Compositions, methods of making, and methods of using irradiated antigen presenting cells in the treatment of health conditions

Abstract

Embodiments of the present disclosure provide novel compositions, methods of making and methods of using irradiated antigen presenting cells (APCs) to treat a health condition in a subject. In some embodiments, novel compositions, methods and uses relate to antigen exposed, irradiated APCs of use to treat cancer in a subject. Certain embodiments disclosed herein concern using antigen exposed, irradiated APCs or professional APCs alone, or in combination with other therapeutics to treat, prevent or reduce onset of a sarcoma in a subject. In certain embodiments, treatments disclosed herein include personalized treatments of a subject to be treated.

Description

COMPOSITIONS, METHODS OF MAKING, AND METHODS OF USING IRRADIATED ANTIGEN PRESENTING CELLS IN THE TREATMENT OF HEALTH CONDITIONSCROSS-REFERENCED APPLICATION

[0001] This International Application claims priority to U.S. Provisional Application No. 63 / 553,962, entitled, “Vaccine Based on High Dose Irradiated Professional Antigen Presenting Cells,” filed February 15, 2024. This provisional application is incorporated herein by reference in its entirety for all purposes.FIELD

[0002] Embodiments of the present disclosure provide novel compositions, methods and uses for irradiated antigen presenting cells (APCs) to treat a health condition. In some embodiments, novel compositions, methods and uses relate to antigen exposed, irradiated professional APCs of use to treat aberrant cell growth in a subject. Certain embodiments disclosed herein concern using antigen exposed, irradiated APCs or professional APCs alone, or in combination with other therapeutics to treat, prevent or reduce onset of a sarcoma in a subject.

[0003] The Sequence Listing submitted herewith as an XML file named“130118_829322_Sequence_Listing,” created on February 06, 2025, and having a size of 8,192 bytes is hereby incorporated by reference in its entirety for all purposes pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUND

[0004] Antigen presenting cells (APCs) are an important part of an active and passive immune system; they process antigens and present those antigens to other immune cells, such as T-cells. The most important types of professional antigen presenting cells are dendritic cells, macrophages and B-cells; and atypical antigen presenting cells (mast cells, eosinophils ILC3s and basophils). In cancer processes, APCs appear to have lost capacity to initiate and / or elicit an effective immune response against tumor antigens due in part to induction of tolerance and immune suppression by the cancer; thus, it has been previously hypothesized that vaccinations based on ex vivo specifically modified dendritic cells may hold great promise to induce tumor regression and maintain control of tumor growth, potentially through the activation of key effector cells of cellular immunity such as B- and T-lymphocytes and macrophages. Initiation of antigen-specific cellular immune responses is dependent upon appropriate antigen presentation by professional APCs. In their immature stage, professionalantigen presenting cells migrate into essentially every tissue where they capture and process antigens. Following activation by a variety of different stimuli, these cells differentiate into mature dendritic cells (DCs). As mature DCs they migrate into lymphoid organs, where, depending on the context in which antigen is presented, they may trigger an immune response or maintain tolerance against autoantigens. Two key factors involved in the differentiation of DCs from monocytes in vitro are interleukin (IL) 4 and granulocyte-macrophage colonystimulating factor (GM-CSF).

[0005] Vaccination strategies based on dendritic cells that have been developed over the last few decades mainly involve differentiation of DCs from monocytes in vitro and infusing them into cancer patients. However, none of these attempts have resulted in established clinical standard therapies, due to lack of efficacy. There is a need for new effective therapies to treat and / or reduce recurrence or prevent onset of cancer.SUMMARY

[0006] Embodiments disclosed herein generally relate to cell-based compositions or vaccines of use to treat health conditions. In some embodiments, compositions, methods of making and using cell-based compositions or vaccines can include a population of irradiated antigen presenting cells of use to reduce onset or treat cancer in a subject. In accordance with these embodiments, antigen presenting cells can include irradiated antigen presenting cells (APCs) or professional APCs. In certain embodiments, high dose of about 2000 to about 20,000 rad is used to irradiate APCs of use as therapeutics disclosed herein. In certain embodiments, the APCs can include any APC. In a further embodiment, the cells (APCs) can be rendered inactive or altered (e.g., non-proliferative, immunostimulated, induced stimulation of cytokine production, etc.) from irradiation detailed herein. In some embodiments, irradiated cells disclosed herein are still able to migrate and in certain settings, migrate better than an unirradiated cells (e.g., APCs) disclosed herein. In accordance with these embodiments, the irradiated APCs or professional APCs can be used in a pharmaceutically effective composition to create an APC-based vaccine or composition for administration to a subject.

[0007] In some embodiments and further to paragraph

[0006] above, the APC-based vaccine can include a population of irradiated APCs or professional APCs (DCs), where the APCs are obtained by incubating APCs in vitro in the presence of at least one target antigen; and subsequently irradiating the antigen-exposed APCs with at least 2000 rad to about 20,000 rad of irradiation. One of skill in the art can accurately deliver a dose of radiation of about 2000 to about 20,000 rads accurately to cells disclosed herein. In other embodiments, theAPC-based vaccine can include a population of irradiated APCs, where the APCs were incubated with a tumor antigen prior to irradiation with at least 2000 rad to about 20.000 rad of irradiation. In certain embodiments, the tumor antigen(s) of use for exposing to APCs or professional APCs can be obtained from a donor tumor, a tumor antigen from a matched donor, a relative or a patient-specific tumor antigen from a tumor of the patient to be treated with compositions disclosed herein.

[0008] In some embodiments and further to paragraphs

[0006] -

[0007] above, the APC population can include a dendritic cell population. In certain embodiments, the dendritic cell population can include a population of irradiated DCs, where the DCs are obtained by incubating DCs in vitro in the presence of at least one target antigen; and subsequently irradiating the antigen-exposed DCs with at least 2000 rad to about 20,000 rad of irradiation. In other embodiments, the DC-based vaccine can include a population of irradiated DCs, where the DCs were incubated with a tumor antigen prior to irradiation with at least 2000 rad to about 20,000 rad of irradiation. In some embodiments, the dendritic cells of use herein are derived from a subject to be treated or from a matched population of cells (e.g.. tissue matched from an immortalized cell population). In certain embodiments, the tumor antigen(s) of use for exposing to DCs or professional DCs can be obtained from a donor tumor, a tumor antigen(s) from a matched donor, a relative or a patient-specific tumor antigen(s) from a tumor of the patient to be treated with compositions disclosed herein.

[0009] In certain embodiments and further to paragraphs

[0006] -

[0008] above, methods can include obtaining a plurality of APCs by incubating the APCs in vitro in the presence of at least one antigen (e.g., tumor antigen); and subsequently treating the antigen-exposed APCs with an agent to at least partially inactivate the APCs; optionally, where the agent exposed antigen primed APCs are rendered non-proliferative, immunostimulated, induced stimulation of cytokine production, etc. In other embodiments, methods can include obtaining a plurality of APCs by incubating the APCs in vitro in the presence of at least one tumor antigen from a patient to be treated, a donor tumor or a tumor from a relative or matched donor; and subsequently treating the tumor antigen-exposed APCs with an agent to at least partially inactivate the APCs; optionally, where the agent exposed antigen primed APCs are rendered at least one of non-proliferative, immunostimulated, induced stimulation of cytokine production (e.g., induced inflammatory cytokine production), etc.

[0010] In some embodiments and further to paragraphs

[0006] -

[0009] above, methods for treating, reducing the risk of or preventing onset of a health condition in a subject are contemplated. In accordance with these embodiments, a subject in need thereof can beadministered a pharmaceutically effective composition including an irradiated APC population and / or an irradiated antigen-exposed APC population, an agent inactivated or partially inactivated APC population and / or an agent inactivated or partially inactivated antigen-exposed APC population to treat the subject. In some embodiments, the subject is a subject having cancer. In certain embodiments, the APC population is a DC population. In some embodiments, the irradiated antigen-exposed APC population and / or agent inactivated, or partially inactivated APC population is an irradiated or agent inactivated or partially inactivated tumor antigen-exposed APC population. In some embodiments, the tumor antigen is derived from the subject to be treated.

[0011] In certain embodiments and further to paragraphs

[0006] -

[0010] above, methods for preparing a composition and using a composition including a therapeutically effective population of cells or a cell-based vaccine disclosed herein, can include exposing a population of cells to an antigen and inactivating or partially inactivating the cells with radiation and / or an agent to render the cells non-proliferative, immunostimulated, induced stimulation of cytokine production, etc. and administering the composition to a subject having cancer. In accordance with these embodiments, the cancer can include a solid tumor or a bone-related cancer.

[0012] In certain embodiments and further to paragraphs

[0006] -

[0011] above, methods and uses disclosed herein concern methods and uses for generating long-term immunity against a cancer or particular tumor in a cancer patient by administering a cell-based vaccine disclosed herein to a subject having or suspected of developing cancer. In some embodiments, combination treatments of antigen-exposed irradiated APCs disclosed herein and irradiated tumor cells can be used to treat a subject, alone or in combination with other standard treatments. In accordance with these embodiments, the APCs can be derived from a subject to be treated or other source; optionally wherein the irradiated tumor cells originate from the subject to be treated where the irradiated tumor cells are apoptotic (e.g. irradiated at about 12,000 rads

[0013] In other embodiments and further to

[0006] -

[0012] above, kits are contemplated for generating, transporting, storing, and using irradiated or agent-treated cell-based antigen exposed APC populations disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certainembodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0015] FIG. 1 represents exemplary graphs of dendritic cell yield. Four methods for monocyte enrichment and different types of media were used as indicated. Because no significant difference was observed between the medias, the bottom graph illustrates averaged data from all experiments independent of the medium used according to various aspects of the disclosure.

[0016] FIGS. 2A-2B represents an exemplary T-cell receptor clonotyping of patient samples. 2A demonstrates some shared unique nucleotide sequences found in several samples; 2B represents a pairwise comparison of the identified clones of the samples; Samples: sample of tumor of 2nd / 3rd relapse (A), sample of Tumor of 4th relapse (B), blood sample after 2nd relapse (C), blood sample after 4th relapse (D), blood sample 11 years after 1st vaccination and 10 years after 2nd treatment (E) according to various aspects of the disclosure. Appearance and persistence of specific and patient-specific T cell clones demonstrated stimulation of specific immune responses by the dendritic cell vaccine according to various aspects of the disclosure.

[0017] FIG. 3 is a representative table. Table 1, illustrating survival rates of patients undergoing treatments disclosed herein compared to controls according to various aspects of the disclosure.

[0018] FIG. 4 is a representative table, Table 2. illustrating various parameters of patients undergoing treatments disclosed herein compared to controls according to various aspects of the disclosure.

[0019] FIG. 5 is a representative table, Table 3, illustrating relapses and location of relapses and surgeries of patients studied compared to control patients according to various aspects of the disclosure.

[0020] FIG. 6 is a representative table. Table 4, illustrating status and diagnosis of patients undergoing treatments disclosed herein according to various aspects of the disclosure.

[0021] FIG. 7 is a representative table, Table 5. illustrating collection and expansion / differentiation under various conditions of APCs (e.g., DC, dendritic cells) from patients undergoing and that underwent treatments disclosed herein according to various aspects of the disclosure.

[0022] FIG. 8 is a representative table, Table 6. illustrating collection and expansion / differentiation under various conditions including varied medias of APCs (e.g.,DC, dendritic cells) from patients undergoing and that underwent treatments disclosed herein according to various aspects of the disclosure.

[0023] FIG. 9 is a representative graph illustrating survival post relapse over several years of selected patients in a comparative study with poor outcome of patients within a few short years compared to treatments disclosed herein.

[0024] FIG. 10 are representative plots of patients treated under conditions disclosed herein according to various aspects of the disclosure.

[0025] FIG. 11 represents a schematic diagram of cells treated and untreated with irradiation according to various aspects of the disclosure.

[0026] FIG. 12 represents graphs depicting analysis of expression of various factors under conditions of irradiated and unirradiated dendritic cells according to various aspects of the disclosure.DETAILED DESCRIPTION

[0027] In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details may be modified through routine experimentation. In some embodiments, well know n methods or components have not been included in the description.

[0028] Those of skill in the art generally acknowledge that human tumors carry a mutational antigenic (non-self) repertoire of immunogenic potential that may be a target for anti-tumor immune therapy. In studying evidence from mouse experiments, it was discovered that one can immunize prophylactically against cancers similar to immunizing against an infectious agent. However, in contrast to most experimental mouse tumor models, human tumors have in general been harbored within their host organisms for a long time providing them with opportunities to influence their microenvironment and the larger immunological environment. The first animal -experimental approaches of cancer vaccination targeting preexisting tumors have assisted in new treatments of human tumors. Antigens capable of mediating specific tumor rejection were found in humans as well as in mouse tumors.

[0029] Historically, many clinical trials using dendritic-based cancer vaccination techniques w ere designed for the treatment of melanoma. Other cancers in which DC-based cancer vaccination was studied include prostate cancer, B cell lymphoma, renal cell carcinoma, glioma, breast and ovarian cancer, carcino-embryonic antigen expressing malignancies, gastrointestinal cancer, and selected solid pediatric tumors. In most of thesetrials some in vivo and / or in vitro evidence for the generation of anti-tumor immunity was found; however complete or partial remission of the tumor was observed in only a few selected cases. The observed side effects were usually described to be mild and not seen to limit the application of DC vaccines for treatment of cancer.

[0030] As disclosed herein, it was surprisingly found that additional treatment (e.g. by a high dosage of irradiation) of APCs, such as dendritic cells to render the cells inactive provided unexpected benefits in the treatment of cancer.

