CN113577267B

CN113577267B - Pharmaceutical compositions of NK cells and antibodies and their use in the treatment of cancer

Info

Publication number: CN113577267B
Application number: CN202111037406.3A
Authority: CN
Inventors: 王京; 钟桂华
Original assignee: Jiangxi Beizheng Stem Cell Biotechnology Co ltd
Current assignee: Jiangxi Beizheng Stem Cell Biotechnology Co.,Ltd.
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2022-04-08
Anticipated expiration: 2041-09-06
Also published as: CN113577267A

Abstract

The invention relates to a medicine composition of NK cells and antibodies and application thereof in treating cancers, wherein the medicine composition contains VEGFR2 monoclonal antibody specific to VEGFR2 protein, docetaxel and high-activity NK cells, and the medicine composition, the VEGFR2 monoclonal antibody, the docetaxel and the high-activity NK cells can be used together to effectively inhibit growth of liver cancer tumors, so that the medicine composition has a good application prospect.

Description

Pharmaceutical compositions of NK cells and antibodies and their use in the treatment of cancer

Technical Field

The present invention relates to the field of biology, more specifically to pharmaceutical compositions of NK cells and antibodies and their use in the treatment of cancer.

Background

Liver cancer is one of common malignant tumors in the world, the number of patients dying from liver cancer worldwide every year is estimated to be 125 thousands, and China is a high-incidence area of liver cancer, so that the research on the diagnosis and treatment of liver cancer is of great significance. Since the technology of creating B lymphocyte hybridomas by Kohler and Milstein in 1975, various monoclonal antibodies (McAb) have been developed in succession, and they play a positive role in basic research and clinical application of disease diagnosis, and among them, the development of liver cancer monoclonal antibody has brought new hope for the refractory tumor.

Most of the studies on the monoclonal antibody against liver cancer are alpha fetoprotein, the monoclonal antibody against liver cancer is little, and in the preparation of the monoclonal antibody against liver cancer, the traditional method is mostly adopted, i.e. soluble substances or cultured cells of liver cancer are taken as immunogens, and the monoclonal antibody with high affinity and high specificity is difficult to obtain by the method because of the mutation or partial loss of tumor antigens. The method is a good method for preparing the monoclonal antibody, and the specific liver cancer monoclonal antibody can be obtained by fusion by the method, wherein the positive rate of HAb18 liver cancer immunohistochemical staining with good specificity is 75 percent, and the method passes the requirements of the key points of human murine monoclonal antibody preparation and quality control of Chinese pharmaceutical and biological product institute. In addition, a rat anti-human primary liver cancer (PHC) cell membrane antibody Hepama-I is reported by the Shanghai cell of the Chinese academy of sciences, and the research proves that the antibody has higher affinity to the PHC cell. These add confidence to the deep research of the liver cancer monoclonal antibody in our country.

The guide chemotherapy, radiotherapy and radioimmunoassay diagnosis using monoclonal antibody as carrier has made substantial progress in recent years. Chemical medicine-monoclonal antibody cross-linked substance: there are dozens of anti-cancer drugs used for cross-linking at home and abroad, mainly including MTX, MMC, DM, AM, etc., and they are selected according to the tumor types. The IC50 value of Hepama-I IgG-BSA-MTX for liver cancer cells is 8 multiplied by 10 to 8M, while the IC50 value of HeLa cells is only 3 multiplied by 10 to 6M; ② cytotoxin-monoclonal antibody cross-linked substance: ricin, smallpox toxin, diphtheria toxin, phytotoxin, A chain-like ribosome inactivating protein; ③ isotope-monoclonal antibody cross-linker: because of the heterogeneity of tumor antigens, antibodies cannot reach liver cancer cells, so that the effect of specific radiotherapy is better than that of chemotherapy.

