Autoserum antigen sensitized DC-CIK cell as well as preparation method and application thereof

Abstract

The invention provides a method for preparing autoserum antigen-sensitized DC-CIK cells, which comprises the following steps: (1) preparation of autoserum: collecting peripheral blood of a patient, standing and centrifuging, and collecting supernatant to obtain the autoserum; (2) separating the PBMCs: separating the PBMCs from the peripheral blood of the patient by using a Ficoll-Paque separating medium to obtain the PBMCs; (3) preparing DC cells: culturing PBMCs, adding GM-CSF and IL-4 to induce DC differentiation, and then adding TNF-alpha and an autoserum antigen to promote DC maturation; (4) preparation of CIK cells: culturing PBMCs through IFN-gamma, an anti-CD3 monoclonal antibody and IL-2, and inducing generation of the CIK cells; and (5) co-culture of the DC-CIK cells: co-culturing the mature DC cells and the CIK cells in proportion, and adding GM-CSF, IL-2 and anti-CD3 monoclonal antibodies to promote cell proliferation and activation so as to obtain the autoserum antigen sensitized DC-CIK cells. The autoserum antigen sensitized DC-CIK cell provided by the invention has higher specificity and safety, and the effect of tumor immunotherapy is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing autologous serum antigen-sensitized dendritic cells (DC) and cytokine-induced killer cells (CIK) and their application in anti-tumor immunotherapy. Background Art

[0002] Dendritic cells (DC) are the most effective antigen-presenting cells in the body, which can activate initial T cells and initiate and regulate immune responses. CIK cells are a group of cells with high anti-tumor activity, which exert their effects by releasing a large number of cytokines (such as IFN-γ). Traditional DC-CIK cell therapy has achieved certain therapeutic effects in tumor treatment, but there are problems such as poor antigen specificity and weak targeting.

[0003] The invention uses tumor-related antigens in autologous serum to sensitize DC-CIK cells, thereby improving the antigen specificity and anti-tumor activity of the cells. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a method for sensitizing DC-CIK cells by antigens in autologous serum, which significantly improves the antigen specificity and anti-tumor activity of DC-CIK cells. The present invention also relates to a method for preparing such cells and their application in tumor immunotherapy.

[0005] Therefore, the present invention discloses a method for preparing DC-CIK cells sensitized with autologous serum antigens, the method comprising the following steps:

[0006] (1) Preparation of autologous serum: collect peripheral blood from the patient, let it stand and centrifuge it, and collect the supernatant to obtain autologous serum;

[0007] (2) Isolation of PBMCs: PBMCs were isolated from the patient's peripheral blood using Ficoll-Paque separation medium;

[0008] (3) Preparation of DC cells: PBMCs were cultured and GM-CSF and IL-4 were added to induce DC differentiation, followed by the addition of TNF-α and autologous serum antigens to promote DC maturation;

[0009] (4) Preparation of CIK cells: PBMCs were cultured with IFN-γ, anti-CD3 monoclonal antibody, and IL-2 to induce CIK cell production;

[0010] (5) Co-culture of DC-CIK cells: Mature DC cells are co-cultured with CIK cells in a certain proportion, and GM-CSF, IL-2 and anti-CD3 monoclonal antibodies are added to promote cell proliferation and activation, thereby obtaining DC-CIK cells sensitized with autologous serum antigens.

[0011] Preferably, the concentrations of GM-CSF, IL-4 and TNF-α used in the preparation of DC cells in step (3) of the present invention are 1000 U / ml, 500 U / ml and 1000 U / ml respectively.

[0012] Preferably, the concentration of the autologous serum antigen used in the preparation of DC cells in step (3) of the present invention is 10% by volume.

[0013] Preferably, in the preparation of CIK cells in step (4) of the present invention, the concentrations of IFN-γ, anti-CD3 monoclonal antibody and IL-2 are 1000 U / ml, 50 ng / ml and 1000 U / ml respectively.

[0014] Preferably, in the step (5) of the present invention, the co-culture ratio of DC cells to CIK cells during the co-culture of DC-CIK cells is 1:5 to 1:10.

