WO2004055053A1

WO2004055053A1 - Tumor vaccine

Info

Publication number: WO2004055053A1
Application number: PCT/CN2003/001018
Authority: WO
Inventors: Ge Chen; Peng Cai
Original assignee: Pel-Freez Biotechnology (Beijing) Ltd.
Priority date: 2002-11-29
Filing date: 2003-12-01
Publication date: 2004-07-01
Also published as: CN1446583A; AU2003289603A1

Abstract

The application disclosed a vaccine complex for preventing or treatment tumor, include antigen present cell / the extraction hybridization cell of tumor cell. The vaccine complex have ability to stimulating living body to immune reaction. The present invention relates to the antigen present cell/the extraction hybridization cell the useful in the preparation, and a method of using the extraction hybridization cell to preventing or treatment tumor.

Description

Tumor vaccine Technical field

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

First, the specific recognition mechanism of anti-tumor immunity

A large number of laboratory, animal tests and clinical research data show that: The occurrence, growth, and formation of tumors are related to functional defects in the immune system of the living body. In normal healthy organisms, when normal cells start to differentiate into tumor cells due to genetic mutations and other factors, the immune system will recognize these mutant cells early and launch an immune attack on these "non-self" cells. This specific immune recognition mechanism and specific immune attack effect constitute the antitumor immunity of the normal body.

The main reason why tumor cells can grow and reproduce uncontrollably in a living organism is that 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 The second signal for cell-specific recognition. The complete "dual-signaling system" described above is a necessary prerequisite for anti-tumor-specific immune recognition. The "dual-signaling system" for most tumor cells is missing or incomplete, thus causing " "Immunity escape" (immunity escape); Activation of tumor cells mount an immune attack response.

In the above dual signal system: 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. 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.

2. "Immune escape" mechanism of tumor cells

It is generally believed that in normal organisms: 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. When the body's immune function is low or suppressed or the normal immune balance is disturbed, the incidence of tumors increases; and after tumorigenesis, the progressive "immune escape" proliferation of tumor cells makes the body's immune system function further suppressed; the two are mutually reinforcing Cause and effect, the rise and fall of various comprehensive factors on both sides determine the final outcome of the tumor.

In the occurrence and development of tumors, in addition to the disorder of the body's own immune system, it is closely related to the immune escape mechanism of tumor cells, which are mainly manifested in the following aspects:

1. 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.

2. 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.

4. Defects in the "dual signal" system of tumor cells; most tumor cells show a lack of expression or no expression or incomplete function of the "dual signal" system components, making them unable to completely present the tumor antigen information to the immune system. Make the body antitumor immune

"Immune tolerant state", resulting in low antitumor immune response or immune incompetence.

5. Tumor cells secrete certain immunosuppressive factors: such as IL-10, PGE-2, TGF-β, 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.

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. .

The important role of Antigen- Presenting Cells (APC).

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.

There are three main antigen presenting cells: dendritic cells (DCs), macrophages and B cells. Among them, 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]. 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;

(2) Follicular DC (FDC): It is present in the primary and secondary follicles of the B cell area of lymph nodes, spleen and mucosa-associated lymphoid tissue (MALT), and can present antigens to B cells. FDCs are non-migrating cell populations that establish a strong network of cells through the desmosomes of cells to form a stable network. FDC lacks MHC class II molecules and binds antigens via complement receptors (CD21 or CD35);

(3) germinal center DC (GCDC): 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;

(4) 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]. However, 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 ^.

DC- Once mature, it loses the ability to capture antigens, but has the following effects [Cella M, Sallusto F, and Lanzavecchia A., Origin, maturation and antigen presenting function of dendritic cells, Curr Opin. Immunol. 1997; 9: 10- 16; 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]: (1) Significantly enhanced by up-regulating class I and II molecules and costimulatory molecules (CD80, CD86) T-cell stimulating activity. At this time, 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. This is important for the design of vaccine treatments [Mailliard RB, Egawa S, Cai Q, Kalinska A, Bykovskaya SN, Lotze MT, Kapsenberg ML, Storkus WJ, Kalinski P., Complementary dendritic cell-activating function of CD8 + and CD4 + T cells : helper role of CD8 + T cells in the development of T helper type 1 responses. J Exp Med 2002; 195: 473-483]; (4) In addition to expressing molecules such as CD40 ligand, activated T cells also release such as γ- Fine factors such as interferon and granulocyte-macrophage colony-stimulating factor (GM-CSF), in turn, promote the antigen presentation of DCs.

