CN115124619A

CN115124619A - Preparation method and application of clinical blood immune cells

Info

Publication number: CN115124619A
Application number: CN202210729772.3A
Authority: CN
Inventors: 王正好; 赵理
Original assignee: Beijing Shunyuantian Biological Products Co ltd
Current assignee: Guangdong Qimei Life Medicine Technology Research Institute; Guangdong Qimei Pharmaceutical Biotechnology Group Co ltd; Guangdong Stanfu International Stem Cell Medical Research Institute; Zhuhai Qimei Stem Cell Bank Co ltd
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2022-09-30
Anticipated expiration: 2042-06-24
Also published as: CN115124619B

Abstract

The invention relates to a preparation method and application of clinical blood immune cells. The invention provides a method for preparing and amplifying NK cells and the NK cells obtained by the method, and verifies that the NK cells have better tumor killing capacity, and after the NK cells are combined with beta-catenin monoclonal antibodies, the growth of tumors in mice can be remarkably inhibited, so that the NK cells have an excellent tumor treatment effect.

Description

Preparation method and application of clinical blood immune cells

Technical Field

The invention relates to the field of biology, in particular to the field of immune cells, and relates to a preparation method and application of clinical blood immune cells.

Background

NK cells were discovered 40 years ago, mainly originated from bone marrow, mainly present in blood and lymphoid organs, do not require prior sensitization, do not depend on antibodies and complement, are not limited by Major Histocompatibility Complex (MHC), can directly kill tumor cells, and have important roles in early anti-tumor and immune surveillance of the body. In tumor immunity, NK cells belong to nonspecific immune cells, surface markers mainly comprise CD3, CD56, CD16 and the like, the surface markers account for about 5% -15% of the total number of lymphocytes in peripheral blood of healthy people, a first immune killing line of an organism is formed, the organism is usually in a dormant state, and after a signal factor is activated, the signal factor can permeate into tissues to attack tumor cells and virus infected cells.

The NK cell has 4 receptors including a killer cell activating receptor (KAR), a killer cell inhibiting receptor (KIR), a killer lectin-like receptor (KLR) and an Fc gamma receptor (CD16), the first 3 receptors can inhibit or activate the NK cell to enable the NK cell to recognize the capability of self-tissue cells and in-vivo abnormal tissue cells, the 4 th receptor mainly recognizes IgG1 and IgG3 markers on the surface of the NK cell and mediates the NK cell to recognize target cells coated by the antibody, so that antibody-dependent cell-mediated cytotoxicity (ADCC) is exerted, and further tumors specifically bound with the IgG antibody are killed. NK cells have a wide antitumor spectrum, can kill homologous, homologous or heterologous tumor cells, and can kill target cells by a mechanism that (i) perforin and granzyme are released to cause necrosis or apoptosis of the target cells. ② the target cell apoptosis is induced by death receptor regulation. ③ secreting a plurality of effector cytokines to resist the metastatic tumors.

And fourthly, stimulating the immune effect of the secondary tumor.

