A culture medium capable of improving the anti-tumor activity of NK cells and a culture method thereof

Abstract

This invention discloses a culture medium that can significantly improve the in vitro expansion efficiency and anti-tumor activity of NK cells. The culture medium comprises: (1) a basal medium: RPMI 1640 medium containing 10% human AB serum, 1% GlutaMAX, and 0.1% β-mercaptoethanol; (2) a fusion polypeptide: with a final concentration of 150 ng / mL, which achieves synergistic activation of the IL-15R and NKG2D dual pathways by linking the IL-15 mutant with the NKG2D ligand ULBP2; and (3) an anti-PD-1 antibody: with a final concentration of 30 μg / mL, which blocks the PD-1 / PD-L1 inhibitory signal. Experiments show that after 7 days of culture in this medium, NK cells can expand by 39.8 times, the CD69 activation rate increases to 83.6%, and the apoptosis rate decreases to 6.5%. The in vitro killing rate of PD-L1+ tumor cells is increased to 64.7%, the tumor volume inhibition rate in tumor-bearing mouse models reaches 66.4%, and the median survival time is extended to >60 days. This invention overcomes the limitations of existing NK cell expansion and activity through multi-pathway synergy, providing a highly efficient solution for immunotherapy of solid tumors.

Description

Technical Field

[0001] This invention relates to immune cell engineering technology, specifically to a culture medium and its culture method that can enhance the anti-tumor activity of NK cells. Background Technology

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

[0003] 1. Low amplification efficiency and easy cell depletion: Traditional methods require 14-21 days to achieve therapeutic levels (>1×10⁻⁶). 9 NK cells, long-term IL-15 stimulation easily leads to CD57 + The proportion of exhausted NK cells increased (>40%), and the apoptosis rate increased, reducing cell viability and anti-tumor efficacy.

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

[0005] 3. Metabolic restriction affects cell survival: NK cell function depends on mitochondrial oxidative phosphorylation (OXPHOS), but OXPHOS is blocked during expansion, and ROS accumulation (>500nM) leads to DNA damage, inhibits proliferation and accelerates cell senescence.

[0006] To address the aforementioned issues, existing research has attempted gene editing (such as CRISPR knock-in of the IL-15 receptor) or antibody-coupled cytokine (such as IL-15-Fc fusion protein), but these methods are costly, complex, and pose safety risks, limiting their clinical translation.

[0007] This invention innovatively combines peptide regulation and metabolic optimization strategies to develop a highly efficient NK cell activation culture medium that can be produced on a large scale without genetic modification. It can improve amplification efficiency, enhance anti-tumor activity, and improve NK cell survival, providing a better solution for NK cell immunotherapy. Summary of the Invention

[0008] This invention provides a culture medium and culture method for activating NK cells through multi-pathway synergistic activation.

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

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

[0011] In one aspect, the present invention also discloses a culture medium for enhancing the antitumor activity of NK cells, the culture medium comprising the following components:

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

[0013] (2) Fusion peptide: 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 of the present invention are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0016] In another aspect, the present invention also discloses the application of the fusion peptide in the preparation of a culture medium that enhances the antitumor activity of NK cells.

[0017] In another aspect, the present invention also discloses the application of the aforementioned anti-PD-1 antibody in the preparation of a culture medium that enhances the anti-tumor activity of NK cells.

[0018] In one aspect, the present invention also discloses the application of the culture medium in NK cell culture.

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

[0020] 1. Highly efficient amplification and activation: After 7 days of culture, the amplification fold reached 39.8 times (compared to 12.5 times in the control group), the CD69+ ratio increased to 83.6% (compared to 44.8% in the control group), and the NKG2D+ ratio reached 89.3% (compared to 56.3% in the control group).

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

[0022] 3. Significant in vivo therapeutic effect: Tumor volume inhibition rate in tumor-bearing mice was 66.4% (PBS group: 865±132 mm). 3vs. experimental group 291±89mm 3 The median survival was extended to >60 days (33 days in the PBS group). Attached Figure Description

[0023] Figure 1 The SDS-PAGE results of the fusion peptides are shown, where 1 represents the fusion peptide.

