A method for preparing immune cells and its use in treating diseases

Abstract

The present invention discloses a method for preparing a novel immune cell, which improves the proliferation, activity and function of immune cells by combining the stimulation of multiple cytokines, activating antibodies and small chemical molecules. The specific steps include isolating PBMCs from peripheral blood, and obtaining highly active immune cells after appropriate treatment and stimulation. Experimental results show that the prepared immune cells have a significant inhibitory effect on tumor cells in a mouse model and improve the survival rate of mice. The present invention combines the comprehensive use of growth factors, activating factors, small chemical molecules and strong stimulants to greatly improve the activity and therapeutic effect of immune cells. By optimizing the cell activation conditions, the present invention makes the prepared immune cells have stronger adaptability and effect in treatment, especially the application potential in tumor immunotherapy is significant.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for preparing novel immune cells and application thereof in immunotherapy. Background Art

[0002] In recent years, immune cell therapy has received widespread attention as an emerging tumor treatment method. Studies have shown that immune cells, especially T cells and NK cells, can effectively recognize and kill tumor cells, and therefore have great potential for application in cancer treatment. However, traditional immune cell preparation methods still have some limitations, such as insufficient cell activity and proliferation ability, resulting in unsatisfactory therapeutic effects. Existing technologies generally rely on a single stimulating factor for activation after cell culture, lacking comprehensive consideration of the cell microenvironment and multiple stimulation conditions. For example, IL-2 and IL-15 are two commonly used cytokines that can promote the proliferation and survival of T cells and NK cells, but may not fully activate the cell's killing ability when used alone. In addition, although the use of CD3 / CD28 antibody combined with antibody stimulation can effectively activate T cells, cell activity may be affected during in vitro expansion and long-term storage, resulting in unstable efficacy of the final product.

[0003] Therefore, there is an urgent need for a novel immune cell preparation method that can combine multiple stimulatory factors, optimize cell culture conditions, and enhance immune cell proliferation and function. This will provide a more effective strategy for the treatment of cancer and other immune-related diseases and promote the development of immunotherapy. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing immune cells and use thereof in treating diseases.

[0005] Therefore, in one aspect, the present invention discloses an anti-CD3+CD28 monoclonal antibody, 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. 3 and SEQ ID NO. 4, respectively.

[0006] Preferably, the monoclonal antibody of the present invention specifically binds to the CD3 protein at aa26 to aa36, and its amino acid sequence is TCPQYPGSEIL.

[0007] Preferably, the monoclonal antibody of the present invention specifically binds to the CD28 protein at aa146 to aa156, and its amino acid sequence is TCPPCPAPELL.

[0008] In another aspect, the present invention further discloses a method for preparing immune cells, comprising the following steps:

[0009] (1) Isolate PBMCs from the peripheral blood of healthy volunteers;

[0010] (2) resuspending the PBMCs in culture medium and adding growth factors IL-2 and IL-15;

[0011] (3) cell activation by anti-CD3+CD28 monoclonal antibodies;

[0012] (4) Adding the small chemical molecule Plerixafor;

[0013] (5) After 48 hours of culture, highly active immune cells were obtained.

[0014] Preferably, the concentrations of the growth factors IL-2 and IL-15 of the present invention are 20 ng / ml and 10 ng / ml, respectively.

[0015] Preferably, the concentration of the anti-CD3+CD28 monoclonal antibody of the present invention is 1 μg / ml.

[0016] Preferably, the concentration of the chemical small molecule Plerixafor of the present invention is 100 nM.

[0017] Preferably, in step (4) of the present invention, lipopolysaccharide may be further added to enhance stimulation, wherein the concentration of lipopolysaccharide is 1 μg / ml.

[0018] In another aspect, the present invention also discloses a use of the anti-CD3+CD28 monoclonal antibody in the preparation of immune cells.

[0019] The core of the present invention is to optimize the preparation process of immune cells by comprehensively using multiple stimulating factors, and to solve the problem of insufficient immune cell function in the prior art. Specifically, IL-2 and IL-15 are selected as cell growth factors, combined with anti-CD3+CD28 monoclonal antibodies for cell activation, Plerixafor is used to improve cell migration ability, and LPS is added to enhance stimulation when necessary. The experimental results show that the immune cells prepared by the present invention have significant proliferation and activity in vitro. In a mouse tumor model, after injection of the prepared immune cells, tumor growth was significantly inhibited and the survival rate was significantly improved. This provides new ideas and methods for the development of immune cell therapy, which has important application value and market prospects.