In the process of tumorigenesis and development, a very noteworthy aspect is: The anti-tumor immunity of the tumor patient's body has been in the struggle with the tumor growth protection mechanism. However, when the tumor cells grow vigorously, the body's anti-tumor immune response is low, and it is in a state of negative balance of immune "tolerance" or "inability"; that is, tumor patients have potential anti-tumor immunity. And, its function can be restored under appropriate conditions. This is the theoretical basis and premise of clinical immunotherapy for tumor patients.

In summary, the immune dysfunction characteristics of cancer patients can be summarized as follows:

1. The state of immunosuppression or tolerance in the microenvironment of the patient;

2. The antigen presentation ability of tumor cells and other defects of low APC function are not enough to cause the body to have an effective anti-tumor immune response;

3. "Overload" of antitumor immune response in cancer patients;

4. Strategy and plan of tumor immunotherapy:

According to the specificity of the body's anti-tumor immune function combined with the latest advances in immunology, molecular biology and a large number of in vitro and animal research results, 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.

Removal of tumors by surgery can reduce the "excessive immune load" caused by tumors to the immune system. Through artificial changes and adjustments to the APC function of patients in vitro, the immune suppression microenvironment in tumor patients is avoided, and the APC antigen is significantly increased Presentation function; by activating CTL in vitro and giving it specific recognition ability, and then directly returning it to the patient to exert specific tumor-killing effects. To achieve the correction of the "immunity tolerance state", re-stimulate and strengthen the clinical efficacy of patients' own anti-tumor immune function.

In recent years, in accordance with the above principles, scientists in various countries have conducted a large number of in vitro and in vivo studies on various tumor immunotherapy schemes (tumor vaccine design), and have achieved satisfactory results, especially for the anti-tumor treatment of DCs in APC. The important role in the process was given unanimous full affirmation.

The following table lists the major tumor vaccine design schemes commonly used internationally in recent years:

Cancer vaccine types and applications

Tumor vaccine type vaccine preparation animal model clinical trial

Melanoma,

Dead whole tumor cells Melanoma, colon cancer, etc. Tumor cells Colon cancer, etc.

Tumor cell lysate sarcoma melanoma melanoma antigen melanoma melanoma purified tumor antigen

Heat shock protein (HSP) multiple tumors Melanoma, kidney cancer, sarcoma Cytokines and B7 genes against tumors

Kidney cancer, sarcoma

Transfection of tumor cells

Cytokine plus synergy

Melanoma, sarcoma, etc. Stimulating factor Cytokine gene transfection APC B asthma leukemia,

APC lung cancer stimulated with tumor antigen

DNA vaccine tumor antigen gene plasmid melanoma melanoma peptide vaccine tumor peptide or synthetic peptide melanoma and other melanoma and other adenovirus, tumor antigen gene-containing melanoma,

Virus vaccine melanoma

The virus ± cytokine sarcoma

DC + whole tumor cell or its melanoma

Solution co-culture melanoma prostate cancer liver cancer tumor gene (DNA) transfection kidney cancer, lung cancer colon cancer

DC vaccine

DC Colon Cancer Kidney Cancer Glioblastoma Tumor Cell Full RNA Transfection Sarcoma Sarcoma

DC [1] Prostate cancer Non-Hodgkin's lymphoma tumor Breast cancer Prostate cancer Tumor / DC hybrid Tumor cells + DC fusion hybrid

B parcel leukemia breast cancer cell vaccine

B fine moon package leukemia [lJBoczkowski D, Nair SK, Nam JH, Lyerly HK, and Gilboa E. Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells. Cancer Res 2000; 60: 1028-1034.

