CN118652343A - A clinical blood immune cell and its application in treating diseases - Google Patents

Abstract

The present invention provides a clinical blood immune cell and its application in treating diseases. Furthermore, the present invention particularly provides immune cell NK cells combined with the PD-1 monoclonal antibody 10G3 prepared by the present invention for the treatment of liver cancer, which can significantly inhibit the growth of tumors and has good application prospects.

Description

A clinical blood immune cell and its application in treating diseases

Technical Field

The present application relates to the field of biological therapy, and specifically to a clinical blood immune cell and its application in treating diseases.

Background Art

Primary liver cancer ranks fourth in the incidence of malignant tumors in my country and third in the cause of death from tumors. In recent years, immune cell-based treatments have achieved remarkable results in prolonging the survival of liver cancer patients and reducing cancer metastasis and recurrence. Dendritic cells (DCs), natural killer cells (NK cells), and natural killer T cells (NKTs), as the main functional cells in the liver, are considered the first line of defense against liver cancer.

Dendritic cells (DCs) recognize, process, and present tumor antigens, which is a prerequisite for effective anti-tumor immune response. Compared with healthy people, HCC patients' DCs express fewer human leukocyte antigen (HLA)-I molecules, have weakened endocytosis, and secrete less interleukin IL-12, suggesting that DCs have maturation defects when HCC occurs. Studies have also shown that the proportion of activated CD83+DC cells in the peripheral blood of HCC patients is less than that of cirrhotic patients and healthy controls. In addition, the proportion of CD83+DC cells in the peripheral blood of HCC patients themselves is significantly lower than that in liver tissue, suggesting that activated DCs are difficult to infiltrate human cancer nodules, making it difficult to recruit tumor-specific lymphocytes to this area. Recent studies have found a new type of DCs subset, CDl4+CTLA-4+regulatory DCs, which can inhibit CD4+T cell responses through cytotoxic T lymphocyte antigen 4 (CTLA-4)-dependent IL-10 and indoleamine 2,3-dioxygenase, thereby causing tumor immune escape.

Natural killer (NK) cells account for 25% to 40% of lymphocytes in human liver. According to the expression of CD56 molecules, they can be divided into two subgroups: CD56 ^bright and CD56 ^dim . Among them, the CD56 ^bright subgroup can be amplified after IL-2 stimulation, and about 10% express killer cell immunoglobulin-like receptors (KIR), which can secrete and synthesize TNF-related apoptosis-inducing ligand (TRAIL); while the CD56 dim subgroup is insensitive to IL-2 stimulation. 85% of CD56 ^dim are KIR+, secreting and synthesizing perforin and granzyme B. When HCC occurs, on the one hand, liver cancer cells express Rael on the surface. This factor, as a ligand for the NK cell activation receptor NKG2D, can activate NK cells and promote their anti-tumor immunity. On the other hand, the immune function of NK cells is limited. The CD56 ^dim NK cell subgroup in the peripheral blood of HCC patients is significantly less than that of healthy controls. At the same time, CD56 ^dim NK cells in the tumor area of HCC patients expressed less IFN-1 than non-tumor areas. In vitro experiments have shown that this is related to CD4+CD25+ regulatory T cells (Treg). When liver cancer occurs, changes in the extracellular matrix microenvironment and TGF-β secreted by HSC can inhibit the activity and function of NK cells, thereby weakening their surveillance function on liver cells.

Natural killer T cells (NKTs) express α, β-T cell antigen receptors (TCR) and NK cell receptors, and secrete cytokines such as IL-4, IFN-γ and TNF-α. In tumor immunity, NKT cells have a dual role and can be divided into two categories, CD4+NKT cells and CD4-NKT cells, according to the presence or absence of CD4 molecule expression. Both accumulate in the tumor environment. The former can inhibit the immune response of tumor-specific CD8+T cells to tumor cells by secreting Th2 cytokines such as IL-4, IL-5, and IL-10, while the latter mainly exerts anti-tumor effects by inhibiting β-catenin. Studies have confirmed that the inflammatory response caused by β-eatenin gene mutation in the occurrence of liver cancer can determine the invasion of HCC. CD4-NKT cells and leukocyte-derived chemotactic factor 2 (LECT2) are important factors in the inflammation caused by β-eatenin. When the NKT gene is knocked out, the invasion and metastasis of HCC are enhanced, suggesting that it has an anti-tumor effect.

