WO2017079881A1 - Method for enhancing capacity to kill abnormal cell and pharmaceutical composition - Google Patents

Abstract

Disclosed are a method for enhancing capacity to kill an abnormal cell, a pharmaceutical composition and a method for enhancing ADCC in a patient undergoing an antibody drug therapy. The method of treating a patient comprises a step of administering to the patient an NK cell in combination with an antibody drug. The NK cell comprises an HANK cell. Acquisition of the HANK cell comprises: using an empty cell envelope carrying a cytokine to perform in vitro activation of an in vivo NK cell. The NK cell comprising the HANK cell is administered to the patient in combination with the antibody drug. The HANK cell and the antibody drug can mutually enhance their capacities to kill abnormal cells, thereby significantly enhancing treatment effect.

Description

Method and pharmaceutical composition for enhancing the lethality of abnormal cells Technical field

The present invention relates to the field of biomedical, and in particular, to a method and a pharmaceutical composition for enhancing the lethality of abnormal cells.

Background technique

Natural killer (NK) cells are important immune cells of the body, not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in hypersensitivity and autoimmune diseases in some cases.

The role of NK cells is broad-spectrum and non-specific; for specific tumor cells, the targeting is not strong; like rockets, some shells may not hit the target and are wasted.

Monoclonal (monoclonal antibody) targeted drugs, including but not limited to rituximab for the treatment of leukemia and Herceptin for the treatment of breast cancer, have been used clinically for many years and have achieved good results.

The therapeutic mechanism of these monoclonal antibodies is mainly through mediating the killing effect of NK cells, ie, antibody dependent cytotoxicity (ADCC). However, because cancer patients' own NK cell activity is often low, coupled with conventional treatment such as chemotherapy or radiotherapy, NK cell activity is usually severely damaged, which naturally limits the efficacy of monoclonal antibody targeting drugs.

How to effectively or more effectively use NK cells and antibody drugs to treat diseases is of great significance.

Summary of the invention

The present invention is directed to solving one of the above technical problems at least to some extent or to provide a commercial choice.

According to an aspect of the invention, a method of treating a patient is provided. The patient has a disease, the method comprising the step of administering a combination of NK cells and an antibody drug, the NK cells comprising highly active NK (HANK) cells, the obtaining of the HANK cells comprising: utilizing cells with cytokines The empty shell expands in vitro to activate NK cells from the body.

The so-called HANK cells were obtained by the inventors by activating in vitro culture to activate NK cells derived from the body. It should be noted that the so-called HANK cells are not ordinary in vivo NK cells. Ordinary NK cells are not only small in number, but are usually in a state of inhibition, and the activity of killing abnormal cells is low, for example, the activity of killing cancer cells or virus-infected cells is low. In addition, the present invention does not limit the antibody drugs to be administered in combination, and may be a monoclonal antibody targeted drug currently available on the market, or may be a newly developed monoclonal antibody targeted drug in the future.

It should be noted that the cytokine carried by the cell vacant shell may be a cytokine which is naturally expressed on the surface of the cell itself, or may be expressed by a genetic engineering method, for example, by transient transfection or stable expression. On the cell surface, it can also be a cytokine that adsorbs or crosslinks on the cell surface. Further, the cells for preparing the cell vesicle may be primary cells such as peripheral blood mononuclear cells (PBMC), or may be passage cells such as K562 cells.

By using the method of this aspect of the invention, the combination of NK cells containing the HANK cells and the antibody drug can enhance the therapeutic effects of the NK cells and the antibody drugs, and the specific expressions include: (1) the antibody drug imparts specificity to the HANK cell. Sex, such as in combination with rituximab, allows HANK cells to kill leukemia cells, and combination with Herceptin monoclonal antibody allows HANK cells to kill breast cancer cells; (2) HANK cells can be monoclonal antibodies Provide enough active factors to effectively kill cancer cells, such as a large number of granzymes, perforin and so on.

According to an embodiment of the invention, the method of this aspect of the invention may also have at least one of the following additional technical features:

The so-called patient has at least one of a cancer, a viral infection, and an immune disease. According to one embodiment of the invention, the so-called patient is a cancer patient and the so-called antibody drug is a monoclonal antibody-targeted drug. Monoclonal targeting drugs confer specificity to HANK cells. For example, in combination with rituximab, HANK cells can kill leukemia cells. Combined with Herceptin monoclonal antibody, HANK cells can kill breast cancer cells; HANK The cells provide the monoclonal antibody with enough active factors to effectively kill cancer cells, such as a large amount of granzymes, perforin and the like. The combined use of HANK cells and monoclonal antibody-targeted drugs can increase the efficacy of each other to better treat cancer.

The antibody drug is a monoclonal antibody or a polyclonal antibody. The two antigen binding sites on one antibody molecule may be the same or different, ie, bispecific antibodies, located at the ends of the arms, called antigen-binding fragments (Fab). According to one embodiment of the invention, the so-called antibody drug is a complete monoclonal antibody drug.

According to a preferred embodiment of the present invention, the ratio of HANK cells to antibody drugs in the NK cells administered in combination is 2 × 10 ⁵ to 5 × 10 ⁵ : 1 (pieces / μg). When the antibody drug is administered, the doctor or the drug instruction will give the amount according to the patient's condition. The in vitro test and the animal test show that the combined administration of the ratio of HANK cells at the time of administration can significantly enhance the effect of treating cancer.

