the expression levels of immunomodulators. Single-cell suspensions of immunomodulator/GFP-transfected tumor cells can be selected for desired expression levels by FACS based on fluorescence intensity. Any fluorescent protein can be used in this regard. For example, any of the following recombinant fluorescent proteins can be used: EBFP, ECFP, EGFP, YFP, mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry, mGrape, mRasberry, mGrape2, mPlum.

Alternatively, recombinant immunomodulatory factor expression can be observed directly by fluorescent antibodies that are specific for the respective immunomodulatory factor or specific for tags engineered onto the respective immunomodulatory factor. For example, according to some embodiments, the extracellular region of an immunomodulator sequence can be fused with a FLAG or HA tag. Anti-FLAG or anti-HA antibodies can be used with fluorophores conjugated to primary or secondary antibodies to detect the expression of immunomodulators on the surface of transfected tumor cells. Tumor cells expressing desired levels of immunomodulators can be selected by FACS sorting and cultured separately.

Sequential Addition of New Plasmid Constructs to Clones According to some embodiments, tumor cell lines or tumor cell line variants expressing one or more immunomodulator sequence(s) are sequentially stabilised. are transfected with additional immunomodulators for enhanced expression. By adding recombinant immunomodulators sequentially in a sequential manner, cells of a tumor cell line or tumor cell line variant can be generated that simultaneously express several immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express two immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express three immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express four immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express five immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express six immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express seven immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express eight immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express nine immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 10 immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 11 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 12 immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 13 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 14 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 15 immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 16 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 17 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 18 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 19 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 20 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 21 immunomodulatory factors. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 22 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 23 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 24 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 25 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 26 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 27 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 28 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 29 immunomodulators. According to some embodiments, tumor cell lines or tumor cell line variants can be generated that simultaneously express 30 immunomodulators.

Variable Expressing Clones According to one aspect of the disclosed invention, multiple recombinant immunomodulatory peptides can be expressed in a single clonally derived tumor cell line or tumor cell line variant. According to some embodiments, the amount (or level) of an individual immunomodulator expressed in each cell is the same as the expression level of all other immunomodulator peptides. However, according to some embodiments, the level of individual immunomodulators expressed in each cell differs from the expression level of other immunomodulators expressed in the cell. According to some embodiments, clonally derived tumor cell lines or tumor cell line variants that express the same complement of immunomodulators stably express those immunomodulators in varying amounts relative to one another.

The relative amount of recombinant immunomodulatory factor expressed within each clonally derived tumor cell line or tumor cell line variant and between tumor cell lines or tumor cell line variants can be measured at the transcriptional or translational level. . For example, relative amounts of recombinant immunomodulators can be quantified by Western blot, RT-PCR, flow cytometry, immunofluorescence immunoassay, and Northern blot, among others.

According to some embodiments, the mutual difference in the amount of expressed immunomodulatory factor is randomized to regions of higher or lower transcriptional activity in the genome of the tumor cell line or tumor cell line variant. It can be the result of built-in. According to some embodiments, the relative difference in the amount of expressed immunomodulatory factor is manipulated into the transfected or transduced DNA used to generate the tumor cell line or tumor cell line variant. can be achieved by the factor

For example, according to some embodiments, the expression level of exogenous immunoregulatory molecules is altered at the transcriptional level by manipulating stronger or weaker gene promoter sequences to control the expression of immunoregulator genes. can be achieved. According to some embodiments, one or more of the following promoters can be used to control the expression of immune modulators: simian virus 40 early promoter (SV40), cytomegalovirus immediate early promoter (CMV ), the human ubiquitin C promoter (UBC), the human elongation factor 1α promoter (EF1A), the mouse phosphoglycerate kinase 1 promoter (PGK), and the chicken β-actin promoter (CAGG) coupled with the CMV early enhancer.

According to some embodiments, expression levels of exogenous immunomodulatory molecules can be achieved at the translational level by engineering stronger or weaker Kozak consensus sequences near the start codon of the immunomodulator transcript. According to some embodiments, the following nucleotide sequence can be provided to control translation of an immunomodulatory factor: GCCGCC(A/G)CCAUGG (SEQ ID NO: 15). According to some embodiments, a sequence that is at least 60% identical to SEQ ID NO: 15 can be provided to control translation of an immunomodulatory factor. According to some embodiments, a sequence that is at least 70% identical to SEQ ID NO: 15 can be provided to control translation of an immunomodulatory factor. According to some embodiments, a sequence that is at least 80% identical to SEQ ID NO: 15 can be provided to control translation of an immunomodulatory factor. According to some embodiments, a sequence that is at least 90% identical to SEQ ID NO: 15 can be provided to control translation of an immunomodulatory factor. According to some embodiments, a sequence that is at least 95% identical to SEQ ID NO: 15 can be provided to control translation of an immunomodulatory factor. According to some embodiments, a sequence that is at least 96% identical to SEQ ID NO: 15 can be provided to control translation of an immunomodulatory factor. According to some embodiments, a sequence that is at least 97% identical to SEQ ID NO: 15 can be provided to control translation of an immunomodulatory factor. According to some embodiments, a sequence that is at least 98% identical to SEQ ID NO: 15 can be provided to control translation of an immunomodulatory factor. According to some embodiments, a sequence that is at least 99% identical to SEQ ID NO: 15 can be provided to control translation of an immunomodulatory factor.

Non-viral approaches can also be used for the introduction of vectors encoding one or more immunomodulatory molecules into cells derived from patients with tumors or tumor cell lines or variants. For example, nucleic acid molecules encoding immunomodulatory molecules can be obtained by lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. al of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989) or by microinjection under surgical conditions (Wolff et al. , Science 247:1465, 1990). Preferably, nucleic acids are administered in combination with liposomes and protamine.

Methods for achieving transfection in vitro include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA to cells.

IV. Therapeutic Compositions Immunogenic compositions of the invention, including allogeneic tumor cell vaccines, are useful as therapeutic and prophylactic agents for the treatment of certain types of cancer. Advantageously, these vaccines can be tailored to treat cancer in specific individuals by generating vaccines that target specific tumor antigens expressed in the tumor of interest. Allogeneic vaccines of the invention typically comprise inactivated tumor cells or cells expressing tumor antigens that have been genetically modified to express exogenous immunomodulatory molecules, as described herein. . According to some embodiments, an allogeneic tumor cell vaccine may comprise a dose of a tumor cell line or tumor cell line variant that contains two or more genes encoding immunomodulatory molecules. According to some embodiments, clones of tumor cell lines or tumor cell line variants that maximally express immunomodulatory molecules are identified and selected. According to some embodiments, expression of immunomodulatory molecules by a population of tumor cell lines or tumor cell line variants is determined by flow cytometry. According to some embodiments, flow cytometry is used to gate the maximal expressing population(s) of the tumor cell line or tumor cell line variant.

According to some embodiments, the immunogenic amount is effective to stimulate an anti-tumor immune response against one or more tumor-specific antigens. According to some embodiments, the immunogenicity amount can be titrated to provide both safety and efficacy.

According to some embodiments, an immunogenic composition comprises a pharmaceutically acceptable carrier.

According to some embodiments the immunogenic composition further comprises an adjuvant.

According to some embodiments, a tumor cell line or tumor cell line variant may comprise tumor cells derived from an established cell line. According to some embodiments, the tumor cell line or tumor cell line variant comprises tumor cells derived from a cancer patient, wherein the tumor cells are derived from solid tumors.

According to some embodiments, the tumor cell line or tumor cell line variant comprises an immunogenic amount of a disrupted tumor cell line or tumor cell line variant. Examples of methods of physical disruption include, without limitation, sonication, cavitation, dehydration, ion depletion, or toxicity by exposure to one or more salts.

According to some embodiments, an immunogenic amount of an immunogenic composition may comprise at least 1×10 ³ cells of an intact or disrupted tumor cell line or tumor cell line variant. According to some embodiments, the amount of immunogenic composition may comprise at least 1×10 ⁴ cells of a intact or disrupted tumor cell line or tumor cell line variant. According to some embodiments, the amount of immunogenic composition may comprise at least 1×10 ⁵ cells of a intact or disrupted tumor cell line or tumor cell line variant. According to some embodiments, the amount of immunogenic composition may comprise at least 1×10 ⁶ cells of a intact or disrupted tumor cell line or tumor cell line variant. According to some embodiments, the amount of immunogenic composition may comprise at least 1×10 ⁷ cells of a intact or disrupted tumor cell line or tumor cell line variant. According to some embodiments, the amount of immunogenic composition may comprise at least 1×10 ⁸ cells of a intact or disrupted tumor cell line or tumor cell line variant. According to some embodiments, the amount of immunogenic composition may comprise at least 1×10 ⁹ cells of a intact or disrupted tumor cell line or tumor cell line variant. According to some embodiments, an immunogenic amount can be a therapeutic amount.

According to some embodiments, the immunogenic amount is (1) of cytotoxic T cell populations, natural killer cell populations, antibodies, APCs, T cell populations, B cell populations, and dendritic cell populations effective in inducing an immune response that reduces tumor burden, including one or more; and (2) the subject's progression free survival, disease free survival, time to progression, distant Effective in improving a clinical outcome parameter selected from one or more of time to metastasis and overall survival.

According to some embodiments, the immunogenic composition is administered once a week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month Once, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months once every 9 months, once every 10 months, once every 11 months, or once a year. According to some embodiments, the administration is for one day, or for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days. , 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or more. According to some embodiments, administration may comprise two or more administrations on the same day.

Combination Therapy According to some embodiments, the present disclosure provides methods further comprising administering an additional agent to the subject. According to some embodiments, the present invention relates to co-administration and/or co-formulation.

According to some embodiments, administration of an immunogenic composition acts synergistically when co-administered with another agent, and generally when such agents are used as monotherapy. administered at lower doses than those used for

According to some embodiments, including but not limited to cancer applications, the present invention relates to chemotherapeutic agents as additional agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cytoxancyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, metledopa, and uredopa; ethyleneimines and methylameramines, including, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (e.g., bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan); CC-1065 (including its adzelesin, carzelesin, vizelesin synthetic analogues); cryptophycins (eg, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (synthetic analogue, KW-2189) sarcodictin; spongistatin; chlorambucil, chlornafadine, clophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan Nitroureas such as Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, and Ranimustine dynemycins, including dynemycin A; bisphosphonates such as clodronate; esperamycin; , azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardinophylline, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin doxorubicin (morpholino-doxorubicin, cyanomorpholino -doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, ethorubicin, idarubicin, marceromycin, mitomycins such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potofilomycin, puromycin, keramycin, rhodorubicin , streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as , 6-mercaptopurine, thiamipurine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; carsterone, dromostanolone propionate, epi androgens such as thiostanol, mepitiostane, testolactone; anti-adrenal agents such as minoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; elformitin; elliptinium acetate; epothilone; etogluside; gallium nitrate; hydroxyurea; lentinan; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; lazoxan; rhizoxin; schizofuran; spirogermanium; triethylamine; trichothecenes (eg, T-2 toxin, veracrine A, roridin A, and angidin); urethane; vindesine; dacarbazine; thiotepa; taxoids such as TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.O.; J. ), Abraxane, an albumin-engineered nanoparticle formulation of paclitaxel without cremophore, and taxoteredoxetaxel; chlorambucil; gemzar gemcitabine; 6-thioguanine; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); ); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation, and any of the above pharmaceutical agents. non-limiting examples thereof include, but are not limited to, legally acceptable salts, acids or derivatives. Additionally, the therapeutic method can further include the use of radiation.

Checkpoint Blockade/Block of Tumor Immunosuppression Some human tumors can be eliminated by the patient's immune system. For example, administration of monoclonal antibodies that target immune "checkpoint" molecules results in complete responses and tumor remissions. The mode of action of such antibodies is through inhibition of immunomodulatory molecules that tumors have selected to protect against anti-tumor immune responses. By inhibiting these "checkpoint" molecules (eg, with antagonist antibodies), the patient's CD8+ T cells can proliferate and destroy tumor cells.

According to some embodiments, the allogeneic vaccine composition may be effective in preventing premature termination of an effective immune response once an effective immune response has been initiated by inhibiting one or more checkpoints. further comprising an agent.

For example, by way of example and not limitation, administration of monoclonal antibodies targeting CTLA-4 or PD-1 can result in complete responses and tumor remissions. The mechanism of action of such antibodies is through inhibition of CTLA-4 or PD-1, which tumors select for protection against anti-tumor immune responses. By inhibiting these "checkpoint" molecules (eg, with antagonist antibodies), the patient's CD8+ T cells can proliferate and destroy tumor cells.

Accordingly, the allogeneic vaccine compositions provided herein can be used in combination with one or more blocking antibodies that target immune "checkpoint" molecules. For example, according to some embodiments, the allogeneic vaccine compositions provided herein are used in combination with one or more blocking antibodies that target molecules such as CTLA-4 or PD-1. can do. For example, the allogeneic vaccine compositions provided herein block binding of PD-1 and PD-L1 or PD-L2, and/or PD-1 to PD-L1 or PD-L2. , reducing and/or inhibiting agents (non-limiting examples include nivolumab (ONO-4538/BMS-936558, MDX1106, Opdivo, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL328OA (ROCHE)). According to some embodiments, the allogeneic vaccine compositions provided herein block CTLA-4 activity and/or binding of CTLA-4 to one or more receptors, It can be used in combination with agents that reduce and/or inhibit (eg, CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For example, according to some embodiments, the immunomodulatory agent is an antibody such as, without limitation, ipilimumab (MDX-010, MDX-101, Yervoy, BMS) and/or tremelimumab (Pfizer). Blocking antibodies to these molecules are available from, for example, Bristol Myers Squibb (New York, N.Y.), Merck (Kenilworth, N.J.), Medlmune (Gaithersburg, Md.), and Pfizer (New York, N.Y.). ) are available from

In addition, the allogeneic immunization compositions provided herein contain immune "checkpoint" molecules such as BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4, CD160 (also called BY55), CGEN. -15049, CHK1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), GITR, GITRL, galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, TMIGD2, and various B-7 family ligands (B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7- H7), etc.) can be used in combination with one or more blocking antibodies.

adjuvant

According to some embodiments, the compositions of the present invention, due to their adjuvant properties, stimulate the immune system to respond to cancer antigens present on inactivated tumor cell(s). It may further contain one or more additional substances capable of acting. Such adjuvants include lipids, liposomes, inactivated bacteria that induce innate immunity (e.g., inactivated or attenuated Listeria monocytogenes), Toll-like receptors (TLR), (NOD)-like receptors (NLR), Compositions that mediate innate immune activation via retinoic acid-inducible gene-based (RIG)-I-like receptors (RLR), and/or C-type lectin receptors (CLR), including but not limited to . Examples of PAMPs include lipoproteins, lipopolypeptides, peptidoglycans, zymosan, lipopolysaccharides, neisseriaporins, flagellins, profilins, galactoceramides, muramyl dipeptides. Peptidoglycan, lipoproteins, and lipoteichoic acid are Gram-positive cell wall components. Lipopolysaccharides are expressed by most bacteria, with MPL as an example. Flagellins refer to structural components of bacterial flagella that are secreted by pathogenic and commensal bacteria. α-galactosylceramide (α-GalCer) is an activator of natural killer T (NKT) cells. Muramyl dipeptide is a bioactive peptidoglycan motif common to all bacteria. This list is not exhaustive.

According to some embodiments, the treatment regimen includes standard anti-tumor therapies, such as surgery, radiotherapy, targeted therapies that precisely identify and attack cancer cells, hormonal therapies, or combinations thereof. can contain. According to some embodiments, standard anti-tumor therapies are effective in treating tumors while maintaining pre-existing anti-tumor immune responses. According to some embodiments, the immunogenic composition is not applied after chemotherapy. According to some embodiments, the immunogenic composition is applied after low dose chemotherapy.