In the above tumor vaccine design scheme, 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 The salient features of this vaccine are: 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 various tumor vaccine design schemes listed in the table above have proved their effectiveness through in vitro tests, animal tests, and preclinical studies. However, the following main deficiencies and limitations remain:

1. Complex preparation techniques: such as purification and identification of tumor antigens, gene recombinant expression of tumor antigens, and tumor gene transfection technology for vaccine design.

2. Introduce viral vector molecules: such as tumor DNA vaccine and tumor gene transfection design. It is difficult to control the integration position and expression of genes in the body, and it is difficult to predict whether the introduced viral vector genes have an effect on the body, what the effects may be, and when.

3. Low antigen presentation efficiency: such as RNA introduction into APC, tumor cell or tumor cell lysate and APC or T lymphocyte co-culture (Pulse).

4. Incomplete antigen presentation: such as tumor DNA vaccine and tumor RNA vaccine design, which cannot present tumor antigens through MHC or HLA-II molecules. Co-culture vaccine design of tumor cells and their lysates with APCs or lymphocytes cannot pass effectively MHC or HLA-I molecules perform tumor antigen presentation.

5. Most of them are designed for monovalent tumor vaccines: They are only used for tumor immunotherapy of specific individual patients without preventive immunization programs.

6. It is difficult to switch to biopharmaceuticals for large-scale production. Summary of invention

In order to overcome one or more of the shortcomings of the above-mentioned tumor vaccines, the inventors of the present invention conducted a lot of in-depth studies to obtain the present invention.

The inventors have now discovered that 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.

Therefore, the present invention provides:

1) A vaccine composition for preventing and / or treating a tumor, comprising a cell extract of an antigen-presenting cell / tumor cell hybrid cell.

In the vaccine composition of the present invention, preferably, the hybrid cell has the ability to specifically stimulate an organism's anti-tumor immune response. In the vaccine composition of the present invention, 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.

In the vaccine composition of the present invention, 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. Preferably, the cell extract is a supernatant or pellet after centrifugation of the cell lysate. More preferably, 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.

In a preferred embodiment of the invention, the antigen presenting cells are occupational antibodies The original presentation cells are more preferably dendritic cells (DC cells).

In the vaccine composition of the present invention, 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. Preferably, the hybrid cell is a hybrid cell formed by fusion of an autologous APC cell and an autologous tumor cell.

In a preferred embodiment of the invention, 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.

Preferably, in the vaccine composition of the present invention, 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. '

2) Application of a cell extract of an antigen presenting cell / tumor cell hybrid cell in the preparation of a medicament for preventing and / or treating a tumor. The above explanations and preferred conditions regarding the vaccine composition of the present invention also apply to the application of the present invention.

In the application of the present invention, it is preferred that 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.

3) 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 above explanations and preferred conditions regarding the vaccine composition of the present invention also apply to the method of the present invention.

In the method of the present invention, 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

As described above, 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" according to the present invention 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. For example, 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.) (as measured by ELISA method), enhanced inhibition / killing effect of peripheral blood T cells on tumor cells (as measured by ⁵¹ Cr test, MTT method )Wait. Preferably, 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. This mainly includes occupational antigen-presenting cells: dendritic cells, macrophages, B lymphocytes; non-professional antigen-presenting cells: any other nucleated cells with antigen-presenting functions, such as epithelial cells, endothelial cells, Mesothelial cells, mesenchymal cells, fibroblasts, mononuclear cells, various tumor cells, etc. Preferably, 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. The above 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. 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.

