Culture medium capable of improving antitumor activity of NK cells and culture method thereof

Abstract

The invention discloses a culture medium capable of remarkably improving in-vitro amplification efficiency and antitumor activity of NK (Natural Killer) cells. The culture medium comprises (1) a basic culture medium, namely an RPMI 1640 culture medium containing 10% of human AB serum, 1% of GlutaMAX and 0.1% of beta-mercaptoethanol; (2) fusion polypeptide: the final concentration is 150ng / mL, and the IL-15 mutant is connected with the NKG2D ligand ULBP2, so that dual-channel synergistic activation of IL-15R and NKG2D is realized; and (3) an anti-PD-1 antibody with a final concentration of 30 [mu] g / mL, wherein the anti-PD-1 antibody blocks a PD-1 / PD-L1 inhibition signal. Experiments show that after the culture medium is used for culturing for 7 days, the NK cell amplification multiple can reach 39.8 times, the CD69 activation proportion is increased to 83.6%, and the apoptosis rate is reduced to 6.5%. The killing rate of PD-L1 + tumor cells in vitro is increased to 64.7%, the tumor volume inhibition rate in a tumor-bearing mouse model reaches 66.4%, and the median lifetime is prolonged to gt; and 60 days. According to the invention, the existing NK cell amplification and activity limitation is broken through through a multi-channel synergistic effect, and an efficient solution is provided for solid tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to immunocyte engineering technology, and particularly to a culture medium capable of enhancing the anti-tumor activity of NK cells and a culture method thereof. Background Art

[0002] Natural Killer (NK) cells are important effector cells of the innate immune system, which can recognize and kill tumor cells in a non-MHC-dependent manner and have broad application potential in immunotherapy. Currently, clinical-grade NK cell expansion mainly relies on the co-culture system of cytokines (such as IL-2, IL-15) and feeder cells (such as K562-mbIL21). However, this technology still has the following bottlenecks:

[0003] 1. Low expansion efficiency and cell exhaustion: It takes 14 - 21 days to obtain therapeutic-level (>1×10 9 ) NK cells by traditional methods. Long-term IL-15 stimulation easily leads to an increase in the proportion of CD57 + exhausted NK cells (>40%) and an increase in apoptosis rate, reducing cell viability and anti-tumor efficacy.

[0004] 2. Tumor microenvironment inhibition: The PD-L1 / PD-1 axis in the solid tumor microenvironment inhibits the function of NK cells. For example, in a melanoma model, high expression of PD-L1 can reduce the killing efficiency of NK cells by 60%. However, the existing expansion system does not integrate the immune checkpoint blockade strategy, resulting in a rapid decline in the activity of NK cells after reinfusion.

[0005] 3. Metabolic limitation affecting cell survival: The function of NK cells depends on mitochondrial oxidative phosphorylation (OXPHOS), but OXPHOS is blocked during the expansion process, and the accumulation of ROS (>500 nM) leads to DNA damage, inhibiting proliferation and accelerating cell senescence.

[0006] In response to the above problems, some studies have attempted gene editing (such as CRISPR knock-in of the IL-15 receptor) or antibody-conjugated cytokines (such as IL-15-Fc fusion protein), but their high production cost, complex process and safety risks limit clinical translation.

[0007] The present invention innovatively combines the polypeptide regulation and metabolic optimization strategies to develop a highly efficient activation medium for NK cells that does not require genetic modification and can be produced on a large scale, which can improve the expansion efficiency, enhance the anti-tumor activity, and improve the survival ability of NK cells, providing a better solution for NK cell immunotherapy. Summary of the Invention

[0008] The present invention provides a culture medium and a culture method for co-activating NK cells through multiple pathways.

[0009] Therefore, on the one hand, the present invention discloses a fusion polypeptide, and the amino acid sequence of the fusion polypeptide is as shown in SEQ ID NO.1.

[0010] Preferably, the nucleotide sequence of the fusion polypeptide after codon optimization according to the present invention is as shown in SEQ ID NO.2.