Abnormalities in the Wnt/β -eatenin signaling pathway are associated with human diseases, including tumors, osteoporosis, aging and degenerative disorders, among others. Therefore, the research on the Wnt/beta-eatenin signal transduction pathway not only helps to understand the occurrence mechanism of human diseases, but also provides a series of new targets for the treatment of diseases. At least 6 Wnt proteins (Wntl, Wnt2, Wnt3, Wnt3a, Wnt8, and Wnt8b) have been shown to activate the Wnt/β -eatenin signaling pathway. The activation of the Wnt/β -eatenin signalling pathway is determined by the levels of β -eatenin proteins in the cytoplasm. Normally, β -eatenin in cytoplasm is mediated by ubiquitin proteasome, which is a multi-protein complex composed of axin (axin), coliform protein, glycogen synthesis kinase 3 β, casein kinase let, etc., including ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3), and is in a degraded state and maintains low level expression. GSK-beta is used for marking protein by phosphorylating Ser3, Ser3 and Thr4, CK1 alpha is used for marking protein by phosphorylating Ser45 only, so that the protein is recognized by E3 subunit beta-transducin repeated protein, the degradation of beta-catenin molecules is mediated, and the proteasome of 26S is degraded. Wnt proteins initiate the aggregation of intracellular β -catenin by binding to the cell surface receptor complex transmembrane protein/co-receptor low density lipoprotein receptor-related proteins. However, the mechanism of cell signaling by binding of Wnt proteins to Fz/LRP receptors is not yet fully understood. Most scholars think that after 2 persons are combined, the downstream protein of the receptor complex, namely cytoplasmic dishevelled protein is phosphorylated, the activity of GSK-3 beta and CK1 alpha is inhibited, axin protein is fixed, the formation of a multi-protein complex is damaged, finally unphosphorylated beta-catenin is aggregated in cytoplasm, the recognition of beta-TrCP is escaped, the degradation process is avoided, and the translocation to nucleus is further carried out. In the nucleus, beta-catenin forms a complex with TCF. When no beta-catenin exists, TCF interacts with Groucho to form a complex to inhibit the transcription activity; when beta-catenin is present, it interferes with the interaction of TCF with Groucho, and co-initiates transcription of downstream target genes with TCF and other transcription cofactors such as CREB Binding Protein (CBP).

The expression level of beta-catenin of tumors with abnormal Wnt signals is up-regulated, so that the treatment targeting the beta-catenin attracts great attention. Currently, antisense oligonucleotide technology, RNA interference technology, and protein knock-out technology are under investigation. The antisense oligonucleotide aiming at the beta-catenin can reduce the expression of tumor beta-catenin protein and effectively inhibit the growth of tumors. The method for screening and obtaining the specific beta-catenin monoclonal antibody by taking the beta-catenin as a target is also an important direction of research. Especially, the combined application of immune cell therapy and beta-catenin monoclonal antibody is not studied sufficiently at present.

Disclosure of Invention

The invention overcomes the defects of the prior art, provides an improved preparation method of NK cells and a method for treating cancer by combining the NK cells and monoclonal antibodies, and has better effect.

In one aspect of the present invention, there are provided a highly active NK cell and a method for preparing the same.

Specifically, the preparation method of the NK cell comprises the following steps:

adding Ficoll-PaquePLUS mononuclear cell separation liquid into a centrifuge tube, slowly adding heparin anticoagulated peripheral blood along the tube wall, avoiding damaging the water phase of the Ficoll separation liquid, centrifuging at room temperature and 3500rpm for 15min, carefully sucking the upper plasma into another centrifuge tube, inactivating at 56 ℃ for 25min, for later use, sucking the yellowish mononuclear cell layer into the centrifuge tube, washing with sterile PBS for 3 times, discarding the supernatant, precipitating to obtain peripheral blood mononuclear cell PBMC, suspending the precipitated cells with serum-free PRMI-1640 medium into a plastic plate coated with goat anti-mouse IgG (25 μ g of goat anti-mouse IgG and 5ml of 0.05M-HCL buffer solution (PH PBS 9), incubating at room temperature for 2h, washing for 3 times, sealing with 1% FCS at room temperature for 15min, washing with PBS for 3 times), adding cells incubated with anti-CD3 into the Tris coated plate, incubating for 2h, slightly sucking the cell suspension to obtain NK cells at 4 ℃ for 2 hours; the mononuclear cells were resuspended in NK MACS GMP medium (Miltenyi), recombinant human IL-2 and IL-15 were added, mixed well and injected into small culture bags, and incubated at 37 ℃ in incubator. On day 3, NK MACS GMP culture medium and recombinant human IL-2 and IL-15 were supplemented, and the culture was continued after mixing. On day 4, the culture medium in the small culture bag was equally distributed to two large culture bags and the culture was continued, and NKMACS GMP culture medium and recombinant human IL-2 were supplied to each large culture bag every 3 days for a total of 14 days, i.e., the proliferated NK cells were collected.