According to some embodiments, an immunogenic composition comprises two or more clonally derived tumor cell lines or tumor cell line variants. According to some embodiments, the two or more tumor cell lines or tumor cell line variants contain the same complement of recombinant immunomodulators. According to some embodiments, the two or more tumor cell lines or tumor cell line variants comprise different panels of recombinant immunomodulators.

According to some embodiments, the tumor cell line or tumor cell line variant is treated with an agent that prevents cell division prior to administration to the subject. According to some embodiments, the tumor cell line or tumor cell line variant is irradiated. According to some embodiments, the tumor cell line or tumor cell line variant is treated with a chemical agent that prevents proliferation.

According to some embodiments, the tumor cell line or tumor cell line variant may be administered parenterally. According to some embodiments, a tumor cell line or tumor cell line variant can be administered locally to a surgical resection cavity. According to some embodiments, tumor cell variants may be administered by intradermal injection. According to some embodiments, the tumor cell line or tumor cell line variant may be administered by subcutaneous injection. According to some embodiments, the tumor cell line or tumor cell line variant may be administered by intramuscular injection.

V. Methods of Treatment According to some embodiments, the allogeneic tumor cell vaccines described herein are capable of recognizing and acting on tumor cells containing target tumor antigens in vivo without systemic inflammation. enhance immune activation, reduce immunosuppression in the tumor microenvironment of tumor cells containing the target tumor antigen, reduce tumor burden, or increase cell death of tumor cells expressing the target tumor antigen. effective for According to some embodiments, the allogeneic tumor cell vaccines described herein are effective to induce immune activation without systemic inflammation. According to some embodiments, the allogeneic tumor cell vaccine is effective to elicit an immune response that improves progression-free survival, overall survival, or both compared to a placebo control.

According to some embodiments, the allogeneic vaccine composition is administered to a subject diagnosed with cancer in combination with an agent that inhibits immunosuppressive molecules produced by tumor cells.

According to some embodiments, the described invention comprises an allogeneic tumor cell vaccine for active immunotherapy that can be universally administered to all patients with a particular type of cancer. According to some embodiments, the allogeneic vaccine comprises genetically modified allogeneic tumor type-specific cells or modified allogeneic tumor cells formulated in a pharmaceutically acceptable carrier. Contains membrane lysates derived from type-specific cells. According to some embodiments, the modified allogeneic tumor type-specific cells are derived from previously established cell lines.

According to some embodiments, allogeneic vaccines are self-recognition and responses to non-self through minimal residual disease and innate (tolerant) and adaptive (specific) immunity, including humoral and cellular immunity. adapted to treat patients with a functional immune system comprising For example, according to some embodiments, the allogeneic vaccine is adapted to treat patients with minimal residual disease obtained shortly after the primary lesion is surgically removed. According to some embodiments, the allogeneic vaccine is adapted for subcutaneous administration of the vaccine. According to some embodiments, doses and schedules for administering allogeneic vaccines are determined by using the immune response to the vaccine as a guide for eventual enhancement of overall survival.

According to some embodiments, the present disclosure provides a method of inducing an immune response against cancer in a subject, comprising administering an allogeneic tumor cell vaccine described herein to the subject parenterally or intratumorally. The allogeneic tumor cell vaccine is type-matched to the subject's cancer.

A tumor cell line or tumor cell line variant provided herein is incorporated into a composition for administration to a subject (e.g., a research animal or a mammal, such as a human, having a clinical condition such as cancer or an infectious disease) be able to. For example, a tumor cell line or tumor cell line variant genetically engineered to stably express a core group of three immunomodulatory molecules, and a pharmaceutically acceptable carrier, comprising: are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), can be administered to a subject for treatment of cancer. In another example, allogeneic tumor cell vaccines containing tumor type-specific cell line variants have been used to demonstrate effective efficacy as reflected in improved progression-free survival, overall survival, or both compared to placebo controls. Delivers a broad spectrum of tumor antigens in the context of an immunomodulatory signal sufficient to elicit an anti-tumor response, the immunomodulatory signal comprising a core group of at least three immunomodulatory molecules, the core group of immunomodulatory molecules , OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), optionally from 3 to 25 immunomodulators selected from those shown in Table 2 (“R group”) An additional number of immunomodulatory molecules, including

Accordingly, the described invention provides methods of treating clinical conditions such as cancer with the allogeneic tumor vaccines provided herein.

According to various embodiments, the described invention relates to cancer and/or tumors, eg, treatment or prevention of cancer and/or tumors. The phrase "cancer or tumor" refers to the uncontrolled proliferation of cells and/or abnormal increased cell survival and/or inhibition of apoptosis that interferes with the normal functioning of the body's organs and systems. Benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases are included. Also included are cells with abnormal growth that are not prevented by the immune system (eg, virus-infected cells). Cancer can be primary or metastatic. A primary cancer can be an area of cancer cells at the site of origin that becomes clinically detectable and can be a primary tumor. In contrast, metastatic cancer can be the spread of disease from one organ or part to another non-adjacent organ or part. Metastatic cancer can be caused by cancer cells that have acquired the ability to penetrate and invade normal tissue around a localized area and form new tumors, which can result in local metastases. Cancer can also be caused by cancer cells that have acquired the ability to penetrate the walls of lymphatics and/or blood vessels, after which cancer cells travel through the bloodstream to other sites and tissues in the body. able to circulate (and thus become circulating tumor cells). Cancer may result from processes such as lymphatic or hematogenous spread. Cancer can also be caused by tumor cells that become quiescent at another site, re-infiltrate through blood vessels or walls, continue to grow, and eventually form another clinically detectable tumor. Cancer may be with this new tumor, and it may be a metastatic (or secondary) tumor.

Cancer may be caused by tumor cells that have metastasized, which may be secondary or metastatic tumors. Cells of a tumor may resemble cells of the original tumor. As an example, if breast or colon cancer metastasizes to the liver, the secondary tumor is present in the liver but is made up of abnormal breast or colon cells rather than abnormal hepatocytes. Therefore, tumors in the liver may be metastatic breast or colon cancer rather than liver cancer.

Examples of cancers that can be treated include carcinoma (various subtypes including adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcoma (including bone and soft tissue), Leukemia (including e.g. acute myeloid, acute lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell), lymphoma and myeloma (e.g. Hodgkin and non-Hodgkin's lymphoma, light chain, non-secretory , MGUS, and plasmacytoma), and cancers of the central nervous system (e.g., brain (e.g., glioma (e.g., astrocytoma, oligodendroglioma, and ependymoma)), Includes, but is not limited to, pituitary adenoma, neuroma, and spinal cord tumors (eg, meningioma and neurofibroma).

According to certain embodiments, the treatable cancer/tumor is that chemotherapy is known to interfere with the immune response, which is expected to occur during successful vaccination protocols. Therefore, the standard of care is no longer chemotherapy. Exemplary tumor types include tumor types treated with hormonal therapy such as prostate and breast cancer (e.g. Abiraterone for prostate cancer and Tamoxifen for breast cancer), antibody (e.g. B cell malignant tumor types treated with targeted therapies such as Rituxan® for tumors, Herceptin® for breast cancer, tumor types treated with kinase inhibitors such as Gleevec® for chronic myelogenous leukemia, and tumor types treated with other immune system-sparing or enhancing modalities such as checkpoint inhibitors, oncolytic viruses, and CAR-T cells.

Representative cancers and/or tumors of the invention are described herein.

The disclosure also includes two or more stably expressed recombinant exogenous immunomodulatory molecules selected from cytokines, TNF family members, secretory receptors, chaperones, members of the IgG superfamily, and chemokine receptors. , provides a composition comprising an allogeneic tumor cell vaccine comprising a tumor cell line or tumor cell line variant. The disclosure also includes two or more stably expressed recombinant exogenous immunomodulatory molecules selected from cytokines, TNF family members, secretory receptors, chaperones, members of the IgG superfamily, and chemokine receptors. , compositions comprising allogeneic tumor cell vaccines comprising two, three, four or more tumor cell lines or tumor cell line variants.

The disclosure also provides an allogeneic tumor cell vaccine comprising a tumor cell line or tumor cell line variant comprising two or more stably expressed recombinant membrane-bound immunomodulatory molecules selected from those shown in Table 2. and a pharmaceutically acceptable carrier in combination with a physiologically and pharmaceutically acceptable carrier as described herein. Physiologically and pharmaceutically acceptable carriers can include any of the well-known ingredients useful for immunization. A carrier can promote or enhance the immune response to the antigen administered in the vaccine. The cell preparation can include buffers to maintain a preferred pH range, salts, or other components that present the antigen to the individual in a composition that stimulates an immune response to the antigen. A physiologically acceptable carrier can also contain one or more adjuvants that enhance the immune response to the antigen. Pharmaceutically acceptable carriers include, for example, pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert vehicle for delivering a compound to a subject. Pharmaceutically acceptable carriers can be liquid or solid, and have a desired bulk, consistency, or consistency when combined with one or more therapeutic compounds and any other ingredients of a given pharmaceutical composition. The planned mode of administration can be chosen with in mind to provide the degree, as well as other suitable transport and chemical properties. Exemplary pharmaceutically acceptable carriers include, without limitation, water, saline, binders such as polyvinylpyrrolidone or hydroxypropylmethylcellulose, bulking agents such as lactose or dextrose and other sugars, gelatin, or calcium sulfate), lubricants (eg, starch, polyethylene glycol, or sodium acetate), disintegrants (eg, starch or sodium starch glycolate), and wetting agents (eg, sodium lauryl sulfate). The compositions may be formulated for subcutaneous, intramuscular, or intradermal administration, or in any acceptable manner for immunization.

"Adjuvant" means, when added to an immunogenic agent, such as a tumor cell expressing a secreted vaccine protein, non-specifically enhances or enhances the immune response to the agent in the recipient host upon exposure to the mixture. Refers to a substance that strengthens. Adjuvants can include, for example, oil-in-water emulsions, water-in-oil emulsions, alum (aluminum salts), liposomes, and microparticles such as polystyrenes, starches, polyphosphazenes, and polylactides/polyglycosides.

Adjuvants also include, for example, squalene mixtures (SAF-I), muramyl peptides, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunits, polyphosphazenes, and derivatives, and Takahashi et al. , Nature 1990, 344:873-875. For veterinary use and antibody production in animals, the mitogenic components of Freund's adjuvant (both complete and incomplete) can be used. In humans, incomplete Freund's adjuvant (IFA) is a useful adjuvant. A variety of suitable adjuvants are well known in the art (see, e.g., Warren and Chedid, CRC Critical Reviews in Immunology 1988, 8:83; and Allison and Byars, in Vaccines: New Approaches to Immunological P roblems, 1992, Ellis, ed. ., Butterworth-Heinemann, Boston). Additional adjuvants include, for example, Bacillus Calmette-Guerin (BCG), DETOX, which includes the cell wall skeleton of Mycobacterium phlei (CWS) and monophosphoryl lipid A from Salmonella minnesota (MPL), and the like (see, e.g., Hoover et al.). al., J Clin Oncol 1993, 11:390; and Woodlock et al., J Immunother 1999, 22:251-259).

According to some embodiments, the allogeneic tumor cell vaccine is administered to the subject one or more times (eg, 1, 2, 2-4, 3-5, 5-8, 6 times to 10 times, 8 times to 12 times, or more than 12 times). Allogeneic tumor cell vaccines provided herein may be administered once or more daily, once or more weekly, biweekly, once or more monthly, once every 2-3 months, 3- It can be administered once every 6 months, or once every 6-12 months. Allogeneic tumor cell vaccines can be administered over any suitable period of time, such as for a period of about 1 day to about 12 months. According to some embodiments, for example, the administration period is about 1-90 days, about 1-60 days, about 1-30 days, about 1-20 days, about 1-10 days, It can be about 1 to 7 days. According to some embodiments, the administration period is about 1 week to 50 weeks, about 1 week to 50 weeks, about 1 week to 40 weeks, about 1 week to 30 weeks, about 1 week to 24 weeks, about 1 weeks to 20 weeks, about 1 week to 16 weeks, about 1 week to 12 weeks, about 1 week to 8 weeks, about 1 week to 4 weeks, about 1 week to 3 weeks, about 1 week to 2 weeks, 2 weeks or more It can be 3 weeks, about 2-4 weeks, about 2-6 weeks, about 2-8 weeks, about 3-8 weeks, 3-12 weeks, or about 4-20 weeks.

According to some embodiments, after an initial dose (sometimes referred to as a "priming" dose) of an allogeneic tumor cell vaccine is administered and a maximal antigen-specific immune response is achieved, one or more Booster doses can be administered. For example, boosting doses can be administered about 10-30 days, about 15-35 days, about 20-40 days, about 25-45 days, or about 30-50 days after the priming dose.

According to some embodiments, the methods provided herein can be used to control growth and/or metastasis of solid tumors. The method can comprise administering to a subject in need thereof an effective amount of an allogeneic tumor cell vaccine described herein.

The vectors and methods provided herein can be useful for stimulating an immune response against tumors. Such immune responses are useful in treating or ameliorating signs or symptoms associated with tumors. Practitioners will appreciate that the methods described herein are used in conjunction with ongoing clinical evaluation by a skilled practitioner (physician or veterinarian) to determine subsequent treatment. Such assessments are helpful and informative in evaluating whether to increase, decrease, or continue a particular therapeutic dose, dosing regimen, or the like.

Thus, the methods provided herein can be used to treat tumors, including cancer, for example. This method can be used to inhibit tumor growth, for example, by preventing further tumor growth, by slowing tumor growth, or by causing tumor regression. Thus, this method can be used, for example, to treat cancer. It is understood that the subject to whom the compound is administered need not suffer from a particular traumatic condition. Indeed, the allogeneic tumor cell vaccines described herein can be administered prophylactically before symptoms develop (eg, patients in remission from cancer).

Anti-tumor and anti-cancer effects include, but are not limited to, modulation of tumor growth (e.g., retardation of tumor growth), tumor size, or metastasis, reduction of toxicity and side effects associated with certain anti-cancer agents. amelioration or minimization of clinical damage or symptoms of cancer, prolongation of survival of a subject beyond that expected in the absence of such treatment, and reduction of tumor growth in animals lacking pre-administration tumorigenesis. Prophylaxis, ie prophylactic administration, is included.

A therapeutically effective amount can be determined, for example, by starting with a relatively low amount and using incremental increments while concurrently assessing beneficial effects. Thus, the methods provided herein can be used alone or in combination with other known tumor therapies to treat patients with tumors. Those skilled in the art are familiar with the allogeneic tumor cell vaccines provided herein, e.g., in extending the life expectancy of cancer patients and/or improving the quality of life of cancer patients (e.g., lung cancer patients). and will readily appreciate the advantageous use of the method.

According to some embodiments, a subject (ie, a subject diagnosed with cancer) is treated with checkpoint inhibitor therapy prior to or concurrently with administration of an allogeneic vaccine composition. In some embodiments, the cancer is melanoma.

Subjects The methods described herein are intended for use with any subject who may experience the benefits of these methods. Thus, "subject,""patient," and "individual" (used interchangeably) include human and non-human subjects, particularly domestic animals.

According to some embodiments, the subject and/or animal is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as be a monkey, chimpanzee, or baboon. In other embodiments, the subject and/or animal is non-mammal, eg, zebrafish. According to some embodiments, the subject and/or animal may contain fluorescently labeled cells (eg, with GFP). According to some embodiments, the subject and/or animal is a transgenic animal comprising fluorescent cells.

According to some embodiments, the subject and/or animal is human. According to some embodiments, the human is a pediatric human. In other embodiments, the human is an adult. In other embodiments, the human is elderly. In other embodiments, humans may be referred to as patients.

