Mononuclear cell culture P-DC cell for tumor treatment, preparation and culture method

Abstract

The invention belongs to the field of tumor immune cell therapy, and discloses a mononuclear cell culture P-DC cell for tumor therapy, a preparation and a culture method, and the culture method comprises the following steps: singly collecting mononuclear cells; the volume is 20 to 30 ml; culturing P-DC cells in an immature state in a laboratory for about 7 days; an immune synergist P-L is added in the process of culturing the DC cells. Reagents for culturing the DC cells are completely localized, so that the cost for treating tumors is greatly reduced, and huge medical insurance cost can be saved for the country. As the P-DC cells are exact in curative effect, free of side effects and free of dependence, the conditions of numerous middle and late stage cancer patients can be rescued and reversed. The effects of relieving the pain of the cancer patient and prolonging the life can be achieved to the minimum extent according to the illness state and different development stages of the cancer patient, and the social benefits cannot be measured by money.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor immune cell therapy, and in particular relates to a method for culturing P-DC cells by using mononuclear cells and an application thereof. Background Art

[0002] It has been 111 years since humans first discovered cancer cells in 1912. Cancer was a rare disease in the past. As humans enter the industrialized society, due to changes in social environment, dietary structure and other factors, the incidence of cancer has increased year by year, and it is getting younger and younger. Big data statistics show that the incidence of various cancers in my country is on a clear upward trend. Various tumors (cancers) are endangering the health and life of the public. Traditional chemotherapy treatments for cancer have obvious harmful effects on normal human cells and are already an outdated treatment method. The emerging various types of expensive imported targeted drugs generally have drug resistance within 2-3 courses of treatment. Now humans have entered a new era of cell therapy. As Academician Wu Zuze said: "Cell therapy will be the ultimate treatment for viral infectious diseases and tumors!" And dendritic cell (DC) immunotherapy shows a definite therapeutic effect and bright prospects for cancer treatment!

[0003] The latest research proves that the immune escape mechanism of tumors is the "deficiency of dendritic cells (DC) function" in the human body. The correct way to treat cancer is to cultivate a type of DC cell with normal function and restore the body's immune self-healing function to eliminate cancer cells.

[0004] At present, various tumors are traditionally treated with the following treatment options: 1. Surgical removal of cancerous lesions; 2. Chemotherapy; 3. "Targeted drug" treatment; 4. Immune checkpoint inhibitors; PD-1PD-L1. Although surgical removal of cancerous lesions can reduce the patient's cancer burden, it cannot remove free cancer cells and tiny lesions in the blood. Chemotherapy drugs are chemically synthesized cytotoxic drugs. While killing cancer cells, they also cause significant damage to normal cells in various organs of the human body; targeted drugs are anti-tumor drugs that target mutation sites of cancer genes. Since oncogenes are mutable genes, drug resistance is generally inevitable within 2-3 courses of treatment: immune checkpoint inhibitors PD-1PD-L1 are based on the fact that tumors can use "checkpoints" to form immune escape. This drug blocks the immunosuppressive function of immune "checkpoints" and destroys the body's immune balance. It has great side effects and can easily induce autoimmune diseases. The above commonly used cancer treatments cannot improve and target the basic cause of cancer: immune dysfunction-dendritic cell dysfunction, so it is easy to metastasize and relapse;

[0005] Through the above analysis, the problems and defects of the existing technology are as follows: the existing technology cannot improve the basic cause of cancer, and while killing cancer cells, it also causes obvious damage to normal cells in various organs of the human body, resulting in drug resistance: it destroys the immune balance of the human body, has great side effects, and is easy to induce autoimmune diseases. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method and application of culturing DC cells using mononuclear cells.

[0007] The present invention is implemented in this way, which is a method for collecting mononuclear cells (PBMC) and culturing immature P-DC cells for treating tumors, comprising the following steps:

[0008] The first step is to use a German blood cell separator to collect the patient's own or his / her biological children's mononuclear cells (PBMC)

[0009] The second step is to culture immature P-DC cells in the laboratory;

[0010] Step 3: Add the immunopotentiator PL on the 4th day of P-DC culture. PL stock solution 2mg / 2ml / tube + 23ml injection water to dilute to 25ml, refrigerate for later use, use 0.5ml each time;

[0011] Furthermore, in the first step, single collection of mononuclear cells; 20-30ml.

