Cancer vaccine and method of use thereof

Abstract

Disclosed is a cancer vaccine that contains dendritic cells. The dendritic cells are prepared by: culturing immature dendritic cells to maturation; co-culturing the matured dendritic cells with attenuated cancer cells, Mannan – BAM (Mannan, a yeast-derived polysaccharide, and BAM, a Biocompatible Anchor for Cell Membrane), and TLR (Toll like receptor) agonists; and optionally sorting the dendritic cells from the co-cultured attenuated cancer cells.

Description

[0001] CANCER VACCINE AND METHOD OF USE THEREOF

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

[0003] DEVELOPMENT

[0004] This invention was made with Government support under project number ZIA BC011773 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention.

[0005] FIELD OF THE INVENTION

[0006] The present invention relates generally to the preparation and use of a cancer vaccine derived from dendritic cells, more specifically, a personalized cancer vaccine, for cancer immunotherapy.

[0007] BACKGROUND OF THE INVENTION

[0008] The mammalian immune system includes elaborate humoral and cellular networks employing an array of defense strategies against foreign agents. Among its cellular networks, dendritic cells (DCs) play a unique role in triggering the immune response. DCs are specialized antigen-presenting cells (APCs) having the capability to capture and process antigens, migrate from the periphery to a lymphoid organ, and present the antigens to resting T cells.

[0009] DCs' role as APCs has fueled efforts to explore DC-based immunization and vaccination. However, research shows maturation of DCs is required in order for DCs to develop into potent T cell stimulators. Because it is difficult to isolate mature DCs from peripheral blood as only less than 1% of the white blood cells count as so, alternative methods to produce mature DCs from immature dendritic cells need to be developed. Once mature DCs are harvested, there are other technical challenges such as loading them with specific antigens to achieve desired therapeutic effects.

[0010] On the other hand, cancer, as one of the deadliest diseases in the world, has emerged as a promising target for immunotherapeutic treatments in the past decade. There are ongoing and intensifying research efforts to harness the power of anti-tumor immunity to improve clinical outcomes in cancer patients.

[0011] Accordingly, there is a need to develop methods for preparing DC-based vaccines for cancer treatment. The present invention satisfies this need and provides additional advantages as well.

[0012] SUMMARY OF THE INVENTION

[0013] Nowadays one method for preparing dendritic cells (DCs) is to collect peripheral blood mononuclear cells (PBMCs) from a subject, then separate monocytes from the rest of the PBMCs, and develop the monocytes into mature DCs. A method was discovered which allows the preparation of a dendritic cell-derived cancer vaccine as follows.

[0014] One aspect of this invention relates to a cancer vaccine including dendritic cells that can be prepared by : i) culturing immature dendritic cells to maturation; ii) co-culturing the matured dendritic cells with attenuated cancer cells, Mannan - BAM (Mannan, a yeast-derived polysaccharide, and BAM, a Biocompatible Anchor for Cell Membrane), and TLR (Toll like receptor) agonists; and optionally iii) sorting the dendritic cells from the co-cultured attenuated cancer cells. Further, the immature dendritic cells can be prepared by harvesting monocytes from a subject. The dendritic cell-derived cancer vaccine thus obtained can be a personalized cancer vaccine for the subject.

[0015] Also, the immature dendritic cells can be prepared from bone marrow aspirate of the subject.

[0016] For preparing the above cancer vaccine, the attenuated cancer cells can be prepared by: i) harvesting cancer cells from a biopsy of a site of tumor from the subject, ii) culturing the harvested cancer cells to a relevant amount, and optionally iii) irradiating the cultured cancer cells.

[0017] Another aspect of this invention relates to a method of treating a cancer. The method includes administering to a subject having the cancer an effective amount of a composition, and the composition contains the above described cancer vaccine. For the cancer vaccine used in the method, the immature dendritic cells are prepared by harvesting monocytes from the same subject. Also, the attenuated cancer cells are prepared by harvesting cancer cells from a biopsy of a site of tumor from the same subject as well.

