CN108379569B - DC vaccine for efficiently loading tumor antigen and method for inducing and amplifying tumor antigen specific CTL (cytotoxic T lymphocyte) - Google Patents

Abstract

The invention provides a DC vaccine for efficiently loading tumor antigens and a method for inducing and amplifying tumor antigen specific CTL (cytotoxic T lymphocyte) by using the same. The DC vaccine is prepared by the following method: (1) preparing a tumor antigen; (2) collecting, separating and efficiently culturing and amplifying the DC cells; (3) sensitizing and activating the DC cells by using tumor antigens and an activator; and (4) blocking the PD-L1 molecules on the surface of the DC cells. The invention also provides a method for in vitro induction amplification of CTL by the DC vaccine through sequential blood drawing, and closure of PD-1 molecules on the surface of the in vitro induction amplification CTL. The invention solves the problems of low immunogenicity, incomplete antigen target, difficult obtainment of tumor antigens of patients and insufficient amplification quantity of DC and CTL in vitro of the existing tumor antigens. The DC vaccine can effectively induce tumor antigen specific CTL in vitro and in vivo, can efficiently generate specific killing to tumors, has no obvious toxic or side effect, and has wide clinical application prospect.

Description

DC vaccine for efficiently loading tumor antigen and method for inducing and amplifying tumor antigen specific CTL (cytotoxic T lymphocyte)

Technical Field

The invention relates to the field of medical and biotechnology, in particular to a method for efficiently preparing autologous or other tumor antigen-bearing DC vaccine and tumor antigen-specific CTL induced amplification by the same by using peripheral blood mononuclear cells with low initial amount.

Background

Tumors are a necessary product of human evolution and are also a serious disease threatening human health and life. At present, the incidence of tumors in China is increased year by year, and the traditional operations, chemotherapy, radiotherapy and the like have great limitations. The operation can only remove the tumor mass seen by naked eyes, can not completely remove tumor cells, and does not change the unbalanced in-vivo environment of the tumor patient; chemotherapy and radiotherapy can kill part of tumor cells, but a part of solid tumors are not sensitive to radiotherapy and chemotherapy, and a plurality of side effects of the solid tumors bring a series of negative effects on the immune system and the life quality of a patient and cannot prevent the recurrence and metastasis of the tumors; the existing molecular targeted drugs have limited application range and are very easy to generate drug resistance. Therefore, there is a great need to develop new therapeutic approaches and means.

Numerous studies have shown that restoring/reconstituting a patient's own anti-tumor immune function is one of the indispensable strategies for tumor therapy. In recent years, tumor immunotherapy has become the fourth leading means of tumor therapy, and tumor-specific immunotherapy is an important factor, and several factors affecting tumor-specific immunotherapy include: 1) Capable of correctly inducing and expanding a sufficient number of Dendritic Cells (DCs) having a potent antigen-presenting function and tumor-specific cytotoxic T Cells (CTLs); 2) An optimal tumor antigen; 3) Infiltrating the chemotactic tumor specific cytotoxic T cells into tumor tissues; 4) Cutting off the immunosuppressive mechanisms of the tumor microenvironment.

The source of the DC may vary, but it is most convenient to isolate mononuclear cells from peripheral blood. Because of the difficulty in DC and CTL expansion, measures are usually taken on the initial number of cells in culture to meet the number of cells required for therapy, including: 1) (ii) bone marrow mobilization by injection of GM-CSF 2-3 days prior to monocyte collection to increase the rate and absolute count of peripheral blood monocytes; 2) A large amount of mononuclear cells are collected by circulating several thousand ml of peripheral blood using a hemocytometer. Such treatment actually carries out powerful external intervention on bone marrow and peripheral blood of the patient, and further causes great interference to the in-vivo environment such as disordered immune system after the operation, radiotherapy and chemotherapy of the tumor patient. Therefore, there is a need for a DC vaccine and its induced expansion effector cells CTL that have no significant effect on the body, start with a small initial amount of cell culture, and satisfy the therapeutic requirements in terms of cell number and phenotype after culture expansion and antigen-sensitized activation.