[0012] Furthermore, in the second step, immature DC cells are cultured in the laboratory for 7 days.

[0013] Another object of the present invention is to provide P-DC cells obtained by the method of culturing DC cells using mononuclear cells.

[0014] The main purpose of the present invention is to provide a targeted immunotherapy preparation for treating solid tumors, wherein the targeted immunotherapy preparation for treating solid tumors comprises the P-DC cells.

[0015] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0016] First, the present invention utilizes the characteristics of immature DC cells with strong ability to absorb tumor antigens. They can actively absorb tumor antigen information during the migration of non-lymphoid tissues, and are naturally processed into tumor antigen peptides in the body, transmitted to T lymphocytes, and produce specific and targeted CTL immune effects to eliminate tumors; in the SOP of culturing immature DC cells; it is the first to collect mononuclear cells from young children of patients to culture P-DC cells, which have stronger immune effects and are used to treat cancer with more significant therapeutic effects; the use of PL immune enhancers in the process of culturing P-DC cells.

[0017] Second, the purpose of the present invention is as follows: the basic understanding of the immune escape mechanism of tumors in modern medicine is that dendritic cells (DC) are functionally deficient, and cannot detect, capture and transmit tumor mutation genes and cancer cell antigen information to T lymphocytes, cannot activate NK cells, and cannot stimulate normal immune responses against tumors. Tumors develop from gene canceration and cell canceration in vivo to cancer mass formation until mid- and late-stage cancer. Take away the patient's life. Based on the above understanding of the mechanism of tumor formation, a functional P-DC cell is cultured in vitro, which can identify, capture, and transmit tumor antigen information to T lymphocytes, activate NK cells, stimulate the human body's specific and targeted immune response against tumors, remove cancer cells and micro-lesions in the blood, or contain unresected lesions to localize and further reduce them, so as to save the cancer patient's condition. This technology conforms to the natural laws of human physiology, and is different from the radiotherapy and chemotherapy and targeted drug treatment based on the "dualism confrontation theory" of Western medicine. P-DC cell immunotherapy conforms to the concept of natural medicine of "strengthening the body and expelling evil spirits" of Oriental medicine, so it has vitality.

[0018] The P-DC cell immunotherapy of the present invention achieves definite efficacy in treating various tumors. It breaks the limitations and side effects of traditional chemotherapy, targeted drugs, and "immune checkpoint inhibitors PD-1PD-L1" in treating tumors. As Academician Wu Zuze said: "Cell therapy will be the ultimate treatment for viral infection and tumors." The widespread promotion of this technology will save the country a lot of foreign exchange costs for importing foreign targeted drugs and other anti-tumor drugs, and reduce dependence on foreign anti-cancer drugs. Moreover, the reagents for culturing P-DC cells are completely localized, and the cost of treating tumors is greatly reduced, which can save the country huge medical insurance costs. Because P-DC cells have definite efficacy, no side effects, and no dependence, the condition of many patients with advanced cancer can be saved and reversed. According to the severity of the cancer patient's condition and different stages of development, the minimum effect of alleviating the pain of cancer patients and prolonging their lives can be achieved, and the social benefits cannot be measured in money.

[0019] Third, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0020] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: P-DC cells will be promoted as the ultimate cell therapy for tumor treatment in clinical practice, which will have immeasurable social benefits, and the development of the equipment, reagents, cytokines and other industries driven by them will also have immeasurable commercial value.

[0021] (2) The technical solution of the present invention fills the technical gap in tumor-specific targeted cell immunotherapy in the domestic and international medical industries, and has revolutionary and groundbreaking clinical application significance.

