WO2004055053A1 - Vaccin antitumoral - Google Patents

Vaccin antitumoral Download PDF

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Publication number
WO2004055053A1
WO2004055053A1 PCT/CN2003/001018 CN0301018W WO2004055053A1 WO 2004055053 A1 WO2004055053 A1 WO 2004055053A1 CN 0301018 W CN0301018 W CN 0301018W WO 2004055053 A1 WO2004055053 A1 WO 2004055053A1
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tumor
cell
cells
hybrid
cancers
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PCT/CN2003/001018
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English (en)
Chinese (zh)
Inventor
Ge Chen
Peng Cai
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Pel-Freez Biotechnology (Beijing) Ltd.
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Priority to AU2003289603A priority Critical patent/AU2003289603A1/en
Publication of WO2004055053A1 publication Critical patent/WO2004055053A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer

Definitions

  • the invention relates to the field of tumor biological immunotherapy and tumor prevention and immunity. It mainly relates to the preparation of tumor vaccines, especially the preparation of a non-cellular tumor vaccine and its application in specific anti-tumor immunotherapy and tumor prevention immunity. Background technique
  • tumor cells can grow and reproduce uncontrollably in a living organism.
  • the body's anti-tumor immune mechanism is defective and incomplete, so that the body is in a kind of "silence” or “resistant” immunity to tumor cells. "Tolerance” status. This is particularly reflected in the fact that the immune cells in the body cannot specifically recognize tumor cells, that is, anti-tumor specific immune recognition disorders.
  • the specific recognition mechanism of immune cells for tumor cells is mainly composed of MHC or HLA (main tissues) Compatible complexes, human leukocyte antigens) and tumor peptides form complexes to form the first signal for T lymphocyte specific recognition; and other co-stimulatory factors such as B7 (B7-1, B7-2) constitute T-lymph
  • B7 B7-1, B7-2
  • B7 B7-1, B7-2
  • MHC-I or HLA-I molecular tumor polypeptide complex can be recognized by CD8 + lymphocyte receptor (T Cell Receptor; TCR); MHC-II or HLA-II molecular tumor peptide complexes are recognized by TCR of CD4 + T lymphocytes, and the CD28 receptor of the T lymphocytes can bind to the B7 molecule, which in turn activates and proliferates CD4 + and CD8 + T lymphocytes, inducing specificity Sexual cellular immune effect.
  • CD8 + T lymphocytes can produce specific killing effects directly on tumor cells in the anti-tumor immune effect.
  • T lymphocytes Such cells are called Cytotoxic T Lyphocytes (CTL); 'CD4 + T lymphocytes are usually called T helper cells (T Helper; T H), which is secreted by a variety of factors as IL-12, INF-R, IL-2 and the like to promote secondary CTL response B lymphocytes to produce anti-tumor antibodies.
  • CTL Cytotoxic T Lyphocytes
  • T H T helper cells
  • the immune system has the function of immune surveillance (Immune Surveillance): it can identify normal cells that have mutated at an early stage, so as to eliminate them and prevent their differentiation and proliferation to tumor cells [Satthapom S, Eremin 0., Dendritic cells (II): role and therapeutic implications in cancer. JR Coll Surg. Edinb 2001; 46: 159-167]. Therefore, whether the body's immune function is normal or not is closely related to the occurrence and development of tumors.
  • Tumor cells originate from normal cells with mutations in the body. Therefore, most of the structural components of tumor cells are "Self" normal components; the body's immune system does not produce an immune response to such autoantigen components.
  • Tumor cells have a "shielding effect" on their tumor antigen expression.
  • Some tumor antigens are a component of a polysaccharide protein shield complex, which are poorly immunogenic or because the corresponding antibodies produced by the body bind to the tumor antigen, "blocking," its antigenic site, making it difficult for the body to immunize it. Identify. 3.
  • the genotype of tumor antigen is extremely unstable and the random mutation rate is high. Therefore, the phenotype of its antigen changes rapidly and diversely, causing the body to specifically recognize tumor antigens and impede the immune response.
  • Tumor cells secrete certain immunosuppressive factors: such as IL-10, PGE-2, TGF- ⁇ , etc. These factors have a significant inhibitory effect on APC, making APC antigen presentation difficult or directly inhibiting T and B lymphocytes Proliferation and activation of macrophages; In addition, some tumors can secrete FasL (Fas ligand; Fas Ligand), which binds to the death receptor (Fas) on the surface of corresponding immune cell membranes, resulting in immune cells with potential anti-tumor capabilities "Apoptotic Death” occurs, which weakens the body's ability to resist tumor immunity.
  • FasL Fas ligand
  • Fas Ligand Fas Ligand
  • the proliferation, growth, metastasis, and dissemination of tumor cells in the body are extremely rapid, causing a heavy "immunity load" on the body's immune system, making the body's anti-tumor immune function relatively low, which is not enough to completely remove many tumor cells and cause the tumor to grow out of control. .
  • APC is a class of immune cells with important functions in the immune system of the body.
  • APC has the functions of exposing, processing, and processing antigens, and presenting the processed antigen information to specific lymphocytes through a certain recognition mechanism, thereby inducing specific cellular and humoral immune responses.
  • DCs dendritic cells
  • macrophages are the most widely distributed and are the main cells that process antigens.
