TW200533754A - Preparation method of cytokine-induced killer cells and the application thereof - Google Patents

Abstract

The present invention relates to a kind of preparation method of cytokine-induced killer (CIK) cells, which is capable of identifying and directly killing cancer cells, and to a preparation method of auto-CIK, which is capable of identifying cancer cells so as to treat sarcoma cancer, comprising offering of cancer cell antigen, dendritic cell and CIK cells. Dendritic cells are co-incubated together with cancer cell antigen and then these dendritic cells, which are co-incubated with cancer cell antigen, are co-incubated with CIK cells, so that CIK cells can acquire the ability to identify cancer cells. The CIK cells are further injected back into patient body by means of intravenous injection, or directly injected into the tumor region of patient body, so as to kill cancer cells and tissues of patient particularly efficiently. This invention also relates to the product derived from the above method and to preparation method of dendritic cells and CIK cells.

Description

200533754 发明 Description of the invention: [Technical field to which the invention belongs] The present invention relates to a method for preparing and cultivating autologous hormone-induced killer cells from cancer patients, and a method for treating autologous hormone-induced killer cells for treating cancer. Preparation. [Previous technology] In human tissues, there is already an immune system that inhibits the growth of abnormal tissues in the body and resists the invasion of viral bacteria. The immune system includes immune organs (such as lymphatic tissue) and immune cells (such as B cells, T cells). And lymphocytes, killer cells (NK), etc., but because the patient will become ill, mostly due to the weakened immune system function, the number of various types of immune cells is reduced or the function becomes worse, etc. Cytokine induced killer cells (CIK) were injected into the body of the patient in vitro, and then injected back into the body to improve the immunity. In addition, only the dendritic cells were induced in vitro. , DC) phagocytose the pathogen's antigen and then inject it back into the body, so that the CIK cells in the body can identify the therapeutic experiment to attack the target. However, these two separate methods of operation are often because CIK cells have not been signaled by DC transplantation. Cancer cells cannot be effectively identified, so although there are a large number of CIK cells, they can only improve immunity but not kill them directly The efficacy of cancer cells. As for the injection of dendritic cells co-cultivated with pathogen antigens in the body, it is expected to combine with the free CIK cells in the body, so that the combined CIK cells have the ability to identify and attack cancer cells. This method is often because CIK in the body 200533754 The dagger itself / poor tongue result in poor binding effect with DC, or the number of I cells is too small to effectively remove cancer cells in the body. [Summary of the Invention] One of the preferred specific facts of the present invention It consists of using the patient ’s own white blood cells and the patient ’s own cancer cell pack to artificially convert white blood cells into CIK cells that are more toxic than white blood cells in vitro, but because the CIK cells themselves cannot directly interact with the patient ’s antigen In combination, the gene transfection method must be used to carry the disease antigen signal in order to accurately identify attacking disease tissues and free cancer cells. Therefore, the present invention additionally uses the patient's own white blood cells in an artificial manner. It will be converted into DC, and the patient's own cancer cell antigen will be artificially cultivated with DC, and then will be combined with cancer DCs co-cultivated with cell antigens are co-cultivated with CIK cells 'so that artificially reproduced a large number of CIK cells with the identification number of the cancer cell antigen flood number, the identification rate of cancer cells from patients in vivo will be nearly 100%' and When applied to treatment, it does not produce a rejection condition. Compared with other cultured single cells in vitro, which is injected into the body and expected to interact with the immune cells in the body, 'the method of producing a therapeutic effect', the present invention will be particularly directly effective. In a preferred specific fact of the present invention, the present invention provides a method for preparing a CIK cell capable of recognizing cancer cells, including: providing a cancer cell antigen; providing dendritic cells (DC); and providing cytokine-induced killer cells (CIK); co-cultivation of dendritic cells with cancer cell antigens; 200533754 co-cultivation of dendritic cells co-cultivated with cancer cell antigens and CIK cells; and payment of cik cells with the ability to recognize cancer cell antigen information. Preferably, in the preparation method described in the present invention, the cancer cell antigen ' is artificially produced to avoid the difficulty in directly collecting and isolating the antigen from the patient's body fluid or tissue.