The present invention is not limited to the individual source used to amplify activated NK cells. According to an embodiment of the present invention, the HANK cell is obtained by in vitro activation of at least one of NK cells derived from the body: the patient's own NK cells, the patient's semi-matched NK cells, and an allogeneic NK cell. The so-called patient's haplotype NK cells refer to NK cells from the relatives of the patient. For example, collecting the patient's own peripheral blood NK cells, preferably at ordinary times Before the treatment, the peripheral blood NK cells or the umbilical cord blood NK cells of the patient's relatives, that is, the semi-matched peripheral blood NK cells or unrelated individuals, are not subject to the treatment limitation, as long as the blood transfusion infectious blood screening test is acceptable.

The so-called cell vesicles can be derived from natural cells or from engineered cells. Engineering cells refer to the modification or recombination of the genetic material of the host cell by genetic engineering technology or cell fusion technology to obtain cells with stable inheritance and unique traits. According to an embodiment of the invention, the cytokine carried by the cell vacant shell comprises at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL. Preferably, the cell vacant shell carries IL-15, 4-1BBL and IL-21 cytokines. Thus, it can be used to efficiently activate NK cells derived from the body in vitro to obtain HANK cells.

According to an embodiment of the present invention, preparing the cell empty shell comprises: washing the cells to obtain washed cells; passing the washed cells through a hypotonic treatment or other methods commonly used in the industry to obtain the The cell is empty. The inventors have found that the cell empty shell can be obtained quickly and efficiently by using the method, and the method is simple in operation, easy to control, and easy to realize mass production.

It should be noted that the cytokine carried by the cell vacant shell may be a cytokine expressed on the surface of the cell itself, or may be expressed in a cell by genetic engineering methods, for example, by transient transfection or stable expression. The surface may also be a cytokine that adsorbs or crosslinks on the cell surface. Further, the cell for preparing the cell vesicle may be a primary cell such as PBMC or a passage cell such as K562 cell or the like.

According to an embodiment of the present invention, the washing treatment comprises: suspending the cells in an isotonic solution to obtain a cell suspension; and centrifuging the cell suspension to obtain the washed cells.

According to a preferred embodiment of the invention, the isotonic solution is pre-cooled to 4 degrees Celsius before suspending the cells in an isotonic solution.

According to one embodiment of the invention, the isotonic solution is an isotonic phosphate buffer (PBS) having a pH of 7.4.

According to an embodiment of the present invention, the hypotonic treatment comprises: suspending the washed cells in a hypotonic solution according to a predetermined volume ratio, and allowing the obtained cell suspension to stand for 2 hours to obtain a cell hypotonic treatment. The cell hypotonic treatment is subjected to centrifugation to obtain the cell empty shell.

According to an embodiment of the invention, the predetermined volume ratio is 1:40, and the hypotonic solution is a hypotonic Tris hydrochloric acid buffer. Thereby, it is beneficial to increase the efficiency of hypotonic lysis of cells.

According to one embodiment of the invention, the hypotonic solution is pre-cooled to 4 degrees Celsius before suspending the washed cells in a hypotonic solution.

According to an embodiment of the present invention, obtaining the HANK cell comprises: isolating a monocyte from a peripheral blood, the PBMC comprising the in vivo NK cell; culturing the PBMC with a medium to which the cell vacant is added To Amplification is performed to activate NK cells in the PBMC to obtain the HANK cells, and the cell empty shell is obtained by the method for preparing a cell empty shell in any of the above embodiments. According to an embodiment of the present invention, activation of NK cells derived from the body by a large amount of expansion in vitro by the technique, obtaining HANK, and then returning to the patient for HANK cell therapy have been proven to have a good effect on various cancers.

According to an embodiment of the invention, the NK cells comprise at least 50% of said HANK cells. Preferably, the NK cells comprise at least 90% of the HANK cells.

In order to achieve the above HANK content, according to a preferred embodiment of the present invention, the PBMC is cultured in a medium supplemented with a cell empty shell to amplify the in vivo NK cells in the activated PBMC to obtain HANK cells, including: The PBMCs were cultured in an X-Vivo15 serum-free medium containing 200 IU/ml IL-2, the cell empty shell and 5% autologous plasma for 12-20 days, the number of empty cells added to the monocytes The ratio of the number is 1:1.

Preferably, in order to efficiently obtain high content of HANK cells, the X-Vivo15 serum-free medium and the cell vesicles are added at least once on days 4-8 of the culture.

According to an embodiment of the present invention, the step of administering the NK cells in combination with the antibody drug is: administering the NK cells and the antibody drug sequentially or simultaneously.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising an antibody drug and NK cells, the NK cells comprising HANK cells, the obtaining of the HANK cells comprising: utilizing a cytokine Expansion of the NK cells from the body is activated by expansion outside the cell empty envelope.

The HANK cells and antibody drugs in this combination are mutually synergistic, and in vitro cell assays and in vivo mouse experiments indicate that treatment of the disease with the pharmaceutical composition can achieve significant therapeutic effects. The diseases mentioned include cancer, viral infections and immune diseases. The above description of the technical features and advantages of the method of treating a patient in one or any of the embodiments of the present invention is equally applicable to the pharmaceutical composition of this aspect of the present invention, and will not be described herein.