[0024] Figure 2 The results of Western blot detection of recombinant PD-1 protein by anti-PD-1 antibody, where 1 is the recombinant PD-1 protein with a molecular weight of approximately 17 kDa. Detailed Implementation

[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 invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated 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 testing of fusion peptides

[0028] 1. Fusion Peptide Design and Optimization

[0029] 1.1 Design Goals

[0030] (1) Functional synergy: It combines the proliferative signal of IL-15 with the NK cell activation signal of ULBP2 to achieve dual-pathway activation of NK cells.

[0031] (2) Autocrine effect: Using a flexible linker ensures that the structural domains fold independently and function, reducing spatial steric hindrance.

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

[0033] 1.2 Sequence Design

[0034] (1) IL-15 mutant (UniProt ID: P40933): First, 48 amino acids at the N-terminus were removed, and then the remaining sequence was mutated to change Asn(N) at position 72 to Asp(D) to reduce IL-15Rα dependence and enhance autocrine signaling.

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

[0036] (3) Flexible Linker: (GGGGS)3 provides flexibility and reduces interference between structural 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. Its 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: The amino acid sequence (SEQ ID NO.1) of the designed fusion polypeptide was codon-optimized, and a CD33 signal peptide (MLPVAALGLLALAQTANA) was added to its N-terminus, and a His-tag (6×His) was added to its C-terminus. The optimized nucleotide sequence is shown in SEQ ID NO.2. The optimized nucleotide sequence was then synthesized by a third-party company for future use.

[0041] 2.1.2 Vector Construction: The synthesized nucleotide sequence was cloned into the pcDNA3.4 vector, with two restriction enzyme sites (EcoRI and XhoI) added. The specific steps are briefly described as follows: The pcDNA3.4 vector was digested with EcoRI and XhoI (37℃, 2h). The gene fragment was ligated to the vector (T4 DNA ligase, 16℃, 12h). The plasmid was transformed into Stbl3 *E. coli* (electroplation, 1.8kV). Single clones were picked, and positive clones were screened by colony PCR. Sanger sequencing was used for verification. The sequencing results were correct, and subsequent experiments could be performed.

[0042] 2.2 Cell transfection and expression

[0043] 2.2.1 Cell culture: ExpiCHO-S, 37℃, 5% CO2, 125rpm shaking culture.

[0044] 2.2.2 Transient transfection: Take 6×10 6 CHO-S cells / mL were seeded into 125mL culture flasks (50mL / flask). DNA-transfection reagent mixture was added according to the ExpiCHO standard procedure. The cells were incubated at 32°C for 14 days, and the culture supernatant was collected.

[0045] 3. Protein purification (Ni-NTA affinity chromatography)

[0046] (1) Supernatant treatment: The collected supernatant was sterilized by passing it through a 0.22μm filter membrane.

[0047] (2) Ni-NTA column purification: The equilibration buffer consisted of 20 mM Tris-HCl, 300 mM NaCl, and 20 mM imidazole, pH 8.0. The elution buffer consisted of 20 mM Tris-HCl, 300 mM NaCl, and 250 mM imidazole, pH 8.0. The peak elution fraction was collected. Imidazole was removed by dialysis, and the fraction was sterilized by passing through a 0.22 μm filter membrane. It was then aliquoted and stored at -80°C for later use.

[0048] (3) SDS-PAGE detection: The purified fusion peptide was subjected to SDS-PAGE detection, and the results showed ( Figure 1 The molecular weight of the fusion peptide is approximately 35.2 kDa, and its purity can reach over 95%.

[0049] 4. Verification of biological functions

[0050] 4.1 IL-15 activity assay: CTLL-2 cells (IL-15-dependent growth) were seeded in 96-well plates (2 × 10⁻⁶ cells / wells). 4 Cells / well were incubated with a fusion peptide (10-100 ng / mL) and wild-type IL-15 (ab259403). Cells were cultured at 37°C for 72 h, and cell proliferation was detected using a CCK-8 assay kit. The results showed that the EC50 of the fusion peptide was 1.8 nM, and the EC50 of wild-type IL-15 was 2.1 nM, indicating that the fusion peptide of this invention has activity comparable to or better than wild-type IL-15.