Preferably, in the preparation method described in the present invention, the white blood cell culture is converted into DC cells by artificial means, so as to avoid too few or poor quality DC cells isolated from the patient's blood. Preferably, the DC is converted, so that in the preparation method of the present invention, when DC cells and cancer cells are co-cultivated, miniature magnetic beads are used. If white blood cells are mixed, the effects may be affected and the effect may be poor. Extraction of blood from the patient by artificial means' If the patient is exempted, it is better to use artificial means to induce, in the preparation method of the present invention, white blood cell culture is transformed into CIK cells, so as to avoid a large number of CIK cells from being isolated, their activity may be Decreased epidemic function declines, but it is easy to cause other diseases. Preferably, in the preparation method of the present invention, DC cells are induced to engulf cancer cell antigens, and DC cells carrying part of the cancer cell antigens are obtained. Purification and separation of DC cells carrying some cancer cell antigens from other excess cancer cell antigens by magnetic beads. Preferably, the DCs and CIK cells described in the present invention are co-cultivated for direct and effective transplantation into CIK fine cancer cells, and Kill it. In the preparation method, the part of the cancer cell antigens of such DCs is obtained by artificial means so that CIK cells can recognize in vivo 200533754. Preferably, in each of the preparation methods described in the present invention, the cultured beauty is RPMI1 640 and Patient autologous serum was further added. Preferably, in each of the preparation methods described in the present invention, a generally serum-free medium can also be used. Preferably, in the preparation method described in the present invention, the DC and CIK cells co-cultivated with the cancer cell antigen are co-cultivated by artificial means, and the ratio is 1:10 (1 DC cell to 10 CIK cells). Cells are mixed for optimal results. In a preferred specific fact of the present invention, in the preparation method of the present invention, the step of supplying the cancer cell antigen includes the method of repeatedly freezing and thawing the cancer cells 3 to 5 times, so that the cancer cells naturally fragment and act as Cancer cell antigen material. Preferably, in the preparation method of the present invention, the cancer cell antigen is further frozen for storage. Preferably, in the preparation method of the present invention, the step of providing dendritic cells comprises: culturing white blood cells; adding white blood cells attached to the wall of the culture container to a medium containing a cytokine; and obtaining dendritic cells. Preferably, in the preparation method of the present invention, the cytokines include granules and macrophage growth hormone (GM-CSF) and interleukin 4 (IL-4). Preferably, in the preparation method described in the present invention, the medium is replaced with fresh medium every three 200533754 days, so that adherent white blood cells are transformed into dendritic cells. Preferably, in the preparation method according to the present invention, the dendritic cell line is purified using micromagnetic beads having a specific monoclonal antibody recognizing DC on the surface. Preferably, in the preparation method of the present invention, when the dendritic cells are cultured on the seventh day, the defrosted cancer cell fragment antigen is added, so that the purified dendritic cells can engulf the fragment antigen, and then supplemented with fresh culture medium. , Gm_csf and IL-4 culture. Preferably, in the preparation method of the present invention, the cytokine-induced killer cell line is transformed into 0 by white blood cells not attached to the wall of the culture container. Preferably, in the preparation method of the present invention, the cell In the hormone-induced killer cell line, unattached white blood cells were cultured in a medium, and interferon (r-interferon) was added. After cultured for 24 hours, interleukin 1 (IL-i) was added. 2 (_xian ^ 2, μ), and CD3 monoclonal antibodies, and supplemented with new medium and IL-2 every two to three days, so that unattached white blood cells can be transformed into cells. Preferably, in the preparation method of the present invention, the dendritic cells and the shrew cells that are co-cultivated with the cancer cell antigen are separately cultured on the tenth day of the culturing of the thallium cells and the CIK cells that are co-cultivated with the cancer cell antigen. ι: ι〇: The ratio is mixed into the same culture flask, so that the cytokine-induced killer cells that have not yet contained the cancer cell antigen Xinxin are formed into CIK cells with identifying cancer cells. 200533754