The so-called patient has at least one of a cancer, a viral infection, and an immune disease. According to one embodiment of the invention, the so-called patient is a cancer patient and the so-called antibody drug is a monoclonal antibody-targeted drug. Monoclonal targeting drugs confer specificity to HANK cells. For example, rituximab allows HANK cells to kill leukemia cells. Herceptin monoclonal antibody allows HANK cells to kill breast cancer cells; HANK cells are the monoclonal antibodies. The drug provides enough active factors to effectively kill cancer cells, such as a large number of granzymes, perforin and the like. In combination with NK cells and monoclonal antibody-targeted drugs containing HANK cells, HANK and monoclonal antibodies can increase the therapeutic effect of each other to achieve better treatment of cancer.

The antibody drug is a monoclonal antibody or a polyclonal antibody. The two antigen binding sites on one antibody molecule may be the same or different, ie, bispecific antibodies, located at the ends of the arms, called antigen-binding fragments (Fab). According to one embodiment of the invention, the so-called antibody drug is Fc-including The complete monoclonal antibody of the segment.

According to a preferred embodiment of the present invention, the ratio of HANK cells to antibody drugs in NK cells in the pharmaceutical composition is 2 × 10 ⁵ to 5 × 10 ⁵ : 1 (pieces / μg). When the antibody drug is administered, the doctor or the drug instruction will give the amount according to the patient's condition. In vitro tests and animal tests show that the specific ratio of HANK cells administered at the time of administration can significantly enhance the effect of treating cancer.

The present invention does not limit the source of the individual used to amplify the activated NK cells, as long as the transfusion infectious disease screening is acceptable. According to an embodiment of the present invention, the HANK cell is obtained by in vitro activation of at least one of NK cells derived from the body: NK cells of the patient, semi-matched NK cells of the patient, and NK cells which are not related to allogeneic . The so-called patient's haplotype NK cells refer to NK cells from the relatives of the patient. For example, the patient's own PBMC is collected to prepare NK cells, preferably before conventional treatment; or the patient's relatives, that is, the semi-matched PBMCs are prepared to prepare NK cells; or the unrelated individual PBMCs are used to prepare NK cells or cord blood to prepare NK cells, It is not subject to this restriction.

According to an embodiment of the invention, the cytokine carried by the cell vacant shell comprises at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL. Preferably, the cell vacant shell carries IL-15, 4-1BBL and IL-21 cytokines. In this way, NK cells in vivo can be efficiently expanded in vitro to obtain HANK cells.

According to still another aspect of the present invention, there is provided a method of enhancing an ADCC effect in a patient being treated with an antibody drug, the method comprising the step of administering a combination of NK cells and an antibody drug, the NK cells comprising HANK cells, The acquisition of HANK cells involves the activation of NK cells in vivo using an empty envelope of cells with cytokines. The above description of the technical features and advantages of the method of treating a patient in one or any of the embodiments of the present invention is equally applicable to the method of enhancing the ADCC effect of this aspect of the present invention, and will not be described herein.

The so-called patient has at least one of a cancer, a viral infection, and an immune disease. According to one embodiment of the invention, the so-called patient is a cancer patient and the so-called antibody drug is a monoclonal antibody-targeted drug. Monoclonal targeting drugs confer specificity to HANK cells. For example, rituximab allows HANK cells to kill leukemia cells. Herceptin monoclonal antibody allows HANK cells to kill breast cancer cells; HANK cells are the monoclonal antibodies. The drug provides enough active factors to effectively kill cancer cells, such as a large number of granzymes, perforin and the like. In combination with NK cells and monoclonal antibody-targeted drugs containing HANK cells, HANK cells and monoclonal antibodies can increase the therapeutic effect of each other to better treat cancer.

The antibody drug is a monoclonal antibody or a polyclonal antibody. The two antigen binding sites on one antibody molecule may be the same or different, ie, bispecific antibodies, located at the ends of the arms, called antigen-binding fragments (Fab). According to one embodiment of the invention, the so-called antibody drug is an intact monoclonal antibody comprising an Fc segment.

According to a preferred embodiment of the present invention, the ratio of HANK cells to antibody drugs in the NK cells administered in combination is 2 × 10 ⁵ to 5 × 10 ⁵ : 1 (pieces / μg). When administered with an antibody drug, the doctor or the drug manual will give an amount according to the patient's condition. In vitro tests and animal tests show that the combined administration of this ratio of HANK cells at the time of administration can significantly enhance the effect of treating cancer.

The present invention does not limit the PBMC-derived individuals used to amplify activated NK cells in vivo, as long as the transfusion infectious disease is screened. According to one embodiment of the invention, the HANK cells are obtained by at least activating one of the following NK cells in vitro: NK cells of the patient, semi-matched NK cells of the patient, and NK cells that are not allogeneic. The so-called patient's haplotype NK cells refer to NK cells from the relatives of the patient. For example, NK cells in the patient's own PBMC are collected, preferably prior to conventional treatment; NK cells in the patient's relatives, ie, semi-matched PBMCs, or NK cells or cord blood NK cells in PBMCs of unrelated individuals are collected. It is not subject to this restriction.