[0018] The cancer can be any clinically treatable by a cancer vaccine. Examples of the cancer include brain cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, melanoma, head and neck squamous cell carcinoma, merkel cell carcinoma, and lymphoma.

[0019] As pointed out above, the immature dendritic cells and cancer cells are derived autologously from the same subject. Alternatively, the above method can be used to prevent cancer. Under the situation, the immature dendritic cells are from the subject who would receive the vaccine, whereas the cancer cells can be derived from a different subject who has the cancer.

[0020] Yet another aspect of this invention relates to a method for inducing an immunological memory against a cancer. The method includes administering to a subject in need thereof an effective amount of the above described cancer vaccine. For the cancer vaccine used in the method, the immature dendritic cells are prepared by harvesting monocytes from the same subject. Also, the attenuated cancer cells are prepared by harvesting cancer cells from a biopsy of a site of tumor from the same subject as well.

[0021] In one embodiment, the described methods could target primary tumors. In another embodiment, the described methods could be applied against distal, untreated tumors.

[0022] In another embodiment, the described methods could delay the progression of a solid tumor in a subject. Further, the solid tumor can be selected from the group consisting of brain cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, and prostate cancer.

[0023] The delivery of the cancer vaccine can be through an intratumoral injection.

[0024] Both the above methods can be applied to a mammal, e.g., a human or a mouse.

[0025] The details of the invention are set forth in the drawing and the description below. Other features, objects, and advantages of the invention will be apparent to those persons skilled in the art upon reading the drawing and the description, as well as from the appended claims.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The embodiments exemplified in the FIGs 1-16 are intended to illustrate the present invention and are not to be considered as limiting.

[0028] The accompanying drawings are not intended to be drawn to scale. The figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every' component may be labeled in every' drawing.

[0029] FIG. 1 is the design: a vaccine consisting of dendritic cells primed to whole tumor cells (WTC) with anchored mannan enhances antitumor immunity and augments tumor infiltrating lymphocytes to treat established tumors and prevents metastasis.

[0030] FIGs. 2-5 are plots of co-stimulatory ligands CD80 and CD86 percentage and intensity' as measured by flow cytometry following co-culture of dendritic cells with tumor antigen source and maturation cocktails.

[0031] FIGs. 6-9 show cytokine time course analysis of dendritic cell co-culture with tumor antigen source and TLR ligands over 48 hours.

[0032] FIGs. 10 and 11 illustrate the design and results for a B 16 melanoma prophylactic DC therapy described in this invention.

[0033] FIGs. 12 and 16 illustrate the design and results of intracranial GL261 glioblastoma therapeutic model with DC cancer vaccines described in this invention. DETAILED DESCRIPTION

[0034] Before the present invention is described in more details, it is to be understood that this invention is not limited to a particular method described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0035] In the present invention, as illustrated in FIG. 1, mannan-BAM would be mixed with the resected tumor cells and co-cultured with patient-derived dendritic cells and the maturation cocktail of TLR ligands. The subsequently harvested dendritic cells would be used as a cancer vaccine.

[0036] The terms "subject," "host," "patient," and "individual" are used interchangeably herein to refer to any mammalian subject for whom diagnosis or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

[0037] This invention relies on preparing mature DCs. Monocytes, a subset of PBMCs, are cultured in a medium that induces the differentiation of monocytes into immature dendritic cells. Following differentiation of monocytes into immature dendritic cells, the immature dendritic cells can be matured into mature DCs. Methods for maturing DCs are known to those of skill in the art.

[0038] The term “dendritic cells (DCs)” refers to a diverse population of morphologically similar cell types found in a variety7of lymphoid and non-lymphoid tissues, Steinman (1991) Ann. Rev. Immunol. 9:271-296. Dendritic cells constitute the most potent and preferred APCs in the organism. While the dendritic cells can be differentiated from monocytes, they possess distinct phenotypes. For example, a particular differentiating marker, CD 14 antigen, is not found in dendritic cells but is possessed by monocytes. Also, mature dendritic cells are not phagocytic, whereas the monocytes are strongly phagocytosing cells. It has been shown that mature DCs can provide all the signals necessary for T cell activation and proliferation.