Preferably, in the preparation method described in the present invention, the CIK cells of the cytoantigen, in each of the three methods, the method for identifying cancer cells is supplemented with new medium and IL_2 in the method for identifying cancer cells. Bulk culture in a flask-like manner. On the other hand, in a preferred specific fact of the present invention, the present invention provides a kind of autologous CIK cells prepared by the preparation method as described above and having the ability to recognize cancer cells. On the other hand, in a preferred specific fact of the present invention, the present invention provides an autologous CIK cell for recognizing cancer cells prepared by the preparation method described above. In another aspect, in a preferred specific fact of the present invention, the present invention provides a method for preparing autologous CIK cells for treating cancer, including: providing cancer cell antigens; providing dendritic cells (DC); providing cytokines Induced killer cells (CIK); co-cultivation of dendritic cells and cancer cell antigens; co-cultivation of dendritic cells co-cultivated with cancer cell antigens and CIK cells, and obtaining CIK cells with recognition of cancer cell antigens. Preferably, in the preparation method of the present invention, the cancer cell antigen, the dendritic cells and the CIK cell line are derived from the autologous body. Preferably, in the preparation method of the present invention, the dendritic cells and CIK cell lines are cultured from autologous white blood cells in vitro. 200533754 On the other hand, in one of the preferred specific facts of the present invention, the present invention provides a method for preparing dendritic cells, including: providing white blood cells; culturing white blood cells in a culture solution; separating the attached white blood cells; and The attached white blood cells are cultured in a culture solution containing granules and macrophage growth hormone (GM-CSF) and interlukin-4 (IL-4); and dendritic cells are obtained. On the other hand, in one of the preferred specific facts of the present invention, the present invention provides a method for preparing cytokine-induced killer cells, comprising: providing white blood cells; culturing white blood cells in a culture solution; separating unattached white blood cells; and The unattached white blood cells were cultured in a culture medium containing r_interferon, and after a period of time, interleukin was added (such as this 卜)], interleukin, and 抗体 single antibody Yupei # Solution; and obtaining cytokine-induced killer cells. With the transformation culture method of the present invention, only a small number of patient's own white blood cells are needed, which is enough to cultivate a large number of CIK cells capable of recognizing cancer cells and capable of attacking cancer cells. Not only can they directly attack cancer cells during treatment. Tissue, and because it is using patient's own cells, although it is transformed and replicated, it will hardly cause any allergic reactions to cause discomfort to the patient, no 10 200533754 like ordinary chemotherapy can cause a lot of hair loss or damage the immune system Symptoms of adverse side effects; and the use of transformation methods to turn white blood cells into DC and CIK cells, not only can detect the activity of these cells in vitro, screen the best quality DC and CIK cells, and then transform them into cells that recognize cancer cell antigens. CIK cells can be cultured in the number required to treat cancer. The CIK cells prepared by the invention and having the ability to recognize cancer cell antigens can be applied to any stage (early, middle and advanced stage) cancer patients, but for large solid tumors, in principle, they should be removed as much as possible by surgery before they are called. By irradiating CIK cells, the highest effect of inhibiting cancer cell metastasis and completely eradicating cancer cell tissue in the original site can be obtained. The term "cancer cell" used in the present invention means various malignant neoplasms. The term "cancer cell antigen" used in the present invention means various fragmented cancer cells.

The term "self" used in the present invention means the same individual. [Embodiment] Other features and advantages of the present invention will be apparent from the following preferred specific facts and scope of patent application. Examples The following examples are provided to illustrate the invention. These examples are inadmissible

The methods are intended to limit the scope of the invention, but are used to indicate how to implement the materials and methods of the invention. X 11 200533754 Preparation of antigen signal ctk cells (the following steps need to be performed aseptically) 1 · 1 · ii The field cell antigen is used to pick up the 1. 1. Collect solid tumor tumor (solidtumor) tissue blocks; 2. Rinse with PBS solution After the cancerous tumor tissue block has no blood stains, it is cut to a size of 002x0.2mm in the cultured JBL. 3. Add it to the D-Hanks solution containing collagenase, hyaluronidase, and dNa enzyme to digest the tissue block to make the cells free. State; 4 · / Single filter out a single cell with a 100-mesh sieve, separate the cancer cells and other fibroblasts with η⑶η solution, collect the cancer cells and wash them with pBs, then calculate the number of cancer cells; 5. Take an appropriate amount of cancer Cells are added to sterile pure water, frozen and thawed 3 to 5 times repeatedly to allow the cancer cells to fragment naturally; and 6. Store frozen at -20 ° C until use. 1 · 2 · Preparation of Dendritic Cells (DCs) 1. After separating the white blood cells from the patient's blood, place them in a culture flask and culture at 37 ° C for about 30 minutes; 2.Transfer the non-adherent white blood cells to another In a culture bottle, retain the adherent white blood cells in the original bottle and add medium to culture;