According to certain embodiments, the human is about 0 months to about 6 months, about 6 to about 12 months, about 6 to about 18 months, about 18 to about 36 months, about 1 to about 5 months. years old, about 5 to about 10 years old, about 10 to about 15 years old, about 15 to about 20 years old, about 20 to about 25 years old, about 25 to about 30 years old, about 30 to about 35 years old, about 35 to about 40 years old , about 40 to about 45 years old, about 45 to about 50 years old, about 50 to about 55 years old, about 55 to about 60 years old, about 60 to about 65 years old, about 65 to about 70 years old, about 70 to about 75 years old, Has an age ranging from about 75 to about 80 years, about 80 to about 85 years, about 85 to about 90 years, about 90 to about 95 years, or about 95 to about 100 years.

According to another embodiment, the subject is a non-human animal and thus the invention relates to veterinary uses. According to certain embodiments, the non-human animal is a domestic pet. According to another particular embodiment, the non-human animal is a livestock animal. According to certain embodiments, the subject is a human cancer patient who cannot undergo chemotherapy. For example, the patient does not respond to chemotherapy or is too sick to have a suitable therapeutic window for chemotherapy (eg, too many dose- or regimen-limiting side effects). In certain embodiments, the subject is a human cancer patient with progressive and/or metastatic disease.

When a range of values is provided, unless the context clearly dictates otherwise, a Each intermediate value in is encompassed by the present invention up to one tenth of the lower unit. The upper and lower limits of these smaller ranges that may independently be included in the smaller ranges are also encompassed within the invention, subject to any specifically excluded limit in the stated range. . Where the stated range includes one or both of the limits, ranges excluding either of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" refer to plural referents unless the context clearly dictates otherwise. Note that it contains All technical and scientific terms used herein have the same meaning.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are intended to limit the scope of what the inventors regard as their invention. It is not intended to represent that the following experiments were all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1.
Examples utilize, but are not limited to, the methods described below.

Western Blotting Briefly, cells are lysed with cold lysis buffer and centrifuged to pellet cell debris. The protein concentration of the supernatant is determined by a protein quantitation assay (eg Bradford Protein Assay, Bio-Rad Laboratories). The lysate supernatant is then combined with an equal volume of 2×SDS sample buffer and boiled at 100° C. for 5 minutes. Equal amounts of protein in sample buffer are loaded into wells of SDS-PAGE gels along with molecular weight markers and electrophoresed at 100V for 1-2 hours. The proteins are then transferred to nitrocellulose or PVDF membranes. The membrane is then blocked for 1 hour at room temperature using 5% non-fat dry milk in TBST blocking buffer. The membrane was then incubated with a 1:500 dilution of primary antibody in 5% non-fat dry milk in TBST blocking buffer, followed by 20 mM Tris, Ph7.5; 150 mM NaCl, 0.1% Tween 20 (TBST ) for 5 minutes three times. The membrane is then incubated with conjugated secondary antibody diluted 1:2000 in 5% non-fat dry milk in TBST blocking buffer for 1 hour at room temperature, followed by 3 washes of TBST for 5 minutes each. Images of blots are acquired using darkroom development techniques for chemiluminescent detection or using image scanning techniques for colorimetric or fluorescent detection.

Real-time PCR
Real-time PCR techniques can be performed as described to analyze mRNA expression levels (Zhao Y. et al., Biochemical and Biophysical Research Communications 360 (2007) 205-211). Briefly, total RNA is extracted from cells using the Quiagen kit (Valencia Calif.) followed by first strand cDNA synthesis using random hexamer primers (Fermentas, Hanover Md.). Real-time PCR is performed for 40 cycles using validated gene-specific RT-PCR primer sets for each gene of interest using the Mx3000p Quantitative PCR System (Stratagene, La Jolla, Calif.). The relative expression level of each transcript is corrected for the expression level of the housekeeping gene beta-actin as an internal control.

Immunofluorescence Briefly, adherent tumor cell line variant cells are fixed with 4% formaldehyde diluted in warm PBS for 15 minutes at room temperature. Aspirate the fixative and wash the cells with PBS three times for 5 minutes each. Cells are blocked with 5% BSA blocking buffer for 60 minutes at room temperature. The blocking buffer is then aspirated and a solution of primary antibody (eg, 1:100 dilution) is incubated with the cells overnight at 4°C. Cells are then rinsed three times with PBS for 5 minutes each, followed by incubation with a solution of fluorochrome-conjugated secondary antibody (eg, 1:1000 dilution) for 1-2 hours at room temperature. Cells are then washed with PBS three times for 5 minutes each and visualized by fluorescence microscopy.

Flow Cytometry Flow cytometry analysis can be performed as described (Zhao Y. et al., Exp. Cell Res., 312, 2454 (2006)). Briefly, tumor cell line mutant cells, either treated with trypsin/EDTA or left untreated, are harvested by centrifugation and resuspended in PBS. Cells are fixed with 4% formaldehyde for 10 minutes at 37°C. For extracellular staining with antibodies, cells are not permeabilized. For intracellular staining, cells are permeabilized by adding ice-cold 100% methanol to pre-chilled cells to a final concentration of 90% methanol and incubating on ice for 30 minutes. Cells are immunostained by first resuspending them in incubation buffer and adding dilutions of primary antibody. Cells are incubated with primary antibody for 1 hour at room temperature and then washed 3 times with incubation buffer. Cells are then resuspended in incubation buffer containing dilutions of conjugated secondary antibody for 30 minutes at room temperature, followed by three washes with incubation buffer. Stained cells are then analyzed by flow cytometry.

Enzyme-linked immunosorbent assay (ELISA)
Briefly, the wells of a microplate are coated with a capture antibody specific for the protein of interest. Standards containing the protein of interest, control samples, and samples containing the unknown protein are pipetted into microplate wells where the protein antigen binds to the capture antibody. After four washes, the detection antibody is added to the wells for 1 hour and binds to the immobilized protein captured during the first incubation. After removing excess detection antibody and washing four times, a horseradish peroxidase (HRP) conjugate (secondary antibody or streptavidin) is added for 30 minutes to bind to the detection antibody. After 4 additional washes to remove excess HRP conjugate, substrate solution is added for 30 minutes in the dark and converted to an enzymatically detectable form (color signal). Stop solution is added to each well of the microplate and evaluated within 30 minutes of stopping the reaction. The intensity of the colored product may be directly proportional to the concentration of antigen present in the original specimen.

Human Mixed Lymphocyte Tumor Response (MLTR) Test The Mixed Lymphocyte Tumor Response (MLTR) is an all-human in vitro assay designed to optimize lead candidates. In MLTR, optimization is achieved through qualitative and quantitative assessment of human peripheral blood mononuclear cell (PBMC) responses to engineered allogeneic tumor cells. MLTR measures proliferation and differentiation by flow cytometry and mass cytometry (CyTOF), by cytotoxicity, by lactate dehydrogenase (LDH) release assay, and by cytokine profile. According to some embodiments, allogeneic cell pools expressing a single immunomodulatory protein are used in the MLTR. According to some embodiments, allogeneic cell pools expressing one or more, two or more, three or more, four or more, or five or more immunomodulatory proteins are used in the MLTR.

A basic MLTR daily routine runs as follows.

Thaw a vial of PBMCs (20 MN cells). Cells are then washed with dPBS. PMBC cells are resuspended at 2.5×10 ⁶ cells/ml in X-VIVO (approximately 8 ml). Cells are characterized by flow cytometry to document the properties of the cell population.

Usage in MLTR is performed as follows.
2.5×10 ⁵ cells of PBMC (100 μl stock)
0.5×10 ⁵ allogeneic cells (100 μl stock), 0.5×10 ⁵ allogeneic cells (100 μl stock) when used. These cells are inactivated with mitomycin C.
50 μl of positive control 6× stock (anti-CD28/CD3)
300 μl total volume for 96-well flat bottom—96-well total volume is 360 μl.
CyTOF with 100 μl removed for cytokine analysis by Luminex incubating for 4 days is performed on the remaining 200 μl.
Supernatants for cytokine profiling are removed after one day.

CyTOF is described, for example, in Bendall et al. (Science, Vol. 332, 6 May 2011) and Bendall and Nolan ((Nature Biotechnology, Vol. 30 No. 7, July 2012), both of which are incorporated herein by reference in their entirety. Human markers used in CyTOF staining are shown in Table 6 below.

Luminex Multiplex Assay Luminex xMAP technology (formerly LabMAP, FlowMetrix) uses digital signal processing that can sort polystyrene beads (microspheres) stained with different ratios of red and near-infrared fluorophores. These ratios define the "spectral address" of each bead population. As a result, up to 100 different detection reactions can be run simultaneously on different bead populations with very small sample volumes (Earley et al. Report from a Workshop on Multianalyte Microsphere Arrays. Cytometry 2002; 50:239-242 Oliver et al., Clin Chem 1998;44(9):2057-2060;Eishal and McCoy, Methods 38(4):317-323, April 2006, all of which are incorporated herein by reference in their entirety. ).

Luminex multiplex assays are commercially available and are available from thermofisher. com/us/en/home/life-science/protein-biology/protein-assays-analysis/luminex-multiplex-assays. html world wide web, which is incorporated herein by reference in its entirety.

Cellular Mitomycin C Preparation Mitomycin C was prepared from dry powder (2 mg per vial) using 400 μl DMSO (500× stock = 5 mg/ml), completely dissolved and dispensed into 25 ul volumes, − Store at 80°C. One aliquot of 20 μl is used in 10 ml of warmed C5 to produce a final working solution of 10 μg/ml. The solution is sterile filtered.

This solution can be used for resuspended or adherent cells in flasks.

Cells are incubated in the dark at 37° C. for 30 minutes, then washed 3 times with warmed C5. Resuspend the cells in 1 ml of X-VIVO. 40ul counts to 200ul on the plate. Cells are resuspended in X-VIVO (serum-free medium, Lonza) at a final concentration of 1×10 ⁶ /ml.

Example 2
The described invention provides an approach for restoring immune balance, eg in the treatment of cancer, by targeting multiple immunomodulators on a single cell platform. This approach allows the simultaneous modification of multiple signals and provides a spatially restricted site of action, a key feature that has limited previous approaches to restore immunological balance.

According to one aspect of the disclosed invention, tumor cell line variants can be generated that express five or more recombinant peptides for use as tumor cell vaccines to treat cancer. For example, a tumor cell line can be selected for modification and lentiviral transfection of recombinant immunomodulator sequences can be used to stably integrate the immunomodulator into the cell's genome. Example 3 below describes seven lentiviral vectors (vector 1, vector 2, vector 3, vector 4, vector 5, vector 6, and vector 7) is described.

According to some embodiments, two recombinant immunomodulatory proteins are co-transfected, followed by co-transfection of two more recombinant immunomodulatory proteins, followed by transfection of a single recombinant immunomodulatory protein. A total of 5 recombinant peptides for use as tumor cell vaccines can be achieved. According to some embodiments, two recombinant peptides are transfected simultaneously, followed by transfection of a single recombinant peptide, followed by transfection of a single recombinant peptide, followed by transfection of a single of recombinant peptides to achieve a total of 5 recombinant peptides for use as tumor cell vaccines. According to some embodiments, a single recombinant immunomodulatory protein is co-transfected, followed by co-transfection of two recombinant immunomodulatory proteins, followed by transfection of two recombinant immunomodulatory proteins. Thus, a total of 5 recombinant peptides for use as tumor cell vaccines can be achieved.

Example 3 below describes lentiviral vectors (Vector 44, Vector 97, Vector 84, Vector 29, Vector 107, Vector 116, Vector 86, Vector 18) that can be used to stably integrate immunomodulatory factors into the genome of cells. , vector 17, vector 98, vector 5, vector 30, vector 109, vector 3, vector 4, vector 106, vector 16, vector 83, vector 31, vector 12, vector 99, vector 121, vector 105, vector 32, vector 37, vector 22, vector 19, vector 20, vector 89, vector 21, vector 23, vector 108, vector 15, vector 124, vector 65, vector 64, vector 88, vector 96, vector 14, vector 119, vector 120, Vector 45, Vector 60, Vector 59, Vector 8, Vector 128, Vector 35, and Vector 6) are described.

According to one embodiment, vector 44 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 29 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 18 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 17 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 5 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 16 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 99 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 15 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 14 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 45 comprises one or more TNF family member immunomodulators. According to one embodiment, vector 6 comprises one or more TNF family member immunomodulators. According to one embodiment, the one or more TNF family immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 44 comprises 3-14 TNF family member immunomodulators. According to one embodiment, vector 29 comprises 3-14 TNF family member immunomodulators. According to one embodiment, vector 18 comprises 3-14 TNF family member immunomodulators. According to one embodiment, vector 17 comprises 3-14 TNF family member immunomodulators. According to one embodiment, vector 5 comprises 3-14 TNF family member immunomodulators. According to one embodiment, vector 16 comprises 3-14 TNF family member immunomodulators. According to one embodiment, vector 99 comprises 3-14 TNF family member immunomodulators. According to one embodiment, vector 15 comprises 3-14 TNF family member immunomodulators. According to one embodiment, vector 14 comprises 3-14 TNF family member immunomodulators. According to one embodiment, vector 45 comprises 3-14 TNF family member immunomodulators. According to one embodiment, Vector 6 comprises 3-14 TNF family member immunomodulators. According to one embodiment, the 3-14 TNF family immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 97 comprises one or more Ig family member immunomodulators. According to one embodiment, vector 84 comprises one or more Ig family member immunomodulators. According to one embodiment, vector 107 comprises one or more Ig family member immunomodulators. According to one embodiment, vector 98 comprises one or more Ig family member immunomodulators. According to one embodiment, vector 30 comprises one or more Ig family member immunomodulators. According to one embodiment, vector 83 comprises one or more Ig family member immunomodulators. According to one embodiment, vector 121 comprises one or more Ig family member immunomodulators. According to one embodiment, vector 119 comprises one or more Ig family member immunomodulators. According to one embodiment, the one or more Ig family member immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 97 comprises 3-14 Ig family member immunomodulators. According to one embodiment, vector 84 comprises 3-14 Ig family member immunomodulators. According to one embodiment, vector 107 comprises 3-14 Ig family member immunomodulators. According to one embodiment, vector 98 comprises 3-14 Ig family member immunomodulators. According to one embodiment, vector 30 comprises 3-14 Ig family member immunomodulators. According to one embodiment, vector 83 comprises 3-14 Ig family member immunomodulators. According to one embodiment, vector 121 comprises 3-14 Ig family member immunomodulators. According to one embodiment, vector 119 comprises 3-14 Ig family member immunomodulators. According to one embodiment, the 3-14 Ig family member immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 116 includes one or more chemokine immunomodulators. According to one embodiment, the one or more chemokine immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 116 comprises 3-14 chemokine immunomodulators. According to one embodiment, the 3-14 chemokine immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 109 includes one or more growth factor immunomodulators.

According to one embodiment, vector 109 comprises 3-14 growth factor immunomodulators.

