Ginsenoside liposome loaded PD-L2 neutralizing antibody preparation as well as preparation method and application thereof

Abstract

The invention relates to a preparation method of a ginsenoside Rg3 liposome loaded PD-L2 neutralizing antibody preparation, which comprises the following steps: dissolving ginsenoside Rg3 and phospholipid into a mixed organic solvent of absolute ethyl alcohol and trichloromethane according to a certain mass ratio, carrying out rotary evaporation to form a lipid membrane, carrying out vacuum drying, then adding a PBS (Phosphate Buffer Solution) of an alphaPD-L2 antibody, and carrying out vacuum drying to obtain the ginsenoside Rg3 liposome loaded PD-L2 neutralizing antibody preparation. And carrying out ultrasonic treatment and filtering with a filter membrane, so as to obtain the Rg3-lipo-alphaPD-L2 preparation. The medicine has great clinical application value, firstly, the awkward situation that a PD-L2 high-expression intestinal cancer patient has a poor curative effect on existing immunotherapy (such as a PD-1 / PD-L1 inhibitor) is broken through; 2, double-effect collaborative treatment of'synchronous inhibition of tumor metastasis' and'reversal of immune depletion ', which cannot be achieved by the existing therapy, is realized; and 3, the targeting of the medicine is enhanced to reduce the systemic toxicity and improve the therapeutic window.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a ginsenoside liposome loaded PD-L2 neutralizing antibody preparation and a preparation method and application thereof. BACKGROUND

[0002] Colorectal cancer is the third most common malignant tumor in the world, and distant metastasis is the main reason for the survival limitation of patients with the disease. In recent years, immunotherapy has made remarkable progress in solid tumors, but due to tissue specificity and immunogenicity, immunotherapy in colorectal cancer is not satisfactory. Among the many immune checkpoints, the role of programmed death ligand-2 (PD-L2) has attracted more and more attention. It has been reported that PD-L2 is highly expressed in about 40% of tumor cells in patients with colorectal cancer. In the process of metastasis of colorectal cancer, PD-L2 also plays multiple key roles in the tumor microenvironment. On the one hand, it has been found that the expression level and precise localization of PD-L2 in cells are directly related to the invasion and metastasis ability of tumor cells. For example, a vesicle transport protein named GOLT1B plays a key role in tumor cells, which can specifically mediate the co-transport of PD-L2 and another signaling molecule DVL2 and anchor them on the cell membrane of tumor cells. This membrane localization is crucial, which activates the related signal pathways (such as Wnt / β-catenin pathway) in cells that promote cell migration and invasion, ultimately leading to tumor cells gaining stronger motility, breaking through the basement membrane barrier, invading surrounding tissues and blood vessels, thereby significantly enhancing the distant metastasis potential of tumors. This process reveals the molecular basis of PD-L2 directly driving tumor metastasis through a non-immune-dependent mechanism. On the other hand, PD-L2 is a core immune escape molecule. PD-L2 carried by tumor cells on the surface or exosomes released by them significantly inhibits the activation and function of T cells by binding to the receptors on the T cells, allowing tumor cells to escape the attack of the immune system and create conditions for metastasis. In addition, the expression of PD-L2 is not limited to tumor cells. Immune cells in the tumor microenvironment, especially tumor-associated macrophages, also often express PD-L2, which further inhibits T cell function and promotes the formation of an immunosuppressive microenvironment. The deletion of certain genes has also been found to promote the transformation of macrophages into specific types and increase PD-L2 expression, exacerbating immune escape. Therefore, PD-L2 not only directly endows tumor cells with strong migration and invasion ability through a non-immune-dependent mechanism to drive the metastasis process, but also creates an immune-exempt microenvironment for tumor cells through a key immune suppression function, making it a core therapeutic target for overcoming the metastasis problem of colorectal cancer, which has great significance for breaking through the existing treatment bottleneck.