[0011] On the one hand, the present invention also discloses a culture medium for enhancing the anti-tumor activity of NK cells, and the culture medium comprises the following components:

[0012] (1) Basal medium: RPMI 1640 medium containing 10% human AB serum, 1% GlutaMAX, and 0.1% β-mercaptoethanol;

[0013] (2) Fusion polypeptide: final concentration 150 ng / mL;

[0014] (3) Anti-PD-1 antibody: final concentration 30 μg / mL.

[0015] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-PD-1 antibody according to the present invention are as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0016] On the one hand, the present invention also discloses the application of the fusion polypeptide in the preparation of a culture medium for enhancing the anti-tumor activity of NK cells.

[0017] On the one hand, the present invention also discloses the application of the anti-PD-1 antibody in the preparation of a culture medium for enhancing the anti-tumor activity of NK cells.

[0018] On the one hand, the present invention also discloses the application of the culture medium in the culture of NK cells.

[0019] The beneficial effects of the present invention are summarized as follows:

[0020] 1. High-efficiency amplification and activation: The amplification multiple reaches 39.8 times after 7 days of culture (12.5 times in the control group), the proportion of CD69+ increases to 83.6% (44.8% in the control group), and the proportion of NKG2D+ reaches 89.3% (56.3% in the control group).

[0021] 2. Enhanced anti-tumor activity: The killing rate against PD-L1+ A549 cells increases to 64.7% (24.1% in the control group), the expression level of granzyme B increases by 3.1 times, and the secretion amount of IFN-γ reaches 489.1 pg / mL (234.6 pg / mL in the control group).

[0022] 3. Significant in vivo efficacy: The tumor volume inhibition rate of tumor-bearing mice is 66.4% (865 ± 132 mm in the PBS group) 3vs experimental group 291 ± 89 mm 3 ), and the median survival period was extended to > 60 days (33 days in the PBS group). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 SDS-PAGE detection results of the fusion polypeptide, where 1 is the fusion polypeptide.

[0024] Figure 2 Western blot detection results of the anti-PD-1 antibody against the recombinant PD-1 protein, where 1 is the recombinant PD-1 protein with a molecular weight of approximately 17 kDa. DETAILED DESCRIPTION OF THE INVENTION

[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 technical field to which this invention belongs. The terms used in the description of the invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0026] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0027] Example 1: Design, Preparation, and Detection of the Fusion Polypeptide

[0028] 1. Design and Optimization of the Fusion Polypeptide

[0029] 1.1 Design Objectives

[0030] (1) Functional Synergy: Combine the proliferative signal of IL-15 and the NK cell activation signal of ULBP2 to achieve dual-pathway activation of NK cells.

[0031] (2) Autocrine Effect: Use a flexible Linker to ensure that the domains fold independently and function, reducing steric hindrance.

[0032] (3) Stability Optimization: The IL-15 mutant reduces dependence on IL-15Rα and increases the half-life.

[0033] 1.2 Sequence Design

[0034] (1) IL-15 Mutant (UniProt ID: P40933): First, remove 48 amino acids at the N-terminus, and then mutate the remaining sequence by changing Asn (N) at position 72 to Asp (D) to reduce IL-15Rα dependence and enhance the autocrine signal.

[0035] (2) Extracellular domain of ULBP2 (UniProt ID: Q9BZM5): First, remove the 25 amino acids at the N-terminus, and then select the amino acids at positions 31-216 from the remaining sequence to retain the α1 / α2 domain and ensure high-affinity binding ability to NKG2D.

[0036] (3) Flexible Linker: (GGGGS)3, which provides flexibility and reduces interference between domains.

[0037] (4) Final sequence: [IL-15N72D]-(GGGGS)3-[ULBP2(31-216)], with a total of 315 amino acids and a theoretical molecular weight of 35.2 kDa. The specific amino acid sequence is shown in SEQ ID NO.1.

[0038] 2. Gene construction and protein expression

[0039] 2.1 Gene synthesis and vector construction

[0040] 2.1.1 Gene synthesis: Optimize the codons of the amino acid sequence of the fusion polypeptide designed above (SEQ ID NO.1), and add a CD33 signal peptide (MLPVAALGLLALAQTANA) at its N-terminus and a His-tag (6×His) at its C-terminus. The optimized nucleotide sequence is shown in SEQ ID NO.2. Entrust a third-party company to synthesize the optimized nucleotide sequence for later use.