[0031] Abbreviations as used herein can include: alloMLR Allogeneic mixed leukocyteAPC Antigen presenting cellCR Complete response d DaysDC Dendritic cellFISH fluorescence in situ hybridizationGM-CSF Granulocyte-macrophage colony-stimulating-factor h Hours iDC Immature dendritic cellIFN InterferonIL InterleukinKLH Keyhole limpet haemocyaninLPS Lipopolysaccharide mDC Mature dendritic cellMHC major histocompatibility complex n Number of independent experiments nd Not different np Not possible.OP OctaplasPBMC Peripheral blood mononuclear cellPBS Phosphate buffered salinePG Prostaglandin rpm Rotations per minuteSEA / SEB Staphylococcus enterotoxin A and B smDC Semi-mature dendritic cellSOP Standard operating procedureTh T helperTNF Tumor necrosis factorTT Tetanus ToxoidUV Ultraviolet y Y ears

[0032] Embodiments of the present disclosure and further to paragraphs

[0028] -

[0031] above, compositions, methods of preparing compositions and methods for treating a subject for a health condition are disclosed. In certain embodiments, compositions and methods for treating cancer using cell-based compositions are disclosed. In some embodiments, antigen presenting cells (e.g. dendritic cells) contacted with or exposed to an antigen and then inactivated (e.g. make at least one of non-proliferative, immunostimulated, inducedstimulation of cytokine production, etc.) provide an unexpected anti-tumor benefit to a subject having cancer receiving such a treatment.

[0033] In certain embodiments and further to paragraphs

[0028] -

[0032] above, antigen presenting cells (APCs) of use herein include, but are not limited to, dendritic cells (DCs). In accordance with these embodiments, the dendritic cells are exposed to or incubated in vitro with at least one antigen to created antigen-conditioned DCs and then irradiating the antigen- conditioned DCs with at least about 2000 to at least about 20.000 rads of radiation for less than one minute up to about 30 minutes; or for less than a minute to up to about 20 minutes.

[0034] In another embodiment and further to paragraphs

[0028] -

[0033] above, a cell-based composition disclosed herein includes, but is not limited to, a population of irradiated dendritic cells, where the dendritic cells were incubated with at least one tumor antigen prior to irradiation with at least 2000 rads or at least 6000 rad of irradiation. In accordance with these embodiments, the at least one tumor antigen includes at least one patient-specific tumor antigen, donor tumor antigen, compatibility7typed-tumor antigen, tumor antigen-derived from a relative or any combination thereof.Antigen presenting cell population

[0035] In some embodiments and further to paragraphs

[0028] -

[0034] above, cell populations suitable for use in compositions, methods of making, kits, and methods of using are disclosed herein. In accordance w ith these embodiments, a cell population of use herein includes an immortalized, non-immortalized or artificial cell line population including at least partially matured cells, matured cells or artificial cells which have been incubated in the presence of at least one antigen and then subsequently at least partially inactivated. In some embodiments, partially or fully inactivated partially matured cells, matured cells or artificial cells can be by irradiation. In some embodiments, the radiation is high dosage irradiation for a predetermined period to partially or fully immortalize the partially matured cells, matured cells or artificial cells.

[0036] In one embodiment and further to paragraphs

[0028] -

[0035] above, compositions and methods disclosed herein relate to a cell population of antigen presenting cells (APCs). In certain embodiments, the APCs are a population of dendritic cells. In some embodiments, the APCs (e g., dendritic cells) of use herein can be derived from a subject to be treated or from a matched population of cells (e.g., tissue matched from an immortalized cell population). In certain embodiments, APCs derived from a subject to be treated can be superior to other APC populations. In some embodiments, a plurality of APCs can be incubated in vitro with at least one antigen (e.g. at least one tumor antigen) to create antigen-conditioned APCs; andsubsequently exposing antigen-conditioned APCs to at least one agent to render the antigen- conditioned APCs at least partially inactive; optionally, where the agent-exposed antigen- conditioned APCs are non-proliferative and / or immunostimulated, induced stimulation of cytokine production, etc. In certain embodiments, compositions and methods disclosed herein relate to incubating a population of APCs (e.g., dendritic cells), in vitro with at least one antigen (e.g., at least one tumor antigen) and subsequently, exposing the APCs to an agent rendering the APCs non-proliferative.

[0037] Tn other embodiments and further to paragraphs

[0028] -

[0036] above, compositions and methods described herein relate to populations of APCs (e.g., dendritic cells), preincubated with an antigen (e.g., a tumor antigen); and subsequently treating these APCs with an agent capable of rendering the APCs non-proliferative. In certain embodiments, the APCs can be derived from a primary or established cell line or a mixture of different primary or established cell lines. In some embodiments, at least one cell line of the cell population can be immortalized, non-immortalized, or artificial cell line. In certain embodiments, the cell line can include semi-matured cells or artificial cells, pre-incubated with at least one antigen and then subsequently partially to fully inactivated; for example, by irradiating the cells with a high dosage of irradiation. In accordance with these embodiments, high dosage radiation can be about 2000 to about 20,000, or about 4000 to about 10,000 or about 6000 rad. In some embodiments, the antigen is a patient specific antigen. In other embodiments, the antigen is a patient specific tumor antigen or tumor lysate or tumor cell lysate containing antigens from a patient-specific tumor. In another embodiment, the antigen or lysate can be tumor specific.

[0038] In some embodiments and further to paragraphs

[0028] -

[0037] above, APCs can be provided in any suitable media or composition. For example, the APCs can be dispersed in a buffered or non-buffered aqueous medium. In one embodiment. APCs can be provided in a nutrient-rich medium. In an alternative embodiment, the APCs can be provided in a minimally defined medium or other medium with a defined composition (e.g., AIM-V, for example, supplemented pooled human AB plasma and / or GM-CSF). In certain embodiments, APCs where at least a portion of the APCs are in a semi-differentiated state (e.g., treated with IL-4 and / or with GM-CSF or similar agent to differentiate or semi-differentiate APCs as known in the art) can be exposed to at least one antigen (e g. tumor antigen) and incubated to generate an antigen-primed population of APCs suitable for partial or full inactivation by irradiation or by an inactivating agent.

[0039] In one embodiment and further to paragraphs

[0028] -

[0038] above, the antigen exposed, inactivated / irradiated APCs can be part of a composition. In certain embodiments,the composition is a pharmaceutical composition further including a pharmaceutically acceptable excipient, buffer or medium (e.g., AIM V supplemented with LPS, and / or IFN-y) and human plasma to a final concentration of 2% to facilitate dendritic cell differentiation. In one embodiment, the antigen exposed, agent-inactivated / irradiated APCs can be provided as a pharmaceutically acceptable composition suitable for injection or infusion: for example, as a bolus. In certain embodiments, the antigen exposed, inactivated / irradiated APCs can be provided in a pharmaceutically acceptable form suitable for intravenous, intradermal, subcutaneous, intratumoral, intranodal, locoregional, transdermal, intradermal, intranasal, by inhalation, or intramuscular administration, injection and / or infusion. For example, the antigen exposed, agent-inactivated / irradiated APCs can be delivered to a subject in need thereof, in a pharmaceutically acceptable form suitable for subcutaneous, intradermal, or intratumoral injection. In a further embodiment, the antigen exposed, agent- inactivated / irradiated APCs can be in a pharmaceutically acceptable form suitable for subcutaneous, renal, or bolus infusion, as appropriate.

[0040] In other embodiments and further to paragraphs

[0028] -

[0039] above, suitability for inj ection or infusion can mean a pharmaceutically acceptable composition having a high level of purity, e.g., essentially absent of chemical, biological and / or microbiological impurities above an acceptable level. In one embodiment, compositions disclosed herein incorporates inactive ingredients or agents safe for parenteral use. Examples of suitable inactive ingredients include, but are not limited to, salts (e.g. sodium chloride); pH-modifying agents (e.g., acid or base agents), surfactants, (e.g., phospholipids or synthetic nonionic surfactants); stabilizers or supplements (e.g., antioxidants or amino acids); sugars or sugar alcohols or pharmaceutically acceptable cell stabilizing agents (e.g., surfactants, physiologic pH, salt, etc.). In certain embodiments, the antigen exposed, agent-inactivated / irradiated APCs or the antigen exposed APCs can be quick frozen or stored at refrigeration or freezing temperatures. For example, the antigen exposed, agent-inactivated / irradiated APCs can be frozen and / or stored and / or transported at a temperature of approximately -80° C or lower (e.g., approximately -196° C in liquid nitrogen) until administered. Alternatively, the antigen exposed APCs can be frozen and / or stored and / or transported at a temperature of approximately -80° C or lower until inactivated or partially inactivated by an agent (e.g., chemical agent) and / or irradiated prior to generating a pharmaceutically acceptable composition for administration to a subject.

[0041] In some embodiments and further to paragraphs

[0028] -

[0040] above, APCs are used for compositions disclosed herein. APCs are an important part of the immune system;they process antigens and present these antigens to other immune cell types, such as T-cells, In accordance with these embodiments, APCs are incubated in vitro with at least one antigen and the APC processes the at least one processed antigen and re-presents the at least one antigen on the surface of the APCs, for example to present to other immune cells as a non-self at least one antigen. In some embodiments, the at least one antigen can be taken up by the APCs and complexed with MHCs (major histocompatibility complexes) and the re-presented on the surface of the cells. In certain embodiments, some of the most important types of APCs include dendritic cells, macrophages and B-cells. In certain embodiments, APCs of use herein can include a dendritic cell line, a macrophage cell line, or a B cell line or any combination thereof. In other embodiments, the APCs can be a mixed population of APC lines including a plurality of different APC lines, such as a dendritic cell line, a macrophage cell line and / or a B cell line. In one embodiment, the APCs are a dendritic cell line. In some embodiments, APCs can be from a healthy subject or a genetically related healthy subject or are generated from induced pluripotent stem cells (iPSCs) from the subject to be treated or other subject. Dendritic cells derived from either of these sources can then be used the same as dendritic cells (APCs) obtained from the subject to be treated. In some embodiments, the APCs are a single cell line or a single population of primary APCs. In one embodiment, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or more of the APCs of the cell population are a single cell ty pe or a single cell line.

[0042] In some embodiments and further to paragraphs

[0028] -

[0041] above, APC populations of use herein are in suspension that include a plurality of single cells. In other embodiments, APC populations of use herein are in monolayers adhered to a surface. In some embodiments, the APCs include dendritic cells, macrophages or B-cells, or a combination thereof. In one embodiment, the APCs are dendritic cells alone or can further include macrophages and / or B-cells. In one embodiment, the cell population are dendritic cells. In one embodiment, the APCs are limited to dendritic cells.

[0043] In some embodiments and further to paragraphs

[0017] -

[0042] above, APCs including dendritic cells of use herein can be prepared by incubating the dendritic cells in vitro in the presence of at least one antigen; and subsequently treating the dendritic cells with a physical or chemical agent capable of at least partially inactivating the dendritic cells in a manner similar to radiation; optionally, where the treated cells are non-proliferative. In certain embodiments, physical agents such as bleomycin or other similar alkylating agent can be used to alter APCs as disclosed herein. In accordance with these embodiments, bleomycincan be used at about 0.01 to about 20 pg / ml; or about 0.01, 0.1, 1.0 and 10 pg / ml or concentration in between for about 10 minutes to about 12 hours or about 3 hours to render APCs non-proliferative but immunostimulated as indicated herein (e g., immune-charged, APCs where inflammatory cytokines are induced). In other embodiments, the dendritic cells can be exposed to at least one physical agent for at least one minutes up to 12 hours. In one embodiment. APCs can be inactivated (e.g., rendering the cells non-proliferative) by in vitro treatment with a chemical agent; for example, a DNA damaging agent. In accordance with these embodiments, DNA damaging agents can include, but are not limited to, ionizing radiation, platinum drugs (e.g., Cisplatin, oxaliplatin, and carboplatin); UV light: UV-A and UV-B radiation from the sun can cause DNA damage and skin cancer; nitrogen mustards; antimetabolites; topoisomerase poisons or the like. In certain embodiments. DNA damaging agents can include platinum drugs.

[0044] In some embodiments and further to paragraphs

[0028] -

[0043] above, dendritic cells of use herein can be prepared by irradiating antigen exposed dendritic cells. In accordance with these embodiments, dendritic cells can be incubated in vitro in the presence of at least one antigen; and subsequently irradiating the dendritic cells with at least about 2000 rads, or at least about 6000 rads or greater than 7,000 rads of irradiation.

[0045] In certain embodiments and further to paragraphs

[0028] -

[0044] above, the APCs (e.g. dendritic cells) used to prepare the APC-based composition or vaccine are human or other mammalian derived APCs. In some embodiments, the APCs are from a pet (e.g., dog or cat), livestock, a horse, wild animal or other mammal. In some embodiments, the cell population comprises antigen presenting cells that are obtained from the same patient (autologous) that will be administered with the treated (e.g. irradiated) cells.

[0046] In certain embodiments and further to paragraphs

[0028] -

[0045] above, the APCs of use herein for a subject contemplated herein can be obtained from the subject or patient, autologous cells from self. In accordance with these embodiments, APCs obtained from the subject / patient for autologous use can be isolated and / or enriched from the subject / pati ent’s blood or serum, or harvested from lymph nodes, spleen, skin or other tissue. Methods for isolating and enriching APCs from blood are known in the art and described herein. Any other method known by one of skill in the art for obtaining APCs is contemplated herein.

[0047] In other embodiments and further to paragraphs

[0028] -

[0046] above, the APCs of use herein for a subject contemplated herein can be obtained from a donor subject (allogeneic). In some embodiments, the APCs can be a mix of APCs from the subject / patientand from a donor if additional APCs are necessary. As used herein, the term “donor subject’" refers to any individual who is not the subj ect / patient. Accordingly, the APCs can be allogeneic to the subj ect / patient. In certain embodiments, the donor subject can be a relative of the subj ect / patient, including, but not limited to a child of the subject, sibling or parent. In some embodiments, the APCs obtained from the donor subject for allogeneic use can be isolated and / or enriched from the donor's blood or serum, or harvested from lymph nodes, spleen, skin or other tissue. In certain embodiments, methods for isolating and enriching APCs are known in the art and contemplated of use herein. Alternatively, the APCs of the donor subject can be from an unknown matched or unmatched donor. For example, APCs can be obtained from blood from a blood bank.