[0020] This invention combines growth factors, activating factors, small chemical molecules, and strong stimulants to significantly enhance immune cell activity and therapeutic efficacy. By optimizing cell activation conditions, the prepared immune cells have enhanced adaptability and efficacy in treatment, with significant potential for application in tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Standard curve of IFN-γ and TNF-α.

[0022] Figure 2 The results of SDS-PAGE detection of anti-CD3+CD28 monoclonal antibodies are shown in Figure 1, where 1 is the marker and 2 is the monoclonal antibody. DETAILED DESCRIPTION

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0024] 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.

[0025] Example 1: Preparation and testing of immune cells

[0026] 1. Cell Source

[0027] Peripheral blood of healthy volunteers (in compliance with ethical requirements) was selected and PBMCs (peripheral blood mononuclear cells) were isolated using Ficoll-Paque density gradient centrifugation, as briefly described below:

[0028] (1) Sample preparation: Dilute the peripheral blood sample with PBS buffer at a ratio of 1:1. For example, take 10 ml of whole blood sample and add 10 ml of PBS buffer.

[0029] (2) Ficoll-Paque density gradient centrifugation: Carefully add 4-5 ml of Ficoll-Paque to a 15 ml centrifuge tube. Slowly layer the diluted whole blood on top of the Ficoll-Paque liquid. Centrifuge at 400 g for 30 minutes at room temperature.

[0030] (3) Collect the PBMC layer: After centrifugation, carefully aspirate the white PBMC layer with a pipette and transfer the PBMC layer to a new centrifuge tube. The specific separation is as follows:

[0031] Upper layer: transparent plasma liquid (containing platelets),

[0032] Middle layer: white misty PBMC layer,

[0033] Lower layer: Ficoll-Paque liquid,

[0034] Bottom layer: red blood cell pellet.

[0035] (4) Washing: Add 10 ml of PBS buffer to the centrifuge tube containing PBMCs and mix gently. Centrifuge at 300 g for 10 minutes and discard the supernatant. Repeat the washing step twice.

[0036] (5) Counting and Storage: Washed PBMCs were resuspended in complete culture medium (RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin) and counted. The cell concentration was adjusted to 1-5 × 10^6 cells / ml for subsequent experiments.

[0037] According to the above method, approximately 5×10^6 PBMCs were obtained in each experiment, and the cell activity was approximately 90%.

[0038] 2. Initial Cell Treatment

[0039] PBMCs were collected and cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2 for 24 hours.

[0040] 3. Immune cell activation and expansion

[0041] The treated PBMCs were divided into five groups (1×10^6 cells per group):

[0042] Group A: IL-2 (20 ng / ml) and IL-15 (10 ng / ml);

[0043] Group B: anti-CD3+CD28 monoclonal antibody (1 μg / ml) (prepared in Example 3);

[0044] Group C: Group A + Group B;

[0045] Group D: Group A+B+Plerixafor (100 nM);

[0046] Group E: Group A+B+Plerixafor+LPS (1 μg / ml) served as a strong stimulation group.

[0047] All groups were cultured at 37°C and 5% CO2 for 48 hours; cells were collected for cell function tests or animal treatment studies or frozen.

[0048] 4. Cell Function Assay

[0049] (1) Cell surface markers (CD3, CD8, CD25) were detected by flow cytometry. The results of cell surface markers (percentage) are shown in Table 1.

[0050] Table 1 Detection results of cell surface markers in each group

[0051] Group CD3+ CD8+ CD25+ Group A 58.5％ 20.4％ 15.6％ Group B 57.6％ 26.3％ 12.8％ Group C 68.7％ 35.2％ 26.1％ Group D 75.2％ 41.3％ 32.5％ Group E 81.6％ 46.3％ 36.6％

[0052] (2) ELISA kit was used to detect the secretion of cytokines (IFN-γ, TNF-α). Figure 1 The standard curve of cytokines is shown in Table 2.