  • DC cells are the cells with the strongest antigen presenting function in the body [Cella M, Sallusto F, and Lanzavecchia A., Origin, maturation and antigen presenting function of dendritic cells, Curr Opin. Immunol. 1997; 9: 10-16].
  • DC cells There are three types of DC cells: (1) and refers to DC (IDC), which exists in the epithelium, exists as Langerhans cells, contains characteristic Birbeck particles, and acts as immature "veiled cells”. Lymphatic vessels migrate into the paracortical area (T cell area) of draining lymph nodes, and Cell interaction to become mature DC;
  • FDC Follicular DC
  • germinal center DC exists in the secondary follicular germinal center of the B cell area, is a group of migrating cells, and interacts with T cells after reaching the germinal center;
  • Thymus IDC It exists in the thymic medulla and plays a major role in T cell development and maturation.
  • the DCs that play a role in tumor immunity are mainly IDCs.
  • Immature DCs can effectively capture antigens. This process is accomplished through the following mechanisms: (1) uptake; (2) macropinocytosis; (3) adsorption and (4) receptors (mannose receptor, C Lectin receptor, FC receptor) -mediated endocytosis. Due to these mechanisms, DCs can capture antigens at very low antigen concentrations.
  • Soluble or granular antigens are processed in DC cells and are degraded into antigenic peptides by the proteolytic enzymes of phagolysosomes in the cells, which are combined with MHC class II molecules and then presented to T cells [Banchereau J , Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, and Palucka K. Immunobiology of dendritic cells. Annu Rev Immunol. 2000; 18: 767-811].
  • DCs can also cross-present apoptotic bodies, bacteria, or soluble antigens that are swallowed through the MHC class I pathway with MHC class I molecules and cross-present them to CD8 + T cells.
  • This pathway is called the internal pathway. It is believed that the presentation of crossover depends on the type of substance swallowed. Only apoptotic cells can enter the internal pathway, while necrotic cells do not. In addition, DC cells can also present endogenous antigens of their cells ⁇ .
  • the conversion rate of the MHC-peptide complex on the cell surface is reduced, and stably exists for several days; (2) Up-regulation of a series of cytokines, such as IL-12, IL-15, these factors can up-regulate T cell Allergic / activate or directly enhance the response of CD4 + T helper cells (Th); (3) DC can also antagonize the lysis of T cells by tumor cells, thereby extending the life of T cells in tumors.
  • cytokines such as IL-12, IL-15
  • the current strategic design of anti-tumor clinical immunotherapy is to reduce the tumor immune load and relieve anti-tumor immunity Inhibited State; re-stimulate and restore the patient's own anti-tumor immune function to achieve a new positive immune system balance; fully reflect the characteristics of tumor-specific individualized immunotherapy.
  • Dead whole tumor cells Melanoma, colon cancer, etc.
  • Tumor cells Colon cancer, etc.
  • HSP Heat shock protein
  • the virus ⁇ cytokine sarcoma The virus ⁇ cytokine sarcoma
  • DC / tumor fusion cell is a new type of tumor vaccine developed in recent years.
  • the clinical treatment scheme designed with this vaccine is currently considered to be the most promising tumor immunotherapy scheme [Gottfried E, Krieg R , Eichelberg C, Andreesen R, Mackensen A, Krause SW., Characterization of cells prepared by dendritic cell-tumor cell fusion. Cancer Immunother 2002; 2: 15
  • Tumor cells are fused to form hybridoma cells; this cell has both the ability of tumor cells to produce all tumor antigens (both known and unknown) and the ability of DC cells to comprehensively (including MHC-I / II) present tumor tumor antigens.
  • the immune-active cells provide a complete dual-signal recognition system, which can effectively stimulate the immune-active cells to produce a specific recognition and killing effect on tumors, thereby achieving clinical therapeutic effects.
  • the inventors of the present invention conducted a lot of in-depth studies to obtain the present invention.
  • the APC / tumor hybrid cell extract has at least the same efficacy as the intact hybrid cells in activating the body's anti-tumor specific immunity, which can fully activate the human anti-tumor specific immunity, and in particular, can strongly and effectively stimulate CD4 + ( T H , helper T cells) and CD8 + (CTL, killer T cells) lymphocytes are activated, exerting a specific immune killing effect on tumor cells.
  • T H helper T cells
  • CTL killer T cells
  • the present invention provides:
  • a vaccine composition for preventing and / or treating a tumor comprising a cell extract of an antigen-presenting cell / tumor cell hybrid cell.
  • the hybrid cell has the ability to specifically stimulate an organism's anti-tumor immune response.
  • the hybrid cell has or does not have a division and proliferation function, but preferably the hybrid cell has a division and proliferation capability, and more preferably, the hybrid cell is an immortalized hybrid cell line.
  • the cell extract is a cell lysate or any part or component thereof which has an activity to stimulate the antitumor immune response of a living body.
  • the cell extract is a supernatant or pellet after centrifugation of the cell lysate.
  • the cell extract is one or more components selected from the group consisting of a cell membrane component and a cytoplasmic component, which are derived from a cell lysate of the hybrid cell and have an activity of stimulating an anti-tumor immune response of the living organism. Substances, nuclear components, protein molecules and fragments thereof, protein shield polypeptide complexes, lipoproteins, glycoproteins, protein complexes, DNA molecules and fragments thereof, and RNA molecules.