The invention further provides application of the NK cells in preparing a pharmaceutical composition for treating cancer.

In addition, in another aspect, the invention also provides a monoclonal antibody specifically aiming at the beta-catenin.

Further, the antibody is a beta-catenin monoclonal antibody beta-catenin-2D 13, and the light chain variable region sequence of the beta-catenin-2D 13 monoclonal antibody is as follows:

DIVITQRPALMAASPGEKVTITCAEYKHHAELGHVWYQQKSGISPKPWIYRSFIYKGGVPARFSGSGSGTSYSLTITSMEAEDAATYYCNTWDWFTLPFGAGTKLELK

the heavy chain variable region sequence is:

EVQLEESATELARPGASVKLSCKASGYIFSFPGFNWIKQRPGQGLEWIGPGLQRLFHHCCFDMRWGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGPDDVCQHWGLGTTLAVSS。

furthermore, the invention also provides application of the beta-catenin monoclonal antibody in preparing a pharmaceutical composition for treating cancer.

Further, the specific kind of cancer in the use is lung cancer.

Further, the cancer is a cancer caused by PC9 human lung cancer cells.

Furthermore, the invention also provides the application of the beta-catenin monoclonal antibody and the NK cell in preparing a kit for treating cancer.

Furthermore, the beta-catenin monoclonal antibody in the medicine box is in the form of a pharmaceutical composition, and the beta-catenin monoclonal antibody also contains a pharmaceutically acceptable carrier.

Pharmaceutical carriers are known to those skilled in the art. These are most typically standard carriers for administering drugs to humans, including solutions at physiological pH, such as sterile water, saline, and buffered solutions. The composition can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard methods used by those skilled in the art.

In addition to the selected molecule, the pharmaceutical composition may include carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like. The pharmaceutical compositions may also include one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, and the like.

Certain compositions can potentially be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid,

further, the NK cells of the present invention are used at a concentration of 1X 10 in terms of cell density ⁷ /mL-1×10 ⁹ mL, preferably, may be 2X 10 ⁷ /mL。

Further, the NK cells and the mabs of the invention are administered simultaneously or substantially simultaneously, or half an hour apart.

Any route of introducing or delivering an agent to a subject is included. Administration can be by any suitable route, including orally, topically, intravenously, subcutaneously, transdermally, intramuscularly, intraarticularly, parenterally, by inhalation, via implanted reservoirs, parenterally (e.g., subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrastemal, intrathecal, intraperitoneal, intrahepatic, enteral and intracranial injection or infusion techniques), and the like, intraarterially, intradermally, intraventricularly, intracranial, intraperitoneal, intranasal, rectal, intravaginally. As used herein, "simultaneous administration," "co-administration," "simultaneous administration," or "substantially simultaneous administration" refers to administration at the same time point or substantially immediately following each other. In the latter case, the two compounds are administered close enough in time that no difference is observed in the results observed from the results obtained when administered at the same time point.

