CIK immune cell and application thereof in cancer treatment

Abstract

The invention relates to a cellular immunotherapy method, in particular to cellular immunotherapy based on cytokine induction. The CIK cell culture method with efficient amplification and immune activation is provided through accurate cell factor combination, low-oxygen environment and metabolic regulation, and the amplification efficiency, the immunophenotype and the anti-tumor activity of the CIK cells are remarkably improved. According to the method, amplification conditions of the CIK cells are optimized through a three-stage dynamic culture system, the cell survival rate is increased while the immune effect is enhanced, and the method is suitable for tumor immunotherapy and other related immunotherapy application. Experiments show that the CIK cells cultured by the method disclosed by the invention have higher efficacy in anti-tumor immunotherapy, and the tumor inhibition effect is further improved by combining the CIK cells with an immune checkpoint inhibitor.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for efficiently amplifying cytokine-induced killer cells (CIK cells) and an application thereof in cancer treatment. Background Art

[0002] With the progress of cancer treatment, immunotherapy has gradually become an important treatment strategy, especially cell immunotherapy. Among many immune cells, Cytokine-Induced Killer (CIK) cells have attracted widespread attention due to their strong anti-tumor ability and low side effects. CIK cells are a type of immune cells obtained by cytokine-induced amplification in vitro. They are mainly composed of T cells and natural killer cells (NK cells) and have high tumor cell killing ability. The immune activity of CIK cells mainly comes from their unique immune phenotype, including CD3+CD56+ double positive cells and CD8+ cells, which have a strong ability to target and recognize tumor cells.

[0003] However, CIK cells still face some challenges in clinical applications. First, the expansion efficiency and immune activity of CIK cells may be affected by culture conditions, and the existing cytokine combinations and culture methods have not yet reached the optimal level. Secondly, the response of CIK cells to the tumor microenvironment is more complex. The tumor's immune escape mechanism, immune tolerance, and the presence of tumor immunosuppressive factors may inhibit the anti-tumor effect of CIK cells. Therefore, how to improve the expansion efficiency of CIK cells, enhance their anti-tumor activity, and overcome the immunosuppressive effects of the tumor microenvironment is still a difficulty in current immune cell therapy research.

[0004] In recent years, combined immunotherapy has gradually become a research hotspot. The combined use of immune checkpoint inhibitors (such as anti-PD-1 and anti-CTLA-4 antibodies) and CIK cell therapy has shown good anti-tumor effects. Therefore, developing new CIK cell expansion and activation methods to improve their clinical efficacy is an important issue that needs to be solved urgently. Summary of the invention

[0005] The purpose of the present invention is to provide a method for efficiently expanding CIK cells, which significantly improves the expansion multiple, immune phenotype and anti-tumor activity of CIK cells by precisely controlling the concentration and combination of cytokines, optimizing metabolic regulation means and setting a hypoxic environment.

[0006] Therefore, the present invention discloses a method for efficiently expanding CIK cells, comprising the following steps:

[0007] (1) Collect peripheral blood samples and separate PBMCs by density gradient centrifugation;

[0008] (2) PBMCs are cultured using a three-stage dynamic culture system, wherein:

[0009] Phase I: Use RPMI-1640 medium supplemented with 1000 IU / mL IFN-γ and induce initial cell activation under hypoxic conditions;

[0010] Phase II: Add 1000 IU / mL IL-2, 50 ng / mL IL-15, 50 ng / mL anti-CD3 single domain antibody and 10 mM sodium pyruvate to expand cells and enhance immune activity, and culture under oxygen-rich conditions;

[0011] Phase III: CIK cells are continuously expanded and immune activity is enhanced by intermittently supplementing IL-2 and maintaining cytokine concentrations, that is, 500 IU / mL IL-2 is added every 48 hours, and culture is carried out under normal oxygen conditions.

[0012] Preferably, the oxygen concentration of the hypoxic condition in stage I of the present invention is 5%.

[0013] Preferably, the amino acid sequence of the anti-CD3 single domain antibody in Phase II of the present invention is as shown in SEQ ID NO.1, and the nucleotide sequence of the anti-CD3 single domain antibody after codon optimization is as shown in SEQ ID NO.2.