[0015] Preferably, the concentrations of GM-CSF, IL-2 and anti-CD3 monoclonal antibody used in the co-culture of DC-CIK cells in step (5) of the present invention are 500 U / ml, 1000 U / ml and 10 ng / ml respectively.

[0016] Preferably, the amino acid sequences of the heavy chain and light chain variable regions of the anti-CD3 monoclonal antibody of the present invention are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0017] In another aspect, the present invention also discloses a use of the autologous serum antigen-sensitized DC-CIK cells in the preparation of anti-tumor drugs.

[0018] Compared with traditional DC-CIK treatment regimens, the autologous serum antigen-sensitized DC-CIK cells provided by the present invention have higher specificity and safety, significantly improve the effect of tumor immunotherapy, have good application prospects, and provide a good reference and research direction for the treatment research of autologous serum antigen-sensitized DC-CIK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 3. Anti-CD3 monoclonal antibody SDS-PAGE detection results. DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] Example 1: Preparation and functional detection of DC-CIK cells sensitized with autologous serum antigens

[0023] 1. Preparation of autologous serum

[0024] A 50-year-old male liver cancer patient (with the patient's consent and in compliance with ethical requirements) was collected, and 50 ml of peripheral blood was collected. The collected blood was allowed to stand at 4°C for 30 minutes, centrifuged (3000 rpm, 10 min), and the supernatant was collected as autologous serum.

[0025] 2. Isolation of PBMCs

[0026] (1) Dilute the collected blood with sterile PBS at a ratio of 1:1. For example, take 20 ml of blood, add 20 ml of sterile PBS, and mix gently.

[0027] (2) Take a clean 50ml centrifuge tube and add 15ml of Ficoll-Paque separation solution. Carefully add the diluted blood slowly onto the upper layer of Ficoll solution, taking care to keep the interface between the two layers clear and avoid mixing.

[0028] (3) Centrifuge at 800 g for 20-30 minutes, set the temperature to room temperature (18-25°C), and set the centrifuge to low acceleration and low braking. After centrifugation, the following four layers will be formed: the top layer (plasma and PBS layer), the middle milky white layer (PBMCs (mononuclear cell layer)), the Ficoll layer (separation fluid), and the bottom (red blood cells and granulocytes).

[0029] (4) Use a Pasteur pipette or a pipette to carefully aspirate the middle milky white PBMC layer and transfer it to a new 15 ml centrifuge tube. Add 10 ml of PBS to the collected PBMC layer, mix gently, and centrifuge at 1000 rpm for 5 minutes. Repeat this step 2-3 times to remove residual plasma, Ficoll separation solution, and other impurities.

[0030] (5) Finally, PBMCs were resuspended in RPMI-1640 medium (containing 10% FBS). PBMCs were counted using the trypan blue exclusion method to ensure that the cell viability was above 90%. Usually, about 5×10 6 Up to 2×10 7 PBMCs.

[0031] (6) If cryopreservation is required, PBMCs were suspended in a cryopreservation solution containing 10% DMSO and 90% FBS at a volume of 1 × 10 7 The cells were divided into aliquots at a concentration of 10 cells / ml, gradually cooled to -80°C, and then transferred to liquid nitrogen for long-term storage.

[0032] 3. DC cell preparation

[0033] (1) The isolated PBMCs were divided into 1×10 6 Inoculate the cells at a density of 100 / ml in RPMI-1640 medium containing 10% FBS. Add GM-CSF at a final concentration of 1000U / ml and IL-4 at 500U / ml, mix gently, and place the culture flask at 37°C and 5% CO 2 Culture in an incubator.

[0034] (2) On the third day of culture, the supernatant was carefully discarded, fresh RPMI-1640 medium containing 10% FBS was added, and GM-CSF and IL-4 were supplemented to the original concentrations. The cells were placed at 37°C and 5% CO. 2 The cells were cultured in the incubator for 3 days.

[0035] (3) On the sixth day, the semi-suspended cells were collected from the culture flask. At this time, the cells showed the morphology of immature DCs. TNF-α with a final concentration of 1000 U / ml and autologous serum antigen (serum concentration was 10% by volume) were added to the culture medium and cultured for 48 hours to induce DC maturation.

