WO2021119376A1 - Personalized tumor vaccine and use thereof for cancer immunotherapy - Google Patents

Personalized tumor vaccine and use thereof for cancer immunotherapy Download PDF

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Publication number
WO2021119376A1
WO2021119376A1 PCT/US2020/064412 US2020064412W WO2021119376A1 WO 2021119376 A1 WO2021119376 A1 WO 2021119376A1 US 2020064412 W US2020064412 W US 2020064412W WO 2021119376 A1 WO2021119376 A1 WO 2021119376A1
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Prior art keywords
dose
tumor
cancer cells
personalized
cells
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PCT/US2020/064412
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English (en)
French (fr)
Inventor
Zhengping Zhuang
Rogelio MEDINA
Herui Wang
Jan ŽENKA
Karen PACAK
Winson Ho
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South Bohemia In Ceske Budejovice, University of
Ne1 Inc
National Institutes of Health NIH
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South Bohemia In Ceske Budejovice, University of
Ne1 Inc
National Institutes of Health NIH
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Priority to JP2022536653A priority Critical patent/JP2023508852A/ja
Priority to EP20899103.4A priority patent/EP4072580A4/en
Priority to CN202080086575.3A priority patent/CN114929265A/zh
Priority to AU2020403013A priority patent/AU2020403013A1/en
Priority to US17/783,866 priority patent/US20230054318A1/en
Priority to KR1020227023239A priority patent/KR20220141280A/ko
Publication of WO2021119376A1 publication Critical patent/WO2021119376A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
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    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
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    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
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    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2815—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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Definitions

  • Immune checkpoint inhibitors such as anti-PD1/PD-L1 and anti-CTLA-4
  • ICIs have vastly improved outcome of various types of advanced cancers, including melanoma and non-small cell lung cancer (see Nat Rev Cancer.2018;18(3):139-147. doi:10.1038/nrc.2017.117).
  • melanoma and non-small cell lung cancer
  • One of the strongest predictors of objective response to ICIs is the tumor mutation burden (see N Engl J Med. 2017;377(25):2500-2501. doi:10.1056/NEJMc1713444).
  • tumors including glioblastoma are less likely to respond to single agent ICI therapy presumably due to the lack of neoantigens necessary to activate an adaptive immune response.
  • These tumors are often found to be “immunologically cold” with low T-cell infiltration.
  • An “immunologically hot” tumor with high T- cell infiltration suggests the presence of an active antitumor immune response and predicts a favorable response to ICIs.
  • conversion of a “cold” to “hot” immunogenic phenotype is an important step to make a successful immunotherapy.
  • novel treatments or immunotherapeutic agents capable of converting an “immunologically cold” tumor to an “immunologically hot” tumor.
  • a personalized tumor vaccine comprises: (a) a phagocytosis stimulating agent, (b) an immunostimulatory adjuvant, and (c) attenuated cancer cells, wherein the personalized tumor vaccine, when administered to an individual in need thereof, is effective to activate an immune response.
  • the attenuated cancer cells are obtained from a tumor of the individual.
  • the tumor is a solid tumor.
  • the tumor is a liquid tumor.
  • the attenuated cancer cells are prepared by (a) harvesting cancer cells from a biopsy of a site of tumor from the individual, (b) culturing the harvested cancer cells to a therapeutically relevant amount, and (c) irradiating the cultured cancer cells. [0010] In some embodiments, the attenuated cancer cells are present in an amount from about 1.0 ⁇ 10 ⁇ 3 to about 1.0 ⁇ 10 ⁇ 7.
  • the phagocytosis stimulating agent comprises a mannan. In some embodiments, the mannan is present in an amount from about 2 mg/dose to about 200 mg/dose. In some embodiments, the phagocytosis stimulating agent is conjugated to a biocompatible anchor for cell membrane (BAM).
  • the BAM is present in an amount from about 0.2 mg/dose to about 20 mg/dose.
  • the BAM comprises Formula I: [0011] [0012] wherein n is the number of ethylene oxide (EO) unit repeats in the PEG chain.
  • the immunostimulatory adjuvant comprises a Toll like receptor (TLR) agonist.
  • the TLR agonist comprises R-848, poly (I:C), lipoteichoic acid (LTA), or combinations thereof.
  • the TLR agonist comprises R-848.
  • the R-848 is present in an amount from about 0.05 mg/dose to about 5 mg/dose.
  • the TLR agonist comprises poly (I:C). In some embodiments, the poly (I:C) is present in an amount from about 0.05 mg/dose to about 5 mg/dose. In some embodiments, the TLR agonist comprises lipoteichoic acid (LTA). In some embodiments, the LTA is present in an amount from about 0.05 mg/dose to about 5 mg/dose. In some embodiments, the immunostimulatory adjuvant comprises an anti-CD40 antibody. In some embodiments, the anti-CD40 antibody is present in an amount from about 0.04 mg/dose to about 4 mg/dose. In some embodiments, the immune response comprises an adaptive immune response specific to the attenuated cancer cells.
  • a personalized tumor vaccine comprises: (a) a mannan, wherein the mannan is conjugated to a BAM, (b) a R-848, a poly (I:C), and an LTA, (c) an anti-CD40 antibody, and (d) irradiated cancer cells, wherein the personalized tumor vaccine, when administered to an individual in need thereof, is effective to activate an immune response.
  • a personalized tumor vaccine comprises: (a) a mannan, wherein the mannan is conjugated to a BAM, (b) a R-848, a poly (I:C), and an LTA, (c) an anti-CD40 antibody, and (d) irradiated cancer cells, wherein the personalized tumor vaccine, when administered to an individual in need thereof, is effective to activate an immune response.
  • a method for treating cancer comprises administering to an individual in need thereof an effective amount of pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) a phagocytosis stimulating agent, (b) an immunostimulatory adjuvant, and (c) attenuated cancer cells.
  • the attenuated cancer cells are obtained from a tumor of the individual.
  • the tumor is a solid tumor.
  • the tumor is a liquid tumor.
  • the attenuated cancer cells are prepared by (a) harvesting cancer cells from a biopsy of a site of tumor from the individual, (b) culturing the harvested cancer cells to a therapeutically relevant amount, and (c) irradiating the cultured cancer cells.
  • the attenuated cancer cells are present in an amount from about 1.0 ⁇ 10 ⁇ 3 to about 1.0 ⁇ 10 ⁇ 7.
  • the phagocytosis stimulating agent comprises a mannan.
  • the mannan is present in an amount from about 2 mg/dose to about 200 mg/dose.
  • the phagocytosis stimulating agent is conjugated to a biocompatible anchor for cell membrane (BAM).
  • BAM is present in an amount from about 0.2 mg/dose to about 20 mg/dose.
  • the BAM comprises Formula I: [0018] [0019] wherein n is the number of ethylene oxide (EO) unit repeats in the PEG chain.
  • the immunostimulatory adjuvant comprises a Toll like receptor (TLR) agonist.
  • TLR Toll like receptor
  • the TLR agonist comprises R-848, poly (I:C), lipoteichoic acid (LTA), or combinations thereof.
  • the TLR agonist comprises R-848.
  • the R-848 is present in an amount from about 0.05 mg/dose to about 5 mg/dose.
  • the TLR agonist comprises poly (I:C).
  • the poly (I:C) is present in an amount from about 0.05 mg/dose to about 5 mg/dose.
  • the TLR agonist comprises lipoteichoic acid (LTA).
  • the LTA is present in an amount from about 0.05 mg/dose to about 5 mg/dose.
  • the immunostimulatory adjuvant comprises an anti-CD40 antibody.
  • the anti-CD40 antibody is present in an amount from about 0.04 mg/dose to about 4 mg/dose.
  • the personalized tumor vaccine is administered subcutaneously.
  • the personalized tumor vaccine activates an immune response specific to the attenuated cancer cells.
  • the immune response comprises an adaptive immune response.
  • FIGUREs 1A-B show a mouse model of subcutaneous primary and metastatic distant tumors.
  • FIGURE 1A depicts a cartoon schematic of a CT26 tumor-bearing mouse model of subcutaneous primary and metastatic distant tumors. The mouse is viewed from the dorsal side. A representative primary tumor and metastatic distant tumor is established at 101 on the right flank and 102 on the left flank, respectively.
  • FIGURE 1B shows representative pictures of CT26 tumor- bearing mice 10 days after injection of saline.
  • FIGUREs 2A-H show that in situ MBTA injection at representative primary tumors induces a potent and systemic subject-specific adaptive immune response.
  • FIGURE 2A depicts a schematic timeline of the MBTA immunotherapy.
  • FIGURE 2B shows a schematic timeline of the MBTA immunotherapy.
  • FIGURE 2C shows representative pictures of CT26 tumor-bearing mice 10 days after the start of the MBTA immunotherapy. The locations of the representative primary and metastatic tumors are shown by arrows. The mouse is viewed from the dorsal side.
  • the primary tumors of most mice with the in situ MBTA injection did not grow, as compared to those of the control mice injected with saline.
  • FIGURE 2F depicts the cumulative survival curves of the CT26 tumor- bearing mice with the in situ injection at the primary tumor.
  • *** p 0.0002 by Log-rank (Mantel-Cox) test.
  • FIGURE 2G depicts 2-dimensional flow cytometry plots confirming CD4 + (left panel) or CD8 + T-cell depletion (middle panel) in the spleen of the CT26 tumor-bearing mice.
  • the primary tumors of most mice with the in situ MBTA injection did not grow, as compared to that of the control mice injected with saline.
  • FIGURE 2J depicts the cumulative survival curves of the CT26 tumor-bearing mice with the in situ injection at the primary tumor.
  • FIGUREs 3A-I show that MBTA stimulates the innate immune system to elicit subject- specific rejection of primary and distant CT26 tumors.
  • FIGURE 3A depicts a schematic timeline of the MBTA immunotherapy and tumor immunophenotyping experiments (I.P.).
  • FIGURE 3B depicts a dot plot showing the percentage of CD45 + cells in total live tumor dissociated cells. I.P. day 10 and I.P. day 16 analyses demonstrate that MBTA-treated mice had significantly higher immune cells within the primary tumor than saline-treated control mice did. **p ⁇ 0.01 by Mann–Whitney U test.
  • FIGURE 3C depicts a dot plot showing the percentage of CD45 + cells in total live tumor dissociated cells from the primary tumors in the CT26 tumor-bearing mice.
  • I.P. day 10 and I.P. day 16 analyses demonstrate that MBTA- treated mice had significantly higher immune cells within the metastatic distant tumor than saline- treated control mice did. **p ⁇ 0.01 by Mann–Whitney U test.
  • Data is shown as individual data points (dots) with median (line).
  • FIGURE 3D depicts a dot plot showing the percentage of innate immune cells in total live tumor dissociated cells from the primary tumors in the CT26 tumor- bearing mice.
  • FIGURE 3E depicts a dot plot showing the percentage of MHC II+ monocytes in total live tumor dissociated cells from the metastatic distant tumors in the CT26 tumor-bearing mice.
  • I.P. day 10 and I.P. day 16 analyses demonstrate that MBTA-treated mice had more MHC II+ monocytes within the primary tumor than saline-treated control mice did. **p ⁇ 0.01 by Mann–Whitney U test.
  • FIGURE 3F depicts a dot plot showing the percentage of innate immune cells in total live tumor dissociated cells from the metastatic distant tumors in the CT26 tumor- bearing mice.
  • I.P. day 16 analyses demonstrate that MBTA-treated mice had more dendritic cells, monocytes, and neutrophils within the metastatic distant tumor than saline-treated control did. *p ⁇ 0.05, **p ⁇ 0.01 by Mann-Whitney U test.
  • Data is shown as individual data points (dots) with median (line).
  • FIGURE 3G depicts a dot plot showing the percentage of MHC II+ monocytes in total live tumor dissociated cells from the metastatic distant tumors in the CT26 tumor-bearing mice.
  • I.P. day 10 and I.P. day 16 analyses demonstrate that MBTA-treated mice (pink) had more MHC II+ monocytes within the metastatic distant tumor than saline-treated control mice (black) did. **p ⁇ 0.01 by Mann–Whitney U test. Data is shown as individual data points (dots) with median (line).
  • FIGURE 3H depicts a dot plot showing the percentage of alternatively activated macrophages in total live tumor dissociated cells from the primary tumors in the CT26 tumor-bearing mice. I.P.
  • FIGURE 3I depicts a dot plot showing the percentage of alternatively activated macrophages in total live tumor dissociated cells from the metastatic distant tumors in the CT26 tumor-bearing mice.
  • I.P. day 10 and I.P. day 16 analyses demonstrate that MBTA-treated mice had fewer alternatively activated macrophages within the distant tumor than saline-treated control mice did.
  • FIGUREs 4A-F show that MBTA stimulates the adaptive immune systems to elicit subject- specific rejection of primary and distant CT26 tumors.
  • FIGURE 4A depicts a dot plot showing the percentage of adaptive immune cells in total live tumor dissociated cells from the primary tumors in the CT26 tumor-bearing mice.
  • I.P. day 10 and I.P. day 16 analyses demonstrate that the MBTA- treated mice had significantly more CD8 + T-cells and B cells within the primary tumor in day 16 than the saline-treated control mice did.
  • FIGURE 4B depicts a dot plot showing the percentage of adaptive immune cells in total live tumor dissociated cells from the primary tumors in the CT26 tumor-bearing mice.
  • I.P. day 10 and I.P. day 16 analyses demonstrate that the MBTA- treated mice had significantly more CD8 + T-cells and B cells within the primary tumor in I.P. day 16 than the saline-treated control mice did. There were also more CD8 + and fewer CD4+ T-cells in the distant tumor from the MBTA-treated mice than the control mice in I.P. day 10.
  • FIGURE 4C depicts a dot plot showing the percentage of IFN gamma or TNF alpha positive CD4 + or CD8 + T-cells per total CD4 + or CD8 + T-cells, respectively.
  • Harvested left flank CD4 + and CD8 + T-cells corresponding to Day 10 and 16 analyses were stimulated for 5 h with PMA/Ionomycin ex- vivo and intracellular IFN ⁇ and TNF ⁇ was determined by flow cytometry.
  • the MBTA-treated mice had significantly more TNF alpha CD8 + T-cells in I.P. day 16 and more IFN gamma CD8 + T-cells in I.P.
  • FIGURE 4D depicts a dot plot showing the percentage of Granzyme B+ CD8 + T-cells per total CD8 + T-cells in the distant tumor from the CT26 tumor-bearing mice.
  • the MBTA-treated mice had significantly more Granzyme B+ CD8 + T- cells in I.P. day 10 and 16 than the saline-treated mice did.
  • FIGURE 4E depicts a dot plot showing the percentage of AH-1-specific CD8 + T-cells extracted from whole blood of the CT26 tumor- bearing mice.
  • FIGURE 4F depicts a dot plot showing the percentage of AH-1-specific CD8 + T-cells extracted from the metastatic distant tumors in the CT26 tumor-bearing mice. Four mice were in the saline treatment arm and five mice were in the MBTA treatment arm.
  • FIGUREs 5A-F show that rCT26-MBTA vaccines generate a potent subject-specific antitumor immune response resulting in improved tumor growth control and survival in CT26- bearing mice.
  • FIGURE 5A depicts a cartoon schematic of the development of the rCT26-MBTA vaccine.
  • FIGURE 5B depicts a bar graph showing the percentage of apoptotic CT26 cells after irradiation.
  • FIGURE 5C depicts a schematic timeline of the rCT26-MBTA vaccination.