According to an embodiment of the invention, the cytokine carried by the cell vacant shell comprises at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL. Preferably, the cell vacant shell carries IL-15, 4-1BBL and IL-21 cytokines. In this way, it is possible to efficiently activate and activate NK cells in vivo in vitro to obtain HANK cells.

The various aspects of the invention described above combine the use of HANK cells with antibody drugs, particularly monoclonal antibody targeting agents, to increase the lethality of monoclonal antibodies, and to enable a broad spectrum of non-specific HANK cells to target tumor cells and viruses. The function of infecting cells, the two can synergize each other.

DRAWINGS

The described and/or additional aspects of the invention and its advantages will be more apparent and more readily understood from the following description of the accompanying drawings in which:

Figure 1 shows the ADCC effect of HANK cells in combination with rituximab on SUDHL-4 lymphoma cells in one embodiment of the invention.

Figure 2 shows the efficacy of HANK cells in combination with rituximab in one embodiment of the invention on mouse lymphoma.

Figure 3 shows the ADCC effect of HANK cells in combination with Herceptin monoclonal antibody on MDA-MB-435 breast cancer cells in one embodiment of the invention.

Figure 4 shows the efficacy of HANK cells in combination with Herceptin monoclonal antibody in mouse breast cancer in one embodiment of the invention.

Figure 5 shows the ADCC effect of HANK cells in combination with GPC3 mAb on HepG2 liver cancer cells in one embodiment of the invention.

Figure 6 shows the effect of HANK cells in combination with GPC-3 monoclonal antibody on liver cancer in mice in one embodiment of the present invention.

Figure 7 shows the ADCC effect of HANK cells in combination with mC3 monoclonal antibody on JEV-infected BHK cells in one embodiment of the invention.

Figure 8 shows the protective effect of HANK cells in combination with mC3 monoclonal antibody on JEV-infected mice in one embodiment of the invention.

detailed description

Embodiments of the present invention are described in detail below. The embodiments described below are illustrative only and are not to be construed as limiting the invention. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in the art or in accordance with the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

Embodiment 1

Preparation of RPMI 8866 cell empty shell

Cell emptyings were prepared using the passage cell line RPMI 8866, which is a human B lymphoblastoid cell line according to the following procedure. details as follows:

RPMI8866 cells were suspended in 3-fold amount of isotonic PBS pre-cooled to 4 ° C and pH 7.4, centrifuged at 1500 rpm for 10 minutes at 4 ° C, the supernatant was removed, and washing was repeated 1-3 times to obtain washed RPMI 8866. Cells; then washed RPMI 8866 cells were added to a hypotonic Tris-HCl buffer pre-cooled to 4 ° C and a concentration of 10 mmol/L at a ratio of 1:40, while slowly stirring, then, The mixture was allowed to stand in a refrigerator at 4 ° C for 2 hours to completely lyse the cells; then, the cells were centrifuged at 9000 rpm for 10 minutes at 4 ° C to precipitate the cells, and the washing was further repeated and centrifuged 3-5 times. RPMI8866 cell empty shell, namely RPMI 8866-empty shell. Then, it is divided into 2×10 ⁷ cell empty shells/ml, and is frozen in a refrigerator at minus 80 degrees Celsius; or after being freeze-dried, it is stored frozen in a refrigerator at 4 degrees Celsius.

Embodiment 2

1, HANK cell preparation, the general method is as follows:

1) collecting peripheral blood coagulation in healthy people, separating PBMC with lymphocyte separation solution;

2) PBMC can be cultured in X-Vivo15 serum-free medium supplemented with IL-2 and RPMI 8866-shell and 5% autologous plasma, which can activate and activate NK cells in large quantities;

3) When cultured for about 14 days, the number of NK cells can be expanded by hundreds to thousands of times, and the purity of NK cells can be increased from 10% in PBMC to above 80-90%;

The activated NK cells expanded in vitro are HANK cells, which can be used freshly or frozen in a -80 degree refrigerator or liquid nitrogen for use as effector cells in subsequent in vitro killing assays, in animal experiments alone or Combined with monoclonal antibody, it plays a role in anti-cancer and anti-virus.

2. Using RPMI 8866-empty shell to stimulate NK cell expansion in PBMC, a total of 3 normal human peripheral blood mononuclear cells were tested, as follows:

1) Preparation of autologous plasma: 50 ml of anticoagulated peripheral blood was taken, 700 G, centrifuged at room temperature for 20 min; the plasma was aspirated, placed in a water bath at 56 ° C for 30 min; then allowed to stand at 4 ° C for 15 min; finally 4 ° C, 900 G, centrifuged for 30 min, taken from the body The plasma was stored at 4 ° C for later use.

2) The cell layer after taking the plasma was added to D-PBS and mixed, and the PBMC was separated by using the lymphocyte separation solution 800G for 20 minutes;

3) One T75 flask was selected, and 40 ml of lymphocyte complete medium (containing about 200 IU/ml IL-2 and autologous plasma 1-10%) was added to the isolated PBMC to prepare a cell suspension (about 5×10 ⁶ cells). Lymphocytes), added to the T75 flask; simultaneously added 2 × 10 ⁷ RPMI 8866 - empty shell, placed in a saturated humidity, 37 ° C, 5.0% CO ₂ incubator;

4) adding about 40 ml of lymphocyte complete culture solution on the fourth day of the culture;

5) Transfer the cells in the T75 flask to the culture bag about the seventh day of the culture, add the complete lymphocyte culture solution to about 400 ml, and add about 8×10 ⁷ front tissues. Obtained cell empty shell;

6) The culture is passaged into two culture bags on the tenth day of the culture, wherein each culture bag contains about 640 ml of the complete lymphocyte culture solution;

7) The culture product was collected on the twelfth day of the culture, and the effect of activating and activating the NK cells in PBMC by different methods was tested. Among them, NK cells in PBMC can be cultured at different times depending on the number of cells required, for example, continuous culture for 18 days or 20 days.

