A method for preparing DC cells and use thereof in the preparation of an antitumor cell preparation

Abstract

The application provides a preparation method of DC cells, and is especially suitable for rapid obtaining of high-purity and high-functionality DC cells. The method comprises the steps of separation from peripheral blood mononuclear cells (PBMC), induced culture, maturation treatment and the like. The maturation of the DC cells is promoted by introducing an anti-CD47 single-domain antibody or LPS, and the immunocompetence of the DC cells is significantly improved. The method is further applied to preparation of an anti-tumor cell preparation, the ability of the DC cells loaded with tumor antigens to activate tumor-specific T cells is significantly enhanced, and the killing activity of the T cells to tumor cells is improved. The preparation method of the application overcomes the defects in the prior art, has the advantages of high efficiency, safety and stability, and can provide a more reliable cell treatment scheme for tumor immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cellular immunology, in particular to a preparation method of dendritic cells (DC cells), and further applied to the preparation of anti-tumor cell preparations. BACKGROUND

[0002] Dendritic cells (DC cells) are important immune system cells, playing a bridging role between innate immunity and acquired immunity. DC cells can take up and process antigens, display antigens through their unique MHC molecules on the surface, and activate T cell-mediated immune responses, so they are widely used in tumor immunotherapy, vaccine development and immune tolerance research. In recent years, dendritic cells in tumor immunotherapy have 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 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 (PBMCs) to differentiate into dendritic cells. These methods can successfully induce dendritic cell differentiation, but there are often problems in the preparation process, such as low cell purity, weak functionality, long culture time, etc. In addition, the immune activity and stability of mature dendritic cells are still technical problems in existing methods.

[0004] In order to improve the yield, purity and functional activity of dendritic cells, researchers have tried to optimize different culture media, cytokine combinations, culture times, etc., but have not yet achieved ideal results in clinical treatment. Therefore, developing a more efficient, safe and higher purity dendritic cell preparation method can better play its role in anti-tumor immunotherapy, which has become a technical problem to be solved. SUMMARY

[0005] The purpose of the present application is to provide a new dendritic cell (DC) preparation method that can obtain high-purity, high-functionality DC cells in a short time, and the method can play a role in the preparation of anti-tumor cell preparations.

[0006] Therefore, one aspect of the present application discloses a preparation method of DC cells, which comprises the following steps:

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

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

[0009] (3) DC cell maturation: on the 7th day of culture, when the DC cell culture reaches about 80% confluence, 1 μg / mL of anti-CD47 single-domain antibody or 1 μg / mL of LPS is added, and the cells are treated for 24 hours to induce the maturation of DC cells, i.e., the prepared DC cells.

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

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

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

[0013] (2) Co-incubating the mature DC cells prepared in claim 1 with tumor antigens, wherein the ratio of mature DC cells to total protein of tumor antigens is 1 x 10 6 cells: 100 μg; the incubation time is 24 hours, and the temperature is 37°C;

[0014] (3) Co-culturing the tumor antigen-loaded DC cells with tumor-specific T cells to promote the proliferation and killing activity of T cells.

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

[0016] Preferably, the T cells of the present application are purified by immunomagnetic bead separation method and activated by anti-CD3 single-domain antibody.

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

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

[0019] The dendritic cell preparation method of the present application 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 demand of clinical application. By introducing the mature induction method of anti-CD47 single domain antibody, 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 mature DC cells are used to load tumor antigens in the present application, 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 the traditional method, the DC cell preparation method provided by the present application is simple to operate, can obtain high-quality dendritic cells on a large scale and quickly, and is helpful to improve the production efficiency of therapeutic products. In addition to the application in tumor immunotherapy, the preparation method can also be widely applied in vaccine research and development, immune tolerance research and other immunotherapy fields, and provides a reliable cell preparation platform for related research. BRIEF DESCRIPTION OF DRAWINGS

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

[0023] Figure 2 SDS-PAGE detection results of anti-CD47 single domain antibody, wherein 1 and 2 are anti-CD47 single domain antibody.

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

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0026] Unless specifically noted, the reagents, methods, and equipment employed in the present application are the conventional reagents, methods, and equipment in the art. Unless specifically noted, 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 with a concentration of 1.077 g / mL, centrifuge at 1000 x g for 20 minutes, collect the middle mononuclear cell layer after centrifugation, which is PBMC.

[0030] (2) Use Trypan blue staining method, the cell count is 5 x 10 6 cells / mL, and the purity is about 90%.

