CN113368226A

CN113368226A - Pharmaceutical composition containing DC tumor vaccine and application thereof in treating cancer

Info

Publication number: CN113368226A
Application number: CN202110734774.7A
Authority: CN
Inventors: 亓爱杰; 李少波; 马宏磊
Original assignee: Nuosa Union Beijing Biomedical Technology Co ltd
Current assignee: Shanghai Liwo Biotechnology Co., Ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2021-09-10
Anticipated expiration: 2041-06-30
Also published as: CN113368226B

Abstract

The invention relates to a pharmaceutical composition containing DC tumor vaccine and application thereof in treating cancer. The tumor vaccine has a good effect of inhibiting the proliferation of the liver cancer cells, can remarkably inhibit the growth of the tumor cells and improve the secretion of related cytokines by combining with a PD-1 monoclonal antibody and cisplatin, and has good application value.

Description

Pharmaceutical composition containing DC tumor vaccine and application thereof in treating cancer

Technical Field

The invention belongs to the field of biology, and particularly relates to a pharmaceutical composition containing DC tumor vaccine and application thereof in treating cancer.

Background

Dendritic Cells (DC) are the antigen presenting cells with the strongest functions at present, and can stimulate the proliferation of primary T cells and stimulate the immune response of organisms. In the anti-tumor immune response of the body, cellular immunity is the predominant form two and the amount of signal reception required for the cytotoxic T cells producing CD8+ to be the core T cell activation of effective anti-tumor cellular immunity is based on three factors, the level of peptide-MHC complexes that trigger signal transduction, the level of co-stimulatory molecules that enhance the signaling process, and the stability of the immunological synapse formed by Antigen Presenting Cells (APC) and T cells, the latter determining the duration of the signaling process. The effectiveness of immune synapses varies with the stage of APC and T cell development. Immature DC can capture antigen efficiently through endocytosis, form a complex with MHI-I, II after intracellular processing treatment, express on DC surface, provide a first signal mature DC high-level expression costimulatory molecule, provide a second signal for T cell activation, induce to generate a large amount of effector T cells and synthesize important cytokines such as IL-12 and IFN-a, and activate various immune-related cells.

Researches show that the number of DCs in a tumor patient is obviously reduced, and the phenotype and the function of the DCs are obviously deficient. In recent years, research on DC tumor vaccine is greatly advanced, and the DC tumor vaccine is expected to become a new method for tumor immunotherapy. At present, the commonly used tumor vaccine preparation method mainly comprises the following modes.

Tumor cell lysate to DC: the DC is sensitized after the tumor cells are repeatedly frozen and thawed or ultrasonically crushed, and specific CTL and protective immune response can be generated. The method can induce wide T cell response without defining tumor antigen. Compared with the influence of a method for preparing 3 tumor cells by radiation, boiling and repeated freeze thawing on a DC presentation antigen in the prior art, the result shows that the DC loaded with the radiated tumor cells can effectively stimulate antigen-specific CTL, and the DC loaded with the radiated tumor cells can not induce to generate IFN-y after boiling or repeated freeze thawing, and in vitro and in vivo experiments show that the preparation method of the tumor cells obviously influences the capability of the DC to present the antigen to T cells. Since the tumor cell extract contains the autologous normal antigen, it is possible to induce an autoimmune response after immunizing the body.

Antigenic peptide in vitro sensitized DC: antigenic peptides are currently the most widely used tumor antigens for priming DCs. It can induce body to generate antigen specificity CTL obviously and excite body's antitumor cell immunoreaction through the combination of specific antigen epitope and DC specific MHC molecule. Since only a few tumors currently define tumor epitopes recognized by T cells, such as the gP100/Pmel-17, tyrosinase, MART-1, gP75/trp-1, and MACE/BALE/GAGE gene families associated with melanoma; the prostate specific membrane antigen PSMA; carcinoembryonic antigen CEA idiotypic protein (Id) of multiple myeloma, and the like.