The frequency of administration (e.g., NK cells) of the compositions disclosed herein includes: but are not limited to, at least once every 12 months, once every 11 months, once every 10 months, once every 9 months, once every 8 months, once every 7 months, once every 6 months, once every 5 months, once every 4 months, once every 3 months, once every 2 months, once every month; or at least once every three weeks, once every two weeks, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, or once per day. In some embodiments, the interval between each administration is less than about 4 months, less than about 3 months, less than about 2 months, less than about 1 month, less than about 3 weeks, less than about 2 weeks, or less than about 1 week, for example less than any of about 6, 5, 4, 3, 2, or 1 day. In some embodiments, the frequency of administration of the composition (e.g., expanded NK cells) includes, but is not limited to, at least once, twice, once or three times daily. In some embodiments, the interval between each administration is less than about 48 hours, 36 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, or 7 hours. In some embodiments, the interval between each administration is less than about 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, or 6 hoursThen (c) is performed. In some embodiments, the interval between each administration is constant. For example, administration may be daily, every second day, every third day, every fourth day, every fifth day, or weekly. Administration may also be continuous and adjusted to maintain the level of the compound within any desired and specified range. Each dose may comprise at least about 1 × 10 ⁸ NK cells/kg (e.g., at least about 1 × 104, 1 × 10 ⁵ ，1×10 ⁶ ，1×10 ⁷ ，1×10 ⁸ ，1×10 ⁹ ，1×10 ¹⁰ ，1×10 ¹¹ ，1×10 ¹² NK cells/kg).

Advantageous effects

The invention provides a method for preparing and amplifying NK cells and the NK cells obtained by the method, and verifies that the NK cells have better tumor killing capacity, and after the NK cells are combined with beta-catenin monoclonal antibodies, the growth of tumors in mice can be remarkably inhibited, so that the NK cells have an excellent tumor treatment effect.

Drawings

FIG. 1 is a diagram showing the identification result of the specific western blot of the beta-catenin monoclonal antibody

FIG. 2 is a graph showing the results of the inhibition rate of the beta-catenin monoclonal antibody on cancer cells

FIG. 3 is a graph showing the effect of monoclonal antibody on the activity of Wnt/beta-catenin pathway

FIG. 4 result graph of NK cell and/or monoclonal antibody effect on mouse tumor weight

Detailed Description

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

EXAMPLE 1 preparation of beta-catenin immunogen

Designing a primer according to a human beta-catenin gene sequence, wherein an upstream primer PF is 5'-AGGATCCAACTTGATTAACTATCAA-3'; downstream primer PR5'-ACTCGAGCAGGTCAGTATCAAACCA-3'. Amplifying beta-catenin fragment by using human blood DNA as a template. The PCR reaction conditions were 94 ℃ denaturation for 5min, 94 ℃ denaturation for 30s, 55 ℃ annealing for 30s, and 72 ℃ extension for 60s, and after 30 cycles, total extension at 72 ℃ for 10 min. The PCR amplification result was checked by 1.0% agarose gel electrophoresis to obtain a specific fragment of about 1932 bp. And connecting the fragment with a T vector, sequencing and identifying the fragment correctly, carrying out double enzyme digestion on the fragment and a pET-30a (+) vector, and connecting the fragment and the pET-30a (+) vector overnight at 4 ℃ by using T4 DNA ligase to construct a recombinant plasmid pET-30a (+) -beta-catenin. The recombinant plasmid is transformed into E.coli DH5 alpha competent cell, after kanamycin resistance screening, the identified plasmid is extracted, the recombinant plasmid is transformed into E.coli Rosetta (DE3) competent cell, LB solid medium is coated, and the culture is carried out overnight at 37 ℃. The single colony which is positive in identification is picked up and inoculated into 5ml LB liquid culture medium (containing 50ug/ml kanamycin), beta-catenin expression is induced (1mmol/L IPTG,25 ℃ induction for 10h), the supernatant is taken out, after 25 percent of saturated ammonium sulfate solution is used for precipitation, a proper amount of balanced solution (25mmol/L Tris, 0.5mol/L NaCl, 50mmol/L imidazole pH7.8) is used for dilution to prepare a crude extract. HisTrap ^TM The column was washed and equilibrated with 10 bed volumes of equilibration solution and loaded at a flow rate of 0.5 ml/min. Finally, gradient elution is carried out by eluent (25mmol/L Tris, 0.5mol/L NaCl, 0.5mol/L imidazole pH7.8) and the target protein is collected. After SDS-PAGE analysis of the purified β -catenin protein, the eluate containing the target protein was dialyzed against TBS (25mmol/L Tris, 0.15mol/L NaClpH8.0) and then quantified by BCA method at a concentration of 1.4 mg/ml.

