CN111481662A

CN111481662A - Individual specific therapeutic vaccine for tumor and preparation method thereof

Info

Publication number: CN111481662A
Application number: CN202010201235.2A
Authority: CN
Inventors: 李彬
Original assignee: Hebei Bio High Technology Deve Co ltd
Current assignee: Hebei Bio High Technology Deve Co ltd
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2020-08-04

Abstract

The invention relates to the field of immunology, and discloses a therapeutic vaccine with individualized and specific tumor and a preparation method thereof.

Description

Individual specific therapeutic vaccine for tumor and preparation method thereof

Technical Field

The invention relates to the field of immunology, in particular to a therapeutic vaccine with individualized and specific tumor and a preparation method thereof.

Background

Malignant tumors are one of the major diseases that seriously affect human health and threaten human life. It is very important for early diagnosis and timely treatment. In the last 20 years, the development of medical diagnosis technology is very rapid, new diagnosis methods are emerging continuously, not only tumors as small as 0.5-1.0 cm can be found, but also the range of the tumors can be judged correctly, and nevertheless, doctors can only make clinical diagnosis on middle and late stage cancers.

Current diagnosis of tumors relies primarily on physical examination, imaging, pathology, and related laboratory tests. The physical examination is an important part of tumor diagnosis, and the local examination of key organs is carried out by combining the medical history on the basis of comprehensive and systematic examination. With the development of medical diagnostic techniques and the updating of diagnostic instruments, various imaging examinations also play an important role in the diagnosis of tumors. Including X-ray fluoroscopy, radiography, tomography, ultrasonography, radionuclide scanning, and selective angiography, among others, can provide a definitive localized diagnosis of tumors. Laboratory tests have an important diagnostic aid for tumors, including enzymatic and immunological tests. The immunological examination is based on that the metabolism and chemical composition of cancer cells are different from those of normal cells, and new antigen substances can appear, such as Alpha Fetoprotein (AFP) appearing in the serum of a primary liver cancer patient, serum carcinoembryonic antigen (CEA) of colon cancer, gastric juice sulfur glycoprotein (FSA) of gastric cancer, gastric cancer related antigen (GCAA), alpha 2 glycoprotein (alpha 2GP) and the like.

Surgery, radiotherapy and chemotherapy are conventional treatment means for tumors, but the problems of metastasis and recurrence of the tumors are difficult to break through. For the operation, the local treatment is thorough, but the wound is large, the operation is not effective on tiny focuses and metastatic focuses, the postoperative recurrence or metastasis rate is high, and about 70 percent of tumor patients lose the operation chance when finding the tumor; more than 60% of patients will relapse within 2-5 years after surgery. For radiotherapy and chemotherapy, postoperative radiotherapy and chemotherapy can reduce the recurrence rate of patients, but the clinical application of radiotherapy and chemotherapy is limited because toxic and side effects cannot be tolerated; excessive radiotherapy and chemotherapy damages and destroys the immune system of the patient, and accelerates the death of the tumor patient.

Emerging tumor treatment regimens:

1. targeted drug therapy under gene sequencing guidance

Tumor production results from the immortalization of cells due to mutations in the genes. The gene detection can find the mutated gene, and then the mutated gene is used for selecting the targeted drug for the patient. However, the latest research finds that the targeted drug administration efficiency under the guidance of gene sequencing is too low, and the proportion of patients effectively treated is only 1.5%, which becomes the pain point of targeted drug treatment; the difficulty of targeted drug therapy under the guidance of gene sequencing is reflected by tumor heterogeneity. There is strong tumor heterogeneity, whether between tumor primary foci, metastases, or between tumor-internal subclones, or between metastasis-internal subclones. The gene detection only aims at EGFR, KRAS, ERCC1, RRM1, HER2 and the like to carry out mutation site detection, has low positive detection rate, can select limited antibody medicines, mostly aims at single target spot targeted medicines, is easy to generate drug resistance, can not realize multi-target spot long-acting strike, and is also the main reason of unobvious targeted medicine treatment effect and relapse after treatment.