  • the antigen presenting cells are occupational antibodies
  • the original presentation cells are more preferably dendritic cells (DC cells).
  • the hybrid cell may be one or more hybrid cells selected from the following: a hybrid cell formed by fusion of an autologous APC cell and an autologous tumor cell; an autologous APC cell and an allogeneic tumor cell or tumor cell Hybrid cells formed by cell fusion; hybrid cells formed by fusion of autologous APC cells with tumor cells or tumor cell lines from multiple allogenes; hybrid cells formed by fusion of allogeneic APC cells with heterologous tumor cells or tumor cell lines; and multiple allogeneic cells A hybrid cell formed by fused APC cells with multiple allogeneic tumor cells or tumor cell lines.
  • the hybrid cell is a hybrid cell formed by fusion of an autologous APC cell and an autologous tumor cell.
  • the vaccine composition of the invention further comprises one or more immune adjuvants.
  • the immune adjuvant may be selected from BCG, QS-21, HSP, ISCOMS, Freund's complete adjuvant and Freund's incomplete adjuvant.
  • the tumor or tumor cell is or is derived from various cancers, sarcomas, gliomas, blastomas, fibromas, various lung cancers, including colorectal cancer, colon cancer, and rectum.
  • cancer of intestine various liver cancer, various gastric cancer, various kidney cancer, prostate cancer, various breast cancer, ovarian cancer, cervical cancer, various skin cancer, melanoma, various nasopharyngeal cancer, various Esophageal cancer. '
  • the hybrid cell extract and one or more cytokines are reinfused with cytotoxic T lymphocytes having specific tumor-killing effects after in vitro culture, and / or with Combination of anti-tumor specific antibody therapy or other immune-enhancing antibodies.
  • a method for preventing and / or treating tumors including administering to a subject in need of treatment a prophylactic or therapeutically effective amount of cell extracts of antigen-presenting cells / tumor cell hybrid cells.
  • the cell extract is preferably administered by the following route: subcutaneous injection, intradermal injection, peri-lymph node injection, intra-lymph node injection, intramuscular injection, intravenous injection Intravenous injection, thoracic injection, intraperitoneal injection, intrathecal spinal injection, local injection around the tumor, intratumor injection, systemic multipoint injection.
  • the tumor vaccine, the hybrid cell application and the antitumor method of the present invention can be used not only for the treatment of various tumors, but also for the use of multivalent or broad-spectrum tumor vaccines for preventive antitumor treatment in normal populations or certain high-risk populations of corresponding tumors. Immunity. Detailed description of the invention
  • the cell extract of the antigen-presenting cell / tumor cell hybrid cell has the ability to specifically stimulate an organism's anti-tumor immune response. Therefore, the extract can be used for preparing a vaccine composition for preventing and / or treating tumors of the present invention.
  • the composition can be used to treat or prevent tumors by administering an effective amount to the subject.
  • the "specific ability to stimulate an organism's anti-tumor immune response” refers to an increase in the number of any immune-functioning cells or subgroups thereof or an increase in immune function as determined by any method known in the art.
  • the number of killer T cells in peripheral blood increased proliferative activity (such as 3 H-labeled thymidine incorporation assay, flow cytometry), the number of CD4 + T cells, increased proliferative activity, and enhanced T cell immune activity
  • Increased release of cytokines such as IFN Y, TNF, IL-2, etc.
  • the above-mentioned antitumor immune response is increased by at least 10%, more preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.
  • the antigen presenting cell (APC cell) in the present invention may be any cell having an antigen presenting function in a living body.
  • dendritic cells are used.
  • the APC cells used may be autologous cells or allogeneic cells.
  • the APC cells of the present invention may also be transformed or induced to transform cells that did not have APC functions into cells with APC functions, such as various stem cells, embryonic stem cells, various stem cells in blood, tissue stem cells, and fibers. Mother cell, DC Precursor cells, epithelial cells, mesothelial cells, endothelial cells, mesenchymal precursor cells, mesenchymal cells, tumor cells, etc.
  • APC cells can be co-cultured with specific tumor antigen components (Pulse) in vitro, and under the combined action of various auxiliary biological factors, they can have specific antigen presentation function against corresponding tumor antigens.
  • the tumors and tumor cells according to the present invention may be any primary, secondary and metastatic tumors and tumor cells of benign and malignant tumors.
  • the tissue source of these tumors and tumor cells can be epithelial tissue, mesenchymal tissue, blood tissue, etc .; for example: various cancers, sarcomas, gliomas, blastomas, fibromas, various lung cancers, colon cancers (colorectal cancers) , Colon cancer, rectal cancer, etc.); all kinds of liver cancer; all kinds of gastric cancer; all kinds of kidney cancer; prostate cancer; all kinds of breast cancer, ovarian cancer, cervical cancer; all kinds of skin cancer, melanoma; all kinds of nasopharynx Cancer; various esophageal cancers.
  • the tumor cell of the present invention may be a tumor cell that is present in an organism without being modified or induced, or a tumor cell that has been modified or induced, thereby enhancing its tumor antigen presenting ability or tumor immunogenicity to stimulate specific resistance Tumor immune response.