[0036] (4) If storage is required, DC cells can be cryopreserved in a freezing solution of 10% DMSO and 90% FBS. Gradually freeze to -80°C and transfer to liquid nitrogen for long-term storage.

[0037] 4. CIK cell preparation

[0038] (1) PBMCs were cultured at 1×10 6 The cells were inoculated at a density of 1000 / ml into a culture flask containing RPMI-1640 medium containing 10% FBS, and IFN-γ was added at a final concentration of 1000 U / ml. After mixing, the cells were placed at 37°C and 5% CO 2 Incubate in an incubator for 24 hours.

[0039] (2) After 24 hours, anti-CD3 monoclonal antibody (prepared and identified for this study, see Example 2 for specific methods, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively) or commercial anti-CD3 monoclonal antibody (ab135372) was added at a final concentration of 50 ng / ml and 1000 U / ml of IL-2 was continued to culture.

[0040] (3) Fresh culture medium was added every 3 days, and IL-2 was added to a final concentration of 1000 U / ml. The cell density should be maintained at 1×10 6 Up to 2×10 6 If necessary, the cell suspension can be diluted by adding fresh culture medium.

[0041] (4) CIK cells reach their peak on the 14th to 21st day of culture, showing significant killing activity. The culture is terminated at this stage, and all cells are collected for subsequent application or analysis.

[0042] (5) Use freezing solution containing 10% DMSO and 90% FBS, and add 1×10 7 After the cells were divided into aliquots, they were rapidly cooled to -80°C and transferred to liquid nitrogen for long-term storage.

[0043] 5. Co-culture of DC-CIK cells

[0044] (1) DC cells and CIK cells are mixed at a ratio of 1:5 to 1:10. For example, 1×10 5 DC cells and 5×10 5 Up to 1×10 6 Add the mixed cell suspension into a T25 or T75 culture flask and mix gently.

[0045] (2) In the co-culture system, GM-CSF with a final concentration of 500 U / ml, IL-2 with a final concentration of 1000 U / ml, and anti-CD3 monoclonal antibody with a final concentration of 10 ng / ml were added (addition or non-addition was optional, and the test results of the cells prepared in this way are shown in Tables 1 and 2 for details) to promote cell proliferation and activation.

[0046] (3) Place the culture flask at 37°C and 5% CO 2 The cells were cultured in an incubator for 3 to 5 days. The culture medium was replaced every 2 to 3 days and supplemented with fresh GM-CSF with a final concentration of 500 U / ml, 1000 U / ml IL-2, and 10 ng / ml anti-CD3 monoclonal antibody.

[0047] (4) Around the fifth day of culture, the DC-CIK cells in the co-culture system have strong anti-tumor activity. The cells are washed with PBS and the co-culture cell population is collected.

[0048] (5) Use trypan blue staining or other viability detection methods to evaluate the number and viability of DC-CIK cells, which generally requires a viability of more than 85%.

[0049] (6) If long-term storage is required, DC-CIK cells can be cryopreserved in a freezing solution of 10% DMSO and 90% FBS. Gradually freeze to -80°C and then transfer to liquid nitrogen for long-term storage.

[0050] 6. Functional detection: The LDH release method was used to detect the specific killing activity of DC-CIK cells against donor tumor cells, as shown below:

[0051] (1) Culture the donor tumor cell line (such as HepG2) in RPMI-1640 medium containing 10% FBS at 37°C and 5% CO 2 When the cells reached the logarithmic growth phase, they were digested with trypsin, washed with PBS, and resuspended in RPMI-1640 medium to adjust the cell concentration to 1×10 6 In a 96-well plate, 100 μl of tumor cell suspension (about 1×10 5 cells / well) and cultured overnight to ensure cell attachment.

[0052] (2) The DC-CIK cells prepared above were washed with PBS and the cell concentration was adjusted to a suitable effector-target ratio (eg, 10:1, 20:1, 40:1, etc.). In this study, 40:1 was selected.

[0053] (3) According to the set effector-target ratio, add the DC-CIK cell suspension to the plated donor tumor cells, 200 μl per well, and set up at least 3 replicate wells per group. Place the 96-well plate at 37°C, 5% CO 2 Incubate in an incubator for 4-6 hours. As a control, set up the following groups:

[0054] Target cell spontaneous release group: only donor tumor cells were added without effector cells.