Regarding 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. Regarding 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. In order to more effectively isolate DC directly from blood, some people have suggested that it should be treated with C-fms-like tyrosine ligand 3 (Fit3 ligand) to expand the circulating DC. This method can increase the amount of DC by 10-15 times [Pulendran B , Banchereau J, Burkeholder S, Kraus E, Guinet E, Chalouni C, Car on D, Maliszewski C, Davoust J, Fay J, and Palucka K. Flt3 -ligand and 01018

granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo. J Immunol 2000; 165: 566-572.]. (2) Isolate 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. Currently, DCs from both sources are considered to have the same role in tumor immunotherapy [Rosenzwajg M, Canque B, and Gluckman JC. Human dendritic cell differentiation pathway from CD34 + hematopoietic precursor. Blood 1996; 87: 535-544 ·]. (3) Use of calcium ionophore (CI): This method is simple and convenient, and can quickly separate CD34 + precursor cells, CD14 + mononuclear cells or CML precursor cells from mature DCs, which is an effective method for separating mature DCs. method. The ability of 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.

Regarding the tumor cells to be used for fusion, it is only necessary that the tumor cells are dispersed. For tumors in leukemia or pleural and ascites fluid and cerebrospinal fluid, 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. For 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.

Regarding the method for fusion of APC cells and tumor cells, it 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:

a) fusion of autologous APC cells with autologous tumor cells;

b) fusion of autologous APC cells with allogeneic tumor cells or tumor cell lines;

c) fusion of autologous APC cells with tumor cells or tumor cell lines from multiple allogenes; d) fusion of allogeneic APC cells with allogeneic tumor cells or tumor cell lines;

e) APC cells from multiple foreign bodies are fused with tumor cells or tumor cell lines from multiple foreign bodies.

As described above, 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 . In a preferred embodiment of the present invention, 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. On the one hand, 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.

As mentioned above, 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. Preferably, the cell extract is a supernatant or a precipitate after centrifugation of the cell lysate. More preferably, 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.

Regarding the method for preparing a hybrid cell extract, it 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. 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. Alternatively, 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.

In the present invention, 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. 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. In a preferred embodiment of the present invention, unit dosage forms of the present invention, 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.

In practical clinical applications, 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. Among them, 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. At the same time or before and after the infusion of tumor-specific CTLs, 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:

(1) use methods well known in the art to measure the number of killer T cells in the peripheral blood and whether the proliferative activity is increased (such as flow cytometry, ³ H-labeled thymine incorporation);

(2) measuring the number of CD4 + T cells and whether their proliferative activity is increased by methods well known in the art (such as flow cytometry, etc.);

(3) whether the cytokines released when the T cell immune activity is enhanced, such as IFN Y, TNF (tumor necrosis factor), IL-2, etc .;

(4) ⁵¹ Cr release method or MTT method is used to measure whether the inhibitory and killing effect of peripheral blood T cells on tumor cells is enhanced;

(5) 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;

(6) 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. Thereby, individualized and specific biological immunotherapy for the tumor patient is realized. 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. T / CN2003 / 001018

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. For example, 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. Through the common tumor antigen presenting mechanism or / and specific tumor antigen cross-presenting mechanism, the above-mentioned hybrid hybrid cell extract can still stimulate or partially stimulate the patient's anti-specific tumor immune effect. Therefore, 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. According to actual needs, 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. Through the specific recognition mechanism, 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. When the same individual is vaccinated a second time with the same tumor vaccine (secondary vaccination or booster immunization) or the individual tumor cell growth occurs, and 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.

As described above, 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.

Because 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.

The present invention is further described below with reference to the accompanying drawings and specific embodiments, but these drawings and embodiments are only used to illustrate the present invention and do not limit the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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.

Among them, PBNC (Peripheral blood mononuclear cell) refers to peripheral i monocytes as a control; 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. Specific embodiments of the invention

Example 1

(1) 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. Supplement the appropriate amount of medium and cytokines (GM-CSF, IL4) on the 4th or 5th day; add TNFα and IFNγ 1000U / ml each on the 6th day to promote DC cell maturation. The cultivation process lasted for 7 days.

(2) Preparation of 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.

(3) Fusion of DC cells and 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.