  • tumor cells Before APC and tumor cells are fused, tumor cells can be inactivated by radiation and drugs to make them lose their ability to proliferate; or radioactive inactivation or other inactivation treatment is not required for tumor cells to ensure that after the fusion cells are formed, The hybrid cell still maintains the original tumor antigen and effective tumor antigen presentation ability and cell division and proliferation activity.
  • the method for isolating APC cells in vitro there can be various methods for isolation using physical characteristics such as cell adhesion, density, and other immunological characteristics such as membrane antigens or special conditions for cell growth. For example: adherence method, density gradient centrifugation, cell separator, flow cytometer, magnetic bead separation, APC cell culture solution, etc. Since DC cells are the APC cells with the strongest antigen presentation function, DC cells are preferably used in the present invention.
  • the method for separating DC cells it may be: (1) Density purification method, directly separating DC precursor cells from blood or bone marrow, and then culturing them into mature DCs in vitro.
  • CD14 + mononuclear cells or CD34 + precursor cells from blood or bone marrow (CD14 is expressed in monocytes, macrophages, and weakly expressed in granulocytes, and is mainly used to label monocytes; CD34 expression In early lymphoid hematopoietic stem cells and precursor cells), CD14 + mononuclear cells were cultured together with IL-4 and GM-CSF and differentiated into DCs.
  • DCs isolated by this method to stimulate T cells is exactly the same as that of DCs cultured with CD40L [Engels FH, Kreisel D, Fades MB, Bedrosian I, Koski GK, Cohen PA, and Czerniecki BJ. Calcium ionophore activation of chronic myelogenous leukemia progenitor cells into dendritic cells is mediated by calcineurin phosphatase. Leuk Res 2000; 24: 795-804.].
  • DCs isolated from cancer patients have no difference in type and functional activity in vitro from DCs isolated from healthy people. Freezing and thawing have no effect on the function of both immature and mature DCs, so a large number of DCs can be prepared at one time for multiple treatment sessions.
  • the tumor cells are dispersed.
  • the tumor cells exist in the blood or pleural and ascites fluid, which is in a dispersed state. It only needs to be separated from other blood or liquid components.
  • solid tumors those skilled in the art will be familiar Method to disperse it into single cells, for example: first physically dissipate the tumor entity into small pieces, and then digest it with an enzyme to collect a single tumor cell.
  • APC / tumor hybrid cells can be any physical, chemical, and biological techniques and methods known to those skilled in the art to make two biologically active cells form a new hybrid cell; including electrofusion and PEG fusion , Microwave cell fusion, receptor-mediated fusion and other methods.
  • the fusion methods of APC / tumor hybrid cells mainly include the following types:
  • APC cells from multiple foreign bodies are fused with tumor cells or tumor cell lines from multiple foreign bodies.
  • the APC / tumor hybrid cell formed after fusion in the present invention has or does not have a division and proliferation function, but preferably the hybrid cell has a division and proliferation capability, and more preferably, the hybrid cell is an immortalized hybrid cell line .
  • the tumor cells are not inactivated, so that the tumor cells retain their good differentiation, proliferative activity, and ability to continuously produce the original tumor-specific antigen.
  • the cell fusion efficiency is significantly improved; on the other hand, hybridoma cells formed after APC / tumor cell fusion can still maintain good differentiation and proliferation activity, and can be subjected to short-term or long-term continuous subculture in vitro to establish permanent hybridoma cell lines. It provides abundant cell sources for tumor vaccine preparation, meets a large number of tumor vaccine preparation requirements, and is suitable for large-scale production of biopharmaceuticals.
  • the cell extract according to the present invention may be a cell lysate or any part or component thereof having an activity to stimulate the antitumor immune response of a living body.
  • the cell extract is a supernatant or a precipitate after centrifugation of the cell lysate.
  • the cell extract is one or more components selected from the group consisting of a cell membrane component and a cytoplasmic component, which are derived from a cell lysate of the hybrid cell and have an activity of stimulating an anti-tumor immune response of the living organism. Substances, nuclear components, protein molecules and fragments thereof, protein-polypeptide complexes, lipoproteins, glycoproteins, protein complexes, DNA molecules and fragments thereof, and RNA molecules.
  • the method for preparing a hybrid cell extract can be any physical, chemical, enzymatic, biochemical, or biological method that disrupts the cells, such as freeze-thaw cells, ultrasonic disruption, microwave, electromagnetic waves, laser, pulping, centrifugation, gradient density Centrifugation, chromatography, affinity chromatography, precipitation, dialysis, ultrafiltration, enzymatic digestion, etc .; the obtained extract contains at least one subcellular component capable of activating and enhancing organism-specific antitumor immune function.
  • the APC / tumor hybrid cell extracts described in the present invention may be well known in the art.
  • the technology is further purified to obtain the corresponding single purification component or multiple purification components, which are used individually or in combination according to the needs of clinical applications. It mainly includes but is not limited to the following: cell membrane component, cytoplasm component, cell nuclear component; HLA component, HLA-tumor peptide complex, HLA-I-tumor peptide complex, HLA-II-tumor peptide complex , Heat Shock Protein (HSP), HSP-tumor peptide complex, protein components, RNA, DNA molecules and fragments thereof, polypeptide components, tumor-associated antigens and polypeptide components, tumor-specific antigens and polypeptides Components, cell membrane receptor components, various CD molecular components, etc.