[0031] 2. DC induction culture

[0032] (1) The separated PBMC is inoculated in a culture dish, and the culture medium is RPMI-1640 containing 10% FBS, 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 inoculation density is 1 x 10 6 cells / mL, and the culture medium is replaced every 2-3 days until the cells reach 80%-90% confluence.

[0034] 3. Maturation of DC cells

[0035] (1) On the 7th day, when the DC cells are cultured to a certain density (about 80% confluence), add the commonly used LPS (with a final concentration of 1 μg / mL) for 24 hours to induce the maturation of DC cells, which are the prepared DC cells.

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

[0037] (3) On the 7th day, when the DC cells are cultured to a certain density (about 80% confluence), add commercial anti-CD47 monoclonal antibody (ab300124) (with a final concentration of 1 μg / mL) for 24 hours to induce the maturation of DC cells.

[0038] 4. Identification of DC cells

[0039] (1) The expression levels of mature markers CD80, CD86, and HLA-DR of DC cells induced by different methods in step 3 are detected by flow cytometry. The results are shown in Table 1, and the anti-CD47 single-domain antibody group has good effect.

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

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

[0042] (2) By observing the cell morphology by optical microscope, the anti-CD47 single domain antibody group mature DC cells presented dendritic morphology (as shown in Table 1), the number of cells doubled within 7 days, and the purity reached more than 90%. Figure 1

[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 0.5 mg of CD47 protein was contained in each milliliter of vaccine. The vaccine was injected subcutaneously into the shark, with each injection amounting to 0.5 mL. Two weeks after the first immunization, a second immunization was performed using the same dose of recombinant CD47 protein emulsified with FIA as the first immunization. Two weeks later, a third immunization was performed in the same manner and dose. Within seven days after the third immunization, the peripheral blood of the shark was collected and the lymphocytes were isolated. The total RNA of B cells 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, with the following PCR conditions: pre-denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, and finally extension 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 positive clones, plasmids were extracted and sent for sequencing, and the obtained VHH sequence was analyzed. Through sequence analysis, the VHH sequence of the anti-CD47 single domain antibody is shown as 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 and is the framework region 1, which is a major structural stabilizing region. It does not have a specific functional role, but maintains the stability of the antibody structure.

[0050] ​(2) CDR1 (Complementarity Determining Region 1): ITKETGSLTI, CDR1 is usually involved in the recognition of antigens, especially the contact with the antigen epitope. In this region, the change of amino acid plays an important role in the specificity of the antibody.

[0051] (3) FR2 (Framework Region 2): NCVLKDSKGT, this is another framework region of the antibody, responsible for maintaining the overall structure of the antibody, especially in cooperation with CDR2.

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

[0053] (5) FR3 (Framework Region 3): KWDKASLSTS, FR3 is a framework region of the antibody, supporting the stable structure of CDR3.

[0054] (6) CDR3 (Complementarity Determining Region 3): GRYSGTKVNK, CDR3 is the most important complementarity determining region, which is usually the key region for the antibody to recognize and bind to the antigen. It usually has high variability to adapt to different antigens.

[0055] (7) Framework Region (FR4): GSKSFSLRTY HCEAYSLRPE ANDCDDRRCG WDDDCHGLGR GTFTGGGTILTV, this is another framework region of the VHH antibody, which 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 E. coli BL21 (DE3) competent cells. When the cells were cultured at 37°C to OD600 of 0.6, 1 mM IPTG was added for induction, and the cells were collected after 4 hours of culture. The soluble protein was obtained by ultrasonic crushing method, and the His-tag was used for nickel column affinity chromatography to purify the anti-CD47 single-domain antibody. The purified antibody was dialyzed, and after removing the salt, it was sterilized by 220 nm filter membrane and aliquoted and stored 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, and the results are shown in Figure 2 Subsequently, the antibody was used for Western blot detection of recombinant CD47 protein, and the results showed that the single-domain antibody could specifically bind to CD47 protein (CD47 protein molecular weight about 17 kDa), as shown inFigure 3 Based on the purification record, the expression amount of the single-domain antibody was calculated, and it was found that the expression amount reached more than 1 g / L, indicating that it had a high expression yield and was suitable for large-scale application.

[0060] Example 3: Preparation of Anti-tumor Cell Preparation

[0061] 1. Tumor Antigen Loading

[0062] (1) Tumor Antigen Source: Human lung cancer cell line A549 was used as an experimental model, and tumor antigens were extracted from A549 cells. The specific process is as follows:

[0063] A549 cells were cultured to 80% confluence, washed with cold PBS, and then 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 lysis buffer was 1.2 mg / mL.