The antibody-directed chemotherapy can change the natural distribution of the drug in vivo, cause the concentration of the drug in the tumor area, and thus play a specific killing role. Zhangshang right and the like research anti-liver cancer monoclonal antibody Hepama-I mitomycin conjugate, and tumor-bearing nude mouse animal experiments show that the targeted chemotherapeutic drug has obvious affinity compared with simple chemotherapy, and is the premise and key of the application.

Radioimmunoassay and therapeutic agent prepared by using radionuclide labeled monoclonal antibody have become one of the powerful means for early diagnosis, comprehensive treatment and postoperative cleaning of tumor. At present, nearly ten imaging agents and therapeutic agents pass through FDA in the United states and enter the clinical verification stage I and II, but no preparation aiming at liver cancer exists, and the research on the aspect in China is originally disclosed. Style reports the 131I-Hepama-I through the femoral artery interventional radioimmunoassay and the study of 30 cases of primary liver cancer patients who lose the operative indication, the imaging rate is 14/18, the excellent imaging time is 96h after the injection, the AFP reduction rate is 75.0%, and the tumor partial shrinkage rate is 66. 6 percent, compared with a control group, the survival time is obviously prolonged, and the nuclide iodine labeled heparan I monoclonal antibody can be used as one of the comprehensive therapies for treating late-stage liver cancer. The liver cancer monoclonal antibody HAb18 has also been widely used and studied, wherein 131I-HAb18 radioimmunoassay and targeted therapeutic agent have been approved by national class I new drug, and clinical lot numbers are obtained to enter phase I and II clinics, and become the first nuclide-antibody radiopharmaceuticals in China. Clinical application shows that the positive imaging rate of the in vivo double-nuclide subtraction in liver cancer patients reaches 90.9 percent (40/44), and the diameter of the detected small liver cancer tumor mass is 0.5 cm. The complete remission rate of the targeted therapy of the tumor is 3.7 percent (1/27), the partial remission rate is 48.1 percent (13/27), the stability is 14.3 percent (4/27), the ineffectiveness is 33.3 percent (9/27), the survival rate of more than 2a is 46.2 percent (6/13), and the targeted therapy has no obvious influence on the hematology function, the renal function and the thyroid function of a patient.

In recent years, a 'double-bullet' immune-oriented therapeutic medicament integrating chemotherapy and internal irradiation therapy has been reported, the liver cancer double-bullet combined monoclonal antibody immune-oriented conjugate 131I-HAb18-ADM is prepared by taking liver cancer monoclonal antibody HAb18 as a carrier and 131I-ADM as a bullet according to an inertial prescription, and the result shows that the double-bullet integrates the advantages of chemotherapy and radiotherapy, the two synergies improve the effect of liver cancer-oriented therapy, and the medicament improves the inhibiting and killing effects on tumor cells.

AU2016232104B2 discloses an anti-Claudin 1 monoclonal antibody for the prophylaxis and treatment of hepatocellular carcinoma, which is capable of inhibiting the hepatocellular carcinoma cell line HUH-7.5.1, but the inhibitory effect is not particularly prominent.

US10927170B2 discloses a method for preventing and/or treating hepatocellular carcinoma by administering such monoclonal antibodies or pharmaceutical compositions thereof, giving experimental results for the liver cancer cell line HUH-7.5.1. Likewise, the effect of the monoclonal antibody is not as pronounced.

The prior art knows that apatinib can be combined with vascular endothelial growth factor receptor 2(VEGFR2) in a highly selective manner, block signal conduction and strongly resist tumor angiogenesis, thereby inhibiting the growth, metastasis and dissemination of tumors. However, the number of monoclonal antibodies against VEGFR2 is not sufficient, and the number of selectable species is not sufficient, and thus, there is a great market demand.