[0003] However, for colorectal cancer patients, the current immunotherapy regimen is mainly anti-PD-1 / PD-L1, but the current clinical treatment faces multiple challenges. First, there is a deficiency in controlling metastasis, and traditional chemotherapy and targeted drugs are difficult to effectively inhibit the enhanced migration ability of tumor cells mediated by PD-L2, resulting in high recurrence and metastasis risk of patients. Second, there is still room for improvement in the local targeting and local infiltration concentration of existing immunotherapy. The commonly used blocking agent often has poor effect or even no effect on colorectal cancer with high expression of PD-L2, and the traditional method of giving antibodies systemically is limited by safety and patient adverse reactions, and the drug is difficult to accumulate to a sufficient concentration inside the tumor. Finally, there is a serious lack of precise intervention means, and there is currently a lack of a dual-function therapy that can block tumor cell metastasis and relieve immune cell functional exhaustion, and existing methods cannot effectively deal with the dual pathological role of PD-L2 in the tumor microenvironment. Therefore, there is an urgent need in the art to develop a preparation capable of loading PD-L2 neutralizing antibodies, which can effectively inhibit the migration ability of tumor cells, improve the targeting and local infiltration concentration of drugs, and at the same time relieve immune suppression, improve treatment effect and reduce toxicity. SUMMARY

[0004] The present application aims to provide a ginsenoside Rg3 liposome loaded with PD-L2 neutralizing antibody preparation and its preparation method and application. Specifically, the following technical solutions are provided:

[0005] The present application provides a preparation method of a ginsenoside Rg3 liposome loaded with PD-L2 neutralizing antibody, comprising the following steps:

[0006] Ginsenoside Rg3 and phospholipid are dissolved in a mixed organic solvent of anhydrous ethanol and chloroform in a certain mass ratio, a lipid film is formed by rotary evaporation and vacuum drying, then a PBS solution of αPD-L2 antibody is added, and after ultrasonic treatment and membrane filtration, Rg3-lipo-αPD-L2 preparation is obtained;

[0007] Further, the phospholipid is selected from one or more phospholipids in phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), sphingomyelin (SM) and hydrogenated derivatives thereof; and more further, the phospholipid is dioleoylphosphatidylcholine (DOPC);

[0008] Further, the mass ratio of ginsenoside Rg3 to phospholipid is 8:12-18; and more further, the mass ratio of ginsenoside Rg3 to phospholipid is 8:15;

[0009] Further, the volume ratio of anhydrous ethanol and chloroform in the mixed solvent is 1:0.8-2; more further, the volume ratio of anhydrous ethanol and chloroform in the mixed solvent is 1:1;

[0010] Further, the mass volume ratio of the sum of ginsenoside Rg3 and phospholipid to the mixed organic solvent is 20-25 mg:10-20 mL;

[0011] Further, the temperature of the reduced pressure evaporation is 20-40 DEG C; more further, the temperature of the reduced pressure evaporation is 25 DEG C;

[0012] Further, the time of the reduced pressure rotary evaporation is 10-20 min; more further, the time of the reduced pressure rotary evaporation is 15 min;

[0013] Further, the time of the vacuum drying is 2-4 h; more further, the time of the vacuum drying is 3 h;

[0014] Further, the concentration of the alpha PD-L2 solution is 1-6 mg / mL; more further, the concentration of the alpha PD-L2 solution is 3 mg / mL;

[0015] Further, the mass ratio of ginsenoside Rg3 to alpha PD-L2 is 2-8:3; more further, the mass ratio of ginsenoside Rg3 to alpha PD-L2 is 4:3;

[0016] Further, the ultrasonic treatment condition is 15-25 DEG C, power 150-300 W, and time 20-40 min; more further, the ultrasonic treatment condition is 20 DEG C, power 200 W, and time 30 min;

[0017] The second aspect of the application provides a ginsenoside Rg3 liposome loaded PD-L2 neutralizing antibody preparation prepared by any one of the above methods;

[0018] The third aspect of the application provides a ginsenoside Rg3 liposome loaded PD-L2 neutralizing antibody preparation in the preparation of an antitumor drug;

[0019] Further, the antitumor drug is an anticancer drug;

[0020] Further, the antitumor effect is an inhibition of tumor metastasis or / and a prolongation of patient survival;

[0021] Compared with the prior art, the beneficial effects of the application at least include:

[0022] 1.The ginsenoside Rg3 liposome of the present application utilizes ginsenoside to modify the liposome, and the hydrophobic parent nucleus structure thereof can be inserted into the inside of the lipid film, while the hydrophilic sugar group is exposed on the surface of the liposome. Such a biomimetic modification can effectively improve the stability of the liposome, evade the clearance of the immune system to prolong the blood circulation time, and actively target tumor cells to reduce the side reactions caused by non-targeted release of drugs. In various animal models such as gastric cancer, glioma and breast cancer, the ginsenoside liposome as a carrier of chemotherapeutic drugs has shown significant improvement in efficacy and reduction in side effects.