[0041] 2.1.2 Vector construction: Clone the synthesized nucleotide sequence into the pcDNA3.4 vector, and add two restriction enzyme sites (EcoRI and XhoI) at the same time. The specific steps are briefly described as follows: Digest the pcDNA3.4 vector with EcoRI and XhoI (37 °C, 2 h). Ligate the gene fragment with the vector (T4 DNA ligase, 16 °C, 12 h). Transform the plasmid into Stbl3 Escherichia coli (electroporation, 1.8 kV). Pick monoclonal colonies and screen positive clones by colony PCR. Verify by Sanger sequencing. If the sequencing result is correct, subsequent experiments can be carried out.

[0042] 2.2 Cell transfection and expression

[0043] 2.2.1 Cell culture: ExpiCHO-S, cultured at 37 °C, 5% CO2, with shaking at 125 rpm.

[0044] 2.2.2 Transient transfection: Take 6×10 6 cells / mL of CHO-S cells and inoculate them into a 125 mL culture flask (50 mL / flask). Add the DNA-transfection reagent mixture according to the ExpiCHO standard operation. Incubate at 32 °C for 14 days and collect the culture supernatant.

[0045] 3. Protein Purification (Ni-NTA Affinity Chromatography)

[0046] (1) Supernatant Treatment: Sterilize the collected supernatant by passing it through a 0.22 μm filter membrane.

[0047] (2) Ni-NTA Column Purification: The equilibration buffer is 20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0. The elution buffer is 20 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0. Collect the peak elution fraction. Dialyze to remove imidazole, sterilize by passing through a 0.22 μm filter membrane, and aliquot and store at -80 °C in the refrigerator for later use.

[0048] (3) SDS-PAGE Detection: Perform SDS-PAGE detection on the purified fusion polypeptide above. The results show that ( Figure 1 ) the molecular weight of this fusion polypeptide is approximately 35.2 kDa, and the purity can reach over 95%.

[0049] 4. Biological Function Verification

[0050] 4.1 IL-15 Activity Detection: CTLL-2 cells (IL-15-dependent growth) are seeded in a 96-well plate (2×10 4 cells / well), and the fusion polypeptide (10 - 100 ng / mL) and wild-type IL-15 (ab259403) are added. Incubate at 37 °C for 72 h, and use a CCK-8 detection kit to detect cell proliferation. The results show after calculation that the EC50 of the fusion polypeptide of the present invention is 1.8 nM, and the EC50 of wild-type IL-15 is 2.1 nM, indicating that the fusion polypeptide of the present invention has an activity equivalent to or better than that of wild-type IL-15.

[0051] 4.2 NKG2D Binding Ability: Biacore T200, immobilize human NKG2D protein onto a CM5 chip. Perform binding kinetics determination on the fusion polypeptide and pure ULBP2 (ab151636). The results show after calculation that the KD of the fusion polypeptide is 9.8 nM, and the KD of pure ULBP2 is 12.3 nM, indicating that the fusion peptide of the present invention has an activity equivalent to or better than that of pure ULBP2.

[0052] 4.3 NK cell activation: NK cells (CD56+CD3-) were sorted from PBMC. The fusion polypeptide (100 ng / mL) and wild-type IL-15 (ab259403, 100 ng / mL) were added. After 24 h, the expression of CD69 was detected by flow cytometry. The results showed that the proportion of CD69+ cells in the fusion polypeptide group was 78.5%, while that in the wild-type IL-15 group was 42.3%. This indicates that the fusion polypeptide prepared in the present invention can significantly enhance NK cell activation and has the potential for application in NK cell therapy.