Furthermore, Natural Killer (NK) cells have a broad spectrum of tumor killing including the interaction of FasL with Fas, perforin/granzyme interaction, antibody-dependent cell-mediated cytotoxicity (ADCC) with tumor-specific IgG via surface CD16, secretion of tumor apoptosis-related factors (TNF-. alpha., IFN-. gamma.). Besides the function of killing target cells by dissolving NK cells, the NK cells also have important functions in the immune response of the organism. NK cells can control innate immunity through the regulatory effects on M phi, granulocytes, dendritic cells. NK cells can act on CD4+ T cells and CD8+ T cells to boost adaptive immunity. However, the way in which NK cells act in combination with other antitumor drugs is still relatively rare and is to be studied further.

Disclosure of Invention

In one aspect of the invention, there is provided a use of a monoclonal antibody that specifically inhibits VEGFR2, docetaxel, and NK cells in the preparation of a kit for treating liver cancer.

A monoclonal antibody that specifically inhibits VEGFR2 is provided, the heavy chain variable region of which (SEQ ID NO:1)

EVQLEESATELARPSASVKLSCKASGYIFSFNSYNWIKQRPGQGLEWIGVLIMPWPKQVLRYPMGGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGWFWLKHQWGLGTTLAVSS

Light chain variable region (SEQ ID NO:2)

DIVITQRPALMAASPGAKVTITCMVHVTTEMWWGGWYQQKSGISPKPWIYTRKKNHEGVPARFSGSGSGTSDSLTITSMEAEDAATYYCRIYKHCYQWFGAGTKLELK。

Further, the NK cells are prepared by the following method: centrifuging peripheral blood at 2000r/min for 10min, carefully sucking out plasma, inactivating in 56 deg.C water bath30min for later use; diluting blood cells with normal saline, adding into a centrifuge tube filled with a Ficoll solution, centrifuging at 2000r/min for 20min, collecting leucocyte cells, washing with normal saline, and counting for later use; 2X 10 of the culture medium GT-T551H3⁷After the PBMCs are resuspended, the PBMCs are inoculated into a T75 cell culture flask, and then IL-2, resveratrol, NK trophoblasts and autologous plasma are added into the system, wherein the IL-2 is 500U/ml, the final concentration of the resveratrol is 2 mu mol/L, and the trophoblasts are 1 multiplied by 10⁶ml-¹Plasma volume 5%, 5% CO at 37 ℃₂In vitro co-culturing for 15d under the condition, wherein, the NK expanded trophoblasts are supplemented once at the 7 th day of culture, and the number of the supplemented NK expanded trophoblasts is 1/4 of the number of the NK cells; observing and counting NK cells every day during the culture process, and when the NK cell density exceeds 2.5 multiplied by 10⁶cells/ml supplemented Medium GT-T551H3 medium with 240U/ml IL-2, density maintained at 1.0X 10 after fluid infusion⁶cells/ml, co-culturing for 15 days, and collecting cells to obtain high-activity NK cells; wherein, the NK cell: monoclonal antibodies: the dosage of docetaxel is 5 multiplied by 10 for NK cells⁹Kg, 100 mug/kg of monoclonal antibody and 100 mug/kg of docetaxel.

In another aspect of the invention, the kit further comprises a pharmaceutically acceptable carrier.

Further, the kit comprises at least one excipient.

In some embodiments, the monoclonal antibody to VEGFR2 is a human immunoglobulin that specifically binds human VEGFR 2. In some embodiments, the monoclonal antibody to VEGFR2 is selected from an antibody fragment of a Fab, Fab ', F (ab') 2, scFv, minibody, or diabody.

In some embodiments, the TSLP-binding molecules are FABs, e.g., human or humanized FABs, that specifically bind to human VEGFR 2.