[0048] In other embodiments and further to paragraphs

[0028] -

[0047] above, APCs of use in compositions and methods disclosed herein can be incubated in the presence of at least one antigen. In one embodiment, the APCs have been incubated in the presence of one antigen of the same antigen type. In other embodiments, APCs can be incubated in the presence of a plurality of antigens, antigens of more than one type. Any antigen is contemplated of use herein. In some embodiments, the at least one antigen includes, but is not limited to, proteins, lipids, carbohydrates, such as polysaccharides or mixtures thereof (e g., such mixture as found in a tumor cell lysate). In certain embodiments, the at one least antigen can be a selfantigen, or tumor or cancer-derived antigen from the subject. In one embodiment, APCs can be incubated in the presence of a plurality of the same antigens, or combination of antigens. In one embodiment, the antigens making up a combination can be derived from the same origin or derived from the same source. In one embodiment, the at least one antigen can be a combination of cancer or tumor antigens originating from the same cancer type or tumor region. In another embodiment, the at least one antigen can be obtained or isolated from the same subject or the same patient as the APCs (autologous). In other embodiments, the APC population can be dendritic cells, of a patient and the at least one antigen originating from the patient, e.g. a tumor antigen from the patient. In another embodiment, the APCs can be dendritic cells of a patient described herein and the at least one antigen is a tumor antigen from the same patient.

[0049] In another embodiment and further to paragraphs

[0028] -

[0048] above, the at least one antigen is obtained or isolated from the patient to be treated but the APCs are obtained or isolated from a donor subject and are allogeneic to the patient. For example, the at least one antigen can be obtained or isolated from a tumor of the patient. In one embodiment, the at least one antigen can be obtained or isolated from a child such as a pediatric cancer patientand the APCs can be obtained or isolated from a donor subject or a donor parent subject or other related subject to the pediatric patient. In some embodiments, the at least one antigen can be a complex mixture of antigens.

[0050] In some embodiments and further to paragraphs

[0028] -

[0049] above, the at least one antigen of use in compositions and methods disclosed herein can be obtained from a cell lysate. In some embodiments, the lysate is a tumor cell lysate. In another embodiment, the at least one antigen can be isolated from a tumor cell lysate of the patient or related or unrelated subject. In certain embodiments, the tumor cell lysate can be obtained from a solid tumor, a blood-derived cancer. In certain embodiments, the tumor can be a sarcoma; or a cancer or tumor resulting from one or more chromosomal translocations. In certain embodiments, the at least one antigen is obtained from a tumor cell lysate of a solid tumor including, but not limited to, melanoma or other skin cancer, bone cancer, kidney cancer, ovarian cancer, prostate cancer, liver cancer (e.g., hepatocellular carcinoma (HCC)), pancreatic cancer, breast cancer, lung cancer (e.g., non- small cell lung cancer), brain cancer, stomach cancer, esophageal cancer or other solid tumor or combinations thereof. In some embodiments, the solid tumor includes, bone cancer, ovarian cancer, lung cancer, pancreatic cancer and liver cancer. In some embodiments, the cancer includes bone cancer. In other embodiments, a health condition for treatment contemplated herein includes aberrant cell or bone growth. In another embodiment, the tumor cell lysate can be obtained from any solid tumor or other cancerous cell disclosed herein. In certain embodiments, the tumor cell lysate can be obtained from a sarcoma, including, but not limited to, Ewing sarcoma or osteosarcoma. In some embodiments, the Ewing sarcoma, can be a primary or metastatic Ewing sarcoma-derived tumor cell lysate for obtaining one or more antigen. In one embodiment, the tumor cell lysate can be obtained from a relapsed metastatic Ewing sarcoma. In accordance with these embodiments, the tumor cell lysate can be from an osteosarcoma, such as a primary or metastatic osteosarcoma. In another embodiment, the tumor cell lysate can be obtained from a relapsed metastatic osteosarcoma.

[0051] In some embodiments and further to paragraphs

[0028] -

[0050] above, the tumor cell lysate can be from a solid tumor including, but not limited to. a pediatric patient-derived solid tumor. In one embodiment, the tumor cell lysate can be from a solid tumor from bone cancer, such as a pediatric bone cancer. In one embodiment, the tumor cell lysate for obtaining at least one antigen or a mixture thereof can made up of immunogenic antigens capable of producing an immune response in a patient or subject. In certain embodiments, atumor cell lysate can be from a tumor of a pediatric cancer patient. In some embodiments, the tumor cell lysate is derived from the pediatric patient or a similar tumor in a donor subject.

[0052] In some embodiments and further to paragraphs

[0028] -

[0051] above, APCs can be incubated with the at least one antigen derived from a tumor cell lysate and then the APCs are inactivated in vitro (e.g., rendered non-proliferative, immunostimulated, induced stimulation of cytokine production, etc.) with a physical agent. In certain embodiments, the physical agent can be a chemical agent, as described herein. In some embodiments, the cells are rendered inactive (e g., non-proliferative, immunostimulated, induced stimulation of cytokine production, etc.).

[0053] In other embodiments and further to paragraphs

[0028] -

[0052] above, the antigen- exposed APCs can be treated with irradiation in vitro. In accordance with these embodiments, the APCs can be irradiated with a high dosage of irradiation. In some embodiments, high dose radiation can be at least about 2000 rad, or at least about 3000 rad, or at least about 4000 rad. or at least about 5000 rad, or at least about 6000 rad, or at least about 7000 rad, or at least about 8000 rad, or at least about 9000 rad, or at least about 10000 rad, or at least about 11000 rad, or at least about 12000 rad, up to about 20,000 rad or any dose in between. In one embodiment, APCs can be irradiated with a high dosage of irradiation in the range of about 2000 to about 20000 rad, or from about 3000 to about 18000 rad, or from about 2000 to about 15000 rad, or from about 3000 to about 10000 rad, or about 3000 to about 8000 rad, or about 6000 to about 10000 rad. or about 6000 to about 20000 rad. In certain embodiments, APCs can be irradiated with at least about 6000 rads of irradiation. It is understood by one of skill in the art that this dosage of irradiation can inactivate the APCs and render them nonproliferative, immunostimulated, induced stimulation of cytokine production (e.g., inflamed cells), etc., a desired feature(s) of embodiments disclosed herein. It is further understood by one of skill in the art that source of radiation dictates the length of time of exposure to reach a certain level of radiation which can range from less than a minute to several hours.

[0054] In other embodiments and further to paragraphs

[0028] -

[0053] above, APCs of use herein and exposed to antigens as disclosed can be rendered inactive (e.g., non-proliferative, immunostimulated, induced stimulation of cytokine production, etc.) by at least one of: UV radiation, X-ray irradiation or / -radiation. In accordance with these embodiments, APCs can be irradiated with y- radiation or x-ray irradiation. In one embodiment, APCs can be irradiated with x-ray irradiation. In another embodiment, APCs can be irradiated with y- radiation. In certain embodiments. APCs can be rendered inactive (e.g.. non-proliferative, immunostimulated, induced stimulation of cytokine production, etc.) by y-radiation, using adose of y-radiation of at least about 2000 rad, or at least about 3000 rad, or at least about 4000 rad. or at least about 5000 rad, or at least about 6000 rad, or at least about 7000 rad, or at least about 8000 rad, or at least about 9000 rad, or at least about 10000 rad, or at least about 11000 rad, or at least about 12000 rad or more. In some embodiments, APCs can be rendered inactive (e.g., non- proliferative) by a dose of y-radiation from about 2000 to about 20000 rad. or from about 3000 to about 12000, or from about 2000 to about 8000 rad, or from about 3000 to about 6000 rad, or from about 6000 to about 10000 rad. or from about 6000 to about 20000 rad, respectively, such as at about 2000 rad, 3000 rad, 4000 rad, 5000 rad, 6000 rad, 7000 rad, 8000 rad, 9000 rad, 10000 rad, 11000 rad, or 12000 rad. In one embodiment, the APCs can be irradiated with at least 6000 rad of y-radiation.

[0055] In one embodiment and further to paragraphs

[0028] -

[0054] above, a high dosage of irradiation as used herein for use on antigen-incubated APCs disclosed herein can refer to a dose of y-radiation of at least about 2000 rad, or at least about 3000 rad, or at least about 4000 rad, or at least about 5000 rad, or at least about 6000 rad, or at least about 7000 rad, or at least about 8000 rad, or at least about 9000 rad, or at least about 10000 rad, or at least about 11000 rad. or at least about 12000 rad or more. In one embodiment, a high dosage of irradiation as contemplated herein can be about 2000 to about 20000 rad, or about 3000 to about 20000 rad, or about from 2000 to about 12000 rad, or about 3000 to about 12000 rad, or from about 6000 to about 20000 rad, or about 6000 to about 10000 rad, or about 3000 to about 6000 rad or about 2000 rad, about 3000 rad, about 4000 rad. about 5000 rad, about 6000 rad, about 7000 rad, about 8000 rad, about 9000 rad, about 10000 rad, about 11000 rad, or about 12000 rad or any condition in between these doses.

[0056] In some embodiments and further to paragraphs

[0028] -

[0055] above, APCs previously incubated with at least one antigen can be inactivated (e.g., by rendering the cells non-proliferative, immunostimulated, induced stimulation of cytokine production, etc.) by treatment with elevated temperatures for a predetermined period. In accordance with these embodiments, elevated temperatures can include mildly elevated temperatures of about 39° C to about 45° C; or about 39° C to about 43° C. In certain embodiments, APCs can be exposed to temperatures of about 39° C to about 43° C, or about 39° C, or about 40° C, or about 41° C, or about 42° C, or about 43° C, or about 44° C, or about 45° C for at least 10 minutes up to a few days; or about 1 hour to about 1 day; or about 2 hours to about 12 hours.

[0057] As used herein, reference to “inactivated cells” or “inactive cells” following treatment with a physical agent or exposure to radiation can mean that the cells are no longer able to proliferate, expand, multiply but are able to migrate to a different location from whereplaced compared to untreated cells contemplated here. In certain embodiments, irradiated cells can have increased migratory ability and can have increased inflammatory cytokine production of use to treat conditions contemplated herein. Accordingly, inactivated cells (e.g. APCs) can be non-proliferative. In certain embodiments, inactivated APCs can be both nonproliferative and immunostimulated. In other embodiments, inactivated APCs can be nonproliferative, immunostimulated, induced stimulation of cytokine production, etc. In accordance with these embodiments, irradiation with a high dosage of radiation (e.g.. at least 2000 rads to at least 10000 rad) can render the cells non-proliferative and / or immunostimulated and / or stimulate inflammatory cytokine production, etc. In some embodiments, irradiated cells can be rendered apoptotic. In accordance with these embodiments, irradiation at a dosage of at least about 12000 rad can cause cell death and render the cells apoptotic. Some embodiments disclosed herein relate to combination therapies that include, but are not limited to, creating a combination therapeutic, separately administering and / or treating a subject with irradiated antigen-exposed APCs disclosed herein and irradiated tumor cells (e.g., irradiated to generate apoptotic tumor cells). In certain embodiments, the APCs and tumor cells are from the subject to be treated. In other embodiments the APCs are dendritic cells. It is contemplated herein that apoptotic APCs (e.g., previously incubated with at least one antigen) can be used in compositions and methods disclosed herein to treat a condition in a subject (e.g., cancer). Any test for assessing whether a cell or cell population is rendered at least one of non-proliferative, immunostimulated, induced stimulation of cytokine production, etc. by treatments described herein are contemplated and one of skill in the relevant art would be familiar with routine tests (e.g. alamar blue, MTT assay, DNA damage assays, cell migration assays, etc.).

[0058] In some embodiments and further to paragraphs

[0028] -

[0057] above, APCs of use herein can be obtained by differentiating peripheral monocyte cells or stem cells into APCs. In certain embodiments, APCs can include dendritic cells that have been obtained by differentiating peripheral monocyte cells or stem cells derived from the patient to be treated or a donor subject and further differentiated in vitro. In another embodiment, APCs can be prepared by in vitro differentiation of peripheral monocyte cells or stem cells. In some embodiments, the peripheral monocyte cells can be CD14+ APCs. In certain embodiments, peripheral monocyte cells or stem cells can be autologous to the patient. In some embodiments, peripheral monocyte cells or stem cells can be obtained or isolated from a donor related or unrelated subject (allogeneic).

[0059] As referenced herein, “stem cells’" can include stem cells obtained from any one of bone marrow, blood, teeth, umbilical cord or other source of stem cells from the patient (autologous) or a donor subject (allogeneic) or a combination if deemed necessary. In one embodiment, APCs can be obtained by enriching monocytes from a population of white blood cells and incubating the enriched monocytes with cytokines known in the art to differentiate the monocytes into APCs where differentiation is performed in vitro. In certain embodiments, cytokines suitable for differentiating peripheral monocyte cells or stem cells into APCs can include, but are not limited to, interleukin-4 (IL-4) and / or Granulocytemacrophage colony-stimulating factor (GM-CSF). In some embodiments, cytokines of use to differentiate monocytes or stem cells to APCs can include, for example, IL-4 and GM-CSF or a combination thereof; optionally, followed by incubation ith LPS. In certain embodiments, peripheral monocyte cells or stem cells can be incubated with cytokines of use herein for at least 1 hour or up to about 2 hours, or up to 4 hours, or up to 6 hours, or up to 12 hours, or up to 24 hours, or up to 48 hours, or up to 72 hours, or up to 96 hours or up to 120 hours or more. In one embodiment, incubation with cytokines can be about 48 hours, about 72 hours, or about 120 hours, or more, or any time between 1 hour and 120 hours. In one embodiment, incubation is for about 48 hours.

[0060] In some embodiments and further to paragraphs

[0028] -

[0059] above, APCs can include APCs that are at least semi-matured cells or partially differentiated cells. In accordance with these embodiments, APCs can be semi-matured cells. In other embodiments, APCs can be matured APCs (e.g., dendritic cells) or a mixture of partially matured and matured APCs. In certain embodiments, APCs can include dendritic cells that are at least semi- or partially- matured dendritic cells (e g., an intermediary population of cells). In some embodiments, APCS include dendritic cells that are semi- or partially- matured dendritic cells. In a further embodiment, APCS can include dendritic cells that are completely or essentially all mature dendritic cells.