[0051] 4.2 NKG2D Binding Ability: Human NKG2D protein was immobilized onto a CM5 microarray using a Biacore T200. Binding kinetics of the fusion peptide and pure ULBP2 (ab151636) were determined. Calculations showed that the KD of the fusion peptide was 9.8 nM, and the KD of pure ULBP2 was 12.3 nM, indicating that the fusion peptide of this invention has activity comparable to or better than pure ULBP2.

[0052] 4.3 NK Cell Activation: NK cells (CD56+CD3-) were sorted by PBMC. A fusion peptide (100 ng / mL) and wild-type IL-15 (ab259403, 100 ng / mL) were added. After 24 h, CD69 expression was detected by flow cytometry. The results showed that the proportion of CD69+ cells in the fusion peptide group was 78.5%, while that of wild-type IL-15 was 42.3%. This indicates that the fusion peptide prepared in this 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: NK cell isolation

[0055] 1.1 PBMC Separation (Density Gradient Centrifugation)

[0056] (1) Under aseptic conditions, 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 clear interface;

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

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

[0060] (5) Wash PBMCs with RPMI 1640 (400×g, 10min, room temperature), repeat twice;

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

[0062] 1.2 Sorting of CD56 Magnetic Beads + CD3 - NK cells

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

[0064] (2) Incubate at 4℃ for 20 min, gently turning and mixing once every 5 min during the process;

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

[0066] (4) Use magnetic sorting columns to sort and collect CD56. + CD3 - NK cells;

[0067] (5) Cells were suspended in RPMI 1640, cell concentration was counted, and purity was detected by flow cytometry. Results showed CD56... + CD3 - The result is >95%, indicating that the prepared NK cells are of good quality and can be further tested.

[0068] Step 2: Preparation of NK cell culture medium

[0069] 2.1 Basic culture 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 phenotypic analysis

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

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

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

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

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

[0079] 2. Cultivation conditions

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

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

[0082] 3. Testing

[0083] (1) Cell expansion fold: Live cells were counted using the trypan blue exclusion method. The calculation formula is as follows:

[0084]

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

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

[0087] Method: Take 2×10 5 Cells were washed once with PBS; incubated with antibodies (CD69-PE, NKG2D-APC) at 4°C for 30 min in the dark; centrifuged at 300×g at 4°C for 5 min, and the supernatant was removed; the precipitate was resuspended in 500 μL PBS and detected and analyzed using FACS.

[0088] (3) Apoptosis rate

[0089] Analysis standard: Annexin V + PI - Annexin V represents early apoptosis. + PI + This represents late-stage apoptosis;

[0090] Method: Take 1×10 6 Cells were washed once with PBS; resuspended in 100 μL Annexin V binding buffer; Annexin V-FITC (5 μL) + PI (5 μL) were added, and incubated at room temperature in the dark for 15 min; flow cytometry was performed immediately for detection and analysis.

[0091] 4. The results are shown in Table 1.

[0092] (1) NK cell expansion ratio: The expansion efficiency of experimental group D (the culture medium of this invention) was significantly improved, which was 3.2 times that of the basic group A, and higher than that of the fusion peptide 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 experimental group D was significantly reduced to 6.5%, while that of control group A was as high as 23.8%, indicating that the culture medium of the present invention can improve cell survival rate.

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

[0096] Table 1 Experimental Data and Statistical Analysis

[0097]

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

[0099] I. 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, without PD-L1 expression); A549-PD-L1 (human lung adenocarcinoma cell line, stably and highly expressing PD-L1, transfected with lentivirus, with a positive rate of >90% by flow cytometry).

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

[0103] II. Experimental Procedure

[0104] 1. Target cell plating: Cell concentration 1×10⁻⁶ 4 / well, 96-well plate, with 5 replicates per group. Culture conditions: 37℃, 5% CO2, 24-hour adherent culture.