In addition to cell number, optimal tumor antigens are critical to ensure tumor-specific immunotherapy accuracy, and three aspects need to be met: 1) The specificity and the stronger immunogenicity of tumor antigens; 2) Multiple target points; 3) Individuation is carried out. To date, several Tumor Associated Antigens (TAAs) have been found that are low expressed in normal tissues and high expressed in tumor tissues; a few tumor-specific antigens (TSA) that are not expressed in normal tissues (except testis and placenta) and are expressed only in tumor tissues; tumor specific polypeptides screened in large scale by a tissue specimen bank, a tissue chip and a monoclonal antibody; neoantigens (individualized Neo-Antigen) obtained based on gene sequencing and bioinformatic analysis; however, for most tumors, well-recognized tumor-specific antigens with broad representatives are not yet available. The tumor antigens in the existing tumor vaccines are all thousands of years: the antigen coverage of the whole tumor cell tumor vaccine and the tumor lysate tumor vaccine is wide, the immunogenicity is strong, but the antigen contains more impurities and the specificity is poor; the tumor-related polypeptide has strong specificity but weaker immunogenicity; anti-idiotype antibody vaccines tend to be single-targeted; genetically modified tumor vaccines attempt to increase the immunogenicity of the tumor or the activity of antigen presenting cells, but are often single-targeted, and transgenic procedures are themselves a type of injury to antigen presenting cells. The vesicle antigen (exosome) discharged by the tumor cell can meet the requirements of multiple target spots and strong immunogenicity of the particulate antigen, but only the antigen in the cell culture supernatant is collected in the preparation process, and a large amount of antigen on the surface of the tumor cell is lost. Given the multigenic changes of tumor cells, the high heterogeneity of tumor tissues, and the high degree of personalization of tumor patients, tumor-specific immunotherapy remains a significant challenge. In addition, as a biological agent, tumor vaccines are produced with consideration given to the efficiency of the production process of the obtained tumor antigen. The development of a tumor antigen preparation method with wide target coverage (multiple targets), high specificity and strong immunogenicity is urgently needed to meet the requirements of individual novel DC vaccine antigen load and the strong induction and amplification of multi-target tumor specific cytotoxic T cells in vitro and in vivo.

Tumor patients usually have different degrees of immune tolerance, and the key of tumor immunotherapy is how to break the immune tolerance and repair or stimulate the strong antigen presenting capacity of Dendritic Cells (DC) so as to fully induce and activate tumor-specific cytotoxic T Cells (CTL). Recently, the role of immunodetection points in tumor therapy has been receiving increasing attention, and PD-1 antibodies and PD-L1 antibodies are being used in tumor therapy. However, it is generally contemplated that blocking of PD-1 molecules on the surface of T cells with PD-1 antibodies and blocking of PD-L1 expressed by tumor cells with PD-L1 antibodies activates or enhances T cell function. In fact, not only tumor cells can express PD-L1, but also DCs can express PD-L1. Especially, the activated mature DC can highly express PD-L1 after loading antigen, which is also a 'brake' mechanism of DC induced specific immunity, and the 'brake' is loosened and effectively applied to the treatment of clinical diseases, the PD-L1 receptor on the surface of the DC cell is sealed, the antigen presentation of the DC and the CTL activation capability are realized to the maximum extent, the PD-1 molecule on the surface of the CTL is sealed in a combined manner, the tumor specific cytotoxicity level of the CTL is improved, and the method is a valuable and explored way for the immunotherapy of breaking DC tolerance and tumor specificity.

Disclosure of Invention

The invention aims to provide a DC vaccine for efficiently loading tumor antigens and a method for inducing and amplifying tumor antigen specific CTL (cytotoxic T lymphocyte), wherein the DC vaccine utilizes low initial amount of peripheral blood mononuclear cells to efficiently prepare loaded autologous or other source tumor antigens, effectively induces and amplifies multi-target tumor specific cytotoxic T Cells (CTL) in vitro and in vivo in a large scale, and is used for clinical treatment of tumor patients.

In order to achieve the purpose of the invention, the invention firstly provides a preparation method of a DC vaccine loaded with tumor antigens with high efficiency, which comprises the following steps:

(1) Preparing a tumor antigen;

(2) Collecting peripheral blood to separate mononuclear cells, and directionally inducing and amplifying DC cells by adherent low initial cell;

(3) Sensitizing and activating DC cells by using tumor antigens and an activator;

(4) And (3) blocking the PD-L1 molecules on the surface of the DC cell, and harvesting the DC vaccine.

In the above preparation method, the tumor antigen of step (1) and the peripheral blood of step (2) are derived from the same individual or different individuals, depending on the feasibility of clinical obtaining autologous tumor tissue or cells and the type of tumor antigen.

The tumor antigen type in the step (1) is selected from one or more of tumor tissue lysate, tumor cell surface protein antigen, tumor cell membrane protein antigen, tumor cell exosome, other tumor-related or specific protein, tumor specific polypeptide, and highly personalized new antigen prediction and preparation based on gene sequencing and bioinformatics analysis.