[0055] Effector cell spontaneous release group: only DC-CIK cells were added without target cells.

[0056] Maximum release group: Lysis buffer was added to the target cell wells to maximize LDH release.

[0057] (4) After the incubation, carefully aspirate the supernatant in the 96-well plate and transfer it to a new 96-well plate for LDH detection. Follow the instructions of the LDH release detection kit, add the detection reagent to the new 96-well plate, incubate for an appropriate time, and measure the absorbance at a wavelength of 490 nm using an ELISA reader, with a wavelength of 600 nm as the reference wavelength.

[0058] (5) The killing rate of DC-CIK cells against donor tumor cells at different effector-target ratios can be used to evaluate their specific killing activity. The calculation formula is:

[0059]

[0060] (6) The results showed (as shown in Table 1) that the cytotoxic activity of sensitized DC-CIK cells against tumor cells was significantly enhanced compared with that of unsensitized DC-CIK cells (P<0.01), and the results of sensitized DC-CIK cells prepared using the anti-CD3 monoclonal antibody prepared in this study were even better.

[0061] Table 1 Functional test results

[0062]

[0063] Example 2: Preparation and testing of anti-CD3 monoclonal antibodies

[0064] 1. Select 8-10 week old SPF grade Balb / c mice, healthy and free of specific pathogens. Mix recombinant human CD3 protein (ab114153) with Freund's complete adjuvant in a 1:1 ratio and inject intraperitoneally into the mice to immunize them, 0.2 ml / mouse (the concentration of recombinant protein in the vaccine is 50 μg). Perform three immunizations on days 0, 14, and 28, with an interval of 14 days each. Three days after the fourth immunization, collect the spleen of the mice for cell fusion.

[0065] 2. Collect spleens from immunized mice and separate spleen cells. Mix spleen cells with SP2 / 0 myeloma cells at a ratio of 10:1. Add appropriate amount of PEG-1500 and incubate at 37°C for 2 minutes for cell fusion. Transfer fused cells to HAT medium for screening and select positive clones.

[0066] 3. Select positive clones one by one, expand and culture. Use indirect ELISA to screen cell lines that produce high-affinity anti-CD3 antibodies. Obtain stable and high-yield monoclonal cell lines through subcloning. Collect the supernatant and purify the antibody by Protein G affinity chromatography. After purification, dialyze to remove salts and detect purity by SDS-PAGE (such as Figure 1After the above steps, an anti-CD3 monoclonal antibody was prepared. The amino acid sequences of the heavy chain variable region and the light chain variable region were shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0067] 4. The antibody titer was detected by ELISA method, which is briefly described as follows: the recombinant human CD3 protein was coated on a 96-well plate (1 μg / ml) and incubated at 4°C overnight. After washing, anti-CD3 monoclonal antibodies of different dilution multiples were added (the concentration of the monoclonal antibodies was adjusted to 1 mg / ml before dilution) for incubation. HRP-labeled secondary antibody was added, and TMB substrate was added for color development after incubation at room temperature. The OD value was read at a wavelength of 450 nm using an enzyme reader to calculate the antibody titer. The results showed that the OD450nm value of the anti-CD3 monoclonal antibody prepared by the present invention was still 2.12 when diluted 20,000 times, while the OD450nm value of the commercial anti-CD3 monoclonal antibody (ab135372) was only 0.78 when diluted 20,000 times, which shows that the anti-CD3 monoclonal antibody prepared by the present invention has better activity.

[0068] 5. Flow cytometry was used to detect the presence of HepG2 cells. The cells were washed and diluted to a certain concentration (usually 1×10 6 / ml) and then incubated with anti-CD3 monoclonal antibody. Add fluorescent labeled secondary antibody and incubate in the dark. Detect antibody binding by flow cytometry and analyze CD3 expression. The results show that the anti-CD3 monoclonal antibody prepared by the present invention can specifically bind to the CD3 antigen on the surface of HepG2 cells, with a positive rate of more than 85%, while the positive rate of commercial anti-CD3 monoclonal antibody (ab135372) is only 68%, indicating that the anti-CD3 monoclonal antibody prepared by the present invention has better specificity and activity.