[0023] 2.The PD-L2 neutralizing antibody loaded in the ginsenoside Rg3 liposome of the present application can block the non-immune dependent autonomous migration signal pathway mediated by PD-L2, directly inhibit metastasis, and specifically, the drug can significantly inhibit tumor metastasis, reduce the number of lung metastatic nodules by more than 60%, and reduce the area of metastatic foci by more than 50%. On the other hand, the signal transmission of PD-L2 to PD-1 is relieved, which can effectively reverse T cell exhaustion and restore its anti-tumor activity, thereby achieving the purpose of synergistic treatment, and finally significantly prolonging survival.

[0024] 3.The drug exhibits excellent safety, and has no pathological damage to major organs such as heart, liver, spleen and kidney, and the whole body toxicity is significantly lower than that of the traditional systemic administration scheme. The drug has great clinical application value. First, it breaks through the blind area that PD-L2 highly expressed in patients with intestinal cancer is ineffective for existing immunotherapy (such as PD-1 / PD-L1 inhibitors); second, it realizes the double-effect synergistic treatment of "synchronous inhibition of tumor metastasis" and "reversal of immune exhaustion" which cannot be achieved by existing therapies; third, it significantly reduces the whole body toxicity through targeted drug delivery, and improves the treatment window. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to the drawings without creating any inventive labor.

[0026] Figure 1 To enhance the in vitro migration ability of MC3 cells overexpressing PD-L2;

[0027] Figure 2 To compare the HE staining of lung nodules in the control group and the PD-L2 overexpression group in the mouse tail vein lung metastasis model;

[0028] Figure 3 To compare the drug (Rg3-lipo here is labeled with FITC in advance) cell uptake of each treatment group after 24h of intervention of MC38 cell line and mouse primary DC cells.

[0029] Figure 4 HE staining of lung metastatic nodules in PD-L2 overexpression group of mouse tail vein lung metastasis model after different treatments;

[0030] Figure 5 Splenocyte CD8 + PD-1 expression level of T cells;

[0031] Figure 6 Heart, liver, spleen, kidney, brain tissue sections in PD-L2 overexpression group of mouse tail vein lung metastasis model after different treatments. DETAILED DESCRIPTION

[0032] The embodiments of the present application will be described in detail below with reference to the drawings.

[0033] The embodiments of the present application will be described in detail below with reference to the drawings.

[0034] Example 1: Preparation of Rg3-lipo-αPD-L2 and FITC-Rg3-lipo

[0035] Prepare 8 mg of ginsenoside Rg3 and 15 mg of DOPC in a 25 mL tomato-shaped bottle; add 15 mL of organic solvent (absolute ethanol: chloroform = 1:1, v / v); rotary evaporation at 25°C under reduced pressure for 15 min to form a lipid film, vacuum drying for 3 h; add 3 mL of PBS solution containing αPD-L2 antibody (2 mg / mL); ultrasonic treatment at 200 W power for 30 min; filter through a 0.22 μm microporous filter to obtain Rg3-lipo-αPD-L2 preparation with a particle size of 128.3 ± 5.6 nm (zeta potential -12.4 mV).

[0036] Prepare 8 mg of ginsenoside Rg3, 15 mg of DOPC, and 0.5 mg of coumarin (C6) and place them in a 25 mL tomato flask; add 15 mL of organic solvent (absolute ethanol: chloroform = 1:1, v / v); form a lipid film by rotary evaporation at 25°C under reduced pressure for 15 min, and dry in vacuum for 3 h; add 3 mL of PBS solution; treat with ultrasonic waves at a power of 200 W for 30 min; filter through a 0.22 μm microporous filter to obtain a C6-Rg3-lipo preparation.

