Immune cell activation composition and its application

Abstract

The present invention relates to an immune cell activation composition and its application, wherein the composition comprises an antigen peptide combination, wherein the antigen peptide combination is selected from any one of antigen peptide blend 1, antigen peptide blend 2, antigen peptide blend 3 and antigen peptide blend 4 or a combination of two or more thereof. The present invention also relates to an immune cell activation method, which comprises the step of contacting immune cells with the immune cell activation composition of the present invention. Using the immune cells activated by the present invention, recurrence and metastasis can be effectively prevented, quality of life can be improved, tumors can be stabilized, tumor stability can be maintained for a long time, quality of life can be improved, and side effects of chemotherapy can be eliminated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an immune cell activation composition and application thereof. Background Art

[0002] Malignant tumors are one of the major diseases that threaten human health and life, and their incidence rate is increasing year by year. According to the 2015 Global Cancer Statistics, there were approximately 14.1 million new cancer cases and 8.2 million deaths in 2015 worldwide. In China, there were 4.29 million new cancer cases and 2.81 million deaths in 2015. Surgery, radiotherapy and chemotherapy have become the three conventional methods of tumor treatment, but they all have the characteristics of insufficient specificity, resulting in varying degrees of damage to normal tissues and organs while clearing tumor cells.

[0003] DC cells, also known as dendritic cells, are a heterogeneous cell population with highly efficient killing activity. The proportion of DC cells in human peripheral blood lymphocytes is 1% to 5%. Clinically, DC cells have significant activity in killing tumors and clearing viruses after a large number of amplifications, and DC cells play an important regulatory role in the body's cellular immunity and humoral immunity. DC cell immunotherapy is the most important component of biological therapy. The process is to extract the patient's own immune cells, activate, modify, and proliferate them in vitro, and then infuse them back into the patient's body to induce the body to produce specific or non-specific immune responses, and play a role in killing tumor cells and antiviral effects in the patient's body.

[0004] Studies have reported that adding antigen peptides to DC cell culture can specifically secrete high concentrations of IFN-γ. However, the above method can only be used for specific indications, and has low activation efficiency, long culture time, and potential risks such as tumor vaccine imbalance and tumor spread. Therefore, there is an urgent need for more efficient, safer, and faster-cultured antigen-activated immune cells in clinical practice. Summary of the invention

[0005] The object of the present invention is to provide an immune cell activation composition, wherein the composition comprises an antigen peptide combination, wherein the antigen peptide combination is selected from any one of antigen peptide blend 1, antigen peptide blend 2, antigen peptide blend 3 and antigen peptide blend 4 or a combination of two or more thereof, wherein

[0006] The antigen peptide blend 1 is formed by mixing peptide segments with amino acid sequences of SEQ ID NO: 1-10 in equal mass proportions;

[0007] The antigen peptide blend 2 is formed by mixing peptide segments with amino acid sequences of SEQ ID NO: 11-40 in equal mass proportions;

[0008] The antigen peptide blend 3 is formed by mixing peptide segments with amino acid sequences of SEQ ID NO: 41-64 in equal mass proportions;

[0009] The antigen peptide blend 4 is formed by mixing peptide segments with amino acid sequences of SEQ ID NO: 65-91 in equal mass proportions.

[0010] In the preferred technical scheme of the present invention, the antigen peptide combination is a combination of antigen peptide blend 2 and antigen peptide blend 3; as one of the preferred embodiments, the immune cells activated by antigen peptide blend 2 and antigen peptide blend 3 are mixed in a ratio of 1:1 and re-infused into the patient.

[0011] In the preferred technical scheme of the present invention, the preparation method of the antigen peptide blend is: taking the same mass of each peptide segment lyophilized powder, adding a solvent to fully dissolve it to obtain each peptide segment solution, and then mixing equal volumes of each peptide segment solution evenly, and filtering with a 0.2μm filter to obtain.

[0012] In a preferred technical solution of the present invention, the antigen peptide blend 1 is obtained by dissolving the lyophilized powders of peptide segments with amino acid sequences of SEQ ID NO: 1-10 in appropriate amounts of solvents and then mixing them.

[0013] In a preferred technical solution of the present invention, the antigen peptide blend 2 is obtained by dissolving the lyophilized powder of the peptide segments with the amino acid sequences of SEQ ID NO: 11-40 in appropriate amounts of solvents and then mixing them.

[0014] In a preferred technical solution of the present invention, the antigen peptide blend 3 is obtained by dissolving the lyophilized powder of the peptide segments with the amino acid sequences of SEQ ID NO: 41-64 in appropriate amounts of solvents and then mixing them.

[0015] In a preferred technical solution of the present invention, the antigen peptide blend 4 is obtained by dissolving the lyophilized powder of the peptide segments with the amino acid sequence of SEQ ID NO: 65-91 in appropriate amounts of solvents and then mixing them.

[0016] In a preferred technical solution of the present invention, the solvent is DMSO and / or serum-free culture medium.

[0017] In a preferred technical solution of the present invention, the working concentration of the antigen peptide combination is 1-5 ug / mL, preferably 3-4 ug / mL.

[0018] In a preferred technical solution of the present invention, the immune cell activation composition further comprises cytokines and / or serum substitutes.

[0019] In the preferred technical solution of the present invention, the cytokine is GM-CSF, and the working concentration is 200-1000 IU / mL, preferably 500 IU / mL.

[0020] In a preferred technical solution of the present invention, the serum substitute is an immune cell serum substitute, and the working concentration is 5-25% (v / v), preferably 10-20% (v / v).

[0021] Another object of the present invention is to provide a method for preparing an immune cell activation composition, wherein the composition comprises an antigen peptide combination, wherein the antigen peptide combination is selected from any one of antigen peptide blend 1, antigen peptide blend 2, antigen peptide blend 3 and antigen peptide blend 4, or a combination of two or more thereof, wherein the antigen peptide blend 1 is formed by mixing peptide segments with amino acid sequences of SEQ ID NOs: 1-10 in an equal mass ratio; the antigen peptide blend 2 is formed by mixing peptide segments with amino acid sequences of SEQ ID NOs: 11-40 in an equal mass ratio; the antigen peptide blend 3 is formed by mixing peptide segments with amino acid sequences of SEQ ID NOs: 41-64 in an equal mass ratio; the antigen peptide blend 4 is formed by mixing peptide segments with amino acid sequences of SEQ ID NOs: 65-91 in an equal mass ratio; the preparation method comprises the following steps: taking the same mass of each peptide segment lyophilized powder, adding a solvent to fully dissolve, obtaining each peptide segment solution, then mixing equal volumes of each peptide segment solution, and filtering with a 0.2 μm filter to obtain.

[0022] In a preferred technical solution of the present invention, the preparation method further comprises adding cytokines and / or serum substitutes.