Apoptotic Tumor Cell (ATCs) sensitized DCs: DCs effectively present antigens to apoptotic cells, stimulating CD8+ CTLs. The apoptosis melanoma cell is used for sensitizing DC, the levels of IL-1, IL-6, TNF-a, GM-CSF, MHC-II, CD86 and the like can be adjusted up, the DC is promoted to mature, the capability of the mature DC migrating to local lymph nodes is enhanced, and effective anti-tumor reaction is induced. The research finds that ATC and immune complex (ATC-IC) can be combined with FcyR expressed by DC, induce DC maturation and enhance the rabbit plague response of the ATCs-DC vaccine. Of course, there are tumor cell fusion with DC, exosome corpuscles in DC, and the like.

In the prior art, although a plurality of methods for preparing the DC tumor vaccine exist, the DC tumor vaccine specific to the liver cancer is not abundant enough, and particularly, the method for jointly using the DC tumor vaccine and other medicines is not enough, so that the method is worthy of further attention.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a DC tumor vaccine specific to liver cancer and a corresponding preparation method and application.

On one hand, the invention provides a DC tumor vaccine specific for liver cancer, and the preparation method of the DC cell comprises the following steps:

(1) peripheral blood mononuclear cells were prepared.

(2) Peripheral blood mononuclear cells are suspended in a culture medium, the concentration is adjusted, and the cells are attached to the wall on a cell culture plate. Collecting suspension cells, washing adherent cells twice with PBS (phosphate buffer solution) to obtain DC precursor cells, observing and counting under a microscope, and adding RPMI1640 culture medium: 10% FBS, resveratrol 5mmol/L, GM-CSF (800U/m1) and IL-4(1000U/m1), cell concentration approximately 10⁶Ml and incubation continued for 24h, after washing, RPMI1640 medium was added: culture was continued for 3d with 10% FBS, GM-CSF (800U/m1) and IL-4(1000U/m 1).

(3) Regulating cell concentration of SMMC-7721 hepatocarcinoma cells in logarithmic growth phase to 5 × 10⁶Loading into test tubes (2 ml per tube), locating high-intensity focused ultrasound focus at the center of cell suspension, and starting system at 1000W/cm²Irradiating SMMC-7721 hepatocarcinoma cells at x 30s, centrifuging irradiated cell lysate at 2000 rpm for 10min, collecting supernatant, filtering with 0.2 μm filter, and sterilizingAnd then collecting for later use.

(4) Adding the SMMC-7721 liver cancer cell antigen prepared by the method into the prepared DC mature suspension for co-culture for 24h, wherein the ratio of DC to antigen is 1: 15 (the amount of the antigen is calculated by the number of the tumor cells before the antigen preparation), and obtaining the DC tumor vaccine prepared by the tumor antigen loaded by the HIFU irradiation method.

The addition of resveratrol in the preparation of DC tumor vaccine can better improve the maturity of DC cells and has better effect.

In another aspect of the invention, a specific PD-1 monoclonal antibody is provided, wherein the monoclonal antibody is a monoclonal antibody which is obtained by immunizing a mouse with PD-1 protein and specifically binds to PD-1 through a hybridoma screening technology. The variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO: 1, and the heavy chain variable region is shown as SEQ ID NO: 1, the monoclonal antibody has a good binding effect with a binding constant of 5.9nM to PD-1 protein, and has good specificity and no reaction with other proteins.

Light chain variable region (SEQ ID NO: 1)

DIVITQSPALMAASPGEKVTITCTCYSDEGYHNCNWYQQKSGISPKPWIYLPEFYQCGVPARFSGSGSGTSYSLTITSMEAEDAATYYCINLILGCERFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 2)

EVQLEESGTELARPGASVKLSCKASGYIFSFWALGWIKQRPGQGLEWIGNFTTQAVERTKHDFQFGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGGAKRCLNWGLGTTLAVSS。

The influence of chemotherapeutic drugs on immune function suggests that it is feasible to initiate active anti-tumor immunity of a host for continuous killing after appropriate dose chemotherapy in combination with immunotherapy such as DC vaccine, and is likely to be more effective and safer than the method of killing all tumor cells by purely relying on high dose chemotherapy. The cisplatin obtained by screening of the invention can be used together with DC tumor vaccine, has better treatment effect, and is improved by more than 20% compared with the combined effect of other chemotherapy drugs.

In another aspect of the invention, a pharmaceutical composition is provided, which comprises DC tumor vaccine and PD-1 monoclonal antibody and cisplatin.

In another aspect of the invention, the application of DC tumor vaccine, cisplatin and PD-1 monoclonal antibody in preparing a pharmaceutical composition for treating liver cancer is also provided.