[0014] Preferably, the oxygen concentration of the oxygen-rich condition in stage II of the present invention is 21%.

[0015] Preferably, the oxygen concentration of the normal oxygen condition in stage III of the present invention is 10%.

[0016] Preferably, the culture time of stage I of the present invention is from day 0 to day 3, the culture time of stage II is from day 4 to day 7, and the culture time of stage III is from day 8 to day 14.

[0017] In one aspect, the present invention further discloses a composition for tumor immunotherapy, comprising the CIK cells prepared according to claim 1 and an anti-PD-1 single domain antibody.

[0018] Preferably, the concentration of CIK cells in the composition of the present invention is 2×10 6 Cells / times were injected via tail vein once a week for 4 weeks.

[0019] Preferably, the amino acid sequence of the anti-PD-1 single domain antibody of the present invention is as shown in SEQ ID NO.3, wherein the anti-PD-1 single domain antibody is used at a concentration of 10 mg / kg / time, injected once a week for 4 weeks.

[0020] The method and application of the present invention have the following beneficial effects:

[0021] (1) Significantly improve the expansion efficiency of CIK cells: Through optimized cytokine combination and hypoxic culture conditions, the expansion multiple of CIK cells during the culture process can reach 82.5 times, which is much higher than the 18 times of traditional methods.

[0022] (2) Enhanced cellular immune phenotype: Among CIK cells cultured by the method of the present invention, the proportion of CD3+CD56+ double positive cells is increased to more than 65%, and this phenotype has a strong anti-tumor activity.

[0023] (3) Enhanced cytotoxicity: CIK cells amplified by the method of the present invention showed significantly enhanced killing ability in cytotoxicity detection, with a killing rate of 78% against A549 lung cancer cells, which was significantly higher than that of the traditional amplification method.

[0024] (4) Improving cell survival rate: Through metabolic regulation measures, such as the addition of sodium pyruvate, lactic acid accumulation is reduced, the cell apoptosis rate is significantly reduced, thereby improving cell survival rate.

[0025] (5) The combined treatment with immune checkpoint inhibitors has significant effects: In tumor models, the combined use of CIK cells and immune checkpoint inhibitors (anti-PD-1 single domain antibodies) significantly enhanced the tumor suppression effect, promoted immune cell infiltration, and increased the survival rate of mice.

[0026] In summary, the CIK cell culture method provided by the present invention has significant advantages in tumor immunotherapy, can greatly improve the treatment effect, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 SDS-PAGE test results of anti-CD3 single domain antibodies, where 1 is an anti-CD3 single domain antibody. DETAILED DESCRIPTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0029] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] Example 1: Efficient expansion of CIK cells

[0031] 1. Collect peripheral blood samples: Collect 10 mL of peripheral blood from healthy volunteers and perform density gradient centrifugation using Ficoll-Hypaque liquid to separate peripheral blood mononuclear cells (PBMCs).

[0032] 2. Cultivate according to the three-stage dynamic cultivation system:

[0033] (1) Phase I (0-3 days): Initial activation phase

[0034] Medium preparation: At this stage, RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mM L-glutamine was used.

[0035] Cell seeding: The isolated PBMCs were seeded in 6-well plates at a cell density of 1×10 6 Cells were cultured under normal conditions of 37°C and 5% CO2.

[0036] Cytokine stimulation: IFN-γ (1000 IU / mL) was added to the culture medium to induce initial activation of cells. IFN-γ can activate T cells and NK cells and enhance their anti-tumor potential.

[0037] Hypoxic conditions: The incubator was set to a hypoxic environment (5% O2) to simulate the hypoxic state of the tumor microenvironment, which helps to improve the tumor targeting and tolerance of CIK cells.

[0038] Observation and adjustment: Check the morphology and density of cells daily to confirm that the cells are not contaminated. Add fresh culture medium as needed to maintain the best growth environment for cells.

[0039] (2) Phase II (4-7 days): Amplification and activity enhancement phase

[0040] Cytokine combination: At this stage, IL-2 (1000 IU / mL), IL-15 (50 ng / mL) and anti-CD3 single domain antibody (50 ng / mL, as shown in Example 3) were added.

[0041] IL-2: As the main expansion factor, it promotes T cell proliferation and function enhancement.