However, current studies have shown that immune cell therapy alone has the disadvantages of low efficacy and unobvious tumor cell killing. Therefore, in actual treatment, immune cells and antibodies are usually used for corresponding liver cancer treatment. NK cells expressing PD-1 show characteristics such as impaired toxicity and reduced proliferation ability, and PD-1 blockade can reverse these adverse effects. In clinical studies, the safety and efficacy of the PD-1 inhibitor nivolumab in the treatment of liver cancer have been confirmed, but this is not dominated by NK cells. The combination of PD-1 inhibitors and NK cell infusions has achieved effective anti-tumor activity in patients with non-small cell lung cancer, and the efficacy is better than that of NK cell infusion alone, which clarifies the potential of PD-1 blockade in NK cell-based immunotherapy. Blocking TIGIT can increase the toxicity of peripheral blood NK cells to in vitro cultured liver cancer cells, which suggests that targeted blockade of TIGIT may be an effective way to restore NK cell function in patients with liver cancer. In addition, TIGIT is usually co-expressed with PD-1, and the combined blockade of the two can more effectively reverse NK cell exhaustion.

Summary of the invention

The present invention provides a monoclonal antibody specific to PD-1, specifically the 10G3 monoclonal antibody.

Furthermore, the 10G3 monoclonal antibody was commissioned to Detai Bio to conduct antibody sequence sequencing. After sequence analysis, the light chain variable region sequence of the 10G3 monoclonal antibody was obtained as shown in SEQ ID NO: 1, and the heavy chain variable region sequence was obtained as shown in SEQ ID NO: 2.

In some embodiments, the light chain variable region of the present invention is further:

i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 1; or

ii) comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO: 1, preferably, the amino acid changes do not occur in the CDR region. Conservative substitution tables providing functionally similar amino acids are well known to those skilled in the art. For example, the characteristics of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains with the following common functional groups or characteristics: aliphatic side chains (G, A, V, L, I, P); hydroxyl groups containing side chains (S, T, Y); sulfur atoms containing side chains (C, M); carboxylic acids and amino compounds containing side chains (D, N, E, Q); bases containing side chains (R, K, H); and aromatic hydrocarbons containing side chains (H, F, Y, W).

In some embodiments, the heavy chain variable region of the present invention is further:

i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 2; or

ii) comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO: 2, preferably, the amino acid changes do not occur in the CDR region. Conservative substitution tables providing functionally similar amino acids are well known to those skilled in the art. For example, the characteristics of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains with the following common functional groups or characteristics: aliphatic side chains (G, A, V, L, I, P); hydroxyl groups containing side chains (S, T, Y); sulfur atoms containing side chains (C, M); carboxylic acids and amino compounds containing side chains (D, N, E, Q); bases containing side chains (R, K, H); and aromatic hydrocarbons containing side chains (H, F, Y, W).

Furthermore, the present invention also provides the use of clinical blood immune cells NK cells in treating cancer.

Furthermore, the present invention also provides use of the PD-1 monoclonal antibody 10G3 in preparing a pharmaceutical composition for treating cancer.

Furthermore, the present invention also provides the use of PD-1 monoclonal antibody 10G3 and NK cells in preparing a pharmaceutical composition for treating cancer.

Furthermore, the pharmaceutical composition of the present invention also contains a pharmaceutically acceptable carrier.

Specifically, examples of suitable pharmaceutically acceptable polymers include, but are not limited to, cellulose derivatives (e.g., hydroxypropyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose polymers, hydroxyethyl cellulose, sodium carboxymethyl cellulose, carboxymethylene hydroxyethyl cellulose and carboxymethyl hydroxyethyl cellulose or any combination thereof), acrylates (e.g., acrylic acid, acrylamide and maleic anhydride polymers, copolymers or mixtures thereof), and mixtures thereof. Polymer blends may also be used. A preferred pharmaceutically acceptable polymer is hydroxyethyl cellulose.