Further, the pharmaceutical composition may also comprise other commonly used therapeutic agents.

Further, in general, a therapeutically effective amount of an antibody, small molecule or other compound or composition described herein can range from 0.01mg/kg to 100mg/kg, and all values therebetween. For example, it may be 0.1mg/kg to 100mg/kg, 0.1mg/kg to 50mg/kg, 1mg/kg to 50mg/kg, etc.

Further, typically, a therapeutically effective amount of a DC tumor vaccine as described herein may be in the range of 1 x 10⁷-1*10¹¹In the range of/kg, and all values in between.

Further, the PD-1 antibody may be simultaneously added or replaced with Small Molecule Inhibitors (SMIs) affecting PD-1/PD-L1 including BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001 and BMS-1166, SB415286, Vorinostat, Panobistat, azacytidine, decitabine, entinostat, JQ1, 1-beta 151, GSK 503.

The compositions may comprise a pharmaceutically acceptable carrier or excipient which does not normally produce an adverse, allergic, or unwanted reaction when administered to an individual, such as a human subject. The pharmaceutically acceptable carrier or excipient may be a filler (solid, liquid, semi-solid), diluent, encapsulating material or the like. Examples include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and the like.

The pharmaceutical compositions may be in the form of solutions, suspensions, emulsions and solid injectable compositions which are dissolved or suspended in a solvent immediately prior to use. The injection may be prepared by dissolving, suspending or emulsifying one or more active ingredients in a diluent. Examples of diluents are distilled water for injection, physiological saline, physiological buffer, vegetable oil, alcohol and combinations thereof. In addition, the composition may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives and the like. The pharmaceutical compositions may be formulated as sterile solid or powder preparations, for example, by lyophilization, and may be sterilized immediately prior to use or dissolved in sterile water for injection or other sterile diluent for use. The composition may include one or more standard pharmaceutically acceptable carriers.

The pharmaceutical compositions of the present invention may be administered by any suitable route, including but not limited to oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal administration or combinations thereof. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intratumoral, intramuscular, intrathecal, and intraarticular. The agent may also be administered in the form of an implant, which allows for slow release of the compound, as well as slow controlled intravenous administration. And (5) transfusion.

Advantageous effects

The invention obtains the DC tumor vaccine which improves the DC maturity by resveratrol aiming at the preparation of the liver cancer cell specificity, the tumor vaccine has better effect of inhibiting the proliferation of the liver cancer cell, and can obviously inhibit the growth of the tumor cell and improve the secretion of related cell factors by combining with the PD-1 monoclonal antibody and the cisplatin for use, thereby improving the effect of tumor treatment and having better application value.

Drawings

FIG. 1 is a graph showing the result of DC cell maturation degree under different concentrations of resveratrol

FIG. 2 is a graph showing the killing effect of tumor vaccine under different target ratios

FIG. 3 is a diagram showing the result of DC tumor vaccine specificity

Detailed Description

To further illustrate the objects, aspects and advantages of the present invention, we shall now describe the invention with reference to the following specific examples, which are only for better illustrating the patent of the present invention and are not intended to limit the scope of the present invention. All other embodiments that can be obtained by a person skilled in the art without making any inventive step based on the examples of the present invention belong to the protection scope of the present invention.

Example 1 preparation of DC cells

Human peripheral blood was mixed with PBS at 1: 4, uniformly mixing, and mixing the diluted blood in a ratio of 1: slowly adding into the upper part of the lymphocyte separation solution at a ratio of 1, and centrifuging at 4 ℃ for 15min at 2000 r/min. The mononuclear cell layer was carefully aspirated, added to another centrifuge tube, washed with 4 volumes of PBS and centrifuged at 1000r/min for 5min, and the lymphocyte separation solution was removed by centrifugation. After two times of PBS washing, peripheral blood mononuclear cells are obtained.