2. Cellular immunotherapy

The tumor cell immunotherapy is a novel anticancer therapy method, and adopts biotechnology and biological preparation to carry out sorting, induction, culture and amplification on immune cells collected from a patient in vitro and then return the immune cells to the patient, so as to directly act on the tumor cells or achieve the purpose of treating tumors by exciting and enhancing the autoimmune function of the body. However, although tumor cell immunotherapy has been recognized as the fourth therapy following three major traditional tumor treatments, it still presents a non-negligible problem:

(1) technology laggard behind

Currently, 80% of hospitals in China develop first-generation and second-generation nonspecific autoimmune cell treatment technologies, such as NK and CIK cells; and less specific cellular immunotherapy techniques are applied.

(2) Few tumor-specific antigens

The most important problem of the antigens is that no specific screening is carried out, namely no specific screening is carried out on a normal tissue bank and a tumor tissue bank, most of the antigens have cross reaction and poor specificity, the DC cells are difficult to efficiently amplify and mature in vitro, and the specific CT L is difficult to induce.

(3) The preparation period is long

The DC vaccine of tumor neoantigen is prepared through sequencing the whole gene in individual tumor cell, finding out mutation site, synthesizing corresponding antigen, culturing DC cell and re-transfusion. This treatment protocol has high specificity, but it takes a long time for gene sequencing, protein synthesis by gene translation, approximately 1 to 2 months from the start of detection to culture of qualified DC cells, and a long waiting time for a patient in clinical use.

With the continuous and intensive research of molecular biology and immunology, researchers have recognized that the detection and treatment of tumors have risen to the epitope peptide level, and safe and effective multi-target, high-specificity, individualized tumor targeted therapy techniques are becoming the main direction of tumor treatment.

Disclosure of Invention

Because the specificity of the traditional tumor treatment mode is poor, the gene detection can be used for tumor gene screening, but the application range is limited, and the targeted drug is easy to have drug resistance; the present invention provides a therapeutic vaccine and its use in the detection of CTCs in a current situation where the type of tumor cannot be determined.

The preparation method of the therapeutic vaccine provided by the invention comprises the following steps:

step 1: hydrolyzing blood serum of a tumor patient to obtain a peptide segment, and acquiring an amino acid sequence of the peptide segment by ultra-high performance liquid chromatography and time-of-flight mass spectrometry;

step 2: comparing the amino acid sequence with a peptide library, and selecting a peptide segment which is not expressed with negative tissues in the peptide library and is only expressed by the specific tumor tissue as a molecular target;

and step 3: co-culturing the molecular target and dendritic cells separated from serum of a tumor patient to load the molecular target, and preparing the therapeutic vaccine.

The killing of the immune system to the tumor is mainly completed by cell immunity, antigen presenting cells represented by DC cells acquire and process tumor antigens, the presenting to T lymphocytes is completed, the T cells receiving the antigen information are activated, proliferated and differentiated into a large number of specific cytotoxic T cells (CT L) to play a tumor killing effect, most of CT L is apoptotic after the antigen is cleared to maintain a self-stable basic state, a small number of T cells are differentiated into memory cells, and the quick immune response effect is played when antigen stimulation is carried out again.

In the process, the immune system plays a key role in the recognition and presentation of tumor specific antigens, and the antigens capable of being recognized and presented are called molecular targets and are polypeptides consisting of 6-15 amino acids. The molecular target is finally presented on the cell surface for recognition by CD8+ T cells, and the tumor killing is carried out.

The invention comprises two parts of target screening and vaccine preparation, detects a positive molecular target in a detected body, namely a tumor specific antigen, through peripheral blood, cultures immune cells of the detected body in vitro to prepare a therapeutic vaccine, and kills tumor cells in the body in a targeted manner after reinfusion.

Preferably, the molecular target is a polypeptide of 6-15 amino acids; more preferably, the molecular target is one or more.

Specifically, the negative tissues in step 2 are normal tissues and different tumor tissues.

More preferably, the method also comprises the following steps that after a part of dendritic cells are loaded with the molecular target, the dendritic cells are cultured with CT L, and MCT L loaded with the molecular target is separated.

More preferably, the method further comprises a detection step, and the load is detected to have a positive rate of more than 90%.

The invention also provides a therapeutic vaccine prepared according to the preparation method.