The tumor antigens may be used alone or in combination. In a preferred embodiment of the present invention, the tumor antigen is a mixture of tumor cell surface protein antigen, tumor cell membrane protein antigen and tumor cell exosome at a mass ratio of 1: 2.

The tumor antigen in the step (1) is obtained by the following method of sequentially carrying out chemical treatment and physical heating: adding arsenic trioxide into tumor cells, culturing, and culturing at 40-46 deg.C to induce expression of heat shock protein when the tumor cells shrink and become round, adherence rate decreases, and cytoplasm bleeds.

Preferably, arsenic trioxide is added to tumor cells isolated or cultured from tumor tissue of a patient to a final concentration of 1-15 umol/L37 ℃,5% CO ₂ Culturing for 4-24 hr until tumor cells shrink and become round, anchorage rate decreases, and cytoplasm bleeds, 43 deg.C, 5% CO ₂ Culturing for 1-12 hours.

In the preparation method of the DC vaccine provided by the invention, the method for collecting and separating and culturing the DC cells in the step (2) is as follows:

1) Separation of peripheral blood mononuclear cells: collecting anticoagulated blood from peripheral vein, adding into lymphocyte separation solution, centrifuging to separate mononuclear cells, adding into serum-free DC cell culture solution, and resuspending cells to cell concentration of 3 × 10 ⁶ -1.5×10 ⁷ Per ml;

2) Separation, directional induction and expansion of DC cells: culturing the mononuclear cells in an adherent way for 2 to 6 hours; sucking out non-adherent cells, wherein the obtained adherent cells are DC precursor cells; and adding the rh-IL-4 with the final concentration of 1000IU and th-GM-CSF with the final concentration of 1000IU into the DC precursor cells, directionally inducing and amplifying the DC precursor cells for 7-9 days, and using the obtained immature DC cells for sensitization and activation.

One key point in the step (2) of the method for preparing the DC vaccine is that 40-50ml of peripheral anticoagulation blood is only required to be collected for preparing the DC vaccine of each person, and a large amount of DC is induced and amplified by using a low initial number of mononuclear cells.

In step (2) of one embodiment of the present invention, the method for collecting and isolating cultured DC cells is as follows:

1) Separation of peripheral blood mononuclear cells: extracting peripheral anticoagulation 40-50ml (preferably 45m 1), obtaining mononuclear cells by conventional density gradient centrifugation, washing, adding serum-free DC cell culture solution for resuspending cells, counting cells, and adjusting cell concentration to 3 × 10 ⁶ -1.5×10 ⁷ One per ml.

The serum-free DC cell culture medium is a special culture medium (BYN-PD 701), and the lymphocyte separation solution can be obtained from commercial products in the market, and is preferably obtained from Sigma company products.

2) Separation, directional induction and expansion of DC cells: cell at 37 ℃ 5% CO ₂ In an incubator, carrying out adherent culture for 2-6 hours by using a serum-free DC cell culture medium (BYN-PD 701); sucking out nonadherent cells, and performing amplification culture on cytotoxic T cells (DC-AT) activated by tumor antigen sensitized DCs or killer Cells (CIK) induced by cytokines to obtain adherent cells, namely DC precursor cells; adding DC cell-specific medium, rh-IL-4 at a final concentration of 1000IU, and rh-GM-CSF at a final concentration of 1000IU to DC precursor cells, and removing CO 5% at 37 deg.C ₂ In an incubator, the DC precursor cells are directionally induced and amplified for 7-9 days, and the obtained immature DC cells are ready to be sensitized and activated. According to the growth and amplification of cells, the individual serum-free DC cell special culture medium (BYN-PD 701) containing rh-IL-4 with the final concentration of 1000IU and rh-GM-FCS with the final concentration of 1000IU is supplemented in a proper amount at proper time.

In the preparation method of the DC vaccine for efficiently loading the tumor antigen, the method for sensitizing and activating the DC cells in the step (3) comprises the following steps: adopting antigen from autologous tumor source of peripheral blood donor, or adding mixed tumor antigen of corresponding tumor matched with pathological detection result according to pathological detection result of patient, simultaneously adding activating agent, and incubating to obtain sensitized and activated DC cell; the activator is bacterial product, composite immunoglobulin, monoclonal antibody and small molecular compound.