EXAMPLE 2 preparation of beta-catenin monoclonal antibodies

Using the beta-catenin protein prepared in example 1 as immunogen, BALB/c female mice of 6 weeks old were selected for subcutaneous multi-point immunization, and the immunization dose was 50. mu.g/mouse. And finally screening 2 cell strains with the strongest positive by adopting a conventional cell fusion and screening method, wherein the cell strains are beta-catenin-1F 2 and beta-catenin-2D 13. The two hybridoma cells are respectively prepared into ascites type antibody by a mouse in vivo induction method, purified by a Protein G affinity chromatography column, and adjusted to the concentration of 1mg/ml for standby. Two monoclonal antibodies beta-catenin-1F 2 and beta-catenin-2D 13 and beta-catenin protein are respectively subjected to Westernblot detection. Specifically, PC9 human lung cancer cells (autumn living things-passing, cargo number: H086) are cracked, total proteins are extracted, the proteins are transferred to a PVDF membrane after SDS-PAGE electrophoresis, 2 diluted purified antibodies (1: 4000) are respectively added after 5% defatted milk powder seals the membrane, the membrane PBST is taken out after overnight reaction at 4 ℃ and washed for 3 times, HRP enzyme-labeled secondary antibody (1: 8000) is added for reaction at room temperature for 1.5H, washing is carried out for 6 times, chemiluminescent liquid is added for reaction, and imaging is carried out by a gel imager. The results are shown in FIG. 1.

As can be seen from the results in FIG. 1, 2 monoclonal antibodies prepared by the present invention all have good binding to the beta-catenin protein in tumor cells.

The SBACLonyping System-HRP kit is adopted to determine the class and subclass of the selected antibody Ig, and all the operations are strictly operated according to the kit instructions. The result shows that the beta-catenin-1F 2 and the beta-catenin-2D 13 are IgG 1.

Example 3 characterization and Performance analysis of the beta-catenin monoclonal antibody beta-catenin-2D 13

The non-competitive enzyme immunoassay determines the Affinity constant (Affinity constant), Ka value. According to the Method of a computer program, according to the formula Ka ═ 1/2 (n [ Ab']t－[Ab]t) calculating the value of the affinity constant Ka. The results show that the Ka value of beta-catenin-2D 13 is 8.71 multiplied by 10 ⁹ L/mol, has better affinity property.

Amplification of V by PCR _L And V _H The gene PCR amplification kit is purchased from Takara company, the amplified product is sequenced and identified, the light chain variable region sequence of the beta-catenin-2D 13 monoclonal antibody is as follows:

the light chain variable region sequence is:

the heavy chain variable region sequence is:

example 4 Activity analysis of the beta-catenin monoclonal antibody beta-catenin-2D 13

Placing PC9 human lung cancer cell in RPMI1640 culture solution containing 10% fetal calf serum, and adding 5% CO ₂ And (5) incubating for 48h in an incubator, detecting the cell state before experiment, and selecting cells in logarithmic growth phase with good state for EDTA digestion for later use.

Take 5X 10 ⁴ PC9 human lung cancer cells per mL are inoculated in a 96-well plate, and the culture medium is sucked out after adherent culture is carried out for 12 h. Adding 100.0 mu g/mL cetuximab into a DMEM medium (a positive control group), adding 1, 10, 50 and 100.0 mu g/mL beta-catenin-2D 13 monoclonal antibodies into the DMEM medium (corresponding to an experimental group 1-4), taking a pure DMEM medium as a blank group, after 48 hours of drug treatment, dripping 8 mu l of MTT solution into each hole, incubating for 8 hours at room temperature, sucking out the solution in the holes, dripping 80 mu l of dimethyl sulfoxide into each hole, oscillating for 30s, dissolving purple crystals, measuring the light absorption value by using a full-automatic enzyme standard analyzer, and calculating the cell proliferation inhibition rate, wherein the calculation formula is as follows: the cell inhibition ratio (%) × 1- (treatment group OD value/blank group OD value) × 100%. The results are shown in FIG. 2.