[0061] In some embodiments and further to paragraph

[0060] above, antigen incubated APCs of use herein that are ‘partially matured’ or 'semi-matured' can mean partially but not fully matured or differentiated cells; and 'at least semi-matured' can mean semi-matured or fully matured or differentiated cells. In accordance with these embodiments, a population of APCs of use herein can include a mixture of mature and partially mature APCs. As contemplated herein, biological markers of at least semi-mature or partially mature APCs can include, but are not limited to, one or more of CD 14 (low presence or negative); expression of CD80; CD86; CDla; CD1 lb, CDl lc, CD103, CD205, BDCA-1, BDCA-2, BDCA-4,CD45RA, CD123, ILT-7, TLR7, TLR9, MHC I; CDllc and MHC II. In one embodiment, a partially mature dendritic cell population can include cells having two or more of the following markers: CD14 (low or negative); CD80; CD86; CDla; MHC I; and MHC II. In one embodiment, a partially mature dendritic cell population can include cells having three or more of the following markers: CD14 (low or negative); CD80; CD86; CDla; MHC I; and MHC II. In one embodiment, a partially mature dendritic cell population can include cells having four or more of the following markers: CD14 (low or negative); CD80; CD86; CDla; MHC I; and MHC II. In one embodiment, a partially mature dendritic cell population can include cells having five or more of the following markers: CD14 (low or negative); CD80; CD86; CDla; MHC I; and MHC II. In one embodiment, a dendritic cell population can include cells having include each of the following markers: CD14 (low or negative); CD80; CD86; CDla; MHC I; and MHC II. In some embodiments, a dendritic cell population of use are MHC II positive dendritic cells. In some embodiments, the APCs can be at least semi- or partially- matured APCs expressing at least one membrane protein including, but not limited to, CD14 (low or negative), CD80, CD86, CDla, MHC I and MHC II.

[0062] In other embodiments and further to paragraphs

[0037] -

[0061] above, APCs can be at least partially matured, fully matured or a mix of maturities and derived from peripheral monocyte cells or stem cells. In certain embodiments, APCs can be dendritic cells that are at least semi matured from peripheral monocyte cells or stem cells. In some embodiments, peripheral monocyte cells can be CD 14+ monocytes. In one embodiment, APCs that are dendritic cells can be at least semi matured from a starting population of peripheral monocyte cells. In one embodiment, peripheral monocyte cells or stem cells can be obtained or isolated from the patient. Accordingly, the peripheral monocyte cells or stem cells are autologous to the patient. Alternatively, the peripheral monocyte cells or stem cells can be obtained or isolated from a donor subject. Accordingly, the peripheral monocyte cells or stem cells are allogeneic to the patient. For example, the cell population can include dendritic cells that are at least semi matured from peripheral monocyte cells or stem cells isolated from the patient, or from an allogenic stem cell pool, cell line, or primary source.

[0063] In other embodiments and further to paragraphs

[0037] -

[0062] above, APCs can be matured in vitro by any suitable method. In one embodiment, APCs can be differentiated to generate at least semi-matured cells. In a further embodiment, APCs can be differentiated to at least semi -maturity by incubating the cells in vitro with at least one liposaccharide (e.g., LPS, about 50 to about 400; or about 200 U / ml) and / or interferon-y (e.g., about 10 to about 250; or about 50 ng / ml) In one embodiment, the APCs can be incubated with at least oneliposaccharide and / or interferon-y for up to about 2 hours, up to about 4 hours, up to about 6 hours, up to about 8 hours, up to about 10 hours, up to about 12 hours, up to about 24 hours, up to about 48 hours, or up to about 72 hours. In one embodiment, APCs can be incubated in vitro with at least one at least one liposaccharide and / or interferon-y for about 48 hours.

[0064] In other embodiments and further to paragraphs

[0037] -

[0063] above, APCs can be matured to at least semi-maturity by incubation with at least one liposaccharide and / or interferon-y after exposure to one or more antigen and prior to treating the cells with a physical agent for inactivation (e.g., by rendering the cells non-proliferative, immunostimulated, induced stimulation of cytokine production, etc.). For example, APCs can be matured to at least semi-maturity by incubation with at least one liposaccharide and / or interferon-y prior to irradiation of the APCs. In some embodiments, antigen-exposed APCs can be matured to at least semi-maturity by incubation with at least one liposaccharide and interferon-y prior to irradiation of the APCs. In one embodiment, when maturation of APCs included incubating the APCs for at least 6 hours with at least one liposaccharide and / or interferon-y, the APCs can further be incubated in the presence of IL-4 and GM- CSF before, during or after incubating APCs with at least one liposaccharide and interferon-y prior to irradiation of the APCs. In other embodiments, the APCs can be incubated in the presence of at least one stimulant and / or adjuvant, at the same time as, prior to and / or after treating the APCs with a physical agent to inactivate the APCs (e.g., rendering them at least one of nonproliferative. immunostimulated, induced stimulation of cytokine production, etc.).

[0065] As used herein, the term “stimulant” or “adjuvant” can mean any useful pharmaceutically acceptable stimulant or pharmaceutically acceptable adjuvant. In one embodiment and further to the previous paragraphs, the at least one pharmaceutically acceptable stimulant or adjuvant can include at least one of KLH (Keyhole Limpet Hemocyanin) and / or tetanus toxoid. In certain embodiments, the at least one stimulant or adjuvant can include both KLH and tetanus toxoid. In one embodiment, APCs can be incubated in the presence of at least one pharmaceutically acceptable adjuvant or stimulant prior to treatment of the cells with a physical agent to render the cells inactive (such as nonproliferative). In some embodiments, APCs can be incubated in the presence of the at least one adjuvant or stimulant prior to irradiation of the APCs (e.g., antigen exposed APCs). In another embodiment, APCs can be incubated in the presence of at least one adjuvant or stimulant at the same time and / or after as the at least one antigen incubation of the APCs.

[0066] In other embodiments and further to paragraphs

[0028] -

[0065] above, APCs are dendritic cells and the dendritic cells can be incubated in the presence of at least onestimulant and / or adjuvant and at least one antigen simultaneously or sequentially, and subsequently matured to obtain at least semi-mature dendritic cells. In certain embodiments, the cells are dendritic cells, incubated in the presence of at least one antigen and at least one stimulant and / or adjuvant including but not limited to, KLH (e.g., about 0.01 to about 10; or about 1.0 pg / ml for about 30 minutes to about 6 hours; or about 2 hours) and tetanus toxoid, and subsequently matured to obtain at least semi-mature dendritic cells. In another embodiment, APCs can be obtained by a method including, but not limited to, enriching monocytes from a population of white blood cells, optionally where the monocytes are CD14+ monocytes; incubating the enriched monocytes with at least one cytokine to differentiate the monocytes into APCs; incubating APCs in the presence of at least one antigen, and optionally, at least one stimulant and / or adjuvant (e.g.. KLH and / or tetanus toxoid); optionally incubating the APCs with at least one lipopolysaccharide and / or interferon-y, obtaining at least semi-matured APCS; and subsequently; incubating the APCs with a physical agent to render them at least partially inactive, optionally wherein the APCs are at least one of non-proliferative, immunostimulated, induced stimulation of cytokine production, etc. It is contemplated herein that all processes for these APCs are performed in vitro. In one embodiment, the monocytes are autologous. In another embodiment, the monocytes are allogeneic to the patient (e.g., obtained or isolated from a donor subject). In one embodiment, the monocytes enriched can be CD14+ monocytes. In another embodiment, incubating the enriched monocytes with cytokines differentiates the monocytes into dendritic cells. In one embodiment, the methods include incubating the at least partially differentiated or partially mature APCs in the presence of at least one antigen and at least one adjuvant or stimulant. In certain embodiments, the at least one antigen can be a cell lysate, part of a cell lysate or an antigen isolated from a cell lysate; for example, a tumor cell lysate of a tumor of the patient, as defined herein. In some embodiments, methods can include irradiating the at least partially differentiated or partially mature antigen-exposed APCs using a radiation dosage as defined herein. For example, irradiating the APCs with at least about 2000 or at least about 6000 rad of irradiation.

[0067] In other embodiments and further to paragraphs

[0028] -

[0066] above, methods can further include harvesting inactivated antigen-exposed APCs and combining the inactivated antigen-exposed APCs with a second ty pe of cells for compositions and methods disclosed herein. For example, combining the inactivated antigen-exposed APCs with tumor cells that have been inactivated (e.g. irradiated). In one embodiment, inactivated tumor cellscontemplated herein can include tumor cells that have been irradiated with at least 12000 rad. Irradiation with at least 12000 rad causes cell death. In one embodiment, the cells irradiated with at least 12000 rad are apoptotic. In one embodiment, methods can further include harvesting inactivated dendritic cells and combining the inactivated antigen-exposed dendritic cells with a second type of cells for compositions and methods disclosed herein. For example, combining the inactivated antigen-exposed dendritic cells with tumor cells that have been inactivated (e.g. irradiated). In one embodiment, inactivated tumor cells contemplated herein can include tumor cells that have been irradiated with at least 12000 rad. In one embodiment, the tumor cells irradiated with at least 12000 rad are apoptotic. In one embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more of the cells in the combined cell population are APCs / dendritic cells as defined herein; optionally where at least 50% or more of the cells in the combined cell population are APCs / dendritic cells as defined herein.

[0068] In other embodiments and further to paragraphs

[0028] -

[0067] above, methods can include dendritic cells obtainable by a method including, but not limited to. enriching monocytes from a population of white blood cells, optionally wherein the monocytes are CD 14+ monocytes; incubating the enriched monocytes with cytokines to differentiate the monocytes into dendritic cells; incubating the differentiated dendritic cells in the presence of an antigen, and optionally, in the presence of at least one stimulant and / or adjuvant (e.g., KLH and / or tetanus toxoid); optionally, maturing the dendritic cells with at least one of a liposaccharide and interferon-y to obtain at least semi-matured dendritic cells; and subsequently treating the population of dendritic cells with a physical agent to render the dendritic cells at least partially inactive, optionally where the treated cells are nonproliferative, immunostimulated, induced stimulation of cytokine production, etc. It is contemplated herein that all processes are performed in vitro. In one embodiment, the white blood cells are autologous to the patient. In another embodiment, the white blood cells are allogeneic to the patient (e.g., obtained or isolated from a donor subject). In one embodiment, the monocytes are CD14+ monocytes. In certain embodiments, the at least one antigen can be a cell lysate, part of a cell lysate or an antigen isolated from a cell lysate; for example, a tumor cell lysate of a tumor of the patient, as defined herein. In some embodiments, methods can include irradiating the at least partially differentiated or partially mature antigen-exposed dendritic cells using a radiation dosage as defined herein. For example, irradiating the dendritic cells with at least 6000 rad of irradiation.

[0069] In one embodiment and further to paragraphs

[0028] -

[0068] , the methods can further include maturing the dendritic cells prior to inactivating the cells using treatments defined herein to obtain at least semi-matured dendritic cells. For example, the above method can further include maturing the dendritic cells prior to inactivating the cells with liposaccharides and interferon-y to obtain at least semi-matured dendritic cells. In another embodiment, methods can further include maturing the dendritic cells prior to inactivating the cells with liposaccharides and interferon-y to obtain semi-matured dendritic cells. For example, methods can include maturing the dendritic cells prior to inactivating the cells with liposaccharides and interferon-y to obtain fully matured dendritic cells. In one embodiment, antigen exposed dendritic cells can be irradiated using a radiation dosage as defined herein. For example, irradiating the dendritic cells with at least about 2000 to about 20000; or about 2000 to about 10000; or about 6000 rad of irradiation.Pharmaceutical compositions

[0070] In one embodiment and further to paragraphs

[0028] -

[0069] , compositions disclosed herein include pharmaceutical compositions including at least one APC population as defined herein. In one embodiment, compositions disclosed herein include a pharmaceutical composition including a population of APCs obtainable incubating APCs in vitro in the presence of at least one antigen; and subsequently treating the antigen-exposed APCs with a physical agent so as to render them at least partially inactive, optionally, where the treated cells are at least one of non-proliferative. immunostimulated, induced stimulation of cytokine production, etc. and the treated antigen-exposed APCs form part of a composition of use to treat a condition disclosed herein. In one embodiment, pharmaceutical compositions include, but are not limited to, a population of irradiated APCs as defined herein; and optionally, include a pharmaceutically acceptable excipient. In certain embodiments, the APCs are dendritic cells rendered non-proliferative and / or immunostimulated, having induced stimulation of cytokine production, etc. In one embodiment, pharmaceutical compositions can include, but are not limited to, a population of irradiated dendritic cells, where the dendritic cells were incubated with at least one tumor antigen prior to irradiation with at least 2000 to about 12000 rad of irradiation; or about 6000 rad of irradiation.

[0071] In some embodiments and further to paragraphs

[0028] -

[0070] , pharmaceutical compositions can include APC populations as defined herein and optionally, one or more constituents selected from pharmaceutically acceptable carriers, media, buffers, and excipients. In other embodiments, pharmaceutical compositions can include APC populations as defined herein and further include at least one adjuvant to enhance a subject’s immuneresponse before, during or after administration of a pharmaceutical composition disclosed herein. In certain embodiments, the pharmaceutical composition an APC-based vaccine formulation. In accordance with these embodiments, the APC-based vaccine formulation can include, but is not limited to, in addition to an inactivated or irradiated, antigen-primed APC preparation as described herein, but one or more constituents including, but not limited to, pharmaceutically acceptable carriers, media, buffer and excipients; optionally one or more adjuvant. In other embodiments, the pharmaceutical composition can include at least a second, differently prepared APC population (e.g., exposed to different antigens; inactivated and / or activated in part by a different process or agent (e.g., chemical, radiation)) or at least a second cell population. In certain embodiments, at least a second type of cells can be stem cells. In accordance with these embodiments, stem cells can be from the patient or donor subject, including, but not limited to, harvested from bone marrow, blood or umbilical cord (if available). In another embodiment, the second type of cells can be tumor cells or tumor cell line or tumor cell lysate, or stem cells harvested from a tumor. In one embodiment, the at least a second type of cells are at least partially inactivated (e.g., by rendering the nonproliferative. immunostimulated, and / or induced stimulation of cytokine production, etc.) by a physical agent, such as a physical agent as defined herein. In one embodiment, the physical agent is radiation, such as, UV-, x-ray or y-radiation. Irradiation is performed in vitro. The dosage amount of radiation used to irradiate the at least second type of cells can be the same as that defined herein for the APCs. For example, the second type of cells can be irradiated with at least about 2000 rad, or at least about 3000 rad, or at least about 4000 rad, or at least about 5000 rad, or at least about 6000 rad, or at least about 7000 rad, or at least about 8000 rad, or at least about 9000 rad, or at least about 10000 rad, or at least about 11000 rad, or at least about 12000 rad or more. In one embodiment, the cells can be irradiated with radiation in the range of from about 2000 to about 20000 rad, or from about 3000 to about 12000 rad, or from about 2000 to about 8000 rad, or from about 3000 to about 6000 rad, or from about 6000 to about 10000 rad, or from about 6000 to about 20000 rad, respectively, such as at about 2000 rad, 3000 rad, 4000 rad, 5000 rad, 6000 rad, 7000 rad, 8000 rad, 9000 rad, 10000 rad. 11000 rad. or 12000 rad. Irradiation with at least 12000 rad causes cell death. In one embodiment, the cells irradiated with at least 12000 rad are apoptotic. In accordance with these embodiments, APCs and the at least second cells can be combined in a single pharmaceutical composition disclosed herein. Alternatively, APCs disclosed herein can be in a separate pharmaceutical formulation than the at least second cells.