In one embodiment, the pharmaceutically acceptable polymer is present in an amount of about 0.01% to about 5.0% (weight/volume), preferably about 0.05% to about 2% (weight/volume), more preferably about 0.1% to about 1.0% (weight/volume), for example about 0.1%, 0.2%, 0.5% or 1.0% (weight/volume).

Examples of suitable pharmaceutically acceptable wetting agents or surfactants include, but are not limited to, amphoteric, nonionic, cationic or anionic molecules. Suitable surfactants include, but are not limited to, polysorbates, sodium dodecyl sulfate (sodium lauryl sulfate), dodecyl dimethylamine oxide, sodium docusate, cetyl trimethyl ammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, N,N-dimethyldodecylamine-N-oxide, cetyl trimethyl ammonium bromide, polyoxyethylene (10) lauryl ether, surfactants (polyoxyethylene fatty alcohol polyoxyethylene ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol), bile salts (e.g., sodium deoxycholate and sodium cholate), polyoxyethylene castor oil, nonylphenol ethoxylate, cyclodextrin, lecithin, methylbenzethonium chloride, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils and fats, sulfated esters, sulfated alkanolamides, alkylphenols (ethoxylated and sulfated), ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylates, polyethylene glycol esters, sorbitan esters and ethoxylated derivatives thereof, fatty acid glycol esters, carboxamides, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, amines having amide bonds, polyoxyethylene alkylamines and polyoxyethylene alicyclic amines, N,N,N,N-tetrasubstituted ethylenediamines, 2-alkyl-1-hydroxyethyl-2-imidazolines, N-cocoyl-3-aminopropionic acid/sodium salt, N-tallow-3-iminodipropionic acid disodium salt, N-carboxymethyl-N,N-dimethyl-N-9-octadecenyl ammonium hydroxide, N-cocoylamideethyl-N-hydroxyethylglycine sodium salt, etc., polyoxyethylene, sorbitan monolaurate and stearate, (polyethoxylated castor oil), (ethylene oxide/12-hydroxystearic acid), polysorbate, tyloxapol, and any combination thereof. Preferred pharmaceutically acceptable surfactants include tyloxapol and (sorbitan monooleate) or mixtures thereof.

Specifically, the dosage of the monoclonal antibody of the present invention is 1 mg/kg, or 2 mg/kg, 3 mg/kg.

Further, the dosage of the NK cells is 1×10 ⁶ to 1×10 ^9. Preferably, it is 1×10 ⁷ to 1×10 ⁹ , more preferably, it is 1×10 ⁸ to 1×10 ⁹ , and more preferably, it is 1×10 ⁹ /time.

Beneficial Effects

The present invention provides a clinical blood immune cell and its application in treating diseases. Furthermore, the present invention particularly provides immune cell NK cells combined with the PD-1 monoclonal antibody 10G3 prepared by the present invention for the treatment of liver cancer, which can significantly inhibit the growth of tumors and has good application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Monoclonal antibody specificity identification results

Figure 2 Effects of each group on HepG2 cell activity

DETAILED DESCRIPTION

The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

Example 1 Preparation, Screening and Identification of PD-1 Monoclonal Antibodies

Recombinant PD-1 protein (catalog number GMP-TL756) was purchased from Tongli Haiyuan Biotechnology Co., Ltd. BALB/c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

Three 6-8 week old female BALB/c mice were selected for the first immunization. 3 mg/mL PD-1 protein was mixed with Freund's complete adjuvant in a ratio of 1:1 and emulsified. The mice were immunized subcutaneously at multiple points. After 2 weeks of immunization, Freund's incomplete adjuvant was mixed with PD-1 protein in a ratio of 1:1 and emulsified. The second to fourth immunizations were performed in sequence. The mice were immunized subcutaneously on both sides of the neck and back. The interval between each immunization was 2 weeks, and the injection dose for each immunization was 110 μg/mouse. The serum titer of the immunized mice was measured, and the mouse No. 1 with the highest titer was selected for cell fusion.