  • FIGURE 5D depicts tumor growth curves in the CT26 tumor-bearing mice injected with saline (left), the rCT26 vaccine (middle), or the combination of the rCT26 vaccine and MBTA (right).
  • FIGURE 5E depicts cumulative survival curves of the mice in FIGURE 5D.
  • all saline-treated and rCT26-vaccinated mice died by day 30, while only 25 % of mice vaccinated with the rCT26 and MBTA combination died. At least 10% survived 100 days post vaccination.
  • FIGURE 5F depicts a dot plot showing the percentage of AH-1-specific CD8 + T-cells extracted from whole blood of the CT26 tumor-bearing mice with saline treatment, rCT26 vaccination, and rCT26-MBTA vaccination.
  • FIGURE 5G depicts a dot plot showing the percentage of AH-1-specific CD8 + T-cells extracted from the tumors of the CT26 tumor-bearing mice with saline treatment, rCT26 vaccination, and rCT26-MBTA vaccination.
  • FIGURE 5I shows a line plot of the mean body weight (g) of the mice subjected to saline and rCT26-MBTA treatment. rCT26-MBTA treated mice did not demonstrate a significant difference in mean body weight to that of the saline-treated mice.
  • FIGUREs 6A-D show MBTA treatment induces subject-specific immunological memory against CT26 cells.
  • FIGURE 6B shows cumulative survival curves of mice in FIGURE 6A.
  • FIGURE 6D shows cumulative survival curves of intracranially re-challenged mice over time.
  • cell refers to one or more mammalian cells.
  • the term includes progeny of a cell or cell population.
  • cells include progeny of a single cell, and there are variations between the progeny and its original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a “cancer cell” as used herein refers to a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth and/or development in an immunocompromised non-human animal model, and/or any appropriate indicator of cellular transformation.
  • “Cancer cell” may be used interchangeably herein with “tumor cell” or “cancerous cell,” and encompasses cancer cells of a solid tumor and a liquid tumor.
  • Immunotherapy refers to treatment of a disease (e.g., cancer) by modulating an immune response.
  • immunotherapy refers to providing an anti-cancer immune response in a subject by administration of a personalized tumor vaccine that elicits an anti-tumor immune response in the subject.
  • An “effective amount” is an amount sufficient to effect beneficial or desired clinical results.
  • An effective amount can be administered in one or more administrations.
  • an effective amount of reagent antibodies is an amount that is sufficient to diagnose, palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.
  • a “TLR” refers to a toll-like receptor of any species origin, e.g., human and rodent.
  • TLR agonist refers to a molecule that acts as an agonist of at least one TLR.
  • a “CD40 agonist” herein refers to a molecule that functions as a CD40 agonist signal such as a CD40 agonistic antibody (e.g., an anti-CD40 monoclonal antibody). It can also refer to a CD40L polypeptide, fragment, or conjugate thereof. In general, ligands that bind CD40 may act as a CD40 agonist.
  • CD40 agonists according to the invention may include aptamers that bind CD40.
  • pathogen associated molecular pattern or “PAMP,” as used herein, refers to any component of a microorganism that directs the targeted host cell to distinguish “self” from “non- self”, e.g., infected pathogen, and promotes signals associated with innate immunity.
  • damage/danger associated molecular pattern or “DAMP,” as used herein, refers to any molecule from damaged or dying cells that activates the innate immune system to target these damaged cells from undamaged cells.
  • Solid tumor refers to tumors that usually do not contain cysts or liquid areas. Solid tumors can include brain and other central nervous system tumors (including but not limited to tumors of the meninges, brain, spinal cord, cranial nerves and other parts of central nervous system, e.g.
  • glioblastomas or medulla blastomas head and/or neck cancer
  • breast tumors including but not limited to circulatory system tumors (including but not limited to heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue); excretory system tumors (including but not limited to tumors of kidney, renal pelvis, ureter, bladder, other and unspecified urinary organs); gastrointestinal tract tumors (including but not limited to tumors of esophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus and anal canal, tumors involving the liver and intrahepatic bile ducts, gall bladder, other and unspecified parts of biliary tract, pancreas, other and digestive organs); oral cavity tumors (including but not limited to tumors of lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands,
  • small cell lung cancer or non-small cell lung cancer skeletal system tumors (including but not limited to tumors of bone and articular cartilage of limbs, bone articular cartilage and other sites); skin tumors (including but not limited to malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and tumors involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneum and peritoneum, eye and adnexa, thyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites.
  • skeletal system tumors including but not limited to tumors of bone and articular cartilage of limbs, bone articular cartilage and other sites
  • liquid cancer or “liquid tumor” as used herein refers to cancer cells that are present in body fluids, such as blood, lymph and bone marrow.
  • Liquid cancers include leukemia, myeloma, myelodysplastic syndrome (MDS), and liquid lymphomas.
  • Liquid lymphomas include lymphomas that contain cysts or liquid areas.
  • Liquid cancers as used herein do not include solid tumors, such as sarcomas and carcinomas or solid lymphomas that do not contain cysts or liquid areas.
  • An “attenuated” cell as used herein refers to a cell that is alive but replication deficient. The attenuated cell may be alive but unable to complete its cell-cycle.
  • the attenuated cell may have a limited capacity to replicate, express proteins, and to develop through some life cycle stages, for example, an attenuated cell may be arrested at a particular life cycle stage and is unable to developmentally progress beyond that stage.
  • the term “attenuated cancer cell” as used herein refers to a cancer cell that is attenuated and with reduced oncogenicity.
  • the attenuated cancer cell may be unable to cause or give rise to a tumor.
  • the attenuated cancer cell may also be unable to metastasize or increase a tumor burden of a subject with the tumor.
  • the attenuated cancer cell may comprise damages in their DNA.
  • the attenuated cancer cell can be obtained by various means, for example, by physical and chemical treatments.
  • the attenuated cancer cell can be obtained by irradiation treatments.
  • the term “personalized tumor vaccine” as used herein refers to a tumor vaccine that can direct a subject-specific immune response to a tumor of a subject. Such a response may be specific to a specific type of tumor from a specific subject.
  • the personalized tumor vaccine as used herein comprises attenuated cancer cells.
  • the personalized tumor vaccine may elicit an adaptive immune response to a tumor or tumor cells of a subject.
  • Personalized Tumor Vaccine Formulations and Compositions [0043]
  • the immunotherapeutic strategy, introduced in this invention leverages the use of phagocytosis stimulating agents and immunostimulatory adjuvants for directing an immune response against a subject-specific cancer or cancer cells.
  • the personalized tumor vaccine described here having immunotherapeutic potential delivered as part of a whole attenuated cancer cell vaccine generates a potent adaptive immune response capable of preventing or controlling tumor growth and inducing tumor regression in a subject-specific subset of primary or metastatic tumors.
  • Personalized tumor vaccines are used to train the immune system to find and destroy, attack, kill, or inhibit subject-specific cancer cells.
  • a personalized tumor vaccine can direct the immune system of a subject to target subject-specific cancer cells.
  • a personalized tumor vaccine can direct the immune system of a subject to discriminate subject-specific cancer cells from other cancer cells not of the same subject.
  • a personalized tumor vaccine can direct the immune system of a subject to discriminate subject-specific cancer cells from cancer cells of other subjects.
  • Such vaccine can make use of a phagocytosis stimulating agent or agents specific to the subject-specific cancer cells.
  • a phagocytosis stimulating agent or agents can allow the immune system to discriminate the subject-specific cancer cells from the non-cancer cells, cancer cells from other tumors, or cancer cells from other subjects.
  • the immune system When an immune system is presented with a phagocytosis stimulating agent or agents specific of a subject-specific cancer, the immune system will be activated to target the cancer cells sharing the same subject-specificity, even if a cancer is not present. Such activation of the immune system will create long lasting subject-specific immune memory.
  • the immune system encounters a cancer with the same or similar subject-specificity in the future, it will immediately activate a subject-specific immune response against the cancer cell.
  • a personalized tumor vaccine can be a prophylactic or preventative vaccine.
  • a personalized tumor vaccine can be a therapeutic or treatment vaccine.
  • a personalized tumor vaccine can be administered to a subject in need thereof before the subject is diagnosed with a cancer.
  • a personalized tumor vaccine can be administered to a subject in need thereof after the subject is diagnosed with a cancer.
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof; an immunostimulatory adjuvant or derivative herein and thereof; and from about 1.0 ⁇ 10 ⁇ 3 to about 1.0 ⁇ 10 ⁇ 7 attenuated cancer cells.
  • a personalized tumor vaccine can comprise a mannan; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise about 2 mg/dose to about 200 mg/dose mannan; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof conjugated to biocompatible anchor for cell membrane (BAM); an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • BAM biocompatible anchor for cell membrane
  • a personalized tumor vaccine can comprise a mannan conjugated to BAM an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a mannan conjugated to from about 0.2 mg/dose to about 20 mg/dose BAM; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a mannan conjugated to BAM comprising Formula I described herein and thereof; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof; a Toll like receptor (TLR) agonist; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • TLR Toll like receptor
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof; R-848, poly (I:C), lipoteichoic acid (LTA), or combinations thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof; from about 0.05 mg/dose to about 5 mg/dose R-848, poly (I:C), lipoteichoic acid (LTA), or combinations thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof; R-848, from about 0.05 mg/dose to about 5 mg/dose poly (I:C), lipoteichoic acid (LTA), or combinations thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof; R-848, poly (I:C), from about 0.05 mg/dose to about 5 mg/dose lipoteichoic acid (LTA), or combinations thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof; an anti-CD40 antibody; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a phagocytosis stimulating agent or derivative herein and thereof; from about 0.04 mg/dose to about 4 mg/dose anti-CD40 antibody; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a personalized tumor vaccine can comprise a mannan attached to BAM, a R-848, a poly (I:C), an LTA, an anti-CD40 antibody, and irradiated cancer cells.
  • a personalized tumor vaccine can comprise from about 0.05 mg/dose to about 5 mg/dose mannan attached to from about 0.05 mg/dose to about 5 mg/dose BAM, from about 0.05 mg/dose to about 5 mg/dose R-848, from about 0.05 mg/dose to about 5 mg/dose poly (I:C), from about 0.05 mg/dose to about 5 mg/dose LTA, from about 0.04 mg/dose to about 4 mg/dose anti-CD40 antibody, and from about 1.0 ⁇ 10 ⁇ 3 to about 1.0 ⁇ 10 ⁇ 7 irradiated cancer cells.
  • Phagocytosis stimulating agents [0051]
  • a personalized tumor vaccine described herein and thereof can comprise one phagocytosis stimulating agent.
  • a phagocytosis stimulating agent can comprise a nucleic acid, amino acid, nucleotide, carbohydrate, lipid, small molecule, ion, compound, any derivatives herein and thereof, or any combinations herein and thereof.
  • a phagocytosis stimulating agent may not be expressed by a cancer cell.
  • a phagocytosis stimulating agent can be exogenous to a cancer cell.
  • a phagocytosis stimulating agent can be specific to a cancer cell.
  • a phagocytosis stimulating agent may not be specific to a cancer cell.
  • a phagocytosis stimulating agent can be linked to an attenuated cancer cell.
  • a phagocytosis stimulating agent can be attached to an attenuated cancer cell.
  • a phagocytosis stimulating agent can comprise a PAMP or DAMP described herein or thereof.
  • PAMPs pathogen- associated molecular patterns
  • PAMPs can comprise secretory molecules, extracellular molecules, or intracellular molecules.
  • PAMPs can activate pattern-recognition receptors, such as Toll-like receptors (TLRs), to trigger antimicrobial and proinflammatory response against the pathogens.
  • TLRs Toll-like receptors
  • Such responses include differential gene expression, production of cytokines, chemokines, and/or phagocytosis.
  • another type of innate immune response–stimulatory agents comprise Danger-Associated Molecular Patterns (DAMP), molecular patterns, structures, or entities associated with stressed, injured, infected, or transformed cells not found in normal cells.
  • DAMP Danger-Associated Molecular Patterns
  • a phagocytosis stimulating agent can comprise a PAMP or DAMP molecule that, when recognized by an immune system, triggers a phagocytosis response against the phagocytosis stimulating agent or any molecule or cell linked to the phagocytosis stimulating agent.
  • a phagocytosis stimulating agent can have an origin in viruses, gram-positive bacteria, gram- negative bacteria, fungi, protozoa, protists, nematodes, plant cells, animal cells, any derivatives herein and thereof, or any combinations herein and thereof.
  • a phagocytosis stimulating agent can also be synthesized in vitro, such as but not limited to organic synthesis.
  • a phagocytosis stimulating agent can be an intracellular, extracellular, lysosomal, endosomal, nuclear, cytoplasmic, mitochondrial, ER-bound, Golgi-bound, membrane-associated, or integrated membrane component.
  • a phagocytosis stimulating agent can also comprise triacyl lipopeptide, diacyl lipopeptide, lipoteichoic acid, lipoprotein, peptidoglycan, lipoarabinomannan, porin, envelope glycoprotein, GPI-mucin, phospholipomannan, zymosan, beta-glycan double-stranded (ds) RNA, double-stranded DNA, single-stranded (ss) RNA, single-stranded DNA, lipopolysaccharide, arabinogalactan, glycoinositolphospholipid, heat shock proteins (HSPs), flagellin, CpG DNA, methylated DNA, 5′-triphosphate RNA, diaminopimelic acid, triacyl lipopeptides, muramyl dipeptide (MDP), surface glycoprotein (GP), membrane components, lipoteichoic acid (LTA), phosphorylcholine (PC), PE, PI, mycolic acid, aden
  • a dsDNA can be long or short.
  • CpG DNA can comprise methylated or unmethylated CpG DNA.
  • Arabinogalactan can comprise D-arabinose or D-galactose.
  • a phagocytosis stimulating agent can comprise glucan or mannan.
  • a phagocytosis stimulating agent can comprise mannan or its derivatives.
  • Mannan [0055] Mannan is often found on the yeast cell wall. It can comprise a series of mannose units linked by alpha (1-6) linkages. Mannan can also have alpha (1-2) and alpha (1-3) branched linkages. Detection of mannan leads to cell lysis or phagocytosis in the mannan-binding lectin (MBL) pathway.
  • Mannans can also be found in plants, algae, fungus, or bacteria. They are synthesized from activated nucleotide sugars such as GDP-mannose, GDP-glucose, and UDP-galactose. Glycosyltransferases, localized in Golgi, utilize the activated nucleotide sugars to synthesize the polymer by facilitating the linkage between mannose monomers.
  • Mannan can comprise the polysaccharide moiety of glycoproteins. Mannan can comprise a linear, branched, or a linear and branched polymer of linked mannose resides or molecules. In some cases, mannan can have beta (1-4) linkages.
  • Mannan can be cytoplasmic or extracellular.
  • Mannan can have a molecular weight of 666.6 g/mol. In some embodiments, mannan can have 14 hydrogen bond donors and 21 acceptors. In other cases, mannan can have 10 rotatable bonds. In some instances, mannan can have a monoisotopic mass of 666.221858 g/mol. In other cases, mannan can have a topological polar surface area of 348 ⁇ 2. In some cases, mannan can have 45 heavy atoms and 0 formal charge.
  • mannan can be (2S,3S,4S,5S,6R)-2-[(2R,3S,4R,5R,6S)-6-[(2R,3S,4R,5S,6S)-4,5-dihydroxy-2- (hydroxymethyl)-6-[(2R,3R,4R,5S,6R)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3- yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol.
  • mannan can be C 24 H 42 O 21 .