[0023] In the preferred technical solution of the present invention, the cytokine is GM-CSF, and the working concentration is 200-1000 IU / mL, preferably 500 IU / mL.

[0024] In a preferred technical solution of the present invention, the serum substitute is an immune cell serum substitute, and the working concentration is 5-25% (v / v), preferably 10-20% (v / v).

[0025] Another object of the present invention is to provide a method for activating immune cells, comprising the step of contacting immune cells with the immune cell activating composition of the present invention.

[0026] In a preferred technical solution of the present invention, the immune cells are peripheral blood mononuclear cells.

[0027] In a preferred technical solution of the present invention, the immune cell activation composition includes any one or a combination of antigen peptide combinations, cytokines, and serum substitutes.

[0028] In a preferred technical solution of the present invention, the working concentration of the antigen peptide combination is 1-5 ug / mL, preferably 3-4 ug / mL.

[0029] In the preferred technical solution of the present invention, the cytokine is GM-CSF, and the working concentration is 200-1000 IU / mL, preferably 500 IU / mL.

[0030] In a preferred technical solution of the present invention, the serum substitute is an immune cell serum substitute, and the working concentration is 5-25% (v / v), preferably 10-20% (v / v).

[0031] In a preferred technical solution of the present invention, the method for activating immune cells comprises the following steps:

[0032] (1) Peripheral blood mononuclear cells (PBMC) were collected from the patient and resuspended in serum-free medium. The total number of viable cells was (1-4)×10 9 / mL; the serum-free medium is selected from any one of KBM 581, GT-T551 H3, X-VIVO15, IMSFDC or a combination thereof;

[0033] (2) adding the immune cell activation composition to the serum-free culture medium of step (1) to obtain a cell culture system, and culturing under conditions of 5% CO2 and 37°C for 1.5-3 days to obtain a cell culture fluid;

[0034] The immune cell activation composition includes any one or a combination of antigen peptide combination, cytokine, and serum replacement;

[0035] The antigen peptide combination is any one of antigen peptide blend 1, antigen peptide blend 2, antigen peptide blend 3, antigen peptide blend 4, and combinations thereof; the final concentration of the antigen peptide combination in the cell culture system is 1-10 ug / mL, preferably 3-4 ug / mL;

[0036] The cytokine is GM-CSF, and the final concentration of the cytokine in the cell culture system is 200-1000 IU / mL, preferably 500 IU / mL;

[0037] The serum substitute is an immune cell serum substitute, and the volume concentration of the serum substitute in the cell culture system is 5-25%, preferably 10-20%;

[0038] (3) The cell culture medium of step (2) is centrifuged and the supernatant is discarded. The remaining cells are washed by centrifugation with 0.1% human albumin solution and the supernatant is discarded. The precipitate is added with a sodium chloride solution containing 1-5% human albumin, mixed and filtered through a sieve to obtain the product.

[0039] In the preferred technical scheme of the present invention, in step (1), the preparation method of the peripheral blood mononuclear cells (PBMC) is: mixing the blood components obtained from peripheral blood or single blood collection with physiological saline at room temperature to obtain a cell suspension; adding a separation solution to the cell suspension, centrifuging, collecting the buffy coat cells, resuspending them with physiological saline, centrifuging, and discarding the supernatant to obtain.

[0040] In a preferred technical solution of the present invention, in step (1), the volume ratio of the cell suspension: separation solution is 1-5:1, preferably 2-3:1.

[0041] In the preferred technical scheme of the present invention, in step (1), the separation liquid is selected from any one of hydroxyethyl starch 40 sodium chloride injection (HES), Ficoll, Lymphoprep, Lymphocyte Separation Media, Cell Separation Media or a combination thereof.

[0042] In the preferred technical solution of the present invention, in step (1), the centrifugation conditions are 1000-3000 rpm, 10-40 min, preferably 1500-2500 rpm, 15-30 min, preferably 2000-2500 rpm, 20-25 min.

[0043] In the preferred technical solution of the present invention, in step (2), the culture temperature is 33-38°C, preferably 36-38°C.

[0044] In a preferred technical solution of the present invention, in step (2), the culture environment contains 2-9% CO2, preferably 4-6% CO2.

[0045] In a preferred technical solution of the present invention, in step (2), the pH of the culture medium is 6.9-7.9, preferably 7.2-7.7.

[0046] In a preferred technical solution of the present invention, in step (2), the culturing time is 1-3 days, preferably 1-2 days.

[0047] In the preferred technical solution of the present invention, in step (3), the centrifugation conditions are 1000-3000 rpm, 1-10 min, preferably 1500-2800 rpm, 2-8 min, more preferably 2000-2500 rpm, 5-6 min.

[0048] In the preferred technical solution of the present invention, in step (3), the centrifugation conditions are as follows: the first centrifugation is 2000 rpm*8 min, the second centrifugation is 2000 rpm*5 min; and the third centrifugation is 1600 rpm*8 min.

[0049] In the preferred technical solution of the present invention, in step (3), the sieve has a pore size of 100 μm.

[0050] Another object of the present invention is to provide an activated immune cell prepared by the method described above; preferably, the immune cell is a DC cell and / or a cytotoxic T cell.

[0051] In the preferred technical solution of the present invention, the immune cells include the following components: CD54 + CD86 + Dendritic cells, CD8 + Cytotoxic T lymphocytes and B cells CD19 + 、CD3 + CD56 + Cells, NK cells.

[0052] In the preferred technical solution of the present invention, the total number of activated immune cells is ≥ 1-4×10 9 indivual.

[0053] In the preferred technical solution of the present invention, the CD54 + The number of cells is ≥5×10 7 indivual.

[0054] In a preferred technical solution of the present invention, the cell activity of the antigen-activated immune cells within 12 hours is ≥70%, preferably ≥80%.

[0055] Another object of the present invention is to provide a pharmaceutical composition comprising an effective dose of the immune cell activation composition or activated immune cells.

[0056] In a preferred technical solution of the present invention, the pharmaceutical composition comprises an effective dose of immune cells activated by antigen peptide blend 2 or antigen 3; more preferably, it comprises an effective dose of immune cells activated by antigen peptide blend 2 or antigen 3 in a 1:1 ratio.