[0064] (2) Antigen Loading Method: Mature DC cells (1 x 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 as follows:

[0065] Experimental group: Tumor antigen-loaded DC cells 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 antigen bound to the surface of antigen-loaded DC cells. The results showed that about 80% of DC cells could uptake and display tumor antigens, and the antigen binding rate was significantly increased compared with DC cells without antigen loading (P < 0.01).

[0068] 2. Co-culture of DC Cells with Tumor-specific T Cells

[0069] (1) Isolation and activation of T cells: CD8 + T cells were isolated from the peripheral blood of a healthy donor, and T cells were purified using immunomagnetic bead separation (Miltenyi Biotec). The activation method is as follows:

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

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

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

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

[0074] (1) T cell proliferation detection: T cells were labeled with CFSE staining method (5 pM 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 experimental group T cells (activated by anti-CD3 single domain antibody) was 40%, the proliferation rate of experimental group T cells (activated by commercial anti-CD3 antibody) was 29% (P < 0.05), and the control group was only 15% (P < 0.01). This result showed that tumor antigen-loaded DC cells could effectively activate the proliferation of tumor-specific T cells, and the effect of using anti-CD3 single domain antibody was better.

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

[0076] Experimental group 1: DC-T cell (activated by anti-CD3 single domain antibody) 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. Mouse model establishment

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

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

[0083] (3) Tumor growth monitoring: The tumor volume of the mice was measured every 3 days (V = 0.5 x length x width

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

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

[0086] (2) Control group: On the 7th day after tumor inoculation in mice, DC-T cell co-culture preparation without tumor antigen loading (1 x 10 6 DC-T cells) was injected. Injection was performed once a week for 4 weeks.

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

[0088] (4) Positive control group: On the 7th day after tumor inoculation in mice, the chemotherapeutic drug cisplatin (5 mg / kg, i.p.) was injected 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 the experimental group mice was 350 mm 3 , while the tumor volume of the control group mice was 800 mm 3 , and the tumor volume of the blank group mice was 900 mm 3 (P < 0.01). On the 28th day of treatment, the tumor volume growth rate of the experimental group mice was 20%, that of the control group was 60%, and that of the blank group was 70%.

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

[0092] 4. Experimental conclusion

[0093] The tumor-specific immune response in mice was significantly enhanced by the co-culture preparation of tumor antigen-loaded DC-T cells, and the tumor growth was effectively inhibited. The combination of tumor antigen-loaded DC cells and T cells significantly improved the survival rate of mice and effectively inhibited the growth of tumors, and the treatment effect was significantly better than that of the control group and the blank group.

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

[0095] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method of preparing DC cells, characterized by, The method comprises the following steps: (1) Isolation of peripheral blood mononuclear cells: take 30 mL of whole blood, add Ficoll-Paque with a concentration of 1.077 g / mL, centrifuge at 1000 x g for 20 minutes, collect the middle mononuclear cell layer after centrifugation, and the PBMC is obtained; (2) DC cell induction culture: the separated PBMC is inoculated in a culture dish, and the culture medium is RPMI-1640 containing 10% FBS, 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, and the cell inoculation density is 1 × 10 6 cells / mL, and the culture medium is replaced every 2-3 days until the cells reach 80%-90% confluence; (3) Maturation of DC cells: on the 7th day of culture, when the DC cells are cultured to 80% fusion, add anti-CD47 single-domain antibody with a final concentration of 1 μg / mL, treat for 24 hours, induce the maturation of DC cells, and the prepared DC cells are obtained; the VHH sequence of the anti-CD47 single-domain antibody is shown as SEQ ID NO.

1.

2. A method of preparing an antitumor cell preparation, characterized by, The method comprises the following steps: (1) Extract tumor antigen from human lung cancer cell line A549, the tumor antigen is extracted by RIPA lysis buffer and quantified by BCA method, wherein the total protein concentration in the lysis buffer is 1.2 mg / mL; (2) co-incubating the mature DC cells prepared in claim 1 with tumor antigens, wherein the ratio of mature DC cells to total protein of tumor antigens is 1 x 10 6 cells : 100 μg; the incubation time is 24 hours, and the temperature is 37°C; (3) Co-culture the tumor antigen-loaded DC cells with tumor-specific T cells to promote the proliferation and killing activity of T cells; the co-culture ratio of tumor antigen-loaded DC cells and T cells is 1:10, and the culture time is 72 hours; the T cells are purified by immunomagnetic bead separation method and activated by anti-CD3 single-domain antibody; the VHH sequence of the anti-CD3 single-domain antibody is shown as SEQ ID NO.

2.

3. Use of the anti-CD47 single-domain antibody of claim 1 in the preparation of DC cells.

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