  • Manan can comprise a backbone of alpha (1-6) linked mannose unites with alpha (1-2) and alpha (1-3) linked side chains.
  • the side chains can have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sugar units in length.
  • a side chain can comprise a mannan-oligosaccharide (MOS).
  • a mannose can be a D-mannose or L-mannose.
  • a mannose can have a molecular formula of C 6 H 12 O 6 .
  • a mannose can have a molecular weight of 180.16 g/mol.
  • a mannose can comprise a D-manno-hexopyranose.
  • a mannose can also be (3S,4S,5S,6R)-6- (hydroxymethyl)oxane-2,3,4,5-tetrol.
  • a mannose can comprise two different-sized rings, a six- membered pyranose form and a five-membered furanose form. In some instances, a ring can have an alpha or beta configuration at the anomeric position.
  • Mannan can be synthesized by yeast.
  • Mannan-producing yeasts can comprise Hansenula holstii, Rhodotorula acheniorum, Sporobolomyces salmonicolor, Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Meyerozyma guilliermondii, Brewers dried yeast, or Candida utilis. Mannan can be synthesized by plants.
  • Mannan-producing plants can comprise the Ebenaceae family, Arabidopsis thaliana, the Leguminoseae family, Caesalpinia spinosa Kuntze, the Annonaceae family, Amorphophallus konjac, Ceratonia siliqua, the Convolvulaceae family, Cyamopsis tetragonoloba, the Loganiaceae family, Senna tora, Trigonella foenum-graecum L., the Palmae family, Picea abies, Cercis siliquastrum, or Nicotiana plumbaginifolia.
  • a phagocytosis stimulating agent can comprise a linker that link the phagocytosis stimulating agent to an attenuated cancer cell by a linker.
  • a linker can comprise a chemical linkage, physical linkage, association, attachment, connection, junction, placement, fusion, interaction, ligation, chemical bond, physical bond, crosslink, joint, coupling, clamping, tie, or any derivatives herein and thereof, or any combinations herein and thereof.
  • a linker can comprise a chemical or physical linker.
  • a chemical linker can comprise a membrane linker.
  • a membrane linker can comprise a myristate, palmitate, farnesyl, geranylgeranyl, oleate, isoprenoid, fatty acid, diacylglycerol, long-chain acyl group, long-chain prenyl group, cholesterol, stearyl, phosphatidyl-ethanolamine (PE), phosphatidylinositol (PI), glycosyl phosphatidyl inositol (GPI) anchor, chelator lipid anchor, polypeptide, derivatives herein and thereof, or any combinations herein and thereof.
  • a chelator lipid anchor can comprise nitrilotriacetic acid ditetradecylamine (NTA ⁇ DTDA).
  • a linker can also comprise Biocompatible Anchors for Membrane (BAM).
  • BAM can comprise a lipid anchor.
  • a lipid anchor can comprise any lipid anchors described herein and thereof.
  • BAM can comprise a lipid anchor and a Polyethylene glycol (PEG) chain.
  • PEG Polyethylene glycol
  • BAM can be used to link a chemical, molecule, polypeptide, nucleic acid, lipid, carbohydrate, any moieties described herein and thereof, any derivatives herein and thereof, any combinations herein and thereof to a cell.
  • BAM can comprise an NHS reactive ester group.
  • a PEG chain can be hydrophilic.
  • BAM can comprise a succinylated poly(ethylene glycol) oleyl ether at the hydroxyl end of a PEG chain.
  • BAM can comprise a N ⁇ hydroxysuccinimide (NHS) at the succinyl PEG end.
  • NHS N ⁇ hydroxysuccinimide
  • BAM comprising an NHS end can bind most proteins.
  • a BAM can comprise a Oleyl ⁇ O ⁇ poly(ethylene glycol) ⁇ succinyl ⁇ N ⁇ hydroxy ⁇ succinimidyl esters.
  • BAM can comprise Formula I (Oleyl- O(CH 2 CH 2 ) n CO-CH 2 CH 2 -COO-NHS): [0063] Formula I: Wherein n can comprise the number of ethylene oxide (EO) unit repeats in the PEG moiety.
  • EO ethylene oxide
  • n can be from 1-500,
  • the number of EO unit repeats in the PEG moiety can be from 0 to 9, from 1 to 10, from 2 to 11, from 3 to 12, from 4 to 13, from 5 to 14, from 6 to 15, from 7 to 16, from 8 to 17, from 9 to 18, from 10 to 19, from 11 to 20, from 12 to 21, from 13 to 22, from 14 to 23, from 15 to 24, from 16 to 25, from 17 to 26, from 18 to 27, from 19 to 28, from 20 to 29, from 21 to 30, from 22 to 31, from 23 to 32, from 24 to 33, from 25 to 34, from 26 to 35, from 27 to 36, from 28 to 37, from 29 to 38, from 30 to 39, from 31 to 40, from 32 to 41, from 33 to 42, from 34 to 43, from 35 to 44, from 36 to 45, from 37 to 46, from 38 to 47, from 39 to 48, from 40 to 49, from 41 to 50, from 42 to 51, from 43 to 52, from 44 to 53, from 45 to 54, from 46 to 55, from 47 to 56,
  • BAM can comprise a single lipid anchor.
  • BAM can comprise more than one lipid anchors.
  • BAM can comprise two lipid anchors.
  • BAM can comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 lipid anchors.
  • a lipid anchor can comprise a myristate, palmitate, farnesyl, geranylgeranyl, oleate, isoprenoid, fatty acid, diacylglycerol, long-chain acyl group, long-chain prenyl group, cholesterol, stearyl, phosphatidyl-ethanolamine (PE), phosphatidylinositol (PI), glycosyl phosphatidyl inositol (GPI) anchor, chelator lipid anchor, polypeptide, derivatives herein and thereof, or any combinations herein and thereof.
  • a chelator lipid anchor can comprise nitrilotriacetic acid ditetradecylamine (NTA ⁇ DTDA).
  • a lipid anchor can comprise methoxy ⁇ poly(ethylene glycol) ⁇ succinyl ⁇ N ⁇ hydroxy ⁇ succinimidyl ester.
  • a lipid anchor can comprise an oleyl chain.
  • a lipid anchor can comprise dioleylphosphatidylethanolamine (DOPE).
  • DOPE-PEG Bam can be pegylated and succinylated at the hydroxyl end of PEG and modified with N ⁇ hydroxysuccinimide (NHS) at the succinyl PEG end.
  • BAM can comprise Formula II. Wherein n can comprise the number of ethylene oxide (EO) unit repeats in the PEG moiety.
  • BAM can have a concentration of 100 M, 10 M, 1 M, 1x10 ⁇ -1 M, 1x10 ⁇ -2 M, 1x10 ⁇ -3 M, 1x10 ⁇ -4 M, 1x10 ⁇ -5 M, 1x10 ⁇ -6 M, 1x10 ⁇ -7 M, 1x10 ⁇ -8 M, 1x10 ⁇ -9 M, 1x10 ⁇ -10 M, 1x10 ⁇ -11 M, 1x10 ⁇ -12 M, 1x10 ⁇ -13 M, 1x10 ⁇ -14 M, 1x10 ⁇ -15 M, 1x10 ⁇ -16 M, 1x10 ⁇ -17 M, 1x10 ⁇ -18 M, 1x10 ⁇ -19 M, 1x10 ⁇ -20 M, 1x10 ⁇ -21 M, 1x10 ⁇ -22 M, 1x10 ⁇ -23 M, 1x10 ⁇ -24 M, 1x10 ⁇ -25 M, 1x10 ⁇ -26 M, 1x10 ⁇ -27 M, 1x10 ⁇ -28 M, 1x10 ⁇ -29 M, or 1x10 ⁇ - 30 M in aqueous solution.
  • BAM can have a concentration from 9.9 x 10 ⁇ -31 M to 1x10 ⁇ -29 M, from 9.9 x 10 ⁇ -30 M to 1x10 ⁇ -28 M, from 9.9 x 10 ⁇ -29 M to 1x10 ⁇ -27 M, from 9.9 x 10 ⁇ -28 M to 1x10 ⁇ -26 M, from 9.9 x 10 ⁇ -27 M to 1x10 ⁇ -25 M, from 9.9 x 10 ⁇ -26 M to 1x10 ⁇ -24 M, from 9.9 x 10 ⁇ -25 M to 1x10 ⁇ -23 M, from 9.9 x 10 ⁇ -24 M to 1x10 ⁇ -22 M, from 9.9 x 10 ⁇ -23 M to 1x10 ⁇ -21 M, from 9.9 x 10 ⁇ -22 M to 1x10 ⁇ -20 M, from 9.9 x 10 ⁇ -21 M to 1x10 ⁇ -19 M, from 9.9 x 10 ⁇ -20 M to 1x10 ⁇ -18 M, from 9.9 x 10 ⁇ -19 M to 1x10 ⁇ -17 M, from 9.9 x 10 ⁇ -18 M to 1x
  • BAM can be conjugated to a biotin, fluorescein, polypeptide, chemical, small molecule, nucleic acid, drug compound, polysaccharide, any derivatives herein and thereof, or any combinations herein and thereof.
  • a polysaccharide can comprise monosaccharide or oligosaccharide units bound together by glycosidic linkages.
  • a BAM can be conjugated to a mannan. This can be achieved by reacting NHS group of a BAM with a primary amine of another molecule at pH 7-9 to form amide bond. In the case of conjugating mannan to BAM, aminated mannan is prepared by reductive amination.
  • Mannan solution in an environment of ammonium acetate (300 mg/ml) is reduced by 0.2 M sodium cyanoborohydride at pH 7.5 and 50 °C for five days.
  • Solution is further dialyzed using MWCO 3500 dialysis tubing (Serva, Heidelberg, Germany) against PBS at 4 °C overnight.
  • Binding of BAM on amino group of mannan is performed at pH 7.3 according to Kato et al., 2004.
  • N-hydroxysuccinimide (NHS) group of BAM reacts with amino group of mannan. Solutions obtained after dialysis as above are stored frozen at -20 °C until use.
  • a personalized tumor vaccine can comprise 1x10 ⁇ 0 M, 2x10 ⁇ 0 M, 3x10 ⁇ 0 M, 4x10 ⁇ 0 M, 5x10 ⁇ 0 M, 6x10 ⁇ 0 M, 7x10 ⁇ 0 M, 8x10 ⁇ 0 M, 9x10 ⁇ 0 M, 1x10 ⁇ -1 M, 2x10 ⁇ -1 M, 3x10 ⁇ -1 M, 4x10 ⁇ -1 M, 5x10 ⁇ -1 M, 6x10 ⁇ -1 M, 7x10 ⁇ -1 M, 8x10 ⁇ -1 M, 9x10 ⁇ -1 M, 1x10 ⁇ -2 M, 2x10 ⁇ -2 M, 3x10 ⁇ -2 M, 4x10 ⁇ -2 M, 5x10 ⁇ -2 M, 6x10 ⁇ -2 M, 7x10 ⁇ -2 M, 8x10 ⁇ -2 M, 9x10 ⁇ -2 M, 1x10 ⁇ -3 M, 2x10 ⁇ -3 M, 3x10 ⁇ -3 M, 4x10 ⁇ -3 M, 5x10 ⁇ -3 M, 6x10 ⁇ -3 M, 7x10 ⁇ -3 M, 8x10 ⁇ -3 M,
  • a personalized tumor vaccine can comprise from 5x10 ⁇ 0M to 1x10 ⁇ 1 M, from 1x10 ⁇ 0M to 5x10 ⁇ 0 M, from 5x10 ⁇ -1M to 1x10 ⁇ 0 M, from 1x10 ⁇ -1M to 5x10 ⁇ -1 M, from 5x10 ⁇ -2M to 1x10 ⁇ -1 M, from 1x10 ⁇ -2M to 5x10 ⁇ -2 M, from 5x10 ⁇ -3M to 1x10 ⁇ -2 M, from 1x10 ⁇ -3M to 5x10 ⁇ -3 M, from 5x10 ⁇ -4M to 1x10 ⁇ -3 M, from 1x10 ⁇ -4M to 1x10 ⁇ -3 M, from 1x10 ⁇ -4M to 5x10 ⁇ -4 M, from 5x10 ⁇ -5M to 1x10 ⁇ -4 M, from 1x10 ⁇ -5M to 5x10 ⁇ -5 M, from 5x10 ⁇ -6M to 1x10 ⁇ -5 M, from 1x10 ⁇ -6M to 5x10 ⁇ -6 M, or from 5x10 ⁇ -7M to 1x10 ⁇ -7 M mannan- BAM.
  • a personalized tumor vaccine can also comprise about 0.2 mM mannan-BAM.
  • a personalized tumor vaccine can also comprise about 0.95 mg mannan.
  • a personalized tumor vaccine can also comprise about 0.098 mg BAM.
  • a personalized tumor vaccine can comprise about 23.1 mg/day mannan.
  • a personalized tumor vaccine can also comprise about 2.38 mg/day mannan.
  • Immunostimulatory adjuvant [0070] An immunostimulatory adjuvant can comprise any substance that acts to accelerate, prolong, or enhance phagocytosis stimulating agent–specific immune responses when used in combination with a phagocytosis stimulating agent linked to an attenuated cancer cell.
  • an immunostimulatory adjuvant can also potentiate, activate, prime, stimulate, or increase an immune response to a phagocytosis stimulating agent linked to an attenuated cancer cell.
  • a personalized tumor vaccine can comprise one immunostimulatory adjuvant.
  • a personalized tumor vaccine can comprise more than one immunostimulatory adjuvant.
  • a personalized tumor vaccine can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more than 100 immunostimulatory adjuvants.
  • a personalized tumor vaccine can comprise from 1 to 10, from 5 to 15, from 10 to 20, from 15 to 25, from 20 to 30, from 25 to 35, from 30 to 40, from 35 to 45, from 40 to 50, from 45 to 55, from 50 to 60, from 55 to 65, from 60 to 70, from 65 to 75, from 70 to 80, from 75 to 85, from 80 to 90, from 85 to 95, from 90 to 100 immunostimulatory adjuvants.
  • a personalized tumor vaccine can comprise 2 immunostimulatory adjuvants.
  • a personalized tumor vaccine can comprise 3 immunostimulatory adjuvants.
  • a personalized tumor vaccine can comprise 4 immunostimulatory adjuvants.
  • an immunostimulatory adjuvant can engage the immune system by mimicking a phagocytosis stimulating agent, the phagocytosis stimulating agent and the immunostimulatory adjuvant can activate or signal through an overlapping set of receptors in the immune system.
  • an immunostimulatory adjuvant can comprise a PAMP or DAMP.
  • an immunostimulatory adjuvant can comprise an organic or inorganic immunostimulatory adjuvant.
  • an immunostimulatory adjuvant can activate a Toll-like receptor (TLR) by a TLR agonist.
  • an immunostimulatory adjuvant can activate a CD40 receptor by an anti- CD40 antibody.
  • An immunostimulatory adjuvant can comprise any PAMPs, DAMPs, TLR agonists, CD40 agonists, any derivatives herein and thereof, or any combination herein and thereof.
  • an immunostimulatory adjuvant can also comprise any nucleic that encodes any PAMPs, DAMPs, TLR agonists, CD40 agonists, any derivatives herein and thereof, or any combination herein and thereof.
  • an immunostimulatory adjuvant can also comprise any nucleic that encodes a protein that can increase, stimulate, activate, or initiate the production of any PAMPs, DAMPs, TLR agonists, CD40 agonists, any derivatives herein and thereof, or any combination herein and thereof.