Preferably, the activator is a polyinosinic injection, an anti-human CD40 monoclonal antibody, TNF-d or a drug (injection) of bacterial product origin on the market. In the embodiment of the invention, the selected mixed tumor antigen contains tumor cell surface antigen, cell membrane antigen and tumor cell exosome. One skilled in the art will appreciate that mixed tumor antigens include, but are not limited to, a mixture of any two or more of the following tumor antigens: tumor tissue lysates, tumor cell surface protein antigens, tumor cell membrane protein antigens, tumor cell exosomes, other tumor-associated or specific proteins, tumor-specific polypeptides, highly personalized neoantigens based on gene sequencing.

Specifically, the method for sensitizing and activating DC cells by using tumor antigens and activators in the step (3) is as follows: the concentration to DC cells was 5X 10 ⁶ -1.5×10 ⁷ 1/10 volume of mixed tumor antigen (the mass ratio of tumor cell surface protein antigen to tumor cell membrane protein antigen to tumor cell exosome is 1: 2) is added into serum-free DC cell culture solution per ml, meanwhile, polyinosinic cell injection with final concentration of 0.2-0.8 mu g/ml and activator with final concentration of 2-10 mu g/ml are added, and the mixture is incubated overnight, so as to obtain mature DC cells.

More preferably, the activator is selected from anti-human CD40 monoclonal antibodies.

In the preparation method of the DC vaccine with high-efficiency loaded tumor antigen, the step (4) is to add anti-human PD-L1 antibody into sensitized and activated DC cells, incubate for 0.5-2 hours at 30-40 ℃ to seal PD-L1 molecules on the surface of the DC cells.

Specifically, in step (4), anti-human PD-L1 antibody was added to mature DC cells at a final concentration of 1-10ug/ml, and the CO was 5% at 37 ℃% ₂ And (3) incubating for 0.5-2 hours under the condition, sealing the PD-L1 antigen molecules on the surface of the DC cell, and harvesting the sealed DC cell, namely the DC vaccine with high-efficiency tumor antigen loading, which can be directly used for active immunotherapy, namely CTL induction and amplification in vivo.

On the other hand, the invention also provides the application of the prepared high-efficiency load tumor antigen DC vaccine in-vitro induction amplification of tumor antigen specific CTL. The use comprises two key steps: (1) Sequentially extracting a small amount of peripheral blood to separate non-adherent mononuclear cells, inducing the non-adherent mononuclear cells in vitro by using a DC vaccine, and culturing and amplifying CTL (cytotoxic T lymphocyte) by using a starting culture medium and an amplification culture medium; (2) In vitro induction of closure of the amplified CTL surface PD-1 molecules. The method comprises the following specific steps:

(1) Tumor antigen sensitized and activated DC in vitro induction amplification tumor specific CTL

The tumor antigen sensitized and activated DC prepared by the method has high expression CD80, CD83, CD86 and HLA-DR on the surface (figure 1A and figure 1B), which shows that the maturity is higher, and can induce tumor specific CTL in vitro, therefore, on the day of harvesting the tumor antigen sensitized DC prepared by first blood drawing, 40-50mL of anticoagulation blood of the same tumor patient is extracted for the second time (namely, sequential blood drawing), the mononuclear cells are separated and cultured in adherence, the nonadherent mononuclear cells are sucked out and mixed with the tumor antigen sensitized and activated DC harvested on the day according to the proportion of 3-7: 1, a CTL serum-free start culture medium (BYN-PT 331) is added into a culture bottle, the temperature is 37 ℃, and the CO content is 5% ₂ Incubating in incubator, adding anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody and rh-IL-2 for continuous culture. Then, according to the growth and amplification conditions of cells, an individual serum-free amplification culture medium (BYN-PT 332) containing rh-IL-2 is supplemented in a proper amount at proper time for CTL amplification culture.

(2) CTL surface PD-1 molecule blocking and CTL harvesting

CTL culture to day 11-14, at the time of peak of logarithmic cell growth, before CTL harvest, 1-10ug/ml (final concentration) of anti-human PD-1 monoclonal antibody was added to block PD-1 molecules on CTL surface, 37 ℃,5% ₂ Incubating in an incubator for 0.5-2 hours. Collecting cell suspension and centrifugally washing for 2 times; a quality control sample is reserved; the obtained CTL is resuspended by 100mL of normal saline, and 0.5% of human serum albumin is added for preparing for intravenous drip of human body; or resuspending with 30mL or 2mL according to clinical requirements, and preparing for injection in thoracic cavity and abdominal cavity of human body, or local injection of part of tumor. Trypan blue staining is carried out, cells are counted, and the cell survival rate is over 95 percent; total cell number 1X 10 ¹⁰ Left and right; the cytophenotypic assay by flow cytometry showed that CD3+ T cells accounted for more than 90% and were dominated by CD8+ cells (cytotoxic T cells) (fig. 2A, 2B).

