Preparation method of DC cell and application of DC cell in preparation of anti-tumor cell preparation

Abstract

The invention provides a preparation method of DC cells. The preparation method is especially suitable for rapidly obtaining high-purity and high-functional-activity DC cells. The method comprises the steps of separation from peripheral blood mononuclear cells (PBMC), induction culture, maturation treatment and the like, maturation of the dendritic cells is promoted by introducing an anti-CD47 single-domain antibody or LPS, and the immunocompetence of the dendritic cells is remarkably improved. The method is further applied to preparation of an anti-tumor cell preparation, the capability of activating tumor specific T cells by tumor antigen-loaded DC cells can be remarkably enhanced, and the killing activity of the T cells to tumor cells is improved. The preparation method provided by the invention overcomes the defects in the prior art, has the advantages of high efficiency, safety and stability, and can provide a more reliable cell therapy scheme for tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of cellular immunology, and in particular to a method for preparing dendritic cells (DC cells), which are further applied to the preparation of anti-tumor cell preparations. Background Art

[0002] Dendritic cells (DC cells) are important immune system cells that serve as a bridge between innate immunity and acquired immunity. DC cells can take up and process antigens, display antigens through the unique MHC molecules on their surface, and activate T cell-mediated immune responses. Therefore, they are widely used in tumor immunotherapy, vaccine development, and immune tolerance research. In recent years, the research on dendritic cells in tumor immunotherapy has gradually gained attention, especially in the preparation of tumor vaccines and immunotherapy preparations, which have great potential.

[0003] However, current methods for preparing dendritic cells mostly rely on the addition of exogenous cytokines, usually using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to induce peripheral blood mononuclear cells (PBMC) to differentiate into dendritic cells. Although these methods can successfully induce dendritic cell differentiation, there are often multiple problems in the preparation process, such as low cell purity, weak functionality, and long culture time. In addition, the immune activity and stability of mature dendritic cells remain technical difficulties in existing methods.

[0004] In order to improve the yield, purity and functional activity of dendritic cells, researchers have tried to optimize conditions such as different culture media, cytokine combinations, and culture time, but have still not achieved the desired effect in clinical treatment. Therefore, developing a more efficient, safe and purer dendritic cell preparation method that can better play its role in anti-tumor immunotherapy has become a technical problem that needs to be solved urgently. Summary of the invention

[0005] The purpose of the present invention is to provide a novel method for preparing dendritic cells (DC), which can obtain high-purity and high-functional activity DC cells in a short time, and the method can play a role in the preparation of anti-tumor cell preparations.

[0006] Therefore, the present invention discloses a method for preparing DC cells, which comprises the following steps:

[0007] (1) Isolation of peripheral blood mononuclear cells: Take 30 mL of whole blood, add Ficoll-Paque at a concentration of 1.077 g / mL, centrifuge at 1000 × g for 20 min, and collect the middle mononuclear cell layer after centrifugation, which is PBMC;

[0008] (2) DC cell induction culture: The isolated PBMCs were inoculated in a culture dish with a culture medium of RPMI-1640 containing 10% FBS and 1% penicillin-streptomycin, supplemented with cytokines at a final concentration of 50 ng / mL IL-4 and 100 ng / mL GM-CSF for DC cell induction culture. The cell inoculation density was 1×10 6 cells / mL, and the medium was replaced every 2-3 days until the cells reached 80%-90% confluence;

[0009] (3) Maturation of DC cells: On the 7th day of culture, when the DC cells were cultured to about 80% confluence, anti-CD47 single domain antibody with a final concentration of 1 μg / mL or LPS with a final concentration of 1 μg / mL was added and treated for 24 hours to induce the maturation of DC cells, thereby preparing DC cells.

[0010] Preferably, the VHH sequence of the anti-CD47 single-domain antibody of the present invention is as shown in SEQ ID NO.1.

[0011] In one aspect, the present invention further discloses a method for preparing an anti-tumor cell preparation, the method comprising the following steps:

[0012] (1) extracting tumor antigens from human lung cancer cell line A549, wherein the tumor antigens are extracted using RIPA lysate and quantified using the BCA method, wherein the total protein concentration in the lysate is 1.2 mg / mL;

[0013] (2) The mature DC cells prepared in claim 1 are co-incubated with tumor antigens, wherein the ratio of the total protein of the mature DC cells to the total protein of the tumor antigen is 1×10 6 Cells: 100 μg; incubation time: 24 hours, temperature: 37°C;

[0014] (3) Co-culture DC cells loaded with tumor antigens with tumor-specific T cells to promote T cell proliferation and cytotoxic activity.