In another aspect, provided herein are pharmaceutical compositions comprising at least one monoclonal antibody to VEGFR2 described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the monoclonal antibody to VEGFR2 is a pharmaceutical composition of about 5% to about 95%, or about 10% to about 90%, or about 15% to about 85%, or about 20% to about 80%, or about 25% to about 75%, or about 30% to about 70%, or about 40% to about 60%, or about 40-50% (w/w). In some embodiments, the kit comprises a shell forming agent, such as trileucine or leucine. In some embodiments, trileucine or leucine is about 10-75% (w/w) of the composition. In some embodiments, trileucine is about 10-30% (w/w) of the composition. In other embodiments, leucine is about 50-75% (w/w) of the composition. In some embodiments, the kit comprises at least one glass forming excipient, wherein the glass forming excipient is selected from histidine, trehalose, mannitol, sucrose, or sodium citrate. In some embodiments, the at least one glass forming excipient is trehalose or a mixture of trehalose and mannitol. In some embodiments, the glass forming excipient is about 15-35% (w/w) of the kit. In some embodiments, the kit comprises a buffer, such as a histidine, glycine, acetate, or phosphate buffer. In some embodiments, the buffer is about 5-13% of the kit.

In some embodiments, the kits provided herein are formulated as a dry powder formulation, for example, a dry powder formulation suitable for inhalation.

Advantageous effects

The invention provides a kit which contains a VEGFR2 monoclonal antibody specific to VEGFR2 protein, docetaxel and high-activity NK cells, and the kit, the VEGFR2 monoclonal antibody, the docetaxel and the high-activity NK cells can be used together to effectively inhibit growth of liver cancer tumors, so that the kit has a good application prospect.

Drawings

FIG. 1 is a Western test result chart

FIG. 2NK cell killing effect graph

FIG. 3 is a graph showing the effect of tumor inhibition rate

FIG. 4 is a graph showing the effect of tumor inhibition rate of the combination of the kit

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation and characterization of VEGFR2 monoclonal antibody

For the first immunization, 100. mu.g of VEGFR2 protein (recombinant human vascular endothelial growth factor receptor-2, cat # PKA-242, Eimei technologies, Inc.) was injected subcutaneously into the back and neck of mice. Day 21 post prime, 2 nd immunization was performed, with 100 μ g VEGFR2 protein injected subcutaneously in the back and neck of each mouse. The 3 rd immunization was performed 14 days after the 2 nd immunization, and each mouse was injected intraperitoneally with 100 μ g of VEGFR2 protein and blood was collected from the orbital venous plexus of the mouse. On day 14 after the 3 rd immunization, the 4 th immunization was performed, and the mice were injected with 50 μ g of VEGFR2 protein in the proximal spleen and abdominal cavity, respectively. Blood is collected through orbital venous plexus after 7 days of 4 th immunization, and the blood serum titer is detected by ELISA together with the three-immune serum. Mice were injected with 100 μ g of VEGFR2 diluted in saline 3-4 days before fusion. Spleen cells of the immunized mice were fused with Sp2/0 cells according to a conventional method. Screening by an indirect ELISA method and cloning by a limiting dilution method.

2 hybridoma cell strains which can stably secrete VEGFR2 monoclonal antibodies are obtained through cell fusion, screening and cloning, and the clone numbers are 6C11 and 4E09 respectively. Preparing mouse ascites from the hybridoma cell strain according to a conventional method, and purifying the ascites to obtain the monoclonal antibody.

(1) Monoclonal antibodies were detected using the mouse Ig class/subclass/subtype detection kit, and the results are shown in table 1.

TABLE 16C 11 monoclonal antibody subtype detection results

Clone number	Subclass of heavy chain	Light chain subtype
			6C11	IgM	κ

(2) Identification of monoclonal antibodies-Western blot: and (3) preparing the VEGFR2 protein, the VEGFR and the BSA protein into Western blot samples respectively for detection, carrying out electrophoresis and membrane conversion, sequentially incubating the antibodies, reacting overnight at 4 ℃, and developing after incubating goat anti-mouse IgG secondary antibody marked by HRP for 40min at room temperature. As shown in FIG. 1, monoclonal antibody clone No. 6C11 was able to specifically bind to VEGFR2 protein, but not to VEGFR and BSA, with better specificity.