  • HSP Heat Shock Protein
  • Extracts from a variety of APC / tumor hybridoma cells and their various components described above can be combined into various active vaccines with multivalent or broad-spectrum antitumor properties based on the tumor type of the particular patient.
  • Cell extracts can be derived from one type of APC / tumor cell hybrid cells or multiple types of APC / tumor cell hybrids.
  • the hybrid cell extract itself obtained as described above can be used as the antitumor vaccine composition of the present invention for a subject in need of treatment.
  • the above-mentioned hybrid cell extract may be mixed with a pharmaceutically acceptable carrier to prepare the antitumor vaccine composition of the present invention.
  • the pharmaceutically acceptable carrier can be selected by those skilled in the art according to the specific route of administration and standard pharmaceutical practice. For example, physiological saline, Hanks solution, phosphate buffered saline, water, and the like can be used.
  • the dose of the vaccine composition of the present invention can be determined by the clinician with reference to the dose of the complete APC / tumor hybrid cell according to the weight, age of the patient, the type and severity of the disease to be treated, and the like.
  • the ratio between the cell extract and the pharmaceutically acceptable carrier in the vaccine composition of the present invention depends on the specific composition of the cell extract, the stability of the composition, the expected dosage level, and the like.
  • the cell extract and the optional drug carrier can be used alone as a tumor vaccine, or with one or more other immune adjuvants such as BCG, QS-2K HSP, ISCOMS, Phosphorus Complete adjuvant, Freund's incomplete adjuvant; or used in combination with one or more cytokines, there is no restriction on the type of cytokines, as long as it can activate or strengthen the body's immune function, granulocyte macrophage colonies are preferred Stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin 12 (IL-12), gamma-interferon (INFy), etc .; preferably in vitro culture with tumor vaccine , Stimulate, induce, activate, proliferate cytotoxic T lymphocytes with specific tumor killing effect Cytotoxicity T Cells (CTL) infusion therapy; combined with anti-tumor specific antibody therapy or other immune-enhancing antibodies such as anti-CD40 antibody and anti-CD28 antibody.
  • CTL Cytotoxicity T
  • the above-mentioned components which can be used in combination with the hybrid cell extract of the present invention may be mixed with the cell extract into the vaccine composition of the present invention, or administered to the subject to be treated simultaneously and sequentially with the cell extract of the present invention. To use in combination.
  • the antitumor vaccine composition of the present invention is administered to a subject in need of treatment in an amount effective for preventing or treating tumors.
  • the vaccine composition contains the equivalent of about 105 to about 108 cells of the hybrid cell extract, equivalent to about 106 and more preferably to about 107 hybrid cells a cell extract, equivalent to about most preferably to about 3X10 6 8X10 6 cells of the hybrid cell extracts.
  • the administration route of the vaccine composition of the present invention can be: subcutaneous intracutaneous injection, intravenous injection, peri-lymph node injection, intra-lymph node injection, intramuscular injection, thoracic injection, intraperitoneal injection, spinal cord intrathecal injection, local injection around the tumor, intratumor injection Multi-point injections throughout the body.
  • the above-mentioned introduction routes may be used alone or in combination according to specific circumstances. Can be used once or multiple times.
  • the tumor vaccine composition mentioned in the present invention can be used simultaneously or successively with other various tumor treatment methods, such as surgical resection, chemotherapy, radiotherapy, various biological factor treatments, and other immunotherapy such as antibody treatment. Wait.
  • the present invention is preferably used in combination with a specific cellular immunization method.
  • the basic design of this scheme is: co-culture with the tumor patients themselves T lymphocytes with the tumor vaccine or other tumor vaccines described in the present invention with the participation of other stimulating factors such as IL-2 in vitro to detach the T lymphocytes
  • the patient's immunosuppressed internal environment receives specific stimulation and activation of the tumor vaccine under appropriate culture conditions in vitro, forms tumor-specific CTLs and undergoes large-scale proliferation, and then returns directly to the patient to directly exert the specific tumor-killing effect.
  • vaccination with tumor vaccine can strengthen and extend the patient's body-specific anti-tumor immune effect ability.
  • the effect of the tumor vaccine of the present invention can be evaluated by observing the number of any immune functioning cells or subpopulations and / or an increase in their immune function after stimulation by the vaccine of the present invention. Price. This evaluation can be performed in vitro, ex vivo or in vivo. After applying the tumor vaccine composition of the present invention to a treatment subject, the treatment effect can also be evaluated by observing the molecular level (DNA, RNA, etc.), cell level or histopathological level. For example, the following non-limiting examples can be cited:
  • cytokines released when the T cell immune activity is enhanced, such as IFN Y, TNF (tumor necrosis factor), IL-2, etc .;
  • Whether the clinical indications of the tumor have improved such as the reduction or disappearance of tumor metastases, the decrease in the volume of tumor entities, the improvement of the function of patients' damaged organs (such as liver function, renal function, etc.), and certain tumor-specific markers Levels of protein (protein level or nucleic acid) such as PSA (prostate cancer), CEA (carcinoembryonic antigen for detecting gastrointestinal tumors), AFP (fetal protein for detecting liver cancer), CA199 (for detecting pancreatic cancer), etc. Level) whether to decrease;
  • composition of the present invention When the composition of the present invention is used in a high-risk / high-risk population, it is detected and followed up whether the tumor incidence of the treated subject is reduced.