[0072] In some embodiments and further to paragraphs

[0028] -

[0071] , when the at least second type of cells are APCs, the APCs can be irradiated with at least about 2000 to about 20000 or about 2000 to about 10000; or about 6000 rad of irradiation; optionally at least about 2000 rad of y-radiation. Alternatively, when the at least second type of cells include tumor cells or cancer stem cells, the cells can be irradiated with at least about 12000 rad; optionally, about 12000 rad of y-radiation. Irradiation with at least 12000 rad causes cell death. In one embodiment, the cells irradiated with at least 12000 rad are apopt otic. In one embodiment of the technology described herein, the at least second type of cells originate from the patient in addition to the APCs. For example, the at least second type of cells can be tumor cells obtained from the patient. In another example, the at least second type of cells can be tumor cells obtained from the same tumor as a tumor cell lysate used to prepare the at least one antigen or a plurality of tumor antigens for incubating with the antigen presenting cells. In one embodiment, the irradiated tumor cells can be from a cancer or tumor as described herein. For example, the tumor cells can be from a solid tumor or non-solid tumor. In certain embodiments, a cancer can include sarcoma; or cancer from one or more chromosomal translocations. In certain embodiments, the tumor cells are derived or originate from a solid tumor. In one embodiment, the tumor cells are from a solid tumor, from an adult, a child, an adolescent or an infant. In other embodiments, the solid tumor is a bone cancer. In one other embodiments, cancer contemplated herein can include, but is not limited to, melanoma, kidney cancer, brain cancer, liver cancer (e.g.. hepatocellular carcinoma (HCC)), pancreatic cancer, breast cancer, lung cancer (e.g., non- small cell lung cancer) or other solid tumor or combinations thereof. In certain embodiments, the cancer in a patient to be treated by compositions disclosed herein is Ewing sarcoma or osteosarcoma. In accordance with these embodiments, the cancer is Ewing sarcoma and can be primary or metastatic Ewing sarcoma. In one embodiment, the tumor cells are from a relapsed metastatic Ewing sarcoma. In another embodiment, tumor cells can be derived from a patient having osteosarcoma and can be primary or metastatic osteosarcoma. In one embodiment, the tumor cells are from a relapsed metastatic osteosarcoma.APC-based vaccines

[0073] In some embodiments and further to paragraphs

[0028] -

[0072] , an APC-based vaccine or pharmaceutical composition for administration to a subject can include, but is not limited to, an APC population; and at least one pharmaceutically acceptable excipient. In certain embodiments, the APC-based composition can be administered to a subject by bolus administration, renal implant, intravenously, intratumorally, subcutaneously or the like, insome embodiments, the cell-based vaccine may comprise irradiated antigen presenting cells obtainable by a method as defined herein. For example, the cell-based vaccines can include a population of irradiated APCs obtainable by incubating the APCs in vitro in the presence of at least one antigen; and subsequently irradiating the APCs with at least about 2000 to about 10000; about 2000 to about 8000; or about 20000 rad of irradiation. For example, the antigen can be a tumor. As disclosed herein, irradiation used to render APCs at least partially inactive (e.g., non- proliferative) can be y-radiation or x-ray radiation, as defined herein. Further, APCs can be autologous or allogeneic to the patient, as defined herein. In certain embodiments, the APCs can be B- cells and / or macrophages, as defined herein. APCs can be a mixed cell population including a plurality of different APCs, such as dendritic cells, macrophages and / or B-cells. In some embodiments. APCs (such as dendritic cells) can be obtained by differentiating peripheral monocyte cells or stem cells as defined herein. Differentiation is performed in vitro. For example, APCs can be prepared by in vitro differentiation of peripheral monocyte cells or stem cells. In other embodiments, APCs can be at least semi- or partially matured by maturing the cells using the in vitro incubation steps defined herein. In some embodiments, the APCs can be at least semi- matured following treatment with liposaccharides and interferon-y prior to treating said cells with a physical agent to render them inactivate (e.g., non-proliferative, immunostimulated, and / or induced stimulation of cytokine production, etc.), as defined herein. In some embodiments, the APCs can be at least semi-matured following treatment with at least one liposaccharide and / or interferon-y prior to irradiation.Methods for Preparing APC-based Compositions

[0074] In certain embodiments and further to paragraphs

[0028] -

[0073] , methods for preparing cell-based compositions for use in a subject to reduce onset of, treat or ameliorate a health condition are disclosed. In one embodiment, methods for preparing a cell-based composition can include, but is not limited to, incubating APCs in vitro in the presence of at least one antigen; and incubating the APCs with a physical agent to render them at least partially inactive; optionally, where the partially inactive cells are non-proliferative. In another embodiment, methods for preparing APC-based compositions can include a population of irradiated APCs, where the irradiated APCs are as defined herein. In certain embodiments, methods for preparing APC-containing compositions can include a population of irradiated APCs; for example, incubating the APCs in vitro in the presence of at least one antigen; and irradiating the antigen-exposed APCs with at least 2000 or at least 4000 or at least 6000 rad of irradiation delivered as a single dose or multiple fractionated doses. In someembodiments, methods for preparing APCs can include methods for preparing dendritic cells and can further include B-cells and / or macrophages, as defined herein. In accordance with these embodiments, a mixed population of APCs including a plurality of different APC types, for example: dendritic cells, macrophages and / or B-cells can be prepared by compositions and methods disclosed herein and formulated for administration to a subject. In some embodiments, methods can include initially obtaining APCs (e.g., obtained from a subject to be treated) for incubating with at least one antigen for example, by enriching monocytes from a population of white blood cells; incubating the enriched monocytes with cytokines to differentiate the monocytes into APCs of use herein. In accordance with these embodiments, the APCs can include, but is not limited to dendritic cells, B-cells and / or macrophages. In certain embodiments, the APCs are differentiated from monocytes into dendritic cells using in vitro techniques known in the art.

[0075] In certain embodiments and further to paragraphs

[0028] -

[0074] , methods for preparing APC-based compositions and / or vaccines for use in a subject can be performed in vitro. In certain embodiments, monocytes for generating APCs can be CD14+ monocytes. In other embodiments, white blood cells can be autologous or allogeneic to the subject (patient) of use to generate the APCs. In certain embodiments, methods can be performed using APCs that are at least semi- matured following exposure to inactivating or partially inactivating agents as defined herein. In some embodiments, the APCs can be at least semi-matured following incubation with liposaccharides and interferon-y prior to exposing the APCs with a physical agent to render them at least partially inactive (such as non-proliferative, immunostimulated, and / or induced stimulation of cytokine production, etc.), as defined herein. For example, the APCs can be at least semi-matured following treatment with liposaccharides and interferon-y prior to irradiation. In certain embodiments, the method can include maturing the APCs with liposaccharides and interferon-y prior to exposing the APCs to a physical agent to render them at least partially inactive (e.g., non-proliferative, immunostimulated, and / or induced stimulation of cytokine production, etc.), as defined herein. In accordance with these embodiments, methods can further include maturing the APCs with liposaccharides and interferon-y prior to exposing the APCs to irradiation, as defined herein. In some embodiments, the at least semi-matured APCs can be semi-matured cells or fully matured APCs. In some embodiments, the APCs can include semi-matured dendritic cells, or fully matured dendritic cells. In other embodiments, the at least one antigen of use in media or compositions for incubation of APCs can include incubation with a cell lysate or incubation with at least one antigen derived from or isolated from a cell lysate, asdefined herein. In certain embodiments, KLH or similar agent can be included during incubation of APCs with one or more antigen or cell lysate disclosed herein. In some embodiments, the at least one antigen can be a tumor cell lysate and / or derived from or isolated from a tumor cell lysate, such as a tumor cell lysate from a subject to be treated.

[0076] In certain embodiments and further to paragraphs

[0028] -

[0075] , methods for preparing APC-based compositions and / or vaccines of use herein can include incubating APCs in the presence of at least one pharmaceutically acceptable stimulant or adjuvant as defined herein. For example, methods can include incubating APCs with at least one antigen and at least one stimulant or adjuvant. In some embodiments, the at least one stimulant or adjuvant can include KLH and / or tetanus toxoid. In other embodiments, methods can further include combining APCs with at least a second type of cells as defined herein. In certain embodiments, a second type of cells can include, but is not limited to, other APCs, stem cells, tumor cells, inactivated tumor cells, or cancer stem cells. In another embodiment, tumor cells can include tumor cells from a solid tumor (e.g., sarcoma, kidney cancer, brain cancer, liver cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, stomach cancer, esophageal cancer or other solid cancer). In other embodiments, tumor cells can include tumor cells from a sarcoma including, but not limited to, Ewing sarcoma or osteosarcoma or another sarcoma. In accordance with these embodiments, the Ewing sarcoma or osteosarcoma tumor cells can be primary or metastatic Ewing sarcoma tumor cells or primary or metastatic osteosarcoma tumor cells.Methods of treating

[0077] In certain embodiments and further to paragraphs

[0028] -

[0076] above, compositions and methods for preparing and using APC-based formulations can be used to treat cancer in a subject in need thereof. In accordance with these embodiments, APCs prepared by methods disclosed herein can be part of a pharmaceutical composition and further include at least one pharmaceutically acceptable excipient. In certain embodiments, methods for treating or ameliorating cancer in a subject can include administering an APC- containing formulation or vaccine as described.

[0078] In certain embodiments and further to paragraphs

[0028] -

[0077] above, the cancer is a solid cancer. In certain embodiments, the solid cancer is a solid cancer of a pediatric patient. In some embodiments, the pediatric cancer is cancer of the bone. In one embodiment, the cancer is a sarcoma: optionally osteosarcoma or Ewing sarcoma. In one embodiment, the cancer is osteosarcoma, such as an osteosarcoma in a pediatric patient. In accordance withthese embodiments, the osteosarcoma can be a primary7or metastatic osteosarcoma; optionally the osteosarcoma can be a primary or metastatic osteosarcoma of a pediatric patient. For example, the cancer can be a relapsed metastatic osteosarcoma. In one embodiment, the cancer is Ewing sarcoma, such as Ewing sarcoma in a pediatric patient. In accordance with these embodiments, the osteosarcoma can be a primary7or metastatic Ewing sarcoma; optionally, a primary or metastatic Ewing sarcoma of a pediatric patient. For example, the cancer can be a relapsed metastatic Ewing sarcoma. In certain embodiments methods for treating cancer as defined herein can include treating a pediatric cancer patient or an adolescent or young adult.

[0079] In other embodiments and further to paragraphs

[0028] -

[0078] above, cancer can be another solid tumor, a non-solid tumor, an immune-related cancer or the like. In some embodiments, the cancer can include, but is not limited to, melanoma, brain cancer, colon cancer, stomach cancer, esophageal cancer, throat cancer, a kidney cancer (e.g., hepatocellular carcinoma), breast cancer, liver cancer, blood-related cancer, ovarian cancer, prostate cancer, or lung cancer (e.g., non-small cell lung cancer) or other cancer. In certain embodiments, the cancer can be a cancer or tumor that results from one or more chromosomal translocations.

[0080] In some embodiments and further to paragraphs

[0028] -

[0079] above, compositions described herein relate to a cell-based formulations or vaccines as described herein for use in treatment or reducing the risk of onset or recurrence of cancer. In certain embodiments, the technology described herein relates to a cell-based vaccine for use as a medicament, such as a cancer medicament or a vaccine, preferably a cancer vaccine, where a cell-based composition or vaccine includes, but is not limited, a population of APCs of semi-matured dendritic cells that have been at least partially inactivated (e.g.. rendered at least one of non- proliferative, immunostimulated, induced stimulation of cytokine production, etc.) by irradiation; the antigen can be a tumor antigen from a tumor of a cancer patient, for example, a tumor cell lysate; optionally, where inactivation of the cells is by irradiation. In one embodiment, methods can include administering a cell population as defined herein. In other embodiments, tumor antigen conditioned APCs as disclosed herein can be combined with tumor cells which have been inactivated (e.g. rendered at least one of non-proliferative, immunostimulated, induced stimulation of cytokine production, etc); optionally, where the tumor cells originate from the same tumor as the tumor antigen: and administering the combination composition cancer patient. In one embodiment, a dendritic cell population is provided to a subject in need thereof in form of a pharmaceutical composition as defined herein.

[0081] In some embodiments and further to paragraphs

[0027] -

[0069] above, the cellbased composition includes dendritic cells prepared by methods disclosed herein cells are at least partially inactivated by exposing the cells to a physical agent. Exposed cells can be rendered at least one of non-proliferative, immunostimulated, induced stimulation of cytokine production, etc. In some embodiments, chemical treatment, heat treatment or treatment by radiation as defined herein can be used to at least partially inactivate the cells. In some embodiments, inactivation is by irradiation and dosage can be as defined herein. In some embodiments, radiation is at least one of UV-, x-ray or y- radiation or combination thereof. In one embodiment, the physical agent is y- radiation, and exposure is at least about 2000 rad, or at least about 3000 rad. or at least about 4000 rad, or at least about 5000 rad, or at least about 6000 rad. or at least about 7000 rad, or at least about 8000 rad, or at least about 9000 rad, or at least about 10000 rad, or at least about 11000 rad, or at least about 12000 rad. In one embodiment, the physical agent is y-gamma radiation in the range of about 2000 to 20000 rad; or about 3000 to 12000 rad, or about 6000 to 10000 rad, or about 2000 to about 8000 rad, or about 3000 to 6000 rad, respectively, such as about 3000 rad, 4000 rad, 5000 rad, 6000 rad. 7000 rad, 8000 rad, 9000 rad or 10000 rad. In one embodiment, the cells are irradiated with at least 2000 or more or at least 6000 or more rad; for example, at least 6000 rad of y- gamma radiation.