(3) Dissociation constant determination of monoclonal antibody 6C11

For monoclonal antibody 6C11, the dissociation constant (Kd value) was determined by the following method. The VRGFR2 protein was diluted with 10mM sodium acetate solution (pH5.0) to prepare a ligand solution at a final concentration of 50. mu.g/mL. For immobilization of the ligand and calculation of Kd values Biacore T200 was used. The ligand solution was immobilized on a sensor chip CM5 using an amine coupling kit. Then, a 6C11 antibody dilution solution of 0.1-50 nM was prepared using the electrophoresis buffer HBS-P +, and a sensor line was obtained. As a fitting model, 1:1 binding is adopted, and a sensorgram is analyzed, so that the Kd value of the 6C11 monoclonal antibody is 8.73nM, and the monoclonal antibody has better binding property.

(4) Identification of antibody variable region sequences: total RNA was extracted from 6C11 hybridoma cells using a total RNA extraction kit and reverse transcribed to cDNA using antibody subtype specific primers and universal primers. Murine immunoglobulin heavy and light chain V-region fragments were then amplified by PCR kit, the resulting PCR fragments were subcloned into the pMD19-T vector system, and the inserts were sequenced using vector-specific primers. The unique V-region nucleotide/amino acid sequence of clone 6C11 was finally obtained. Wherein, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

Heavy chain variable region (SEQ ID NO:1)

Light chain variable region (SEQ ID NO:2)

Example 26 inhibition experiment of C11 on liver cancer cells

(1) Plate preparation, digesting liver cancer SMMC-7721 cells in logarithmic growth phase with trypsin, blowing with a pipette (avoiding bubbles), mixing cell suspension, counting with a cell counting plate, diluting with low serum (5%) complete culture solution to density of 1 × 10⁵Taking out the cell suspension per mL, taking out 96-well plate, adding complete culture medium without cell suspension into 3-5 wells as zero setting well, accurately adding 100uL cell suspension into the rest wells, uniformly plating, and ensuring that the cell concentration inoculated into each well is 1 × 10⁴Respectively, placing at 37 deg.C and 5% CO₂The culture box is used for culturing for 24 hours to make the suspension cells adhere to the wall.

(2) Adding medicine: after the cells adhere to the wall, the cells are washed for 1-2 times by sterile PBS or normal saline, PBS solution is added into 36 holes on the outer circle, only complete culture medium is added into the zero adjustment holes, only 100uL of RPMI-1640 complete culture medium is added into 3-5 holes, no medicine is added to serve as a control hole, and 100uL of RPMI-1640 complete culture medium containing different medicine concentrations is added into the rest holes by using a pipette gun to serve as an experimental hole. 3 monoclonal antibody pre-concentrations (5ug/mL, 10ug/mL, 20ug/mL) were set separately and intervened at two time points, 24h and 72h, respectively, and retested twice at each time point. Apatinib 20ug/mL was used as a positive control;

(3) MTT interaction with cells: washing with physiological saline or PBS for 1-2 times after 24h and 72h respectively, adding 100uL of RPMI-1640 complete culture medium into a 96-well plate, then adding 20uL of prepared MTT solution into each well, continuing to incubate in an incubator for 4h, after 4h, slightly absorbing the solution in the 96-well plate, respectively adding 150uL of DMSO solution into the 96-well plate, shaking the 96-well plate on a shaker away from light for 10min at a slow speed, and fully and uniformly mixing formazan and DMSO solution.

(4) Absorbance was measured and calculated: the absorbance value (OD value) at 490nm was measured using a microplate reader. Counting according to a formula: cell growth inhibition (%) × (0D value control-OD value experimental group)/(OD value control-OD value zero-adjusted group) × 100%. The results are shown in Table 2.