Non-immune mice were killed 1 day before fusion, and disinfected by immersion in 75% alcohol for 5 minutes. 8 mL of HAT medium was injected into the mouse peritoneum aseptically to obtain feeder cells, which were plated on 96-well plates at 1×10 ⁴ /well for standby use. SP2/0 cells were revived 1 week before fusion and expanded 2 days before fusion. Blood was collected from the orbits of immune mice to prepare immune serum, and the mice were killed after anesthesia. After immersion and disinfection, the spleen was taken and placed on a plate containing 1640 medium. The medium was repeatedly blown into the spleen until the membrane gradually became transparent. The spleen cells obtained by blowing and aspiration were collected and centrifuged, and the precipitated cells were resuspended and counted. SP2/0 cells and spleen cells were mixed at a ratio of 1:10, centrifuged and discarded the supernatant, and preheated PEG1500 was added for suspension. Fusion was terminated with 1640 medium for about 1 minute. After centrifugation at 1500r/min, the cells were resuspended with HAT medium and added to the cell wells with feeder cells in advance (100μL/well), and cultured in a 5% CO ₂ incubator. 7 days after cell fusion, half of the HT cells containing 10% fetal bovine serum were replaced. When the hybridoma cells grew to about 1/3 of the bottom of the well, the cell culture supernatant was taken and the culture supernatant was detected by indirect ELISA. The 18 cell wells with the strongest positive reaction were selected and cloned by limiting dilution until the positive rate of the cell supernatant in the monoclonal well was 100%. The monoclonal cells with the strongest positive reaction were expanded and frozen in liquid nitrogen and named 10G3. The supernatant of the positive clone cells was collected and diluted from 1:800 to 1:25600. The antibody titer was determined by indirect ELISA, and the antibody titer before and after freezing was compared. The results are shown in Table 1.

Table 110G3 hybridoma supernatant ELISA titer comparison

Group/Titer 10G3 Before cryopreservation 1:12800 After cryopreservation 1:12800

As can be seen from Table 1, the 10G3 hybridoma supernatant has a good titer, and the titer before and after freezing is basically similar, indicating that the 10G3 monoclonal antibody has good stability and the activity is not significantly affected even after freezing.

Six-week-old female BALB/c mice were selected, and 500 μL sterilized liquid paraffin was first injected into the mouse's peritoneal cavity. After 7 days, 0.5 mL of cell suspension (about 1×10 ⁶ cells) was injected into the mouse's peritoneal cavity. After about 10 days, ascites was extracted and centrifuged at 12000 r/min for 10 minutes. The supernatant was taken for antibody column purification. After testing the protein purity, the protein concentration was adjusted to 1 mg/mL for storage. The subclass of the prepared ascites monoclonal antibody was identified according to the instructions of the monoclonal antibody subclass identification kit. The titer of hybridoma cell ascites antibody was determined by indirect ELISA, and the results showed that the monoclonal antibody subclass was identified as IgG2b subclass.

Monoclonal antibody Western blot identification: PD-1 recombinant protein and mouse serum were identified by 12% SDS-PAGE and transferred to NC membrane, blocked with 5% skim milk for 2 hours, and washed 5 times with PBST; 10G3 monoclonal antibody was used as the primary antibody and incubated overnight at 4°C, the NC membrane was washed 5 times with PBST, transferred to IRDye800CW goat anti-mouse IgG solution, incubated in the dark for 1 hour, exposed in the dark room, and photographed. The results are shown in Figure 1.

As can be seen from FIG1 , the 10G3 monoclonal antibody of the present invention has good specificity and can only specifically bind to the PD-1 recombinant protein but not to mouse serum.

Example 210G3 monoclonal antibody affinity determination

The affinity of 10G3 antibody was detected by SPR technology commonly used in the field. The anti-mouse IgG secondary antibody was fixed on the CM5 chip, and the 10G3 antibody was captured by BiacoreT200. The PD-1 antigen was used as the analyte and diluted to 0, 7.5, 15 and 30 nM concentration gradients with buffer, and the binding of different antibodies to MB antigen was detected by single cycle kinetics. The final data was analyzed by kinetics fitting according to the 1:1 model using Biacoreevaluation software 3.0. The results are shown in Table 2.