In particular, the invention discloses the application of the therapeutic vaccine, namely an individual-specific tumor treatment method, which comprises the following steps:

step 1, hydrolyzing blood serum of a tumor patient to obtain a peptide fragment, and obtaining a sequence of the peptide fragment through ultra-high performance liquid chromatography and time-of-flight mass spectrometry (UP L C-QTOF);

step 2: comparing the peptide segment sequence with a peptide library, and selecting a peptide segment which is not expressed with negative tissues in the peptide library and is only expressed by the specific tumor tissue as a molecular target;

and step 3: co-culturing the molecular target and dendritic cells separated from serum of a tumor patient, and loading the molecular target to prepare a therapeutic vaccine for transfusion back into the patient.

Preferably, the molecular target is a polypeptide of 6-15 amino acids. More preferably, the molecular target is one or more.

Preferably, the negative tissue in step 2 is a normal tissue or a tumor tissue of a different type.

More preferably, the method also comprises the following steps that after a part of dendritic cells are loaded with the molecular target, the dendritic cells are cultured with CT L, and MCT L loaded with the molecular target is separated and is infused back into the patient.

Compared with the most advanced neoantigen DC vaccine at present, the therapeutic vaccine has the obvious advantages of simple operation, short treatment period and outstanding treatment effect, the neoantigen DC vaccine needs to be subjected to the processes of neoantigen design and synthesis and DC cell culture, wherein the process of neoantigen design and synthesis comprises the steps of obtaining tumor DNA, tumor cell whole exon sequencing, analyzing tumor cell somatic mutation, calculating mutant neoantigen combined with H L A and synthesizing neoantigen, the whole neoantigen treatment process approximately needs 1-2 months, and cannot provide timely diagnosis and treatment measures for tumor patients, and MCT L shortens the diagnosis and treatment time to 20 days through the detection and synthesis of targets, greatly shortens the waiting time of the tumor patients and provides more opportunities for clinical diagnosis and treatment of the tumor patients.

The DC-CIK stimulates DC cells by utilizing tumor-related antigens such as tumor cell lysate, tissue homogenate and the like, the related antigens do not have cross reaction with normal tissues, inflammatory tissues and the like after being screened by a tissue bank, and the antigens are holoproteins and directly influence the presenting efficiency of the DC.MCT L applies small molecular polypeptide antigens screened by a target peptide bank to accurately position the tumor cells, and meanwhile, the small molecular polypeptides meet the presenting requirement of the DC cells on the antigens, thereby greatly improving the presenting efficiency and strengthening the killing effect of immune cells on the tumor cells.

In the specific implementation mode of the invention, DC activated by a melanoma molecular target is detected by a target monoclonal antibody, the load positive rate is more than 90%, and the activated DC highly expresses CD11C, CD86 and H L A-DR molecules and presents T lymphocytes to become CT L cells for specifically identifying the melanoma cells.

The therapeutic vaccine provided by the invention is co-cultured with autoimmune cells of a detected person, can be used for accurately positioning and directly killing tumor cells at multiple targets, and can be used for enhancing the immune response of an organism by acting on an immune system, so that the life cycle of a patient is finally prolonged, and the life quality is improved.

Defining:

therapeutic vaccines: in organisms infected with pathogenic microorganisms or suffering from certain diseases, natural, artificially synthesized or expressed by gene recombination technology for treating or preventing disease progression is achieved by inducing specific immune responses.

MCT L Multi-target cytotoxic T cells

DC-SCT (specific Cluster Target of Dendritic cell): specific dendritic cell cluster target

Dc (dendritic cell): dendritic cell

CIK (Cytokine-Induced Killer): cytokine-induced killer cells

Pbmc (peripheral blood mononary cell): peripheral blood mononuclear cells

Detailed Description

The invention discloses a therapeutic vaccine with individualized and specific tumor and a preparation method thereof, and a person skilled in the art can use the content of the vaccine to realize the vaccine by properly improving process parameters. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

Example 1: molecular target detection, analysis and synthesis

1. Collecting samples: collecting 5ml of blood sample to be tested according to clinical blood collection requirement, separating serum, 2-8 deg.C

And (4) transporting, and ensuring that the laboratory is reached within 72 hours.