According to one embodiment, vector 3 comprises one or more cytokine immunomodulators. According to one embodiment, vector 4 comprises one or more cytokine immunomodulators. According to one embodiment, vector 32 includes one or more cytokine immunomodulators. According to one embodiment, vector 22 comprises one or more cytokine immunomodulators. According to one embodiment, vector 19 comprises one or more cytokine immunomodulators. According to one embodiment, vector 20 comprises one or more cytokine immunomodulators. According to one embodiment, vector 89 comprises one or more cytokine immunomodulators. According to one embodiment, vector 21 comprises one or more cytokine immunomodulators. According to one embodiment, vector 23 comprises one or more cytokine immunomodulators. According to one embodiment, vector 121 includes one or more cytokine immunomodulators. According to one embodiment, vector 65 includes one or more cytokine immunomodulators. According to one embodiment, vector 64 includes one or more cytokine immunomodulators. According to one embodiment, vector 88 comprises one or more cytokine immunomodulators. According to one embodiment, vector 96 includes one or more cytokine immunomodulators. According to one embodiment, vector 60 includes one or more cytokine immunomodulators. According to one embodiment, vector 59 comprises one or more cytokine immunomodulators. According to one embodiment, vector 128 includes one or more cytokine immunomodulators. According to one embodiment, the one or more cytokine immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 3 comprises 3-14 cytokine immunomodulators. According to one embodiment, Vector 4 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 32 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 22 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 19 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 20 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 89 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 21 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 23 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 121 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 65 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 64 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 88 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 96 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 60 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 59 comprises 3-14 cytokine immunomodulators. According to one embodiment, vector 128 comprises 3-14 cytokine immunomodulators. According to one embodiment, the 3-14 cytokine immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 37 comprises one or more receptor immunomodulators. According to one embodiment, vector 124 includes one or more receptor immunomodulators. According to one embodiment, vector 88 includes one or more receptor immunomodulators. According to one embodiment, vector 8 comprises one or more receptor immunomodulators. According to one embodiment, the one or more receptor immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 37 comprises 3-14 receptor immunomodulators. According to one embodiment, vector 124 comprises 3-14 receptor immunomodulators. According to one embodiment, vector 88 comprises 3-14 receptor immunomodulators. According to one embodiment, vector 8 comprises 3-14 receptor immunomodulators. According to one embodiment, the 3-14 receptor immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 86 includes one or more other immunomodulators. According to one embodiment, vector 106 includes one or more other immunomodulators. According to one embodiment, vector 107 includes one or more other immunomodulators. According to one embodiment, vector 31 includes one or more other immunomodulators. According to one embodiment, vector 12 includes one or more other immunomodulators. According to one embodiment, vector 105 includes one or more other immunomodulators. According to one embodiment, vector 108 includes one or more other immunomodulators. According to one embodiment, vector 120 includes one or more other immunomodulators. According to one embodiment, vector 35 includes one or more other immunomodulators. According to one embodiment, the one or more other immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment, vector 86 includes 3-25 other immunomodulators. According to one embodiment, vector 106 includes 3-25 other immunomodulators. According to one embodiment, vector 107 includes 3-25 other immunomodulators. According to one embodiment, vector 31 comprises 3-25 other immunomodulators. According to one embodiment, vector 12 comprises 3-25 other immunomodulators. According to one embodiment, vector 105 includes 3-25 other immunomodulators. According to one embodiment, vector 108 includes 3-25 other immunomodulators. According to one embodiment, vector 120 includes 3-25 other immunomodulators. According to one embodiment, vector 35 includes 3-25 other immunomodulators. According to one embodiment, the 3-25 other immunomodulators are selected from those listed in Table 2 or Table 3.

According to one embodiment of the disclosed invention, a single cell expressing multiple immunomodulatory proteins can be produced using a combination of allogeneic cell pools each expressing a single immunomodulatory protein. model the nature of interactions (e.g., additivity, synergy, interference).

According to one aspect of the disclosed invention, one, two, three, four, or five or more recombinant peptides for use as a tumor cell vaccine to treat skin cancer. Expressing tumor cell line variants can be generated. For example, the SK-MEL2 human melanoma cell line (ATCC HTB-68) can be selected for modification and lentiviral transfection of recombinant immunomodulator sequences can be used to transfect the immunomodulator into the cell's genome. can be stably incorporated into

According to one aspect of the disclosed invention, one, two, three, four, or five or more recombinant peptides are used as a tumor cell vaccine to treat prostate cancer. Expressing tumor cell line variants can be generated. For example, the DU-145 human prostate cancer cell line can be selected for modification, and lentiviral transfection of recombinant immunomodulator sequences is used to stably integrate the immunomodulator into the cell's genome. can be done. According to some embodiments, two recombinant immunomodulatory proteins are co-transfected, followed by co-transfection of two more recombinant immunomodulatory proteins, followed by transfection of a single recombinant immunomodulatory protein. A total of 5 recombinant peptides for use as tumor cell vaccines can be achieved. According to some embodiments, two recombinant peptides are transfected simultaneously, followed by transfection of a single recombinant peptide, followed by transfection of a single recombinant peptide, followed by transfection of a single of recombinant peptides to achieve a total of 5 recombinant peptides for use as tumor cell vaccines. According to some embodiments, a single recombinant immunomodulatory protein is co-transfected, followed by co-transfection of two recombinant immunomodulatory proteins, followed by transfection of two recombinant immunomodulatory proteins. Thus, a total of 5 recombinant peptides for use as tumor cell vaccines can be achieved.

According to another aspect of the invention, two or more tumor cell line variants can be generated that express one or more recombinant peptides for use as tumor cell vaccines to treat prostate cancer. . For example, the DU-145 and PC-3 human prostate cancer cell lines can be selected for modification, and lentiviral transfection of recombinant immunomodulator sequences can be used to introduce immunomodulators into the genome of the cells. It can be installed stably.

CD40L Immunomodulator The CD40L Immunomodulator cDNA sequence can be cloned into the lentiviral transfer plasmid construct pLenti-puro (Addgene catalog number 39481) driven by a CMV promoter containing a puromycin selectable marker. The CD40L immunomodulatory factor cDNA sequence can be engineered to be non-cleavable (eg, SEQ ID NO: 7) that ultimately retains the translated CD40L protein in a membrane-bound state. A human influenza hemagglutinin tag (HA tag) can also be cloned into the extracellular portion of the CD40L sequence. The translated HA tag has the peptide sequence YPYDVPDYA (SEQ ID NO:28). The packaging plasmid psPAX2 (AddGene Catalog No. 12260) and the envelope plasmid pLTR-RD114A (AddGene Catalog No. 17576) can also be selected for lentiviral systems.

Each of the lentiviral transfer, packaging, and envelope plasmids can be transfected into exponentially growing 293T cells using Lipofectamine 2000 (ThermoFisher catalog number 11668027). Briefly, cells are seeded at 70%-90% confluency. On the day of transfection, dilute 12 μl Lipofectamine reagent in 150 μl serum-free cell culture medium. 5 μg of DNA for transfection is also diluted in 150 μl of serum-free medium. The diluted DNA is then added to the diluted lipofectamine and incubated for 5 minutes at room temperature. The entire volume of the mixture is then added dropwise to the medium of the seeded 293T cells while swirling. Cells are then incubated at 37 degrees for 1-3 days.

293T cell culture medium containing viral particles is harvested three times every 8-12 hours and centrifuged to pellet detached cells and cell debris. Media containing virus particles are used directly to infect the DU-145 cell line.

The DU-145 cell line is cultured to approximately 70% confluence in Eagle's Minimum Essential Medium (EMEM) containing 10% fetal bovine serum. Hexadimethrine bromide (Sigma-Aldrich catalog number H9268) is then combined with the medium containing the virus particles to a final concentration of 8 μg/mL of hexadimethrine bromide. The culture medium of DU-145 cells is aspirated and replaced with medium containing virus particles and 8 μg/mL hexadimethrine bromide. After culturing DU-145 cells for 18-20 hours, the medium is changed.

Infected DU-145 cells are then grown in medium containing 1 μg/mL puromycin (ThermoFisher Cat# A1113802) until the cells begin to die after approximately one week. Multiple surviving colonies of transfected cells are picked for expansion and tested for CD40L expression by Western blot. Western blots were probed with mouse monoclonal anti-HA primary antibody (Abcam Cat #ab18181) and goat anti-mouse HRP (Abcam Cat #ab205719) secondary antibodies to quantify the relative amount of recombinant CD40L expressed in each clonal line. do. The most stably expressing DU-145 strain is labeled DU145-Gen1 and selected for further manipulation.

TNF-α/GM-CSF
DU145-Gen1 cells transfected to express CD40L are further transfected with a bicistronic lentiviral vector containing TNF-α and GM-CSF sequences. Each of the TNF-α and GM-CSF cDNAs is first cloned into the pEF1α-IRES bicistronic mammalian expression vector (Clontech catalog number 631970) under the control of the human elongation factor 1α (EF1α) promoter. . A variant of TNF-α is used that cannot be cleaved by TACE so that the translated protein remains membrane bound. The TNF-α sequence is provided with a FLAG tag sequence in the extracellular region of TNF-α for easy detection of the translated protein. The FLAG tag peptide sequence is DYKDDDDK (SEQ ID NO:29). A GM-CSF sequence is used that is capable of forming soluble GM-CSF. The entire pEF1 promoter, TNF-α, IRES, and GM-CSF sequences are then cloned into the pLenti-puro (Addgene catalog number 39481) lentiviral vector (the original CMV promoter from the vector was removed during this process). removed). The packaging plasmid psPAX2 (AddGene Catalog No. 12260) and the envelope plasmid pLTR-RD114A (AddGene Catalog No. 17576) are also selected.

Each of the lentiviral transfer, packaging, and envelope plasmids are transfected into exponentially growing 293T cells using Lipofectamine 2000 (ThermoFisher catalog number 11668027). Briefly, cells are seeded at 70%-90% confluency. On the day of transfection, dilute 12 μl Lipofectamine reagent in 150 μl serum-free cell culture medium. 5 μg of DNA for transfection is also diluted in 150 μl of serum-free medium. The diluted DNA is then added to the diluted lipofectamine and incubated for 5 minutes at room temperature. The entire volume of the mixture is then added dropwise to the medium of the seeded 293T cells while swirling. Cells are then incubated at 37 degrees for 1-3 days.

293T cell culture medium containing viral particles is harvested three times every 8-12 hours and centrifuged to pellet detached cells and cell debris. Medium containing virus particles is used directly to infect the DU145-Gen1 cell line.

The DU145-Gen1 cell line is cultured until approximately 70% confluent. Hexadimethrine bromide (Sigma-Aldrich catalog number H9268) is then combined with the medium containing the virus particles to a final concentration of 8 μg/mL of hexadimethrine bromide. The culture medium of DU145-Gen1 cells is aspirated and replaced with medium containing viral particles and 8 μg/mL hexadimethrine bromide. After culturing DU145-Gen1 cells for 18-20 hours, the medium is changed.

Transduced DU145-Gen1 cells are then selected for clones stably expressing the recombinant immune modulator. The selection process is performed by fluorescence-activated cell sorting using the TNF-α FLAG tag to identify cells that have integrated the immunomodulator. Live cells are probed with mouse monoclonal anti-FLAG antibody (Sigma Aldrich F3040) and rabbit anti-mouse FITC conjugated secondary antibody (Sigma Aldrich ASB3701170) in PBS containing blocking buffer. The highest expressing cells are sorted, isolated and cultured for further processing. Expression of soluble GM-CSF is confirmed by Western blot after sorting based on the presence of the FLAG tag. Concentrated media of the sorted cultured cells are separated by SDS-PAGE and probed by Western blot with mouse anti-GM-CSF antibody (ThermoFisher cat#3092) and goat anti-mouse HRP-conjugated secondary antibody. Cell lysates can also be separated by SDS-PAGE and probed for the FLAG tag to confirm the presence of TNF. Cell cultures expressing high levels of recombinant GM-CSF and TNF-α are designated DU145-Gen2 and selected for further processing.

Flt-3L
DU145-Gen2 cells transfected to express CD40L, GM-CSF, and TNF are further transfected with a lentiviral vector containing Flt-3L immunomodulator sequences. The Flt-3L cDNA is cloned into the pEF1α-IRES bicistronic mammalian expression vector (Clontech catalog number 631970) along with the GFP protein sequence used as an integration and expression marker. The Flt-3L sequence is translated into a membrane-bound peptide, whereas GFP remains in the cytoplasm. The entire pEF1 promoter, Flt-3L, IRES, and GFP sequences are then cloned into the pLenti-puro (Addgene catalog number 39481) lentiviral vector (the original CMV promoter from the vector was removed during this process). ). The packaging plasmid psPAX2 (AddGene Catalog No. 12260) and the envelope plasmid pLTR-RD114A (AddGene Catalog No. 17576) are also selected.

Each of the lentiviral transfer, packaging, and envelope plasmids are transfected into exponentially growing 293T cells using Lipofectamine 2000 (ThermoFisher catalog number 11668027). Briefly, cells are seeded at 70%-90% confluency. On the day of transfection, dilute 12 μl Lipofectamine reagent in 150 μl serum-free cell culture medium. 5 μg of DNA for transfection is also diluted in 150 μl of serum-free medium. The diluted DNA is then added to the diluted lipofectamine and incubated for 5 minutes at room temperature. The entire volume of the mixture is then added dropwise to the medium of the seeded 293T cells while swirling. Cells are then incubated at 37 degrees for 1-3 days.

The 293T cell culture medium containing viral particles is harvested three times every 8-12 hours and centrifuged to pellet the detached cells and cell debris. Medium containing virus particles is used directly to infect the DU145-Gen2 cell line.

The DU145-Gen2 cell line is cultured until approximately 70% confluent. Hexadimethrine bromide (Sigma-Aldrich catalog number H9268) is then combined with the medium containing the virus particles to a final concentration of 8 μg/mL of hexadimethrine bromide. The culture medium of DU145-Gen2 cells is aspirated and replaced with medium containing viral particles and 8 μg/mL hexadimethrine bromide. After culturing DU145-Gen2 cells for 18-20 hours, the medium is changed.

DU145-Gen2 cells are then selected for cells stably expressing the Flt-3L sequence using the GFP marker. The selection process is performed by fluorescence-activated cell sorting (FACS) using a GFP marker to identify cells that have integrated the immunomodulator. The highest expressing cells are sorted, isolated and cultured for further processing. After sorting based on the presence of the GFP marker, expression of Flt-3L is confirmed by Western blot. Lysates of cultured cells are separated by SDS-PAGE and probed by Western blot with a rabbit polyclonal anti-Flt-3L antibody (AbCam Cat#ab9688) and a goat anti-rabbit HRP conjugated secondary antibody (AbCam Cat#ab205718). Cell cultures expressing high levels of recombinant Flt-3L are designated DU145-Gen3 and selected for further processing.

IgG heavy chain DU145-Gen3 cells transfected to express CD40L, GM-CSF, TNF-α, and Flt-3L were transfected with lentivirus containing IgG1 (SEQ ID NO: 1), a membrane-bound IgG1 heavy chain fragment. Further transfect with the vector. The IgG1 heavy chain cDNA is cloned into the pEF1α-IRES bicistronic mammalian expression vector (Clontech catalog number 631970) along with the RFP protein sequence used as an integration and expression marker. The IgG1 heavy chain sequence is translated into a membrane-bound peptide, while the RFP remains in the cytoplasm. The entire pEF1 promoter, IgG1 heavy chain sequence, IRES sequence, and RFP sequence are then cloned into the pLenti-puro (Addgene catalog number 39481) lentiviral vector (the original CMV promoter from the vector was removed during this process). ). The packaging plasmid psPAX2 (AddGene Catalog No. 12260) and the envelope plasmid pLTR-RD114A (AddGene Catalog No. 17576) are also selected.

Each of the lentiviral transfer, packaging, and envelope plasmids are transfected into exponentially growing 293T cells using Lipofectamine 2000 (ThermoFisher catalog number 11668027). Briefly, cells are seeded at 70%-90% confluency. On the day of transfection, dilute 12 μl of Lipofectamine reagent in 150 μl of serum-free cell culture medium. 5 μg of DNA for transfection is also diluted in 150 μl of serum-free medium. The diluted DNA is then added to the diluted lipofectamine and incubated for 5 minutes at room temperature. The entire volume of the mixture is then added dropwise to the medium of the seeded 293T cells while swirling. Cells are then incubated at 37 degrees for 1-3 days.

293T cell culture medium containing viral particles is harvested three times every 8-12 hours and centrifuged to pellet detached cells and cell debris. Medium containing virus particles is used directly to infect the DU145-Gen3 cell line.