[0015] Preferably, the co-culture ratio of DC cells loaded with tumor antigens and T cells of the present invention is 1:10, and the culture time is 72 hours.

[0016] Preferably, the T cells of the present invention are purified by immunomagnetic separation and activated by anti-CD3 single domain antibodies.

[0017] Preferably, the VHH sequence of the anti-CD3 single domain antibody of the present invention is as shown in SEQ ID NO.2.

[0018] In one aspect, the present invention also discloses a use of the anti-CD47 single domain antibody in preparing DC cells.

[0019] The dendritic cell preparation method of the present invention significantly improves the purity and functional activity of DC cells by optimizing the culture conditions, and can obtain mature DC cells in a short time to meet the needs of clinical applications. By introducing the maturation induction method of anti-CD47 single-domain antibodies, the immune activity of DC cells is enhanced, the proliferation and activity of tumor-specific T cells are effectively promoted, and the effect of tumor immunotherapy is improved.

[0020] The present invention uses mature DC cells to load tumor antigens, which can significantly improve the presentation efficiency of tumor antigens, activate tumor-specific T cells, and significantly enhance the tumor-killing effect of T cells, showing good anti-tumor immunotherapy potential.

[0021] Compared with traditional methods, the DC cell preparation method provided by the present invention is easy to operate, can obtain high-quality dendritic cells on a large scale and quickly, and helps to improve the production efficiency of therapeutic products. In addition to its application in tumor immunotherapy, the preparation method can also be widely used in vaccine development, immune tolerance research and other immunotherapy fields, providing a reliable cell preparation platform for related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Microscopic observation results of mature DC cells.

[0023] Figure 2 SDS-PAGE test results of anti-CD47 single domain antibodies, where 1 and 2 are both anti-CD47 single domain antibodies.

[0024] Figure 3 Western blot test results of anti-CD47 single domain antibody, where 1 is CD47 protein. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] Example 1: Preparation method of DC cells

[0028] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0029] (1) Take 30 mL of whole blood, add Ficoll-Paque at a concentration of 1.077 g / mL, centrifuge at 1000 × g for 20 min, and collect the middle mononuclear cell layer after centrifugation, which is PBMC.

[0030] (2) Using the Trypan blue staining method, the cell count was 5×10 6 cells / mL, with a purity of approximately 90%.

[0031] 2. DC induction culture

[0032] (1) The isolated PBMCs were inoculated into a culture dish in a culture medium containing RPMI-1640 containing 10% FBS and 1% penicillin-streptomycin, and supplemented with cytokines with a final concentration of 50 ng / mL IL-4 and 100 ng / mL GM-CSF for DC cell induction culture;

[0033] (2) The cell seeding density is 1×10 6 cells / mL, and the medium was replaced every 2-3 days until the cells reached 80%-90% confluence.

[0034] 3. Maturation of DCs

[0035] (1) On the 7th day, when the DC cells were cultured to a certain density (about 80% fusion), conventional LPS (final concentration of 1 μg / mL) was added for 24 hours to induce the maturation of DC cells, thus preparing DC cells.

[0036] (2) On the 7th day of culture, when the DC cells were cultured to about 80% confluence, anti-CD47 single domain antibody was added at a final concentration of 1 μg / mL for 24 hours to induce DC cell maturation.

[0037] (3) On day 7, when DC cells were cultured to a certain density (about 80% fusion), commercial anti-CD47 monoclonal antibody (ab300124) (final concentration of 1 μg / mL) was added for 24 hours to induce DC cell maturation.

[0038] 4. Identification of DCs

[0039] (1) Flow cytometry was used to detect the expression levels of CD80, CD86, and HLA-DR, the maturation markers of DC cells induced by different methods in step 3. The results are shown in Table 1. The anti-CD47 single domain antibody group had a good effect.

[0040] Table 1 Detection results of CD80, CD86 and HLA-DR expression levels

[0041] Group CD80 CD86 HLA-DR Cell morphology changes LPS group 93％ 92％ 93％ Obvious dendritic morphology Anti-CD47 single domain antibody panel 96％ 95％ 95％ Dendritic morphology is very obvious Anti-CD47 monoclonal antibody panel 85％ 82％ 87％ Slightly dendritic morphology

[0042] (2) Cell morphology was observed under an optical microscope. Mature DC cells in the anti-CD47 single-domain antibody group showed a dendritic morphology ( Figure 1 and Table 1), the cell number doubled within 7 days and the purity reached more than 90%.