  • the basic method for realizing the present invention is to prepare a monovalent tumor vaccine: fusion of autologous APC cells and autologous tumor cells, obtaining autologous APC / tumor hybrid cells, and culturing the hybrid cells under specific conditions in vitro to make them express MHC (HLA)-at high levels Tumor antigen polypeptide and co-stimulatory factor B7 and other related cytokines such as ICAM-1, IFA-3, CD40 and so on.
  • a tumor vaccine containing hybrid cell extracts is then made.
  • a part of the hybrid cells is reserved for continuous culture to establish cell lines.
  • the vaccine prepared by this method is highly targeted, can specifically induce the same tumor patient to produce specific anti-tumor immune effects, and selectively kill and clear tumor cells in the patient.
  • the hybrid cell line thus obtained can be used not only for subsequent treatment of the tumor patient, but also as a component of the preparation of a multivalent tumor vaccine.
  • the invention also includes the preparation of a multivalent tumor vaccine:
  • Autologous APC cells are fused with one or more allogeneic tumor cells and / or homogeneous tumor cell lines.
  • autologous APC cells from patients with melanoma have been fused with various allogeneous melanoma cells or cell lines to form autologous APC / multiple allogeneous melanoma tumor hybrid cells.
  • This hybrid cell has multiple melanoma tumor peptide-HL A complexes. It also presents a variety of melanoma cell-specific antigens.
  • the mixed hybridoma cells were made into cell extracts, and the patients' melanoma antigen-specific T cells were activated by the Cross Priming mechanism of common melanoma tumors, and immunotherapy was performed on the corresponding patients.
  • One or more allogeneic APC cells are fused to multiple allogeneic homogeneous or heterogeneous tumor cells and / or tumor cell lines.
  • the tumor cells used for preparing the tumor vaccine of the present invention can be homogeneous tumor cells derived from multiple allogenes, or multi-type tumor cells derived from multiple allogenes, and a broad-spectrum, multivalent tumor vaccine can be established.
  • the tumor vaccine of the present invention has the dual functions of tumor immunotherapy and tumor preventive immunity.
  • the above-mentioned multivalent or broad-spectrum tumor vaccine is used to preventively immunize normal populations or some corresponding high-risk populations of tumors, so that immune cells under normal physiological conditions can obtain complete tumor antigen stimulation; that is, HLA-tumor Specific peptide complexes and co-stimulatory factors.
  • the resting T-lymphocyte immune system of a normal body immune to the tumor vaccine is activated and generates specific immune memory cells.
  • the above process is very similar to the effect of the body's immune system's first acceptance of a certain alloantigen component.
  • the tumor antigen component of the tumor cell can be specific memory lymphocytes produced by the primary tumor vaccine Upon recognition, it can quickly and fully induce the anti-tumor response of the body's cellular immunity (memory T cells) and humoral immune system (memory B cells), and specifically kill and clear the corresponding cells, thereby preventing the tumor cells from continuing to grow. Prevent the formation of tumors.
  • the active ingredient of the tumor vaccine composition of the present invention is a cell extract of APC / tumor cell hybrid cells.
  • the invention shows for the first time that: activating the body's anti-tumor specificity Immunization does not require the use of intact APC / tumor hybrid cells as a vaccine.
  • the extract of APC / tumor hybrid cells has the same tumor vaccine activity as intact hybrid cells, which can fully activate the human anti-tumor specific immunity, and can particularly effectively stimulate CD4 + (T H, T helper cells) and CD8 + (CTL, killer T cells) lymphocyte activation, exert effects on killing tumor specific immune cells.
  • the vaccine composition of the present invention uses cell extracts of APC / tumor cell hybrid cells, it has no tumor proliferative activity, does not require inactivation of tumor cells, does not introduce heterologous genes, and hybridizes cells before preparing the extract. Constant proliferation fully expresses tumor cell antigens, not only using tumor cell membrane antigens, but also cytoplasm and nuclear antigens, so the preparation method is simple, efficient, safe to use, and has high titer.
  • Figure 1 Dendritomas lysates, Dendritomas lysates solution, and Dendritomas lysates depositions of peripheral blood lymphocytes and DC-tumor cell hybrid cells are co-cultured. And its subtype percentage change.
  • PBNC Peripheral blood mononuclear cell
  • CD3 is a T cell common antigen marker
  • CD4 is a helper T cell antigen marker
  • CD8 is a killer T cell antigen marker
  • CD56 is an antigen marker of natural killer cells .
  • Figure 2 Shows the results of MTT assay for cytotoxic activity.
  • DT refers to DC-tumor cell hybrid cells;
  • DT-lysates refers to lysate of DC-tumor cell hybrid cells;
  • DT-lysates S refers to lysate supernatant;
  • DT-lysates D refers to lysate precipitate.
  • A549 is a human lung adenocarcinoma cell line, and K562 is a human chronic myelogenous leukemia cell line.
  • Isolation and culture of DC cells using a blood collection machine (Baxter, USA) CS3000plus) Isolate peripheral blood lymphocytes, count, centrifuge at 2000 rpm, and then wash the cells twice with serum-free medium for 10 minutes each time; use serum-free medium (American GIBCO clinical treatment grade, AIM V type) to reduce cell concentration Adjust to 4 x 10 6 / ml; add GM-CSF1000U / ml to the culture medium overnight; collect suspended lymphocytes the next day and freeze them by conventional methods for later CTL culture, add an appropriate amount of medium to the culture flask, GM-CSF was supplemented and 1000 U / ml of IL-4 was added to continue to culture adherent cells.