The DU145-Gen3 cell line is cultured until approximately 70% confluent. Hexadimethrine bromide (Sigma-Aldrich catalog number H9268) is then combined with the medium containing the virus particles to a final concentration of 8 μg/mL of hexadimethrine bromide. The culture medium of DU145-Gen2 cells is aspirated and replaced with medium containing virus particles and 8 μg/mL hexadimethrine bromide. After culturing DU145-Gen3 cells for 18-20 hours, the medium is changed.

DU145-Gen3 cells are then selected for cells stably expressing IgG1 heavy chain sequences using the RFP marker. The selection process is performed by fluorescence-activated cell sorting (FACS) using the RFP marker to identify cells that have integrated the immunomodulator. The highest expressing cells are sorted, isolated and cultured for further processing. After sorting for the presence of the RFP marker, expression of IgG1 heavy chain is confirmed by Western blot. Cell cultures expressing high levels of recombinant IgG1 heavy chain are designated DU145-Gen4 and selected for further processing.

DU145-Gen4 tumor cell lines transfected to express CD40L, GM-CSF, TNF, Flt-3L, and IgG1 heavy chain were characterized by RT-PCR, immunofluorescence, and Western blotting, all Ensure that the recombinant immunomodulatory factor is expressed by the cells and is in the proper location (eg, the membrane of the cell, etc.).

Human Mixed Lymphocytic Tumor Response (MLTR) Test DU145-Gen4 cells were tested against primary and other generations of modified and unmodified DU145 cells (i.e., DU145-Gen2 and DU145-Gen3), respectively. It is tested for immunomodulatory potential by secondary MLTR assays.

Peripheral blood mononuclear cells (PBMC) are obtained from the peripheral blood of healthy individuals and prostate cancer patients, and blood cells are separated using Ficoll-Paque gradients. Anticoagulant-treated blood is diluted with PBS/EDTA ranging from 1:2 to 1:4 to reduce red blood cell aggregation. The diluted blood is then layered on top of the Ficoll-Paque solution in the centrifuge tube without mixing. The layered blood/Ficoll-Paque is centrifuged at 400×g for 40 minutes at 18° C.-20° C. without centrifugal brake to form a blood fraction. Fractions containing mononuclear cells are selected for further processing.

Cells of each of the transfected tumor cell line variants and the parental tumor cell line DU-145 (control) were co-cultured with PBMCs for 7 days under standard tissue culture conditions, followed by immune cell proliferation, flow cytometry and Immune cell differentiation as measured by CyTOF, cytokine release profile, and cytotoxicity as measured by LDH release assay are assessed.

Example 3.
Vectors used herein are described in detail below:
vector1. Immunomodulator: scFv-anti-biotin-G3hinge-mIgG1 (to generate surface IgG).

A schematic representation of the construction of Vector 1 used for the immunomodulator scFv-anti-biotin-G3hinge-mIgG1 is shown in FIG. Table 7 below shows the name of the vector component, the corresponding nucleotide position in SEQ ID NO:47, the full name and description of the component.

When using Vector 1, anti-IgG is used for flow detection. Biotin plus fluorescently labeled oligodeoxynucleotides (ODN) is used as a secondary detection method.

Below is a description of the immunomodulator scFv-anti-biotin-G3hinge-IgG1-Tm.

Type: Immunoglobulin

Annotation:
H7 heavy chain leader Anti-biotin variable heavy chain (VH) allows loading of CpG labeled biotin Interdomain disulfide bonds VH44 (G->C) and VL100 (G->C)
IgG3 hinge to strengthen FcyR interaction Ligation is canonical IgG1 (CH2-CH3-Tm-Cyt) used for interaction with FcyR/FcRn and membrane anchoring
- T233A mutation that enhances the interaction between FcRn and FcyR

リンカー（配列番号５６）
ＧＧＧＧＳＧＧＧＧＳ ＧＧＧＧＳ
軽鎖可変（ヒトラムダ可変）（配列番号５７）

ＦｃｙＲに対するより優れたアクセスのためのＩｇＧ３ヒンジ（配列番号５８）
ＬＫＴＰＬＧＤＴＴＨＴＣＰＲ ＣＰＥＰＫＳＣＤＴＰ ＰＰＣＰＲＣＰＥＰＫ ＳＣＤＴＰＰＰＣＰＲ
ＣＰＥＰＫＳＣＤＴＰ ＰＰＣＰＲＣＰ
ＩｇＧ１のＣＨ２、ＣＨ３、Ｔｍ、及び細胞質尾部（Ｔ２５６Ａ）（配列番号５９）

The array is shown below:
H7 heavy chain leader (SEQ ID NO:54)
MEFGLSWVFLVALFRGVQC
Anti-biotin mouse vH (SEQ ID NO: 55) with Cys inserted for interdomain linkage

Linker (SEQ ID NO:56)
GGGGSGGGGGSGGGGGS
Light chain variable (human lambda variable) (SEQ ID NO: 57)

IgG3 hinge for better access to FcyRs (SEQ ID NO:58)
LKTPLGDTTTHTCPPRCPEPKSCDTPPPCPRCPEPK SCDTPPPCPR
CPEPKSCDTPPPCPRCP
IgG1 CH2, CH3, Tm, and cytoplasmic tail (T256A) (SEQ ID NO: 59)

TVTFFKVKWI FSSVVDLKQT IIPDYRNMIG QGA*
scFv-anti-biotin-G3 hinge-IgG1-Tm(598 ORF1) (SEQ ID NO: 60)
MEFGLSWVFLVALFRGVQCQVKLQESGPGLVAPSQSLSITCTVSGFSLTA
YGVDWVRQPPGKCLEWLGVIWGGGRTNYNSGLMSRLSIRKDNSKSQVFLT
MNSLQTDDTAKYYCVKHTNWDGGFAYWGQGTTVTVSSGGGGGSGGGGSGGG
GSGSPGQSVSISCSGSSSNIGNNYVYWYQHLPGTAPKLLIYSDTKRPSGV
PDRISGSKSGTSASLAISGLQSEDEADYYCASWDDSLDGPVFGCGTKLTV
LLKTPLGDTTTHTCPRCEPEPKSCDTPPPPCPRCPEPPKSCDTPPPCPRCPEPK
SCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRAPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCVMHEALHNHYTQKSLSLSPELQLEESCAEAQDGELDGLWTTI
TIFITLFLLSVCYSATVTFFKVKWIFSSVVVDLKQTIIPDYRNMIGQGA*

Vector2. Immunomodulator: full length anti-biotin-G3 hinge-mIgG1 (using heavy chain/ires/light chain)
A schematic representation of the construction of vector 1 used for immunomodulator full-length-anti-biotin-G3 hinge-mIgG2 is shown in FIG. Vector 2 is bicistronic. Table 8 below shows the name of the vector component, the corresponding nucleotide position in SEQ ID NO:48, the full name and description of the component.

When using vector 2, anti-IgG is used for flow detection. Biotin plus fluorescently labeled ODN is used as a secondary detection method.

Below is a description of the immunomodulator full-length anti-biotin-G3hinge-mIgG1 (using heavy chain/ires/light chain).

Type: membrane-bound immunoglobulin

Annotation:
- H7 heavy chain leader - IgG3 hinge to enhance FcyR interaction - T233A mutation to enhance interaction between FcRn and FcyR - Anti-biotin variable H allows loading of CpG labeled biotin - CH1 (generic)
・ LC variable (hit lambda variable)
- LC constant region 1 from lambda (http://www.uniprot.org/uniprot/P0CG04)
- Interdomain disulfide bonds VH44 (G->C) and VL100 (G->C) (ref)
Ligation is standard IgG1 (CH2-CH3-Tm-Cyt) for interaction with FcyR/FcRn and membrane anchoring
- L1 light chain leader (modified for IRES) MATDMRVPAQLLGLLLLLWLSGARC (SEQ ID NO: 61)

The array is shown below:
H7 heavy chain leader (SEQ ID NO:54)
MEFGLSWVFLVALFRGVQC
anti-biotin vH (mouse) (SEQ ID NO: 62)
QVKLQESGPG LVAPSQSLSI TCTVSGFSLT AYGVDWVRQP PGKGLEWLGV
IWGGGRTNYN SGLMSRLSIR KDNSKSQVFL TMNSLQTDDT AKYYCVKHTN
WDGGFAYWGQGTTVTVSS
CH1 (generic) (SEQ ID NO: 63)
PSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV
HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVE
IgG3 hinge for better access to FcyRs (SEQ ID NO: 64)

LKTP LGDTTHTCPPR CPEPKSCDTPPPCPRCPEPK SCDTPPPCPR

CPEPKSCDTPPPCPRCP
IgG1 CH2, CH3, Tm, and cytoplasmic tail (T256A) (SEQ ID NO: 65)

APELLGGPSVFLF PPKPKDTLMI SRAPEVTCVV VDVSHEDPEV KFNWYVDGVE
VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE
KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES
NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSV MHEALH
NHYTQKSLSL SPELQLEESC AEAQDGELDG LWTTITIFIT LFLLSVCYSA
TVTFFKVKWI FSSVVDLKQT IIPDYRNMIG QGA*
Summary (578 ORF2a) (SEQ ID NO: 66)
MEFGLSWVFLVALFRGVQCQVKLQESGPGLVAPSQSLSITCTVSGFSLTA
YGVDWVRQPPGKGLEWLGVIWGGGRTNYNSGLMSRLSIRKDNSKSQVFLT
MNSLQTDDTAKYYCVKHTNWDGGFAYWGQGTTVTVSSPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVELKTPLGDTTTHTCPRCPEPK
SCDTPPPCPRCPEPKSCDTPPPPCPRCPEPKSCDTPPPPCPRCPAPELLGGP
SVFLFPPKPKDTLMISRAPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVMHEALHNHYTQ
KSLSLSPELQLEESCAEAQDGELDGLWTTITIFITLFLLSVCYSATVTFF
KVKWIFSSVVDLKQTIIPDYRNMIGQGA*
IRES (SEQ ID NO: 67)
L1 signal (modified for IRES compatibility) (SEQ ID NO: 61)
MATDMRVPAQLLGLLLLLWLSGARC
LC variable (human lambda variable) (SEQ ID NO: 69)
GSPGQSVSIS CSGSSSNIGN NYVYWYQHLP GTAPKLLIYS DTKRPSGVPD
RISGSKS GTS ASLAISGLQS EDEADYYCAS WDDSLDGPVF GGGTKLTVL
LC constant region 1 from lambda (http://www.uniprot.org/uniprot/P0CG04) (irrelevant) (SEQ ID NO: 70)
GQPKANPTTV LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK
AGVETTKPSK QSNNKYASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV
APTECS*
Summary (229 ORF2b) (SEQ ID NO: 71)
MATDMRVPAQLLGLLLLLWLSGARCGSPGQSVSISCSGSSSNIGNNYVYWY
QHLPGTAPKLLIYSDTKRPSGVPDRISGSKSGTSASLAISGLQSEDEADY
YCASWDDSLDGPVFGGGTKLTVLGQPKANPTTVTLFPPSSEELQANKATLV
CLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPE
QWKSHRSYSCQVTHEGSTVEKTVAPTECS*

Vector3. Immunomodulator: sGM-CSF/ires/mFLT3L
A schematic diagram of the construction of Vector 3 used for the immunomodulator sGM-CSF/ires/mFLT3L is shown in FIG. Vector 3 is bicistronic. Table 9 below shows the name of the vector component, the corresponding nucleotide position of SEQ ID NO:49, the full name and description of the component.

When using vector 3, anti-FLT3L is used for flow detection. Those expressing the highest surface FLT3L show the highest secreted GM-CSF expression.

Below is a description of the immunomodulator sGM-CSF/ires/mFLT3L.

Type: Cytokines, proliferation and differentiation factors

ＦＬＴ３Ｌ（配列番号７６）

要約（１４４ ＯＲＦ３ａ）（配列番号７７）
ＭＷＬＱＳＬＬＬＬＧＴＶＡＣＳＩＳＡＰＡＲＳＰＳＰＳＴＱＰＷＥＨＶＮＡＩＱＥＡＲＲＬＬＮＬＳＲＤＴＡ
ＡＥＭＮＥＴＶＥＶＩＳＥＭＦＤＬＱＥＰＴＣＬＱＴＲＬＥＬＹＫＱＧＬＲＧＳＬＴＫＬＫＧＰＬＴＭＭＡＳＨＹＫＱＨＣＰＰＴＰＥＴＳＣＡＴＱＩＩＴＦＥＳＦＫＥＮＬＫＤＦＬＬＶＩＰＦＤＣＷＥＰＶＱＥ＊
要約（２３６ ＯＲＦ３ｂ）（配列番号７８）
ＭＡＴＶＬＡＰＡＷＳＰＴＴＹＬＬＬＬＬＬＬＳＳＧＬＳＧＴＱＤＣＳＦＱＨＳＰＩＳＳＤＦＡＶＫＩＲＥＬＳ
ＤＹＬＬＱＤＹＰＶＴＶＡＳＮＬＱＤＥＥＬＣＧＧＬＷＲＬＶＬＡＱＲＷＭＥＲＬＫＴＶＡＧＳＫＭＱＧＬＬＥＲＶＮＴＥＩＨＦＶＴＫＣＡＦＱＰＰＰＳＣＬＲＦＶＱＴＮＩＳＲＬＬＱＥＴＳＥＱＬＶＡＬＫＰＷＩＴＲＱＮ
ＦＳＲＣＬＥＬＱＣＱＰＤＳＳＴＬＰＰＰＷＳＰＲＰＬＥＡＴＡＰＴＡＰＱＰＰＬＬＬＬＬＬＬＰＶＧＬＬＬＬ
ＡＡＡＷＣＬＨＷＱＲＴＲＲＲＴＰＲＰＧＥＱＶＰＰＶＰＳＰＱＤＬＬＬＶＥＨ＊ Annotations: The wild-type sequence sequence is shown below:
GM-CSF signal sequence (SEQ ID NO:72)
MWLQSLLLLG TVACSIS
Wild-type GM-CSF sequence (SEQ ID NO:73)
APA RSSPPSTQPW EHVNAIQEAR RLLNLSRDTA
AEMNETVEVI SEMFDLQEPT CLQTRLELYK QGLRGSLTKL KGPLTMASH
YKQHCPPTPE TSCATQIITF ESFKENLKDF LLVIPFDCWE PVQE*
IRES (SEQ ID NO:74)
FLT3L signal (modified to be IRES friendly) (SEQ ID NO:75)

FLT3L (SEQ ID NO:76)

Summary (144 ORF3a) (SEQ ID NO: 77)
MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTA
AEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGGPLTMMASHYKQHCPPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE*
Summary (236 ORF3b) (SEQ ID NO:78)
MATVLAPAWSPTTYLLLLLLLLSSGL SGTQDCSFQHSPISSDFAVKIRELS
DYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQN
FSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLLPVGLLLL
AAAWCLHWQRTRRRRTPRPGEQVPPVPSPQDLLLVEH*

vector4. Immunomodulator: sFLT3L/ires/(FLT3 signal-GM-CSF-Tm)
A schematic representation of the construction of Vector 4 used for the immunomodulator sFLT3L/ires/(FLT3 signal-GM-CSF-Tm) is shown in FIG. Vector 4 is bicistronic. Table 10 below shows the name of the vector component, the corresponding nucleotide position of SEQ ID NO:50, the full name and description of the component.

When using Vector 4, anti-GM-CSF is used for flow detection. Those expressing the highest surface GMCSF show the highest secreted FLT3L expression.

Below is a description of the immunomodulator sFLT3L/ires/(FLT3 signal-GM-CSF-Tm).