[0053] Example 2: Isolation of NK cells and preparation of culture medium

[0054] Step 1: Isolation of NK cells

[0055] 1.1 Isolation of PBMC (density gradient centrifugation method)

[0056] (1) Under sterile conditions, the peripheral blood collected from healthy volunteers was diluted 1:1 to 50 mL (RPMI 1640 without serum);

[0057] (2) Take 15 mL of Ficoll-Paque Plus and gently add 30 mL of diluted blood to a 50 mL centrifuge tube to form a distinct interface;

[0058] (3) Centrifuge at 2000 rpm for 30 min at room temperature (braking set to the lowest);

[0059] (4) Use a Pasteur pipette to remove the PBMC layer (white cloudy layer) and transfer it to a new 15 mL tube;

[0060] (5) Wash the PBMC with RPMI 1640 (400×g, 10 min, room temperature) and repeat 2 times;

[0061] (6) Count the total number of cells and adjust the concentration to 1×10 7 cells / mL.

[0062] 1.2 Magnetic bead sorting of CD56 + CD3 - NK cells

[0063] (1) Take 1×10 7 PBMC prepared above and add 100 μL of NK Cell Isolation Kit magnetic bead mixture, and gently mix;

[0064] (2) Incubate at 4°C for 20 min, and gently invert and mix every 5 min during incubation;

[0065] (3) Wash once with RPMI 1640 (300×g, 5 min, 4°C);

[0066] (4) Use a magnetic sorting column for sorting and collect CD56 + CD3 - NK cells;

[0067] (5) Suspend the cells in RPMI 1640, count the cell concentration, and detect the purity by flow cytometry. The results show that CD56 + CD3 - > 95%, indicating that the prepared NK cells are good and can be further used for subsequent experiments.

[0068] Step 2: Preparation of NK cell medium

[0069] 2.1 Basic medium: RPMI 1640 medium containing 10% human AB serum, 1% GlutaMAX, and 0.1% β-mercaptoethanol.

[0070] 2.2 Added components

[0071] (1) Fusion polypeptide (prepared in Example 1): final concentration 150 ng / mL;

[0072] (2) Anti-PD-1 antibody (prepared in Example 6): final concentration 30 μg / mL.

[0073] Example 3: NK cell expansion and phenotype analysis

[0074] 1. Experimental design, experimental groups (n = 6)

[0075] (1) Control group A: Basic medium + IL-15 100 ng / mL;

[0076] (2) Control group B: Basic medium + IL-15 (100 ng / mL + fusion polypeptide (150 ng / mL);

[0077] (3) Control group C: Basic medium + IL-15 (100 ng / mL) + anti-PD-1 antibody (30 μg / mL);

[0078] (4) Experimental group D: Basic medium + IL-15 (100 ng / mL) + fusion polypeptide (150 ng / mL) + anti-PD-1 antibody (30 μg / mL).

[0079] 2. Culture conditions

[0080] (1) Initial cell density: 1×10 6 cells / mL;

[0081] (2) Cell culture: 6-well plates, 3 mL of culture medium per well; replace half of the medium every 48 hours, and supplement with fresh cytokines and reagents; culture at 37 °C, 5% CO2, and 95% humidity for 7 days.

[0082] 3. Detection

[0083] (1) Cell expansion multiple: Use the trypan blue exclusion method to count viable cells. The calculation formula is:

[0084]

[0085] (2) Expression of NK cell activation markers

[0086] Detection indicators: CD69 (early activation marker), NKG2D (NK cell killing receptor);

[0087] Method: Take 2×10 5 cells, wash once with PBS; incubate with antibodies (CD69-PE, NKG2D-APC) at 4 °C for 30 min in the dark; centrifuge at 300×g for 5 min at 4 °C to remove the supernatant; resuspend the pellet in 500 μL of PBS and detect and analyze using FACS.

[0088] (3) Cell apoptosis rate

[0089] Analysis criteria: Annexin V + PI - represents early apoptosis, Annexin V + PI + represents late apoptosis;

[0090] Method: Take 1×10 6 cells, wash once with PBS; resuspend in 100 μL of Annexin V binding buffer; add Annexin V-FITC (5 μL) + PI (5 μL), incubate at room temperature in the dark for 15 min; immediately perform flow cytometry detection and analysis.

[0091] 4. Results, as shown in Table 1 specifically.

[0092] (1) NK cell expansion multiple: The expansion efficiency of experimental group D (the culture medium of the present invention) was significantly improved, which was 3.2 times that of the basic group A, and higher than that of the fusion polypeptide alone group (B) or the anti-PD-1 antibody alone group (C).