[0057] Another object of the present invention is to provide the immune cell activation composition of the present invention, the use of the activated immune cells or the pharmaceutical composition in the preparation of a drug for preventing or treating a malignant proliferative disease; preferably, the malignant proliferative disease is selected from lung cancer, lymphoma, leukemia, gastric cancer, adenocarcinoma, liver cancer, colon cancer, melanoma, breast cancer, nasal cancer, lung adenocarcinoma, moderately differentiated adenocarcinoma of the right lower lobe of the lung, carcinoma with lymph node metastasis, moderately differentiated squamous cell carcinoma of the cervix, T lymphocyte hyperplasia disease, primary Epstein-Barr virus Infection, myoma, colorectal cancer, nasopharyngeal cancer, ovarian cancer, kidney cancer, bladder cancer, breast cancer, malignant blood disease, brain tumor, head and neck cancer, glioma, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Penile cancer, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, any one or a combination of environmentally induced cancers; preferably, the malignant proliferative disease is selected from one or more of hematological tumors, lymphomas and solid tumors; preferably, the malignant proliferative disease is selected from one or more of hematological tumors, lymphomas and solid tumors; preferably, the malignant The proliferative disease is selected from one or more of colon cancer, malignant melanoma, esophageal melanoma, lung cancer, lymphoma, Burkitt lymphoma, AML-M2 leukemia (acute myeloid leukemia M2 type), acute lymphocytic leukemia, gastric adenocarcinoma, gastric cancer, small cell lung cancer, hepatocellular carcinoma, prostate cancer, melanoma, breast cancer, nasal cancer, lymph node metastasis, cervical squamous cell carcinoma, lung adenocarcinoma, T lymphocyte proliferative disease, myoma, rectal cancer, and nasopharyngeal carcinoma.

[0058] In the preferred technical scheme of the present invention, the tumor is selected from any one or a combination of prostate cancer, hepatocellular carcinoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia, well-differentiated adenocarcinoma of gastric cancer, right lung adenocarcinoma (stage IV), rectal cancer after gastric cancer surgery, hepatitis B positive, chronic or acute leukemia.

[0059] In a preferred technical solution of the present invention, the lung cancer is selected from any one of small cell lung cancer, non-small cell lung cancer or their complications.

[0060] In a preferred technical solution of the present invention, the leukemia is selected from any one of acute lymphocytic leukemia, acute myeloid leukemia, B-cell chronic lymphocytic leukemia, chronic myeloid leukemia or their complications.

[0061] In the preferred technical scheme of the present invention, the acute myeloid leukemia is selected from any one of acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute megakaryocytic leukemia, acute erythroleukemia, and acute panmyelosis with myelofibrosis or a combination thereof.

[0062] Another object of the present invention is to provide the immune cell activation composition of the present invention, and the use of the activated immune cells or the pharmaceutical composition in the preparation of immunotherapy drugs.

[0063] In a preferred technical solution of the present invention, the immunotherapy is selected from any one of anti-tumor immunotherapy and post-radical tumor immunotherapy or a combination thereof.

[0064] In a preferred technical solution of the present invention, the immunotherapy is selected from immunotherapy after radical resection of primary hepatocellular carcinoma.

[0065] In a preferred technical solution of the present invention, the drug is used for adult patients or pediatric patients.

[0066] Another object of the present invention is to provide the immune cell activation composition of the present invention, and the use of the activated immune cells or the pharmaceutical composition in the preparation of immunity-enhancing drugs. Preferably, the immunity-enhancing drugs are antiviral and anti-infective drugs.

[0067] Another object of the present invention is to provide the immune cell activation composition of the present invention, and the use of the activated immune cells or the pharmaceutical composition in the preparation of drugs for treating autoimmune diseases.

[0068] In a preferred technical solution of the present invention, the autoimmune disease is selected from systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, idiopathic thrombocytic purpura, autoimmune hemolytic anemia, ulcerative colitis, any one of skin diseases or their complications.

[0069] Another object of the present invention is to provide a pharmaceutical composition comprising an immune cell activation composition, or activated immune cells, and an anti-tumor drug.

[0070] Another object of the present invention is to provide the immune cell activation composition of the present invention, and the use of the activated immune cells or the pharmaceutical composition in the preparation of anti-tumor drugs.

[0071] Another object of the present invention is to provide the use of the antigen-activated immune cells of the present invention in combination with any one of surgery, radiotherapy and chemotherapy in anti-tumor or immunotherapy.

[0072] The present invention also provides a treatment kit, which comprises an immune cell activation composition, or the above-mentioned pharmaceutical composition, and an organic solvent for dissolving the antigen peptide. In one embodiment, the organic solvent is DMSO.

[0073] Another object of the present invention is to provide a dosage regimen for immunotherapy using the antigen-activated immune cells of the present invention, comprising the following steps:

[0074] (1) obtaining a biological sample containing lymphocytes;

[0075] (2) using the preparation method of the present invention to obtain the antigen-activated immune cells of the present invention;

[0076] (3) Returning the antigen-activated immune cells of the present invention or the pharmaceutical composition thereof to the individual.

[0077] In a preferred technical solution of the present invention, obtaining the biological sample containing lymphocytes includes obtaining it from an individual.

[0078] In the preferred technical solution of the present invention, for patients treated with the immune cells of the present invention alone, each reinfusion is performed at an interval of 2-6 weeks, with 4 reinfusions per course of treatment, and at least two courses of treatment.

[0079] In the preferred technical scheme of the present invention, for patients undergoing chemotherapy, if it is a three-week chemotherapy regimen, when chemotherapy is on the first day, then the drug is infused once on the 7th and 14th days of chemotherapy; when chemotherapy is on the first and eighth days, then the drug is infused once on the 2nd and 10th days of chemotherapy; if it is a four-week chemotherapy regimen, then the drug is infused once on the 7th and 17th days of chemotherapy.

[0080] In the preferred technical solution of the present invention, for patients undergoing antibody drug targeted therapy, depending on the treatment plan of the targeted drug, cell reinfusion is usually performed 24-48 hours after the administration of the targeted drug.

[0081] Unless otherwise specified, "antigenic peptide" herein refers to a polypeptide or antigen-derived peptide having immunogenicity. The antigenic peptide of the present invention can be obtained by any known method for preparing a polypeptide. For example, the antigenic peptide of the present invention can be synthesized by conventional chemical methods, can be made by commercial customization, or can be obtained by genetic engineering methods, such as inserting a nucleic acid sequence encoding the antigenic peptide into a suitable expression vector and transforming a host cell, culturing the transformed host cell, and then recovering the antigenic peptide from the culture.

[0082] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0083] The antigen peptide blends 1-4 are formed by mixing peptide segments of different amino acid sequences in equal mass proportions.

[0084] The term "antigenic peptide blend" refers to a composition or mixture formed by mixing or compounding a plurality of polypeptides used as antigenic peptides in a certain proportion; in the present invention, it specifically refers to a mixture formed by mixing overlapping peptides derived from the same protein in a certain proportion or manner; in an embodiment according to the present invention, the synthesized overlapping peptide lyophilized powder is first dissolved in an appropriate amount of DMSO, and then the DMSO solutions containing different overlapping peptides are mixed. In some embodiments, the overlapping peptide lyophilized powder can also be dissolved in a serum-free medium, and the serum-free medium is selected from KBM 581, GT-T551 H3, IMSFDC and X-VIVO15. In some embodiments according to the present invention, the antigenic peptide blend can be first formulated into a mother solution with a concentration of about 2-5 mg / ml (preferably a concentration of about 3 mg / ml); after the antigenic peptide blend is applied to the immune cell culture system, the final concentration of the antigenic peptide blend in the immune cell culture system is 1-10ug / mL, preferably 3-4ug / mL.