Type: Cytokines, proliferation and differentiation factors

Annotation: wild-type sequence

野生型ＧＭ－ＣＳＦ配列（天然のシグナルを除く）（配列番号８２）
ＡＰＡ ＲＳＰＳＰＳＴＱＰＷ ＥＨＶＮＡＩＱＥＡＲ ＲＬＬＮＬＳＲＤＴＡ
ＡＥＭＮＥＴＶＥＶＩ ＳＥＭＦＤＬＱＥＰＴ ＣＬＱＴＲＬＥＬＹＫ ＱＧＬＲＧＳＬＴＫＬ ＫＧＰＬＴＭＭＡＳＨＹＫＱＨＣＰＰＴＰＥ ＴＳＣＡＴＱＩＩＴＦ ＥＳＦＫＥＮＬＫＤＦ ＬＬＶＩＰＦＤＣＷＥ ＰＶＱＥ
ＣＤ８α膜貫通及び細胞質ドメイン（配列番号８３）

要約（１８３ ＯＲＦ４ａ）（配列番号８４）
ＭＴＶＬＡＰＡＷＳＰＴＴＹＬＬＬＬＬＬＬＳＳＧＬＳＧＴＱＤＣＳＦＱＨＳＰＩＳＳＤＦＡＶＫＩＲＥＬＳＤ
ＹＬＬＱＤＹＰＶＴＶＡＳＮＬＱＤＥＥＬＣＧＧＬＷＲＬＶＬＡＱＲＷＭＥＲＬＫＴＶＡＧＳＫＭＱＧＬＬＥＲＶＮＴＥＩＨＦＶＴＫＣＡＦＱＰＰＰＳＣＬＲＦＶＱＴＮＩＳＲＬＬＱＥＴＳＥＱＬＶＡＬＫＰＷＩＴＲＱＮＦ
ＳＲＣＬＥＬＱＣＱＰＤＳＳＴＬＰＰＰＷＳＰＲＰＬＥＡＴＡＰＴＡＰＱ＊
ＣＹＡＧＥＮについての要約（２５３ ＯＲＦ４ｂ）（配列番号８５）
ＭＡＴＶＬＡＰＡＷＳＰＴＴＹＬＬＬＬＬＬＬＳＳＧＬＳ ＡＰＡＲＳＰＳＰＳＴＱＰＷＥＨＶＮＡＩＱＥＡＲ
ＲＬＬＮＬＳＲＤＴＡＡＥＭＮＥＴＶＥＶＩＳＥＭＦＤＬＱＥＰＴＣＬＱＴＲＬＥＬＹＫＱＧＬＲＧＳＬＴＫＬＫＧＰＬＴＭＭＡＳＨＹＫＱＨＣＰＰＴＰＥＴＳＣＡＴＱＩＩＴＦＥＳＦＫＥＮＬＫＤＦＬＬＶＩＰＦＤＣＷＥ
ＰＶＱＥＰＴＴＴＰＡＰＲＰＰＴＰＡＰＴＩＡＳＱＰＬＳＬＲＰＥＡＣＲＰＡＡＧＧＡＶＨＴＲＧＬＤＦＡＣＤ
ＩＹＩＷＡＰＬＡＧＴＣＧＶＬＬＬＳＬＶＩＴＬＹＣＮＨＲＮＲＲＲＶＣＫＣＰＲＰＶＶＫＳＧＤＫＰＳＬＳＡＲＹＶ＊ The array is shown below:
FLT3L sequence with transmembrane removed (SEQ ID NO: 79)
MTVLAPAWSP TTYLLLLLLLL SSGLSGTQDC SFQHSPISSD FAVKIRELSD YLLQDYPVTV ASNLQDEELC GGLWRLVLAQ RWMERLKTVA GSKMQGLLERVNTEIHFVTK CAFQPPPSCL RFVQTNISRL LQETSEQLVA LKPWITRQNFSRCLELQCQP DSSTLPPPWS PRPLEATAPT APQ*
IRES (SEQ ID NO:80)
FLT3L signal (modified to be IRES friendly) (SEQ ID NO:81)

Wild-type GM-CSF sequence (minus the native signal) (SEQ ID NO:82)
APA RSSPPSTQPW EHVNAIQEAR RLLNLSRDTA
AEMNETVEVI SEMFDLQEPT CLQTRLELYK QGLRGSLTKL KGPLTMMASHYKQHCPPTPE TSCATQIITF ESFKENLKDF LLVIPFDCWE PVQE
CD8α transmembrane and cytoplasmic domains (SEQ ID NO:83)

Summary (183 ORF4a) (SEQ ID NO: 84)
MTVLAPAWSPTTYLLLLLLLLSSGL SGTQDCSFQHSPISSDFAVKIRELSD
YLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNF
SRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQ*
Summary for CYAGEN (253 ORF4b) (SEQ ID NO: 85)
MATVLAPAWSPTTYLLLLLLLLSSSGLS APARSPSPSTQPWEHVNAIQEAR
RLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGGPLTMMASHYKQHCPPPTPETSCATQIITFESFKENLKDFLLLVIPFDCWE
PVQEPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
IYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPPVVKSGDKPSLSARYV*

vector5. Immunomodulator: mCD40L
A schematic diagram of the construction of Vector 5 used for the immunomodulator mCD40L is shown in FIG. Vector 5 is monocistronic. Table 11 below shows the name of the vector component, the corresponding nucleotide position of SEQ ID NO:51, the full name and description of the component.

When using Vector 5, anti-CD40L is used for flow detection.

Below is a description of the immunomodulator mCD40L.

Type: TNF type II transmembrane protein

Annotation: mutation (underlined) introduced to create an uncleavable version

要約（２６１ ＯＲＦ５）（配列番号８７）
ＭＩＥＴＹＮＱＴＳＰＲＳＡＡＴＧＬＰＩＳＭＫＩＦＭＹＬＬＴＶＦＬＩＴＱＭＩＧＳＡＬＦＡＶＹＬＨＲＲＬ
ＤＫＩＥＤＥＲＮＬＨＥＤＦＶＦＭＫＴＩＱＲＣＮＴＧＥＲＳＬＳＬＬＮＣＥＥＩＫＳＱＦＥＧＦＶＫＤＩＭＬ
ＮＫＥＥＴＫＫＥＮＳＦＥＭＰＲＧＥＥＤＳＱＩＡＡＨＶＩＳＥＡＳＳＫＴＴＳＶＬＱＷＡＥＫＧＹＹＴＭＳＮ
ＮＬＶＴＬＥＮＧＫＱＬＴＶＫＲＱＧＬＹＹＩＹＡＱＶＴＦＣＳＮＲＥＡＳＳＱＡＰＦＩＡＳＬＣＬＫＳＰＧＲ
ＦＥＲＩＬＬＲＡＡＮＴＨＳＳＡＫＰＣＧＱＱＳＩＨＬＧＧＶＦＥＬＱＰＧＡＳＶＦＶＮＶＴＤＰＳＱＶＳＨＧ
ＴＧＦＴＳＦＧＬＬＫＬ＊ The array is shown below:
Modified sequence to stop cleavage (SEQ ID NO:86)

Summary (261 ORF5) (SEQ ID NO: 87)
MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRL
DKIEDERNLHEDFVFMKTIQRCNTGERSLLLLNCEEIKSQFEGFVKDIML
NKEETKKENSFEMPRGEEDSQIAAHVISEASSKTTSVLQWAEKGYYTMSN
NLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGR
FERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHG
TGFTSFGLKL*

ベクター６を使用する場合、フロー検出には抗ＴＮＦαが使用される。
以下は、免疫調節因子ｍＴＮＦαの説明である。
種類：ＴＮＦタイプＩＩ膜貫通タンパク質
アノテーション：切断不可能なバージョンを作成するために変異が導入された vector6. Immunomodulator: mTNFα (TNFa)
A schematic diagram of the construction of Vector 6 used for the immunomodulator mTNFα is shown in FIG. Vector 6 is monocistronic. Table 12 below shows the name of the vector component, the corresponding nucleotide position of SEQ ID NO:52, the full name and description of the component.

When using Vector 6, anti-TNFα is used for flow detection.
Below is a description of the immunomodulatory factor mTNFα.
Type: TNF type II transmembrane protein Annotation: mutagenized to create an uncleavable version

ベクター７を使用する場合、フロー検出には抗ＲＡＮＫＬが使用される。抗Ｖ５ ｍＡｂは、二次検出方法として使用される。
以下は、免疫調節因子ｍＲＡＮＫＬ／ｉｒｅｓ／ＦＬＴ３シグナル－Ｖ５－ｓｃＦＶ抗－ビオチン－Ｔｍの説明である。
種類：ＴＮＦタイプＩＩ膜貫通タンパク質
アノテーション：野生型配列 Vector 7 . Immunomodulator: mRANKL/ires/FLT3 signal-V5-scFV anti-biotin-Tm
A schematic representation of the construction of vector 7 used for the immunomodulator mRANKL/ires/FLT3 signal-V5-scFV anti-biotin-Tm is shown in FIG. Table 13 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

When using vector 7, anti-RANKL is used for flow detection. Anti-V5 mAb is used as a secondary detection method.
Below is a description of the immunomodulator mRANKL/ires/FLT3 signal-V5-scFV anti-biotin-Tm.
Type: TNF type II transmembrane protein Annotation: wild-type sequence

Vector 44
FIG. 9 shows a schematic diagram of vector 44 .
Table 14 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 97
FIG. 10 shows a schematic diagram of vector 97 .
Table 15 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 84 .
FIG. 11 shows a schematic diagram of vector 84 .
Table 16 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 29.
FIG. 12 shows a schematic diagram of vector 29 .
Table 17 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 107
FIG. 13 shows a schematic diagram of vector 107 .
Table 18 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 116
FIG. 14 shows a schematic diagram of vector 116 .
Table 19 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 86
FIG. 15 shows a schematic diagram of vector 86 .
Table 20 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

vector 18
FIG. 16 shows a schematic diagram of vector 18 .
Table 21 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

vector 17
FIG. 17 shows a schematic diagram of vector 17. FIG.
Table 22 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 98
FIG. 18 shows a schematic diagram of vector 98 .
Table 23 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

vector 30
FIG. 19 shows a schematic diagram of vector 30 .
Table 24 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 109
FIG. 20 shows a schematic diagram of vector 109 .
Table 25 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 106
FIG. 21 shows a schematic diagram of vector 106 .
Table 26 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

vector 16
FIG. 22 shows a schematic diagram of vector 16 .
Table 27 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 83
FIG. 23 shows a schematic diagram of vector 83 .
Table 28 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 31
FIG. 24 shows a schematic diagram of vector 31 .
Table 29 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

vector 12
FIG. 25 shows a schematic diagram of vector 12 .
Table 30 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 99
FIG. 26 shows a schematic diagram of vector 99. FIG.
Table 31 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 121
FIG. 27 shows a schematic diagram of vector 121 .
Table 32 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 105
FIG. 28 shows a schematic diagram of vector 105 .
Table 33 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

vector 32
FIG. 29 shows a schematic diagram of vector 32 .
Table 34 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 37
FIG. 30 shows a schematic diagram of vector 37 .
Table 35 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 22
FIG. 31 shows a schematic diagram of vector 22 .
Table 36 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

vector 19
FIG. 32 shows a schematic diagram of vector 19. FIG.
Table 37 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

vector 20
FIG. 33 shows a schematic diagram of vector 20 .
Table 38 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 89
FIG. 34 shows a schematic diagram of vector 89. FIG.
Table 39 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 21
FIG. 35 shows a schematic diagram of vector 21 .
Table 40 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 23
FIG. 36 shows a schematic diagram of vector 23 .
Table 41 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 108
FIG. 37 shows a schematic diagram of vector 108 .
Table 42 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 15
FIG. 38 shows a schematic diagram of vector 15. FIG.
Table 43 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 124
FIG. 39 shows a schematic diagram of vector 124 .
Table 44 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 65
FIG. 40 shows a schematic diagram of vector 65 .
Table 45 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 64
FIG. 41 shows a schematic diagram of vector 64 .
Table 46 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 88
FIG. 42 shows a schematic diagram of vector 88 .
Table 47 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 96
FIG. 43 shows a schematic diagram of vector 96 .
Table 48 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 14
FIG. 44 shows a schematic diagram of vector 14 .
Table 49 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 119
FIG. 45 shows a schematic diagram of vector 119 .
Table 50 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 120
FIG. 46 shows a schematic diagram of vector 120 .
Table 51 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 45
FIG. 47 shows a schematic diagram of vector 45 .
Table 52 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 60
FIG. 48 shows a schematic diagram of vector 60 .
Table 53 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 59
FIG. 49 shows a schematic diagram of vector 59. FIG.
Table 54 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 8
FIG. 50 shows a schematic diagram of Vector 8. FIG.
Table 55 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 128
FIG. 51 shows a schematic diagram of vector 128 .
Table 56 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

Vector 35
FIG. 52 shows a schematic diagram of vector 35 .
Table 57 below shows the name of the vector component, the corresponding nucleotide position, the full name of the component and a description.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using Vector 2, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulators are stably integrated into the cell genome using Vector 3, at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulators are stably integrated into the cell genome using Vector 4, at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using Vector 5, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulators are stably integrated into the cell genome using Vector 6, at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using vector 14, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using vector 18, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using vector 30, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using vector 15, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using vector 19, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using vector 22, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using vector 23, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using vector 29, and at least 3 immunomodulators are OX40L , CD27L and CD28L, and optionally additional immunomodulators selected from R ¹ to R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 2 and vector 3, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 2 and vector 4, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 2 and vector 5, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 2 and vector 6, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 3 and vector 4, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 3 and vector 5, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 3 and vector 6, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-14 immunomodulatory factors are stably integrated into the cell genome using Vector 2, Vector 3, and Vector 4, and at least The three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulators are stably integrated into the cell genome using Vector 2, Vector 3, and Vector 5, and at least The three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using Vector 2, Vector 3, and Vector 6, and at least The three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using Vector 2, Vector 3, and Vector 6, and at least The three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulatory factors are stably integrated into the cell genome using Vector 3, Vector 4, and Vector 5, and at least The three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using Vector 3, Vector 4, and Vector 6, and at least The three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulatory factors are stably integrated into the cell genome using Vector 2, Vector 3, Vector 4, and Vector 5. wherein the at least three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulatory factors are stably integrated into the cell genome using Vector 2, Vector 3, Vector 4, and Vector 6. wherein the at least three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulatory factors are stably integrated into the cell genome using Vector 2, Vector 3, Vector 5, and Vector 6. wherein the at least three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulatory factors are stably integrated into the cell genome using Vector 3, Vector 4, Vector 5, and Vector 6. wherein the at least three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immune modulators are stabilized in the cell genome using Vector 2, Vector 3, Vector 4, Vector 5 and Vector 6. specifically incorporated, the at least three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 14 and vector 18, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 14 and vector 30, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification, 3-30 immunomodulatory factors are stably integrated into the cell genome using vector 18 and vector 30, and at least 3 immunomodulatory The factors are OX40L, CD27L and CD28L, and optionally additional immunomodulatory factors are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulatory factors are stably integrated into the cell genome using Vector 14, Vector 18, and Vector 30, and at least The three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and 3-30 immunomodulators using one or more of Vector 15, Vector 19, Vector 22, Vector 23 and Vector 29. The factor is stably integrated into the cellular genome and the at least three immunomodulators are OX40L, CD27L and CD28L, and optionally additional immunomodulators are selected from R ¹ -R ⁴⁴ of Table 3.