Table 210G3 monoclonal antibody affinity determination results

Various Measurement results KD(mol/L) 2.22E-10 Ka[1/(mol*s/L)] 1.57E+05 Kd(1/s) 3.48E-05

As can be seen from Table 2, the 10G3 monoclonal antibody has a very high affinity with the PD-1 recombinant protein.

Example 3 Effect of 10G3 monoclonal antibody on the ability of T lymphocytes to kill tumor cells

Jurkat cells were pre-activated with phytohemagglutinin (PHA) at a final mass concentration of 2 μg/ml for 48 h; HepG2 cells were resuspended and counted at the same time, and inoculated into 96-well cell culture plates at 5×103/well. After the cells adhered to the wall, IFN-γ (10 ng/ml) was added for stimulation and culture for 24 h. Activated Jurkat cells (human T lymphocyte leukemia cells) were inoculated into IFN-γ pre-treated HepG2 cells (human liver cancer cells) culture plates at a target-effect ratio of 1:10 and cultured for 48 h. In addition to the co-culture group, monoclonal antibody blocking group 1 and monoclonal antibody blocking group 2, IgG control group and blank control group were set up at the same time. Monoclonal antibody blocking group 1 was incubated with homemade 10G3 monoclonal antibody (10μg/ml) for 48h, monoclonal antibody blocking group 2 was incubated with homemade 10G3 monoclonal antibody (50μg/ml) for 48h, and monoclonal antibody isotype protein mouse IgG (10μg/ml) was incubated for 48h. The blank control group did not add any interfering factors, but only added the same volume of culture medium for co-culture for 48h. After the co-culture, the supernatant was discarded and washed repeatedly with sterile PBS to remove the residual Jurkat cells. 180μl serum-free culture medium was added to each well, and then 20μl of 5mg/ml MTT solution was added and mixed thoroughly. Incubate and culture for 4h at 37℃; after centrifugation again by plate centrifuge, the cell supernatant in the culture well was gently aspirated. 150μl DMSO solution was added to each well to dissolve the crystals in the well, and oscillation was performed at 37℃ for 10min to promote its dissolution. The optical density (OD) value of each well at λ=490nm was measured on a BIO-RAD enzyme-linked immunosorbent assay. The OD value corresponding to the activated Jurkat cells + HepG2 cells was used as the benchmark and the relative HepG2 cell activity base number 1. The results of each group are shown in FIG2 .

By using the co-culture results of Jurkat cells and HepG2 cells, the effect of PHA-activated Jurkat cells on the killing ability of liver cancer cells HepG2 after 10G3 monoclonal antibody blocked the PD-1/PD-L1 pathway was observed. The results showed that there was no significant change in the survival rate of HepG2 cells in the IgG control group compared with the blank control group, but the 10G3 monoclonal antibody blocking groups 1 and 2 had a significant effect on the survival rate of HepG2 cells, and it was dose-dependent. In particular, the relative HepG2 cell activity of monoclonal antibody blocking group 2 was (38.07±3.03)%, which was significantly lower than that of the IgG control group and the blank control group (P<0.01) (Figure 2).

Example 4 Preparation of NK cells

Peripheral blood donated by healthy people was collected, anticoagulated with heparin, and diluted with an equal volume of PBS. PBMCs were separated by gradient centrifugation of lymphocyte separation medium, washed twice with PBS, and adjusted to a PBMC density of 1X10 ⁶ /L well plate with SCGM (containing 0.05 volume fraction of human AB serum and 500U/mL of IL-2 and 400U/mL of IL-15). Each well had 2mL. 500μg/L mouse anti-human CD3 monoclonal antibody and 50mg/L PHA were added, respectively. After culturing for 3 days in a CO2 incubator at 37℃ and a volume fraction of 0.05, the cells were centrifuged at 2000r/min for 5min to wash away the CD3 monoclonal antibody and PHA in the culture medium. SCGM (containing 0.05 volume fraction of human AB serum and 500U/mL of IL-2 and 400U/mL of IL-15) was added again. The culture was continued, and the medium was half-changed every 3 days. After 14 days, the uncultured cells were used as the control group and cultured at 1X10 10μCD3-FITC and CD56-PE were added to ⁶ cells respectively, combined in dark at room temperature for 20 minutes, washed twice with PBS, suspended in 500μL PBS, and CD3/CD56 expression was detected by flow cytometry. The results showed that NK cells were obtained by culture, of which CD3-CD56+NK cells could reach (71.87±5.09).