As can be seen from FIG. 2, after the monoclonal antibody is treated and cultured for 48h, the inhibition rate of the monoclonal antibody group for treating PC9 human lung cancer cell is significantly lower than that of the blank group (P < 0.05); under the same concentration, the beta-catenin-2D 13 monoclonal antibody has better inhibition effect than the PC9 human lung cancer cell proliferation of a positive control cetuximab group, and the cell inhibition rate reaches (95.9 +/-2.1)%, thus having better inhibition effect.

The activity detection of the Wnt/beta-catenin pathway adopts a dual-luciferase method, wherein a group of samples are placed in a 96-hole white fluorescence detection plate, 30 mu L of LAR II working solution is dripped into each hole, 8ul of each group of cell lysate is dripped into each hole, the solution is absorbed for 10min and uniformly mixed, the solution is immediately placed in a Modulus micropore plate multifunctional photometer for detection, the original value of firefly luciferase excited fluorescence is recorded, the detection plate is taken out, 30 mu LStop & Glo working solution is dripped into each hole, the solution is uniformly mixed and placed in the Modulus micropore plate multifunctional photometer for detection, the original value of renilla luciferase excited fluorescence is recorded, and data processing is carried out according to the following formula, wherein the relative luciferase activity is the luciferase activity of each hole sample/renilla luciferase activity of the same hole sample; activity of each Wnt/β -catenin pathway set TOPFlash relative luciferase activity/FOPFlash relative luciferase activity. The results are shown in FIG. 3.

As can be seen from FIG. 3, the active TOP/FOP values of the Wnt/beta-catenin pathway of each monoclonal antibody group are significantly lower than those of the blank group (P < 0.05) after the monoclonal antibody is cultured for 48 h. Under the same concentration, the beta-catenin-2D 13 monoclonal antibody has better effect of reducing the activity of the Wnt/beta-catenin pathway of the PC9 human lung cancer cells than that of positive control cetuximab, and has better inhibition effect by TOP/FOP value bit (4.21 +/-0.3)% under the concentration of 100.0 mu g/mL.

Example 5 isolation and culture of NK cells of immune cells

Adding 15 mLFicol-PaquePLUS mononuclear cell separating medium into a 50mL centrifuge tube, anticoagulating with heparin, slowly adding 30mL heparin anticoagulated peripheral blood along the tube wall, avoiding damaging the water phase of the Ficoll separating medium, centrifuging for 15min at room temperature and 3500rpm, carefully sucking the upper plasma into another 50mL centrifuge tube, inactivating for 25min at 56 ℃, for later use, sucking the yellowish mononuclear cell layer into the 15mL centrifuge tube, washing with sterile PBS for 3 times, discarding the supernatant, precipitating to obtain peripheral blood mononuclear cells, obtaining PBMC, adding the serum-free PRMI-1640 culture medium suspension precipitated cells into a plastic plate coated with goat anti-mouse IgG, incubating the plate with 25 μ g goat anti-mouse IgG and 5mL 0.05M Tris-HCL buffer solution (PH9), washing PBS for 3 times at room temperature for 2h, sealing with 1% FCS for 15min at room temperature, and washing with PBS for 3 times at room temperature, and adding the cells incubated with the anti-CD3 for 2h into a coated plate, and slightly sucking the cell suspension for 2h at 4 ℃ to obtain the required NK cells.