2. Virus detection and sample retention:

3. enrichment: adding serum to be detected into a phosphoric acid solution and an acetonitrile solution, wherein the volume ratio of the serum to the phosphoric acid to the acetonitrile is 1:1-2: 4-6; mixing, centrifuging, and collecting supernatant;

4. and (3) purification: adding acetonitrile solution into the supernatant, uniformly mixing, wherein the volume ratio of acetonitrile to the supernatant is 0.3-1:2, centrifuging, taking the supernatant, concentrating to 1-50 mu l, and adding water for dilution;

5. and (3) collecting, namely filtering by using a hydrophobic solid phase extraction column Oasis Prime H L B, washing the solid phase extraction column by using methanol, eluting by using a mixed solution of trifluoroacetic acid and acetonitrile in a volume ratio of 0.1: 40-0.1: 70, and collecting eluent.

After the treatment of the steps, the serum to be detected is treated by phosphoric acid and acetonitrile solution to remove phospholipid, fat and high-peak protein, then small molecular interference impurities are removed by a solid phase extraction column, and finally the eluent is obtained by elution of trifluoroacetic acid and acetonitrile solution.

Sending the eluent (detection sample) into a mass spectrum center for liquid phase mass spectrum detection: after a sample enters a high performance liquid chromatography, different substances sequentially enter an ion source of a mass spectrum according to time sequence through effective separation of a chromatographic column, various charged molecules formed by ionizing and fragmenting substances to be detected entering the ion source sequentially reach a mass spectrum detector TOF according to different mass-to-charge ratios, signals of the detector are analyzed by software to obtain specific data such as the molecular weight of each molecule, and the specific data are compared with a peptide library (composed of disclosed peptides related to tumors), so that a detection result is obtained.

6. And according to the detection result, synthesizing and purifying the target by using a polypeptide synthesis platform to obtain the molecular target.

Example 2: tumor specific dendritic cell target in-vitro amplification and activation method

Blood sample collection: collecting 80-120 ml of peripheral blood of a patient according to the clinical blood collection requirement, transporting at 2-8 ℃, and ensuring that the peripheral blood is delivered to a laboratory within 6 hours.

PBMC separation: mononuclear Cells (PBMC) in blood were separated by density gradient centrifugation using lymphocyte lysates.

Culture of DC cells and MCT L cells were adjusted to approximately 1 × 10 using culture medium based on PBMC counts⁷Inoculating each cell per ml, and incubating in a carbon dioxide incubator at 37 ℃; 5% CO₂(ii) a Incubate for 30 min.

And (4) performing DC culture on the incubated adherent cells, namely taking out the culture bottle, slightly shaking to float the suspended cells settled at the bottom, and transferring the suspended cells to a centrifugal tube. Washing adherent wall with normal saline repeatedly for several times until no suspended cells exist, adding 20ml DC cell culture solution containing target (2ml target filtered in 40ml culture solution), culturing in carbon dioxide incubator at 37 deg.C; 5% CO₂。

Adding the collected suspension cells (CT L) into physiological saline to 50ml for re-suspending cells, centrifuging and discarding supernatant, re-suspending the cells with 50ml culture solution and inoculating the cells into a culture bottle, adding 1.8ml of interferon gamma and 2.5ml of self-inactivated plasma, placing the suspension cells in a carbon dioxide incubator at 37 ℃, supplementing the suspension cells with interleukin 2 and interleukin 1 α mixed solution after 5% CO 2.1-2 days, and supplementing the suspension cells with the culture solution containing I L-2 to 100ml after 1.8 ml.2-3 days.

Supplementing DC cells with the culture solution of DC cells containing target, collecting mature DC cells after 5-7 days, and returning half of DC cells to patient, wherein the total number of DC cells is 3 × 10⁷(ii) a The activity rate was 95%.

Adding half of DC cells into suspension cells for culture, sampling and detecting bacteria, fungi and endotoxin, supplementing a culture solution to 200ml (containing I L-2) for the suspension cells, transferring MCT L cells into a culture bag, supplementing the culture solution (containing I L-2), periodically sampling and detecting the bacteria, the fungi and the endotoxin, harvesting all cell suspensions, and 100ml of normal saline(1 bag) resuspending the cells, preserving the sample, sealing, and returning to the ward after sealing, MCT L cell count, wherein the total number of MCT L cells is 1 × 10⁹(ii) a The activity rate was 95%.