Embodiment 3

In vitro killing test, general method:

1) One tube of HANK effector cells was resuscitated one day before the assay, and cultured in NK cell culture medium (NKEM, X-Vivo15 containing IL-2 and 5% autologous plasma);

2) One cell line was used as a target cell for each measurement, and K562 cells were used as a positive control cell. 6X10^5 effector cells and 3X10^5 target cells are required;

3) Dilute Calcein-AM (calcein-AM) with NKEM medium to prepare CAM solution;

4) Suspend 10^6 target cells in 1 mL of CAM solution. Incubate at 37 ° C for 1 hour and shake at the appropriate time. It was then washed twice with NKEM medium and centrifuged at 1200 rpm for 5 min each time. Count, adjust the concentration to 1X10^5/mL;

5) Dilute the effector cells to 1×10^6/mL, set 3 wells on the U-bottom 96-well cell culture plate, add 200 uL of target cells per well, and the corresponding target ratio (E:T) 10:1;

6) The test set the maximum release hole, the negative control hole, the natural killing resistance, and the ADCC hole;

7) Add 100uL 2% Triton X-100 to the largest release well, add the appropriate dilution of the monoclonal antibody to the ADCC well, and the remaining wells. Add 100 uL of complete medium;

8) The effector cells were diluted 5 times, and the ratio of the last dilution (E:T) was 0.3125:1;

9) Add 100 μL of target cells to each well and centrifuge at 100 g for 1 min to direct cell contact. Incubate for 4 hours at 37 ° C in a 5% CO ₂ float tank;

10) Gently pipette the cells with a 100 μL pipette to suspend the released calcein; centrifuge at 100 g for 5 min to pellet the cells. Gently pipet 100 μL of the supernatant and transfer to a new plate to prevent foaming. If there is foam formation, use a needle to pierce;

11) Reading the plate with a fluorescence plate reader (excitation light 485 nm, emission light 530 nm);

12) Calculate the percentage of specific cytotoxicity. [(Test group - natural release group) / (maximum release group - natural release group)] X100.

Embodiment 4

Establish mouse tumor and JE model, the general method:

a) Establishment of a tumor model of human diffuse large B-cell lymphoma: subcutaneous inoculation of 10^7 cells in SCID mice can successfully establish a diffuse large B-cell lymphoma (DLBCL) xenograft model, and the tumor formation rate 70%, the histological performance of the tumor is similar to human DLBCL.

1) Cell culture: SUDHL-4 is a human GCB-like DLBCL cell line. The cells with an initial density of 2.5×10^5/ml were placed in a T25 cell culture flask containing RPMI1640 medium containing 10% FBS, 100 U/ml penicillin, 100 μg/ml glutamine, and 30 μg/ml glutamine. Incubate in °C, 5% CO2, saturated humidity incubator; after the first passage of 2-3d, transfer to T75 cell culture flask, and then transfer to T150 cell culture flask according to cell growth condition;

2) SPF-class SCID mice, female, 5 weeks old, weighing 16-20g, were randomly divided into groups. The feeding and experiment were carried out in a SPF class rat room with constant temperature (20-26 °C) and constant humidity (50%-56%). . The mice were placed in a laminar flow box with a cover mouse box, the air was filtered by medium efficiency, and the standard pellet feed was fed, and all the items in contact with the rats were previously sterilized;

3) Cell seeding: SUDHL-4 cells in logarithmic growth phase were washed twice with serum-free PBS and suspended in serum-free PBS.

Experimental groups of mice (10 per group) cell suspension containing ¹⁰⁷ cells were inoculated subcutaneously on one side of the rib 0.1ml; normal control group (10 per group) were injected subcutaneously in the right side of the rib 0.1ml PBS.

4) Detection of tumor-bearing mice: During the experiment, the general condition, tumor formation and tumor growth of the mice were observed daily. Body weight and tumor length and diameter were measured daily, and tumor volume was calculated (calculation method: π/6×length×width×height); when the tumor reached 1200 mm ³ , it was regarded as the human end point. After the mice were anesthetized and the neck was sacrificed, the tumors were observed at various parts of the body surface; then the animals were dissected and the internal organs and lymph node metastasis were observed.

b) Human breast cancer MDA-MB-435 cell line nude mouse xenograft model: fully humidified with 37 U/ml gentamicin, 10% inactivated calf serum MEM medium at 37 ° C and 5% CO 2 The human breast cancer cell line MDA-MB-435 was used for further use. Adopt BALB/C female nude mice, SPF grade, body weight 20±2g; 35-40 days old, logarithmically grown breast cancer cells MDA-MB-435, the cell concentration was adjusted to 1×10 ⁷ /ml . Under sterile conditions, MDA-MB-435 human breast cancer cells were inoculated into the fat pad of the second nipple on the left side of nude mice, and the inoculum amount was 0.1 ml/cell (the number of cells was 1×10 ⁶ /piece). After 2 weeks, the subcutaneous lesions were clearly randomized when they reached the growth of the mass. The administration time was 10 weeks, and the animals were sacrificed by cervical dislocation 3 days after the withdrawal.