The DC vaccine of the invention induces tumor-associated or specific CTL obtained from peripheral blood in vitro, and belongs to the protection scope of the invention.

The DC vaccine of the invention provides an integral solution aiming at the problems of the existing tumor antigen preparation aspects of weak immunogenicity, incomplete antigen target, difficult acquisition of tumor antigens of tumor patients and the like, the problem of insufficient in-vitro amplification quantity of DC and CTL, and the problem of the closure of activated immunity detection points in the in-vitro culture process of DC and CTL, can effectively induce tumor antigen specific CTL in vitro and in vivo, can efficiently generate specific killing to tumors, and has no obvious toxic or side effect.

The beneficial effects of the invention are embodied in the following aspects:

(1) In the aspect of preparing tumor antigens, the invention uses arsenic trioxide to target and induce the apoptosis of tumor stem cells, and then treats the tumor cells by a heating method, thereby not only inducing the apoptosis of the tumor cells, but also inducing the expression of heat shock proteins of the tumor cells. The heat shock protein can be present in the endoplasmic reticulum of cells and can also be expressed on the surfaces of the cells, is an important molecular chaperone of tumor antigens, and can increase the immunogenicity of the tumor antigens.

(2) In the aspect of tumor antigen selection, the tumor antigens which can be selected by the DC vaccine provided by the invention not only comprise surface proteins, cell membrane proteins and intracellular proteins of tumor cells, but also comprise vesicles which are externally discharged by the tumor cells in a culture solution after the tumor cells are cultured and amplified. Thus, the whole tumor cell antigen can be obtained from different levels of tumor cells to the greatest extent, the antigen target spots are covered comprehensively, soluble protein, lipid protein and rich heat shock protein are taken into consideration as antigen molecular chaperones and the granularity of vesicle antigens, and the immunogenicity of the mixed antigen is increased; meanwhile, the antigen can be obtained from patient self-excision or biopsy tumor tissue and hydrothorax and ascites tumor cells, so that the source of the self-tumor antigen is expanded, and the requirement of individuation is met.

(3) In the aspect of obtaining the peripheral blood collection amount and the DC cell amount, the invention utilizes the characteristics that after the low initial amount of peripheral blood mononuclear cells are cultured and expanded for 8-10 days and are loaded with tumor antigens, the DC with higher amount and higher maturity can be obtained, the clinical operation is simple, the safety and the reliability are realized, the side reaction is small, the effective rate is high, and the like, so that the pain and the adverse effect on the body caused by the extraction of a large amount of peripheral blood of a tumor patient are relieved on one hand; on the other hand, the preparation cost is reduced, and the sufficient dosage of DC cells for treatment is ensured.

(4) In the aspect of blocking DC cell surface PD-L1 to start a T cell apoptosis program so as to inhibit immunity, the CD40 monoclonal antibody and the PD-L1 monoclonal antibody are used in combination to break DC immune tolerance, the CD40 monoclonal antibody is used to activate DC, and the anti-PD-L1 monoclonal antibody is used to block DC surface PD-L1. Currently, in the aspect of the research on the immunosuppressive mechanism of PD-1/PDL-1 checkpoint, the skilled person mainly focuses on the expression of programmed death molecule (PD-1) after the activation of T cells, and the expression of PD-L1 molecule (ligand of PD-1) by tumor cells in tissues, and once the two are combined, the T cells start apoptosis, which is one of the important immunosuppressive mechanisms of tumor immune escape. However, it is often neglected that the DC, when loaded with tumor antigen and activated, expresses PD-L1 molecules at high levels, and when the DC is contacted with T cells, it may initiate the T cell apoptosis process during the process of completing tumor antigen presentation and T cell activation. The invention adopts the anti-PD-L1 monoclonal antibody to seal the PD-L1 loaded on the surface of the tumor antigen DC, blocks the immunosuppressive mechanism, activates the anti-tumor specific immune reaction of an organism, and lays a foundation for inducing and amplifying tumor specific cytotoxic T cells in vivo and in vitro and continuously playing the role of the T cells.