TABLE 2 proliferation inhibition of cells by different concentrations of monoclonal antibodies

Compared with the blank control group, the experimental group has obvious inhibition effect on the proliferation of the liver cancer cells, and the inhibition effect is more obvious along with the increase of time and concentration, and the difference has statistical significance (P is less than 0.05) (shown in table 2). In the concentration and time of the antibody medicament, the antibody has a quantity-effect and time-effect dependence on the proliferation inhibition effect of the cell, which shows that the monoclonal antibody has obvious inhibition effect on the proliferation of the human liver cancer cell HepG 2. Compared with the positive control group apatinib, the antibody drug has the advantage that the inhibition rate effect of the antibody drug is greatly improved in 72h under the condition of the same concentration, and has a better effect.

Example 3 preparation of high Activity NK cells

Centrifuging adult peripheral blood at 2000r/min for 10min, carefully sucking out plasma, and inactivating in 56 deg.C water bath for 30 min; diluting blood cells with normal saline, adding into a centrifuge tube filled with a Ficoll solution, centrifuging at 2000r/min for 20min, collecting leucocyte cells, washing with normal saline for 2 times, and counting for later use. Resuspending 2X 107 PBMCs in 30ml GT-T551H3 medium, inoculating into T75 cell culture flask, and adding IL-2, NK trophoblast and autologous plasma into the system, wherein the IL-2 content is 500U/ml, the final concentration of resveratrol is 2. mu. mol/L, and the number of trophoblast is 1X 10⁶ml-¹The plasma volume was 5%. 5% CO at 37 ℃₂And (3) carrying out in-vitro co-culture for 15d under the condition, wherein the culture day 7 is supplemented with one time of NK expanded trophoblasts, and the number of the supplemented NK expanded trophoblasts is 1/4 which is equal to the number of the NK cells. The culture process is carried out every dayCounting the NK cells, when the density of the NK cells exceeds 2.5 multiplied by 10⁶cells/ml were supplemented with GT-T551H3 medium containing 240U/ml IL-2, and the density was maintained at 1.0X 106cells/ml after the medium was replenished for 15 days. Cell number 4X 10 from initial inoculum size at day 15 of cell expansion⁷About one, the amplification reaches (2.52 +/-0.04) x 10¹⁰The number of the resveratrol is obviously improved, and particularly, compared with the prior art, the addition of the resveratrol can obviously improve the amplification effect.

Take about 5X 10⁶The cells are resuspended in 1ml of physiological saline after centrifugation at 1500r/min for 5min and are divided into two tubes on average, 5. mu.l of anti-CD 3-FITC and 5. mu.l of anti-CD56-PE are added to one tube, the corresponding isotype antibody is added to the other tube as a control, and the percentage of CD 3-CD 56+ cells is detected by a flow cytometer after 30 min. The result shows that the proportion of CD 3-CD 56+ cells reaches over 96.53 percent, which indicates that the culture method can effectively induce the expansion of NK cells and ensure the expansion purity.

Example 4 killing experiment of NK cells

The research on the killing effect of the NK cells on the liver cancer SMMC-7721 takes the liver cancer cells as target cells and the NK cells as effector cells, the effector cells are added into the target cells according to the effect/target ratio of 10: 1, and corresponding target cell holes, effector cell holes and culture medium blank control holes are arranged at the same time. Each set was provided with 3 parallel holes. The number of target cells was 1X 10⁴Respectively inoculating target cells for 6 hr, adding effector cells at 37 deg.C and 5% CO₂And adding 10 mu l of CCK-8 solution into each well when culturing for 0, 12, 24 and 36 hours under the saturated humidity condition, incubating for 1 hour +/-10 min at 37 ℃, and measuring the OD value by using an enzyme-labeling instrument at the wavelength of 450 nm. The killing rate was calculated by the following formula,% killing [ 1- (effect target cell action well OD value-effect cell well OD value ]]OD in target cell wells X100%. The results are shown in FIG. 2.

As can be seen from the time relationship of the NK cell killing effect of FIG. 2, the killing effect of liver cancer shows a certain time dependence, which indicates that the NK cell can kill tumor cells, and the killing rate can reach about 57.2% in 36h, thus having better effect.