The mononuclear cells were resuspended in 90mL NK MACS GMP medium (Miltenyi), 5 ten thousand units of recombinant human IL-2 and IL-15 were added, mixed well and injected into a small culture bag, and placed in a 37 ℃ incubator for static culture. On day 3, 50mL of NK MACS GMP medium and 2.5 ten thousand units of recombinant human IL-2 and IL-15 were supplemented, and the mixture was mixed and cultured. On day 4, the culture medium in the small bag was equally distributed to two large bags and the culture was continued, and each large bag was supplemented with 100mL of NK MACS GMP culture medium and 15 ten thousand units of recombinant human IL-2 every 3 days for a total of 14 days, i.e., the proliferated NK cells were collected. Taking the amplified NK cell smear, drying, and adopting an Envision two-step method to carry out anti-CD56 labeling and anti-CD3 labeling, wherein the working concentration of the antibody is 1: 20. The results showed that CD56+ was 89.7% and CD3+ was 3.72%, indicating that the purity of the prepared NK cells was high.

Example 6 characterisation of immune cell NK cells

Placing the PC9 human lung cancer cell suspension at 37 ℃ and 5% CO ₂ Culturing in incubator, collecting PC9 human lung cancer cell in logarithmic growth phase, adjusting cell number to 2.0 × 10 ⁵ mL ^－1 Add 96 well U plate, 100. mu.L/well. Taking NK cells prepared in example 5, grouping the NK cells according to different effective target ratios, namely, an NK group, an NK + PC9 human lung cancer cell 10:1 and a 50: 1) group, a PC9 human lung cancer cell group and a blank group, culturing for 12 hours in a 96-hole U-shaped plate with the temperature of 37 ℃ and 5% CO2, adding 20ul MTT 5mg/ml into each hole, incubating for 4 hours at the temperature of 37 ℃, adding DMSO 100ul into each hole, fully blowing, uniformly mixing, detecting the OD value of the 570nm wavelength by using a microplate reader, and calculating according to the following formula: the killing rate (%) (1- (experimental well a value-effector cell well a value)/target cell well a value × 100%). The results are shown in Table 1.

TABLE 1 killing Activity of groups on cancer cells

Group of	Killing rate (%)
		10:1 group	83.46±2.02
50:1 group	90.31±3.47

As can be seen from the results in Table 1, NK has a killing effect on PC9 human lung cancer cells, and the killing rate was (90.31. + -. 3.47)%, at an effective target ratio of 50: 1.

Example 7 combination therapy experiment of NK cells and beta-catenin-2D 13 monoclonal antibody

Grouping nude mice: placing BALB/c nude mice in a sterile environment for feeding, and randomly grouping the mice into groups, wherein each group comprises 10 mice; a is a blank control group of a sterile nude mouse, B is a human lung cancer cell group transplanted with PC9 in vivo, C is a group injected with beta-catenin-2D 13 monoclonal antibody in vivo, D is a group injected with NK cells in vivo, E is a group injected with beta-catenin-2D 13 monoclonal antibody and NK cells in vivo, and F is a group injected with cetuximab in vivo.

Establishing a mouse model: digesting cultured PC9 human lung cancer cells with trypsin, centrifuging, preparing a glioblastoma cell suspension by using a serum-free DMEM/F12 culture medium, and injecting the cell suspension into the back subcutaneous of a BALB/c nude mouse by using a 0.2mL syringe through subcutaneous injection, wherein the injection amount is 2 multiplied by 10 ⁶ (0.2 ml). In each experimental group, NK cell treatment was: the NK cell suspension prepared in example 5 was taken and injected into the body through the tail vein of mouse using 0.2mL syringe (cell density 2X 10) ⁷ mL), once more at intervals of 7 d. The monoclonal antibody treatment groups were: the beta-catenin-2D 13 monoclonal antibody or cetuximab is respectively injected into the mouse body by a 0.2mL syringe in the same way, the injection amount is 150.0 mu g/mouse, and the injection is administered 1 time at 3 days and 3 times in total. The above operations are all completed in the same time period and the injection of cancer cells is completed first. The NK cells and the monoclonal antibody are injected at an interval of 30min during the combination treatment. Nude mice were sacrificed at 14d after treatment, tumor tissues of the nude mice were dissected, and their weights were measured. The results are shown in FIG. 4.