[0022] (3) The technical solution of the present invention breaks away from and overcomes the technical bias of China's long-term adherence to European and American tumor treatment guidelines and lack of holistic medicine, and realizes a new treatment method based on the concept of holistic medicine, with the purpose of restoring the human body's immune self-healing function against tumors in accordance with the natural laws of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flow chart of a method for culturing DC cells using mononuclear cells provided in an embodiment of the present invention;

[0024] Figure 2 The immune escape mechanism of tumor provided by the present invention is a schematic diagram of dendritic cell dysfunction;

[0025] Figure 3 This is a schematic diagram of the interaction between DC cells and T cells provided in an embodiment of the present invention, and DC transmitting tumor antigen information;

[0026] Figure 4 Schematic diagram of the effect of PL on T cell proliferation; T: T cells maintained by IL-2 in vitro from patients with chronic hepatitis B; DC-T: T cells stimulated by dendritic cells; PDC-T: T cells stimulated by dendritic cells after PL ("━" indicates the mean);

[0027] Figure 5 Schematic diagram of the effect of PL on the expression of DC cell surface molecules. DETAILED DESCRIPTION

[0028] like Figure 1 As shown, the method for culturing DC cells using mononuclear cells provided in the embodiment of the present invention comprises the following steps:

[0029] S101: Use German blood cell separator to collect 20-30ml of mononuclear cells (PBMC);

[0030] S102: Cultivate immature DC cells in the laboratory for about 7 days;

[0031] S103: On the 4th day during the culture of P-DC cells, the immunopotentiator PL was added.

[0032] Experimental data on the application of DC culture immune enhancer PL:

[0033] Figure 4 The effect of PL on T cell proliferation; T: T cells maintained by IL-2 in vitro from patients with chronic hepatitis B; DC-T: T cells stimulated by dendritic cells; PDC-T: T cells stimulated by dendritic cells after PL (“━” indicates the mean).

[0034] Figure 5 The effect of PL on the expression of DC cell surface molecules; the DC cells using DC cell culture enhancers are named P-DC, which are used to treat tumor patients, and can obviously control the condition, the patient's blood picture gradually improves, the tumor markers continue to decrease and clear, and the patient's mental state and physical strength are significantly improved. The present invention has recently applied dendritic cell (P-DC) immunotherapy to treat many cancer patients and has received a definite therapeutic effect. It is manifested that not only the patient's mental and physical strength are significantly improved, the symptoms such as pain and bleeding are alleviated and disappear, but also the laboratory examination generally shows the improvement of blood picture and T cell flow analysis: the total white blood cell count, lymphocyte count, red blood cell, and hemoglobin are significantly increased, and the monocyte count and tumor markers continue to decrease until normal. The above results can show that the patient's condition is significantly reversed, and the minimum efficacy can also achieve the effect of relieving the patient's pain, prolonging life, alleviating, and reversing the condition! The application of P-DC cell immunotherapy in the present invention is a new hope for the treatment of cancer patients, and it is also a revolutionary new treatment method for curing cancer.

[0035] Cases of DC cell immunotherapy in treating various solid tumors:

[0036] Typical cases of P-DC treatment of tumors:

[0037] Ling Mouhua, female, 52 years old, was diagnosed with pancreatic cancer 5 months ago and had undergone surgery to remove the lesion. She did not receive chemotherapy after surgery and received three courses of P-DC cell immunotherapy. All laboratory indicators showed a significant improvement, indicating that her condition had reversed and her mental and physical strength had improved.

[0038] Table 1 Examination results Ling** test results of pancreatic cancer patients

[0039]

[0040]

[0041] Xue Moubo, male, 34 years old, was diagnosed with rectal cancer 6 months ago and had undergone surgery to remove the lesion. He did not receive chemotherapy after surgery, but received a course of P-DC cell immunotherapy. After that, all laboratory indicators showed a significant improvement, indicating that his condition had reversed and his mental and physical strength had improved.

[0042] Table 2 Inspection results

[0043]

[0044] Ms. Li*, 71 years old, suffers from arteriosclerosis and diabetes. She was diagnosed with adenocarcinoma in the upper lobe of the right lung in April 2019 and underwent surgical resection on June 20. She started DC immune cell therapy on June 17, 2020. After two courses of treatment, her condition improved greatly. She went from weakness, poor sleep and appetite to normal sleep and appetite, and her mental and physical strength recovered. She can walk 7,000-10,000 steps a day, and all laboratory test indicators have improved significantly.

[0045] Table 3 Inspection results

[0046]

[0047] Hou Mouqin, female, 51 years old, had surgery to remove the lesions of thyroid cancer and breast cancer 4 months after surgery; she received chemotherapy after surgery and then three courses of P-DC cell immunotherapy. All tumor markers in the test decreased, indicating that the disease reversed and her mental and physical strength improved; no signs of recurrence or metastasis were found in the follow-up 4 years later.