[0085] Compared with the prior art, the present invention has the following beneficial technical effects:

[0086] 1. The present invention provides an immune cell activation composition, which contains a combination of antigen peptide blends. Through scientific selection, a combination of multiple antigen peptides is used to activate immune cells, so that the immune cells contain multiple activated effector cells with dendritic cells as the core. After being re-infused into the patient's body, it will induce a target-specific anti-tumor immune response, thereby achieving the effect of identifying and clearing tumors.

[0087] 2. The antigen-activated immune cells of the present invention have strong specificity (multiple specific effector cells stimulated by antigen-presenting cells after specific tumor antigen stimulation), short culture time (1.5-3 days from blood collection to transfusion, antigen-presenting cells still survive and play a stimulating role during transfusion), high safety (serum-free culture medium is used throughout the process, and the human body's own cells are used to kill tumor cells, without toxic side effects), strong cell viability (the viability after culture is above 80%), and stable cell quality (the cell viability does not decrease significantly within 12 hours, and the cell viability decreases by about 5% within 24 hours, and the cell preparation can be transported long distances at low temperature). The antigen-activated immune cells of the present invention can effectively prevent recurrence and metastasis, improve the quality of life and stabilize tumors, maintain tumor stability for a long time, improve the quality of life, and eliminate the side effects of chemotherapy.

[0088] 3. The present invention has the advantages of simple operation, controllable quality, and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 This is a graph showing the results of surface factor staining detection of immune cells obtained by co-culturing with the antigen peptide mixture of the present invention. DETAILED DESCRIPTION

[0090] The present invention is further described below in conjunction with embodiments, but the present invention is not limited to the described embodiments.

[0091] Example 1 Preparation of immune activation composition

[0092] The antigen peptide sequences used in this application are shown in Tables 1-4, which are synthesized by Gill Biochemical (Shanghai) Co., Ltd. The synthesized antigen peptides are freeze-dried to prepare lyophilized powder.

[0093] The antigen peptide blend 1 of the present invention is a combination of peptide segments composed of amino acid sequences shown in SEQ ID NO: 1-10, which are uniformly mixed in equal proportions. The specific preparation method is: the peptide segment freeze-dried powder shown in SEQ ID NO: 1-10 is respectively packed into 10 test tubes, 2 mg in each test tube, and the peptide segments in each test tube are different. 0.6 mL DMSO is added to each test tube, and after the freeze-dried powder is fully dissolved, 400 μl of solution is taken out from each test tube, and after being fully mixed, it is filtered with a 0.2 μm filter to obtain a mixed solution. When used, it is diluted with IMSFDC culture medium and mixed evenly until the final concentration of the antigen peptide blend is 3 mg / mL to obtain an antigen peptide blend 1 solution.

[0094] In the same manner, an antigen peptide blend 2 solution containing the peptide segments shown in SEQ ID NOs: 11-40; an antigen peptide blend 3 solution containing the peptide segments shown in SEQ ID NOs: 41-64; and an antigen peptide blend 4 solution containing the peptide segments shown in SEQ ID NOs: 65-91 were prepared (the peptide segments contained in each antigen peptide are shown in Tables 1-4).

[0095] Table 1 Antigenic peptide blend 1

[0096] Sequence Number Peptide Sequence SEQ ID NO: 1 MKRATQIPSYKKLIMYSAHDTTVSGLQMAL SEQ ID NO: 2 KKLIMYSAHDTTVSGLQMALDVYNGLLPPY SEQ ID NO: 3 TTVSGLQMALDVYNGLLPPYASCHLTELYF SEQ ID NO: 4 DVYNGLLPPYASCHLTELYFEKGEYFVEMY SEQ ID NO: 5 ASCHLTELYFEKGEYFVEMYYRNETQHEPY SEQ ID NO: 6 EKGEYFVEMYYRNETQHEPYPLMLPGCSPS SEQ ID NO: 7 YRNETQHEPYPLMLPGCSPSCPLERFAELV SEQ ID NO: 8 PLMLPGCSPSCPLERFAELVGPVIPQDWST SEQ ID NO: 9 CPLERFAELVGPVIPQDWSTECMTTNSHQG SEQ ID NO: 10 GPVIPQDWSTECMTTNSHQGTEDSTD

[0097] Table 2 Antigenic peptide blend 2

[0098]

[0099]

[0100] Table 3 Antigenic peptide blend 3

[0101] Sequence Number Peptide Sequence SEQ ID NO: 41 MQAEGRGTGGSTGDADGPGGPGIPDGPGGN SEQ ID NO: 42 STGDADGPGGPGIPDGPGGNAGGPGEAGAT SEQ ID NO: 43 PGIPDGPGGNAGGPGEAGATGGRGPRGAGA SEQ ID NO: 44 AGGPGEAGATGGRGPRGAGAARASGPGGGA SEQ ID NO: 45 GGRGPRGAGAARASGPGGGAPRGPHGGAAS SEQ ID NO: 46 ARASGPGGGAPRGPHGGAASGLNGCCRCGA SEQ ID NO: 47 PRGPHGGAASGLNGCCRCGARGPESRLLEF SEQ ID NO: 48 GLNGCCRCGARGPESRLLEFYLAMPFATPM SEQ ID NO: 49 RGPESRLLEFYLAMPFATPMEAELARRSLA SEQ ID NO: 50 YLAMPFATPMEAELARRSLAQDAPPLPVPG SEQ ID NO: 51 EAELARRSLAQDAPPLPVPGVLLKEFTVSG SEQ ID NO: 52 QDAPPLPVPGVLLKEFTVSGNILTIRLTAA SEQ ID NO: 53 VLLKEFTVSGNILTIRLTAADHRQLQLSIS SEQ ID NO: 54 NILTIRLTAADHRQLQLSISSCLQQLSLLM SEQ ID NO: 55 DHRQLQLSISSCLQQLSLLMWITQCFLPVF SEQ ID NO: 56 SCLQQLSLLMWITQCFLPVFLAQPPSGQRR SEQ ID NO: 57 MPREDAHFIYGYPKKGHGHSYTTAEEAAGI SEQ ID NO: 58 GYPKKGHGHSYTTAEEAAGIGILTVILGVL SEQ ID NO: 59 YTTAEEAAGIGILTVILGVLLLIGCWYCRR SEQ ID NO: 60 GILTVILGVLLLIGCWYCRRRNGYRALMDK SEQ ID NO: 61 LLIGCWYCRRRNGYRALMDKSLHVGTQCAL SEQ ID NO: 62 RNGYRALMDKSLHVGTQCALTRRCPQEGFD SEQ ID NO: 63 SLHVGTQCALTRRCPQEGFDHRDSKVSLQE SEQ ID NO: 64 TRRCPQEGFDHRDSKVSLQEKNCEPVVPNA