3. Supplement the granules and macrophage growth hormone (OMM_CSF) and interlukin-4 (IL-4) in the medium to stimulate them to transform into DC cells; 200533754 4. Remove the old one every three days After the culture medium, & add fresh culture medium and cell growth factor (GM-CSF and IL-4);-5. On the seventh day, at least 30% of the adherent white blood cells will be transformed into DC cells; 6 · Add micro-magnetic beads with a specific antibody specific to DC on the surface (currently using Dynabeads brand) to bind to cells that have been transformed into DC; 7. Adsorb Dc to the bottle wall from the outside of the culture flask using a magnet, After removing the culture medium, rinse the culture cells 2 or 3 times with PBS buffer solution. After washing other Lufi DC cells and debris, remove the external magnet, separate Dc and magnetic beads, and then rinse with PBS buffer solution. In the culture flask, add new medium; 8 · Add 1 ♦ 1 thawed tumor cell fragments prepared by the method as the antigen 'let DC to this antigen fragment; 9 · continue to add fresh medium and cytokines (GM-CSF and IL_ 4) Culture and transform DC. 1 · 3 · Preparation of cytokine induced killer gillj ^ ciK i. After the other part of the white blood cells not adherent in the second step of the above 1.2 method was transferred to a new culture bottle, Add the medium immediately to start the culture; 2. Add 7-interferon, and then incubate for 24 hours, then add '丨 interlukin-1 (IL-1), interleukin-2 (interlukin-2, 2 CD3 monoclonal antibody (CD3 monoclonal antibody, CD3 13 200533754)

McAb) stimulated the cells to transform into CIK cells; 3. supplemented with new medium and IL-2 every two to three days; 4. on the seventh day, it was observed that at least 50% of the number of white blood cells would be transformed into CIK cells 5. Continue to add new medium and IL-2 to increase the number of CIK cells 1.4. DC and CIK cells co-cultivate 1. On the tenth day, separate DC prepared by method 1.2 and CIK cells prepared by 1.3 separately Out, then mix in the ratio of i: 10 (1 dc cell to 10 CIK cells) into the same new culture flask; 2. Supplement the new medium and il-2 every three days to support the binding and replication of DC and CIK cells Reproduction; 3. Observe the transformation and growth status with a microscope; 4. In the fourteenth to twenty-one days, a large number of transformed CIK replicating cells that can recognize the antigen message of cancer cells can be harvested. Actual jfe example 2: Biological test The cultured cik that can recognize cancer cell antigens

Six times as a course of treatment, the CIK cells were tested for cytotoxicity and toxicity, and then diluted with water and injected back into the patient's body by intravenous injection. The patients were injected once every two weeks and rested. Ji;,, + a.. ^

Total number of CIK cells 200533754 In principle, it should be at least required (early, middle and late)

Uxio 9, this method can be applied to any stage of cancer patients. It is based on the three amendments and changes made by this book. For those who are familiar with the technique, they will not deviate from the scope and spirit of the present invention. In fact, it must be understood that the present invention should not be limited to these special macros ^ B ", 4 and the specific matters. In fact, in terms of implementing the mode described in this month, it is obvious to those skilled in the study of Ergu κ,… I. 4 Different amendments are also covered by the following patents. [Schematic description] (I) Schematic part None (II) Symbols for components None

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Claims (1)