As can be seen from FIG. 4, the tumor weights in the C-F treatment groups were significantly reduced (P < 0.05) relative to the PC9 human lung cancer cell group. After the beta-catenin-2D 13 monoclonal antibody and the NK cells are treated in a combined mode, the tumor weight is only (0.24 +/-0.05) g, while the tumor weight of the PC9 human lung cancer cell group is (2.70 +/-0.12) g, and the combined treatment group has a remarkable inhibition effect compared with a control group.

It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

Sequence listing

<110> Beijing Shuyuansheng biological products Co., Ltd

<120> preparation method of clinical blood immune cells and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ala Glu Tyr Lys His His Ala Glu Leu

20 25 30

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

35 40 45

Ile Tyr Arg Ser Phe Ile Tyr Lys Gly Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Thr Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Asn Thr Trp Asp Trp Phe Thr

85 90 95

Leu Pro Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 2

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Phe Pro

20 25 30

Gly Phe Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Pro Gly Leu Gln Arg Leu Phe His His Cys Cys Phe Asp Met Arg

50 55 60

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

65 70 75 80

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

85 90 95

Ala Gly Pro Asp Asp Val Cys Gln His Trp Gly Leu Gly Thr Thr Leu

100 105 110

Ala Val Ser Ser

115

Claims

1. The beta-catenin monoclonal antibody is characterized in that the antibody is beta-catenin-2D 13, and the variable region sequence of the light chain of the monoclonal antibody is as follows:

the heavy chain variable region sequence is:

2. use of the beta-catenin monoclonal antibody of claim 1 in the preparation of a pharmaceutical composition for treating lung cancer.

3. The use according to claim 2, wherein said composition further comprises a pharmaceutically acceptable carrier.

4. Use according to claim 3, characterized in that the carrier also contains a preservative.

Use of NK cells in combination with beta-catenin monoclonal antibodies for the preparation of a kit for the treatment of cancer lung cancer; wherein, the beta-catenin monoclonal antibody is beta-catenin-2D 13, and the light chain variable region sequence of the monoclonal antibody is as follows:

the heavy chain variable region sequence is:

EVQLEESATELARPGASVKLSCKASGYIFSFPGFNWIKQRPGQGLEWIGPGLQRLFHHCCFDMRWGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGPDDVCQHWGLGTTLAVSS；

the preparation method of the NK cells comprises the following steps: separating peripheral blood mononuclear cells, adding suspension precipitated cells in a serum-free PRMI-1640 culture medium into a plastic plate coated with goat anti-mouse IgG for 15 minutes at room temperature, sealing the plastic plate with PBS containing 1% FCS (FCS), washing the plastic plate with PBS for 3 times, adding cells incubated with anti-CD3 for 2 hours into the coated plate, slightly sucking cell suspension at 4 ℃ for 2 hours, suspending the mononuclear cells by using NK MACS GMP culture medium, adding recombinant human IL-2 and IL-15, uniformly mixing, injecting into a small culture bag, and placing into an incubator at 37 ℃ for standing culture; on the 3 rd day, half amount of NK MACS GMP culture medium and half amount of recombinant human IL-2 and IL-15 are supplemented, and the culture is continued after the mixture is evenly mixed; on day 4, the culture medium in the small culture bag was equally distributed to two large culture bags and the culture was continued, and NK MACS GMP culture medium and recombinant human IL-2 were supplied to each large culture bag every 3 days for a total of 14 days, i.e., the proliferated NK cells were collected.

6. Use according to claim 5, characterized in that the β -catenin monoclonal antibody is itself in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier.

7. Use according to claim 6, characterized in that the carrier also contains a preservative.