[0039] The terms "cell," and "cells," and "cell population," used interchangeably, intend one or more mammalian cells. The term includes progeny of a cell or cell population. Those skilled in the art will recognize that "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.

[0040] The terms "cell proliferation" and "to proliferate" as used herein refer to the amplification of the cell by cell division. 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 grow th 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, a semi-solid tumor, a primary tumor, a metastatic tumor, and the like.

[0041] “Immune response” broadly refers to the antigen-specific responses of lymphocytes to foreign substances. Any substance that can elicit an immune response is said to be “immunogenic” and is referred to as an “immunogen”. All immunogens are antigens, however, not all antigens are immunogenic. An immune response of this invention can be humoral (via antibody activity) or cell-mediated (via T cell activation).

[0042] "Immunotherapy" refers to treatment of disease (e.g., cancer) by modulating an immune response to a disease antigen. In the context of the present application, immunotherapy refers to providing an anti-cancer immune response in a subject by administration of an antibody (e.g., a monoclonal antibody) and / or by administration of an antigen that elicits an anti-tumor antigen immune response in the subject.

[0043] Those skilled in the art understand how to make and apply vaccines, including personalized cancer vaccines.

[0044] The terms "antigen" and "epitope" are well understood in the art and refer to the portion of a macromolecule (e.g., a polypeptide) which is specifically recognized by a component of the immune system, e.g.. an antibody or a T-cell antigen receptor. As used herein, the term "antigen" encompasses antigenic epitopes, e.g., fragments of an antigen which are antigenic epitopes. Epitopes can be recognized by antibodies in solution, e.g. free from other molecules. Epitopes can be recognized by T-cell antigen receptor when the epitope is associated with a class I or class II major histocompatibility complex molecule.

[0045] The terms “major histocompatibility complex” or “MHC” refers to a complex of genes encoding cell-surface molecules that are required for antigen presentation to T cells and for rapid graft rejection. In humans, the WIC is also known as the “human leukocyte antigen” or “HL A” complex. The proteins encoded by the WIC are known as “WIC molecules” and are classified into Class 1 and Class II WIC molecules. Class 1 MHC molecules include membrane heterodimeric proteins made up of an a chain encoded in the WIC noncovalently linked with the P2-microglobulin. Class I WIC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8+ T cells. Class I molecules include HLA- A, B, and C in humans. Class II WIC molecules also include membrane heterodimeric proteins consisting of noncovalently associated a and P chains. Class II WIC molecules are known to function in CD4+ T cells and, in humans, include HLA-DP, -DQ, and -DR.

[0046] The term “antigen presenting cells (APCs)” refers to a class of cells capable of presenting one or more antigens in the form of peptide-WIC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. APCs can be intact whole cells such as macrophages, B- cells, endothelial cells, activated T-cells, and dendritic cells; or other molecules, naturally occurring or synthetic, such as purified MHC Class I molecules complexed to 2-microglobulin. While many types of cells may be capable of presenting antigens on their cell surface for T-cell recognition, only dendritic cells have the capacity7to present antigens in an efficient amount to activate naive T-cells for cytotoxic T-lymphocyte (CTL) responses.

[0047] “Immune response’" broadly refers to the antigen-specific responses of lymphocytes to foreign substances. Any substance that can elicit an immune response is said to be “immunogenic’’ and is referred to as an “immunogen”. All immunogens are antigens, however, not all antigens are immunogenic. An immune response of this invention can be humoral (via antibody activity) or cell-mediated (via T cell activation).

[0048] 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. For purposes of this invention, 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.

[0049] "FACS" (Fluorescence-Activated Cell Sorting) is a specialized type of flow- cytometry used for sorting and analyzing cells based on specific characteristics, such as size, shape, and the presence of fluorescently labeled markers. A "single-point ELISA" (Enzyme-Linked Immunosorbent Assay) refers to a simplified version of the traditional ELISA, where the concentration of an analyte (e.g., a protein or antibody) is determined based on one specific point or dilution of the sample, rather than creating a full standard curve.