[0082] In certain embodiments and further to paragraphs

[0028] -

[0081] above, an APC population, pharmaceutical composition and / or cell-based vaccine disclosed herein can be administered in addition to at least one conventional cancer therapy. For example, the APC population, pharmaceutical composition and / or cell-based vaccine can be administered in addition to chemo- and / or radiation therapy and / or surgery. In one embodiment, the cell population, pharmaceutical composition and / or cell-based vaccine can be administered as a first line treatment, optionally in addition to other cancer therapies, such as chemo- or standard radiation therapies and / or surgery. In another embodiment, the APC population, pharmaceutical composition and / or cell-based vaccine can be administered as a second-line treatment, optionally in addition to other cancer therapy, such as in addition to chemo- or standard radiation therapies. In some embodiments, these treatments can be co-administered. In one embodiment, the APC population, pharmaceutical composition and / or cell-based vaccine can be administered before, in support to, or after partial or full surgical tumor removal.

[0083] In other embodiments and further to paragraphs

[0028] -

[0082] above, a subject to be treated can have a bone-related sarcoma. In certain embodiments, the subject is a youngchild (pediatric patient), an adolescent or young adult human patient. In other embodiments, the subject can be a subject having had a first relapse of a bone-related sarcoma; optionally where the subject has metastasis of the bone-related sarcoma. In some embodiments, an antigen-exposed irradiated APC population can be administered to the subject at a dose of about 1.0 x 106to about 1.0 x 107about 2 times per day to about 1 time every' other week; or about 1 time per week. In certain embodiments, the subject is undergoing surgery , and the APCs can be administered before and / or after surgery. In some embodiments, the APCs are administered to the subject before and after surgery; optionally, one dose per week for three weeks before and after surgery' to remove tumors in the subject. In certain embodiments, the APCs can be administered subcutaneously, intradermally, by any form of bolus administration, intravenously’, intratumorally, or other mode or a combination thereof. In certain embodiments, the antigen-exposed irradiated APC population is a matured antigen exposed population of dendritic cells. In some embodiments, the dendritic cells are derived from cells harvested from the subject to be treated. In other embodiments, the antigens for exposure to the APCs can include tumor antigens or a tumor cell lysate from the subject to be treated. In other embodiments, the dendritic cells are derived from cells harvested from the subject to be treated and the antigens for exposure of the APCs can include tumor antigens or a tumor cell lysate from the subject to be treated. In certain embodiments, the subject has Ewing sarcoma or osteosarcoma. In accordance with these embodiments, overall survival of the subject can be increased.

[0084] In certain embodiments and further to paragraphs

[0028] -

[0083] above, an APC population, pharmaceutical composition and / or APC-based vaccine disclosed herein can be administered to a subject by any suitable mode of administration, including, but not limited to, intravenously, intradermally, subcutaneously, intratumorally, locally, by bolus administration, intrarenally, or intramuscularly injected or infused or a combination thereof. In one embodiment, the APC population, pharmaceutical composition and / or APC-based vaccine can be administered to a subject by subcutaneous injection, by bolus and / or by infusion. In certain embodiments, the APC population is administered subcutaneously, for example, by injection or infusion.

[0085] In certain embodiments and further to paragraphs

[0028] -

[0084] above, an APC population, pharmaceutical composition and / or APC-based vaccine disclosed herein are suitable to generate a long-term immunity in a subject disclosed herein. In some embodiments, methods disclosed herein can generate long-term immunity against a cancer in a subject. In accordance with these embodiments, methods can include administering APCpopulations, pharmaceutical compositions thereof or APC-based vaccines as defined herein to a subject and generate long-term immunity against a specific cancer and / or generally against related cancers. In certain embodiments, the APCs are dendritic cells. In some embodiments, prolonged immunity can occur from a single bolus treatment of APCs disclosed herein or from two or more bolus or other treatments using APCs disclosed in the instant application.

[0086] In certain embodiments and further to paragraph

[0085] above, as used herein, the phrase ‘"long-term immunity7’ can refer to a prolonged immune response that a subject attains against a cancer type or multiple cancer types which reduces or prevents onset or recurrence of the cancer or cancers Long term immunity7in a patient can last for up to about 1 year, up to about 2 years, up to about 3 years, up to about 4 years, up to about 5 years or more, or up to about 10 years or more. In some embodiments, the subject is a subject such as a pediatric subject having at least one of osteosarcoma and Ewing sarcoma and when treated using compositions and methods disclosed herein attains prolonged immunity' against the osteosarcoma and / or Ewing sarcoma. In certain embodiments, the subject can be treated using a single or multiple administrations of APC populations of cells disclosed herein (e.g., 1.0 x 106to about 1.0 x 1010cells / m2body surface per dose administration). Intradermal or subcutaneous treatment can be accompanied by about 10 to about 200 or about 20 to about 100 or about 50 pg / m2body surface IFN-y along with APC cells or supplemented before or after the APCs are delivered to a subject. In one embodiment. APC populations of cells disclosed herein and IFN-y can be delivered to a subject 1 time or up to 14 times: or about 2 times to about 6 times, or about 6 times, or weekly doses, bi-weekly or monthly doses or bimonthly doses or another appropriate regimen.

[0087] Other embodiments and further to paragraphs

[0028] -

[0086] above, relate to kits for storing, transporting, preparing and or treating subjects disclosed herein. In certain embodiments, kits can include at least one APC population. In other embodiments, kits can include at least one APC population exposed to at least one antigen and then at least partially inactivated. In some embodiments, kits can include one or more suitable containers, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe, other delivery device, or other container.EXAMPLES

[0088] The materials, methods, and embodiments described herein are further defined in the following Examples. Certain embodiments are defined in the Examples herein. These Examples, while indicating certain embodiments, are given by way of illustration only. Fromthe disclosure herein and these Examples, one of skill in the art can ascertain the essential characteristics of these embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt it to various usages and conditions.Example 1

[0089] In one exemplary' method, dendritic cells were obtained from by various processes under varying conditions. In this example, dendritic cells of use in creating conditioned cell populations disclosed herein were obtained to assess superior methods for obtaining a desired dendritic cell population (see FIG. 1 ).

[0090] FIG. 1 represents exemplary' graphs of dendritic cell yield. In this example, four methods for monocyte enrichment using 3 different types of media were used as indicated, adherence (no fill), selection (light gray), depletion (dark gray) and elutriation (black) medias. No apparent difference was observed between the different medias, so any media known to work in these conditions is contemplated. The lower right graph illustrates averaged data from all experiments independent of the medium used. It w as observed that depletion methods and elutriation methods were superior to adherence and selection methods. (See below for additional information regarding these methods for comparison)

[0091] For dendritic cell generation one example regarding adherence, monocytes were enriched by plastic adherence from autologous leukocyte apheresis products containing monocytes and cultivated for about 5 days in AIM-V medium (Invitrogen Corporation, Bethesda, MD, USA) supplemented with 2% pooled human AB plasma (Octaplas, Octapharm, Vienna, Austria), 400 U / mL IL-4 (Pan Biotech GmbH, Aidenbach, Germany) and 1000 U / mL GM-CSF (Roche, Basel, Switzerland). On day 5 the cells were recovered, washed with PBS and resuspended in AIM-V without plasma but supplemented with 400 U / mL IL-4 and 1000 U / mL GM-CSF and exposed to 1-10 mg / mL tumor cell-derived soluble Ag and 1 mg / mL KLH for 2 h at 37° C. Subsequently, the DC culture was supplemented to a final concentration of 200 U / mL LPS (US Pharmacopeia, Rockville, MD, USA), 50 ng / mL IFN-y (Boehringer Ingelheim, Vienna, Austria) and human plasma to 2%. The cells were incubated for 48 h (fully mature) or 6 h (semi-mature) at 37° C. After the maturation step, the DC were washed in PBS and kept on ice until application to the patients. For some patients, tumor cell extract-loaded smDCl w ere frozen in appropriate aliquots until application. These smDCl were recovered from liquid nitrogen at the day of treatment, washed, and kept on ice until injection. The absence of viral andmicrobial contamination was examined in specialized clinical diagnosis laboratories. Bacterial contamination was excluded by inoculating culture supernatants into a Bactec culture system (Becton Dickinson, Sparks, MD, USA); samples were analyzed for CMV and EBV by quantitative real-time PCR of culture supernatant, and mycoplasma contamination was excluded by PCR and a biologic assay (see for example Dohnal et al., Cytotherapy (2007) vol. 9, No. 8 pg. 755-770).Example 2

[0092] Tn another exemplary method, T-cell receptor sequences were studied to assess treatment efficacy in a patient and comparison of polynucleotide sequences were assessed over time for homologies. This data indicates there are shared sequences where certain sequences were more common than others. In this example. 2 polynucleotides were found to have commonalities in the various patient samples, A-E. T-cell receptor clonotyping. In this example, it was found that vaccination with a vaccine as described herein leads to a long-term immunity as evidenced by evidence of specific T-cell receptor (TCR) clonoty ping. TCR clonotyping was performed from whom tumor and peripheral blood samples were available at different time points: 1st relapse-tumor (A). 2nd relapse-tumor (B). 1st relapse-blood (C), 2nd relapse-blood (D), blood- 10 years after vaccination using a composition disclosed herein containing irradiated APCs (dendritic cells exposed to antigen(s)) (E).

[0093] TCR clonotyping consists of Next Generation T cell receptor sequencing of all T cell repertoire present in biological specimens. In this analysis, it was observed that certain T cell clones were expanded in tumors compared with peripheral blood, indicating accumulation of specific T cells potentially targeting tumor antigens. Moreover, some of these T cell clones were present in all samples analyzed, including a sample from peripheral blood collected 10- 11 years after Dendritic cell vaccination. This designates not only an immunological response against tumors but also a memory component characteristic of a successful vaccination and decisive evidence of permanent cancer-fighting T cells in immunized patients (See FIGS 2A and 2B).

[0094] FIGS. 2A and 2B illustrate exemplary results of a T-cell receptor clonotyping of patient samples. FIG. 2A illustrates a most shared unique nucleotide sequence found in the samples tested. FIG. 2B illustrates a pairwise comparison of the identified clones of the samples. The samples were of the same patient taken at different time points: (A) sample of tumor of 2nd / 3rd relapse, (B) sample of Tumor of 4th relapse, (C) blood sample taken after 2nd relapse, (D) blood sample taken after 4th relapse, (E) blood sample taken 11 years after the 1st vaccination, corresponding to 10 years after the 2nd treatment.

[0095] Clonotyping was performed as follows: Using the AllPrep DNA / RNA / Protein KIT (Qiagen) the genomic DNA was isolated from peripheral blood and single cell suspension of the tumor. For further analysis, the samples were sent to Adaptive Biotechnology Corp (Seattle, WA) where sequencing was performed by using immunoSEQ sen ice. The survey level was set to 200000 sequences for our experiments.Example 3

[0096] In another exemplary method, clinical studies were performed to test APC compositions in the treatment of cancer patients. In this example, several patients were administered compositions and effects of a dendritic cell-based composition was assessed in cancer treatment. Irradiation was performed as described herein. Protocols as described below were carried out for each patient separately and each patient received a vaccine based on his / her / their own dendritic cells and tumor extracts. One example production for a patient included the following:Isolation and preparation of single cells from solid tumor tissue

[0097] Tumor tissue was obtained by biopsy from a patient’s tumor. Freshly resected tumor tissue was placed in a physiological buffer (e.g., Phosphate Buffered Saline (PBS)) in a plate for preservation. In this example, necrotic areas and connective tissue were removed while keeping the specimen immersed in PBS. Remaining tissue was minced into fragments (e.g., about 0.5 mm) on each side. Then, the pieces of tissue were pressed through a sterile nylon mesh, resulting in a single cell suspension of tumor cells. Tumor cells were irradiated (e.g., 12000 rads of irradiation). Then a screening procedure for identifying cytogenetically abnormal cells w as done by fluorescence in situ hybridization (FISH).Tumor cell lysis

[0098] A single cell suspension of fresh tumor cells was created and used. Tumor cells were collected from the cell suspension by centrifugation (e.g., 1600 revolutions per minute (rpm) for 7 minutes) at refrigeration temperature of about 4°C. Then a fraction of the cell pellet w as recovered in distilled water and remaining cells w ere frozen using a standard freezing medium. The cell suspension obtained in this process was quick frozen in liquid nitrogen until completely frozen. After freezing, the cell suspension was submerged in a w ater bath of about 37° C until thaw ed. This freeze-thaw procedure was repeated (e g., five times in this example) to produce a homogenized cell extract. The freeze-thaw ed cell material was centrifuged (e.g., at 1600 rpm for 7 minutes at 4°C) to remove particulate components. Then, a supernatant containing water-soluble cellular components including tumor antigens was collected from the centrifuged cell material as a tumor cell lysate. In this example, 1 / 10volume of a 1 Ox PBS solution was added to the tumor cell lysate to bring the tumor cell lysate, to a physiologic salt concentration, and this tumor cell lysate was frozen in aliquots at about -80° C and stored for further use. Protein concentration of the tumor cell lysate was determined after the lysis procedure by a standard analytical procedure.Collection, enrichment and cultivation of monocytes

[0099] In another method, mononuclear cells were isolated using standard techniques, for example, from leukocyte apheresis products or heparinized blood (e.g.. by Ficoll- Hypaque; Pharmacia Biotech, Uppsala, Sweden) followed by density gradient centrifugation, washed, and incubated for about 2 hours in plastic flasks containing a standard media, AIM-V medium (ThermoFisher, Waltham. MA) supplemented with 1% human pooled AB plasma (e.g., Octaplas. Octapharm, Vienna, Austria). Non-adherent cells were removed by repeated rinsing of the flasks and adhered cells were used for further study. These adhered cells were further cultivated in AIM-V medium with 2% pooled human AB plasma (Octaplas, OP) at 37° C in a humidified incubator. In this example, culture medium was supplemented with 1000 U / ml recombinant human GM-CSF (e.g., AESCA, Traiskirchen, Austria) and 400 U / ml recombinant human IL-4 (e.g.. Strathmann, Hamburg. Germany). Adherent cells were further cultivated for about 5 days introducing the same volume of AIM- V / 2%OP GM-CSF and IL- 4 on day 3.Loading and maturation of DC

[0100] On day 5 of incubation, the adhered cell population was washed with PBS and new culture medium (AIM- V) without OP (human plasma) was added. Then, the cell culture as supplemented with 10 ug / ml tumor lysate as isolated above, and for example 1 pg / ml Keyhole limpet hemocyanin (KLH) (Calbiochem, San Diego, SA) and 1 pg / ml Tetanus Toxoid (TT) (Calbiochem, San Diego. SA) and incubated for about 2 hours at 37° C. This phase is referred to herein as antigen-exposed APCs. In this example, dendritic cells are incubated with patient specific tumor antigens.