[0042] IL-15: It works synergistically with IL-2 to enhance the survival and proliferation of CIK cells, and has a particularly positive effect on the proliferation of NK cells.

[0043] Anti-CD3 single-domain antibody: By cross-linking T cell receptors (TCR), it further activates T cells and enhances their anti-tumor activity.

[0044] Metabolic regulation: Sodium pyruvate (10 mM) was added as a metabolic regulator to help maintain cell metabolic balance, slow down lactic acid accumulation, and enhance cell survival.

[0045] Culture conditions: Continue to culture at 37°C, 5% CO2, and keep the oxygen concentration in the incubator at 21%. During this stage, the cell density will gradually increase, and the culture medium needs to be replaced every 2 days and cytokines need to be supplemented.

[0046] Cell monitoring: Record cell density daily, observe cell proliferation and morphological changes, and ensure that cells grow under optimal conditions. Adjust the culture medium if necessary to maintain a stable cell environment.

[0047] (3) Phase III (8-14 days): Continuous expansion and immune activation

[0048] Intermittent pulse stimulation: IL-2 (500 IU / mL) is supplemented every 48 hours to promote the continuous expansion and activation of CIK cells. Compared with the high-dose IL-2 in Phase II, a lower dose of IL-2 is used in this phase to avoid excessive cell fatigue and apoptosis.

[0049] Cytokine maintenance: Maintain the previously added low concentrations of IL-2, IL-15, and anti-CD3 single-domain antibodies to maintain the immune activity of the cells. At this stage, the proliferation rate of the cells will be relatively slow, but by intermittently supplementing IL-2, the anti-tumor function of CIK cells can be continuously activated and enhanced.

[0050] Culture conditions: Continue to culture at 37°C, 5% CO2, while maintaining the oxygen concentration in the incubator at 10%.

[0051] Cell monitoring and analysis: Check cell density and morphology every 3 days and record cell growth status. Use flow cytometry to detect cell surface markers and analyze immunophenotypes such as CD3, CD56, and CD8 to ensure high purity and high activity of CIK cells.

[0052] Cell collection and application preparation: On day 14, CIK cells were collected for final phenotypic analysis and functional testing, such as cytotoxicity experiments and cell apoptosis analysis, to ensure that CIK cells have sufficient immune effects.

[0053] (4) Summary: This three-stage dynamic culture system achieves efficient expansion and immune activation of CIK cells by precisely controlling the types and concentrations of cytokines and regulating the metabolic environment (such as the addition of sodium pyruvate and the use of hypoxic conditions). During the entire culture process, the stimulation of cytokines is gradually increased to promote cells from initial activation to expansion and then to the final enhancement of immune activity, ensuring the optimal functionality of CIK cells.

[0054] 3. Cell collection and phenotype analysis: On day 14, cells in the culture medium were collected for flow cytometry analysis. Anti-CD3, anti-CD56, and anti-NKG2D antibodies were used to label the cells and detect the immunophenotype of CIK cells. At least three independent experiments were performed for each sample to ensure the accuracy of the results.

[0055] 4. Cytotoxicity assay: CFSE-labeled A549 lung cancer cells were used as target cells. After CFSE-labeled A549 cells were co-cultured with CIK cells at a ratio of 1:1 for 48 hours, the mortality of A549 cells was detected. The apoptosis ratio of target cells was detected by PI staining (propidium iodide) using flow cytometry.

[0056] 5. Experimental results are shown in Table 1.

[0057] (1) Expansion multiple: The method of the present invention significantly improves the expansion efficiency of CIK cells. During the 14-day culture period, the cell expansion multiple reached 82.5±9.7 times, while the traditional method was only 18.2±3.1 times. This result shows that the use of the method of the present invention can greatly improve the expansion efficiency of CIK cells.

[0058] (2) Immunophenotype: Flow cytometry showed that the proportion of CD3+CD56+ double positive cells in CIK cells cultured by the method of the present invention was significantly increased to 65.8±5.9%, which was significantly higher than 31.4±4.2% in the traditional method. This indicates that the method can more effectively induce CIK cells to transform into an immune phenotype with strong anti-tumor activity.