[0102] Table 4 Antigenic peptide blend 4

[0103] Sequence No. Peptide Sequence SEQ ID NO: 65 MRAAPLLLARAASLSLGFLFLLFFWLDRSV SEQ ID NO: 66 AASLSLGFLFLLFFWLDRSVLAKELKFVTL SEQ ID NO: 67 LLFFWLDRSVLAKELKFVTLVFRHGDRSPI SEQ ID NO: 68 LAKELKFVTLVFRHGDRSPIDTFPTDPIKE SEQ ID NO: 69 VFRHGDRSPIDTFPTDPIKESSWPQGFGQL SEQ ID NO: 70 DTFPTDPIKESSWPQGFGQLTQLGMEQHYE SEQ ID NO: 71 SSWPQGFGQLTQLGMEQHYELGEYIRKRYR SEQ ID NO: 72 TQLGMEQHYELGEYIRKRYRKFLNESYKHE SEQ ID NO: 73 LGEYIRKRYRKFLNESYKHEQVYIRSTDVD SEQ ID NO: 74 KFLNESYKHEQVYIRSTDVDRTLMSAMTNL SEQ ID NO: 75 QVYIRSTDVDRTLMSAMTNLAALFPPEGVS SEQ ID NO: 76 RTLMSAMTNLAALFPPEGVSIWNPILLWQP SEQ ID NO: 77 AALFPPEGVSIWNPILLWQPIPVHTVPLSE SEQ ID NO: 78 IWNPILLWQPIPVHTVPLSEDQLLYLPFRN SEQ ID NO: 79 IPVHTVPLSEDQLLYLPFRNCPRFQELESE SEQ ID NO: 80 DQLLYLPFRNCPRFQELESETLKSEEFQKR SEQ ID NO: 81 CPRFQELESETLKSEEFQKRLHPYKDFIAT SEQ ID NO: 82 TLKSEEFQKRLHPYKDFIATLGKLSGLHGQ SEQ ID NO: 83 LHPYKDFIATLGKLSGLHGQDLFGIWSKVY SEQ ID NO: 84 LGKLSGLHGQDLFGIWSKVYDPLYCESVHN SEQ ID NO: 85 DLFGIWSKVYDPLYCESVHNFTLPSWATED SEQ ID NO: 86 DPLYCESVHNFTLPSWATEDTMTKLRELSE SEQ ID NO: 87 FTLPSWATEDTMTKLRELSELSLLSLYGIH SEQ ID NO: 88 TMTKLRELSELSLLSLYGIHKQKEKSRLQG SEQ ID NO: 89 LSLLSLYGIHKQKEKSRLQGGVLVNEILNH SEQ ID NO: 90 KQKEKSRLQGGVLVNEILNHMKRATQIPSY SEQ ID NO: 91 GVLVNEILNHMKRATQIPSYKKLIMYSAHD

[0104] The antigen peptide blend solution prepared in Example 1 was used to prepare antigen-activated immune cells. In Examples 2-4, the centrifuge was a Heraeus Multifuge X3, Thermo; the Ficoll separation solution (commercially available from GE Health Garé, Cat No. 17-5442-03, density 1.077 g / mL); the serum-free culture medium was IMSF-DC culture medium, commercially available from Immunotech UK Ltd.; CTS TM Immune cell serum substitute was purchased from ThermoFisher; GM-CSF granulocyte-macrophage colony stimulating factor was purchased from Merck; 0.1 human albumin solution was human serum albumin solution purchased from Chengdu Rongsheng Pharmaceutical Co., Ltd.

[0105] GM-CSF solution: Take 1 mg of GM-CSF with a potency of 10 million IU / mg, dissolve it in 100 mL of IMSFDC to prepare a 100,000 IU / mL GM-CSF solution.

[0106] Example 2 Cultivation of immune cells activated by antigens of the present invention

[0107] Patient 1: Melanoma, male, 41 years old.

[0108] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0109] 100 mL of the single blood drawn from the patient was placed in a 250 mL centrifuge tube, and physiological saline was added to 200 mL, and mixed to form a mixed solution; according to the volume ratio of the mixed solution to the Ficoll separation solution of 2:1, the mixed solution was transferred to the centrifuge tube to which the Ficoll separation solution had been added, and centrifuged (2000 rpm*20 min, acceleration rate 7, deceleration rate 4), the cell buffy coat layer at the interface was transferred to a new centrifuge tube, and physiological saline was added to make the volume to 250 mL, and centrifuged (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, and physiological saline was added to the remaining cells to make the volume to 200 mL and mixed, and centrifuged (1600 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, and the precipitate was resuspended in 200 mL of serum-free culture medium IMSF-DC.

[0110] 2. Co-culture with antigen peptide mixture 1

[0111] Add antigen peptide blend 1 solution, 1 mL of GM-CSF solution (100,000 IU / mL) and 4 mL of CTS to the serum-free medium in step 1. TM The final concentration of the immune cell serum substitute and antigen peptide blend 1 in the cell system is 3ug / mL. The cell suspension is mixed and then placed in a cell culture bottle and cultured for 2 days at 5% CO2 and 37°C to obtain a cell culture fluid.

[0112] 3. Harvesting cells

[0113] Pour the cell culture solution of step (2) into a 250mL centrifuge tube, centrifuge (2000rpm, 8min, acceleration rate 9, deceleration rate 9), and discard the supernatant. Add 0.1% human albumin solution to the cell culture bottle and centrifuge for multiple washings (first centrifuge at 2000rpm, 5min; then centrifuge at 1600rpm, 8min) and discard the supernatant; add 1-5% human albumin sodium chloride solution to the precipitate, mix well, filter through a sieve (100μm), and put into a transfer bag to obtain.

[0114] 4. Immune cell infusion

[0115] In February 2010, the patient underwent 3 cycles of systemic chemotherapy after surgery, and radiotherapy + chemotherapy was used 1 year after surgery, but no effect was seen. On January 19, February 8, and March 23, 2011, the immune cells of Example 2 were infused three times, with 100 mL infused each time. Pathology later showed only subcutaneous nodular hyperplasia of fibroblasts and blood vessels, and no melanoma. In the next 4 years, the immune cells of Example 2 were infused again for a total of 19 times. The patient is still alive and has not seen signs of tumor recurrence and metastasis.

[0116] Example 3 Cultivation of immune cells activated by antigens of the present invention

[0117] Patient 2: Breast cancer, female, 53 years old.