  • an immunostimulatory adjuvant can comprise LTA, poly(I:C), R-848, anti-CD40 antibody, any derivatives herein and thereof, any combinations herein and thereof.
  • Toll-like receptors (TLRs) [0074]
  • the innate immune response pathway comprises cellular components that recognize the PAMPs or DAMPs. Such cellular components can comprise the Toll pathway and cytoplasmic pathway.
  • the cellular components of innate immunity can comprise antigen-presenting dendritic cells (DCs), phagocytic macrophages and granulocytes, cytotoxic natural killer (NK) cells, and gamma-delta T lymphocytes.
  • the Toll pathway uses a class of receptors, TLRs, in the PAMP recognition and immune pathway activation. Upon activation of PAMP, TLRs recruit adaptor proteins to propagate PAMP/antigen-induced signal transduction pathway in the inflammatory response.
  • TLRs are type I integral membrane receptors, each with an N-terminal ligand recognition domain, a single transmembrane helix, and a C-terminal cytoplasmic signaling domain.
  • TLRs can comprise TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, any derivatives herein and thereof, any combinations herein and thereof.
  • a TLR can be expressed on the membranes of dendritic cells, macrophages, natural killer cells, T cells, B cells, epithelial cells, endothelial cells, or fibroblasts.
  • TLRs can be activated by PAMPs or DAMPs described herein and thereof. TLRs can also be activated by small molecules described herein and thereof.
  • a TLR ligand can comprise beta-defensin 2, fibronectin, HMGB1, HSP22, HSP70, HSP72, endoplasmin, alpha-crystallin A chain, human cardiac myosin, resistin, S100 proteins, surfactant proteins A, tenascin-C, biglycan, CD138, oligosaccharides of hyaluronan, hyaluronan breakdown fragments, mRNA, small interfering RNA (siRNA), oxidized PAPC (OxPAPC), monosodium urate crystals, etc.
  • a TLR agonist can comprise live, dead, or a fragment of bacteria, fungi, protist, protozoa, nematodes, plant cells or animal cells.
  • a TLR agonist can comprise a virus or an inactivated virus.
  • a TLR agonist can also comprise OX40, OX40 ligand, 4-1-BB, 4-1BB ligand, CD27, CD30, CD30 ligand, HVEM, TROY, RELT, TNF-alpha, TNF-beta, CD70, RANK ligand, LT-alpha, LT-beta, GITR ligand and LIGHT, MALP-2, IRM compounds, derivatives herein and thereof, combinations herein and thereof, and other TLR agonists known in the art.
  • a TLR ligand can be a synthetic ligand.
  • a synthetic ligand can comprise Pam3Cys, CFA, MALP-2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, polyadenosine-polyuridylic acid (poly A:U), AGP, Monophosphoryl lipid A (MPLA), RC-529, MDF2 beta, flagellin, guanosine analogs, imidazaquinolines, or loxoribine.
  • Imidazoquinoles can comprise Imiquimod, Aldara, R-848, ssPolyU, 3M-012, CpG-oligonucleotides, or Resiquimod, polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Poly-ICLC), HLA-A2 restricted peptides, Sialyl-Tn (STn), keyhole limpet haemocyanin (KLH), derivatives herein and thereof, or combinations herein and thereof.
  • Lipoteichoic acid (LTA) [0081]
  • a personalized tumor vaccine can comprise LTA.
  • Lipoteichoic acid (LTA) is a surface- associated adhesion molecule.
  • LTA is a major component of the cell wall of gram-positive bacteria. LTA can be secreted by the bacteria. LTA can be maintained as a lyophilized powder. Different gram-positive bacterial species can have different structures of LTA. LTA is anchored to the cell membrane by diacylglycerol. LTA can also comprise ribitol or plycerol phosphate. LTA can induce a proinflammatory cytokine response in the immune system. An exemplary chemical structure of LTA is shown in Formula III. [0082] [0083] LTA is a TLR agonist: LTA can bind to TLR-2 to induce NF- K B TNF alpha expression. LTA can induce the complement system to induce passive immune kill phenomenon.
  • LTA can also induce release of the reactive oxygen and nitrogen species from neutrophils and macrophage, acid hydrolases, cationic proteinases, or cytotoxic cytokines. LTA can also activate scavenger receptors. In some embodiments, LTA can link to tumor or cancer cells and the fragments of tumor or cancer cell membranes can support scavenger receptors-mediated recognition of cancer antigen by dendritic cells. In some cases, LTA can be a PAMP. In other case, LTA can be an immunostimulatory adjuvant. [0084] LTA can comprise LTA from any gram-positive bacteria.
  • LTA can comprise LTA from Bacillus subtilis, Enterococcus hirae, Staphylococcus aureus, or Streptococcus pyogenes.
  • a personalized tumor vaccine can comprise 1x10 ⁇ 0 g/dose, 2x10 ⁇ 0 g/dose, 3x10 ⁇ 0 g/dose, 4x10 ⁇ 0 g/dose, 5x10 ⁇ 0 g/dose, 6x10 ⁇ 0 g/dose, 7x10 ⁇ 0 g/dose, 8x10 ⁇ 0 g/dose, 9x10 ⁇ 0 g/dose, 1x10 ⁇ -1 g/dose, 2x10 ⁇ -1 g/dose, 3x10 ⁇ -1 g/dose, 4x10 ⁇ -1 g/dose, 5x10 ⁇ -1 g/dose, 6x10 ⁇ -1 g/dose, 7x10 ⁇ -1 g/dose, 8x10 ⁇ -1 g/dose, 9x10 ⁇ -1 g/dose, 1x10 ⁇ -2 g/dose, 2x10 ⁇ -2 g/dose
  • a personalized tumor vaccine can comprise from 5x10 ⁇ 0 g/dose to 1x10 ⁇ 1 g/dose, from 1x10 ⁇ 0 g/dose to 5x10 ⁇ 0 g/dose, from 5x10 ⁇ -1 g/dose to 1x10 ⁇ 0 g/dose, from 1x10 ⁇ -1 g/dose to 5x10 ⁇ -1 g/dose, from 5x10 ⁇ -2 g/dose to 1x10 ⁇ -1 g/dose, from 1x10 ⁇ -2 g/dose to 5x10 ⁇ -2 g/dose, from 5x10 ⁇ -3 g/dose to 1x10 ⁇ -2 g/dose, from 1x10 ⁇ -3 g/dose to 5x10 ⁇ -3 g/dose, from 5x10 ⁇ -4 g/dose to 1x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -4 g/dose, from 5x10 ⁇ -5 g/dose to 1x10 ⁇ -4
  • a personalized tumor vaccine can also comprise about 0.05 to 5 mg/dose LTA.
  • a personalized tumor vaccine can also comprise about 0.05 mg/dose LTA.
  • a personalized tumor vaccine can also comprise about 5 mg/dose LTA.
  • a personalized tumor vaccine can also comprise about 0.024 mg/dose LTA.
  • a personalized tumor vaccine can also comprise about 0.584 mg/dose LTA.
  • Polyinosinic:polycytidylic acid (Poly(I:C)) [0085]
  • a personalized tumor vaccine can comprise Poly (I:C).
  • Poly(I:C) can activate TLR3, expressed at the cell membrane of B-cells, macrophages, and dendritic cells. Because of its structural similarity to dsRNA found in virus, poly(I:C) is a TLR agonist.
  • Poly(I:C) and its derivatives can be used as an agonist or activator of TLR3. Such activation can further activate the transcription factor interferon regulatory factor 3 (IRF3) and produce type I IFN-beta.
  • Poly(I:C) can also generate stable mature dendritic cells.
  • poly(I:C) can be a PAMP.
  • poly(I:C) can be an immunostimulatory adjuvant.
  • Derivatives of poly(I:C) can comprise poly-L-lysine and carboxymethylcellulose (Poly ICLC) or Poly I:C 12 U. Derivatives of poly(I:C) can also comprise any chemical known by an ordinary person in the cancer or immunity biology or therapy art.
  • Poly(I:C) can comprise a mismatched double-stranded nucleic acid.
  • a nucleic acid strand in poly(I:C) can comprise RNA.
  • Poly(I:C) can comprise a polymer of inosinic acid and cytidylic acid.
  • Poly(I:C) can be maintained or administered to a subject as a sodium salt or potassium salt. Poly(I:C) can also be maintained as a lyophilized powder.
  • a personalized tumor vaccine can comprise 1x10 ⁇ 0 g/dose, 2x10 ⁇ 0 g/dose, 3x10 ⁇ 0 g/dose, 4x10 ⁇ 0 g/dose, 5x10 ⁇ 0 g/dose, 6x10 ⁇ 0 g/dose, 7x10 ⁇ 0 g/dose, 8x10 ⁇ 0 g/dose, 9x10 ⁇ 0 g/dose, 1x10 ⁇ -1 g/dose, 2x10 ⁇ -1 g/dose, 3x10 ⁇ -1 g/dose, 4x10 ⁇ -1 g/dose, 5x10 ⁇ -1 g/dose, 6x10 ⁇ -1 g/dose, 7x10 ⁇ -1 g/dose, 8x10 ⁇ -1 g/dose, 9x10 ⁇ -1 g/dose, 1x10 ⁇ -2 g/dose, 2x10 ⁇ -2 g/dose, 3x10 ⁇ -2 g/dose, 4x10 ⁇ -2 g/dose, 5x10 ⁇ -2 g/dose, 6x10 ⁇ -2 g/dose, 7x10 ⁇ -2 g/
  • a personalized tumor vaccine can comprise from 5x10 ⁇ 0g to 1x10 ⁇ 1 g/dose, from 1x10 ⁇ 0 g/dose to 5x10 ⁇ 0 g/dose, from 5x10 ⁇ -1 g/dose to 1x10 ⁇ 0 g/dose, from 1x10 ⁇ -1 g/dose to 5x10 ⁇ -1 g/dose, from 5x10 ⁇ -2 g/dose to 1x10 ⁇ -1 g/dose, from 1x10 ⁇ -2 g/dose to 5x10 ⁇ -2 g/dose, from 5x10 ⁇ -3 g/dose to 1x10 ⁇ -2 g/dose, from 1x10 ⁇ -3 g/dose to 5x10 ⁇ -3 g/dose, from 5x10 ⁇ -4 g/dose to 1x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -4 g/
  • a personalized tumor vaccine can also comprise about 0.05 to 5 mg/dose poly(I:C).
  • a personalized tumor vaccine can also comprise about 0.05 mg/dose poly(I:C).
  • a personalized tumor vaccine can also comprise about 5 mg/dose poly(I:C).
  • a personalized tumor vaccine can also comprise about 0.024 mg/dose poly(I:C).
  • a personalized tumor vaccine can also comprise about 0.584 mg/dose poly(I:C).
  • Resiquimod (R-848) [0093]
  • a personalized tumor vaccine can comprise a Resiquimod (R-848).
  • R-848 is an imidazoquinolinamine TLR agonist.
  • R-848 may have antitumor or antiviral activity. In some cases, R-848 can be a PAMP.
  • R-848 can be used as an immunostimulatory adjuvant.
  • R-848 can activate TLR7/8. Activation of TLR7/8 by R-848 can lead to the production of cytokines, especially interferon-alpha (INF- ⁇ ), and enhanced T-helper 1 (Th1) immune responses.
  • R-848 can also stimulate the maturation of dendritic cells.
  • R-848 can also activate macrophages and B-cells.
  • R-848 is an analogue of imiquimod. Aa exemplary structure of R-848 is listed in Formula V. [0095] Formula V: [0096] R-848 can also comprise C 17 H 22 N 4 O 2 .
  • R-848 can have a molecular weight of 314.4 g/mol.
  • a personalized tumor vaccine can comprise 1x10 ⁇ 0 g/dose, 2x10 ⁇ 0 g/dose, 3x10 ⁇ 0 g/dose, 4x10 ⁇ 0 g/dose, 5x10 ⁇ 0 g/dose, 6x10 ⁇ 0 g/dose, 7x10 ⁇ 0 g/dose, 8x10 ⁇ 0 g/dose, 9x10 ⁇ 0 g/dose, 1x10 ⁇ -1 g/dose, 2x10 ⁇ -1 g/dose, 3x10 ⁇ -1 g/dose, 4x10 ⁇ -1 g/dose, 5x10 ⁇ -1 g/dose, 6x10 ⁇ -1 g/dose, 7x10 ⁇ -1 g/dose, 8x10 ⁇ -1 g/dose, 9x10 ⁇ -1 g/dose, 1x10 ⁇ -2 g/dose, 2x10 ⁇
  • a personalized tumor vaccine can comprise from 5x10 ⁇ 0g to 1x10 ⁇ 1 g/dose, from 1x10 ⁇ 0 g/dose to 5x10 ⁇ 0 g/dose, from 5x10 ⁇ -1 g/dose to 1x10 ⁇ 0 g/dose, from 1x10 ⁇ -1 g/dose to 5x10 ⁇ -1 g/dose, from 5x10 ⁇ -2 g/dose to 1x10 ⁇ -1 g/dose, from 1x10 ⁇ -2 g/dose to 5x10 ⁇ -2 g/dose, from 5x10 ⁇ -3 g/dose to 1x10 ⁇ -2 g/dose, from 1x10 ⁇ -3 g/dose to 5x10 ⁇ -3 g/dose, from 5x10 ⁇ -4 g/dose to 1x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -4 g/
  • a personalized tumor vaccine can also comprise about 0.05 to 5 mg/dose R-848.
  • a personalized tumor vaccine can also comprise about 0.05 mg/dose R-848.
  • a personalized tumor vaccine can also comprise about 5 mg/dose R-848.
  • a personalized tumor vaccine can also comprise about 0.024 mg/dose R-848.
  • a personalized tumor vaccine can also comprise about 0.584 mg/dose R-848.
  • mAb monoclonal antibody
  • Activation of CD40 can induce dendritic cell licensing, T-cell activation, cytokine production, macrophage infiltration of the tumors, and other antitumor responses.
  • CD40 agonists can be used activate CD40 and to induce antitumor immune response.
  • a CD40 agonist can comprise an anti-CD40 monoclonal antibody (mAb).
  • a personalized tumor vaccine can comprise 1x10 ⁇ 0 g/dose, 2x10 ⁇ 0 g/dose, 3x10 ⁇ 0 g/dose, 4x10 ⁇ 0 g/dose, 5x10 ⁇ 0 g/dose, 6x10 ⁇ 0 g/dose, 7x10 ⁇ 0 g/dose, 8x10 ⁇ 0 g/dose, 9x10 ⁇ 0 g/dose, 1x10 ⁇ -1 g/dose, 2x10 ⁇ -1 g/dose, 3x10 ⁇ -1 g/dose, 4x10 ⁇ -1 g/dose, 5x10 ⁇ -1 g/dose, 6x10 ⁇ -1 g/dose, 7x10 ⁇ -1 g/dose, 8x10 ⁇ -1 g/dose, 9x10 ⁇ -1 g/dose, 1x10 ⁇ -2 g/dose, 2x10 ⁇ -2 g/dose, 2
  • a personalized tumor vaccine can comprise from 5x10 ⁇ 0 g/dose to 1x10 ⁇ 1 g/dose, from 1x10 ⁇ 0 g/dose to 5x10 ⁇ 0 g/dose, from 5x10 ⁇ -1 g/dose to 1x10 ⁇ 0 g/dose, from 1x10 ⁇ -1 g/dose to 5x10 ⁇ -1 g/dose, from 5x10 ⁇ -2 g/dose to 1x10 ⁇ -1 g/dose, from 1x10 ⁇ -2 g/dose to 5x10 ⁇ -2 g/dose, from 5x10 ⁇ -3 g/dose to 1x10 ⁇ -2 g/dose, from 1x10 ⁇ -3 g/dose to 5x10 ⁇ -3 g/dose, from 5x10 ⁇ -4 g/dose to 1x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -3 g/dose, from 1x10 ⁇ -4 g/dose to 5x10 ⁇ -4 g/dose, from 5x10 ⁇ -5 g/dose to 1x10 ⁇ -4
  • a personalized tumor vaccine can also comprise about 0.04 mg/dose to about 4 mg/dose anti-CD40 mAb.