(5) In the aspects of enhancing the DC vaccine to induce the tumor specific immunoreaction in vivo and inducing and amplifying the tumor specific CTL in vitro, the invention is used for collecting a small amount of peripheral blood of the same tumor patient for the second time to separate the mononuclear cells when the DC vaccine loaded with the tumor antigen is harvested. From the zero moment of culturing non-adherent mononuclear cells, the DC vaccine prepared by the invention is mixed with pure T cells in a mononuclear cell group in vitro in a higher proportion for culture, and antigen presentation and T cell activation are completed in an in vitro superior microenvironment excluding specific immunosuppressive factors of tumor patients. The method can obtain total CTL cell amount of 1 × 10 from 45ml of initial mononuclear cells of peripheral blood ¹⁰ About, higher than the number of DC-CIK amplified by the conventional method (1X 10) ⁹ Left and right); the cell survival rate is more than 95 percent; the cell surface can highly express CD3 and CD8 molecules. Adoptive transfer of these tumor-specific CTLs to patients can directly kill tumor cells, and can be used in combination with DC vaccines to improve therapeutic effects.

Drawings

FIGS. 1A-1B are typical maps of high expression CD80, CD83, CD86 and HLA-DR on the surface of DC cells according to the present invention.

FIGS. 2A-2B are typical maps of the CTL cell surface high-expression CD3 and CD8 molecules of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and all of the biomaterials used are commercially available.

Example 1 preparation of a DC vaccine that is highly loaded with tumor antigens

1. Tumor cell culture

The tumor cells are from tumor tissues or tumor tissue biopsy specimens which are removed by the patient himself through operation, and tumor cell lines which have the same pathological diagnosis characteristics with the patient. The cell line can be a laboratory self-established line, can also be purchased from China national cell collection center or American ATCC, and is cultured in vitro according to the biological characteristics of the tumor cells respectively. Generally, the content of CO is 5% at 37 ℃ using a DMEM medium containing 10-15% fetal bovine serum ₂ Culturing in incubator, and subculturing and amplifying.

2. Inducing apoptosis of cells and expressing heat shock proteins

After the cells have grown to logarithmic phase and 80% of the cells have spread to the bottom of the flask, treating the cells with the drug, adding arsenic trioxide (1-15 umol/L) and continuing at 37 deg.C with 5% CO ₂ Culturing in incubator for 4-24 hr. Microscopic observation of tumor cell shrinkage and rounding, decrease in anchorage rate and cytoplasmic "blebbing", transferring the cells to 43 ℃ for heating, 5% CO ₂ Incubate for 10 hours.

3. Preparation of tumor cell exosomes

After arsenic trioxide and heating sequential induction, tumor cells are subjected to apoptosis, adherent cells are suspended, and more exosomes are discharged. To harvest more antigen, adherent cells can be further separated with a cell scraper, and then the following procedure is continued:

1) Centrifuging at low speed for 10-15 min at 300-600g, and collecting tumor cell precipitate to prepare tumor cell protein antigen (see steps 4 and 5).

2) Collecting supernatant, centrifuging at high speed for 10000g for 30 min, collecting supernatant, further centrifuging at high speed for 100000g for 60 min, and collecting precipitate, i.e. tumor cell exosome.

4. Preparation of tumor cell surface protein antigens using a surface protein extraction Kit, mem-PEP Plus Kit, purchased from Thermo, inc.

5. Tumor Cell membrane Protein antigen was prepared using a membrane Protein extraction Kit, pierce Cell Surface Protein isolation Kit, purchased from Thermo, inc. Tumor cell membrane protein antigens were prepared according to the manual of commercial use.

6. Preparation of tumor antigen loaded DC vaccine

1) Isolation of peripheral blood mononuclear cells

The method for separating peripheral blood mononuclear cells is easily performed by using a method known in the art. The following method is adopted in the embodiment: 45ml of peripheral blood was aseptically collected, lightly added to a lymphocyte separation medium having a specific gravity of 1.077, and mononuclear cells were separated by gradient density centrifugation. And (3) sucking the white cell layer in the middle of the centrifuge tube, transferring the centrifuge tube into a centrifuge tube containing normal saline, washing cells, removing supernatant, adding a serum-free DC cell culture medium (BYN-PD 701), re-suspending the cells, and counting the cells.

2) Directed induction and amplification of DCs

Cells at 37 ℃,5% ₂ Culturing in incubator while adhering to wall for 2-6 hr, sucking out non-adhering cells, adding rh-IL-4, rh-GM-CSF 1000IU (final concentration), 37 deg.C, 5% CO ₂ And in an incubator, directionally inducing and amplifying the DC cells for 7-9 days. According to the growth and amplification of cells, the individual is supplemented with serum-free medium (BYN-PD 701) containing rh-IL-4, rh-GM-CSF 1000IU (final concentration) in a proper amount at proper time.