According to one embodiment, a tumor cell line is selected for modification and vector 44 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and using vector 97 3-25 immunomodulators are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 84 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and, using vector 29, 3-25 immunomodulatory factors are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 107 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 116 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 86 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 18 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 17 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and using vector 98 3-25 immunomodulators are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and using Vector 5, 3-25 immunomodulatory factors are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 30 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 109 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and using Vector 3, 3-25 immunomodulatory factors are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and using Vector 4, 3-25 immunomodulatory factors are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 106 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 16 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 83 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 31 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and using vector 12, 3-25 immunomodulatory factors are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and 3-25 immunomodulatory factors are stably integrated into the cell genome using Vector99. According to one embodiment, a tumor cell line is selected for modification and vector 121 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 105 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 32 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and, using vector 37, 3-25 immunomodulatory factors are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 22 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 19 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 20 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 89 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 21 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 23 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 108 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 15 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 124 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 65 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 64 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 88 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and, using vector 96, 3-25 immunomodulatory factors are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 14 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 119 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 120 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 45 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 60 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 59 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and using vector 8, 3-25 immunomodulatory factors are stably integrated into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 128 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and vector 35 is used to stably integrate 3-25 immunomodulatory factors into the cell genome. According to one embodiment, a tumor cell line is selected for modification and using Vector 6, 3-25 immunomodulatory factors are stably integrated into the cell genome.

According to one embodiment, a tumor cell line is selected for modification, vector 44, vector 29, vector 18, vector 17, vector 5, vector 16, vector 99, vector 15, vector 14, vector 45 and vector Using one or more of the 6, 3-14 TNF family immunomodulators are stably integrated into the cellular genome. According to one embodiment, the 3-14 TNF family member immunomodulators are selected from those listed in Table 2.

According to one embodiment, a tumor cell line is selected for modification and one or more of Vector 97, Vector 84, Vector 107, Vector 98, Vector 30, Vector 83, Vector 121 and Vector 119 are used. As a result, 3-25 Ig family immunomodulators are stably integrated into the cellular genome. According to one embodiment, the 3-25 Ig family member immunomodulators are selected from those listed in Table 2.

According to one embodiment, a tumor cell line is selected for modification and vector 109 is used to stably integrate 3-25 growth factor immunomodulators into the cell genome.

According to one embodiment, a tumor cell line is selected for modification, vector 3, vector 4, vector 32, vector 22, vector 19, vector 20, vector 89, vector 21, vector 23, vector 121, vector 65 , Vector 64, Vector 88, Vector 96, Vector 60, Vector 59, and Vector 128 are used to stably integrate 3-25 cytokine immunomodulators into the cell genome. According to one embodiment, the 3-25 cytokine immunomodulators are selected from those listed in Table 2.

According to one embodiment, a tumor cell line is selected for modification, and one or more of vector 37, vector 124, vector 88, and vector 8 are used to immunomodulate 3-25 receptor immunomodulators. The factor stably integrates into the cell genome. According to one embodiment, the 3-25 receptor immunomodulators are selected from those listed in Table 2.

According to one embodiment, a tumor cell line is selected for modification and is one of vector 86, vector 106, vector 107, vector 31, vector 12, vector 105, vector 108, vector 120, and vector 35 Using the above, 3-25 other immunoregulatory factors are stably integrated into the cellular genome. According to one embodiment, the 3-25 other immunomodulators are selected from those listed in Table 2.

Example 4.
Experiments were performed to demonstrate that the immunomodulators described herein expressed on the melanoma tumor cell line SK-MEL2 differentially affect human PBMC proliferation and differentiation. Figure 53 is a schematic showing the general experimental design. The following allogeneic cell lines were tested:
・ SK-MEL (parent strain) (“SK”)
・ SK modified with vector 2 only (“2”)
・ SK modified with vector 3 only (“3”)
・ SK modified with vector 4 only (“4”)
・ SK modified with vector 5 only (“5”)
・ SK modified with vector 6 only (“6”)
・ SK modified by vector 2 and vector 3 (“2-3”)
・ SK modified by vector 3, vector 4 and vector 5 (“3-4-5”)
・ SK modified by vector 3, vector 5 and vector 6 (“3-5-6”)
・ SK modified by vector 3, vector 4, vector 5 and vector 6 (“3-4-5-6”)
・ SK modified by vector 2, vector 3, vector 4, vector 5 and vector 6

Functional characterization of allogeneic cell lines was performed using primary MLTR assays as described herein. MLTR assays were set up with 250,000 freshly thawed PBMC and 50,000 selected engineered allogeneic cell lines. The following outputs were measured: 1) proliferation measured by flow over PMBC labeled CFSE; 2) differentiation measured by CyTOF on unlabeled PMBC; Profiling is performed by Luminex.

Flow Cytometry Data "Non-self-recognition" is a term used to define immunological recognition of tissue-incompatible antigens between genetically distinct individuals within the same species. "Direct non-self-recognition" is the mechanism by which recipient T cells recognize determinants on MHC-molecule-peptide complexes presented on the surface of transplanted cells without the need for antigen processing by the recipient APCs. be. A direct response can most easily be demonstrated in vitro by a mixed lymphocyte reaction in which only direct non-self-presentation can occur.

"Indirect non-self recognition" refers to recognition of processed peptides of allogeneic histocompatibility antigens presented by self MHC in a self-restricted manner. Indirect alloantigen presentation always results in non-autoreactivity with a predominance of CD4+ T cells.

Approximately 10% of peripheral blood T cells carry TCRs capable of non-self recognition of allogeneic tumor type-specific cells used for vaccination. This is termed 'direct non-self-recognition' and occurs early in the post-vaccination event. Direct non-self-recognition targets the T-cell-mediated immune response against allogeneic cells, killing them and releasing tumor type-specific neo-antigens and shared normal antigens. These tumor neo-antigens (and normal antigens) are taken up by host antigen-presenting cells, processed and presented in association with host HLA. This 'indirect non-self-recognition' occurs late in the post-vaccination event. Although the TCR activated during indirect nonself-recognition is distinct from that previously involved during direct nonself-recognition, both processes are repeatedly exposed to allogeneic cells carrying high-density immunomodulators. occurring in the local environment (e.g. monthly vaccination).

Epitope spreading is the process of amplifying the immune response to different but closely related T cell epitopes. This is commonly described as maturation of the immune response. The differential maturation of immune responses against tumor neoantigens against self-antigens is caused by the fact that tolerance mechanisms are in place to differentially protect immune responses against self-antigens. Although self-tolerance can be broken, it is more difficult than responding to tumor neoantigens.

Without being bound by theory, since all tumors of a particular type share many antigens, T cell-mediated responses driven primarily by indirect, non-self-recognition of the immune response may be associated with host tumors of the same type. cross-react to According to some embodiments, this approach may be used after debulking therapy (e.g., surgery, radiation, or oncolytic viruses) because the tumor microenvironment may present insurmountable negative immunomodulatory hurdles. May be used in combination with checkpoint inhibitors in the setting of minimal residual disease.

The experiments described here use hPBMC activation by direct non-self-recognition versus pan-stimulation. Primary human pan T cells include CD4 and CD8 T cells, as well as several gamma/delta T cell subsets. For pan-stimulation, non-target cells, i.e., monocytes, neutrophils, eosinophils, B cells, stem cells, dendritic cells, NK cells, granulocytes, or erythroid cells, are treated with a cocktail of biotin-conjugated antibodies. label with T cells are separated from peripheral blood (PB) mononuclear cells (MNC) using a negative immunomagnetic separation technique without the use of columns. Cells are unaffected by the separation process and are ready for downstream use. Activation of hPBMCs by direct non-self-recognition was found to display radically different responses to tumor cells. Three important observations were made regarding the activation of hPBMCs by this method: 1) ~10% of hPBMCs proliferate compared to ~50% with anti-CD3/CD28 treatment; Induces more cell division than CD3/CD28 treatment; 3) Induces more diverse morphologies.

FIG. 54 is a panel of graphs showing the results of flow cytometry experiments. Forward scatter (FSC) and side scatter (SSC) plots of size versus particle size are shown. SK tumor cell lines are represented by number code; SK, parental unchanged; vector 3, secreted GM-CSF and membrane-expressed FLT-3L; vector 4, secreted FLT3L and membrane-expressed GM- CSF; vector 5, uncleaved form of CD40L; vector 6, uncleaved form of TNF-α; 3-4 is a combination of vectors 3 and 4; and 3-4-6 is a combination of vectors 3, 4, and 6. Cell lines 6, 3-4-5, and 3-4-6 exhibit a larger, more granular phenotype, presumably due to the presence of TNF-a and CD40L receptors on cells of epithelial origin.

FIG. 55 is a panel of graphs showing representative flow cytometry staining of the indicated engineered surface markers: GM-CSF, FLT3L, TNF-α, and CD40L. SK tumor cell lines are represented by number code; SK, parental unchanged; vector 3, secreted GM-CSF and membrane-expressed FLT-3L; vector 4, secreted FLT3L and membrane-expressed GM- CSF; 5, uncleaved form of CD40L; Vector 6, uncleaved form of TNF-α; 3-4 is a combination of vectors 3 and 4; and 3-4-6 is a combination of vectors 3, 4, and 6.

FIG. 56 is a panel of graphs showing representative flow cytometry staining of the indicated engineered surface markers: GM-CSF, FLT3L, TNF-α, and CD40L. SK tumor cell lines are represented by number code; SK, parental unchanged; vector 3, secreted GM-CSF and membrane-expressed FLT-3L; vector 4, secreted FLT3L and membrane-expressed GM- CSF; vector 5, uncleaved form of CD40L; vector 6, uncleaved form of TNF-α; 3-4 is a combination of vectors 3 and 4; and 3-4-6 is a combination of vectors 3, 4, and 6.

CyTOF Data The results of CyTOF mass cytometry single-cell phenotypic analysis of hPBMC responses to SK melanoma cells with alterations due to expression of immunomodulators are shown in Figures 14A and 14B. The SK melanoma cell line and hPBMC were cultured for 24 hours. Cells were harvested from culture and stained with a 32-marker CyTOF antibody panel to detect multiple immune cell subsets and cell surface and intracellular phenotypic markers. CyTOF mass cytometry data were generated on a Helios instrument. Data were normalized to signal using equilibration beads. Cell staining data were analyzed using Cytobank, a cloud computing suite for CyTOF data analysis that includes cell gating functionality and a suite of data visualization methods.

The data shown in Figures 57A and 57B were plotted using viSNE, a dimensionality reduction method that converts multidimensional staining signals from single cells into plots for visualization. FIG. 57A shows viSNE density contour plots of CyTOF staining data showing relative changes in abundance and phenotype of immune cell subsets. FIG. 57B shows single cell phenotype analysis. viSNE density contour plots were generated by viSNE from ungated total PBMCs cultured with SK melanoma cells or modified SK melanoma cells. Plots show relative changes in cell density of hPBMC immune cell subsets. The inset viSNE plot identifies immune cell subsets that fall within the clusters of the viSNE density plot. Arrows in density contour plots indicate visible changes in immune cell subsets among hPBMCs, SK cells and modified SK cells. SK tumor cell lines are represented by number code; SK, parental unchanged; vector 3, secreted GM-CSF and membrane-expressed FLT-3L; vector 4, secreted FLT3L and membrane-expressed GM- CSF; vector 5, uncleaved form of CD40L; vector 6, uncleaved form of TNF-α; 3-4 is a combination of vectors 3 and 4; and 3-4-6 is a combination of vectors 3, 4, and 6.

Figures 58A-D show activation markers CD40 (Figure 58A), CD86 (Figure 58B), CD69 (Figure 58C) after 1 day of stimulation with the indicated genetically modified SK lines at a 1:5 cell ratio. ), and CyTOF monocyte cluster analysis of hPBMC showing changes in expression of CD25 (FIG. 58D). FIG. 58E shows CyTOF monocyte cluster analysis of hPBMC showing relative median expression levels of monocyte markers CD40 and CD86. FIG. 58F shows CyTOF monocyte cluster analysis of hPBMC showing relative median expression index (MEI) of CD4 T cell markers CD69 and CD25. SK tumor cell lines are represented by number code; SK, parental unchanged; vector 3, secreted GM-CSF and membrane-expressed FLT-3L; vector 4, secreted FLT3L and membrane-expressed GM- CSF; vector 5, uncleaved form of CD40L; vector 6, uncleaved form of TNF-α; 3-4 is a combination of vectors 3 and 4; and 3-4-6 is a combination of vectors 3, 4, and 6.

Cytokine Data Luminex multiplex cytokine profiling of human PBMC responses to SK parental and genetically modified SK strains is shown in FIG. SK cells or the indicated modified cell lines were cultured with human PBMCs at a cell ratio of 1:5 for 24 hours. Control cultures included SK cells only, hPBMCs only, and hPBMCs stimulated with a mixture of anti-CD3 and anti-CD28 antibodies (1 μg/ml final concentration). IL-1a, IL-1b, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, IL- Supernatants were assayed for cytokine levels using a multiplex Luminex bead array assay to detect 23, TNFa, IFNg, G-CSF, GM-CSF, MIP1b, MCP-1, Rantes, Tweak, and TREM-1. Screened. Cytokines found to be specifically induced by the SK parent strain and the modified SK strain are shown in the plot. Symbols indicate cytokine levels in pg/ml extrapolated from standard curves using recombinant cytokines. Absence of symbols indicates that no cytokine was detected. SK strains are represented by number code; SK, unmodified parent strain; 3, secreted GM-CSF and membrane-expressed FLT-3L; 3, secreted FLT3L and membrane-expressed GM-CSF; 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; 6 is a combination of 3, 4, and 6.

The studies described provide proof of concept that the complex combinatorial space of immunomodulators can be rapidly and efficiently assessed using all human in vitro MLTR assays.

Example 5.
Experiments were performed to determine the effect of the immunomodulators described herein expressed on the tumor cell line SK-MEL2 on the activation and expansion of CD8+ T cells and NK cells.

The following allogeneic cell lines are tested:
SK-MEL (parent strain, used as control)
・ SK modified by vector 14, vector 18 and vector 30 (“14-18-30”)
- SK modified with vector 15 only ("15")
・ SK modified with vector 19 only (“19”)
- SK modified with vector 22 only ("22")
- SK modified with vector 23 only ("23")
- SK modified with vector 29 ("29")

Functional characterization of allogeneic cell lines was performed using primary MLTR assays, as described in Example 1. The following outputs were measured: 1) CD8+ T cell proliferation was measured by flow cytometry (Figure 60); 2) tumor cell killing using live/dead staining on day 9 (Figure 61); 3) Expansion of natural killer (NK) cells, dendritic cells (DC), and B cells was measured by flow cytometry (Figure 62).

An initial series of experiments revealed that stimulation of CD8+ T cells was specifically enhanced by '14-18-30'. FIG. 60 compares the effect of tumor cells modified with Vector 14, Vector 18, and Vector 30 (“14-18-30”) and the parental line. The dotted ellipse in the top panel of the graph indicates the lymphocyte gate. In the parental strain (i) the lymphocytes are quiescent, whereas in the '14-18-30' strain (ii) the lymphocytes are significantly expanded. Dotted circles in the lower panel of the graph indicate CD8 gates. A small number of CD8+ T cells are present in the parental line (iii), whereas the number of CD8+ T cells is greatly increased in the '14-18-30' line (iv). An increase in CD8+ T cell counts was found to be approximately 300-fold over baseline, and the CD8/CD4 ratio, indicative of immune activation, was found to be increased approximately 15-fold over baseline. In addition, the CCR7+/CCR7− ratio (a surrogate for CTL memory morphology) increased approximately 15-fold over baseline.

In addition, when comparing the parent strain on day 9 of culture with the tumor cells modified with vector 14, vector 18, and vector 30 (“14-18-30”), the parent strain (“unmodified parent tumor strain + hPBMC”): Healthy tumor cells and resting lymphocytes can be visualized, whereas vector 14, vector 18 and vector 30 modified tumor cells (“APX-14-18-30 vaccine candidate + hPBMC”) remain alive No tumor cells were visualized, but activated CTLs were seen.

In a further series of experiments, the effect of tumor cells modified with Vector 15, Vector 19, Vector 22, Vector 23, and Vector 29 on immune cell stimulation was determined. In particular, using flow cytometry, it was possible to determine which subsets of immune cells were stimulating each particular immunomodulator, as shown in FIG. Killing potency was determined visually. As shown in Figure 62, vector 15 ("APX/15") stimulates DCs, vector 19 ("APX/19") stimulates NK cells, and vector 22 ("APX/22") stimulates DCs. vector 23 (“APX/23”) stimulated DC cells and vector 29 (“APX/29”) stimulated B cells.