Peripheral blood mononuclear cells were suspended in RPMI1640 medium at a concentration of l08/ml and attached to the cell culture plate. Carrying out adherent culture for 3-6 h in an incubator at 37 ℃ with 5% CO2, collecting suspension cells, washing adherent cells twice with PBS (phosphate buffer solution) to obtain DC precursor cells, observing and counting under a microscope, and adding an RPMI1640 culture medium: 10% FBS, resveratrol (0, 0.5, 1, 5, 10 concentrations were individually applied in sequence) mmol/L, GM-CSF (800U/m1) and IL-4(1000U/m1), cell concentration was about L0⁶Ml and incubation continued for 24h, after washing, RPMI1640 medium was added: culture was continued for 3d with 10% FBS, GM-CSF (800U/m1) and IL-4(1000U/m 1).

CD80, CD86 expression analysis DC cells: after being suspended after being washed, the suspension is labeled with FITC-CD86mAb and PE-CD80mAb for 30min before being detected and analyzed by an up-flow cytometry detector. The results are shown in figure 1 of the drawings,

under different concentrations of resveratrol, the expression of two types of costimulatory molecular markers CD80/CD86 on the cell surface of DC cells has concentration-dependent positive correlation, and the maturity CD80/86 can reach (87.1 +/-3.0)%, when the concentration is 10 mmol/L. DC cells were prepared for use at a concentration of 5mmol/L resveratrol.

EXAMPLE 2 preparation of DC tumor vaccine

Regulating cell concentration of SMMC-7721 hepatocarcinoma cells in logarithmic growth phase to 5 × 10⁶Loading into test tubes (2 ml per tube), locating high-intensity focused ultrasound focus at the center of cell suspension, and starting system at 1000W/cm²Irradiating SMMC-7721 hepatocarcinoma cells at x 30s, centrifuging irradiated cell lysate at 2000 rpm for 10min, collecting supernatant, filtering with 0.2 μm filter, sterilizing, and collecting.

Adding the SMMC-7721 hepatoma cell antigen prepared by the method into the DC mature suspension prepared in the example 1 for co-culture for 24 hours, wherein the ratio of DC to antigen is 1: 15 (the amount of the antigen is calculated by the number of tumor cells before the preparation of the antigen), obtaining DC tumor vaccine prepared by the HIFU irradiation method loading the tumor antigen, and adjusting the cell concentration to be 1 multiplied by 10⁹The volume of the solution is kept for later use.

Example 3DC tumor vaccine induces specific anti-cancer immune effects

SD rat bone marrow-derived T lymphocytes are taken and incubated with the tumor vaccine prepared by DC cells prepared in the embodiment of 25 mmol/L resveratrol concentration, after 5d, the tumor vaccine cells are stimulated again, and rIL-2 is added to promote the survival and proliferation of the T cells. The cells were collected for each group by stimulating again for 5d, and the harvested cells were used as effector T cells. SMMC-7721 liver cancer cells are used as target cells, and the killing capacity of effector T cells to liver cancer cells is detected by a 51Cr release method. SD rat bone marrow-derived T lymphocytes were used as a control, and the results are shown in FIG. 2.

Under the condition of different effective target ratios, the tumor vaccine groups all show high-efficiency and specific killing activity to cancer cells. The killing capacity is in direct proportion to the number of effector cells and is obviously higher than that of a control group. Especially under the condition of 40:1, the killing effect reaches 75 percent, and the effect is better.

Example 4DC tumor vaccine specific detection

UMR108 brain glioma cells, C6 pancreatic cancer cells and SMMC-7721 liver cancer cells are respectively used as target cells, the difference between CTLs effect induced by tumor vaccine is compared, and the change of CTLs effect is tested by inhibiting MHC-I molecules. The results are shown in FIG. 3.

As can be seen from FIG. 3, the tumor vaccine group showed high killing activity against hepatoma cells at a target-to-effect ratio of 40: 1. The effect is obviously higher than that of taking other cancer cells as target cells; furthermore, the present study also shows that CTLs effect is mediated by MHC-I molecules as shown by the results of inhibiting the effect of MHC-I molecules on CTLs.