[0053] Table 2 Cytokine detection results of each group (pg / ml)

[0054] Group IFN-γ TNF-α Group A 58.9 28.7 Group B 56.3 26.2 Group C 101.4 52.5 Group D 155.6 72.5 Group E 226.7 105.4

[0055] (3) The CCK-8 assay was used to detect cell proliferation in each group. The results are as follows (increase times compared to the control group): Group A: 1.55 times; Group B: 1.46 times; Group C: 2.2 times; Group D: 2.8 times; Group E: 3.3 times.

[0056] The above test results show that the cells prepared by group E of the present invention are the best, followed by group D and group C, and group A and group B are basically the same with no significant difference.

[0057] Example 2: Mouse Therapeutic Studies

[0058] 1. Experimental Animal Selection: Six-week-old C57BL / 6 mice, half male and half female, weighing approximately 20-25 g, were selected and adaptively raised for one week to ensure they were in good health.

[0059] 2. Tumor Cell Inoculation: Mouse melanoma cells (B16-F10) were selected as model cells. Cells were cultured until the logarithmic growth phase, harvested, and resuspended in PBS at a concentration of 1×10^7 cells / ml. Each mouse was subcutaneously injected with 0.1 ml of the cell suspension (1×10^6 cells) into the right axilla. The injection time was recorded (experimental day 0).

[0060] 3. Immune cell preparation: Select Group C, Group D, and Group E from Example 1 and prepare immune cells respectively. Resuspend the prepared immune cells in appropriate culture medium and prepare for injection.

[0061] 4. Immune cell injection: 5 days after tumor cell inoculation (day 5 of the experiment), each group of mice was injected with different immune cells:

[0062] Group 1: no immune cells as the control group;

[0063] Group 2: Group C immune cells (1×10^6 cells);

[0064] Group 3: Group D immune cells (1×10^6 cells);

[0065] Group 4: Group E immune cells (1×10^6 cells).

[0066] 5. Monitoring tumor growth: The long and short diameters of the tumor in the right axilla of the mouse were measured every two days using a caliper and the tumor volume (V = 0.5 × long diameter × short diameter) was recorded. 2 The experiment lasted for 30 days, and the behavior and body weight of the mice were observed regularly.

[0067] 6. Data Recording and Analysis: After the experiment, record the tumor volume and survival rate of each group of mice. Perform statistical analysis on the experimental data and compare the tumor growth of each group.

[0068] Tumor volume data (Table 3) show that groups 2 to 4 significantly outperformed the control group (Group 1) in inhibiting tumor growth. Survival data (Table 3) show that immune cells from Group 4 performed best in inhibiting tumor growth and improving mouse survival.

[0069] Table 3 Follow-up results of mice after treatment

[0070]

[0071] The above test results show that the prepared immune cells have good anti-tumor effects, especially Group E in Example 1 has significant effects in tumor inhibition and survival rate improvement.

[0072] Example 3: Preparation and testing of anti-CD3+CD28 monoclonal antibodies

[0073] 1. Antigen Preparation and Immunization

[0074] (1) Antigen preparation: Through analysis, aa26 to aa36 of the human CD3 protein (amino acid sequence shown in SEQ ID NO. 1), whose amino acid sequence is TCPQYPGSEIL; and aa146 to aa156 of the human CD28 protein (amino acid sequence shown in SEQ ID NO. 2), whose amino acid sequence is TCPPCPAPELL, were selected. These two polypeptides were prepared separately by artificial synthesis and simultaneously coupled to BSA to form fusion polypeptide proteins. The concentration of the fusion polypeptide protein was adjusted to 1 mg / ml, and the mixed proteins were mixed in equal mass ratios to serve as the immunogen. The injection volume for immunization was 100 μg of the mixed fusion polypeptide protein.

[0075] (2) First immunization: Mix the mixed fusion polypeptide protein with complete Freund's adjuvant at a ratio of 1:1 and fully emulsify. Administer 0.5 ml of the mixture subcutaneously to each rabbit.

[0076] (3) Booster immunization: Booster immunization is performed at the 2nd, 4th and 6th week after the first immunization. The mixed fusion polypeptide protein is mixed with incomplete Freund's adjuvant and injected. The dosage is the same as the first immunization.

[0077] (4) Final immunization: The final immunization is the injection of the mixed fusion polypeptide protein without the use of adjuvant.

[0078] 2. Isolation of Rabbit Spleen Cells

[0079] (1) Three to four days after the final immunization, euthanize the rabbit and remove the spleen. Under sterile conditions, place the spleen in cold PBS and gently grind the spleen tissue using a cell sieve or syringe mesh to obtain a cell suspension.