[0043] Example 2: Preparation of anti-CD47 single domain antibody

[0044] 1. Immunization and vaccine preparation

[0045] First, the recombinant human CD47 protein (ab220594) was emulsified with FCA to prepare the vaccine, ensuring that each milliliter of the vaccine contained 0.5 mg of CD47 protein. The vaccine was injected subcutaneously into the shark, with each injection volume of 0.5 mL. Two weeks after the first immunization, the second immunization was performed using the same dose of recombinant CD47 protein and FIA emulsified vaccine as the first immunization. Two weeks later, the third immunization was performed in the same manner and dose. Within seven days after the third immunization, shark peripheral blood was collected and lymphocytes were isolated. Total B cell RNA was extracted by the TRIzol method, and the RNA was reverse transcribed into cDNA according to the kit instructions.

[0046] Specific primers were designed to amplify the shark single domain antibody VHH fragment, using the following primers: upstream primer: 5'-gtcctggctcagggagagcgg t-3'; downstream primer: 5'-ggtacggttggcctgttgagg-3'. The VHH fragment was amplified using PCR, and the PCR conditions were: 94°C pre-denaturation for 5 minutes, followed by 35 cycles (94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute), and finally extended at 72°C for 5 minutes.

[0047] 2. Cloning and Sequence Analysis

[0048] The amplified VHH fragment was cloned into the pGEM-T vector and transformed into DH5α E. coli competent cells. After selecting the positive clones, the plasmid was extracted and sent for sequencing to analyze the obtained VHH sequence. Through sequence analysis, the VHH sequence of the anti-CD47 single domain antibody is shown in SEQ ID NO.1. The specific information is as follows:

[0049] (1) FR1 (framework region 1): ARVDTQTPRT, this sequence is usually located at the N-terminus of VHH, is framework region 1, the main structural stabilizing region. It has no special functional role, but maintains the stability of the antibody structure.

[0050] (2) CDR1 (complementarity determining region 1): ITKETGSLTI, CDR1 is usually involved in antigen recognition, especially contact with antigen epitopes. In this region, changes in amino acids play an important role in the specificity of antibodies.

[0051] (3) FR2 (framework region 2): NCVLKDSKGT, which is another framework region of the antibody and is responsible for maintaining the overall structure of the antibody, especially the synergistic effect with CDR2.

[0052] (4) CDR2 (complementarity determining region 2): YWYLTKFDAT, this is the second complementarity determining region, which is directly involved in binding to the antigen. CDR2 plays a key role in antigen selectivity.

[0053] (5) FR3 (framework region 3): KWDKASLSTS, FR3 is the framework region of the antibody and supports the stable structure of CDR3.

[0054] (6) CDR3 (complementarity determining region 3): GRYSGTKVNK, CDR3 is the most important complementarity determining region, usually the key region for antibody recognition and binding to antigens. It usually has high variability to adapt to different antigens.

[0055] (7) Framework region (FR4): GSKSFSLRTY HCEAYSLRPE ANDCDDRRCG WDDDCHGLGR GTFTGGGTILTV, which is another framework region of the VHH antibody and usually plays a supporting role in the structure of the antibody.

[0056] 3. Expression and Purification

[0057] The VHH sequence of the anti-CD47 single domain antibody was cloned into the pET28a vector and transformed into Escherichia coli BL21 (DE3) competent cells. When the cells were cultured at 37°C to an OD600 of 0.6, 1mM IPTG was added for induction, and the cells were collected after 4 hours of culture. Soluble protein was obtained by ultrasonic fragmentation, and the anti-CD47 single domain antibody was purified by nickel column affinity chromatography using His-tag. The purified antibody was dialyzed to remove salt, sterilized through a 220nm filter membrane, and aliquoted and frozen at -80°C for use.

[0058] 4. Purity analysis and functional verification

[0059] The purity of the anti-CD47 single domain antibody was analyzed by SDS-PAGE. Figure 2 Subsequently, the antibody was used to perform Western blot detection on the recombinant CD47 protein, and the results showed that the single-domain antibody could specifically bind to the CD47 protein (the molecular weight of the CD47 protein is about 17 kDa), as shown in FIG. Figure 3 Based on the purification records, the expression level of the single domain antibody was calculated and found to be above 1 g / L, indicating that it has a high expression yield and is suitable for large-scale application.