[0093] (2) CD69 and NKG2D expression: In experimental group D, the activation markers of NK cells were significantly upregulated (p<0.01), indicating that they were in a highly activated state.

[0094] (3) Decreased apoptosis rate: The apoptosis rate of the experimental group D was significantly reduced to 6.5%, while that of the control group A was as high as 23.8%, indicating that the culture medium of the present invention can improve the cell survival rate.

[0095] (4) Synergistic effect: The combination of fusion peptide + anti-PD-1 antibody is superior to the addition of fusion peptide or anti-PD-1 antibody alone in terms of cell proliferation, activation and survival, indicating that there is a synergistic effect between the two.

[0096] Table 1 Experimental data and statistical analysis

[0097]

[0098] Example 4: In vitro tumor cell killing experiment

[0099] 1. Experimental Materials and Reagents

[0100] 1. NK cells: control group A (NK cells cultured with IL-15 alone); experimental group B (NK cells cultured with fusion peptide + anti-PD-1).

[0101] 2. Target cells: K562 (chronic myeloid leukemia cell line, no PD-L1 expression); A549-PD-L1 (human lung adenocarcinoma cell line, stably and highly expressing PD-L1, transfected with lentivirus, flow cytometry-verified positive rate >90%).

[0102] 3. Culture medium: K562 (RPMI-1640 + 10% FBS), A549-PD-L1 (DMEM + 10% FBS).

[0103] 2. Experimental steps

[0104] 1. Target cell plating: cell concentration is 1×10 4 / well, 96-well plate, 5 replicate wells per group. Culture conditions were 37°C, 5% CO2, 24 hours adherent culture.

[0105] 2. NK cell effector target cell co-culture: The effector target ratio (E:T) was set at 5:1, and 5×10 4 NK cells were co-cultured for 4 hours.

[0106] 3. Cytotoxicity assay: After 4 hours of co-culture, pipette 100 μL of supernatant into a new 96-well plate. Add 50 μL of LDH reaction solution and incubate at room temperature in the dark for 30 minutes. Read the OD 490nm value and calculate the killing rate.

[0107] 4. Cell activity detection: After the cells were collected, they were washed once with PBS and centrifuged (300×g, 5 min). Annexin V-FITC / PI double staining was used to detect the proportion of live cells (FACSCalibur).

[0108] 5. Mechanism exploration: Collect NK cell lysate and measure the protein concentration by BCA method. After SDS-PAGE electrophoresis, transfer the membrane and incubate with the blocking solution for 1 hour. Incubate with primary antibodies (perforin, granzyme B, GAPDH) overnight at 4°C. Incubate with secondary antibody for 1 hour, develop color with ECL, and perform quantitative analysis with ImageJ.

[0109] 6. Cytokine detection: Collect the co-culture supernatant and centrifuge (1000×g, 10 min). Detect the contents of IFN-γ and TNF-α according to the ELISA kit instructions.

[0110] III. Experimental Results

[0111] 1. Cytotoxicity detection: The killing rate of experimental group B against PD-L1+A549 cells increased by 2.7 times, indicating that the anti-PD-1 antibody effectively blocked the PD-1 / PD-L1 inhibitory signal and improved the cytotoxic ability of NK cells against PD-L1+ tumors. Specifically, as shown in Table 2.

[0112] Table 2 Cytotoxicity detection results

[0113]

[0114] 2. Cell survival rate: The proportion of live cells in control group A was 76.3% ± 3.1%, and that in experimental group B was 91.4% ± 2.8%. This indicates that the survival rate of experimental group B was significantly improved, suggesting that the optimized medium enhanced the anti-apoptotic ability of NK cells.

[0115] 3. Cytotoxic proteins: The expression level of granzyme B in experimental group B increased by 3.1 times, indicating that the fusion polypeptide enhanced the ability of NK cells to release cytotoxic granules. Specifically, as shown in Table 3.

[0116] Table 3 Cytotoxic protein detection results

[0117]

[0118] 4. Cytokine secretion level: The secretion levels of IFN-γ and TNF-α in experimental group B were significantly increased (p < 0.005), indicating that the system of the present invention enhanced the cytokine secretion ability of NK cells, thereby improving its anti-tumor activity. Specifically, as shown in Table 4.