Example 3: in-vitro amplification activation method for tumor specific Dendritic Cell (DC) target

1. Collecting a specimen:

the hemogram of melanoma patients is within the normal range (WBC: 4-10 × 10)⁹L YM% of 20-40%, the fluctuation is not more than 5%, the circulation amount of mechanical peripheral blood collection is 1000-4000ml, and the number of mononuclear cells is more than 1 × 10⁹The blood drawing mode is 50-100ml, the number of mononuclear cells is more than 1 × 10⁶。

2. Isolation of mononuclear cells

Transferring the PBMC cell suspension sample, pouring into a centrifuge tube for centrifugal separation, balancing the centrifuge tube, 1310g, centrifuging for 10min, and performing RT. The plasma layer was aspirated and discarded. Diluting the cell precipitate with normal saline at a ratio of 1:1, and blowing, beating and mixing. 30ml of the diluted cell suspension was slowly added to a 50ml sterile centrifuge tube containing 15ml of the human lymphocyte separation medium at room temperature. After balancing, the mixture is centrifuged at 750g for 20min and at RT (the centrifugal speed should be slowly increased and decreased).

Extracting mononuclear cells: after the centrifugation is finished, obvious layering appears in the centrifugal tube, which is from bottom to top: red blood cell layer, granulocyte layer, Ficoll layer, mononuclear cell layer and plasma physiological saline layer. The plasma layer was aspirated and discarded until 5 millimeters (mm) from the buffy coat layer. Carefully transferring the leucoderma cells into a 50ml sterile centrifuge tube, supplementing physiological saline with the same volume, and uniformly mixing; washing and centrifuging.

Cell counting: sucking 0.2ml-0.5ml of cell suspension, adding into an EP tube, mixing uniformly, and counting in a cell counter.

And (4) carrying out sterile detection and sample reservation: and washing with physiological saline again, and taking at least 5ml of washed supernatant for sample retention for sterility test.

4. Seeding of cells based on the count, adjusting the cells to 1 × 10 with GT-T551⁷Inoculating each cell per ml, and placing in a carbon dioxide incubator at 37 ℃; 5% CO₂Incubate for 30 minutes.

5. Cell culture

And (4) culturing the incubated adherent cells by using DC, and collecting suspension cells for CIK culture.

And (3) DC cell culture:

and (3) culturing the incubated adherent cells by using DC:

30ml (T175 flasks) of DC cell culture solution GT-T551 containing the specific target (prepared as described in example 1) was added to each flask, and the mixture was further cultured in a carbon dioxide incubator at 37 ℃; saturation humidity; 5% CO₂。

Culturing adherent cells for 5-6 days, changing the culture solution with DC cell culture solution containing target (sucking out 10ml of culture solution and then supplementing 10ml of new DC culture solution), placing in carbon dioxide incubator for continuous culture at 37 deg.C; 5% CO₂. All DC cells were collected. Transferring the culture solution in the culture bottle into a 50ml centrifuge tube, adding 4 ℃ pre-cooled physiological saline into the culture bottle, repeatedly washing, transferring into the 50ml centrifuge tube, and repeating the steps until all cells in the culture bottle are collected into the centrifuge tube. The cell suspension from the previous step was centrifuged at 1200rpm for 10 min. The cells were resuspended in physiological saline and sampled for counting. And centrifuging the cell suspension at 1200rpm for 10min, and then re-suspending the cell suspension by using physiological saline or cell culture solution to obtain the mature DC cell suspension loaded with the antigen.

The collected DC cells are divided into 2 parts, namely part 1 is used for culturing MCT L together with CIK cells, and the other part is used for returning DC tumor vaccine (5 × 10)⁷)。

6. Preparation of specific targets:

melanoma specific targets SAKYGVRKF 150-300 μ g were prepared as described in example 1, with greater than ten of each tumor target dissolved in 200ml of GT-T551 medium.

7. Quality control standard:

7.1 amplification factor of DC cells cultured in vitro.

	100ml	2000ml	4000ml
				1d	﹥1.2*10⁶	﹥1.5*10⁸	﹥2.6*10⁸
3d	﹥2.6*10⁶	﹥3.1*10⁸	﹥5.2*10⁸
				5d	﹥3.5*10⁶	﹥4.9*10⁸	﹥7.1*10⁸
7d	﹥6.9*10⁶	﹥6.0*10⁸	﹥9.2*10⁸
				9d	﹥8.6*10⁶	﹥8.1*10⁸	﹥1.1*10⁹

7.2 Positive Rate of DC cell detection by target monoclonal antibody

The positive rate of the activated DC is more than 90 percent through the detection of the target monoclonal antibody.