[0037] Example 2: Flow cytometry staining procedure

[0038] The prepared fresh spleen single-cell suspension of tumor-bearing mice was subjected to cell counting and viability determination. A total of 1 x 10 6 ~5 x 10 6 The cells were resuspended in 100 μL of pre-cooled flow cytometry staining buffer (e.g., PBS containing 1% fetal bovine serum (FBS) and 0.05% sodium azide) and transferred to a flow cytometry detection tube. To reduce non-specific antibody binding, the cells were incubated with an anti-mouse CD16 / CD32 Fc receptor blocker at room temperature for 10 min in the dark. Subsequently, multi-color antibody surface staining was performed. The cells were incubated with the following fluorescently labeled antibody mixture at 4°C in the dark for 30 min: Live / Dead dye: to distinguish between live and dead cells, to exclude the non-specific staining background caused by dead cells. Anti-mouse CD8a antibody: to label cytotoxic T lymphocytes. Anti-mouse PD-1 antibody: to assess the exhaustion status of T cells. All antibodies were titrated and used according to the manufacturer's recommended proportions. After incubation, the cells were washed twice with an excess of staining buffer (centrifugation conditions: 500 g, 5 min, 4°C) to completely remove unbound antibodies.

[0039] Finally, the cells were resuspended in an appropriate amount (about 100-200 μL) of flow cytometry staining buffer or fixing solution and immediately subjected to machine detection. Flow cytometry data were collected using a BD FACS Symphony, and subsequent analysis was performed using FlowJo V10 software. Cell debris was excluded by setting gates based on forward scatter and side scatter, and the live cell population was defined using a Live / Dead dye-negative gate, and based on this, the expression levels of CD8 + T cells and PD-1 thereof were further analyzed.

[0040] Example 3: Mouse metastasis model and therapeutic effect

[0041] 3.1 Construction of animal model

[0042] (1) Cell line: PD-L2-overexpressing mouse colon cancer cell line MC38 (purchased from ATCC, CRL-27645);

[0043] (2) Modeling: 6-week-old female C57BL / 6 mice (SPF level, Jiangsu Jizu Yakang) were injected with 5 x 10 5 MC38 cells via the tail vein;

[0044] (3) Grouping and administration: Each group (n = 6) was administered by intraperitoneal injection in a volume of 100 μL

[0045]

[0046]

[0047]

[0048] (4) Administration regimen: Starting on day 6 after tumor cell injection, the mice were administered treatment every 2 days for a total of 3 times;

[0049] (5) Detection method:

[0050] a. Metastasis inhibition: On day 14, the lung tissue was fixed with 4% paraformaldehyde and stained with HE to count the metastatic nodules;

[0051] b. Survival analysis: Survival was recorded using the Kaplan-Meier method;

[0052] c. Safety: When the experimental mice reached the preset endpoint (day 14 after tumor inoculation), the mice were sacrificed by cervical dislocation or exsanguination after anesthesia. The thoracic and abdominal cavities were quickly opened, and heart, liver, spleen, and kidney tissue samples were collected. The collected tissue blocks were immediately immersed in sufficient 4% paraformaldehyde phosphate buffer for 24-48 h to ensure adequate fixation of the tissue. The fixed tissue blocks were dehydrated in gradient ethanol and then transparentized using xylene. The transparentized tissue blocks were immersed in molten paraffin for paraffin embedding. The embedded tissue paraffin blocks were sectioned using a rotary microtome to obtain continuous tissue sections with a thickness of about 4-6 μm. The sections were floated in a warm water bath to flatten and then mounted on clean glass slides. The glass slides loaded with tissue sections were placed in xylene to remove paraffin. The sections were rehydrated in gradient ethanol down to distilled water.

[0053] Hematoxylin-eosin (HE) staining: After rehydration, the sections were immersed in Harris hematoxylin staining solution for 5 min. After rinsing with running water, the sections were differentiated with 1% hydrochloric acid ethanol solution for a short time to remove non-specific staining such as cytoplasm. After rinsing with running water again, the sections were immersed in 0.5% ammonia water for rebluing. Further, the sections were immersed in an aqueous eosin solution for 5 min to make cytoplasm, collagen fibers, and the like pink to red. After completion of the staining, the sections were dehydrated with gradient ethanol, and then transparentized with xylene. Finally, neutral balsam was added dropwise, and a cover glass was placed thereon for mounting. The microstructure of each tissue section was observed under an optical microscope. Whether the myocardial fiber arrangement, liver lobule structure, hepatocyte morphology, sinusoidal area, spleen white and red pulp structure, splenic nodule, kidney glomerulus, and kidney tubule (proximal tubule, distal tubule, and collecting duct) structure was clear and complete, and whether the cell morphology was normal, and whether there were signs of pathological damage such as inflammatory cell infiltration, edema, degeneration (such as vacuolar degeneration and fatty degeneration), necrosis, fibrosis, hemorrhage, abnormal hyperplasia, or tumor infiltration were evaluated. By comparison with the control group (such as the blank control group or the solvent control group), the potential toxic effects of the test drug (Rg3-lipo-αPD-L2) on each major organ were systematically evaluated.