[0118] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0119] 100 mL of the single blood sampled from the patient was placed in a 250 mL centrifuge tube and centrifuged (2500 rpm*10 min, acceleration rate 9, deceleration rate 9). After discarding the upper plasma, normal saline was added to the lower cells to make up to 200 mL and mixed to form a mixed solution; according to the volume ratio of the mixed solution to the Ficoll separation solution of 2:1, the mixed solution was transferred to a centrifuge tube to which Ficoll separation solution had been added, centrifuged (2000 rpm, 20 min, acceleration rate 7, deceleration rate 4), the cell buffy coat layer at the interface was transferred to a new centrifuge tube, normal saline was added to make up to 250 mL, centrifuged (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, normal saline was added to the remaining cells to make up to 200 mL and mixed. After the cells were centrifuged (1600 rpm*8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, and the precipitate was resuspended in 200 mL of serum-free culture medium.

[0120] 2. Co-culture with antigen peptide

[0121] Add antigen peptide blend 2 solution, 1 mL of GM-CSF solution and 4 mL of CTS to the serum-free medium in step (1). TM The final concentration of the immune cell serum substitute and antigen peptide blend 2 in the cell system is 3ug / mL. The cell suspension is mixed and then placed in a cell culture bottle and cultured for 2 days at 5% CO2 and 37°C to obtain a cell culture fluid.

[0122] 3. Harvesting cells

[0123] Pour the cell culture solution in step (2) into a 250 mL centrifuge tube, centrifuge (2000 rpm, 8 min, acceleration 9, deceleration 9), and discard the supernatant. Add 0.1% human albumin solution to the cell culture bottle and centrifuge for multiple washings, and discard the supernatant; add 1-5% human albumin sodium chloride solution to the precipitate, mix well, filter through a sieve (100 μm), and put into a transfer bag to obtain.

[0124] 4. Immune cell infusion

[0125] The patient was infused with the above-mentioned immune cells five times from September 2012 to October 2014, which significantly limited tumor proliferation and significantly improved the patient's condition. The patient is still alive today.

[0126] Example 4 Cultivation of immune cells activated by antigens of the present invention

[0127] Patient 3: Gastric cancer, male, 62 years old

[0128] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0129] 100 mL of the single blood sampled from the patient was placed in a 250 mL centrifuge tube and centrifuged (2500 rpm × 10 min, acceleration 9, deceleration 9). After discarding the upper plasma, normal saline was added to the lower cells to make up to 200 mL and mixed to form a mixed solution. According to the volume ratio of the mixed solution to the Ficoll separation solution of 2:1, the mixed solution was transferred to a centrifuge tube to which the Ficoll separation solution had been added and centrifuged (2000 rpm, 20 min, acceleration rate 7, deceleration rate 4). The cell buffy coat layer at the interface was transferred to a new centrifuge tube, normal saline was added to make up to 250 mL, centrifuged (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, normal saline was added to the remaining cells to make up to 200 mL and mixed, centrifuged (1600 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, and the precipitate was resuspended in 200 mL of serum-free culture medium.

[0130] 2. Co-culture with antigen peptide

[0131] Add antigen peptide blend 3 solution, 1 mL of GM-CSF solution and 4 mL of CTS to the serum-free medium in step (1). TM The final concentration of the immune cell serum substitute and antigen peptide blend 3 in the cell system was 3ug / mL. The cell suspension was mixed and then placed in a cell culture bottle and cultured for 2 days at 5% CO2 and 37°C to obtain a cell culture fluid.

[0132] 3. Harvesting cells

[0133] Pour the cell culture solution in step (2) into a 250 mL centrifuge tube, centrifuge (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), and discard the supernatant. Add 0.1% human albumin solution to the cell culture bottle and centrifuge for multiple washings, and discard the supernatant; add 1-5% human albumin sodium chloride solution to the precipitate, mix well, filter through a sieve (100 μm), and put into a transfer bag to obtain.

[0134] 4. Immune cell infusion

[0135] From September 2012 to October 2014, the patient received multiple rounds of infusions of the above-mentioned immune cells for a total of 27 times, which significantly limited tumor proliferation and significantly improved the patient's condition. The patient is still alive today.

[0136] Example 5 Cultivation of immune cells activated by antigens of the present invention

[0137] Patient 4: Gastric cancer, male, 62 years old

[0138] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0139] 100 mL of the single blood sampled from the patient was placed in a 250 mL centrifuge tube and centrifuged (2500 rpm × 10 min, acceleration rate 9, deceleration rate 9). After discarding the upper plasma, normal saline was added to the lower cells to make up to 200 mL and mixed to form a mixed solution; according to the volume ratio of the mixed solution to the Ficoll separation solution of 2:1, the mixed solution was transferred to a centrifuge tube to which Ficoll separation solution had been added, centrifuged (2000 rpm, 20 min, acceleration rate 7, deceleration rate 4), the cell buffy coat layer at the interface was transferred to a new centrifuge tube, normal saline was added to make up to 250 mL, centrifuged (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, normal saline was added to the remaining cells to make up to 200 mL and mixed, centrifuged (1600 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, and the precipitate was resuspended in 200 mL of serum-free culture medium.

[0140] 2. Co-culture with antigen peptide

[0141] Add antigen peptide blend 3 solution, 1 mL of GM-CSF solution and 4 mL of CTS to the serum-free medium in step (1). TM The final concentration of the immune cell serum substitute and antigen peptide blend 3 in the cell system was 3ug / mL. The cell suspension was mixed and then placed in a cell culture bottle and cultured for 2 days at 5% CO2 and 37°C to obtain a cell culture fluid.

[0142] 3. Harvesting cells

[0143] Pour the cell culture solution in step (2) into a 250 mL centrifuge tube, centrifuge (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), and discard the supernatant. Add 0.1% human albumin solution to the cell culture bottle and centrifuge for multiple washings, and discard the supernatant; add 1-5% human albumin sodium chloride solution to the precipitate, mix well, filter through a sieve (100 μm), and put into a transfer bag to obtain.

[0144] 4. Immune cell infusion

[0145] From December 2015 to March 2023, the patient received multiple rounds of infusions of the above-mentioned immune cells for a total of 5 times, which significantly limited tumor proliferation and significantly improved the patient's condition. The patient is still alive today.

[0146] Example 6 Cultivation of immune cells activated by antigens of the present invention

[0147] Patient 4: Colon cancer, female, 47 years old

[0148] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0149] 100 mL of the single blood sampled from the patient was placed in a 250 mL centrifuge tube and centrifuged (2500 rpm*10 min, acceleration rate 9, deceleration rate 9). After discarding the upper plasma, normal saline was added to the lower cells to make up to 200 mL and mixed to form a mixed solution; according to the volume ratio of the mixed solution to the Ficoll separation solution of 2:1, the mixed solution was transferred to a centrifuge tube to which Ficoll separation solution had been added, centrifuged (2000 rpm, 20 min, acceleration rate 7, deceleration rate 4), the cell buffy coat layer at the interface was transferred to a new centrifuge tube, normal saline was added to make up to 250 mL, centrifuged (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, normal saline was added to the remaining cells to make up to 200 mL and mixed, centrifuged (1600 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, and the precipitate was resuspended in 200 mL of serum-free culture medium.