[0050] The immunotherapeutic strategy, introduced in this invention, leverages the antigen presenting ability of mature DCs for directing an immune response against tumor-associated antigens (TAA) or tumor-specific antigens (TSA).

[0051] The term “tumor associated antigen” or “TAA” refers to an antigen that is associated with a tumor. BEST MODE FOR C ARRYING OUT INVENTION

[0052] The following example explains the present invention more concretely, but do not limit the range of the present invention.

[0053] EXAMPLES

[0054] Example 1

[0055] When a patient comes in with primary tumor and dendritic cell vaccination is decided to be the treatment, PBMCs or bone marrow would be collected for dendritic cell differentiation at least 1 week prior to vaccination date and cultured for cell differentiation. The tumor would be resected 1 week later and a portion saved for vaccine generation. mannan-BAM would be mixed with the resected tumor cells and co-cultured with patient-derived dendritic cells and the maturation cocktail of TLR ligands for 24 hours. The dendritic cells would be lifted and washed from the co-culture before being given fresh as the initial vaccine through peripheral injection. The rest of the doses would be frozen and given on a weekly basis for an optimal four week regime and more if possible.

[0056] Details are as follows.

[0057] Generation of bone marrow derived dendritic cells

[0058] Both femurs and tibias were collected from a single mouse and bone marrow was flushed out using a syringe and RPMI media. Collected marrow was dissociated as close to single-cell suspension and filtered using a 70 um cell strainer for any leftover debris. Cells underwent red blood cell lysis and were resuspended in RPMI media with 1000 lU / mL of GMCSF. Media was switched out every two days and suspended cells and lightly -adherent cells were harvested as the immature dendritic cell population on day 5.

[0059] Mannan-BAM attachment to irradiated tumor cells

[0060] Tumor cells that had been irradiated with 100 Gy were mixed with mannan-BAM for 30 minutes at room temperature to allow insertion into the cell membrane. Cells were spun down and washed before use for dendritic cell co-culture.

[0061] Dendritic cell co-culture with mannan-BAM irradiated tumor cells

[0062] Immature dendritic cells were plated in RPMI in either in 6 well dishes or 150 mm dishes with mannan-BAM irradiated tumor cells at a ratio of 2 dendritic cells :1 tumor cell. The TLR ligands poly:IC (5ug / mL) and resiquimod (25ug / mL) were added to the co-culture for dendritic cell maturation over 24 hours

[0063] For vaccination, dendritic cells were lifted upon from the plates and spun down before being counted and diluted to the proper concentration in PBS. Time course analysis of dendritic cell (cdllc+MHCII+)

[0064] Co-stimulatory ligands CD80 and CD86 percentage and intensity as measured by flow cytometry following co-culture of dendritic cells with tumor antigen source and maturation cocktails. MBT demonstrates equivalent CD80% and CD86 MFI as TLT but superior CD80 MFI and CD86 % at 24 hours with differences being amplified at 48 hours in CD80%. CD80 MFI, and CD86%. The use of TLR ligands established superior costimulatory ligands at all timepoints compared to TL. As shown in FIGs. 2-5, Groups: Ctrl = negative control; tlr = poly-ic and resiquimod, irt = irradiated cells w / tlr; mb = mannan-bam coated irradiated cells; mbt = mb w / tlr; tl = tumor lysate; tit = tl w / tlr

[0065] FIGs. 6-9 show cytokine time course analysis of dendritic cell co-culture with tumor antigen source and TLR ligands over 48 hours. MIP-la, MIP-lb, and TNF measurements demonstrate tumor lysate with TLRs primed dendritic cells hinder the release of these factors compared to MBT. In MCP-1. MBT and IRT demonstrated superiority in release of this chemokine starting at 20 hours.