Example 5 establishment and grouping experiment of liver cancer transplantable tumor

Collecting SMMC-7721 liver cancer cell line (2 × 10)⁷/mL) was directly inoculated into 5 mice intraperitoneally, after 7 days, the mice were subjected to peritoneal dialysis to generate a large amount of ascites, 3mL of the mice were taken as a tumor source under aseptic conditions, and 15mL of physiological saline was added thereto for dilution, thereby adjusting the tumor cells to 2.0X 10⁸and/mL, respectively taking 0.3mL of cell suspension to inoculate under the skin of the right forelimb armpit of the mouse, and growing tumor blocks under the skin of the mouse after 3 d. Mice were randomly divided into a control group, a monoclonal antibody-treated group, an NK cell-treated group, a monoclonal antibody-NK cell-combined treated group, and a positive control chloroquine-treated group, each group containing 10 mice. The positive chloroquine-treated mice were injected with chloroquine (100. mu.g/kg) at the beginning of day 2 after modeling, the mab-treated mice were injected with mab (100. mu.g/kg) at the beginning of day 2 after modeling, and the NK cell-treated mice were injected with NK cells (5X 10) prepared in examples at the beginning of day 2 after modeling (5X 10)⁹/kg), mab-combined NK cell-treated group mice were injected intraperitoneally with the NK cells prepared in example (5 × 10) starting on day 2 after modeling⁹/kg) and monoclonal antibody (100. mu.g/kg), the control group was injected with physiological saline as a control, treated continuously for 16 days and closely observed for the survival status of the mice. All mice were intraperitoneally injected with 0.8% pentobarbital (60mg/kg) after 16d, sacrificed after the mice lost consciousness, tumor specimens were collected and processed according to steel's formula (V ═ a × b)²And/2) calculating the tumor volume after modeling and the tumor volume after sacrifice, and calculating the growth inhibition rate of the tumor volume. The results are shown in FIG. 3.

From the results in fig. 3, it can be seen that the NK treatment group, the mab-NK cell-combined treatment group, and the positive control chloroquine treatment group were all able to effectively inhibit tumor growth. Especially, the monoclonal antibody is used together with NK cells, the inhibition rate of the tumor growth can be obviously improved to 94.1 +/-2.03%, and the effect is better.

Example 6 Combined experiment for liver cancer treatment

Collecting SMMC-7721 liver cancer cell line (2 × 10)⁷/mL) was directly inoculated into 5 mice intraperitoneally, and after 7 days, a large amount of ascites was produced in the abdominal cavity of the mice, and 3mL of ascites was extracted as a tumor under aseptic conditionsTumor source, and adding 15mL of physiological saline for dilution to adjust the tumor cells to 2.0X 10⁸and/mL, respectively taking 0.3mL of cell suspension to inoculate under the skin of the right forelimb armpit of the mouse, and growing tumor blocks under the skin of the mouse after 3 d. Mice were randomly divided into a control group, a mab-combined NK cell treatment group, a docetaxel treatment group, and a mab-combined docetaxel and NK cell treatment group, 10 mice per group. Mice in the mab-and-NK cell-treated group were injected intraperitoneally (5 × 10) with NK cells prepared in the examples starting on day 2 after modeling⁹/kg) and mab (100 μ g/kg), mab in combination with NK cells and docetaxel treatment groups mice were injected i.p. with NK cells prepared in the examples (5 × 10) starting on day 2 after modeling⁹(100 μ g/kg) and mab (100 μ g/kg) and docetaxel (100 μ g/kg), docetaxel treated mice were injected intraperitoneally with docetaxel (100 μ g/kg) starting on day 2 after modeling. The control group was injected with physiological saline as a control, treated continuously for 16 days and closely observed for the survival status of the mice. All mice were intraperitoneally injected with 0.8% pentobarbital (60mg/kg) after 16d, sacrificed after the mice lost consciousness, tumor specimens were collected and processed according to steel's formula (V ═ a × b)²And/2) calculating the tumor volume after modeling and the tumor volume after sacrifice, and calculating the growth inhibition rate of the tumor volume. The results are shown in FIG. 4.