[0101] After tumor antigen incubation of the dendritic cells, maturation stimulus w as provided by adding for example, 50 ng / ml IFN- y (e.g., Boehringer Ingelheim, Vienna, Austria), and 200 U / ml LPS (e.g., Promochem, GmbH, Wesel, Germany). In another method, further dendritic cell maturation was performed by incubating cells for about 6 hours with about 400 U / ml IL-4, 1000 U / ml GM-CSF and 2% OP The antigen-exposed dendritic cell culture in this example w as then incubated for about 2- to about 48 hours to obtain semi- or mature antigen-exposed or antigen-conditioned DCs (smDCs / mDCs). After incubation time, the semi-or mature dendritic cells were harvested and washed twice with PBS. Then, thepatient's tumor cells were thawed, washed and combined with these DCs at a ratio of about 1: 1, other ratios are contemplated such as 2: 1 , 1 :2. 3 : 1 , 1 : 3 or other appropriate ratio. Dendritic cells or APCs were irradiated with about 6000 rad and when not immediately administered as a non-frozen DC / tumor cell vaccine, these compositions were frozen (e.g., in liquid nitrogen) and stored for later use. For negative control patients in this study, the DCs were not exposed to irradiation and no patient-specific tumor cells were added to a negative control vaccine. (See Table 1, A: OS (overall survival) from day of enrollment in the study (days); B: OS day of enrollment in the study (years), C: OS from day of initial surgery for the health condition (days) D: OS from initial surgery for the health condition (years), E: OS from diagnosis (days) F: OS from diagnosis (years). Further, see Table 2, (A: number of relapses before vaccination; B: Status at vaccination (CR: Clinical Remission) C: Recurrent disease at time of vaccination with dendritic cell combination (Y es or No); D: Metastatic disease at time of vaccination (Yes or No) E: time of vaccination (day -month, year) and F: EFS (event -free survival) after 1st DC vaccination (days)). Inclusion time as presented herein indicated time of enrollment in the study.Overview of Treatment of Patients

[0102] After the final preparation and quality control, the cells obtained by the method as described above were given subcutaneously in this example to the patients.Overview of Cell Preparation

[0103] Collection of tumor cells after a surgical event: Each tumor was processed, lethally irradiated (12000 rad), and their proteins were extracted, lysed and prepared and set aside for later use. Collection of white blood cells using leucocyte apheresis of blood collection in heparinized blood tubes from the peripheral blood of the cancer patient. Monocyte enrichment: Monocytes (CD14+ cells) from white blood cells were enriched ex vivo by adherence. Monocytes were differentiated in vitro into dendritic cells (DCs) with cytokines (e.g., IL-4 and GM- CSF) for about 5 days. Around Day 5: DCs were first loaded or exposed to patient tumor antigens, KLH, and tetanus toxoid for about 2 hours, and matured with LPS and IFNy for 2-48 hr. The set aside tumor cells, were mixed with DC prepped above and irradiated with about 6000 rad (which can be less than a minute up to several hours, or in minutes to hours, depending on the type of radiation) and frozen in liquid nitrogen. Treatment of the patient: Cancer vaccine was given subcutaneously into patients (about 1 to about 5 million DCs / application).Overview of treatment

[0104] The inventors surprisingly found that four patients treated with dendritic cell vaccines disclosed above had an unexpected improvement in their survival. These four patients were children suffering from relapsed, metastatic bone, or Ewing sarcoma that had exhausted all conventional therapeutic options and all were in complete macroscopic remission at the time of DC-CIT. The long-term follow-up observation period (e.g., greater than 13 years) demonstrates that these end-stage patients survived unexpectedly long compared to published data in metastatic sarcoma. According to literature, the 5-year overall survival is normally low in this patient group. Here, four patients surv ived for prolonged periods with the disclosed vaccine, three are still alive, the fourth patient died about 20 years after diagnosis from an unrelated condition.Example 4Monocyte enrichmentMonocyte enrichment by plastic adherence

[0105] In another exemplary method, mononuclear cells were isolated from leukocy te apheresis or heparinized blood products by for example, Ficoll- Hypaque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation, washed, and incubated for 2 hours in plastic flasks containing AIM-V medium (ThermoFisher, Waltham, MA) supplemented with 1% human pooled AB plasma (Octaplas, Octapharm, Vienna, Austria) or in CellGro medium (CellGenix, Freiburg, Germany) without plasma supplement. Non-adherent cells were removed by repeated rinsing of the plate. The enriched monocytes were differentiated into DCs as described below.Enrichment of monocytes by elutriation

[0106] For the enrichment of monocy tes from PBMCs in the leukocyte apheresis product or pooled heparinized blood the Elutra cell separator (Gambro BCT, Inc. Lakewood, Colorado, U.S.A.) was used. Cells were separated based on sedimentation velocity, which is dependent on cell-size and to a lesser extent on density. After priming the single use plastic set, the leukocyte apheresis product or the pooled heparinized blood was loaded into the elutriation chamber while maintaining the centrifuge speed at 2400 rpm.

[0107] Thereafter, the centrifuge speed was held constant. The flow of elutriation media (PBS, Miltenyi Biotec, Bergisch Gladbach, Germany, HSA 5% Baxter AG, Vienna, Austria) was contained in two 3 1 pooling bags. For this elutriation procedure, the pre-defined machine settings followed instructions of the manufacturer. Cellular components of all collected fractions were counted using the Trucount system for example (Becton Dickinson, New Jersey, NJ). For flow cytometry, cells were labelled with monoclonal antibodies directedagainst CD3, CD56, CD19, CD14, and CD15 to detect total T-lymphocytes, NK- Cells, B- lymphocytes. monocytes, and granulocytes, respectively; CD45 was used for determining total leukocyte numbers. All antibodies were purchased from BD Pharmingen (San Diego, CA) or DAKO (Glostrup, Denmark) and were analysed on a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA). Data analysis was performed using Paint a Gatesoftware (Becton Dickinson, Mountain View, CA). The appropriate negative controls were included in the analysis. The enriched monocytes were differentiated into DCs as described below.Monocyte enrichment by selection using CD 14 antibody beads

[0108] Positive selection of monocy tes was done using the CliniMACS cell selection system (Miltenyi, Bergisch Gladbach, Germany) according to the protocol provided by the manufacturer. Briefly, leukocyte apheresis products or heparinized blood were subjected to a washing step to remove platelets. The remaining leukocytes were incubated with CD14 antibody (Miltenyi, Bergisch Gladbach, Germany) specific for monocytes and the antibody- coated cells were rosetted with magnetic beads. The sensitized cells were connected to the CliniMACS system in which an automated procedure guided all subsequent steps. These steps include the retention in a magnetic column, removal of unbound cells by repeated washing, and the release of enriched monocytes from the magnetic column. The cells were characterized by flow cytometry and cultured as described below.Monocyte enrichment by depletion of T and B lymphocytes using CD2 and CD 19 antibody beads

[0109] In another method, leukocyte apheresis products or heparinized blood were automatically processed for T- and B- lymphocyte depletion with anti-CD2 and anti-CD19 antibodies (both from Nexell, Irvine. CA) using the Isolex 300i Magnetic Cell Selector (Nexell, Irvine, CA) according to the manufacturer’s instructions with some minor experimental modifications. Briefly, platelets were removed by washing and cells were incubated with anti-CD2 and anti-CD19 antibodies. Sensitized T- and B-lymphocytes were rosetted or captured with immuno-magnetic beads (e.g., Dynabeadslz, Nexell, Irvine. CA) inside the magnetic chamber. A negative fraction was drained and collected from the chamber by passage over a secondary magnet into the product bag. The final product, enriched for monocytes, was characterized by flow cytometry and used for further DC generation as described below.DC preparation

[0110] In another method, monocytes isolated by the respective enrichment procedures described above were cultured at a density of 1 x 106 monocytes / cm2 either in AIM-V medium (ThermoFisher, Waltham, MA) with 2% pooled human AB plasma (Octaplas, OP, Octapharm, Vienna, Austria) or in CellGro medium (CellGenix, Freiburg, Germany) at 37° C in a humidified incubator. The culture medium was supplemented with 1000 U / ml recombinant human GM-CSF and 317 U / ml recombinant human IL-4 (both from CellGenix, Freiburg. Germany) for 6 days, with feeding the same volume of AIM-V / 2% OP or CellGro plus GM-CSF and IL-4 on day 3. For loading on day 6 of the cells lOpg / ml tumor lysate, Ipg / ml Keyhole limpet hemocyanine (KLH) (Calbiochem, San Diego, SA) and Ipg / ml Tetanus Toxoid (TT) (Calbiochem, San Diego, SA) was incubated for 2 hours at 37° C. Maturation was carried by adding 50 ng / ml IFN- y (Boehringer Ingelheim, Vienna. Austria) and 200 U / ml LPS (E. coli strain 0111 :B4, Calbiochem, San Diego, CA, USA) to the culture for 6 hours, to obtain semi-mature DCs (smDCs). GM-CSF, IL-4, IFN-y, and LPS biological activity can vary between batches of the compounds. Therefore, these materials were tested before their routine use in DC preparation in a test run to determine an optimal concentration. Recovery of DCs from monocytes in the leukocyte apheresis product[OHl] In another method, monocytes were obtained after enrichment of semi-mature DCs (smDC) using IL-4 and GM-CSF differentiation media as well as mature DCs (mDCs) about two days after exposure to an LPS / IFN-y maturation stimulus to assess effects / outcome of a vaccine inoculated into a patient. To directly determine absolute cell numbers, the Trucount system was used. Two different media, AIM V / Octaplas, or CellGro, for the differentiation of monocytes into DC were used. Statistical analysis revealed that both media were equally potent for the differentiation of monocytes into smDCs and mDCs. The percentages given in the text are mean ± SEM from experiments with AIM V / Octaplas or CellGro as well as one graph that shows averages of all experiments independently of the culture medium used (See FIG.l). Tables 4 and 5 provide supplementary data from a separate analysis of DCs cultivated in the two respective media.

[0112] Table 4 illustrates DC yield. Mean ± SEM values (percentage) for experiments in which AIM / Octaplas or CellGro was used as DC culture medium. Because differences between these two culture conditions are not significantly different, values given in the text below are overall mean ± SEM of AIM V / Octaplas and CellGro experiments using medias disclosed herein.

[0113] Table 5 represents an overview table of DC yield. Significance. The p-values (percentage) for experiments in which AIM / Octaplas or CellGro was used as DC culture media.Monocyte enrichment and culture media

[0114] Recovery' of monocytes by the respective enrichment procedures, adhesion (number of independent experiments, n=15), selection (n=15), depletion (n=15), or elutriation (n=17). was analyzed by assessing monocyte number in leukocyte apheresis product and after enrichment. This analysis could not be performed for monocyte enrichment by adhesion. There is currently no adequate method for counting monocytes sticking to a culture plate without disrupting the culture and thereby introducing artificial effects. Among the other three enrichment procedures elutriation was most efficient resulting in a recovery of about 87 ± 7%. Selection and depletion yielded a recovery of 41 ± 3% and 59 ± 4%, respectively (elutriation versus selection, p<0,01; elutriation versus depletion, p<0,01; selection versus depletion, p<0,05). Then, number of smDCs after differentiation from monocytes was assessed and the yield calculated as a percentage of monocytes in the leukocyte apheresis product. This is an important figure because smDCs are frozen in liquid nitrogen at this stage and applied to the patients after recovery from a freezing process. Using adherence as the enrichment procedure 8 ± 1% smDCs were recovered. Here, the most effective procedure was elutriation (36 ± 4%, elutriation versus adhesion, p<0,01). Selection and depletion yielded 7 ± 1% (selection versus elutriation, p<0,01; selection and adhesion are not different) and 22 ± 4% (depletion versus elutriation, p<0,01; depletion versus adhesion, p<0,05), respectively (depletion versus selection, p<0,05).

[0115] Then, mDC yield was determined as a percentage of monocytes in the leukocyte apheresis product. Complete differentiation of smDC into mDC takes place after in vivo inoculation of the cancer vaccine. To model this final differentiation step, quality control procedures were performed on an aliquot of the cancer vaccine that was thawed and cultivated for 2 days to allow' for measurement of IL-12 secretion, immune phenotyping, and an alloMLR as potency assay. Number of mDCs was determined after these two days of culture and does not necessarily reflect the number of DCs that would reach this time point in vivo. However, it is an important measure as it reflects cell loss due to the freezing procedure as well as cell loss due to the terminal differentiation of smDC into mDC. Using adhesion for monocyte enrichment 6 ± 2% mDCs were recovered, selection yielded 4 ± 1% mDCs (no significant difference), with depletion 15 ± 3% mDCs (adhesion versus depletion, p<0.05; selection versus depletion, p<0,05), and using elutriation 16 ± 2% mDCs (adhesion versuselutriation, p<0,01; selection versus elutriation, p<0,01; depletion versus elutriation, no significant difference was observed). Thus, either elutriation or depletion or a combination preparation can be used to provide superior recovery and is suggested to be used for monocyte enrichment in processes disclosed herein.Example 5Combination of irradiated dendritic cell vaccine with other cancer therapies

[0116] In another exemplary method, combination therapies of antigen-exposed irradiated dendritic cell (DC) compositions can be used in conjunction with other cancer therapies. For example, immunotherapies such as checkpoint inhibitors (e.g., Anti-CTLA4, Anti-PDl) and / or standard cancer chemotherapy can be combined with use of these DC compositions. This multi-modal treatment approach can enhance anti-tumor efficacy and improve patient outcomes. The DC composition can be co-administered in combination with checkpoint inhibitors, such as anti-CTLA4 and anti-PDl antibodies or separately administered such as sequentially or at the same time or one after the other. These checkpoint inhibitors are known to block inhibitory signals that affect or can even prevent T cells from attacking cancer cells, thereby enhancing the immune response elicited by the DC composition.