[0066] Groups: tlr = poly-ic and resiquimod; none = negative control; irradiated = irradiated cells (IR); irradiated + polysaccharide = mannan-BAM anchored irradiated cells (MB)

[0067] B16-F10 tail vein metastasis prevention model

[0068] Design and results for a B16 melanoma prophylactic DC therapy to evaluate the effect of the recipe components of the MBT therapy on melanoma anti-metastatic efficacy. As illustrated in FIG. 10, 100,000 dendritic cells primed on mannan-BAM B16-F10 cells were injected via tail vein into C57 BL / 6 mice as a vaccination one week before 400,000 B16-F10 cells tumor challenge via tail vein. After two weeks, mice lungs were harvested, and metastases were counted before paraffin embedding and H&E and IHC staining. FIG. 1 1 presents the left lung lobe of each mouse that was used to count the metastatic nodules, indicating that MBT therapy was most effective in reducing metastatic nodules.

[0069] Intracranial GL261 glioblastoma therapeutic model

[0070] Design, survival curve, and histology for a GL261 intracranial glioblastoma therapeutic model to assess MBT efficacy in comparison to current TL standard loading method.

[0071] Mice were implanted with GL261 glioblastoma cells before a 2 dose de therapy on day 3 and day 13. PD-1 treatment was administered on days 3-5 and days 13-15 for groups MBT + PD-1 and TL + PD-1. FIG. 13 indicated the survival curve with 5 / 9 mice in MBT+PD-1 and 1 / 9 mouse in MBT achieving complete regression. These completely regressed mice were rechallenged as shown in FIG. 14 with the same dose of GL261 glioblastoma cells on the opposite hemisphere with all surviving rechallenge. FIGs 15-16 demonstrate representative hematoxylin&eosin, CD4+ T cell, and CD8+ T cell staining for endpoint negative control. TL+PD-1, and MBT+PD-1 groups. These indicate greater necrosis and CD4+ and CD8+ T cell infiltration in MBT+PD-1.

[0072] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0073] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent a definition of a term set out in a document incorporated herein by reference conflicts with the definition of a term explicitly defined herein, the definition set out herein controls.

Claims

What is claimed is:

1. A cancer vaccine comprising dendritic cells prepared by: culturing immature dendritic cells to maturation; and co-culturing the matured dendritic cells with attenuated cancer cells, Mannan - BAM(Mannan, a yeast-derived polysaccharide, and BAM, a Biocompatible Anchor for Cell Membrane), and TLR (Toll like receptor) agonists; thereby the cancer vaccine comprising dendritic cells is obtained.

2. The cancer vaccine of claim 1, wherein the immature dendritic cells are prepared byharvesting monocytes from a subject.

3. The cancer vaccine of claim 1, wherein the attenuated cancer cells are prepared by: harvesting cancer cells from a biopsy of a site of tumor from a subject, culturing the harvested cancer cells to a relevant amount, and irradiating the cultured cancer cells.

4. A method of treating a cancer, the method comprising administering to a subject having the cancer an effective amount of a composition, wherein the composition comprises the cancer vaccine of claim 1.

5. A method of treating a cancer, the method comprising administering an effective amount of a cancer vaccine to a subject in need thereof, wherein the cancer vaccine comprises dendritic cells and is prepared by: culturing immature dendritic cells to maturation; and co-culturing the matured dendritic cells with attenuated cancer cells, Mannan - BAM (Mannan, a yeast-derived polysaccharide, and BAM, a Biocompatible Anchor for Cell Membrane), and TLR (Toll like receptor) agonists.

6. The method of claim 5, wherein the immature dendritic cells are prepared by harvesting monocytes from the subject.

7. The method of claim 5, wherein the attenuated cancer cells are prepared by: harvesting cancer cells from a biopsy of a site of tumor from the subject, culturing the harvested cancer cells to a relevant amount, and irradiating the cultured cancer cells.

8. The method of claim 5, wherein the cancer vaccine is administered by intratumoral injection.

9. The method of claim 5, wherein the cancer vaccine is administered by a subcutaneous inoculation.

10. A method for inducing an immunological memory- against a cancer, the method comprising administering an effective amount of the cancer vaccine of claim 1 to a subject in need thereof.