[0150] 2. Co-culture with antigen peptide

[0151] Add antigen peptide blend 2 solution, 1 mL of GM-CSF solution and 4 mL of CTS to the serum-free medium in step (1). TM The final concentration of the immune cell serum substitute and antigen peptide blend 2 in the cell system was 3ug / mL. The cell suspension was mixed and then placed in a T300 cell culture flask and cultured for 2 days at 5% CO2 and 37°C to obtain a cell culture fluid.

[0152] 3. Harvesting cells

[0153] Pour the cell culture solution in step (2) into a 250 mL centrifuge tube, centrifuge (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), and discard the supernatant. Add 0.1% human albumin solution to the cell culture bottle and centrifuge for multiple washings, and discard the supernatant; add 1-5% human albumin sodium chloride solution to the precipitate, mix well, filter through a sieve (100 μm), and put into a transfer bag to obtain.

[0154] 4. Immune cell infusion

[0155] From April to June 2011, the patient received a total of four infusions of the above-mentioned immune cells, which significantly limited tumor proliferation and markedly improved the patient's condition.

[0156] Example 7 Cultivation of immune cells activated by antigens of the present invention

[0157] Patient 5: Prostate cancer, male, 61 years old

[0158] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0159] 100 mL of the single blood sampled from the patient was placed in a 250 mL centrifuge tube and centrifuged (2500 rpm*10 min, acceleration rate 9, deceleration rate 9). After discarding the upper plasma, normal saline was added to the lower cells to make up to 200 mL and mixed to form a mixed solution; according to the volume ratio of the mixed solution to the Ficoll separation solution of 2:1, the mixed solution was transferred to a centrifuge tube to which Ficoll separation solution had been added, centrifuged (2000 rpm, 20 min, acceleration rate 7, deceleration rate 4), the cell buffy coat layer at the interface was transferred to a new centrifuge tube, normal saline was added to make up to 250 mL, centrifuged (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, normal saline was added to the remaining cells to make up to 200 mL and mixed, centrifuged (1600 rpm, 8 min, acceleration rate 9, deceleration rate 9), the supernatant was discarded, and the precipitate was resuspended in 200 mL of serum-free culture medium.

[0160] 2. Co-culture with antigen peptide

[0161] Add the antigen peptide mixture 4 solution, 1 mL of GM-CSF solution and 4 mL of CTS to the serum-free medium in step (1). TM The final concentration of the immune cell serum substitute and antigen peptide blend 4 in the cell system was 3ug / mL. The cell suspension was mixed and then placed in a T300 cell culture flask and cultured for 2 days at 5% CO2 and 37°C to obtain a cell culture fluid.

[0162] 3. Harvesting cells

[0163] Pour the cell culture solution in step (2) into a 250 mL centrifuge tube, centrifuge (2000 rpm, 8 min, acceleration rate 9, deceleration rate 9), and discard the supernatant. Add 0.1% human albumin solution to the cell culture bottle and centrifuge for multiple washings, and discard the supernatant; add 1-5% human albumin sodium chloride solution to the precipitate, mix well, filter through a sieve (100 μm), and put into a transfer bag to obtain.

[0164] 4. Immune cell infusion

[0165] The patient was infused with the above-mentioned immune cells six times from February 2012 to October 2012, which significantly limited tumor proliferation and significantly improved the patient's condition.

[0166] Experimental Example 1 Detection of cell surface factors of immune cells after co-culture with antigen peptides

[0167] 1. All test reagents are commercially available

[0168]

[0169] 2. Test samples

[0170] Cultured cells

[0171] 3. Operation steps

[0172] 1) Cell separation

[0173] Count cells >1×10 6 / mL, centrifuged by density gradient (1500rpm, 3min), and then washed twice to obtain cell suspension.

[0174] 2) Surface factor staining

[0175] 2-1) Take 4 flow cytometry tubes and add antibodies in the following order:

[0176] a.10μl IgG1,10μl IgG2a

[0177] b.10μl CD40-FITC, 10μl CD54-PE

[0178] c.10μl HLA-DPQR-FITC, 10μl CD80-PE

[0179] d.10μl CD209-FITC, 10μl CD19-PE, 10μl CD184-PerCP-Cy5.5, 10μl CD56-APC;

[0180] 2-2) 100 μl of cell suspension was added to each flow cytometry tube, PBMC before culture was used as the control, and immune cells activated with different doses (10 μg, 30 μg, 100 μg, 300 μg, 1000 μg) of antigen peptide mixture 3 were used as the experimental group, and incubated at room temperature in the dark for 15 min after shaking.

[0181] 2-3) Add 2 mL of Stain Buffer (FBS) to each flow cytometry tube.

[0182] 2-4) Centrifuge at 250 g for 3 min and discard the supernatant.

[0183] 2-5) Add 0.5 mL Stain Buffer (FBS) to each flow cytometry tube and perform detection on the flow cytometry machine (medium speed, lymphocyte + monocyte gate, 20,000 cells within the gate).

[0184] 4. Results Analysis

[0185] By flow cytometry, the PBMCs before culture in Example 4 and the immune cells after co-culture with the antigen peptide mixture 3 were stained for cell surface factors to detect changes in cell surface factors before and after culture. The cell surface factors included CD40, CD54, HLA-DPQR, CD80, CD209, and CD184. The results are as follows Figure 1 shown.

[0186] like Figure 1 As shown, the surface factors of immune cells after co-culture with antigen peptide blend 3 changed significantly compared with PBMC before culture, indicating that the antigen peptide of the present invention not only contains antigen presenting cells (mainly dendritic cells) that can induce activation of tumor antigen-specific effector cells, but also contains a large number of tumor antigen-specific cytotoxic T lymphocytes.

[0187] Experimental Example 2 Evaluation of the clinical trial effect of immune cells co-cultured with antigen peptides of the present invention

[0188] 1. Patient enrollment

[0189] A total of 35 patients were enrolled, including 13 females and 22 males. The patients' ages ranged from 4 to 65 years old, with an average age of 38.1 years. There was no statistical difference among the patients.