  • a personalized tumor vaccine can also comprise about 0.04 mg/dose anti-CD40 mAb.
  • a personalized tumor vaccine can also comprise about 4 mg/dose anti-CD40 mAb.
  • a personalized tumor vaccine can also comprise about 0.02 mg/dose anti-CD40 mAb.
  • a personalized tumor vaccine can also comprise about 0.467 mg/dose anti-CD40 mAb.
  • a personalized tumor vaccine can also replace, substitute or combine an anti-CD40 mAb with a CD40 agonist described herein and thereof.
  • a CD40 agonist can also comprise trimer CD40 ligand (CD40L), ectopic expression of CD40L, any derivatives herein and thereof, or any combinations herein and thereof.
  • an anti-CD40 antibody can comprise ABBV-927, ADC-1013, Selicrelumab, APX005M, ChiLob7/4, ADC-1013, SEA-CD40, CDX1140, CP-870893, dacetuzmab, any derivatives herein and thereof, or any combinations herein and thereof.
  • a CD40 agonist can also comprise CD40L, GM.CD40L, MEDI5083 (NCT03089645), HERACD40L, duokine, and derivatives herein and thereof, or any combinations herein and thereof.
  • An antibody can comprise IgA, IgD, IgE, IgG, IgM, any derivatives herein and thereof, or any combinations herein and thereof.
  • an anti-CD40 antibody can comprise a murine, human, chimeric, or humanized antibody. In other cases, an anti-CD40 antibody can comprise a polyclonal or monoclonal antibody.
  • an antibody can comprise an antibody from chickens, goats, guinea pigs, hamsters, horses, mice, rats, sheep, monkeys, chimpanzees, humans, camels, sharks, rabbits, alpaca, llama, or any combinations thereof.
  • an anti-CD40 antibody can comprise an intact antibody or an antibody fragment.
  • an anti-CD40 antibody can comprise IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, any derivatives herein and thereof, or any combinations herein and thereof.
  • an anti-CD40 antibody can comprise a bispecific antibody, monoclonal antibody, single-chain variable fragment (scFv), single-chain antigen-binding fragment (scFab), Dual-variable domains Ig (DVD-Ig), scFv- IgG fusion, scFv-Fc (constant region), heavy chain antibody (HcAb), new antigen receptor antibody (IgNAR), domain antibody (dAb), single-dAb (sdAb), diabody, intrabody, trioMab, F(ab)2 bispecific antibody, F(ab)3 trispecific antibody, BiTE antibody, DART antibody, tand antibody, minibody, Bis-scFv, triabody, tetrabody, camel Ig, shark Ig, fragments herein and thereof, derivatives herein and thereof, or any combinations herein and thereof.
  • scFv single-chain variable fragment
  • scFab single-chain antigen-binding fragment
  • Dual-variable domains Ig VD
  • activation of CD40 and TLRs can independently activate distinct immune response pathways. In some cases, activation of CD40 and TLRs can activate overlapping immune response pathways. In some embodiments, activation of CD40 and TLRs have an additive effect on the activation of immune response pathway. In other cases, activation of CD40 and TLRs have a synergistic effect on the activation immune response pathways. In some embodiments, activation of CD40 and TLRs can have an additive or synergistic effect on an antitumor response.
  • an antitumor response can comprise inhibiting, reversing, stropping, reducing, restraining, suppressing, or prohibiting the growth, metastasis of tumors, differentiation, migration, division, or secretion of tumor or tumor cells.
  • Organic and inorganic adjuvants [0103] In some instances, an immunostimulatory adjuvant can comprise an organic molecule immunostimulatory adjuvant. In some cases, an immunostimulatory adjuvant can comprise an inorganic molecule immunostimulatory adjuvant.
  • An organic immunostimulatory adjuvant can comprise inactivated Mycobacterium tuberculosis, squalene, saponins, Monophosphoryl lipid A (MPL), any derivatives herein and thereof, or any combination herein and thereof.
  • MPL Monophosphoryl lipid A
  • an organic immunostimulatory adjuvant can also comprise nucleic acid sequences encoding immune regulatory lymphokines such as IL-18, IL-1 beta, IL-2, IL-12, IL-15, IL-4, IL10, ⁇ interferon, NF ⁇ B regulatory signal protein, any derivatives herein and thereof, or any combination herein and thereof.
  • immune regulatory lymphokines such as IL-18, IL-1 beta, IL-2, IL-12, IL-15, IL-4, IL10, ⁇ interferon, NF ⁇ B regulatory signal protein, any derivatives herein and thereof, or any combination herein and thereof.
  • an organic immunostimulatory adjuvant can comprise Trehalose-6,6′-dimycolate (TDM), muramyl dipeptide (MDP); AF or SPT (emulsion of squalane (5%); Tween 80 (0.2%); AVRIDINETM (propanediamine); BAY R1005TM ((N-(2-deoxy-2- L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydroacetate); CALCITRIOLTM (1-alpha,25-dihydroxy-vitamin D3); cholera holotoxin; cholera-toxin-A1-protein- A-D-fragment fusion protein; sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide
  • MPLTM (3-Q-desacyl-4′-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L- alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))- ethylamide, monosodium salt); MURAMETIDETM (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINETM and D-MURAPALMITINETM (Nac-Mur-L-Thr-D-isoGln-sn- glyceroldipalmitoyl
  • An organic immunostimulatory adjuvant can also comprise cationic or polycationic compounds.
  • cationic or polycationic compounds can comprise cationic or polycationic peptides or proteins, including protamine, nucleoline, spermin or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin- rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(
  • cationic or polycationic compounds can comprise cationic polysaccharides.
  • cationic polysaccharides can comprise chitosan; polybrene; cationic polymers comprising polyethyleneimine (PEI); cationic lipids comprising DOTMA: 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt); DMRIE; di-C14-amidine; DOTIM; SAINT; DC-Chol; BGTC; CTAP: DOPC; DODAP; DOPE: Dioleyl phosphatidylethanol-amine; DOSPA; DODAB; DOIC; DMEPC; DOGS: Dioctadecylamidoglicylspermin; DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide; DOTAP: dioleoyloxy-3-(trimethylammonio)propane; DC-6-14: O,O-ditetradecano
  • an organic immunostimulatory adjuvant can comprise emulsion adjuvant.
  • an emulsion adjuvant can comprise squalene; DL-a-tocopherol (vitamin E); lipids from the Salmonella minnesota bactrerium or Quillaja saponaria tree; AS01B; AS01E; AS03; AS04; CFA; SAF; IFA; MF59; Provax; TiterMax; Montanide; or Vaxfectin.
  • an organic immunostimulatory adjuvant can comprise derivatives or combinations of any organic stimulatory adjuvants herein and thereof; microspheres and microparticles of any organic adjuvants described herein and thereof, or nanospheres or nanoparticles of any organic adjuvants described herein and thereof.
  • an immunostimulatory adjuvant can comprise an inorganic molecule immunostimulatory adjuvant.
  • An inorganic immunostimulatory adjuvant can comprise salts of metals such as cerium, zinc, iron, aluminum and calcium.
  • an inorganic immunostimulatory adjuvant can comprise an aluminum-containing compound (alum).
  • Alum can comprise materials with a formula KAl(SO 4 ) 2 ⁇ 12H 2 O, (NH 4 + )(Al(SO 4 ) 2 ) ⁇ 12H 2 O any derivatives herein and thereof, or any combinations herein and thereof.
  • Alum can also comprise aluminum oxyhydroxide (AlO(OH)) or aluminum hydroxyphosphate (Al(OH) x (PO 4 ) y ).
  • an aluminum-containing compound can also comprise Imject TM alum.
  • Imject TM alum can comprise crystalline magnesium hydroxide and aluminum hydroxide.
  • Alum can also comprise Alhydrogel®, Adju-Phos®, ADJUMERTM (polyphosphazene), aluminium phosphate gel, calcium phosphate gel, CAPTM (calcium phosphate nanoparticles), cesium alum, phosphate-buffered saline, Rehydragel TM , any derivatives herein and thereof, or any combinations herein and thereof.
  • Attenuated cancer cells [0106]
  • a personalized tumor vaccine can comprise a population or groups of attenuated cancer cells.
  • An attenuated cancer cell can comprise a cancer cell or population or group of cancer cells undergoing or undergone treatments. In some instances, an attenuated cancer cell can be a mammalian cell.
  • an attenuated cancer cell can be a non-mammalian cell. In some instances, an attenuated cancer cell can comprise a mouse cell. In some instances, an attenuated cancer cell can be a human cell. In some instances, an attenuated cancer cell can be a living cell. In some embodiments, an attenuated cancer cell can comprise an aged, stressed, injured, infected, or transformed cells not found in normal or healthy cells. In some instances, an attenuated cancer cell can comprise a tumor or cancer cell. In some instances, an attenuated cancer cell can comprise an overgrown cell. In some instances, an attenuated cancer cell can comprise an over- divided cell. In some cases, an attenuated cancer cell can comprise an over-differentiated cell.
  • an attenuated cancer cell can comprise a stem cell. In some instances, an attenuated cancer cell can comprise a cancer stem cell. In some instances, an attenuated cancer cell can comprise a non-dividing cell. In some instances, an attenuated cancer cell can comprise a mitotic cell. In some instances, an attenuated cancer cell can comprise a meiotic cell. In some cases, an attenuated cancer cell can comprise a growth-arrested cell. In some cases, an attenuated cancer cell can comprise a replication-arrested cell. In some cases, an attenuated cancer cell can comprise a division-arrested cell. In some cases, an attenuated cancer cell can comprise a mitosis-arrested cell.
  • an attenuated cancer cell can comprise a differentiation-arrested cell. In some cases, an attenuated cancer cell can comprise a meiosis-arrested cell. In some embodiments, an attenuated cancer cell can comprise a monkey, chimpanzee, rat, rabbit, pig, guinea pig, horse, camel, alpaca, llama, dog, cat, cow cell. [0107] In some cases, an attenuated cancer cell can comprise a primary tumor or cancer cell. In some cases, an attenuated cancer cell can comprise a cultured cancer cell. In some instances, an attenuated cancer cell can comprise a tumor or cancer cell extracted from an individual and cultured in vitro after the extraction.
  • an extraction can comprise harvesting a tumor or cancer cell from a biopsy of tissues from an individual. In some instances, an extraction can comprise harvesting a tumor or cancer cell from a biopsy of a site of tumor or cancer from the individual. In some cases, an extraction can comprise harvesting a tumor or cancer cell from a biopsy of circulating tumor or cancer cells from an individual.
  • a biopsy can comprise a bone biopsy, a bone marrow biopsy, a breast biopsy, a gastrointestinal biopsy, a lung biopsy, a liver biopsy, a prostate biopsy, a nervous system biopsy, a urogenital biopsy, a lymph node biopsy, a muscle biopsy, a skin biopsy, a blood biopsy, a body fluid biopsy, a cardiac biopsy, an endometrial biopsy, an open biopsy, a sentinel lymph node biopsy, any derivatives herein and thereof, or any combinations herein and thereof.
  • a biopsy can comprise a fine needle aspiration biopsy, a core needle biopsy, a vacuum-assisted biopsy, an excisional biopsy, a shave biopsy, a punch biopsy, an endoscopic biopsy, a laparoscopic biopsy, a bone marrow aspiration biopsy, a liquid biopsy, any derivatives herein and thereof, or any combinations herein and thereof.
  • a biopsy can comprise an incisional biopsy or an excisional biopsy.
  • an attenuated cancer cell can comprise a tumor or cancer cell extracted from an individual without any culturing in vitro after the extraction.
  • a personalized tumor vaccine can comprise any types of cells described herein and thereof.
  • a personalized tumor vaccine can comprise from 1x10 ⁇ 0 to 1x10 ⁇ 1 attenuated cancer cells, from 5x10 ⁇ 0 to 5x10 ⁇ 1 attenuated cancer cells, from 1x10 ⁇ 1 to 1x10 ⁇ 2 attenuated cancer cells, from 5x10 ⁇ 1 to 5x10 ⁇ 2 attenuated cancer cells, from 1x10 ⁇ 2 to 1x10 ⁇ 3 attenuated cancer cells, from 5x10 ⁇ 2 to 5x10 ⁇ 3 attenuated cancer cells, from 1x10 ⁇ 3 to 1x10 ⁇ 4 attenuated cancer cells, from 5x10 ⁇ 3 to 5x10 ⁇ 4 attenuated cancer cells, from 1x10 ⁇ 4 to 1x10 ⁇ 5 attenuated cancer cells, from 5x10 ⁇ 4 to 5x10 ⁇ 5 attenuated cancer cells, from 1x10 ⁇ 5 to 1x10 ⁇ 6 attenuated cancer cells, from 5x10 ⁇ 5 to 5x10 ⁇ 6 attenuated cancer cells, from 1x10 ⁇ 6 to 1x10 ⁇ 7 attenuated cancer
  • a personalized tumor vaccine can comprise 1x10 ⁇ 5, 2x10 ⁇ 5, 3x10 ⁇ 5, 4x10 ⁇ 5, 5x10 ⁇ 5, 6x10 ⁇ 5, 7x10 ⁇ 5, 8x10 ⁇ 5, 9x10 ⁇ 5, 1x10 ⁇ 6, 2x10 ⁇ 6, 3x10 ⁇ 6, 4x10 ⁇ 6, 5x10 ⁇ 6, 6x10 ⁇ 6, 7x10 ⁇ 6, 8x10 ⁇ 6, 9x10 ⁇ 6, or 1x10 ⁇ 7 attenuated cancer cells.
  • a personalized tumor vaccine can comprise 1 x10 ⁇ 6 attenuated cancer cells.
  • a therapeutically relevant amount of attenuated cancer cells can comprise from 1x10 ⁇ 0 to 1x10 ⁇ 1 attenuated cancer cells, from 5x10 ⁇ 0 to 5x10 ⁇ 1 attenuated cancer cells, from 1x10 ⁇ 1 to 1x10 ⁇ 2 attenuated cancer cells, from 5x10 ⁇ 1 to 5x10 ⁇ 2 attenuated cancer cells, from 1x10 ⁇ 2 to 1x10 ⁇ 3 attenuated cancer cells, from 5x10 ⁇ 2 to 5x10 ⁇ 3 attenuated cancer cells, from 1x10 ⁇ 3 to 1x10 ⁇ 4 attenuated cancer cells, from 5x10 ⁇ 3 to 5x10 ⁇ 4 attenuated cancer cells, from 1x10 ⁇ 4 to 1x10 ⁇ 5 attenuated cancer cells, from 5x10 ⁇ 4 to 5x10 ⁇ 5 attenuated cancer cells, from 1x10 ⁇ 5 to 1x10 ⁇ 6 attenuated cancer cells, from 5x10 ⁇ 5 to 5x10 ⁇ 6 attenuated cancer cells, from 1x10 ⁇ 6 to 1xx10 ⁇
  • a therapeutically relevant amount of attenuated cancer cells can comprise 1x10 ⁇ 5, 2x10 ⁇ 5, 3x10 ⁇ 5, 4x10 ⁇ 5, 5x10 ⁇ 5, 6x10 ⁇ 5, 7x10 ⁇ 5, 8x10 ⁇ 5, 9x10 ⁇ 5, 1x10 ⁇ 6, 2x10 ⁇ 6, 3x10 ⁇ 6, 4x10 ⁇ 6, 5x10 ⁇ 6, 6x10 ⁇ 6, 7x10 ⁇ 6, 8x10 ⁇ 6, 9x10 ⁇ 6, or 1x10 ⁇ 7 attenuated cancer cells.