[0059] (3) Cytotoxicity: Through the co-culture experiment of CFSE-labeled A549 lung cancer cells, it was found that the killing rate of CIK cells cultured by the method of the present invention on A549 cells was 78%±5%, which was much higher than 45%±6% of the traditional method. This result shows that the improved culture conditions enhance the cytotoxicity of CIK cells and effectively improve their anti-tumor ability.

[0060] (4) Metabolic regulation effect: In the experiment, sodium pyruvate was used as a metabolic regulator, which can effectively reduce the accumulation of lactic acid and reduce the metabolic burden of cells during the expansion process. Through this method, the apoptosis rate of CIK cells was significantly reduced from 22% in the control group to 8%, further improving the cell survival rate and anti-tumor effect.

[0061] Table 1 Summary of experimental results

[0062]

[0063] 6. Summary

[0064] (1) The method of the present invention significantly improves the expansion efficiency, immune phenotype and anti-tumor activity of CIK cells through the optimized combination and metabolic regulation of cytokines.

[0065] (2) The addition of sodium pyruvate effectively inhibited lactic acid accumulation, reduced the metabolic burden of cells, and further enhanced the activity and survival rate of CIK cells.

[0066] (3) The results showed that CIK cells expanded by this method have higher efficacy in anti-tumor immunotherapy and have broad application prospects.

[0067] Example 2: CIK cells combined with immune checkpoint inhibitors to treat tumors

[0068] 1. Experimental design:

[0069] (1) Tumor model: Mouse HepG2 liver cancer model. BALB / c nude mice (6-8 weeks old, weighing about 20 g) were used to establish an orthotopic transplantation model of HepG2 liver cancer. 5×10 6 HepG2 liver cancer cells were injected into the subcutaneous tissue of mice and the tumors were grown to about 50 mm. 3 When the treatment begins.

[0070] (2) Treatment options:

[0071] CIK cell therapy: Autologous or allogeneic CIK cells (2×10 6 cells / time), injected via tail vein every week for 4 weeks.

[0072] Anti-PD-1 single domain antibody treatment: Anti-PD-1 monoclonal antibody (10 mg / kg injected weekly) was used, and the treatment group was injected once a week for 4 weeks.

[0073] Combination therapy: CIK cells and anti-PD-1 single-domain antibodies were used for combined therapy simultaneously, and both CIK cells and anti-PD-1 single-domain antibodies were administered at the above dosages.

[0074] (3) Experimental groups:

[0075] Control group: No treatment, only injection of saline.

[0076] CIK cell treatment group only: CIK cells were injected once a week.

[0077] Anti-PD-1 single domain antibody only group: Inject anti-PD-1 single domain antibody once a week.

[0078] CIK cells + anti-PD-1 single domain antibody combined treatment group: CIK cells and anti-PD-1 single domain antibodies were injected simultaneously, once a week.

[0079] 2. Experimental steps:

[0080] (1) Mouse model establishment: BALB / c nude mice aged 6-8 weeks were selected to ensure good health. 5×10 6 HepG2 liver cancer cells were resuspended in 200 μL sterile PBS and injected subcutaneously into the back of mice. The tumor growth progress was observed and the tumor volume reached 50 mm. 3 Group treatment started at that time.

[0081] (2) Treatment start: According to the grouping scheme, treatment was started weekly. CIK cells were injected into the tail vein (2×10 6 Cells), anti-PD-1 single domain antibody was injected intraperitoneally (10 mg / kg each time). The treatment lasted for 4 weeks, during which the tumor was measured weekly, the changes in tumor volume were recorded, and the treatment effect was evaluated.

[0082] (3) Tumor volume measurement: The length, width and height of the tumor were measured weekly using a caliper, and the tumor volume was calculated (V = 0.5 × length × width × height). The survival of the mice was regularly observed, and the survival time and health status were recorded.

[0083] (4) Immune cell infiltration analysis: At the end of the experiment, the tumor tissue was subjected to immunophenotyping analysis by flow cytometry (FACS) to evaluate the infiltration of immune cells such as CD8+T cells, CD4+T cells, NK cells, and Treg cells.

[0084] (5) Tumor tissue was sectioned and immunohistochemistry (IHC) was used to detect the distribution and density of immune cells.

[0085] 3. Experimental results are shown in Table 2.