(4) Preparation of DC-tumor cell hybridoma lysate: Take 2/3 of overnight cultured DC-tumor cell hybridoma cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 1 ml of 1640-RPMI medium to the pellet The cell pellet was resuspended, and the cells were repeatedly frozen and thawed 3 times in liquid nitrogen to completely rupture the cells. Centrifuge at 15,000 rpm for 20 minutes, take the supernatant for use, and resuspend the pellet in 1 ml of 1640-RPMI medium.

(5) 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.

(6) 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 . At the same time, 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. During the treatment process, the body's various immunological indicators are monitored dynamically, and the vaccine treatment plan is adjusted in a timely manner.

(7) Evaluation of the therapeutic effect: the number of killer T cells in peripheral blood was measured by flow cytometry; the proliferation activity of killer T cells was measured by ³ H-labeled thymine incorporation; the peripheral blood T cell pair was measured by MTT method Inhibition / killing effect of tumor cells; Changes in clinical indications of tumors.

Example 2

Peripheral blood DC preparation

(1) 10-300ml of human peripheral blood is drawn under sterile conditions.

(2) Mononuclear cells were separated by Ficoll-Paque gradient centrifugation and suspended in RPMI1640 medium.

(3) Place the mononuclear cell culture flask at 37 ° C and incubate in a CO ₂ incubator for 30 minutes-5 hours.

(4) Aspirate non-adherent cells.

(5) Wash the adherent cells with the culture medium and add a certain amount of DC culture medium (RPMI1640, 1%-30% human serum; 50-2000 units / ml of GM-CSF and

IL-4).

(6) Incubate in a 37 ° C, CO ₂ incubator for 3-12 days; add a certain amount of INF before harvest to promote further maturation of the DC. The mature DCs were harvested and suspended in the culture medium for use.

Example 3 Bone marrow DC preparation:

(1) 5-100ml bone marrow is withdrawn from human patella puncture according to clinical routine under sterile and anesthesia conditions. After low-speed centrifugation, aspirate the supernatant, add a certain amount of RBC sedimentation solution, and let it stand for a certain period of time.

(2) Add RBC lysate, disrupt RBC, and collect bone marrow nucleated cells by centrifugation.

(3) Place bone marrow nucleated cells in a culture flask at a certain concentration, and add DC culture solution.

(4) Incubate in a 37 ° C, CO ₂ incubator for 5-12 days. DC cells were harvested for later use. Example 4

DC / tumor cell fusion (PEG fusion):

(1) Mix DC and tumor cells in a certain ratio, and aspirate and discard the supernatant. Slowly add a certain amount of 25-55% PEG solution under incubation at 37 ° C, and mix while adding.

(2) Continue to slowly add 5-10ml of cell culture solution (RPMI1640 containing serum). (3) Wash and centrifuge to harvest fused cells and add cell culture solution. Incubate at 37 ° C in a CO ₂ incubator for 2-48 hours and use it for further tumor vaccine preparation or continuous subculture to establish cell lines.

Example 5

Preparation of DC / tumor hybrid cell extract vaccine

(1) Take a certain amount of DC / tumor fusion cells and place them in a sterile test tube

(2) Place the above test tube below -20 ° C, 10 minutes-2 hours

(3) Remove the test tube and place in a 37C water bath for 5-10 minutes

(4) Repeat steps 2 and 3 a total of 2-5 times.

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.

Example 6

Preparation of DC / tumor hybrid cell extract vaccine

(1) Put a certain amount of DC / tumor hybrid cells into a homogenizer.

(2) The speed (rpm) of the fixed hook pulper is 500-5000 rpm. (3) 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.

(4) All operations for preparing vaccine from plasma should be performed under low temperature and aseptic conditions.

The prepared vaccine is stored in a low temperature environment for future use.

Example 7

DC / Tumor Hybrid Cell Extract Tumor Prevention Immunization Program:

(1) Take a certain amount of the corresponding tumor vaccine (or mix it with an immune adjuvant, such as BCG, etc.) under sterile conditions, and inject subcutaneously, lymph nodes or surrounding tissues into the high-risk population or normal population of the corresponding tumor.

(2) Booster injections are performed 1-4 weeks, a total of 2-3 times.