[0048] Table 4 Inspection results

[0049]

[0050] He**, 50 years old, had advanced breast cancer, lung and liver lymph node metastasis, new cancerous masses in the neck and chest wall, resistance to targeted drugs, unbearable pain, bedridden, unable to eat. A hospital ordered her to be discharged from the hospital and receive palliative treatment with "hydroxycodone acetate" for pain relief. After receiving DC treatment and two cell infusions, the pain basically disappeared. During the two courses of treatment, the large mass of chest wall metastasis was shrunk and shrunken, and the small mass in the neck disappeared; her mental and physical strength improved, and she could eat and get out of bed;

[0051] Chen*, male, 75 years old, was diagnosed with pancreatic cancer in March 2020. He had pancreatic portal hypertension, retroperitoneal lymph node metastasis, abdominal and pelvic implantation metastasis, multiple metastases in both lungs, and metastasis in the third lumbar vertebra. In May, his son's PBMC was collected to culture DCs, and the DCs were infused three times during one course of treatment. All laboratory indicators improved significantly before and after treatment.

[0052] Table 5 Inspection results

[0053]

[0054] Osteosarcoma patient Tian**

[0055] After receiving P-DC treatment, genetic testing showed that the TP53 mutation gene P.C229fs, a cancer cell driver gene, was cleared; Patient Tian** had surgery for osteosarcoma in 2017 and relapsed in October 2019. Pathological examination showed high-grade sarcoma (right hip) with tumor infiltration of surrounding soft tissues. Another surgery was performed and P-DC cell immunotherapy was received after surgery.

[0056] The "Personalized Panoramic Genetic Testing of Tumors" report before DC cell therapy showed: mutation of cancer cell driver gene TP53: p.C229fs mutation positive, mutation abundance 19.8% copies.

[0057] A total of 5 P-DC treatments were performed from January 10, 2020 to May 11, 2020, with a one-month interval between each treatment. After treatment, not only did the pain symptoms disappear, but the patient also went from being unable to move in a wheelchair to walking quickly and returning to normal life. A genetic test was performed again, and the p.C229fs cancer cell driver gene was eliminated.

[0058] This test is of great significance. It not only changes and reverses the condition, but more importantly, it eliminates the basis and possibility of tumor recurrence from the perspective of gene expression and molecular carcinogenesis.

[0059] Patient Liu*, male, 53 years old, had chronic hepatitis B since childhood, cirrhosis for 15 years, decompensation for 4 months, liver cancer was found 3 months ago, and he received 3 courses of P-DC treatment after radiofrequency ablation. His liver function was significantly improved, ascites, liver palms, and spider nevi disappeared, and no metastasis or recurrence was found in the recent enhanced CT examination. On August 6, AFP was 6.06ng / ml;

[0060] Table 7 Inspection results

[0061]

[0062] Huang Mouji, male, 61 years old, cirrhosis, postoperative liver cancer, suffered from hepatitis B cirrhosis for 30 years, underwent liver lobectomy for liver cancer, and received DC cell immunotherapy after surgery. No recurrence after 20 years of follow-up; HBsAg changes: 5540-3627-1962-1374-920-711-<10-<10ng / ml; has turned negative.

[0063] Table 8 Inspection results

[0064]

[0065] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A P-DC cell obtained by a method of culturing DC cells with mononuclear cells.

2. A preparation for targeted immunotherapy for treating solid tumors, characterized in that: The targeted immunotherapy preparation for treating solid tumors comprises the P-DC cells according to claim 1.

3. A method for culturing P-DC cells according to any one of claims 1 to 2, characterized in that: The culture method comprises the following steps: The first step is to collect mononuclear cells; The second step is to culture immature DC cells, namely P-DC, in the laboratory; The third step is to add the immune enhancer-PL during the culture of DC cells.

4. The culture method according to claim 1, characterized in that The first step is to collect mononuclear cells; 20-30ml.

5. The culture method according to claim 1, characterized in that The second step is to culture immature DCs, namely P-DC cells, in the laboratory for 7 days.

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