[0086] (1) Tumor inhibition effect: The tumor volume of the combined treatment group was significantly reduced to 150±30 mm at 4 weeks. 3 , compared with 750±120mm in the control group 3 , the tumor inhibition rate reached 80%. In contrast, the CIK cell treatment group and the anti-PD-1 single domain antibody group only inhibited 46% and 33% of tumor growth, respectively. The combined treatment significantly enhanced the anti-tumor effect and showed stronger tumor control ability.

[0087] (2) Immune cell infiltration: In the tumor tissue of the combined treatment group, the infiltration of CD8+T cells and NK cells increased significantly. The CD8+T cell infiltration rate was 40%±5%, which was significantly higher than the 8%±2% in the control group; the NK cell infiltration rate also reached 28%±6%, which was much higher than the 5%±1% in the control group. This result shows that CIK cell combined with anti-PD-1 single domain antibody treatment effectively promoted the infiltration of immune cells in the tumor microenvironment and enhanced the local immune response.

[0088] (3) Survival rate of mice: The survival rate of mice in the combined treatment group reached 100%, which was much higher than the 50% in the control group. This result further demonstrated the significant effect of combined treatment in improving the survival rate of mice.

[0089] (4) Mechanism of combined therapy: The increase in immune cell infiltration may be related to the release of immune checkpoint inhibition by anti-PD-1 single domain antibodies and the promotion of T cell and NK cell activation. CIK cells significantly improve the immune system's ability to control tumors through direct cytotoxic effects and enhanced immune surveillance functions, coupled with the immune escape inhibition effect of anti-PD-1 single domain antibodies.

[0090] Table 2 Summary of experimental results

[0091]

[0092] Conclusion: This experiment shows that the combined treatment of CIK cells and immune checkpoint inhibitors (anti-PD-1 single domain antibody) significantly enhanced the anti-tumor effect, especially in promoting immune cell infiltration and improving mouse survival. This combined treatment strategy can be used as a cancer immunotherapy with great potential, providing new ideas for clinical cancer treatment.

[0093] Example 3: Preparation of anti-CD3 single domain antibody

[0094] 1. The recombinant human CD3 protein (ab167755) was diluted to 100 μg / mL with shark saline and mixed with adjuvant at a ratio of 1:1 for emulsification. Freund's complete adjuvant (CFA) was used for the initial immunization, and Freund's incomplete adjuvant (IFA) was used for subsequent immunization. A total of 3 to 4 immunizations were performed, with an interval of 2 weeks between each immunization, all by intraperitoneal injection. After immunization, shark serum was collected and transcriptome sequencing was performed to analyze the antibody production in sharks. At the same time, RNA from shark spleens and lymphocytes was extracted for RT-PCR, and high-throughput sequencing data containing only shark VNAR was obtained. The transcriptome sequencing results were compared with the high-throughput database of VNAR to screen out sequences that existed in both databases and had high expression levels. Through this screening, a specific VNAR sequence (SEQ ID NO.1) was obtained, and the protein corresponding to the sequence was the amino acid sequence of the anti-CD3 single-domain antibody.

[0095] 2. The sequence of the single domain antibody was optimized by codon optimization (the optimized nucleotide sequence is shown in SEQ ID NO.2), and the optimized sequence was subcloned into the pET28a vector. The recombinant plasmid was transformed into BL21 (DE3) competent cells, spread on LB agar plates containing kanamycin, and cultured for 16 to 20 hours at 36-37 ° C. After picking a single colony, it was inoculated into LB medium containing kanamycin and cultured until the OD600nm value reached 0.4 to 0.6. The bacterial solution was harvested, transferred to 37 ° C for amplification culture, cooled to 20 ° C, and IPTG was added to induce expression. The bacteria were collected by centrifugation, resuspended, broken, and the cell fragments were separated by centrifugation, and the supernatant was collected.

[0096] 3. Purification was performed by nickel column affinity chromatography. The purified sample was dialyzed with PBS and then filtered with a 0.22 μm filter membrane to obtain the final anti-CD3 single domain antibody. The SDS-PAGE results showed that ( Figure 1 As shown), the purity of the anti-CD3 single domain antibody can reach more than 95%, and its expression level can reach 1.5g / L.