Example 5 verification of therapeutic Effect of tumor vaccine in combination with PD-1 monoclonal antibody on liver cancer

BALB/c mice were randomly divided into 4 groups of 10 mice each. Group A is a model group, group B is a DC tumor vaccine groupThe group C is DC tumor vaccine + monoclonal antibody treatment group, and the group D is monoclonal antibody group. Collecting SMMC-7721 hepatocarcinoma cells in logarithmic growth phase, washing with trypsin-digested PBS, and adjusting cell concentration to l × 10⁷And/ml. SMMC-7721 hepatoma cells were injected subcutaneously into the right chest of four groups of ABCD mice at 0.1 ml/mouse. C, injecting DC tumor vaccine O.1ml to bilateral axilla and inguinal subcutaneous injection 3D before modeling, injecting DC tumor vaccine O.1ml to bilateral axilla and inguinal subcutaneous injection C, simultaneously injecting monoclonal antibody 5 mg/kg.d to 14D, and injecting physiological saline with the same amount to A. The tumor vaccine is injected for 1 time in 7 days by the same method. After 14d, the neck was removed and the tumor was completely detached and weighed. Taking venous blood, centrifuging (1800r/min, 20min, centrifugation radius 15cm), and detecting the IL-2, IL-12 and IFN-Y levels of serum by an ELISA method. The results are shown in tables 1 and 2, respectively.

TABLE 1 comparison of tumor weights for each group

Group of	Tumor weight (g)
		Group A	0.702±0.034
Group B	0.241±0.028
		Group C	0.134±0.017
Group D	0.293±0.025

The results in table 1 show that the group B was the DC tumor vaccine group, the group C was the DC tumor vaccine + monoclonal antibody treatment group, and the group D was the monoclonal antibody group, both of which had better tumor proliferation inhibitory effects than the group a model group. Wherein, the DC tumor vaccine of the C group and the monoclonal antibody treatment group has the minimum tumor mass of 0.134 +/-0.017 g, which shows that the DC tumor vaccine and the monoclonal antibody are used together and have synergistic treatment effect.

TABLE 2 IL-2, IL-12, IFN-. gamma.levels in each group

Group of	IL-2pg/mL	IL-12pg/mL	IFN-γpg/mL
				Group A	48.52±8.19	43.27±3.21	47.52±5.74
Group B	89.31±6.97	84.12±5.24	83.17±6.62
				Group C	101.23±7.69	92.57±9.47	99.45±8.55
Group D	86.23±5.87	81.07±4.85	79.56±5.23

As can be seen from Table 2, the C group DC tumor vaccine together with the monoclonal antibody can generate higher level IL-12, stimulate T cells to generate more IFN-gamma, and reach 99.45 +/-8.55 pg/mL, so as to stimulate stronger Th1 immune response in mice, generate more Th1 cellular immune molecules such as IL-2, IL-12, IFN-gamma and the like, and enhance the anti-tumor immune response of the mice.

Example 6 tumor vaccine combined with PD-1 monoclonal antibody and cisplatin for the treatment of liver cancer

BALB/c mice were randomly divided into 3 groups of 10 mice each. Group A is the model group, group B is the DC tumor vaccine + monoclonal antibody treatment group, and group C is the DC tumor vaccine + monoclonal antibody + cisplatin group. Collecting SMMC-7721 hepatocarcinoma cells in logarithmic growth phase, washing with trypsin-digested PBS, and adjusting cell concentration to l × 10⁷And/ml. SMMC-7721 liver cancer cells were injected subcutaneously into the right chest of four groups of ABC mice at 0.1 ml/mouse. Before modeling, 3d, injecting DC tumor vaccine O.1ml and 5 mg/kg.d of monoclonal antibody into bilateral axilla and inguinal subcutaneous injection of B group for 14d, injecting DC tumor vaccine O.1ml and 5 mg/kg.d of monoclonal antibody into bilateral axilla and inguinal subcutaneous injection of C group for 14d and injecting cisplatin (100 mu g/piece) into abdominal cavity for 14d continuously once a day. The tumor vaccine is injected for 1 time in 7 days by the same method. After 14d, the neck was removed and the tumor was completely detached and weighed. Taking venous blood, centrifuging (1800r/min, 20min, centrifugation radius 15cm), and detecting the IL-2, IL-12 and IFN-Y levels of serum by an ELISA method. The results are shown in tables 3 and 4, respectively.

TABLE 3 comparison of tumor weights for each group

Group of	Tumor weight (g)
		Group A	0.713±0.028
Group B	0.133±0.016
		Group C	0.101±0.004

As can be seen from the results in Table 3, the effect of the group C consisting of the DC tumor vaccine + the monoclonal antibody + cisplatin treatment group was better than the effect of the DC tumor vaccine and the monoclonal antibody alone and the effect of the group A model group in inhibiting tumor proliferation. Wherein, the group C of DC tumor vaccine + monoclonal antibody + cisplatin treatment group has the minimum tumor mass of 0.101 plus or minus 0.004g, which shows that the DC tumor vaccine has synergistic treatment effect when being used together with monoclonal antibody and cisplatin.