Monitor various immunological indicators and evaluate the effect of preventive immunity.

Example 8

Peripheral blood lymphocyte culture and typing

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 γ interferon content.

Take 1 X 10 ⁶ cells, add 1% BSA PBS and centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and use CD4 / CD8, CD3 / 56 (FITC / PE, BD, USA) fluorescently labeled antibodies to react with the cells for 20 minutes in the dark, It was then washed with PBS and centrifuged at 1,000 rpm for 10 minutes. The washing and centrifugation process was repeated twice, and the cells labeled with fluorescent antibodies were analyzed by flow cytometry for typing. The results are shown in Figure 1.

'' The experimental results show that after co-culture of lysates, lysate supernatants, and lysate pellets from peripheral blood lymphocytes and DC-tumor cell hybrid cells, the total T3 cells and CD8 + killer T cells compared to before Have a significant increase, showing that The cellular immunity of the body is significantly activated after the vaccine.

Example 9 ·

Determination of cytotoxic activity by MTT method

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 μL 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 96-wells plate was incubator (37 ° C, saturated humidity, C0 ₂ incubator) culturing 2 ⁴ h; added 4mg / L of MTT (Sigma, Lot61k5138, USA) to each well at 20h, incubation was continued After 4 h, centrifuge at 2000 rpm for 10 min, discard the supernatant, add DMSO 100 L, and dissolve gently after the formazan crystals are dissolved, develop the color at room temperature for 10 min, and measure the absorbance A value with a microplate reader at a wavelength of 570 nm. Calculate each experiment Inhibition rate of tumor cells in wells. Tumor inhibition rate (%) = 1-A test value / control A value. The results are shown in Figure 2.

The results showed that for A549 cells, 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. In practical applications, in the prior art method using intact hybrid cells, 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. However, in this embodiment, the DC-tumor cell hybrid cells used to process the lymphocytes have not been subjected to radiation treatment, and therefore have high activity.

Claims

Rights request

What is claimed is: 1. A vaccine composition for preventing and / or treating tumors, comprising a cell extract of an antigen-presenting cell / tumor cell hybrid cell.

2. The vaccine composition according to claim 1, wherein the hybrid cell has the ability to specifically stimulate an organism's anti-tumor immune response.

3. The vaccine composition according to claim 1, wherein the hybrid cell has a division and proliferation function.

4. The vaccine composition of claim 3, wherein said hybrid cell is an immortalized hybrid cell line.

5. The vaccine composition of claim 1, wherein the cell extract is a cell lysate.

The vaccine composition according to claim 1, wherein the cell extract is a supernatant or a precipitate after centrifugation of a cell lysate.

7. The vaccine composition of claim 1, wherein the cell extract is one or more components selected from the group consisting of a cell lysate from the hybrid cell having a stimulating anti-tumor immune response in the organism: Cell membrane components, cytoplasmic contents, nuclear components, protein molecules and fragments thereof, protein-polypeptide complexes, lipoproteins, glycoproteins, protein complexes, DNA molecules and fragments thereof, and RNA molecules.

8. The vaccine composition of claim 1, wherein the antigen-presenting cells are occupational antigen-presenting cells.

The vaccine composition according to claim 1, wherein said antigen-presenting cells are dendritic cells.

10. The vaccine composition of claim 1, wherein the hybrid cell is selected from one or more of the following:

a) Hybrid cells formed by fusion of autologous APC cells with autologous tumor cells;

b) hybrid cells formed by fusion of autologous APC cells with allogeneic tumor cells or tumor cell lines;

c) Autologous APC cells fused with tumor cells or tumor cell lines from multiple allogenes Formed hybrid cells

d) a hybrid cell formed by fusion of an allogeneic APC cell with an allogeneic tumor cell or tumor cell line;

e) A hybrid hybrid cell formed by fusing multiple allogeneic APC cells with multiple allogeneic tumor cells or tumor cell lines.