[0054] (6) Experimental results:

[0055] a. Figure 1 To perform an in vitro migration experiment using mouse intestinal cancer cells MC38 overexpressing PD-L2, the results showed that the mouse intestinal cancer cells overexpressing PD-L2 exhibited stronger tumor metastasis ability in the scratch test ( Figure 1 A, B), chamber migration test ( Figure 1 C, D), and matrix gel-coated invasion test ( Figure 1 E, F).

[0056] b. Figure 2 To inject the mouse intestinal cancer cells overexpressing PD-L2 and the control cells MC38 into the tail vein of mice to construct a mouse intestinal cancer lung metastasis model. The results showed that the mouse intestinal cancer cells overexpressing PD-L2 exhibited stronger lung metastasis ability ( Figure 2 A, B), and the number of nodules and the invasion area were higher than those of the control group ( Figure 2 C, D).

[0057] c. Figure 3 After successfully constructing FITC-labeled Rg3-lipo liposomes, PBS, FITC-Rg3-lipo, αPD-L2 antibody single drug, FITC-Rg3-lipo and αPD-L2 combination, and αPD-L2-loaded FITC-labeled new ginsenoside Rg3 liposomes were used to treat MC38 ( Figure 3 A), mouse primary DCs ( Figure 3B) Comparison of phagocytosis of different drugs in each group of cells 24 hours after cell treatment.

[0058] d. Figure 4 To inject the above-mentioned PD-L2-overexpressing mouse intestinal cancer cells and control cells MC38 into the tail vein of mice, construct a mouse intestinal cancer lung metastasis model, and on the 6th, 8th, and 10th days, use PBS, Rg3-lipo, aPD-L2 antibody single drug, Rg3-lipo and aPD-L2 combination, and aPD-L2-loaded new ginsenoside Rg3 liposomes to treat mice, and on the 14th day, all mice are sacrificed, the mouse lungs are taken, and then fixed in 4% paraformaldehyde and subjected to HE staining. The results show that the metastasis area ratio of the aPD-L2-loaded new ginsenoside Rg3 liposome group is lower than that of the combination group: 39.3% (combination group: 51.2% VS aPD-L2-loaded new ginsenoside Rg3 liposome group: 31.1%, P<0.05) Figure 4 A, B, C); the survival period of the aPD-L2-loaded new ginsenoside Rg3 liposome group is significantly longer than that of the other groups, and all mice are alive on the day of sampling Figure 4 D);

[0059] e. Figure 5 To inject the above-mentioned PD-L2-overexpressing mouse intestinal cancer cells MC38 into the tail vein of mice, construct a mouse intestinal cancer lung metastasis model, and on the 6th, 8th, and 10th days, use PBS, Rg3-lipo, aPD-L2 antibody single drug, Rg3-lipo and aPD-L2 combination, and aPD-L2-loaded new ginsenoside Rg3 liposomes to treat mice, and on the 14th day, all mice are sacrificed, the mouse spleens are taken, ground to break red, centrifuged to resuspend as a single cell suspension, and then subjected to flow cytometry staining, Figure 5 A is the flow cytometry graph of the CD8 + T cell PD-1 expression level of the spleen of each group of mice, Figure 5 B is the flow cytometry graph of the PD-1 + CD8 + T cell proportion of the spleen of each group of mice.

[0060] f. Figure 6 To Figure 3 The sections of the important organs of each group of mice: the main organs have no pathological damage, A, B, C, and D are the HE section staining of the heart, liver, spleen, and kidney of each group, respectively.

[0061] (7) Experimental conclusion: using lentiviral vector to transfect mouse colorectal cancer cell line MC38 to make it overexpress PD-L2 molecule, the results showed that compared with the control group, the tumor overexpressing PD-L2 showed stronger migration and invasion ability. Further, we constructed a colon cancer lung metastasis model by tail vein injection of the two groups of cells, the results showed that the tumor cells in the PD-L2 overexpression group also showed stronger metastasis in vivo. Then we used the new ginsenoside Rg3 liposome loaded with αPD-L2 to treat the lung metastasis model mice, and found that it could significantly reduce the number and area of lung metastasis nodules in mice, prolong the survival of mice, and had no damage to the main organs.