[0080] (2) Centrifugation and washing: Transfer the spleen cell suspension to a centrifuge tube and centrifuge at 800 g for 5 minutes. Discard the supernatant, wash twice with PBS, and then resuspend the cells in RPMI 1640 medium.

[0081] 3. Cell Fusion

[0082] (1) Cell counting: Spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 5:1.

[0083] (2) PEG fusion: 50% PEG was gradually added to the cell mixture at 37°C and stirred for 1 minute. RPMI 1640 medium was then gradually added to terminate the reaction.

[0084] (3) Centrifugation: Centrifuge at 500 g for 5 minutes, discard the supernatant, and resuspend in RPMI 1640 medium.

[0085] 4. Hybridoma Cell Screening

[0086] (1) The fused cell suspension was placed in HAT medium and inoculated into a 96-well culture plate, with 100 μl per well.

[0087] (2) Place the culture plate in a 37°C, 5% CO2 incubator and observe the growth of cell clones.

[0088] (3) After 1-2 weeks, select hybridoma clones that produce antibodies.

[0089] 5. Antibody Screening

[0090] (1) ELISA screening: Use recombinant human CD3 protein (ab167755) and recombinant human CD28 protein (ab198634) as coating antigens, respectively, to coat ELISA plates and detect the binding activity of antibodies in hybridoma cell culture supernatants to the target proteins. Hybridomas with strong binding activity to both proteins are selected for subcloning.

[0091] (2) Subcloning: Subclone the positive clones to ensure that each clone secretes only a single antibody.

[0092] 6. Antibody Production and Purification

[0093] After the above screening, a promising hybridoma cell line was selected for expansion. Antibodies were extracted from the cell culture supernatant using Protein G affinity chromatography. Ultimately, an anti-CD3+CD28 monoclonal antibody was obtained. This monoclonal antibody was sterilized by filtration through a 220 nm filter and stored at -80°C until further use.

[0094] 7. Antibody testing

[0095] (1) The concentration of the monoclonal antibody was determined using a commercial BCA kit (ThermoFisher). The concentration of the monoclonal antibody was 3.45 mg / ml.

[0096] (2) The purity of the monoclonal antibody was analyzed by conventional SDS-PAGE. The results are shown in Figure 2 , showing that its purity is over 95%.

[0097] (3) mRNA was extracted from the hybridoma cells, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody were analyzed. By sequencing, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody were shown as SEQ ID NO. 3 and SEQ ID NO. 4. The specific analysis results are shown in Table 4.

[0098] Table 4 Sequence analysis of monoclonal antibodies

[0099]

[0100] (4) The binding activity of the monoclonal antibody of the present invention and the commercially available monoclonal antibody was detected using the ELISA detection method for screening positive hybridomas (before the test, the monoclonal antibodies were diluted to 1 mg / ml, and before the test, they were diluted 10,000 times and incubated, and three replicates were performed for each sample). The results are shown in Table 5. The binding activity of the monoclonal antibody of the present invention is better than that of the commercially available monoclonal antibody.

[0101] Table 5 Monoclonal antibody ELISA test results (OD450nm)

[0102]

[0103] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An anti-CD3+CD28 monoclonal antibody, characterized in that: 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. 3 and SEQ ID NO. 4, respectively.

2. A method for preparing immune cells, characterized in that: The method comprises the following steps: (1) Isolate PBMCs from the peripheral blood of healthy volunteers; (2) resuspending the PBMCs in culture medium and adding growth factors IL-2 and IL-15; (3) Activating cells using the anti-CD3+CD28 monoclonal antibody of claim 1; (4) Adding the small chemical molecule Plerixafor; (5) After 48 hours of culture, highly active immune cells were obtained.

3. The method according to claim 2, characterized in that The concentrations of the growth factors IL-2 and IL-15 were 20 ng / ml and 10 ng / ml, respectively.

4. The method according to claim 2, characterized in that The concentration of the anti-CD3+CD28 monoclonal antibody was 1 μg / ml.

5. The method according to claim 2, characterized in that The concentration of the chemical small molecule Plerixafor is 100 nM.

6. The method according to claim 2, characterized in that In step (4), lipopolysaccharide may be further added to enhance stimulation, wherein the concentration of lipopolysaccharide is 1 μg / ml.

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