[0069] Example 3: Anti-tumor effect of DC-CIK cells sensitized with autologous serum antigens in animal models

[0070] 1. Select 4-6 week old BALB / c nude mice and subcutaneously inject human liver cancer cell line HepG2 cells to establish human liver cancer nude mouse model. The brief description is as follows:

[0071] When HepG2 cells reach 80-90% confluence, discard the culture medium and add an appropriate amount of 0.25% trypsin to digest the cells. After the cells fall off, add DMEM culture medium containing 10% FBS to terminate the digestion. Transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend in an appropriate amount of PBS. Use a 1 ml sterile syringe to inject 100 μl of HepG2 cell suspension (containing about 2×10 6The mice were injected subcutaneously with 100 cells of 50 μg / mL of 1 ... 3 When the time is about 2 seconds, you can proceed with subsequent experimental operations.

[0072] 2. When the tumor volume reaches 100 mm 3 The experimental groups were injected with DC-CIK cells sensitized with autologous serum antigens prepared in Table 1 (2×10 7 The control group was injected with unsensitized DC-CIK cells (2×10 7 The blank group was injected with PBS. The treatment was performed once a week for a total of 4 times.

[0073] 3. Tumor inhibition rate: The tumor volume in the experimental group was significantly smaller than that in the control group and the blank group, and the effect of DC-CIK cells sensitized with autologous serum antigen prepared with anti-CD3 monoclonal antibody in this study was better. See Table 2 for details.

[0074] 4. Survival rate: The survival rate of mice in the experimental group was significantly higher than that in the control group and the blank group, and the effect of DC-CIK cells sensitized with autologous serum antigen prepared with anti-CD3 monoclonal antibody in this study was better, as shown in Table 2.

[0075] Table 2 Antitumor effect results in mice

[0076]

[0077] 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-CIK cells sensitized with autologous serum antigens, characterized in that: The method comprises the following steps: (1) Preparation of autologous serum: collect peripheral blood from the patient, let it stand and centrifuge it, and collect the supernatant to obtain autologous serum; (2) Isolation of PBMCs: PBMCs were isolated from the patient's peripheral blood using Ficoll-Paque separation medium; (3) Preparation of DC cells: PBMCs were cultured and GM-CSF and IL-4 were added to induce DC differentiation, followed by the addition of TNF-α and autologous serum antigens to promote DC maturation; (4) Preparation of CIK cells: PBMCs were cultured with IFN-γ, anti-CD3 monoclonal antibody, and IL-2 to induce CIK cell production; (5) Co-culture of DC-CIK cells: Mature DC cells are co-cultured with CIK cells in a certain proportion, and GM-CSF, IL-2 and anti-CD3 monoclonal antibodies are added to promote cell proliferation and activation, thereby obtaining DC-CIK cells sensitized with autologous serum antigens.

2. The method according to claim 1, characterized in that The concentrations of GM-CSF, IL-4 and TNF-α used in the preparation of DC cells in step (3) are 1000 U / ml, 500 U / ml and 1000 U / ml respectively.

3. The method according to claim 1, characterized in that The concentration of the autologous serum antigen used in the preparation of DC cells in step (3) is 10% by volume.

4. The method according to claim 1, characterized in that The concentrations of IFN-γ, anti-CD3 monoclonal antibody and IL-2 used in the preparation of CIK cells in step (4) are 1000 U / ml, 50 ng / ml and 1000 U / ml respectively.

5. The method according to claim 1, characterized in that: In the step (5) of co-culturing DC-CIK cells, the co-culture ratio of DC cells to CIK cells is 1:5 to 1:

10.

6. The method according to claim 1, characterized in that The concentrations of GM-CSF, IL-2 and anti-CD3 monoclonal antibody used in the co-culture of DC-CIK cells in step (5) are 500 U / ml, 1000 U / ml and 10 ng / ml respectively.

7. The method according to claim 1, characterized in that The amino acid sequences of the heavy chain and light chain variable regions of the anti-CD3 monoclonal antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

8. Use of the DC-CIK cells sensitized by autologous serum antigens prepared according to claim 1 in the preparation of anti-tumor drugs.

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