[0062] The above examples are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the essence of the present application should be covered within the protection scope of the present application.

Claims

1. A preparation method of ginsenoside Rg3 liposome loaded PD-L2 neutralizing antibody, characterized in that, The method comprises the following steps: The ginsenoside Rg3 and phospholipid are dissolved in a mixed organic solvent of anhydrous ethanol and chloroform at a certain mass ratio, and a lipid film is formed by rotary evaporation and vacuum drying, and then a PBS solution of αPD-L2 antibody is added, and after ultrasonic treatment and membrane filtration, the Rg3-lipo-αPD-L2 preparation is obtained; preferably, the phospholipid is selected from one or more phospholipids in the group consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), sphingomyelin (SM) and hydrogenated derivatives thereof; further, the phospholipid is dioleoylphosphatidylcholine (DOPC).

2. The method of claim 1, wherein the preparation of the ginsenoside Rg3 liposome-loaded PD-L2 neutralizing antibody formulation is characterized by, The mass ratio of ginsenoside Rg3 to phospholipid is 8:12-18; further, the mass ratio of ginsenoside Rg3 to phospholipid is 8:

15.

3. The method of claim 1, wherein the preparation of the ginsenoside Rg3 liposome-loaded PD-L2 neutralizing antibody formulation is characterized by, The volume ratio of anhydrous ethanol to chloroform in the mixed solvent is 1:0.8-2; further, the volume ratio of anhydrous ethanol to chloroform in the mixed solvent is 1:

1.

4. The method of claim 1, wherein the preparation of the ginsenoside Rg3 liposome-loaded PD-L2 neutralizing antibody formulation is characterized by, The mass / volume of the sum of ginsenoside Rg3 and phospholipid to the mixed organic solvent is 20-25 mg:10-20 mL.

5. The method of claim 1, wherein the preparation of the ginsenoside Rg3 liposome-loaded PD-L2 neutralizing antibody formulation is characterized by, The temperature of the reduced pressure evaporation is 20-40°C; further, the temperature of the reduced pressure evaporation is 25°C.

6. The method of claim 1, wherein the preparation of the ginsenoside Rg3 liposome-loaded PD-L2 neutralizing antibody formulation is characterized by, The time of the reduced pressure rotary evaporation is 10-20 min; further, the time of the reduced pressure rotary evaporation is 15 min.

7. The method of claim 1, wherein the preparation of the ginsenoside Rg3 liposome-loaded PD-L2 neutralizing antibody formulation is characterized by, The time of the vacuum drying is 2-4 h; further, the time of the vacuum drying is 3 h.

8. The method of claim 1, wherein the preparation of the ginsenoside Rg3 liposome-loaded PD-L2 neutralizing antibody formulation is characterized by, The concentration of the αPD-L2 solution is 1-6 mg / mL; further, the concentration of the αPD-L2 solution is 3 mg / mL.

9. The method of claim 1, wherein the preparation of the ginsenoside Rg3 liposome-loaded PD-L2 neutralizing antibody formulation is characterized by, The mass ratio of ginsenoside Rg3 to αPD-L2 is 2-8:3; further, the mass ratio of ginsenoside Rg3 to αPD-L2 is 4:

3.

10. The method of claim 1, wherein the preparation of the ginsenoside Rg3 liposome-loaded PD-L2 neutralizing antibody formulation is characterized by, The ultrasonic treatment conditions are 15-25°C, power 150-300 W, and time 20-40 min; further, the ultrasonic treatment conditions are 20°C, power 200 W, and time 30 min.

11. A ginsenoside Rg3 liposome loaded PD-L2 neutralizing antibody preparation prepared by any one of the methods of claims 1-10.

12. Use of the ginsenoside Rg3 liposome loaded PD-L2 neutralizing antibody preparation of claim 11 in the preparation of an antitumor drug.

13. Use according to claim 12, characterized in that, The antitumor drug is an anti-colon cancer drug; further, the antitumor effect is inhibition of tumor metastasis or / and prolongation of patient survival. The antitumor drug is an anti-colon cancer drug; further, the antitumor effect is inhibition of tumor metastasis or / and prolongation of patient survival.

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