[0097] 4. Verify the specificity and activity of the anti-CD3 single domain antibody by ELISA. Use 50mM carbonate buffer (pH9.6) to dilute the anti-CD3 antibody (ab237721) and the above-purified anti-CD3 single domain antibody to a concentration of 1μg / mL. Add 100μL of the diluted antibody solution to each well and coat the plate overnight at 4°C. After washing, add PBST solution containing 2% BSA to each well and incubate at 37°C for 2 hours. After washing, add recombinant human CD3 protein (1μg / mL, containing 1% BSA) to the well and continue to incubate at 37°C for 1 hour. After washing, add recombinant HRP-labeled anti-CD3 antibody (ab195017) as a secondary antibody, dilute it at a ratio of 1:10000, and incubate it at 37°C for 1 hour. After washing and patting dry, add 100μL of TMB colorimetric solution and react at room temperature in the dark for 10 minutes. After the reaction was completed, 50 μL of 2M H2SO4 was added to terminate the reaction, and the absorbance value (OD450nm) was measured at 450nm by an ELISA reader. The experimental results are shown in Table 3.

[0098] Table 3 ELISA test results

[0099]

[0100] From the ELISA test results, it can be seen that the anti-CD3 single domain antibody prepared by the present invention has a high specificity in binding to the CD3 protein, and its OD value is significantly higher than that of the blank control group and the commercial anti-CD3 antibody group. This shows that the single domain antibody not only has good specificity, but also has good binding activity. It can be inferred that the anti-CD3 single domain antibody can be used as a potential specific binding molecule for application in CD3-related research and the development of related treatments.

[0101] It should be noted that an anti-PD-1 single domain antibody (i.e., used in Example 2) was also prepared according to the above method, and its amino acid sequence is shown in SEQ ID NO.3, and the single domain antibody also has better sensitivity and specificity than existing products (its OD450nm value can reach 2.45, while the control commercial antibody (ab237728) is only 1.65).

[0102] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for efficiently expanding CIK cells, characterized in that: The method comprises the following steps: (1) Collect peripheral blood samples and separate PBMCs by density gradient centrifugation; (2) PBMCs are cultured using a three-stage dynamic culture system, wherein: Phase I: Use RPMI-1640 medium supplemented with 1000 IU / mL IFN-γ and induce initial cell activation under hypoxic conditions; Phase II: Add 1000 IU / mL IL-2, 50 ng / mL IL-15, 50 ng / mL anti-CD3 single domain antibody and 10 mM sodium pyruvate to expand cells and enhance immune activity, and culture under oxygen-rich conditions; Phase III: CIK cells are continuously expanded and their immune activity is enhanced by intermittently supplementing IL-2 and maintaining cytokine concentrations, i.e., 500 IU / mL IL-2 is added every 48 hours, and the cells are cultured under normal oxygen conditions.

2. The method according to claim 1, characterized in that The oxygen concentration of the hypoxic condition in stage I is 5%.

3. The method according to claim 1, characterized in that The amino acid sequence of the anti-CD3 single domain antibody in the phase II is shown in SEQ ID NO.1, and the nucleotide sequence of the anti-CD3 single domain antibody after codon optimization is shown in SEQ ID NO.

2.

4. The method according to claim 1, characterized in that: The oxygen concentration of the oxygen-rich condition in stage II is 21%.

5. The method according to claim 1, characterized in that Normal oxygen conditions in Stage III have an oxygen concentration of 10%.

6. The method according to claim 1, characterized in that The culture time of stage I is from day 0 to day 3, the culture time of stage II is from day 4 to day 7, and the culture time of stage III is from day 8 to day 14.

7. A composition for tumor immunotherapy, characterized in that: The composition comprises the CIK cells prepared according to claim 1 and an anti-PD-1 single domain antibody.

8. The composition according to claim 7, characterized in that The concentration of CIK cells in the composition is 2×10 6 Cells / times were injected via tail vein once a week for 4 weeks.

9. The composition according to claim 7, characterized in that The amino acid sequence of the anti-PD-1 single domain antibody is shown in SEQ ID NO.3, wherein the anti-PD-1 single domain antibody is used at a concentration of 10 mg / kg / time, injected once a week for 4 weeks.

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