[0190] Patient inclusion criteria: patients diagnosed with prostate cancer, melanoma, cervical cancer, colon cancer, malignant melanoma, esophageal melanoma, lung cancer, lymphoma, Burkitt lymphoma, AML-M2 leukemia (acute myeloid leukemia M2 type), non-Hodgkin's lymphoma, acute lymphocytic leukemia, gastric cancer well-differentiated adenocarcinoma, moderately differentiated adenocarcinoma, gastric cancer, moderately differentiated adenocarcinoma of the left lung with neuroendocrine differentiation, small cell lung cancer, hepatocellular carcinoma, hepatitis B positive, breast cancer, nasal cancer, right lung adenocarcinoma (stage IV), moderately differentiated adenocarcinoma of the right lower lobe, plus lymph node metastasis, and moderately differentiated squamous cell carcinoma of the cervix, lung adenocarcinoma, T lymphocytic proliferative diseases, primary Epstein-Barr virus infection, myoma, gastric cancer surgery, rectal cancer, nasopharyngeal carcinoma (poorly differentiated), etc., asymptomatic or with mild symptoms; patients with an expected survival period of more than 3 months. Laboratory examinations must meet the following conditions: lymphocyte absolute value > 0.8, monocyte absolute value > 0.12; hemoglobin > 90g / L, platelets > 80×10 9 / L; creatinine (Cr) ≤1.0×UNL, total bilirubin (TBI) ≤1.0×UNL, aspartate aminotransferase or alanine aminotransferase (ALT or AST) ≤2.5×UNL, alkaline phosphatase (AKP) <5×UNL (except for patients with bone metastasis but not liver disease); patients have self-awareness and can sign informed or voluntary consent; patients have good compliance, are eligible for follow-up, and can cooperate with treatment for toxic and side effects; anti-HIV (-), TPHA (-).

[0191] 2. Treatment plan: Take peripheral blood from patients, refer to the method of Examples 2-5, prepare immune cells after co-culture with each antigen peptide composition, and infuse them once every 2-6 weeks, 4 times per course of treatment, at least two courses of treatment, and the total number of cells harvested after culture is (1-10)×10 9 The average cell viability of immune cells was above 80%.

[0192] Table 2

[0193]

[0194]

[0195] 3. Results: The quality of life of all 35 patients was significantly improved. Among them, 10 patients maintained stable tumors without metastasis. The side effects during the treatment were mainly chills, fever, headache, flu-like symptoms, etc. Most of the observed side effects were grade 1 or 2 and self-limiting, a few were grade 3 but had no tendency to progress, and most were relieved within 24-48 hours. No patient had lung or kidney symptoms, any signs of infection, liver function decline or autoimmune disorder reaction. The antigen peptide of the present invention showed significant effectiveness and safety in the experiment.

[0196] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. An immune cell activating composition, characterized in that: The composition comprises an antigen peptide combination, wherein the antigen peptide combination is selected from any one of antigen peptide blend 1, antigen peptide blend 2, antigen peptide blend 3 and antigen peptide blend 4, or a combination of two or more thereof; wherein, The antigen peptide blend 1 is formed by mixing peptide segments with amino acid sequences of SEQ ID NO: 1-10 in equal mass proportions; The antigen peptide blend 2 is formed by mixing peptide segments with amino acid sequences of SEQ ID NO: 11-40 in equal mass proportions; The antigen peptide blend 3 is formed by mixing peptide segments with amino acid sequences of SEQ ID NO: 41-64 in equal mass proportions; The antigen peptide blend 4 is formed by mixing peptide segments with amino acid sequences of SEQ ID NO: 65-91 in equal mass proportions.

2. A method for activating immune cells, comprising the step of contacting immune cells with the immune cell activating composition according to claim 1, wherein the composition comprises an antigen peptide combination, and the antigen peptide combination is selected from any one of antigen peptide blend 1, antigen peptide blend 2, antigen peptide blend 3 and antigen peptide blend 4, or a combination of two or more thereof; Preferably, the immune cells are peripheral blood mononuclear cells; Preferably, the working concentration of the antigen peptide combination is 1-10 μg / mL, preferably 3-5 μg / mL; Preferably, the method further comprises the step of contacting the immune cells with cytokines and / or serum replacement; Preferably, the cytokine is GM-CSF, and the working concentration of the cytokine is 200-1000 IU / mL, preferably 500 IU / mL; Preferably, the serum substitute is an immune serum substitute, and the working concentration of the immune serum substitute is 5-25%, preferably 10-20%.

3. An activated immune cell prepared by the method according to claim 2; preferably, the immune cell is a DC cell and / or a killer T cell.

4. A pharmaceutical composition comprising an effective dose of the immune cell activation composition according to claim 1, or the activated immune cells according to claim 3.

5. Use of the immune cell activation composition according to claim 1, the activated immune cells according to claim 3 or the pharmaceutical composition according to claim 4 in the preparation of a medicament for preventing or treating a malignant proliferative disease; preferably, the malignant proliferative disease is selected from lung cancer, lymphoma, leukemia, gastric cancer, adenocarcinoma, liver cancer, colon cancer, melanoma, breast cancer, nasal cancer, lung adenocarcinoma, moderately differentiated adenocarcinoma of the right lower lobe of the lung, carcinoma with lymph node metastasis, moderately differentiated squamous cell carcinoma of the cervix, T lymphocyte hyperplasia disease , primary Epstein-Barr virus infection, sarcoma, colorectal cancer, nasopharyngeal cancer, ovarian cancer, kidney cancer, bladder cancer, breast cancer, malignant blood disease, brain tumor, head and neck cancer, glioma, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue muscle Preferably, the malignant proliferative disease is any one of hematologic malignancies, lymphomas, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinomas, squamous cell carcinomas, T-cell lymphomas, and environmentally induced cancers, or a combination thereof; preferably, the malignant proliferative disease is selected from one or more of hematologic malignancies, lymphomas, and solid tumors; preferably, The malignant proliferative disease is selected from one or more of colon cancer, malignant melanoma, esophageal melanoma, lung cancer, lymphoma, Burkitt lymphoma, AML-M2 leukemia (acute myeloid leukemia M2 type), acute lymphocytic leukemia, gastric adenocarcinoma, gastric cancer, small cell lung cancer, hepatocellular carcinoma, prostate cancer, melanoma, breast cancer, nasal cancer, lymphatic metastasis, cervical squamous cell carcinoma, lung adenocarcinoma, T lymphocyte proliferative disease, myoma, rectal cancer, and nasopharyngeal carcinoma.

6. Use of the immune cell activation composition according to claim 1 or the activated immune cells according to claim 3 in the preparation of immunotherapy drugs.

7. Use of the immune cell activation composition according to claim 1 or the activated immune cells according to claim 3 in the preparation of anti-tumor drugs.

8. A pharmaceutical composition comprising the immune cell activating composition according to claim 1, or the activated immune cells according to claim 3, and an anti-tumor drug.

9. Use of the immune cell activating composition according to claim 1 or the antigen-activated immune cells according to claim 3 in the preparation of a drug for enhancing immunity or treating autoimmune diseases.

10. A therapeutic kit comprising the immune cell activation composition according to claim 1, the activated immune cells according to claim 3, or the pharmaceutical composition according to any one of claims 4 and 8, and an organic solvent for dissolving the antigen peptide; preferably, the organic solvent is DMSO.

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