[0117] In another exemplary method, standard chemotherapy regimens commonly used in cancer treatment can be integrated into these antigen-exposed irradiated APC composition therapied. For example, patients can receive a combination of chemotherapeutic agents with these APC compositions. Common chemotherapeutic agents include, but are not limited to, cisplatin, doxorubicin, cyclophosphamide, vincristine, ifosfamide, etoposide, paclitaxel and methotrexate, which are known for their efficacy in various cancers. These agents are known to kill cancer cells and can be used for priming a patient for T cells activated by the APC composition to kill remaining cancer cells that survive chemotherapy.

[0118] Other combination therapies can include administering APC compositions disclosed herein, before, during or after surgical intervention and / or radiation therapy which reduces overall cancer burden in the patient, making it easier for activated T cells to kill remaining cancer cells. In one method, these combination therapies can be used to treat a patient having metastatic tumor cells present or undetected metastatic tumor cells present in the patient.

[0119] These examples demonstrate effective use of an additive or synergistic approach to cancer treatment by combining an irradiated dendritic cell vaccine with checkpoint inhibitors and / or standard chemotherapy.Example 6

[0120] In another exemplary study, patients were treated with compositions disclosed herein. As referenced, DEN2, is Dendritic Cell (DC)-Cancer Immunotherapy (CIT) (DC-CIT) of matured and irradiated DCs able to induce a potent, long-lasting anti-tumor immune stimulation and avoid DC tolerance. In this study, patients survived long after receiving DEN2 compared with other historical data from referenced literature. DEN2 considers natural kinetics of immune responses avoiding the final step of immune tolerance induction by y- irradiation. Further, DEN2 demonstrated a trend towards inflammatory and cytotoxic response in in vitro experiments compared to standard DC methods. DEN2 provides immunological memory7responsible to avoid tumor recurrence and keep patients cancer-free for long time after vaccination. FIG. 9 illustrates as standard cancer treatment where longer- term survival dramatically drops in a year to a few years (this is from Bielack et al 2009) compared to treatments disclosed herein. FIG. 10 illustrates long-term survival of subjects treated with compositions disclosed herein (e.g, DEN2 patients lived an average of 18 years from diagnosis and about 13 years after DC-CIT treatment).

[0121] FIG. 11 is a schematic representation of an in vitro experiment designed to evaluate the effect of y-radiation on DC phenotype in the presence and absence of other immune cell subsets. A gene expression profile of DEN2 (6,000 Rads irradiation) versus DENI (standard DC without irradiation) demonstrated differences in key interleukins and other mediators of inflammation and cytotoxic immune responses like IL-8 (one of the major mediators of the inflammatory response) and Granulysin (e.g., protein present in cytotoxic granules of CTLs and NK cells and released upon antigen stimulation). There were other genes found differentially expressed between DENI and DEN2, which play important roles in anti -tumor immune responses such as IFNp: released upon phenotypic and functional activation of DCs IL36G: a sign of local inflammatory response CCL19 and chemokine that induces pro- inflammatory7differentiation (data not shown but available upon request). FIG. 12 illustrates expression of a few7select genes demonstrating induced inflammatory cytokine production. Overall differences of gene expression profiles between DEN2 and DENI indicate a trend tow ards an inflammatory response following y-irradiation. DEN2 was found to be a superior DC-based therapeutic vaccine w ith a novel irradiation step of dendritic cells lead to at least the following advantages over other unirradiated DC-CITs: potent, effective, durable antitumor immune response at the same time as preventing subsequent immune regulation. DEN2 significantly extended survival and improves quality7of life of refractory bone cancer patients in this study. DEN2 offers higher response rates and management for refractory7cancer andprovides personalized, precise, long-term anti-tumor immune responses while overcoming limitations of current cancer therapies. DEN2 is a new class of active, safe therapy for cancer, specifically targeting aberrant cell growth and in particular cancer cells only, having fewer side effects for patients. Further, due at least in part to memory of immune cells, patients can have continuous protection even upon completion of treatment, displaying long-term immunity.Example 7

[0122] Tn another exemplary method, young adults or adolescent patients are selected having been diagnosed with a bone cancer (bone sarcoma) and in a relapse phase. Six doses of about 1.0-5.0 x 106tumor cell lysate (patient derived or other) pulsed dendritic cells (patient derived or other such as an established cell line matched to the patient) that have been irradiated with at least 2000 rad (e.g., 6000 rad) are given at weekly intervals before and after surgery. Then the patients are followed for survival and long-term immunity to the bone cancer. Interventive cell treatments will be introduced as needed alone or in combination with other standard cancer treatments. In addition, irradiated tumor cells obtained from the subject where the irradiated tumor cells are apoptotic after irradiation (e.g.. 12000 rads) can be mixed with one or more of the cell populations to be administered to the patient per evaluation of a health professional.All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of embodiments, it is apparent to those of skill in the art that variations maybe applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a population of antigen-presenting cells (APCs), wherein the APCs are irradiated antigen-primed APCs.

2. The composition according to claim 1. wherein the APCs are dendritic cells.

3. The composition according to claim 1 or 2, wherein the APCs are autologous or allogeneic to a subj ect to be treated.

4. The composition according to claim 1 or 2, wherein the APCs are semi- or fully matured.

5. The composition according to claim 2. wherein the composition further comprises at least one of B-cells and macrophages; optionally wherein the at least one of B-cells and macrophages are autologous to a subject to be treated.

6. The composition according to any one of claims 1-5, wherein the APCs are dendritic cells originating from in vitro differentiation of peripheral monocyte cells or stem cells differentiated into dendritic cells; optionally, wherein when peripheral monocyte cells, the peripheral monocytes are CD 14+ monocytes.

7. The composition according to any one of claims 1-6, wherein the irradiation is y- radiation or x-ray radiation.

8. The composition according to any one of clams 1-7, wherein the antigen of antigen primed APCs comprises multiple antigens and the multiple antigens comprises immunogenic antigens or cancer-related antigens.

9. The composition according to claim 1 , wherein the antigen of the antigen primed APCs comprise cancer antigens or antigens from a tumor cell lysate originating from a subject to be treated.

10. The composition according to any one of clams 1-7, wherein antigens of antigen primed APCs comprises cancer antigens originating from a solid or non-solid tumor.

11. The composition according to claim 1, wherein antigens of antigen primed APCs comprises cancer antigens from a sarcoma; optionally, wherein the sarcoma comprises Ewing sarcoma or osteosarcoma.

12. The composition according to any one of claims 1-11, further comprising irradiated tumor cells; optionally wherein the irradiated tumor cells and APCs originate from different subjects.

13. The composition according to claim 1, further comprising irradiated tumor cells; optionally, wherein the irradiated tumor cells and APCs originate from a subject to be treated.

14. The composition according to claim 12 or 13, wherein the irradiated tumor cells comprise irradiated tumor cells originating from Ewing sarcoma or osteosarcoma.

15. The composition according to claims 12-14, wherein the irradiated tumor cells are apoptotic or dying.

16. A composition for use in treating a subject comprising a population of irradiated APCs, wherein the APCs w ere obtained by a method comprising; incubating APCs in vitro with at least one antigen; irradiating the APCs with about 2000 to about 20000 rad of irradiation; and further including at least one pharmaceutically acceptable excipient.

17. The composition for use according to claim 16, wherein the irradiation is / -radiation or x-ray radiation; optionally, wherein the APCs are irradiated for about 10 seconds to about 5 minutes.

18. The composition for use according to claim 17, wherein the APCs are autologous or allogeneic to the subject.

19. The composition for use according to claim 18, wherein the APCs are at least semi-matured following incubation in vitro with at least one of liposaccharides and interferon-y prior to irradiation.

20. The composition for use according to any one of claims 16-19, wherein the APCs comprise dendritic cells.

21. The composition for use according to any one of claims 16-20, wherein the APCs comprise APCs incubated in vitro with at least one adjuvant prior to irradiation; optionally, wherein the adjuvant comprises at least one of keyhole limpet hemocyanin (KLH) and tetanus toxoid.

22. The composition for use according to any one of claims 16-21, wherein the composition further comprises irradiated tumor cells; optionally, wherein the tumor cells originate from a tumor of the subj ect to be treated.

23. The composition for use according to any one of claims 16-23, wherein the subject comprises a subject having bone cancer; optionally, wherein the bone cancer has relapsed in the subject.

24. A method for preparing an APC population comprising: incubating the APCs in vitro with at least one antigen; and irradiating the APCs with about 2000 to about 20000 rad of irradiation.

24. The method according to claim 23, wherein said irradiation is y-radiation or x-ray radiation.

25. The method according to claim 23 or 24, wherein the APC comprise autologous or allogeneic to a subject to be treated; optionally, wherein the APCs are dendritic cells.

26. The method according to any one of claims 23-25, wherein prior to incubating the APCs in vitro with at least one antigen; enriching monocytes collected from a population of white blood cells; and incubating in vitro the enriched monocytes with cytokines to differentiate the enriched monocytes into APCs; optionally wherein the APCs comprise dendritic cells.

27. The method according to claim 26, wherein the enriched monocytes comprise enriched CD 14+ monocytes.

28. The method according to any one of claims 23-27, further comprising when incubating the APCs in vitro with at least one antigen further, incubating the APCs with at least one adjuvant; optionally wherein the at least one adjuvant comprises at least one of KLH and tetanus toxoid.

29. The method according to any one of claims 23-28, wherein the method further comprises maturing the APCs with at least one of a liposaccharide and interferon-y prior to irradiating the APCs to obtain at least some semi-matured APCs; optionally wherein the APCs comprise semi-matured dendritic cells.

30. The method according to any one of clams 23-29, wherein the at least one antigen is part of a cell lysate; optionally, wherein the cell lysate is a tumor cell lysate.

31. The method according to claim 23, wherein the at least one antigen is part of a tumor cell lysate from the subject to be treated.

32. The method according to any one of claims 23-31 , wherein the tumor cell lysate is from a solid tumor cell lysate or a non-solid tumor cell lysate.

33. The method according to claim 32, wherein the tumor of the tumor cell lysate comprises a sarcoma.

34. The method according to claim 23, wherein the at least one antigen is part of a tumor cell lysate, and the tumor cell lysate is from at least one tumor of Ewing sarcoma or osteosarcoma.

35. The method according to any one of claims 23-34, further comprising combining the irradiated APCs with irradiated tumor cells; optionally wherein the at least one antigen and the tumor cells of the irradiated tumor cells are from a subject to be treated.

36. The method according to claim 35. wherein said tumor cells have been irradiated with at least about 6,000 to at least about 20,000 rad; or about 12000 rad; or sufficient radiation able to render the tumor cells apoptotic.

37. The method according to claim 23, wherein irradiating the APCs comprises irradiating the APCs with at least 2000 rad to about 6000 rad of y-radiation or x-ray radiation for about 10 seconds to about 20 minutes or about 10 seconds to about 10 minutes; or about 5 minutes or less.

38. The method according any one of claim 23-37, wherein the APCs are autologous or allogeneic to a subject to be treated with the irradiated APCs.

39. The method according to any one of claims 23-38, further comprising obtaining the APCs for incubating in vitro with the at least one antigen by obtaining a population of white blood cells and enriching for monocytes from the population of white blood cells, optionally wherein the enriched monocytes are CD 14+ monocytes; and incubating the enriched monocytes with cytokines to differentiate the monocytes into APCs.

40. A method for treating an aberrant cell growth condition in a subject, the method comprising administering the composition according to any one of claims 1 to 15 to the subject and treating the aberrant cell condition in the subject.

41. The method according to claim 40, wherein the aberrant cell growth condition in the subject comprises cancer.

42. The method according to claim 41, where in the cancer comprises a solid tumor or nonsolid tumor.

43. The method according to claim 40 or 41, wherein the cancer comprises a solid tumor and the solid tumor comprises a sarcoma; optionally, wherein the sarcoma is an Ewing sarcoma or osteosarcoma.

44. The method according to any one of claims 40-43, wherein administering the composition comprises administering the composition to the subject by any suitable method toadminister a cell population; optionally, wherein administering comprises subcutaneously, intradermally, intranodally. intratumorally or by bolus administration to the subject.

45. The method according to claim 44, wherein administering the composition to the subject comprises administering the composition intrarenally, subcutaneously, intratumorally, intravenously, by bolus administration; optionally, subcutaneously administering the composition to the subject.

46. The method according to any one of claims 40-44, wherein administering the composition to the subject comprises administering about 1.0 x 105to about 1.0 x 109; or about 1.0 x 106to about 1.0 x 108irradiated APC-primed cells to the subject per dose; optionally, wherein a dose comprises twice daily, once per day, 6 times per week or less, 3 times per week, weekly, bi-monthly or monthly treatment dose to the subject.

47. A composition comprising a population of irradiated APCs, wherein the APCs were incubated with a tumor antigen prior to irradiation with at least 2000 or at least 6000 rad of irradiation; optionally wherein the irradiation comprises y-radiation or x-ray radiation.

48. The composition according to claim 47, wherein the APCs comprise dendritic cells; and optionally, the composition further comprises at least one of irradiated tumor cells, B- cells and macrophages.

49. The composition according to claim 47 or 48, further comprising at least one pharmaceutically acceptable excipient.

50. The composition according to any one of claims 47-49, wherein the composition comprises a vaccine for reducing onset of, reducing recurrence of, or preventing cancer in a subj ect.

50. A method for treating cancer in a subject comprising administering the composition according to any one of claims 47-50 to the subject.

51. A kit comprising the composition according to any one of claims 1-15; and at least one container.

52. The kit according to claim 51, wherein the composition is frozen, and the kit is stored in a freezer.