  • serum-free medium American GIBCO clinical treatment grade, AIM V type
  • tumor cells Under sterile conditions, the tumor tissue was cut into small pieces of 1-2 mm 3 and placed in PRMI1640 medium to add a certain amount of collagenase and DNase. Incubate at 37 ° C for 30 minutes-4 hours. After removing the bulk tissue by filtration, the cell fractions were collected by centrifugation. Wash the cells and add RBC lysate to destroy RBC. After collecting the nucleated cells by centrifugation, the tumor cells were collected by Ficoll-Paque layered liquid gradient centrifugation. Suspend tumor cells in an appropriate culture medium for future use.
  • DC tumor cells Mix mature DC cells and tumor cells dispersed into single cells according to 6 1 (DC tumor cells), centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and gently blow with a pipette.
  • Cells are pelleted and suspended; the cell suspension is placed in a 37 ° C water bath for 1 minute, and 1 ml of PEG preheated to 37 ° C is added, and the cells are gently mixed, and then 1 ml of 1640-RPMI medium is added every 1 minute for 10 Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 10 mL of 1640-RPMI medium to the pellet, centrifuge at 1000 rpm for 5 minutes, wash the cells twice with 1640-RPMI medium, and add an appropriate amount of serum-free medium overnight.
  • Preparation method of specific anti-tumor CTL induction 10-100 ml of human peripheral blood is collected by routine clinical sterile vein. Collect mononuclear cells by lymphocyte layered liquid gradient centrifugation Components; or collect peripheral blood mononuclear cells with a cell separator. The mononuclear cells are allowed to stand in an appropriate culture medium for 30 minutes to 5 hours, and non-adherent cells are collected. After washing, the cells were suspended in a serum-containing culture medium and added to a culture flask. Add a certain amount of DC / tumor hybrid cell extract and other factors such as IL-2 to the culture medium; continue to culture in a 37 ° C CO 2 incubator for 5-12 days. Washed activated CTLs are ready for use.
  • DC anti-tumor hybrid cell extract clinical anti-tumor immunotherapy scheme take a certain amount of tumor vaccine and / or a certain amount of immune adjuvant mixed under sterile conditions (such as BCG, etc.) to inject the patient around the subcutaneous lymph nodes .
  • sterile conditions such as BCG, etc.
  • a certain amount of interleukins such as IL-2 or other cytokines is given intradermally or intramuscularly as an adjuvant comprehensive treatment; the injection is continued for 3-10 days. Repeat injections every 1-4 weeks depending on clinical conditions to strengthen the immune effect.
  • the body's various immunological indicators are monitored dynamically, and the vaccine treatment plan is adjusted in a timely manner.
  • the DC / tumor hybrid cell mixed extract after repeated freezing and thawing can be directly applied to immunotherapy of clinical tumor patients or used after further purification.
  • the speed (rpm) of the fixed hook pulper is 500-5000 rpm.
  • Start the pulping machine hook the pulp for 10 seconds to 1 minute; suspend the pulping for 1-5 minutes. Repeat the process 3-10 times.
  • the prepared vaccine is stored in a low temperature environment for future use.
  • the frozen lymphocytes were recovered after isolation, and an appropriate amount of serum-free medium, 50u / ml of IL-2 was added overnight.
  • Cell lysate and pellet prepared according to 10 1 (lymphocyte DC-tumor cell fusion cells or corresponding cell number) with DC cells, DC-tumor cell fusion cells, DC-tumor cell fusion cell lysate, DC- Lysate supernatant of tumor cell fusion cells and DC-tumor cell fusion cell lysate pellets (prepared according to the method of Example 1) were mixed and cultured every 3 days in serum-free medium (containing 50u / ml IL-2 ) Change the medium and harvest the cells after 7 days of culture. Immunotype the cells. Save the supernatant and use the ELISA method to determine the ⁇ interferon content.
  • the effector cell was the lymphocyte obtained in Example 8, wherein the tumor cell used for fusion was A549. Take logarithmic growth stage tumor cells A549 at a density of l-5xl0 5 / ml, and load 10 ⁇ L cell suspension per well in a 96-well plate at 20: 1 (cell number ratio, 20 is lymphocytes, 1 is The ratio of target cells (ie, tumor cells) was added to the prepared lymphocyte suspension into the corresponding wells. Another irrelevant cell was selected as a cross-control, and the culture medium containing no cells was used as a blank control. Three MTTs were set for each dose. .
  • the tumor cell inhibition rate of peripheral blood lymphocytes co-cultured with lysate, lysate supernatant, and lysate pellet of DC-tumor cell hybrid cells was co-cultured with DC-tumor cell hybrid cells.
  • the tumor cell suppression rate of blood lymphocytes is comparable. This shows that the lysate, lysate supernatant and lysate pellet of DC-tumor cell hybrid cells have immunostimulatory activity. And the former has no effect on K562, indicating that its inhibitory effect on tumors is more specific.
  • DC-tumor cell hybrid cells are required to inactivate the tumor cells with radiation before the preparation of the tumor vaccine, so that the tumor cells lose the ability to differentiate and proliferate. Before the subject, the hybrid cells need to be inactivated again with radiation.