3) Tumor antigen sensitization and activation of DCs

Culturing DC to 7-9 days, adding 1/10 volume of corresponding mixed tumor antigen (tumor cell surface protein antigen: tumor cell membrane protein) from patient autologous tumor source or according to patient pathological detection resultBai Kangyuan tumor cell exosomes mass ratio 1: 2) together with polyinosinic cell injection (final concentration 0.2-0.8. Mu.g/ml), anti-human CD40 monoclonal antibody (final concentration 2-10. Mu.g/ml), 37 ℃,5% ₂ Incubate overnight.

4) Blocking of PD-L1 molecules on DC surfaces

Culturing and sensitizing DC to 8-10 days, adding anti-human PD-L1 antibody (Junzi organism) 1-10ug/ml (final concentration) before harvesting DC, blocking PD-L1 molecule on DC surface, and removing CO 5% at 37 deg.C ₂ Incubating in an incubator for 0.5-2 hours.

5) Harvesting of DC cells

After the PD-L1 molecules on the surface of the DC are sealed, sucking out a culture solution (containing cells and not discarding), blowing and flushing the remained adherent cells by using ice physiological saline until the cells fall off, collecting all cell suspensions, and centrifugally washing for 2 times; the cells were resuspended in 1ml of physiological saline for therapeutic use. Meanwhile, a quality control sample is reserved, and microorganisms and endotoxin are detected conventionally; the expression of CD80, CD83, CD86 and HLA-DR on the surface of the DC cell is detected by flow cytometry (shown in figures 1A-1B), and the high expression of CD80, CD83, CD86 and HLA-DR on the surface of the DC cell can be seen from figures 1A-1B.

The DC vaccine prepared in this example can separate low initial DC precursor cells in adherent mononuclear cell population from 45ml of peripheral anticoagulation blood, and after culture amplification and antigen sensitization, the total amount of tumor antigen sensitized DC can be finally harvested, wherein the total amount of the tumor antigen sensitized DC is about 1 × 10 ⁸ -2×10 ⁸ Can simultaneously meet the requirements of 1-2 DC vaccine treatments for required dosage and one induction and amplification of tumor specific CTL. The method has the characteristics of simple clinical application and operation, safety, reliability, small side reaction, high efficiency and the like, and provides a preferable DC vaccine and a new way for preparing tumor specific cells for clinical application.

Example 2 preparation of a DC vaccine highly loaded with tumor antigens

Separating tumor cells from pleural fluid and ascites of tumor patients, culturing and amplifying, and preparing tumor antigen from tumor cells and exosomes in tumor cell culture solution in a one-time manner.

1. Collecting hydrothorax or ascites 500-1000ml under aseptic condition, centrifuging at 300g for 8 min, and discarding supernatant.

2. Adding physiological saline to 100ml, resuspending, and making into cell suspension.

3. Double specific gravity density centrifugation: from bottom to top: layer 1 of 100% human lymphocyte isolate (density 1.077 g/ml); layer 2 of 75% lymphocyte separation medium (density 1.077 g/ml); layer 3 cell suspension. Centrifuging: 700g,20 minutes.

4. Cells at interface 1 and interface 2 were collected by aspiration and washed 2 times with physiological saline.

5. And repeating the step 3 and the step 4 once.

6. The cells of interface 2 are tumor cells.

7. If enough tumor cells are obtained, the tumor cells can be directly treated according to example 1, and autologous tumor membrane protein antigens are extracted.

8. Alternatively, the isolated tumor cells may be cultured in vitro and then treated as in example 1 to extract the tumor cell vesicles as well as the autologous tumor cell membrane protein antigen.

After the preparation of the tumor antigen, a tumor antigen-sensitized DC vaccine and CTL can be prepared by the method described in example 1.

Example 3 in vitro Induction of tumor antigen-sensitized activated DCs to amplify tumor-specific CTLs

The DC vaccines prepared in example 1 and example 2 were selected, respectively.

1. On the day of DC vaccine harvest, a second heparin extraction anticoagulation treatment was performed by 40 to 50ml, and freshly isolated nonadherent mononuclear cells were aspirated, mixed with the prepared DC vaccine (the DC vaccine prepared by the method of example 1 or example 2) at a ratio of 5:1, placed in a serum-free CTL initiation medium (BYN-PT 331) containing overnight, and the anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody and rh-IL-2 were added the next day, and 5% CO was added at 37 ℃ according to the procedures of 1) and 2) of step 6 of example 1 ₂ Culturing in an incubator. Then, according to the growth and amplification conditions of cells, an individual serum-free amplification culture medium (BYN-PT 332) containing rh-IL-2 is supplemented in a proper amount at proper time for CTL amplification culture.