11. A method for inducing an immunological memory against a cancer, the method comprising administering an effective amount of a cancer vaccine to a subject in need thereof, wherein the cancer vaccine comprises dendritic cells and is prepared by: culturing immature dendritic cells to maturation; and co-culturing the matured dendritic cells with attenuated cancer cells, Mannan - BAM (Mannan, a yeast-derived polysaccharide, and BAM, a Biocompatible Anchor for Cell Membrane), and TLR (Toll like receptor) agonists.

12. The method of claim 11, wherein the immature dendritic cells are prepared by harvesting monocytes from the subject.

13. The method of claim 11. wherein the attenuated cancer cells are prepared by: harvesting cancer cells from a biopsy of a site of tumor from the subject, culturing the harvested cancer cells to a relevant amount, and irradiating the cultured cancer cells.

14. The method of claim 11, wherein the cancer vaccine is administered by intratumoral injection.

15. The method of claim 11, wherein the cancer vaccine is administered by a subcutaneous inoculation.

16. A cancer vaccine comprising dendritic cells prepared by: culturing immature dendritic cells to maturation; co-culturing the matured dendritic cells with attenuated cancer cells. Mannan - BAM (Mannan, a yeast-derived polysaccharide, and BAM, a Biocompatible Anchor for Cell Membrane), and TLR (Toll like receptor) agonists; and sorting the dendritic cells from the co-cultured attenuated cancer cells, thereby the cancer vaccine comprising dendritic cells is obtained.

17. The cancer vaccine of claim 16, wherein the immature dendritic cells are prepared by harv esting monocytes from a subject.

18. The cancer vaccine of claim 16, wherein the attenuated cancer cells are prepared by: harvesting cancer cells from a biopsy of a site of tumor from a subject, culturing the harvested cancer cells to a relevant amount, and irradiating the cultured cancer cells.

19. A method of treating a cancer, the method comprising administering to a subject having the cancer an effective amount of a composition, wherein the composition comprises the cancer vaccine of claim 16.

20. A method of treating a cancer, the method comprising administering an effective amount of a cancer vaccine to a subject in need thereof, wherein the cancer vaccine comprises dendritic cells and is prepared by: culturing immature dendritic cells to maturation; co-culturing the matured dendritic cells with attenuated cancer cells, Mannan - BAM (Mannan, a yeast-derived polysaccharide, and BAM, a Biocompatible Anchor for Cell Membrane), and TLR (Toll like receptor) agonists; and sorting the dendritic cells from the co-cultured attenuated cancer cells.

21. The method of claim 20, wherein the immature dendritic cells are prepared by harvesting monocytes from the subject.

22. The method of claim 20, wherein the attenuated cancer cells are prepared by: harv esting cancer cells from a biopsy of a site of tumor from the subject, culturing the harvested cancer cells to a relevant amount, and irradiating the cultured cancer cells.

23. The method of claim 20, wherein the cancer vaccine is administered by intratumoral injection.

24. The method of claim 20, wherein the cancer vaccine is administered by a subcutaneous inoculation.

25. A method for inducing an immunological memory against a cancer, the method comprising administering an effective amount of the cancer vaccine of claim 16 to a subject in need thereof.

26. A method for inducing an immunological memory' against a cancer, the method comprising administering an effective amount of a cancer vaccine to a subject in need thereof, wherein the cancer vaccine comprises dendritic cells and is prepared by: culturing immature dendritic cells to maturation; co-culturing the matured dendritic cells with attenuated cancer cells, Mannan - BAM (Mannan, a yeast-derived polysaccharide, and BAM, a Biocompatible Anchor for Cell Membrane), and TLR (Toll like receptor) agonists; and sorting the dendritic cells from the co-cultured attenuated cancer cells.

27. The method of claim 26, wherein the immature dendritic cells are prepared by harvesting monocytes from the subject.

28. The method of claim 26, wherein the attenuated cancer cells are prepared by: harvesting cancer cells from a biopsy of a site of tumor from the subject, culturing the harvested cancer cells to a relevant amount, and irradiating the cultured cancer cells.

29. The method of claim 26. wherein the cancer vaccine is administered by intratumoral injection.

30. The method of claim 26, wherein the cancer vaccine is administered by a subcutaneous inoculation.

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