11. The vaccine composition of claim 1, further comprising one or more immune adjuvants.

12. The vaccine composition of claim 11, wherein the immune adjuvant is selected from the group consisting of BCG, QS-2 HSP, ISCOMS, Freund's complete adjuvant and Freund's incomplete adjuvant.

13. The vaccine composition according to any one of claims 1 to 12, wherein the tumor or tumor cell is or is derived from various cancers, sarcomas, gliomas, blastomas, fibromas, various lung cancers, including colorectal cancer , Colon cancer, rectal cancer, bowel cancer, various liver cancers, various gastric cancers, various kidney cancers, prostate cancers, various breast cancers, ovarian cancers, cervical cancers, various skin cancers, melanoma, various noses Pharyngeal cancer, various esophageal cancers.

14. Use of a cell extract of an antigen presenting cell / tumor cell hybrid cell in the preparation of a medicament for the prevention and / or treatment of a tumor.

15. The use according to claim 14, wherein the hybrid cells have the ability to specifically stimulate an organism's anti-tumor immune response.

16. The use according to claim 14, wherein the hybrid cell has a division and proliferation function.

17. The use of claim 16, wherein said hybrid cell is an immortalized hybrid cell line.

18. The use of claim 14, wherein the cell extract is a cell lysate.

19. The use according to claim 14, wherein the cell extract is a supernatant or a precipitate after centrifugation of the cell lysate.

20. The use according to claim 14, wherein the cell extract is one or more components selected from the group consisting of cell lysates from the hybrid cells, which have a stimulating anti-tumor immune response in the organism: cell membrane components , Cytoplasmic contents, nuclear components, protein molecules and fragments thereof, protein-polypeptide complexes, lipoproteins, glycoproteins, protein complexes, DNA molecules and fragments thereof, and RNA molecules.

21. The use according to claim 14, wherein said antigen presenting cells are occupational antigen presenting cells.

22. The use of claim 14, wherein said antigen presenting cells are dendritic cells.

23. The use of claim 14, wherein the hybrid cell is selected from one or more of the following hybrid cells:

a) hybrid cells formed by fusion of autologous APC cells and autologous tumor cells;

c) hybridization formed by fusion of autologous APC cells with tumor cells or tumor cell lines from multiple allogenes;

24. The vaccine composition of claim 14, further comprising one or more immune adjuvants.

25. The vaccine composition of claim 24, wherein the immune adjuvant is selected from the group consisting of BCG, QS-2K HSP, ISCOMS, Freund's complete adjuvant and Freund's incomplete adjuvant.

26. The use of any one of claims 14 to 25, wherein the tumor or tumor cell is or comes from various cancers, sarcomas, gliomas, blastomas, fibromas, various lung cancers, including colorectal cancer, colon Cancer, rectal cancer, colon cancer, various liver cancers, various gastric cancers, various kidney cancers, prostate cancers, various breast cancers, ovarian cancers, cervical cancers, various skin cancers, melanoma, various nasopharyngeal cancers Various types of esophageal cancer.

27. The use of any one of claims 14 to 25, wherein the hybrid cell extract and one or more cytokines are reinfused with cytotoxic T lymphocytes having specific tumor killing effects after in vitro culture Treatment, and / or in combination with anti-tumor specific antibody therapy or other immune-enhancing antibodies.

28. A method for preventing and / or treating tumors, including pre-treating a subject in need of treatment A preventive or therapeutically effective amount of a cell extract of an antigen presenting cell / tumor cell hybrid cell.

29. The method of claim 28, wherein said hybrid cell has the ability to specifically stimulate an organism's anti-tumor immune response.

30. The method of claim 28 or 29, wherein the cell extract is administered by: subcutaneous injection, intradermal injection, peri-lymph node injection, intra-lymph node injection, intramuscular injection, intravenous injection, thoracic injection, intraperitoneal injection, spinal sheath Intravenous injection, local injection around the tumor, intratumor injection, systemic multipoint injection.

31. The method of claim 28, wherein a multivalent or broad-spectrum tumor vaccine is administered to a subject belonging to a normal population or some corresponding high-risk population of tumors for preventive immunity against tumors.