200533754 Patent application scope: 1. A method for preparing cytokine induced killer cells (CIK) capable of identifying cancer cells and directly killing cancer cells, including: providing cancer cell antigens; Provide dendritic cells (DCs); Provide cytokine-induced killer (CIK) cells; co-cultivate dendritic cells with cancer cell antigens; co-cultivate dendritic cells co-cultivated with cancer cell antigens and CIK cells ; And obtaining CIK cells that recognize cancer cell antigens. 2 · The preparation method described in item 1 of the scope of the patent application, wherein the step of providing cancer cell antigens includes repeatedly applying the cancer cells to the cold; Dong and Jie Kang 3 to 5 times, the cancer cells are naturally fragmented as Cancer cell antigen material 03. The preparation method as described in item 2 of Shenyan's patent scope, wherein the cancer cell antigen is further stored in a cold beam for future use. Lu 4. The preparation method as described in item 1 of Shen Qing's patent scope, wherein the step of providing dendritic cells comprises: culturing white blood cells; adding white blood cells attached to the wall of the culture container to a cytokine-containing medium, and obtaining Dendritic cells. 5. The preparation method as described in item 4 of the scope of the patent application, wherein the 16 200533754 cytokines include granules and macrophage growth hormone (GM-CSF) and interlukin-4 (IL-4). 6. The preparation method according to item 5 of the scope of patent application, wherein the medium is replaced with fresh medium every three days. 7. The preparation method according to item 6 of the scope of the patent application, wherein the dendritic cells are further purified by using micromagnetic beads with specific monoclonal antibodies recognizing dendritic cells on the surface. 8. The preparation method as described in item 7 of the scope of the patent application, wherein the step of co-cultivating the dendritic cells with the cancer cell antigen includes culturing the dendritic cells for at least seven days, adding the thawed cancer cell fragment antigen, and then replenishing the fresh Culture medium, GM-CSF and IL-4 culture dendritic cells. 9. The preparation method according to item 1 of the scope of the patent application, wherein the CIK cell line is cultured from white blood cell cells that are not attached to the wall of the culture container. 10 · The preparation method as described in item 9 of the scope of the patent application, wherein the step of providing CIK cells includes: culturing white blood cells; culturing white blood cells that are not attached to the wall of the culture container to a cell containing interferon (τ-interferon) Incubate in the culture medium for at least 24 hours; add interleukin-1 (IL-1), interlukin-2 (IL-2), and CD3 monoclonal antibody (CD3 mono-antibody, CD3 McAb) to the culture medium; New medium and IL-2 were replenished every two to three days; and CIK cells were obtained. 1 1 · The preparation method as described in item 1 of the scope of patent application, wherein the ratio of dendritic cells and cik cells co-cultured with 17 200533754 "cytoantigens is cultured in a culture container at least ten days later. Head 12 . The preparation method described in item 1 of the scope of the patent application, wherein the CIK cells having the recognition of cancer cell antigens are further cultured in the culture medium. 13 · The preparation method described in item 12 of the patent specification, which is in violation of identification CIK cells of cancer cell antigens are cultured by adding new medium and IL-2 in culture flasks every three days. • 1 4 · Please refer to any of the items 1-1 in item 1-1 Production method 'wherein the medium used is RPMI1640.丄 5. The preparation method as described in item i4 of the patent scope, wherein the medium further comprises 10% of the patient's autologous serum. 16 The preparation method according to any one of claims 1 to 14 in the scope of the Shenyan patent, wherein the cancer cell antigen and the dendritic cell-receiving cik cell line are derived from an autologous body. 17 · The preparation method as described in item 26 of the scope of patent application, wherein 4 canine cells and CIK cell lines are obtained by culturing autologous white blood cells. 18 · An autologous CIK cell prepared as described in any one of claims 1 to 15 of the scope of patent application. 19 · An auto-CIK cell having the ability to recognize cancer cells prepared by the preparation method according to any one of claims 1 to 15 of the scope of patent application for treating cancer. zu · A method for preparing autologous CIK cells with cancer cell recognition for treating cancer, including: providing cancer cell antigens; 18 200533754 providing dendritic cells; providing cytokine-induced killer (CIK) cells; dendritic cells Co-cultivation of cells with cancer cell antigens; Co-cultivation of dendritic cells co-cultured with cancer cell antigens and CIK cells: and obtaining CIK cells with identification of cancer cell antigens. 2 1. The preparation method according to item 20 of the scope of the patent application, wherein the cancer cell antigen, dendritic cells and CIK cell line are derived from autologous body. 2 2 · The preparation method according to item 21 of the scope of the patent application, wherein the dendritic cells and the CIK cell line are obtained by culturing autologous white blood cells. 2 3 · A method for preparing dendritic cells, comprising: providing white blood cells; culturing white blood cells in a culture solution; isolating white blood cells attached to a wall of a culture container; and culturing the attached white blood cells in a cell containing granules and macrophage growth hormone (GM-CSF) and interlukin_4 (IL_4) culture medium; and obtaining dendritic cells. · 2 4.-A method for preparing cytokine-induced killer cells, including providing white blood cells; culturing white blood cells in a culture solution; isolating white blood cells not attached to the wall of a culture container; 7-interferon culture medium 'IL-1 ), Interleukin 2 The unattached white blood cells are cultured in the medium containing interleukin-1 (interlukin-1 19 200533754 interlukin-2, IL-2), and CD3 monoclone antibody (CD3 McAb). Solution; and obtaining cytokine-induced killer cells. First, schema: None 20 200533754 (1) Designated representative map: (1) The designated representative map in this case is: (). (2) Brief description of the representative symbols of the components in this representative drawing: None 捌 If there is a chemical formula in this case, please disclose the chemical formula that can best show the characteristics of the invention