[0105] 2. Co-culture of NK cell effector and target cells: The effector-to-target ratio (E:T) was set at 5:1, and 5 × 10⁶ cells were added to each well. 4 NK cells were co-cultured for 4 hours.

[0106] 3. Cytotoxicity assay: After co-culturing for 4 hours, 100 μL of supernatant was transferred to a new 96-well plate. 50 μL of LDH reaction solution was added, and the plate was incubated at room temperature in the dark for 30 min. The OD value at 490 nm was read, and the killing rate was calculated.

[0107] 4. Cell viability assay: After cell collection, cells were washed once with PBS and centrifuged (300×g, 5 min). The proportion of viable cells was detected by Annexin V-FITC / PI double staining (FACSCalibur).

[0108] 5. Mechanism Investigation: NK cell lysates were collected, and protein concentration was determined using the BCA method. After SDS-PAGE electrophoresis, the cells were transferred to a membrane and incubated with blocking buffer for 1 hour. Primary antibody incubation (perforin, granzyme B, GAPDH) was performed overnight at 4°C. Secondary antibody incubation was completed for 1 hour, followed by ECL staining and quantitative analysis using ImageJ.

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

[0110] III. Experimental Results

[0111] 1. Cytotoxicity assay: Experimental group B showed a 2.7-fold increase in the killing rate of PD-L1+ A549 cells, indicating that the anti-PD-1 antibody effectively blocked the PD-1 / PD-L1 inhibitory signal and enhanced the cytotoxic ability of NK cells against PD-L1+ tumors. See Table 2 for details.

[0112] Table 2. Cytotoxicity test results

[0113]

[0114] 2. Cell viability: The viable cell percentage in control group A was 76.3% ± 3.1%, while that in experimental group B was 91.4% ± 2.8%. This indicates that the viable cell percentage in experimental group B was significantly improved, suggesting that optimized culture 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 peptide enhanced the cytotoxic particle release ability of NK cells. See Table 3 for details.

[0116] Table 3 Results of Cytotoxic Protein Detection

[0117]

[0118] 4. Cytokine secretion levels: The secretion of IFN-γ and TNF-α in experimental group B was significantly increased (p<0.005), indicating that the system of the present invention enhanced the cytokine secretion capacity of NK cells, thereby improving their anti-tumor activity. See Table 4 for details.

[0119] Table 4 Results of Cytokine Secretion Level Detection

[0120]

[0121] IV. Experimental Conclusions

[0122] The killing activity of NK cells against PD-L1+A549 tumor cells was significantly enhanced (killing rate increased by 2.7-fold), indicating that the anti-PD-1 antibody effectively relieved PD-1 / PD-L1 immunosuppression. Increased expression levels of perforin and granzyme B on NK cells (granzyme B increased by 3.1-fold) suggest that the fusion peptide enhanced the cytotoxic function of NK cells. Increased cytokine secretion (IFN-γ and TNF-α increased by approximately 2-fold) further enhanced the immune response. The NK cell survival rate increased by 15.1%, indicating that the optimized culture medium of this invention improved the survival capacity and anti-tumor effect of NK cells.

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

[0124] Example 5: In vivo efficacy verification in tumor-bearing mice

[0125] 1. Model Building

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

[0127] (2) Tumor cell inoculation: SKOV3 (human ovarian cancer cell line, PD-L1 positive) cells were collected during the logarithmic growth phase and washed twice with PBS; after cell counting, they were inoculated at 5 × 10⁻⁶ cells per cell line. 6 The solution was administered subcutaneously to the right axilla of mice at a density of 100 μL PBS; tumor growth was continuously monitored until the tumor volume reached approximately 50 mm². 3 Intervention treatment will begin approximately 7 days later.

[0128] 2. Treatment Plan

[0129] (1) PBS group: An equal volume of PBS was injected via the tail vein once a week for a total of 3 times;

[0130] (2) Experimental group: 1×10⁻⁶ tidal vein injection 6 The NK cells / mouse expanded using the culture medium of this invention were administered once a week for a total of 3 times.