[0060] Example 3: Preparation of anti-tumor cell preparation

[0061] 1. Tumor antigen loading

[0062] (1) Source of tumor antigens: Human lung cancer cell line A549 was used as the experimental model, and tumor antigens were extracted from A549 cells. The details are as follows:

[0063] A549 cells were cultured to 80% confluence, washed with cold PBS, lysed with RIPA lysis buffer, incubated on ice for 30 minutes, centrifuged (12000g, 5 minutes, 4°C), and the supernatant was collected. The antigen concentration was determined using the BCA protein quantification method, and the total protein concentration in the lysate was 1.2 mg / mL.

[0064] (2) Antigen loading method: Mature DC cells (1×10 6 Cells) were incubated with tumor antigens (total protein: 100 μg) at a ratio of 1:10 for 24 hours at 37°C. Two groups of experiments were set up at the same time, as shown below:

[0065] Experimental group: DC cells loaded with tumor antigens were co-cultured with T cells.

[0066] Control group: DC cells without tumor antigen loading were co-cultured with T cells.

[0067] (3) Antigen uptake efficiency detection: Flow cytometry was used to detect the antigens bound to the surface of DC cells loaded with antigens. The results showed that about 80% of DC cells could take up and display tumor antigens, and the antigen binding rate was significantly increased compared with DC cells not loaded with antigens (P<0.01).

[0068] 2. Co-culture of DCs and tumor-specific T cells

[0069] (1) Isolation and activation of T cells: CD8 T cells isolated from peripheral blood of healthy donors were used. + T cells were purified using immunomagnetic separation (Miltenyi Biotec). The activation method was as follows:

[0070] Activation method: T cells were incubated with commercial anti-CD3 antibody (10 μg / mL, ab16669) or anti-CD3 single domain antibody (10 μg / mL, the specific preparation method is shown in Example 2, wherein the VHH sequence of the anti-CD3 single domain antibody is shown in SEQ ID NO.2) for 24 hours, and the activated T cells were used for subsequent experiments.

[0071] (2) Co-culture conditions: DC cells loaded with tumor antigens (1×10 6 Cells) and tumor-specific T cells (1×10 cells) were co-cultured at a ratio of 1:10. The co-culture conditions were as follows:

[0072] Culture conditions: 37°C, 5% CO2, culture medium is RPMI-1640 (10% FBS, 1% penicillin-streptomycin), co-culture time is 72 hours.

[0073] 3. T cell proliferation detection and cell killing activity assessment

[0074] (1) T cell proliferation detection: T cells were labeled using CFSE staining (5 μM CFSE was added per million cells), and the proliferation of T cells was detected by flow cytometry. The results showed that after 72 hours of co-culture, the proliferation rate of T cells in the experimental group (activated by anti-CD3 single domain antibody) was 40%, the proliferation rate of T cells in the experimental group (activated by commercial anti-CD3 antibody) was 29% (P<0.05), and the control group was only 15% (P<0.01). This result shows that DC cells loaded with tumor antigens can effectively activate the proliferation of tumor-specific T cells, and the effect of using anti-CD3 single domain antibodies is better.

[0075] (2) Evaluation of cell killing activity: The LDH release method was used to evaluate the killing activity of T cells against A549 tumor cells. A549 cells were inoculated in a 96-well plate and co-cultured with T cells for 72 hours after 24 hours. The results showed that the killing rate of T cells in the experimental group was significantly higher than that in the control group (P<0.05), indicating that DC cells loaded with tumor antigens can effectively enhance the killing activity of T cells. The details are as follows:

[0076] Experimental group 1: DC-T cell (anti-CD3 single domain antibody activated) co-culture group, the killing rate of T cells on A549 cells was 60%.

[0077] Experimental group 2: DC-T cell (activated by commercial anti-CD3 antibody) co-culture group, the killing rate of T cells on A549 cells was 48%.

[0078] Control group: DC cells without antigen loading were co-cultured with T cells, and the killing rate of T cells on A549 cells was 25%.

[0079] Example 4: Mouse tumor model experiment

[0080] 1. Establishment of mouse model

[0081] (1) Model animals: 6-8 week old C57BL / 6 mice (from the same batch) were selected and divided into 4 groups (10 mice in each group). Adaptive feeding and management were performed one week before the experiment.

[0082] (2) Tumor inoculation: A549 cells were used to construct a tumor mouse model. Each mouse was subcutaneously injected with 1×10 6 A549 cells, tumor cell suspension (200 μL) was inoculated by subcutaneous injection.

[0083] (3) Tumor growth monitoring: The tumor volume of mice was measured every 3 days (V = 0.5 × length × width 2). The growth of tumor volume was used as an indicator of tumor progression.

[0084] 2. Treatment plan for the experimental group and the control group: The best DC-T cell preparation of the experimental group 1 prepared above was used for the experiment, as shown below.