c) Mouse liver cancer model: 10 million HepG2 cells were injected subcutaneously into nude mice, and treatment was started 14 days later. The formula for calculating the tumor size in cubic centimeters (mm ³ ) is: (a) X(b) X 0.5; where a is the length of the tumor and b is the width of the tumor.

d) Mouse JE model:

1) Virus strain: JEV SA14 is administered through the brain of the suckling rat for 3 consecutive generations of poisoning;

2) Infection of mice: 8 g of Balb/c mice of 3 weeks old were used, and the brain suspension of JEV-infected rats was inoculated intraperitoneally, and the measurement was 10^5 LD50;

3) Calculate the efficacy by the survival rate of infected mice.

Embodiment 5

1. Monoclonal antibody

Rituxoxib is an anti-CD20 mAb that binds to the Fc receptor CD16 on NK cells and kills CD20 positive cells, and has been used in clinical treatment of leukemia for many years in many countries.

2, HANK cells combined with rituximab for leukemia

(1) In vitro killing test: Using HANK cells as effector cells, K562 cells and SUDHL-4 cells as target cells, the natural killing effect of HANK cells on two kinds of target cells and rituximab were determined according to the description in Example 3. Anti-mediated ADCC effect.

The results are shown in Figure 1. The killing rate of HANK cells on K562 cells is about 92%, and the killing rate on SUDHL-4 cells is about 52%. Rituximab does not affect the killing effect of HANK cells on K562 cells, but The killing effect of HANK cells on SUDHL-4 cells was greatly increased, from 52% to 87%.

(2) In vivo protection test: On the 15th day after inoculation of SUDHL-4 cells, the tumor volume of the tumor-bearing mice was about 524 mm^3. The tumor-bearing mice were divided into 4 groups of 10 animals each. One group was treated with HANK cells alone, and each mouse was intravenously infused with 1×10^7 HANK cells once a week. The other group was treated with rituximab alone, and each mouse was intravenously infused with 40 μg once a week. Group 3 is treated with HANK cells in combination with rituximab, intravenously infused with 1X10^7HANK cells plus 40 μg. Clitoxib was administered once a week; Group 4 was a saline control group.

The results are shown in Figure 2: 14 days after treatment, the tumor volume of the saline control group was 1129 mm^3, the tumor volume of the HANK cell treatment group was 224 mm^3, and the rituximab-targeted treatment group was 315 mm^3, HANK cells + rituximab. Treatment group 83mm^3. The smaller the tumor volume after treatment, the better the efficacy.

Embodiment 6

1. Monoclonal antibody

Herceptin is an anti-EGFR Her2 monoclonal antibody that binds to Her2 positive cells and kills EGFR-positive cells and has been used in clinical treatment of breast cancer for many years in many countries.

2, HANK cells combined with Herceptin for breast cancer

(1) In vitro killing test: Using HANK cells as effector cells, K562 cells and MDA-MB-435 cells as target cells, the natural killing effect of HANK cells on two kinds of target cells and the ADCC effect mediated by Herceptin monoclonal antibody were determined. .

The results are shown in Figure 3. The killing rate of HANK cells to K562 cells is about 92%, and the killing rate of MDA-MB-435 cells is about 63%. Herceptin monoclonal antibody does not affect the killing effect of HANK cells on K562 cells. However, the killing effect of HANK cells on MDA-MB-435 cells was greatly increased, from 63% to about 91%.

(2) In vivo protection test: On the 15th day after inoculation of MD-MB-435 cells, the tumor volume of the tumor-bearing mice was about 311 mm^3. The tumor-bearing mice were divided into 4 groups of 10 animals each. One group was treated with HANK cells alone, and each mouse was intravenously infused with 1×10^7 HANK cells once a week. The other group was treated with Herceptin monoclonal antibody alone, and each mouse was intravenously infused with 40 μg once a week. Group 3 was treated with HANK cells in combination with Herceptin, intravenously infused with 1X10^7HANK cells plus 40 μg of Herceptin monoclonal antibody once a week; Group 4 was a saline control group.

The results are shown in Figure 4: 14 days after treatment, the saline control group had a tumor volume of 724 mm^3, the HANK cell treatment group had a tumor volume of 121 mm^3, and the Herceptin monoclonal antibody targeted treatment group was 312 mm^3, HANK cells + Herceptin combination. Treatment group 51mm^3. The smaller the tumor volume after treatment, the better the efficacy.

Example 7

1, monoclonal antibody targeted drugs

1G12 is an anti-GPC3 monoclonal antibody that inhibits the proliferation of GPC3-positive hepatoma cells in vitro and has a good therapeutic effect on transplanted hepatocellular carcinoma (HCC) in nude mice.

2, HANK cells combined with GPC3 monoclonal antibody treatment of liver cancer

(1) In vitro killing test: Using HANK cells as effector cells, K562 cells and HepG2 cells as target cells, the natural killing effect of HANK cells on two kinds of target cells and the anti-GPC3 monoclonal antibody 1G12-mediated ADCC effect were determined.