  • a therapeutically relevant amount of attenuated cancer cells can comprise 1 x10 ⁇ 6 attenuated cancer cells [0111]
  • a treatment of obtaining an attenuated cancer cell can comprise a physical or a chemical treatment of a cancer cell.
  • a treatment can stress, arrest, growth-inhibit, or injure a cancer cell.
  • a treatment can change the chemical or physical composition of a cancer cell.
  • a treatment can increase or decrease a component, molecule, chemical, or any derivatives herein and thereof of a cancer cell.
  • a treatment can damage the DNA of a cancer cell.
  • a treatment can stress, arrest, growth-inhibit, or injure 0 %, 1 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 % of a population or group of cells.
  • a treatment can stress, arrest, growth-inhibit, or injure from 1 to 5 %, from 2 to 6 %, from 3 to 7 %, from 4 to 8 %, from 5 to 9 %, from 6 to 10 %, from 7 to 11 %, from 8 to 12 %, from 9 to 13 %, from 10 to 14 %, from 11 to 15 %, from 12 to 16 %, from 13 to 17 %, from 14 to 18 %, from 15 to 19 %, from 16 to 20 %, from 17 to 21 %, from 18 to 22 %, from 19 to 23 %, from 20 to 24 %, from 21 to 25 %, from 22 to 26 %, from 23 to 27 %, from 24 to 28 %, from 25 to 29 %, from 26 to 30 %, from 27 to 31 %, from 28 to 32 %, from 29 to 33 %, from 30 to 34 %, from 31 to 35 %, from 32 to 36 %, from 33 to 37 %, from 34 to 38 %
  • a personalized tumor vaccine can comprise administering a treatment to a cancer cell or population of cancer cells.
  • a treatment can comprise an irradiation treatment.
  • an irradiation treatment can comprise using of radio waves, microwaves, infrared (IR), visible light, ultraviolet (UV), X-rays and gamma rays.
  • a gamma ray irradiation treatment can comprise 1Gy, 10 Gy, 20 Gy, 30 Gy, 40 Gy, 50 Gy, 60 Gy, 70 Gy, 80 Gy, 90 Gy, 100 Gy, 110 Gy, 120 Gy, 130 Gy, 140 Gy, 150 Gy, 160 Gy, 170 Gy, 180 Gy, 190 Gy, 200 Gy, 210 Gy, 220 Gy, 230 Gy, 240 Gy, 250 Gy, 260 Gy, 270 Gy, 280 Gy, 290 Gy, 300 Gy, 310 Gy, 320 Gy, 330 Gy, 340 Gy, 350 Gy, 360 Gy, 370 Gy, 380 Gy, 390 Gy, 400 Gy, 410 Gy, 420 Gy, 430 Gy, 440 Gy, 450 Gy, 460 Gy, 470 Gy, 480 Gy, 490Gy, or 500Gy gamma radiation.
  • a gamma ray irradiation can comprise from 1 to 25 Gy, from 2 to 26 Gy, from 3 to 27 Gy, from 4 to 28 Gy, from 5 to 29 Gy, from 6 to 30 Gy, from 7 to 31 Gy, from 8 to 32 Gy, from 9 to 33 Gy, from 10 to 34 Gy, from 11 to 35 Gy, from 12 to 36 Gy, from 13 to 37 Gy, from 14 to 38 Gy, from 15 to 39 Gy, from 16 to 40 Gy, from 17 to 41 Gy, from 18 to 42 Gy, from 19 to 43 Gy, from 20 to 44 Gy, from 21 to 45 Gy, from 22 to 46 Gy, from 23 to 47 Gy, from 24 to 48 Gy, from 25 to 49 Gy, from 26 to 50 Gy, from 27 to 51 Gy, from 28 to 52 Gy, from 29 to 53 Gy, from 30 to 54 Gy, from 31 to 55 Gy, from 32 to 56 Gy, from 33 to 57 Gy, from 34 to 58 Gy, from 35 to 59 Gy, from 36 to 60 Gy, from 37 to 61 Gy, from 38 to 62 Gy, from 39 to 63 Gy,
  • a gamma ray irradiation can comprise from 50 Gy gamma radiation.
  • a treatment or a gamma ray irradiation treatment can comprise 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, or 1 hr, 55 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minutes, or less than 1 minute.
  • a cancer cell can undergo one treatment.
  • a cancer cell can undergo 2, 3, 4, 5, 6, 7, 8, 9, 10, or more treatments.
  • a personalized tumor vaccine can comprise from 1x10 ⁇ 0 to 1x10 ⁇ 1 gamma ray irradiated cancer cells, from 5x10 ⁇ 0 to 5x10 ⁇ 1 gamma ray irradiated cancer cells, from 1x10 ⁇ 1 to 1x10 ⁇ 2 gamma ray irradiated cancer cells, from 5x10 ⁇ 1 to 5x10 ⁇ 2 gamma ray irradiated cancer cells, from 1x10 ⁇ 2 to 1x10 ⁇ 3 gamma ray irradiated cancer cells, from 5x10 ⁇ 2 to 5x10 ⁇ 3 gamma ray irradiated cancer cells, from 1x10 ⁇ 3 to 1x10 ⁇ 4 gamma ray irradiated cancer cells, from 5x10 ⁇ 3 to 5x10 ⁇ 4 gamma ray irradiated cancer cells, from 5x10 ⁇ 3 to 5x
  • a personalized tumor vaccine can comprise 1x10 ⁇ 5, 2x10 ⁇ 5, 3x10 ⁇ 5, 4x10 ⁇ 5, 5x10 ⁇ 5, 6x10 ⁇ 5, 7x10 ⁇ 5, 8x10 ⁇ 5, 9x10 ⁇ 5, 1x10 ⁇ 6, 2x10 ⁇ 6, 3x10 ⁇ 6, 4x10 ⁇ 6, 5x10 ⁇ 6, 6x10 ⁇ 6, 7x10 ⁇ 6, 8x10 ⁇ 6, 9x10 ⁇ 6, or 1x10 ⁇ 7 gamma ray irradiated cancer cells.
  • a personalized tumor vaccine can comprise 1 x10 ⁇ 6 gamma ray irradiated cancer cells.
  • a personalized tumor vaccine can comprise 2 x10 ⁇ 6 gamma ray irradiated cancer cells.
  • a personalized tumor vaccine can comprise 2.5 x10 ⁇ 6 gamma ray irradiated cancer cells.
  • a personalized tumor vaccine can comprise 5 x10 ⁇ 6 gamma ray irradiated cancer cells.
  • a personalized tumor vaccine can comprise 5.5 x10 ⁇ 6 gamma ray irradiated cancer cells.
  • a treatment of obtaining an attenuated cancer cell can also comprise a chemical treatment.
  • a chemical treatment can comprise reactive oxygen species (ROS) (e.g., superoxide, hydroxyl radicals and hydrogen peroxide), deaminating agents (e.g., nitrous acid, which can cause transition mutations by converting cytosine to uracil.), polycyclic aromatic hydrocarbon (PAH), alkylating agents (e.g., ethylnitrosourea, nitrosamines, mustard gas and vinyl chloride), aromatic amines and amides (e.g., 2-Acetylaminofluorene), alkaloid from plants, bromine and some compounds that contain bromine in their chemical structure, sodium azide, psoralen, benzene, ethidium bromide, proflavine, daunorubicin, and some metals (arsenic, cadmium, chromium, nickel, iron, cobalt).
  • ROS reactive oxygen species
  • deaminating agents e.g., nitrous acid, which can cause transition mutations by
  • a subject-specific pharmaceutical composition can be a prophylactic or preventative composition.
  • a subject-specific pharmaceutical composition can be a therapeutic or treatment pharmaceutical composition.
  • a subject-specific pharmaceutical composition can be administered to a subject in need thereof before the subject is diagnosed with a cancer.
  • a subject-specific pharmaceutical composition can be administered to a subject in need thereof after the subject is diagnosed with a cancer.
  • a subject-specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof; an immunostimulatory adjuvant or derivative herein and thereof; and from about 1.0 ⁇ 10 ⁇ 3 to about 1.0 ⁇ 10 ⁇ 7 attenuated cancer cells.
  • a subject specific pharmaceutical composition can comprise a mannan; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise about 2 mg/dose to about 200 mg/dose mannan; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof conjugated to biocompatible anchor for cell membrane (BAM); an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • BAM biocompatible anchor for cell membrane
  • a subject specific pharmaceutical composition can comprise a mannan conjugated to BAM an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a mannan conjugated to from about 0.2 mg/dose to about 20 mg/dose BAM; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a mannan conjugated to BAM comprising Formula I described herein and thereof; an immunostimulatory adjuvant or derivative herein and thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof; a Toll like receptor (TLR) agonist; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • TLR Toll like receptor
  • a subject specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof; R-848, poly (I:C), lipoteichoic acid (LTA), or combinations thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof; from about 0.05 mg/dose to about 5 mg/dose R-848, poly (I:C), lipoteichoic acid (LTA), or combinations thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof; R-848, from about 0.05 mg/dose to about 5 mg/dose poly (I:C), lipoteichoic acid (LTA), or combinations thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof; R-848, poly (I:C), from about 0.05 mg/dose to about 5 mg/dose lipoteichoic acid (LTA), or combinations thereof; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof; an anti-CD40 antibody; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a phagocytosis stimulating agent or derivative herein and thereof; from about 0.04 mg/dose to about 4 mg/dose anti-CD40 antibody; and an attenuated cancer cell, cell population or derivative herein and thereof.
  • a subject specific pharmaceutical composition can comprise a mannan attached to BAM, a R-848, a poly (I:C), an LTA, an anti-CD40 antibody, and irradiated cancer cells.
  • a subject specific pharmaceutical composition can comprise from about 0.05 mg/dose to about 5 mg/dose mannan attached to from about 0.05 mg/dose to about 5 mg/dose BAM, from about 0.05 mg/dose to about 5 mg/dose R-848, from about 0.05 mg/dose to about 5 mg/dose poly (I:C), from about 0.05 mg/dose to about 5 mg/dose LTA, from about 0.04 mg/dose to about 4 mg/dose anti-CD40 antibody, and from about 1.0 ⁇ 10 ⁇ 3 to about 1.0 ⁇ 10 ⁇ 7 irradiated cancer cells.
  • Described herein are also methods of treating an individual in need thereof with an effective amount of a personalized cancer described herein and thereof.
  • a personalized tumor vaccine or a pharmaceutical composition can comprise mixing an irradiated cell population extracted from a tumor of a subject with a phagocytosis stimulating agent and an immunostimulatory adjuvant simultaneously.
  • a personalized tumor vaccine or a pharmaceutical composition can comprise mixing an irradiated cell population extracted from a tumor of a subject with a phagocytosis stimulating agent and an immunostimulatory adjuvant sequentially.
  • a personalized tumor vaccine or a pharmaceutical composition can comprise mixing an irradiated cell or cell population extracted from a tumor of a subject and a mannan-BAM, R-848, anti-CD40 mAb, poly(I:C), and LTA to form a personalized tumor vaccine.
  • Excipient [0123]
  • a personalized tumor vaccine or a pharmaceutical composition can comprise an excipient.
  • an excipient can comprise a preservative, adjuvant, diluent, or stabilizer.
  • an excipient can prevent contamination.
  • an excipient can stabilize vaccine potency during storage.
  • an excipient can comprise monosodium glutamate, sucrose, D-mannose, D-frusctose, dextrose, human serum albumin, potassium phosphate, plasdone C, anhydrous lactose, microcrystalline, cellulose, polacrilin potassium, magnesium stearate, cellulose acetate phthalate, alcohol, acetone, caster oil, aluminum hydroxide, sodium chloride, benzethonium chloride, formaldehyde, glycerin, asparagine, citric acid, magnesium sulfate, iron ammonium citrate, sodium carbonate, essential amino acid, L-phenylalanine, non-essential amino acids, L-arginine hydrochloride, D-trehalose dihydrate, D-sorbitol, trometamol, thimerosal, gelatin, urea, isotonic sodium chloride, glutaraldehyde, 2-phenoxyethanol, polysorbate 80 (Tween 80), n
  • an excipient of a personalized cancer vaccine or a pharmaceutical composition can comprise amorphous sugars.
  • an amorphous sugar can comprise bulking agents and protein stabilizers.
  • an amorphous sugar can comprise glass- forming sugars.
  • an amorphous sugar can comprise sucrose, raffinose, trehalose, stachyose, lactose, maltose, any derivatives or combinations herein and thereof.
  • a sugar can comprise an amount effective for volume increase or an amount effective for protein stabilization.
  • the sugar can be present in the range of about 1-10 %, 5-15 %, 10-20 %, 15-25 %, 20-30 %, 25-35 %, 30-40 %, 35-45 %, 40-50 %, 45-55 %, 50-60 %, 55- 65 %, 60-70 %, 65-75 %, 70-80 %, 75-85 %, 80-90 %, 85-95 %, or 90-100 %, by weight.
  • an excipient of a personalized cancer vaccine or a pharmaceutical composition can comprise a thickener.
  • a thickener can comprise polymeric materials.
  • a polymeric material can comprise hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), methylcellulose (MC), hydroxyethylmethylcellulose (HEMC), or ethylhydroxyethylcellulose (EHEC) and other cellulose derivatives.
  • an excipient can increase the stability of an antigen, phagocytosis stimulating agent, or cell or cell population in a dry solid formulation as compared to a dry solid formulation of the antigen, phagocytosis stimulating agent, or cell or cell population without any excipients.
  • at least one excipient can be used in a personalized tumor vaccine.
  • excipients can provide a greater effect than that of any excipient alone, and in some cases provides a greater than additive effect of the multiple excipients on the stability of the antigen, phagocytosis stimulating agent, or cell or cell population.
  • An excipient or excipients can be present in the composition in a total amount of about 0 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %, by weight or volume, of a personalized tumor vaccine.
  • an excipient or excipients can be present in the composition in a total amount of 0-10 %, 5-15 %, 10-20 %, 15-25 %, 20-30 %, 25-35 %, 30-40 %, 35-45 %, 40-50 %, 45-55 %, 50-60 %, 55- 65 %, 60-70 %, 65-75 %, 70-80 %, 75-85 %, 80-90 %, 85-95 %, or 90-100 %, by weight or volume, of a personalized tumor vaccine.
  • a personalized tumor vaccine or a pharmaceutical composition can comprise a diluent.
  • a diluent can comprise water, PBS buffer, sodium chloride, MenCWY, DTaP-IPV, calcium carbonate, xanthan, adjuvants described herein and thereof, and derivatives herein and thereof, or any combinations herein and thereof.
  • Administration of a personalized tumor vaccine or pharmaceutical composition [0129]
  • a personalized tumor vaccine can comprise packing the vaccine into a composition or formulation for delivery or administration in a subject.