  • the tumor vaccine composition of the present invention uses hybrid cell extracts, and there is no problem of introducing tumor cells with proliferative capacity into the body, so it is not necessary to use radiation for inactivation treatment, so the actual titer is significantly higher than the use of intact hybrid cells The vaccine.
  • the DC-tumor cell hybrid cells used to process the lymphocytes have not been subjected to radiation treatment, and therefore have high activity.

Abstract

La présente invention concerne un complexe vaccin destiné à la prévention ou au traitement de tumeur. Ce vaccin comprend une cellule présentatrice de l'antigène/une cellule d'hybridation et d'extraction de tumeur. Ce complexe de vaccin a la capacité de stimuler un corps vivant en vue d'une réaction immunitaire. La présente invention concerne la cellule présentatrice de l'antigène/cellule d'hybridation et d'extraction utile dans la préparation de ce complexe et un procédé d'utilisation de cette cellule d'hybridation et d'extraction destinée à prévenir ou à traiter une tumeur.
PCT/CN2003/001018 2002-11-29 2003-12-01 Vaccin antitumoral WO2004055053A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8309485B2 (en) 2009-03-09 2012-11-13 Chevron Phillips Chemical Company Lp Methods for producing metal-containing sulfated activator-supports
CN113368226A (zh) * 2021-06-30 2021-09-10 诺赛联合(北京)生物医学科技有限公司 含有dc瘤苗的药物组合物及其在治疗癌症中的应用

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100392074C (zh) * 2003-10-15 2008-06-04 上海海欣生物技术有限公司 树突状细胞肿瘤疫苗及其制法和用途
CN100354002C (zh) * 2005-05-31 2007-12-12 中国人民解放军第三军医大学第一附属医院 一种新的以异种免疫细胞为细胞载体的细胞疫苗及其制备方法
CN101724011B (zh) * 2008-10-21 2011-11-23 南京瑞尔医药有限公司 一种肿瘤组织全抗原的制备方法和用途
CN102905534B (zh) * 2010-04-06 2016-03-02 埃克瑟赛特医疗私人有限公司 治疗癌症的方法
DE102012213838A1 (de) * 2012-08-03 2014-02-06 Katharina Pachmann Verfahren zur Kultivierung einer Subpopulation zirkulierender epithelialer Tumorzellen aus einer Körperflüssigkeit
CN104651311B (zh) * 2014-09-03 2018-03-13 深圳市茵冠生物科技有限公司 制备dc‑ctl的试剂盒及其应用
CN105132386B (zh) * 2015-09-02 2018-06-26 北京多赢时代科技有限公司 一种外分泌体及其制备方法和其作为肿瘤疫苗的应用
CN105063013B (zh) * 2015-09-02 2018-05-15 北京多赢时代科技有限公司 一种融合细胞及其制备方法和其作为肿瘤疫苗的应用
US20200338173A1 (en) * 2018-01-18 2020-10-29 University Of South Florida Dead antigen stimulated immature heterogenous dendritic cells as therapeutics for diseases
JP7282874B2 (ja) * 2018-08-20 2023-05-29 中国科学院過程工程研究所 エクソソームに基づく抗腫瘍ワクチン
CN113924358A (zh) * 2019-05-27 2022-01-11 锦高投资株式会社 基于树突细胞的癌症疫苗及其制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004603A2 (fr) * 2000-07-10 2002-01-17 Eppendorf Ag Procede pour la modification de cellules biologiques
WO2002072013A2 (fr) * 2001-03-09 2002-09-19 Baylor Research Institute Methode de traitement de malignites par l'induction de reponses immunitaires dans le sang
CN1374869A (zh) * 1999-03-05 2002-10-16 史密丝克莱恩比彻姆生物有限公司 Casb616多肽和多核苷酸在治疗癌症方面的应用
CN1377892A (zh) * 2001-04-04 2002-11-06 上海华晨生物技术研究所 细胞因子基因修饰的抗原提呈细胞/肿瘤细胞偶联物、其制法和用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1374869A (zh) * 1999-03-05 2002-10-16 史密丝克莱恩比彻姆生物有限公司 Casb616多肽和多核苷酸在治疗癌症方面的应用
WO2002004603A2 (fr) * 2000-07-10 2002-01-17 Eppendorf Ag Procede pour la modification de cellules biologiques
WO2002072013A2 (fr) * 2001-03-09 2002-09-19 Baylor Research Institute Methode de traitement de malignites par l'induction de reponses immunitaires dans le sang
CN1377892A (zh) * 2001-04-04 2002-11-06 上海华晨生物技术研究所 细胞因子基因修饰的抗原提呈细胞/肿瘤细胞偶联物、其制法和用途

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8309485B2 (en) 2009-03-09 2012-11-13 Chevron Phillips Chemical Company Lp Methods for producing metal-containing sulfated activator-supports
CN113368226A (zh) * 2021-06-30 2021-09-10 诺赛联合(北京)生物医学科技有限公司 含有dc瘤苗的药物组合物及其在治疗癌症中的应用
CN113368226B (zh) * 2021-06-30 2021-11-30 上海力沃生物科技有限公司 含有dc瘤苗的药物组合物及其在治疗癌症中的应用

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