Example 510G3 monoclonal antibody and NK cell in vivo biological experiment

60 6-week-old, 20-22g female BALB/c nude mice were selected. All animals were kept in the animal room for 1 week before being used in the experiment. HepG2 cells were expanded and cultured before inoculation, and cells in the logarithmic growth phase were collected and the concentration was adjusted to 5×10 ⁷ /ml. They were placed in an ice box and quickly taken to the animal room for use. In the clean bench, 0.1ml of the shaken cell suspension was aspirated with a 1ml syringe, and 0.1ml of the cell suspension (containing 5×10 ⁶ cells) was subcutaneously injected in the left armpit. The tumor formation of the mice was observed. The animal tumor model was successfully established when a tumor of relatively uniform size grew in the left armpit of the mouse.

When the tumor grew to a size of about ²⁰⁰ mm3, 60 nude mice were randomly divided into 6 groups, with 10 mice in each group, and received the following interventions:

(1) PBS 0.2 ml intraperitoneal injection;

(2) 10G3 monoclonal antibody 1 mg/kg intraperitoneal injection;

(3) 1 mg/kg intraperitoneal injection of camrelizumab;

(4) 2×10 ⁶ NK cells were injected into the tail vein;

(5) 10G3 monoclonal antibody + NK cells intraperitoneal injection (1 mg/kg) + tail vein injection (2 × 10 ⁶ cells);

(6) Carrelizumab + NK cell intraperitoneal injection (1 mg/kg) + tail vein injection (2 × 10 ⁶ cells);

The drug was administered by intraperitoneal injection 3 times a week, and the NK cells were administered once a week for 4 consecutive weeks. The NK cells were the NK cells prepared in Example 4.

Three days after the end of the medication, the nude mice were killed by cervical dislocation, the tumors were completely removed and weighed, and the tumor inhibition rate was calculated using the formula: tumor inhibition rate = (average tumor weight of the control group - average tumor weight of the experimental group) / average tumor weight of the control group × 100%. The results are shown in Table 3.

Table 3 Inhibitory effect of each experimental group on the growth of HepG2 transplanted tumors in nude mice

Group Tumor inhibition rate (%) (1) PBS - (2) 10G3 monoclonal antibody 19.87±0.53 (3) Carrelizumab 14.56±0.45 (4) NK cells 9.86±0.21 (5) 10G3 monoclonal antibody + NK cells 58.47±0.75* (6) Carrelizumab + NK cells 42.53±0.64*

Note: * indicates P<0.05 compared with other groups of the same tumor cells.

During the administration of the present invention, the growth and activity of mice were good, and the weight of animals did not change significantly. After the treatment, there was no significant difference between each group and the PBS control group (P>0.05), indicating that the drugs given to each experimental group had no obvious toxicity. As shown in Table 3, at the end of the experiment, the tumor inhibition rate of the 10G3 monoclonal antibody and NK cell combined group in the transplanted tumor was significantly higher than that of the other groups (P<0.05), indicating that the combined treatment of 10G3 monoclonal antibody and NK cells has a better synergistic therapeutic effect than the groups treated alone.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit it; although the present invention is described in detail with reference to the above embodiments, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the above embodiments, or replace part or all of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (4)

1. A monoclonal antibody 10G3 specifically targeting PD-1, characterized in that the light chain variable region sequence of the antibody is as shown in SEQ ID NO: 1, and the heavy chain variable region sequence is as shown in SEQ ID NO: 2. 2. Use of the monoclonal antibody 10G3 specifically targeting PD-1 as claimed in claim 1 in the preparation of a pharmaceutical composition for treating liver cancer. 3. Use of the monoclonal antibody 10G3 specifically targeting PD-1 and immune cells NK cells as claimed in claim 1 in the preparation of a pharmaceutical composition for treating liver cancer. 4. The use according to claim 3, characterized in that the liver cancer is caused by HepG2 cells.