  • an administration of a composition or a pharmaceutical composition provided herein can refer to methods that can be used to enable delivery of the vaccine or pharmaceutical composition to the desired site of biological action. Delivery can include direct application to the affect tissue or region of the body. Delivery can include intracranial injection.
  • Delivery can include a parenchymal injection, an intra-thecal injection, an intra-ventricular injection, or an intra-cisternal injection.
  • a composition provided herein can be administered by any method.
  • a method of administration can be by inhalation, intraarterial injection, intracerebroventricular injection, intracisternal injection, intramuscular injection, infraorbital injection, intraparenchymal injection, intraperitoneal injection, intraspinal injection, intrathecal injection, intravenous injection, intraventricular injection, stereotactic injection, subcutaneous injection, or any combination thereof.
  • Delivery can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion), oral administration, inhalation administration, intraduodenal administration, rectal administration. Delivery can include topical administration (such as a lotion, a cream, an ointment) to an external surface of a surface, such as a skin. In some cases, administration is by parenchymal injection, intra-thecal injection, intra-ventricular injection, intra-cisternal injection, intravenous injection, or intranasal administration or any combination thereof. In some instances, a subject can administer the composition in the absence of supervision.
  • a subject can administer the composition under the supervision of a medical professional (e.g., a physician, nurse, physician’s assistant, orderly, hospice worker, etc.).
  • a medical professional can administer the composition.
  • a cosmetic professional can administer the composition.
  • the methods of treating an individual with cancer described herein can comprise administration of a personalized tumor vaccine or a pharmaceutical composition in an individual with a cancer or suspected of a cancer.
  • the methods of treating an individual with cancer described herein can also comprise administration of a personalized tumor vaccine or a pharmaceutical composition in an individual without a cancer or suspected of a cancer.
  • the cancer comprises Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic tumor necrosis
  • Administration or application of a personalized tumor vaccine or a pharmaceutical composition can be performed for a treatment duration of at least about at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 days consecutive or nonconsecutive days.
  • a treatment duration can be from about 1 to about 30 days, from about 2 to about 30 days, from about 3 to about 30 days, from about 4 to about 30 days, from about 5 to about 30 days, from about 6 to about 30 days, from about 7 to about 30 days, from about 8 to about 30 days, from about 9 to about 30 days, from about 10 to about 30 days, from about 11 to about 30 days, from about 12 to about 30 days, from about 13 to about 30 days, from about 14 to about 30 days, from about 15 to about 30 days, from about 16 to about 30 days, from about 17 to about 30 days, from about 18 to about 30 days, from about 19 to about 30 days, from about 20 to about 30 days, from about 21 to about 30 days, from about 22 to about 30 days, from about 23 to about 30 days, from about 24 to about 30 days, from about 25 to about 30 days, from about 26 to about 30 days, from about 27 to about 30 days, from about 28 to about 30 days, or from about 29 to about 30 days.
  • Administration or application of a composition disclosed herein can be performed for a treatment duration of at least about 1 week, at least about 1 month, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, or more.
  • Administration can be performed repeatedly over a lifetime of a subject, such as once a month or once a year for the lifetime of a subject.
  • Administration can be performed repeatedly over a substantial portion of a subject’s life, such as once a month or once a year for at least about 1 year, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, or more.
  • an administration of any personalized tumor vaccines or pharmaceutical compositions provided herein, including pharmaceutical compositions can be in an effective amount, for example to reduce a symptom of a disease or condition and/or to reduce a disease or condition.
  • an effective amount can be sufficient to achieve a desired effect.
  • the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions.
  • the effective amount is the amount sufficient to result in a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is the amount sufficient to result in antibody generation against the antigen. In some embodiments, the effective amount is the amount required to confer passive immunity on a subject in need thereof. With respect to immunogenic compositions, in some embodiments the effective amount will depend on the intended use, the degree of immunogenicity of a particular antigenic compound, and the health/responsiveness of the subject’s immune system, in addition to the factors described above.
  • Administration or application of personalized tumor vaccines or pharmaceutical compositions disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times a day. In some cases, administration or application of personalized tumor vaccines or pharmaceutical compositions disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times a week.
  • administration or application of personalized tumor vaccines or pharmaceutical compositions disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 times a month.
  • a personalized tumor vaccines or pharmaceutical composition of the present disclosure can be administered/applied as a single dose or as divided doses.
  • the personalized tumor vaccines or pharmaceutical compositions described herein can be administered at a first time point and a second time point.
  • a personalized tumor vaccines or pharmaceutical composition can be administered such that a first administration is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more.
  • An effective amount of a personalized tumor vaccines or pharmaceutical composition can reduce the size of a tumor.
  • a personalized tumor vaccines or pharmaceutical composition can decrease the size of a tumor by 0 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 % of the size of the tumor before the administration of the personalized tumor vaccines or pharmaceutical composition.
  • an effective amount of a personalized tumor vaccines or pharmaceutical composition can decrease the size of a tumor by 1-10 %, 5-15 %, 10-20 %, 15-25 %, 20-30 %, 25-35 %, 30-40 %, 35- 45 %, 40-50 %, 45-55 %, 50-60 %, 55-65 %, 60-70 %, 65-75 %, 70-80 %, 75-85 %, 80-90 %, 85-95 %, or 90-100 % of the size of the tumor before the administration of the personalized tumor vaccines or pharmaceutical composition.
  • An effective amount of a personalized tumor vaccines or pharmaceutical composition can reduce the number of cancer cells.
  • a personalized tumor vaccines or pharmaceutical composition can reduce the number of cancer cells by 0 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 % of number of the cancer cells before the administration of the personalized tumor vaccines or pharmaceutical composition.
  • an effective amount of a personalized tumor vaccines or pharmaceutical composition can decrease the number of cancer cells by 1-10 %, 5-15 %, 10-20 %, 15-25 %, 20-30 %, 25-35 %, 30-40 %, 35-45 %, 40-50 %, 45-55 %, 50-60 %, 55-65 %, 60-70 %, 65- 75 %, 70-80 %, 75-85 %, 80-90 %, 85-95 %, or 90-100 % of the number of the cancer cells before the administration of the personalized tumor vaccines or pharmaceutical composition.
  • An effective amount of a personalized tumor vaccines or pharmaceutical composition can extend the life-span of a subject administered with the personalized tumor vaccines or pharmaceutical composition.
  • an effective amount of a personalized tumor vaccines or pharmaceutical composition can extend the life-span of a subject administered with the personalized tumor vaccines or pharmaceutical composition by 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 days, 28 days, 29 days, 30 days, 1 month,
  • an effective amount of a personalized tumor vaccines or pharmaceutical composition can extend the life-span of a subject administered with the personalized tumor vaccines or pharmaceutical composition by an amount of time from 1 day to 1 month, from 25 days to 6 months, from 5 months to 12 months, from 10 months to 2 years, from 1 year to 5 years, from 4 years to 10 years, from 9 years to 15 years, from 14 years to 20 years, from 19 years to 25 years, from 24 years to 30 years, from 29 years to 35 years, from 34 years to 40 years, from 39 years to 45 years, or from 44 years to 50 years.
  • An effective amount of a personalized tumor vaccines or pharmaceutical composition can delay the onset of a cancer of a subject administered with the personalized tumor vaccines or pharmaceutical composition.
  • an effective amount of a personalized tumor vaccines or pharmaceutical composition can delay the onset of a cancer of a subject administered with the personalized tumor vaccines or pharmaceutical composition by 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 days, 28 days, 29 days, 30 days, 1
  • an effective amount of a personalized tumor vaccines or pharmaceutical composition can delay the onset of a cancer of a subject administered with the personalized tumor vaccines or pharmaceutical composition by an amount of time from 1 day to 1 month, from 25 days to 6 months, from 5 months to 12 months, from 10 months to 2 years, from 1 year to 5 years, from 4 years to 10 years, from 9 years to 15 years, from 14 years to 20 years, from 19 years to 25 years, from 24 years to 30 years, from 29 years to 35 years, from 34 years to 40 years, from 39 years to 45 years, or from 44 years to 50 years.
  • EXAMPLES [0140]
  • Example 1 A Mouse Model of Subcutaneous Primary and Metastatic Distant Tumors.
  • Provided herein are methods for creating a mouse model to study subcutaneous primary and metastatic distant tumors.
  • Cell lines [0143] CT26 colon carcinoma cells were obtained from ATCC. Tumor cells were cultured in complete medium (RPMI 1640, Gibco) containing 10% (vol/ vol) FBS (Gibco), 100 Uml ⁇ 1 penicillin, 100 ⁇ gml ⁇ 1 streptomycin (Gibco). All cell lines used were shown to be negative for mycoplasma contamination using PCR amplification.
  • a CT26 tumor-bearing mouse model [0144] To establish a mouse model to study primary and metastatic distant tumors, tumor cells were injected into the right and left flank, respectively, to form the primary tumor and distant metastatic tumor. [0145] Mice were maintained, and experiments were conducted with the approval of the NCI Animal Use and Care Committees. Female BALB/c (6–8-week-old) were purchased from Charles River Laboratory.5.5 x 10 ⁇ 5 and 2.5 x 10 ⁇ 5 CT26 cells suspended in 100-200 ⁇ L PBS were subcutaneously injected into 101 and 102 on right or left flanks respectively, as shown in FIGURE 1A.
  • FIGURE 1B shows representative pictures of CT26 tumor-bearing mice 10 days after injection of saline.
  • Sample sizes of animal studies were determined empirically. Pre-established starting tumor size used as inclusion criteria for randomization was determined by pilot experiments. Investigators not blinded from the group allocation during outcome measurement performed randomization manually. Survival end points for all animal studies were defined according to the following criteria: (1) tumor volume exceeding 2000 mm 3 , (2) tumor diameter exceeding 2 cm, (3) severe non-healing skin necrosis over the tumor.
  • Example 2 In situ MBTA Injection at Primary Tumors Suppresses Distant Tumor Growth via a T-Cell Dependent Subject-specific Immune Response [0149] Provided herein are methods for using MBTA to treat primary and distant tumor growth.
  • FIGURE 2A shows the general strategy of the MBTA treatment: To bolster mannan-BAM’s inductive effect on innate immune cells—such as dendritic cells, macrophages, monocytes, and neutrophils—three TLR ligands (Lipo-teichoic acid (LTA), polyinosinic-polycytidylic acid (poly(I:C)), Resiquimod (R-848)), and immunostimulatory anti-CD40-mAb (recombinant mouse CD40 fusion protein, Bio X Cell, catalog number: BE0016-2, clone number: FGK4.5/FGK45) were incorporated as adjuvants.
  • LTA Lipo-teichoic acid
  • poly(I:C) polyinosinic-polycytidylic acid
  • Resiquimod R-848
  • immunostimulatory anti-CD40-mAb recombinant mouse CD40 fusion protein, Bio X Cell, catalog
  • TLR2 mediated inflammatory pathways known to increase TNF ⁇ secretion.
  • Poly(I:C) a synthetic analog of viral dsRNA
  • activated TLR3 mediates signaling previously demonstrated to activate APCs, modulate the phenotype of tumor associated macrophages to become increasingly immunosupportive, and induce tumor cell apoptosis.
  • R-848 an imidazoquinolinamine and synthetic analog of viral ssRNA, activates TLR7/8 pathways, resulting in the induction of Th1 cell–mediated immunity.
  • anti-CD40- mAb was introduced to mimic the natural ligand to activate a CD40 receptor, a tumor necrosis factor receptor, on the T-cells.
  • DCs Dendritic cells
  • MHC major histocompatibility complex
  • endogenous antigens on MHC class I molecules In some cases, DCs can also present exogenous antigens on MHC molecules, which is necessary for the generation of the cytotoxic T lymphocytes.
  • DCs can also induce T helper (Th)1, Th2, or Th17 polarization response.
  • Th T helper
  • DCs need to undergo a maturation process in order to primer na ⁇ ve T-cells.
  • Immature DCs can be induced to undergo maturation by a combination of PAMPs, inflammatory cytokines, and TLR agonistics; thymic stromal lymphopoietin; TLR agonists and CD40 agonist; or Wnt-B- cadherin.
  • DCs can be matured by a combination of LTA, Poly(I:C), R-848, and anti-CD40 mAb.
  • DCs represent a link between innate immunity and adaptive immune response: DCs, as innate immune response cells, can engulf or phagocytose pathogens, such as the attenuated cancer cell associated with mannan-BAM They then migrated to the lymph node for the activation of adaptive immune cells T-cells. There, DCs digest and express antigens from the phagocytosed attenuated cancer cells on their cell surface using MHC molecules on the surface of their cell membrane. The presentation of cancer antigens then activates T-cell differentiation and proliferation, activating an immunological memory, long after even if the pathogens are eliminated.
  • pathogens such as the attenuated cancer cell associated with mannan-BAM
  • mannan-BAM Mannan-BAM
  • BAM mannan-BAM
  • BAM subsequently exploited mannan’s recognition by pattern recognition receptors on innate immune cells via the recruitment of the MASP1 & MASP2 proteins to activate the complement components (such as C2, C3, and C4), leading to the opsonphagocytosis of the tumor cells by the innate immune cells.
  • ligation of anti-CD40-mAb to the CD40 receptor activates antigen presenting cells to present the tumor antigens to na ⁇ ve T-cells in the lymph node.
  • the na ⁇ ve T-cells then differentiated into helper CD4 + and cytotoxic CD8 + T-cells for long-lasting adaptive immune response.
  • Mannan and Poly (I:C) were obtained from Sigma-Aldrich (St. Louis, Mo).
  • LTA was obtained from Sigma- Aldrich (St. Louis, Mo) and InvivoGen (San Diego, CA).
  • R-848 was obtained from Tocris Bioscience (Minneapolis MN).
  • anti-mouse CD40 (clone: FGK4.5/GFK45) was obtained from BioXCell (West Lebanon, NH). Biocompatible anchor for cell membrane (BAM) was obtained from NOF America Corporation (White Plains, NY). [0157] Mannan-BAM synthesis was performed as previously reported (Janotová et al. PLoS One 2014, 9, e85222. Aminated mannan was prepared by reductive amination, as previously reported (Torosantucci et al. J. Exp. Med.2005, 202, 597).
  • Mannan solution in an environment of ammonium acetate 300 mg mL -1 was reduced by 0.2 m sodium cyanoborohydride at pH 7.5 and 50 °C for five days.
  • Solution was further dialyzed using MWCO 3500 dialysis tubing (Serva, Heidelberg, Germany) against PBS at 4 °C overnight. Binding of BAM on amino group of mannan was performed at pH 7.3 according to Kato et al. (Biotechnol. Prog.2004, 20, 897).
  • N- hydroxysuccinimide (NHS) group of BAM reacted with amino group of mannan within one hour at room temperature. Solutions obtained after dialysis was stored frozen at -20 °C until use.
  • In situ MBTA Injections 50 ⁇ L of the therapeutic mixture consisting of 0.5 mg R-848 (HCl form), 0.5 mg poly(I:C), 0.5 mg LTA, and 0.4 mg anti-CD40 per mL of 0.2 mM mannan-BAM in PBS was injected intratumorally.
  • MBTA Treatment of Primary and Distant Tumors 50 ⁇ L of the therapeutic mixture consisting of 0.5 mg R-848 (HCl form), 0.5 mg poly(I:C), 0.5 mg LTA, and 0.4 mg anti-CD40 per mL of 0.2 mM mannan-BAM in PBS was injected intratumorally.
  • MBTA Treatment of Primary and Distant Tumors 50 ⁇ L of the therapeutic mixture consisting of 0.5 mg R-848 (HCl form), 0.5 mg poly(I:C), 0.5 mg LTA, and 0.4 mg anti-CD40 per mL of 0.2 mM mannan-BAM in PBS was injected intratumorally.