[0119] Table 4 Cytokine secretion level detection results

[0120]

[0121] IV. Experimental Conclusions

[0122] The killing activity of NK cells against PD-L1+A549 tumor cells was significantly enhanced (the killing rate increased by 2.7 times), indicating that anti-PD-1 antibody effectively relieved PD-1 / PD-L1 immunosuppression. The expression levels of perforin and granzyme B in NK cells increased (granzyme B increased by 3.1 times), suggesting that the fusion polypeptide enhanced the cytotoxic function of NK cells. The cytokine secretion ability increased (IFN-γ and TNF-α increased by about 2 times), further enhancing the immune effect. The survival rate of NK cells increased by 15.1%, indicating that the optimization of the culture medium of the present invention improved the survival ability and anti-tumor effect of NK cells.

[0123] Based on the above results, the culture system of the present invention can effectively promote the activation of NK cells, enhance the killing activity against anti-PD-L1 tumors, and improve the anti-tumor ability of NK cells through multiple mechanisms, having potential clinical application value.

[0124] Example 5: Efficacy verification in tumor-bearing mice

[0125] 1. Model construction

[0126] (1) Experimental animals: NOD / SCID female mice, 6-8 weeks old, weighing 18-22 g; adaptively raised for 1 week in a standard SPF environment (temperature 22-24 °C, relative humidity 50-60%, 12 h light-dark cycle); randomly grouped (n = 10 / group) 1 day before the experiment.

[0127] (2) Tumor cell inoculation: SKOV3 (human ovarian cancer cell line, PD-L1 positive), collected in the logarithmic growth phase, washed 2 times with PBS; after cell counting, subcutaneous injection was performed into the right axilla of the mice at a density of 5×10 6 / 100 μL PBS; continuously monitor tumor growth, and start intervention treatment when the tumor volume reaches about 50 mm 3 (about 7 days).

[0128] 2. Treatment plan

[0129] (1) PBS group: Intravenous injection of an equal volume of PBS once a week for a total of 3 times;

[0130] (2) Experimental group: Intravenous injection of 1×10 6 The above NK cells amplified using the culture medium of the present invention per mouse once a week for a total of 3 times.

[0131] (3) Intravenous infusion was performed under anesthesia (isoflurane inhalation) before treatment to reduce the stress response.

[0132] 3. Observation indexes and detection methods

[0133] (1) Tumor volume measurement: The long diameter (L) and short diameter (W) of the tumor were measured every 3 days using vernier calipers, and the volume was calculated: Volume = 0.5 × L × W 2 .

[0134] (2) Survival time record: The survival days of each mouse were recorded, with the criteria being that the tumor volume > 2000 mm 3 or death due to disease deterioration.

[0135] 4. The test results are shown in Table 5 specifically.

[0136] (1) Tumor growth inhibition: The tumors of the mice in the PBS group grew significantly after 21 days, and the tumor volume reached 865 ± 132 mm 3 . The tumor volume of the mice in the experimental group decreased significantly, only 291 ± 89 mm 3 , a 66.4% decrease compared to the PBS group (p < 0.001).

[0137] (2) Median survival time extension: The median survival time of the mice in the PBS group was 33 days, indicating that the tumor progressed rapidly and ultimately led to the death of the mice due to excessive tumor burden. The mice in the experimental group were still alive at the end of the experiment (> 60 days), showing that NK cell treatment significantly extended the survival time (p < 0.001).

[0138] Table 5 Summary of Experimental Result Tracking

[0139]

[0140] Example 6: Preparation and Testing of Anti-PD-1 Antibody

[0141] 1. Preparation of Anti-PD-1 Antibody

[0142] (1) Immunize mice to prepare monoclonal antibodies: Select 6 - 8-week-old SPF-grade Balb / c mice, and use recombinant PD-1 protein (ab174035) (50 μg / mouse) mixed with Freund's complete adjuvant for the first immunization (subcutaneous injection), and then perform two booster immunizations (Freund's incomplete adjuvant) every two weeks. After 6 weeks of immunization, take spleen cells and fuse them with SP2 / 0 myeloma cells, and use HAT selection medium to screen positive hybridoma cell lines.