TABLE 4 IL-2, IL-12, IFN-. gamma.levels in each group

Group of	IL-2pg/mL	IL-12pg/mL	IFN-γpg/mL
				Group A	48.57±7.33	43.39±3.00	47.48±5.10
Group B	101.42±6.55	92.48±8.12	99.62±8.47
				Group C	117.44±9.59	99.41±10.12	106.29±9.87

As can be seen from Table 2, the C group DC tumor vaccine together with the monoclonal antibody and cisplatin can generate higher level IL-12 and stimulate T cells to generate more IFN-gamma, which reaches 106.29 +/-9.87 pg/mL, so that stronger Th1 immune response in mice is stimulated, and more Th1 cell immune molecules such as IL-2, IL-12, IFN-gamma and the like are generated, thereby enhancing the anti-tumor immune response of the mice.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention. The scope of the invention is defined by the appended claims.

Sequence listing

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Claims

Use of a combination of DC tumor vaccine, PD-1 monoclonal antibody and cisplatin in the preparation of a pharmaceutical composition for the treatment of liver cancer; the preparation method of the DC tumor vaccine comprises the following steps:

(1) human peripheral blood was mixed with PBS at 1: 4, uniformly mixing, and mixing the diluted blood in a ratio of 1: slowly adding the mixture above the lymphocyte separation solution in a ratio of 1, centrifuging at the temperature of 4 ℃ at 2000r/min for 15min, carefully sucking a mononuclear cell layer, adding the mononuclear cell layer into another centrifugal tube, washing by 4 times of volume of PBS, centrifuging at 1000r/min for 5min, centrifuging to remove the lymphocyte separation solution, and washing by PBS twice to obtain peripheral blood mononuclear cells;

(2) the peripheral blood mononuclear cells were suspended in RPMI1640 medium at a concentration of 10⁸Perml, attached to a cell culture plate at 37 ℃ with 5% CO₂Culturing for 3-6 h in an incubator under the condition of adherent culture, collecting suspension cells, washing adherent cells with PBS twice to obtain DC precursor cells, observing and counting under a microscope, adding RPMI1640 culture medium added with FBS with final concentration of 10%, resveratrol 5mmol/L, GM-CSF 800U/ml and IL-41000U/ml, wherein the cell concentration is about 10⁶Performing incubation for 24h, washing, adding RPMI1640 culture medium containing 10% FBS, GM-CSF 800U/ml and IL-41000U/ml, and culturing for 3d to obtain DC mature cell;

(3) liver cancer cells in the logarithmic growth phase,adjusting the cell concentration to 5X 10⁶Loading into test tubes (2 ml per tube), locating high-intensity focused ultrasound focus at the center of cell suspension, and starting system at 1000W/cm²Irradiating the liver cancer cells for 30s, centrifuging the irradiated cell lysate for 10min at 2000r/min, taking the supernatant, filtering and sterilizing the supernatant by a filter with the diameter of 0.2 mu m, and collecting the supernatant for later use;

adding the liver cancer cell antigen prepared by the method (3) into the DC mature cell prepared by the step (2) for co-culture for 24h, wherein the ratio of DC to antigen is 1: 15, calculating the amount of the antigen according to the number of tumor cells before antigen preparation to obtain DC tumor vaccine;

wherein the variable region of the light chain of the PD-1 monoclonal antibody is shown as SEQ ID NO: 1, and the heavy chain variable region is shown as SEQ ID NO: 2, respectively.
2. Use according to claim 1, characterized in that said composition comprises a pharmaceutically acceptable carrier or excipient.
3. Use according to claim 2, characterized in that the pharmaceutically acceptable carrier or excipient is a filler, diluent or encapsulating material.
4. Use according to claim 3, characterized in that the carrier or excipient is buffered saline, dextrose, water, glycerol or ethanol.
5. The use according to claim 4, wherein the pharmaceutical composition is in the form of a solution, suspension, emulsion or solid injectable composition.