The results in fig. 4 show that the monoclonal antibody combined with NK cells and docetaxel treatment group can significantly improve the inhibition rate of tumor growth, and the tumor basically disappears completely, so that the combined use can significantly improve the tumor treatment effect, and has a better application prospect.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Sequence listing

<110> Beijing Biotechnology Ltd

<120> pharmaceutical compositions of NK cells and antibodies and their use in the treatment of cancer

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Glu Val Gln Leu Glu Glu Ser Ala Thr Glu Leu Ala Arg Pro Ser Ala

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Ser Tyr Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Leu Ile Met Pro Trp Pro Lys Gln Val Leu Arg Tyr Pro Met

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Gly Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Gly Trp Phe Trp Leu Lys His Gln Trp Gly Leu Gly Thr Thr Leu

100 105 110

Ala Val Ser Ser

115

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Asp Ile Val Ile Thr Gln Arg Pro Ala Leu Met Ala Ala Ser Pro Gly

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Trp Gly Gly Trp Tyr Gln Gln Lys Ser Gly Ile Ser Pro Lys Pro Trp

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Ile Tyr Thr Arg Lys Lys Asn His Glu Gly Val Pro Ala Arg Phe Ser

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100 105

Claims

1. Use of a combination of a monoclonal antibody that specifically inhibits VEGFR2, docetaxel, and NK cells in the preparation of a kit for the treatment of liver cancer; the monoclonal antibody is characterized in that the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO. 1, and the light chain variable region sequence is shown as SEQ ID NO. 2; the NK cells are prepared by the following method: centrifuging peripheral blood at 2000r/min for 10min, carefully sucking out plasma, and inactivating in 56 deg.C water bath for 30 min; diluting blood cells with normal saline, adding into a centrifuge tube filled with a Ficoll solution, centrifuging at 2000r/min for 20min, collecting leucocyte cells, washing with normal saline, and counting for later use; 2X 10 of the culture medium GT-T551H3⁷After the PBMCs are resuspended, the PBMCs are inoculated into a T75 cell culture flask, and then IL-2, resveratrol, NK trophoblasts and autologous plasma are added into the system, wherein the IL-2 is 500U/mL, the final concentration of the resveratrol is 2 mu mol/L, and the trophoblasts are 1 multiplied by 10⁶mL^-1Plasma volume 5%, 5% CO at 37 ℃₂In vitro co-culturing for 15d under the condition, wherein, the NK expanded trophoblasts are supplemented once at the 7 th day of culture, and the number of the supplemented NK expanded trophoblasts is 1/4 of the number of the NK cells; observing and counting NK cells every day during the culture process, and when the NK cell density exceeds 2.5 multiplied by 10⁶ mL^-1Supplementing the culture medium, wherein the supplemented culture medium is GT-T551H3 culture medium containing 240U/mL IL-2, and the density is maintained at 1.0 × 10 after fluid supplementation⁶ mL^-1Co-culturing for 15d, and collecting cells to obtain high-activity NK cells; wherein, the dosage of the NK cells, the monoclonal antibody and the docetaxel is respectively 5 multiplied by 10 for the NK cells⁹100 mu g/kg of monoclonal antibody and 100 mu g/kg of docetaxel.

2. The use of claim 1, wherein said kit further comprises a pharmaceutically acceptable carrier.

3. Use according to claim 1, characterized in that the kit comprises at least one excipient.

4. Use according to claim 3, characterized in that the excipient in the kit is selected from histidine, trehalose, mannitol, sucrose or sodium citrate.

5. Use according to claim 3, characterized in that the kit comprises a buffer.

6. The use of claim 5, wherein the buffer in the kit is a histidine, glycine or phosphate buffer.