The results are shown in Figure 5: the killing rate of HANK cells to K562 cells is about 92%, for SUDHL-4 cells. The killing rate was about 52%; 1G12 monoclonal antibody did not affect the killing effect of HANK cells on K562 cells, but greatly increased the killing effect of HANK cells on HepG2 cells, from 48% to 85%.

(2) In vivo protection test: On the 15th day after inoculation of HepG2 cells, the tumor-bearing mice had a tumor volume of about 435 mm^3. The tumor-bearing mice were divided into 4 groups of 10 animals each. One group was treated with HANK cells alone, and each mouse was intravenously infused with 1×10^7HANK cells once a week. The other group was treated with 1G12 mAb alone. Each mouse was intravenously infused with 40 μg once a week; The group was treated with HANK cells in combination with 1G12, intravenously infused with 1X10^7HANK cells plus 40 μg of 1G12 mAb once a week; and group 4 was a saline control group.

The results are shown in Fig. 6: 14 days after treatment, the tumor volume of the saline control group was 936 mm^3, the tumor volume of the HANK cell treatment group was 185 mm^3, the target treatment group of 1G12 monoclonal antibody was 423 mm^3, and the HANK cell +1G12 combination treatment group was 77 mm^. 3. The smaller the tumor volume after treatment, the better the efficacy.

Example eight

1, monoclonal antibody targeted drugs

mC3 is an anti-JEV (Japanese encephalitis virus) monoclonal antibody, which has the activity of neutralizing JEV alone and has a protective effect on mice infected with JEV.

2, HANK cells combined with Japanese encephalitis virus monoclonal antibody treatment of Japanese encephalitis

(1) In vitro killing test: HANK cells were used as effector cells, K562 cells and JEV-infected BHK cells were used as target cells, and the natural killing effect of HANK cells on two kinds of target cells and the anti-JEV mC3-mediated ADCC effect were determined.

The results are shown in Figure 7. The killing rate of HANK cells to K562 cells was about 92%, and the killing rate of JEV-BHK cells was about 62%. The mC3 monoclonal antibody did not affect the killing effect of HANK cells on K562 cells, but greatly improved. The killing effect of HANK cells on JEV-BHK cells increased from 62% to about 88%.

(2) In vivo protection test: On the 5th day after JEV infection, some mice were nearly dead. The infected mice were divided into 4 groups of 10 animals each. One group was treated with HANK cells alone, and each mouse was intravenously infused with 1X10^7HANK cells once a week. The other group was treated with mC3 monoclonal antibody alone. Each mouse was intravenously infused with 40 μg once a week; The group was treated with HANK cells combined with mC3 monoclonal antibody, intravenously infused with 1X10^7HANK cells plus 40 μg mC3 monoclonal antibody once a week; group 4 was a saline control group.

The results are shown in Figure 8: 14 days after treatment, all the mice in the saline control group, the survival rate of the mice in the HANK cell treatment group was 72%, and the survival rate of the mC3 monoclonal antibody-targeted group was 62%. HANK cells + mC3 combination therapy The survival rate of the mice in the group was 90%. The higher the protection rate, the better the efficacy.

In the description of the present specification, reference is made to the terms "one embodiment", "some embodiments", "example", "with The description of the embodiments, or "some examples" and the like, is intended to mean that the specific features, structures, materials, or characteristics described in connection with the embodiments or examples are included in at least one embodiment or example of the invention. The singular representations of the terms are not necessarily the same as the embodiments or examples. Also, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. In the case of contradictions, those skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples.

Although the embodiments of the present invention have been shown and described, it is understood that the above-described embodiments are illustrative and are not to be construed as limiting the scope of the invention. The embodiments are subject to variations, modifications, substitutions and variations.

Claims (39)

1. A method of treating a patient suffering from a disease, characterized in that the method comprises the step of administering a combination of natural killer cells and an antibody drug,

   The natural killer cells comprise highly active natural killer cells,

   The obtaining of the highly active natural killer cells comprises: activating natural killer cells in vivo using an empty shell of cells with cytokines.
2. The method of claim 1 wherein said disease is selected from the group consisting of at least one of cancer, viral infection, and immune disease.
3. The method of claim 1 wherein the antibody drug is a monoclonal antibody or a polyclonal antibody.
4. The method of claim 1 wherein said natural killer cells comprise at least 50% of said highly active natural killer cells.
5. The method of claim 1 wherein said natural killer cells comprise at least 90% of said highly active natural killer cells.
6. The method of claim 1, wherein the ratio of the highly active natural killer cells to the antibody drug in the natural killer cells administered in combination is 2 x 10 ⁵ to 5 x 10 ⁵ : 1 (pieces / μg).
7. The method of claim 1 wherein said highly active natural killer cells are obtained by in vitro activation of at least one of the following natural killer cells in vivo: said patient's natural killer cells, said patient's semi-consistent natural killer Cells and natural killer cells that are not related to allogeneic.
8. The method of claim 1 wherein said cellular vesicles are derived from natural cells and/or engineered cells.
9. The method of claim 8 wherein said cytokine carried by said cellular vesicle comprises at least IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL One.
10. The method of claim 8 wherein said cell vacant shell carries IL-15, 4-1BBL and IL-21 cytokines.
11. The method of claim 8 wherein preparing said cellular empty shell comprises:

    Washing the cells to obtain washed cells;

    The washed cells are subjected to hypotonic treatment to obtain the cell empty shell.
12. The method of claim 11 wherein said washing process comprises:

    Suspending the cells in an isotonic solution to obtain a cell suspension;

    The cell suspension is subjected to centrifugation to obtain the washed cells.
13. The method of claim 12 wherein said isotonic solution is pre-cooled to 4 degrees Celsius prior to suspending said cells in an isotonic solution.
14. The method of claim 12 wherein said isotonic solution is an isotonic phosphate buffer having a pH of 7.4.
15. The method of claim 11 wherein said hypotonic treatment comprises:

    The washed cells are suspended in a hypotonic solution according to a predetermined volume ratio, and the obtained cell suspension is allowed to stand for 2 hours to obtain a cell lysate;

    The cell lysate is subjected to centrifugation to obtain the cell empty shell.
16. The method of claim 15 wherein said predetermined volume ratio is 1:40.
17. The method of claim 15 wherein said hypotonic solution is pre-cooled to 4 degrees Celsius prior to suspending said washed cells in a hypotonic solution.
18. The method of claim 15 wherein said hypotonic solution is a hypotonic Tris hydrochloride buffer.
19. The method of claim 1 wherein said highly active natural killer cells are obtained, comprising:

    Mononuclear cells are isolated from peripheral blood, the monocytes comprising the natural killer cells in vivo;

    The monocyte is cultured in a medium to which the cell vacant is added to amplify and activate the natural killer cells in the monocyte to obtain the highly active natural killer cell.

    The cell vesicle is obtained by the method of any one of claims 11-18.
20. The method of claim 19, wherein said mononuclear cells are cultured in a medium to which a cell vacant is added to amplify the natural killer cells in the activated monocytes to obtain highly active natural killer cells, including:

    The monocytes were cultured in X-Vivo15 serum-free medium supplemented with 200 IU/ml IL-2, the cell empty shell and 5% auto plasma for 12-20 days, and the number and addition of the empty shells of the cells were The ratio of monocytes is 1:1.
21. The method of claim 20, wherein said X-Vivo15 serum-free medium and said cell vesicle are added at least once during said fourth to eighth days of said culture.
22. A method according to any one of claims 1 to 21, wherein the step of administering the natural killer cell and the antibody drug in combination is:

    The natural killer cells and the antibody drug are administered sequentially or simultaneously.
23. A pharmaceutical composition comprising an antibody drug and a natural killer cell, the natural killer cell comprising a highly active natural killer cell,

    The obtaining of the highly active natural killer cells comprises: activating natural killer cells in vivo using an empty shell of cells with cytokines.
24. The pharmaceutical composition according to claim 23, wherein the antibody drug is a monoclonal antibody or a polyclonal antibody.
25. The pharmaceutical composition according to claim 23, wherein said natural killer cells comprise at least 50% of said highly active natural killer cells.
26. The pharmaceutical composition according to claim 23, wherein said natural killer cells comprise at least 90% of said highly active natural killer cells.
27. The pharmaceutical composition according to claim 23, wherein the ratio of the highly active natural killer cells to the antibody drug in the natural killer cells in the pharmaceutical composition is 2 × 10 ⁵ to 5 × 10 ⁵ : 1 (pieces / μg ).
28. The pharmaceutical composition according to claim 23, wherein said highly active natural killer cells are obtained by in vitro activation of at least one of natural killer cells in vivo: said patient's natural killer cells, said patient's semi-conforming natural Killer cells and natural killer cells that are not related to allogeneic.
29. The pharmaceutical composition according to claim 23, wherein said cytokine carried by said vacant shell comprises at least IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL. One kind.
30. The pharmaceutical composition according to claim 29, characterized in that the cell vacant shell carries IL-15, 4-1BBL and IL-21 cytokines.
31. A method of enhancing the ADCC effect of a patient being treated with an antibody drug, characterized in that the method comprises the step of administering a natural killer cell and an antibody drug in combination,

    The natural killer cells comprise highly active natural killer cells,

    The obtaining of the highly active natural killer cells comprises: activating natural killer cells in vivo using an empty shell of cells with cytokines.
32. The method of claim 31, wherein the antibody drug is a monoclonal antibody or a polyclonal antibody.
33. The method of claim 31, wherein said natural killer cells comprise at least 50% of said highly active natural killer cells.
34. The method of claim 31, wherein said natural killer cells comprise at least 90% of said highly active natural killer cells.
35. The method according to claim 31, wherein the ratio of the highly active natural killer cells to the antibody drug in the natural killer cells administered in combination is 2 × 10 ⁵ to 5 × 10 ⁵ : 1 (pieces / μg).
36. The method of claim 31, wherein said highly active natural killer cells are obtained by in vitro activation of at least one of the following natural killer cells in vivo: said patient's natural killer cells, said patient's semi-consistent natural killer Cells and natural killer cells that are not related to allogeneic.
37. The method of claim 31, wherein said cellular vesicles are derived from natural cells and/or engineered cells.
38. The method of claim 37, wherein said cytokine carried by said vacant shell comprises at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL One.
39. The method of claim 37, wherein said cell vacant shell carries IL-15, 4-1BBL and IL-21 cytokines.

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