[0085] (1) Experimental group: On the 7th day after tumor inoculation, mice were injected with DC-T cell co-culture preparation loaded with tumor antigen (1×10 6 DC-T cells). Injection once a week for 4 weeks.

[0086] (2) Control group: On the 7th day after tumor inoculation, mice were injected with DC-T cell co-culture preparations (1×10 6 DC-T cells). Injection once a week for 4 weeks.

[0087] (3) Blank group: On the 7th day after tumor inoculation, mice were injected with normal saline.

[0088] (4) Positive control group: On the 7th day after tumor inoculation, mice were injected with the chemotherapy drug cisplatin (5 mg / kg, ip) once a week for 4 weeks.

[0089] 3. Tumor growth monitoring and analysis

[0090] (1) Tumor volume monitoring: On the 14th day after treatment, the tumor volume of mice in the experimental group was 350 mm 3 , while the tumor volume of the control group mice was 800mm 3 The tumor volume of mice in the blank group was 900 mm 3 (P<0.01). On the 28th day of treatment, the tumor volume growth rate of mice in the experimental group was 20%, that of the control group was 60%, and that of the blank group was 70%.

[0091] (2) Survival rate analysis: The survival rate of mice in the experimental group was 80% 28 days after treatment, while that in the control group and the blank group was 40% and 30%, respectively (P<0.01).

[0092] 4. Experimental conclusion

[0093] The DC-T cell co-culture preparation loaded with tumor antigens significantly enhanced the tumor-specific immune response in mice and effectively inhibited tumor growth. The DC cells loaded with tumor antigens and T cells acted together to significantly improve the survival rate of mice and effectively inhibited tumor growth. The therapeutic effect was significantly better than that of the control group and the blank group.

[0094] The above results show that DC cells loaded with tumor antigens effectively activate the proliferation and killing activity of T cells in vitro, significantly inhibit tumor growth in mouse models, and can enhance the infiltration and action of T cells in tumor tissues, thereby improving the effect of anti-tumor immunotherapy.

[0095] 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 preparing DC cells, characterized in that: The method comprises the following steps: (1) Isolation of peripheral blood mononuclear cells: Take 30 mL of whole blood, add Ficoll-Paque at a concentration of 1.077 g / mL, centrifuge at 1000 × g for 20 min, and collect the middle mononuclear cell layer after centrifugation, which is PBMC; (2) DC cell induction culture: The isolated PBMCs were inoculated in a culture dish with a culture medium of RPMI-1640 containing 10% FBS and 1% penicillin-streptomycin, supplemented with cytokines at a final concentration of 50 ng / mL IL-4 and 100 ng / mL GM-CSF for DC cell induction culture. The cell inoculation density was 1×10 6 cells / mL, and the medium was replaced every 2-3 days until the cells reached 80%-90% confluence; (3) Maturation of DC cells: On the 7th day of culture, when the DC cells were cultured to about 80% confluence, anti-CD47 single domain antibody with a final concentration of 1 μg / mL or LPS with a final concentration of 1 μg / mL was added and treated for 24 hours to induce the maturation of DC cells, thereby preparing DC cells.

2. The method according to claim 1, characterized in that The VHH sequence of the anti-CD47 single-domain antibody is shown in SEQ ID NO.

1.

3. A method for preparing an anti-tumor cell preparation, characterized in that: The method comprises the following steps: (1) extracting tumor antigens from human lung cancer cell line A549, wherein the tumor antigens are extracted using RIPA lysate and quantified using the BCA method, wherein the total protein concentration in the lysate is 1.2 mg / mL; (2) The mature DC cells prepared in claim 1 are co-incubated with tumor antigens, wherein the ratio of the total protein of the mature DC cells to the total protein of the tumor antigen is 1×10 6 Cells: 100 μg; incubation time: 24 hours, temperature: 37°C; (3) Co-culture DC cells loaded with tumor antigens with tumor-specific T cells to promote T cell proliferation and cytotoxic activity.

4. The method according to claim 3, characterized in that The co-culture ratio of the tumor antigen-loaded DC cells and T cells was 1:10, and the culture time was 72 hours.

5. The method according to claim 3, characterized in that: The T cells were purified by immunomagnetic separation and activated by anti-CD3 single domain antibodies.

6. The method according to claim 5, characterized in that The VHH sequence of the anti-CD3 single domain antibody is shown in SEQ ID NO.

2.

7. Use of the anti-CD47 single domain antibody as claimed in claim 2 in preparing DC cells.

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