  • mice bearing left flank tumors (average left flank tumor volume – 31.3 mm 3 ) were randomized into two treatment arms: normal saline (control) and in situ injection of MBTA into right flank tumors. Treatments were administered every day for 3 days then repeated weekly for a total of 4 weeks. Tumor growth was assessed twice a week until survival end point. Experiment was performed twice. Representative pictures of these mice were shown in FIGURE 2C.
  • both right and left flank tumor volumes show a statistically significant difference between in situ MBTA injected mice versus saline treated mice (median tumor volumes ⁇ SEM of in situ MBTA versus saline treated mice: right flank treated tumors— 74.4 ⁇ 8.8 versus 150.2 ⁇ 28.1 mm 3 .
  • the MBTA treated mice achieved complete regression (CR) for the duration of the study. Therefore, the in situ injection of MBTA into the primary tumors suppressed the distal tumor growth.
  • antibodies were administered both prior to and during treatment to deplete CD4 + or CD8 + T-cells in the CT26 tumor- bearing mice.
  • mice in the CD8 + and CD4 + depletion group were injected with 250 ⁇ g of CD8- depleting antibodies (clone 53–6.7; BioXcell) and CD4-depleting antibodies (clone GK1.5; BioXcell), respectively. Injections were given -2 day, -1 day, and on the day of therapy initiation (day -2, -1, 0), then weekly thereafter. The T-cell depletion was confirmed via flow cytometry 16 days after initiating treatment, as shown in FIGURE 2G. The CD4 + and CD8 + T-cell depleted mice were subjected to the same in situ MBTA treatment schedule as shown in FIGURE 2B.
  • CD8 + T-cells significantly affect tumor growth control of the primary tumors.
  • No significant difference in distant tumor volumes was detected between CD4 + or CD8 + depleted in situ MBTA treated mice and the saline treated control mice (median tumor volumes ⁇ SEM: CD4 + depleted in situ MBTA treated versus saline treated mice—448.7 ⁇ 291.1 versus 607.4 ⁇ 121.1 mm3.
  • Bonferroni adjusted p > 0.05; CD8 + depleted in situ MBTA treated versus saline treated mice—582.5 ⁇ 75.6 versus 607.4 ⁇ 121.1 mm 3 .
  • Left flank tumor growth data suggest that CD4 + and CD8 + T-cells significantly affect tumor growth control of the metastatic distant tumors.
  • Both CD4 + and CD8 + depleted in situ MBTA treated mice demonstrated no significant improvement in survival over saline treated control mice (median survival ⁇ SEM of CD4 + depleted in situ MBTA versus saline treated mice: 21 ⁇ 3.2 versus 17 ⁇ 0.9 days, Tukey–Kramer adjusted p > 0.05; CD8+ depleted in situ MBTA versus saline treated mice: 17 ⁇ 0.6 versus 17 ⁇ 0.9 days, Tukey–Kramer adjusted p > 0.05), as shown in FIGURE 2J.
  • mice in the non-T-cell depleted in situ MBTA injected treatment group had a decreased median survival time (median survival ⁇ SEM of in situ MBTA injected mice in FIGURE 2F versus non-T-cell depleted in situ MBTA injected mice: 67 ⁇ 11.7 versus 28 ⁇ 2.4 days in FIGURE 2J).
  • Example 3 Immunophenotyping (I.P.) of Tumors with Flow Cytometry
  • I.P. Immunophenotyping
  • mice were sacrificed, both right and left flank tumors were excised and subjected to mechanical disruption using a GentleMACS Dissociator (Miltenyi Biotec) in the presence of enzymatic digestion using Tumor Dissociation Kit (Miltenyi Biotec).
  • a GentleMACS Dissociator Miltenyi Biotec
  • Tumor Dissociation Kit Tumor Dissociation Kit
  • CD45.2 + CD11c + MHCII+ Dendritic cells
  • Macrophages CD45.2 + CD11c-CD11b + Ly6G- Ly6C-/low
  • Monocytes CD45.2 + CD11c- CD11b + Ly6G- Ly6C high
  • MHC class II + Monocytes CD45.2 + CD11c- CD11b + Ly6G-Ly6C high MHCII+
  • Neutrophils CD45.2 + CD11c-CD11b + Ly6G+
  • CD4+ T-cells CD45.2 + TCR ⁇ + CD4+CD8-
  • CD8+ T-cell CD45.2 + TCR ⁇ +CD4-CD8+
  • B cells CD45.2 + TCR ⁇ -CD19+).
  • Intracellular cytokine staining and flow cytometry Suspensions containing T-cells were stained with a fixable live/dead stain (Invitrogen) in PBS followed by surface antibody staining in FACS buffer (PBS with 0.5% BSA and 0.1% sodium azide).
  • FACS buffer PBS with 0.5% BSA and 0.1% sodium azide.
  • cells were first stimulated with Cell Stimulation Cocktail (eBioscience) containing PMA/Ionomycin and protein transport inhibitor for five hours prior to undergoing staining. Next, cells were stained for surface molecules following fixation and permeabilization (eBioscience), and then stained with cytokine antibodies. Stained cells were analyzed by flow cytometry (LSRII; BD Bioscience). Data analysis was performed using FlowJo software (TreeStar).
  • Example 4 Enhanced Subject-specific Innate Immunity against CT26 tumors After MBTA Treatment
  • I.P. Immunophenotyping
  • Example 3 Immunophenotyping of tumors were performed according to Example 3 to further assess the immune profile of the tumor microenvironment of subcutaneous tumor model in Example 1. Both right and left flank tumors were harvested 10 days and 16 days after the start of the MBTA treatment.
  • I.P. analyses were completed at two distinct time points, as shown in FIGURE 3A. The first timepoint was carried out 10 days after first treatment and within 6 hours following in situ injection of MBTA.
  • FIGURE 3D revealed a striking dendritic (CD45.2 + CD11c + MHCII+) and neutrophilic (CD45.2 + CD11c- CD11b + Ly6G+) inflammatory response in the MBTA treated mice versus the control.
  • results from I.P. (Day 16) analysis of the right flank tumors revealed a significant decrease in neutrophils and increase in APCs, including DCs and MHC class II positive monocytes (CD45.2 + CD11c-CD11b + Ly6G-Ly6C high MHC II+), as shown in FIGURE 3D and 3E.
  • Assessments of the innate immune cell populations at the distal, non-treated left flank tumors showed an increased neutrophilic immune response on I.P. (Day 10), followed by a significant increase in APCs (DCs and MHC class II positive monocytes) on I.P. (Day 16) in the tumors of the MBTA treated mice versus the control, as shown in FIGURE 3F and 3G.
  • FIGURE 3D and 3F show that there were no significant changes in the overall quantity of macrophages (CD45.2 + CD11c-CD11b + Ly6GLy6C-/ low), an assessment of the CD206 positive alternatively activated macrophage (AAM) population—also known as M2-macrophages (CD45.2 + CD11c-CD11b + Ly6GLy6C-/ low CD206+), as shown in FIGURE 3H—demonstrated that the AAM population was significantly decreased in the primary (MBTA treated) tumors on both I.P. day 10 and 16.
  • AAM CD206 positive alternatively activated macrophage
  • FIGURE 3I shows that the AAM population in the distal tumors from the MBTA treated mice significantly decreased on I.P. day 10.
  • TLR agonist and anti-CD40-mAb were shown to skew the polarization of tumor associated macrophages (TAM) toward an M1-like phenotype.
  • TAM tumor associated macrophages
  • Example 5 Enhanced Subject-specific Adaptive Immune Response against CT26 Tumors After MBTA Treatment
  • Results from I.P. day 10 and 16, as described in Example 4 also revealed an overall increase in adaptive immune cells in both primary and distant tumors of the MBTA treated mice compared to the control, as shown in FIGURE 4A and 4B.
  • FIGURE 4A shows that CD8+ T-cells (CD45.2 + TCR ⁇ + CD4-CD8+) and B cells (CD45.2 + TCR ⁇ -CD19+) populations statistically increased in the primary tumor of the MBTA treated mice demonstrated on I.P. day 16.
  • CD4 + (CD45.2 + TCR ⁇ +CD4 + CD8-) and CD8 + T-cell populations were extracted from the distant tumors and stimulated with PMA/Ionomycin in vitro to assess for intracellular expression of IFN ⁇ , TNF ⁇ and Granzyme B.
  • T-cells harvested from the MBTA treated mice on I.P. (Day 10) and (Day 16) had higher production of IFN ⁇ and TNF ⁇ than the control, as shown in FIGURE 4C.
  • CD8+ T-cells demonstrated a significant increase in IFN ⁇ on I.P. (Day 10) and TNF ⁇ during the six-day interval between I.P. (Day 10) and (Day 16).
  • FIGURE 4D also shows an increased, though not statistically significant, population of Granzyme B positive CD8+ T cells.
  • the MBTA treatment significantly augments the quantity and activation of CD8+ T-cells within the distant tumors, suggesting their major role in the MBTA-induced tumor growth inhibition.
  • AH-1 loaded H-2Ld tetramer was used to assess if the MBTA therapy generated higher quantities of CD8 + T-cells (CTLs) against the immunodominant epitope of CT26, gp70 423–431 (AH-1) (Huang, et al., Proc. Natl. Acad. Sci. U. S. A.1996, 93, 9730.).
  • Peripheral blood samples and the distant tumor of the in situ MBTA treated and control mice were collected 11 days after the start of treatment for analysis.
  • the in situ MBTA injections increase the AH-1-specific CD8+ T-cell clones circulating in the blood and in the metastatic distant tumors within 11 days from the start of MBTA treatment.
  • the in situ injection of MBTA resulted in significant changes in the immunophenotypes of representative primary and metastatic tumors.
  • the MBTA treatment generated an innate inflammatory response initially characterized by a strong neutrophilic and DC predominance and followed by a significant increase in APC populations (MHC II+ monocytes and DCs).
  • the MBTA treatment strengthened the adaptive immune response by increasing the overall supply of CD8+ T-cells and B cells infiltrating the tumor.
  • T-cells extracted from the distant tumor were not only higher in quantity but demonstrated higher expressions of IFN ⁇ and TNF ⁇ cytokines, relative to the control, validating their enhanced tumoricidal activity.
  • our finding in situ MBTA treatment elicits tumor rejection of representative metastatic tumors.
  • CT26 cells are expanded in vitro and subsequently aliquoted into 1 ⁇ 10 ⁇ 6 million cells per vaccine dose.
  • CT26 cells are sub-lethally irradiated with 50 Gy using a 137 Cs MARK I model irradiator (JL Shepherd & Associates, San Fernando, CA) to induce tumor cell apoptosis and prevent engraftment of tumor cells at the vaccine site.
  • CT26 tumor cells were initially irradiated to induce tumor cell apoptosis and prevent tumor outgrowth when used as a component in the rCT26-MBTA vaccine, as shown in FIGURE 5B.
  • rCT26-MBTA vaccines 100 ⁇ L of the following therapeutic mixture were injected subcutaneously into the right flanks of rCT26-MBTA treated mice: a) 1 ⁇ 10 ⁇ 6 irradiated CT26 cells suspended in 50 ⁇ L PBS and b) 50 ⁇ L of the therapeutic mixture consisting of 0.5 mg R-848 (HCl form), 0.5 mg poly(I:C), 0.5 mg LTA, and 0.4 mg anti-CD40 per mL of 0.2 mM mannan-BAM in PBS.
  • the vaccine dose (a) was then pulsed (incubated) with (b) (50 ⁇ L, dose per mouse) for 1 h, facilitating the in vitro integration of mannan-BAM into tumor cell membranes, to form the therapeutic mixture. It was then subsequently injected subcutaneously to the right flank of treated animals according to the specified therapeutic schedule. Mice receiving the rCT26 vaccine were injected with 1 ⁇ 10 ⁇ 6 irradiated tumor cells suspended in 100 ⁇ L PBS (dose per mouse). Generation of a potent immune response using the rCT26-MBTA vaccine [0187] The rCT26-MBTA vaccinated mice can generate CD8+ T-cell responses against CT26- specific antigens.
  • mice in the rCT26 vaccine treatment group did not have improved survival over the saline treated mice (median survival ⁇ SEM of rCT26 versus saline treated mice: 20 ⁇ 2.1 versus 20 ⁇ 1.9 days, Tukey–Kramer adjusted p > 0.05).1/8, or 12.5% of the rCT26-MBTA vaccinated mice achieved CR for the duration of the study, as shown in FIGURE 5E. Therefore, the treatment with a total of 12 rCT26-MBTA vaccinations can successfully generate a potent antitumor immune response.
  • Peripheral blood samples were collected 10 days after the start of treatment and after receiving a total of two vaccinations. Tumors were harvested 22 days after the start of treatment and after receiving a total of four vaccinations, which is in accordance with the known timeline for T-cell activation after initial antigen stimulation (Pennock et al. Adv. Physiol. Educat.2013, 37, 273).
  • the AH-1-MHC tetramer results suggest that significantly more AH-1-specific CD8+ T-cell clones were found in the whole blood and within the tumors of the rCT26-MBTA vaccinated mice when compared to the control mice, underscoring its potential to generate CD8+ T-cell responses against CT26-specific antigens.
  • the MBTA treatment was overall well-tolerated: The in situ MBTA treated mice demonstrated a significant acute drop in mean body weight 3 days after the start of treatment when compared to the pre-treatment level, as shown in FIGURE 5H. The loss in body weight was recuperated 7 days after the start of treatment. The mice treated with the rCT26-MBTA vaccine had no significant changes in body weight, as shown in FIGURE 5I. None of the in situ MBTA or rCT26-MBTA vaccinated mice died unexpectedly in our investigations. [0192] Example 7: MBTA Treatment Induces Subject-specific Immunological Memory against CT26 Cells Provided herein are methods for inducing immunological memory against CT26 tumors using the MBTA treatment.
  • the MBTA treatment can generate durable and robust immunological memory to prevent the relapse of parental tumors in both the periphery and intracranially.
  • FIGURE 6A and 6B show that all CR mice displayed no evidence of tumor growth, while all control mice developed tumors and reached corresponding end points, confirming the induction of an effective immunological memory against CT26 tumor antigens.
  • Immunological memory generated by the MBTA treatment also extends beyond peripheral circulation and engages CT26 tumors within the central nervous system (CNS).
  • CT26-Luc cells with stable luciferase expression were used to facilitate monitoring of intracranial tumor growth.
  • 1.0 ⁇ 10 ⁇ 4 CT26-Luc cells in 2 ⁇ L HBSS containing 5 ug mL -1 DNase I were stereotactically injected through an entry site at a point 1 mm rostral of the bregma, 2 mm right of midline, and 2 mm deep from the skull surface in the right frontal lobe, using a Hamilton syringe (Hamilton Company), as previously reported, according to Toda et al. (Cancer Gene Ther.2002, 9, 356). Surgical anesthesia was obtained via vaporized isoflurane.
  • mice None of the previously treated mice demonstrated evidence of intracranial tumor formation, while all control mice developed intracranial tumors and reached corresponding end points, as shown in FIGURE 6C and 6D. Taken together, re-challenge experiments revealed that MBTA treated mice developed immunological memory against CT26 cells that is not only durable, but also sufficiently robust to prevent the relapse of parental tumors in both the periphery and intracranially.
  • Example 8 Human Equivalent Dose (HED) Calculation Based on Body Surface Area
  • HED Human Equivalent Dose

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