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

[0132] 3. Observation indicators 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 as follows: Volume = 0.5 × L × W 2 .

[0134] (2) Survival record: The survival days of each mouse were recorded, with the standard being a tumor volume >2000 mm. 3 Or death may result from the worsening of the disease.

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

[0136] (1) Tumor growth inhibition: The tumors in the PBS group of mice increased significantly after 21 days, with a tumor volume of 865±132 mm. 3 The tumor volume in the experimental group mice was significantly reduced, to only 291±89 mm. 3 Compared with the PBS group, the rate decreased by 66.4% (p<0.001).

[0137] (2) Prolonged median survival: The median survival of mice in the PBS group was 33 days, indicating rapid tumor progression, which ultimately led to the death of mice due to excessive tumor burden. Mice in the experimental group were still alive at the end of the experiment (>60 days), showing that NK cell therapy significantly prolonged survival (p<0.001).

[0138] Table 5 Summary of Experimental Results

[0139]

[0140] Example 6: Preparation and testing of anti-PD-1 antibodies

[0141] 1. Preparation of anti-PD-1 antibodies

[0142] (1) Preparation of monoclonal antibodies by immunizing mice: 6-8 week old SPF-grade Balb / c mice were selected and immunized for the first time (subcutaneous injection) with recombinant PD-1 protein (ab174035) (50 μg / mouse) mixed with Freund's complete adjuvant. Subsequently, booster immunizations were performed twice every two weeks (with Freund's incomplete adjuvant). Six weeks after immunization, spleen cells were harvested and fused with SP2 / 0 myeloma cells. Positive hybridoma cell lines were screened using HAT selection medium.

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

[0144] (3) Antibody preparation: Anti-PD-1 monoclonal antibody was prepared in large quantities using the CHO cell culture system and purified using Protein A affinity. The purified antibody was detected and calculated. 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 antibodies

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

[0147] (2) Affinity detection by ELISA: PD-1 protein was coated onto an ELISA plate, and serially diluted versions of the antibody of this invention and a commercially available antibody (ab234444) were added, respectively. The OD450 values ​​were measured. The results showed that the EC50 value of the antibody of this invention (0.5 ng / mL) was superior to that of the commercially available antibody (1.2 ng / mL), indicating that it has higher affinity.

[0148] (3) Cellular experiments to detect 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 this invention or a commercial antibody (ab234444) were added. The antibody blocking effect was evaluated by detecting IL-2 secretion. The results showed that the antibody of this invention could effectively block PD-1 / PD-L1 interaction, and the IL-2 secretion was significantly increased. Specifically, after adding 10 μg / mL of the antibody of this invention, the IL-2 secretion reached 450 pg / mL, while when using the same amount of commercial antibody, the IL-2 secretion was only 290 pg / mL.

[0149] 3. Conclusion

[0150] This invention's anti-PD-1 antibody improves affinity and functional efficacy through optimized immunization strategies and antibody screening. Furthermore, antibody humanization technology enhances its suitability for clinical applications. Compared to commercially available antibodies, this invention's antibody demonstrates significant advantages in binding capacity, PD-1 / PD-L1 blocking ability, and cell function experiments, making it 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 to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within 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. A culture medium for enhancing the antitumor activity of NK cells, characterized in that, The culture medium contains the following components: (1) Basic culture medium: RPMI 1640 medium containing 10% human AB serum, 1% GlutaMAX, and 0.1% β-mercaptoethanol; (2) The fusion polypeptide of claim 1: final concentration 150 ng / mL; (3) Anti-PD-1 antibody: final concentration 30 μg / mL.

3. The culture medium according to claim 2, 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.

4. The use of the fusion polypeptide as described in claim 1 in the preparation of a culture medium that enhances the antitumor activity of NK cells.

5. The use of the anti-PD-1 antibody as described in claim 3 in the preparation of a culture medium that enhances the anti-tumor activity of NK cells.

6. The application of the culture medium as described in claim 2 in NK cell culture.

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