Example 4: cell positive rate of DC cell marker antibody detection

Melanoma patient peripheral blood DC cell surface indirect immunofluorescence positive detection step

1. Melanoma patient peripheral blood DC cells (1X 10) cultured in vitro for 5 days using melanoma DC target (SAKYGVRKF) culture solution⁷Ml) 200. mu.l, divided into two tubes, one tube for each experimental control (100. mu.l/tube), washed 1 time with PBA centrifugation (PBA: adding 1-2% bovine serum albumin into PBS, and adding 0, 1% sodium azide);

2. 20 ul of each of FITC-CD86, PE-H L A-DR and APC-CD11c was added to the experimental tube, 20 ul of each of FITC-mouse IgG1, PE-mouse IgG1 and APC-mouse IgG1 was added to the control tube, and the tubes were incubated for 30min in the absence of light, washed with PBS and then detected by flow cytometry.

3. The results were analyzed as follows:

experiment tube:

HLA-DR⁺cell: 99.74 percent

CD11c⁺Cell: 98.83 percent

CD86⁺Cell: 99.69 percent

HLA-DR⁺CD11c⁺Cell: 97.75 percent

HLA-DR⁺CD86⁺Cell: 99.34 percent

Control tubes were negative.

The above results demonstrate that co-culture of patient DC cells with melanoma DC targets in vitro can activate DC cells by attaching cell membranes to the target in association with MHC in DC cells, and that activated DC cells highly express CD11C, CD86, and H L a-DR molecules and present T lymphocytes, becoming CT L cells specifically recognizing melanoma cells.

Example 5: clinical therapeutic effect

1000 tumor patients are treated by the tumor treatment method of the invention, wherein the tumor treatment method comprises three groups of cases of chemotherapy, chemotherapy and MCT L combined and MCT L applied independently, and the three groups are treated by 3-5 treatment courses according to a clinical scheme.

The treatment results were evaluated with the following evaluation criteria:

complete Remission (CR): all target lesions disappeared and the short diameter of all pathological lymph nodes (including target and non-target nodes) had to be reduced to < 10 mm.

Partial Remission (PR): the sum of the target lesion diameters is reduced by at least 30% from baseline levels.

Disease Stability (SD): the target lesion was decreased to a degree that did not reach PR and increased to a degree that did not reach PD levels, between which the minimum of the sum of the diameters was considered for the study.

Disease Progression (PD): the diameter and relative increase is at least 20% with respect to the minimum of the sum of all measured target lesion diameters throughout the experimental study (baseline values are referenced if the baseline measurement is minimal); in addition to this, it must be satisfied that the absolute value of the sum of the diameters increases by at least 5mm (the appearance of one or more new lesions is also considered as disease progression).

The objective effective rate is CR rate + PR rate.

See table below.

The results show that the CR and PR of the MCT L and the MCT L for treating the intestinal cancer and the adenocarcinoma by combining with the chemotherapeutic medicament are better than the CR and PR of the chemotherapeutic medicament by combining with the chemotherapeutic medicament, and the difference has statistical significance.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method of preparing a therapeutic vaccine, comprising the steps of:

2. The method of claim 1, wherein the molecular target is a polypeptide of 6-15 amino acids.

3. The method of claim 1 or 2, wherein the molecular targets are one or more.

4. The method according to claim 1, 2 or 3, wherein the negative tissue is a normal tissue or a tumor tissue of a different type.

5. The method according to any one of claims 1 to 4, further comprising the step of isolating MCT L loaded with the molecular target by co-culturing a portion of the loaded molecular target with CT L after loading the molecular target on the dendritic cells in step 3.

6. The method according to claims 1 to 4, wherein the load has a positive rate of 90% or more.

7. A therapeutic vaccine prepared according to the method of any one of claims 1 to 6.

8. A method of individual-specific tumor therapy comprising the steps of:

9. The method of claim 8, wherein the negative tissue in step 2 is normal tissue or different tumor tissue.

10. The method of claim 9, wherein the molecular target is a polypeptide of 6-15 amino acids.

11. The method of any one of claims 8-10, wherein the molecular targets are one or more.

12. The method of any one of claims 8-11, further comprising the step of isolating MCT L loaded with the molecular target by co-culturing a portion of the dendritic cells with CT L after loading the molecular target.