2. Harvesting of CTL

CTL culture is carried out until 11-14 days, and the logarithmic growth peak of cellsBefore CTL harvest, 1-10ug/ml (final concentration) of anti-human PD-1 monoclonal antibody was added to block PD-1 molecules on the surface of CTL at 37 ℃ C. And 5% CO ₂ Incubating in an incubator for 0.5-2 hours. Collecting cell suspension and centrifugally washing for 2 times; a quality control sample is reserved; suspending CTL with 100ml of normal saline, adding 0.5% human serum albumin for intravenous drip of human body; or, resuspending with 30ml or 2ml according to clinical requirements, preparing for injection in thoracic cavity or abdominal cavity of human body, or partially injecting for local tumor. Trypan blue staining is carried out, cells are counted, and the cell survival rate is over 95 percent; the total number of cells is generally 1X 10 ¹⁰ Left and right, high up to 2X 10 ¹⁰ (ii) a The CTL cell surface of the tumor patients can highly express CD3 and CD8 molecules (figures 2A-2B).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (1)

1. The application of the DC vaccine of high-efficiency load tumor antigen in the in-vitro preparation of tumor specificity CTL is characterized in that,

the preparation method of the DC vaccine loaded with the tumor antigen efficiently comprises the following steps:

(1) Preparing a tumor antigen;

(2) Preparing DC vaccine of each part requires extracting 40-50ml of anticoagulation blood from peripheral vein and separating mononuclear cells; directionally inducing and amplifying DC cells by adherent low initial quantity cells, wherein the method comprises the following steps: obtaining mononuclear cells, washing, adding serum-free DC cell culture solution to resuspend cells, counting cells, and adjusting the cell concentration to 3 × 10 ⁶ -1.5×10 ⁷ Culturing the cells in an incubator for 2-6 hours in an adherent way, wherein the obtained adherent cells are DC precursor cells, adding a serum-free DC cell culture medium containing rh-IL-4 and rh-GM-FCS into the DC precursor cells, and culturing the DC precursor cells in the incubator for 7-9 days to obtain immature DC cells to be sensitized and activated;

the tumor antigen of step (1) and the peripheral blood of step (2) are from the same individual or different individuals, depending on the feasibility of clinical acquisition of autologous tumor tissue or cells and the type of tumor antigen;

(3) And (3) sensitizing and activating the immature DC cells obtained in the step (2) by using a tumor antigen and an activating agent, wherein the method for sensitizing and activating the DC cells in the step (3) comprises the following steps: adopting an antigen from a peripheral blood donor, or adding a mixed tumor antigen of a corresponding tumor matched with a pathological detection result according to the pathological detection result of a patient, simultaneously adding a polyinosinic injection with the final concentration of 0.2-0.8 ug/ml and an anti-human CD40 monoclonal antibody with the final concentration of 2-10 ug/ml, and incubating to obtain sensitized and activated DC cells; the mixed tumor antigen is prepared from a tumor cell surface protein antigen: tumor cell membrane protein antigens: the tumor cell exosome consists of the following components in a mass ratio of 1;

(4) DC vaccine was harvested after blocking PD-L1 molecules on the DC cell surface: adding an anti-human PD-L1 antibody with the final concentration of 1-10ug/ml into the sensitized and activated mature DC cells, incubating at 37 ℃ for 0.5-2 hours, and harvesting the DC cells to seal PD-L1 molecules on the surfaces of the DC cells;

the method for inducing tumor-specific CTL in vitro is that on the day when a patient draws blood for the first time to prepare DC vaccine and obtains DC, 40-50mL of anticoagulated peripheral blood is drawn for the second time from the same individual, the anticoagulated peripheral blood is separated and cultured in an adherent manner, the nonadherent mononuclear cells are sucked out and mixed with the DC vaccine according to the proportion of 5:1, a CTL serum-free starting culture medium is added, and after in vitro antigen presentation, anti-human CD3 monoclonal antibody, anti-human CD28 monoclonal antibody and rh-IL-2 are added for CTL culture;

when CTL begins to clone and grow, the amplification culture is carried out by changing into an amplification culture medium; when the CTL is cultured to the peak of logarithmic growth of cells, adding an anti-human PD-1 monoclonal antibody with the final concentration of 1-10ug/ml before CTL harvesting, sealing PD-1 molecules on the surface of the CTL, and incubating for 0.5-2 hours; collecting cell suspension and centrifugally washing; a quality control sample is left; the resulting CTLs were resuspended in saline for patient infusion.

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