Figure 65 shows results from another series of experiments that assessed CD8+ T cell expansion and cell killing using flow cytometry. Day 6 and 8 time points were compared in a CD8 expansion assay using the SK parent strain versus modified SK strains expressing immunomodulatory molecules 14, 18, and 30 (“14-18-30”). Lower left panel (iii) represents CD8+ T cells. Comparing panel (iii) with panel (iv) shows that the number of CD8+ T cells increases and the flow cytometry readout extends to the right, indicating an activated morphology. Upper right panel (ii) shows allogeneic cells. Comparing allogeneic cells (panels (i) and (ii)), flow cytometry results are shown in panel (ii), which contains modified SK lines expressing immunomodulatory molecules 14, 18, and 30. was observed to shift to These results represent a transition to dead and dying cells. Thus, not only did the CD8+ T cells expand, but they also killed the allogeneic cells within them. These results indicated that allogeneic tumor cells interacted with blood cells to kill the injected cells. Therefore, in clinical scenarios, allogeneic tumor cell vaccines are used to activate and expand a patient's lymphocytes, particularly a subset of immune killer cells (CD8+ T cells and NK cells), to kill the patient's tumor cells. can be done. Tumor cell vaccines are allogeneic to the subject, but blood cells and tumor cells have the same HLA.

Dendritic Cell (DC) Expansion CyTOF was performed as described in Example 1. SK melanoma cells with modification by expression of immunomodulatory molecules (Vector 3 (“APX/3”), Vector 3 and Vector 4 (“APX/3-4)”; Vector 3, Vector 4, Vector 5 (“APX/3”); CyTOF mass cytometry single-cell phenotypic analysis of hPBMC responses to vector 3, vector 4, vector 6 (“APX/3-4-6”)) and vector 3-4-5”) are shown in FIGS. show. CyTOF offers extreme multiplexing with atomic mass spectrometry resolution compared to flow cytometry and is therefore used here to define different PBMC subpopulations following stimulation with different immunomodulatory molecules. was done. The SK melanoma cell line and hPBMC were cultured for 24 hours. Cells were harvested from culture and stained with a 32-marker CyTOF antibody panel to detect multiple immune cell subsets and cell surface and intracellular phenotypic markers. CyTOF mass cytometry data were generated on a Helios instrument. Data were normalized to signal using equilibration beads. Cell staining data were analyzed using Cytobank, a cloud computing suite for CyTOF data analysis that includes cell gating functionality and a suite of data visualization methods.

The data shown in Figures 63 and 64 were plotted using viSNE, a dimensionality reduction method that converts multidimensional staining signals from single cells into plots for visualization. FIG. 63 shows viSNE density contour plots of CyTOF staining data showing relative changes in abundance and phenotype of immune cell subsets. In Figure 63, dotted circles indicate subpopulation(s) of cells that were not present in the parental strain ("parent"). As shown in Figure 63, populations of NK cells and myeloid cells that were absent in the parental cell line were present in Figure 64, showing CyTOF monocyte cluster analysis of hPBMCs, modified with immunomodulatory molecules. Shows changes in expression of markers PD-L1, CD86, CD25, CD16, CD14, CD141, CD64, CD123, and T-bet after 9 stimulations with the indicated SK strains. As shown in Figure 64, the expression of known DC markers CD123 and CD141 was increased in cell lines modified with immunomodulatory molecules. In addition, expression of the myeloid marker CD14 was increased in cell lines modified with immunomodulatory molecules.

Example 6. In Vivo Xenograft Mouse Experiments Six-week-old female inbred SCID mice are obtained from Charles River Laboratories (Hartford, Conn., USA). Animals are handled according to protocols approved by the institution's Institutional Animal Care and Use Committee. Mice were acclimated to the animal house.

Human tumor xenografts were established in NSG (NOD scid gamma mice (Jackson Laboratory)). Human tumors were laterally implanted in NSG mice. Human tumor cells were laterally implanted in NGS (NOD scid gamma) mice and allowed to grow to 150 mm ³ . Mice were randomly divided into two groups, a control group and a treatment group, with 6 mice per group. Treatment groups were treated with expanded activated PBMC containing expanded activated serial killer cells activated by ENLST™ cells expressing 14-18-30. On day 30 (t=0), mice in the control group were inoculated with vehicle alone and mice in the treatment group were inoculated with 3×10 ⁶ expanded-activated PBMC containing expanded-activated serial killer cells. Tumor size was measured by caliper over time after inoculation in both groups. FIG. 66 is a plot showing mean and standard deviation results of xenograft treatment studies using NGS mice. The ends of each box are the upper and lower quartiles. The median is marked by a vertical line inside the box, and the whiskers are the two lines outside the box, extending to the highest and lowest observations. Human tumor cells were implanted into the flanks of NGS (NOD scid gamma) mice. Tumors were grown to 150 mm ³ . Mice were randomly divided into two groups, a control group and a treatment group, with 6 mice per group. On day 30 (t=0), mice in the control group were inoculated with vehicle alone and mice in the treatment group were activated with 14-18-30-expressing ENLIST™ cells (“SUPLEXA™ cells”). 3×10 ⁶ PBMC were inoculated. Tumor size was measured at intervals up to 36 days post-inoculation. Divergence between the two groups appeared within 5 days. After 22 days, divergence became statistically significant (*P<0.05;**P<005).

Although the present invention has been described with reference to specific embodiments thereof, it will be appreciated by those skilled in the art that various changes can be made and equivalents substituted without departing from the true spirit and scope of the invention. will be understood. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims (22)

An allogeneic tumor cell vaccine comprising:
(1) a population of live, non-proliferative, 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 for inducing one or more subpopulations of PBMCs to proliferate in response to said expressed immunomodulatory molecules and then enter the effector phase to kill tumor cells; , OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both;
said population of PBMC cells comprising one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or B lymphocytes;
(2) a pharmaceutically acceptable carrier;
The allogeneic tumor cell vaccine comprising: (a) the population of live, non-proliferative, genetically engineered tumor cells expressing said one or more tumor-specific antigens is stably treated with one or more additional stably selected from R ¹ -R ⁴⁴ ; or (b) said tumor cells are rendered proliferation-incompetent by irradiation, or (c) said tumor cells are melanoma, colorectal cancer, leukemia, chronic myelogenous Leukemia, prostate cancer, head and neck cancer, squamous cell cancer, tongue cancer, laryngeal cancer, tonsil cancer, hypopharyngeal cancer, nasopharyngeal cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, glioblastoma, and brain cancer or (d) said population of viable, growth-resistant tumor cells is derived from a biological sample derived from a subject, or (e) said viable, growth-resistant tumor cells (f) said immunostimulatory molecule is presented on the external surface of said genetically engineered tumor cells; or (g) said T lymphocyte population is derived from a tumor cell line; wherein induction comprises activation of said subpopulation of T lymphocytes, expansion of said T lymphocytes, or both; or (h) induction of said NK cells comprises activation of said subpopulation of NK cells, said or (i) induction of said DC subpopulation comprises activation of said DC subpopulation, expansion of said DC subpopulation, or both. or (j) the induction of the B lymphocyte subpopulation comprises activation of the B lymphocyte subpopulation, expansion of the B lymphocyte subpopulation, or both.
2. The allogeneic tumor cell vaccine of claim 1. (a) said subpopulation of T lymphocytes comprises a subpopulation of CD8+ cytotoxic T lymphocytes (CTLs), or (b) said subpopulation of T lymphocytes comprises a subpopulation of memory T cells. or (c) said subpopulation of T lymphocytes comprises a subpopulation of regulatory T cells;
(d) said subpopulation of T lymphocytes comprises a subpopulation of helper T cells;
(e) the subpopulation of B lymphocytes comprises a subpopulation of memory B cells;
2. The allogeneic tumor cell vaccine of claim 1. (1) the vaccine enhances immune activation of cells effective to recognize and act against tumor cells containing the target tumor antigen in vivo without systemic inflammation, or (2) said vaccine reduces immunosuppression in the tumor microenvironment of tumor cells containing said target tumor antigen; or (3) said vaccine increases cell death of tumor cells expressing said target tumor antigen; (4) said population of live, growth-resistant tumor cells induces immune activation without systemic inflammation; or (5) said vaccine reduces progression-free survival, overall survival compared to placebo control induce an immune response that improves duration, or both;
2. The allogeneic tumor cell vaccine of claim 1. said melanoma tumor cells preferentially express gp100, tyrosinase, Melan-A, tyrosinase-related protein (TRP-2-INT2), melanoma antigen-1 (MAGE-A1), NY-ESO-1, melanoma 3. The allogeneic tumor cell vaccine of claim 2, characterized by the expression of one or more of the following antigens (PRAME), CDK4 and multiple myeloma oncogene 1 (MUM-1). The colorectal cancer tumor cells are characterized by carcinoembryonic antigen (CEA), MAGE, HPV, human telomerase reverse transcriptase (hTERT), EPCAM, PD-1, PD-L1, p53, and cell surface associated mucin 1 (MUC1) 3. The allogeneic tumor cell vaccine of claim 2, characterized by the expression of one or more of: said one or more additional stably expressed immunomodulatory molecules selected from R ¹ -R ⁴⁴ are cytokines, TNF family members, secretory receptors, chaperones, IgG superfamily members, and/or chemokine receptors be,
3. The allogeneic tumor cell vaccine of claim 2. providing an allogeneic parental tumor cell line comprising a population of viable tumor cells;
introducing into said population of live tumor cells an exogenous nucleic acid encoding a stably expressed immunomodulatory molecule, said immunomodulatory molecule being OX40 ligand (OX40L);
introducing into said population of live tumor cells an exogenous nucleic acid encoding a stably expressed immunomodulatory molecule, said immunomodulatory molecule being CD27 ligand (CD70);
Introducing into said population of live tumor cells an exogenous nucleic acid encoding a stably expressed immunomodulatory molecule, said immunomodulatory molecule being a CD28 ligand (CD28L), including CD80, CD86, or both. ) and stable expression of OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both, wherein one or more subpopulations of PBMC expressed said inducing, introducing, to proliferate in response to immunomodulatory molecules and then enter the effector phase to kill tumor cells;
generating a tumor cell line variant by selecting a tumor cell clone stably expressing an immunogenic amount of an exogenous subset of said immunomodulatory molecule;
selecting a clonally derived cell line variant by one or more parameters selected from cell proliferation, cell subset differentiation, cytokine release profile, and tumor cell lysis in a mixed lymphocyte tumor cell reaction, said selected the clonally derived cell line variant is effective to stimulate activation of one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or B lymphocytes; choosing and
2. The allogeneic tumor cell vaccine of claim 1 produced by a process comprising: 9. The process of claim 8, further comprising introducing into said population of live tumor cells exogenous nucleic acids encoding one or more stably expressed immunomodulatory molecules selected from ^R1 - ^R44 . Allogeneic tumor cell vaccine produced by . (a) the tumor cells are rendered proliferation-incompetent by irradiation; or (b) the parental tumor cell line is derived from melanoma or colorectal cancer; or (c) the exogenous nucleic acid is DNA. or RNA, or (d) said introducing step comprises viral transduction, or (e) said introducing step comprises electroporation, or (f) said introducing step is liposome-mediated transfer, adenovirus, adeno-associated virus, herpes virus, retroviral-based vectors, lipofection, and lentiviral vectors; or (g) the introducing step comprises lentiviral introducing said exogenous nucleic acid by transfection of a vector;
9. An allogeneic tumor cell vaccine produced by the process of claim 8. A method of inducing an immune response against cancer in a subject, comprising:
(a) administering to said subject an allogeneic tumor cell vaccine parenterally or locally into a tumor, said allogeneic tumor cell vaccine comprising: (1) one or more tumor-specific 1. A population of live, proliferation-incompetent, genetically engineered tumor cells expressing an antigen, said population comprising at least three stably expressed immunoregulatory molecules, said at least three immunoregulatory molecules comprising: said population of live proliferation-incompetent genetically engineered tumor cells that are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28 ligand (CD28L), including CD80, CD86, or both;
(2) a pharmaceutically acceptable carrier;
administering the allogeneic tumor cell vaccine to the subject parenterally or locally into the tumor, comprising
(b) induce one or more subpopulations of peripheral blood mononuclear cells (PBMCs) to proliferate in response to said expressed immunomodulatory molecule and then enter the effector phase to kill tumor cells; and
including
said subpopulation of PBMC cells comprises one or more of T lymphocytes, natural killer (NK) cells, dendritic cells (DC), or B lymphocytes;
wherein the allogeneic tumor cell vaccine is type-matched to the subject's cancer;
the aforementioned method. 12. The method of claim 11, wherein said cancer is selected from melanoma or colorectal cancer. 12. The method of claim 11, wherein the subject has an infectious viral disease with progression to cancer. 12. The method of claim 11, further comprising administering a checkpoint inhibitor to said subject. The population of live, non-proliferative, genetically engineered tumor cells expressing said one or more tumor-specific antigens is combined with one or more additional stably expressed immune cells selected from R ¹ to R ⁴⁴ . 12. The method of claim 11, further comprising a regulatory molecule. (a) said population of viable, growth-resistant tumor cells is derived from a biological sample derived from a subject; or (b) said population of viable, growth-resistant tumor cells is derived from a tumor cell line.
12. The method of claim 11. A method of treating cancer in a subject, comprising:
the cancer is melanoma or colorectal cancer, and the method comprises:
(a)(1) a population of live proliferation-incompetent genetically engineered tumor cells expressing one or more tumor-specific antigens;
(2) a pharmaceutically acceptable carrier;
administering to said subject an allogeneic tumor cell vaccine comprising
said population comprises at least three stably expressed immunomodulatory molecules, wherein said at least three immunomodulatory molecules are OX40 ligand (OX40L), CD27 ligand (CD70), and CD28, including CD80, CD86, or both is a ligand (CD28L);
administering the allogeneic tumor cell vaccine to the subject;
(b) induce one or more subpopulations of peripheral blood mononuclear cells (PBMCs) to proliferate in response to said expressed immunomodulatory molecule and then enter the effector phase to kill tumor cells; and
including
wherein said subpopulation of PBMC cells is effective to reduce tumor burden in said subject and improve progression-free survival, overall survival, or both in said subject compared to a placebo control; comprising one or more of lymphocytes, natural killer (NK) cells, dendritic cells (DC), or B lymphocytes;
the aforementioned method. The population of live, non-proliferative, genetically engineered tumor cells expressing said one or more tumor-specific antigens is combined with one or more additional stably expressed immune cells selected from R ¹ to R ⁴⁴ . 18. The method of claim 17, further comprising a regulatory molecule. 18. The method of claim 17, further comprising rendering the population of live, non-proliferative, genetically engineered tumor cells non-proliferative by irradiation. (a) said population of viable, growth-resistant tumor cells is derived from a biological sample derived from a subject; or (b) said population of viable, growth-resistant tumor cells is derived from a tumor cell line.
18. The method of claim 17. 18. The method of claim 17, wherein said immunostimulatory molecule is presented on the external surface of said genetically engineered tumor cells. (a) inducing said T lymphocytes comprises activation of a subpopulation of said T lymphocytes, expansion of said T lymphocytes, or both, or (b) inducing said NK cells , activation of said subpopulation of NK cells, expansion of said subpopulation of NK cells, or both; or (c) inducing said subpopulation of DCs comprises activation of said subpopulation of DCs. , expansion of said subpopulation of DCs, or both; or (d) inducing said subpopulation of B lymphocytes comprises activation of said subpopulation of B lymphocytes, subpopulation of said B lymphocytes, including population expansion, or both;
18. The method of claim 17.

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