[0143] (2) Screening of hybridoma cells, determination of antibody variable region sequences and humanization: Positive clones of PD-1 specific antibodies were screened by ELISA, and the optimal hybridoma cell line was amplified and cultured. Then, the light and heavy chain variable regions (VL and VH) of the antibody in the hybridoma cells were amplified by RT-PCR, and the sequences were confirmed by Sanger sequencing. Further humanization was carried out by optimizing and transplanting the CDR regions of the murine antibody into the human IgG framework, and the antibody structure was optimized by molecular docking simulation. The amino acid sequences of the heavy chain variable region and light chain variable region of the optimized antibody are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively. Finally, it was cloned into an expression vector and stably expressed in CHO cells to prepare a humanized anti-PD-1 antibody.

[0144] (3) Antibody preparation: The anti-PD-1 monoclonal antibody was prepared in large quantities using the CHO cell culture system and purified by Protein A affinity purification. The purified antibody was detected and calculated, and the expression level of the antibody could reach up to 2 mg / mL. Therefore, the antibody has a high expression yield.

[0145] 2. Detection of anti-PD-1 antibody

[0146] (1) Western blot detection: The PD-1 protein was separated by SDS-PAGE, and the binding characteristics of the antibody of the present invention were analyzed by Western blot. The results showed ( Figure 2 ), the antibody of the present invention could specifically recognize the PD-1 protein, and the band was clear.

[0147] (2) ELISA detection of affinity: The PD-1 protein was coated on the ELISA plate, and the antibody of the present invention and the commercial antibody (ab234444) diluted in gradients were added respectively, and the OD450 value was measured. The results showed that the EC50 value (0.5 ng / mL) of the antibody of the present invention was better than that of the commercial antibody (1.2 ng / mL), indicating that it had a higher affinity.

[0148] (3) Cell experiment to detect the PD-1 / PD-L1 blocking ability: Jurkat cells expressing PD-1 and APC cells expressing PD-L1 were co-cultured, and different concentrations of the antibody of the present invention or the commercial antibody (ab234444) were added. The antibody blocking effect was evaluated by detecting the IL-2 secretion. The results showed that the antibody of the present invention could effectively block the PD-1 / PD-L1 interaction, and the IL-2 secretion increased significantly. The specific data showed that after adding 10 μg / mL of the antibody of the present invention, the IL-2 secretion reached 450 pg / mL, while when using the same amount of the commercial antibody, the IL-2 secretion was only 290 pg / mL.

[0149] 3. Conclusion

[0150] The anti-PD-1 antibody of the present invention improves affinity and functional effects by optimizing the immune strategy and antibody screening. At the same time, through antibody humanization technology, it is more suitable for clinical applications. Compared with the commercial antibodies on the market, the antibody of the present invention shows obvious advantages in binding ability, PD-1 / PD-L1 blocking ability and cell function experiments, and is suitable for further development for immunotherapy.

[0151] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A fusion polypeptide, characterized in that, The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO.

1.

2. The fusion polypeptide according to claim 1, wherein The nucleotide sequence of the fusion polypeptide after codon optimization is shown in SEQ ID NO.

2.

3. A culture medium for enhancing the anti-tumor activity of NK cells, characterized in that, The medium comprises the following components: (1) Basal medium: RPMI 1640 medium containing 10% human AB serum, 1% GlutaMAX, and 0.1% β-mercaptoethanol; (2) The fusion polypeptide according to claim 1: final concentration 150 ng / mL; (3) Anti-PD-1 antibody: final concentration 30 μg / mL.

4. The culture medium according to claim 3, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-PD-1 antibody are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

5. Use of a fusion polypeptide according to claim 1 in the preparation of a medium for enhancing the anti-tumor activity of NK cells.

6. Use of an anti-PD-1 antibody according to claim 4 in the preparation of a medium for enhancing the anti-tumor activity of NK cells.

7. Use of a medium according to claim 3 in the culture of NK cells.

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