CN115124619B - Preparation method and application of clinical blood immune cells - Google Patents
Preparation method and application of clinical blood immune cells Download PDFInfo
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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 NK cells obtained by the method, and verifies that the NK cells have better tumor killing capability, and after the NK cells are combined with a beta-catenin monoclonal antibody, the NK cells can remarkably inhibit the growth of tumors in mice and have excellent tumor treatment effect.
Description
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 found 40 years ago to be mainly derived from bone marrow, mainly exist in blood and lymphoid organs, do not need to be sensitized in advance, do not depend on antibodies and complements, are not limited by Major Histocompatibility Complex (MHC), can directly kill tumor cells, and play an important role in early antitumor and immune monitoring of organisms. In tumor immunity, NK cells belong to nonspecific immune cells, the surface markers mainly comprise CD3, CD56, CD16 and the like, and the surface markers account for about 5% -15% of the total number of lymphocytes in peripheral blood of healthy people, so that a first immune killing defense line of an organism is formed, and the NK cells are usually in a dormant state, and after signal factors are activated, the NK cells can permeate into tissues to attack tumor cells and virus infected cells.
NK cells have 4 receptors, namely, a killer cell activation receptor (KAR), a killer cell inhibition receptor (KIR), a killer cell lectin-like receptor (KLR) and an Fc gamma receptor (CD 16), and the first 3 receptors can inhibit or activate NK cells so as to enable the NK cells to recognize own tissue cells and abnormal tissue cells in vivo, and the 4 th receptor mainly recognizes IgG1 and IgG3 markers on the surface of NK cells, and mediates NK cells to recognize target cells coated with antibodies, thereby exerting antibody-dependent cell cytotoxicity (ADCC) and further killing tumors specifically combined with the IgG antibodies. NK cells have a broad anti-tumor spectrum and can kill homologous, allogeneic or xenogeneic tumor cells, and the mechanism for killing target cells can be (1) perforin and granzyme release to cause target cell necrosis or apoptosis. (2) Apoptosis of target cells is induced by death receptor modulation. (3) Multiple effector cytokines are secreted against metastatic tumors.
(4) The secondary tumor immune effect is stimulated.
Abnormalities in Wnt/β -earin signaling pathways are associated with human diseases including tumors, osteoporosis, aging, and degenerative disorders. Therefore, the research on Wnt/beta-eatinin signal transduction pathway is helpful for understanding the occurrence mechanism of human diseases, and can provide a series of new targets for the treatment of diseases. Current studies demonstrate that at least 6 Wnt proteins (Wnt l, wnt2, wnt3a, wnt8, and Wnt8 b) activate Wnt/β -earin signaling pathways. Activation of the Wnt/β -eatinin signaling pathway is determined by β -eatinin protein levels in the cytoplasm. Normally, β -eatinin in the cytoplasm is maintained in low level expression by being degraded in the mediation of ubiquitin proteasomes, which are polyprotein complexes composed of axin (axin), polyposis coli, glycogen synthesis kinase 3 β, and casein kinase let, etc., including ubiquitin activating enzyme (E1), ubiquitin binding enzyme (E2), and ubiquitin ligase (E3). GSK-beta marks the protein through phosphorylating Ser3, ser3 and Thr4, CK1 alpha marks the protein through phosphorylating Ser45 only, so that the protein is recognized by repeated protein of E3 subunit beta-transducin, degradation of beta-catenin molecules is mediated, and a 26S proteasome is also degraded. Wnt proteins initiate the aggregation of intracellular beta-catenin by binding to cell surface receptor complex transmembrane proteins/co-receptor low density lipoprotein receptor-related proteins. However, the mechanism by which Wnt proteins bind to the Fz/LRP receptor to cause cellular signaling is not well understood. Most scholars believe that after 2 are combined, the downstream protein of the receptor complex, namely cytoplasmic random protein, is phosphorylated, inhibits the activity of GSK-3 beta and CK1 alpha, and damages the formation of a multiprotein complex by fixing axin protein, finally leads to the aggregation of non-phosphorylated beta-catenin in cytoplasm, and escapes the recognition of beta-TrCP, thereby avoiding the degradation process and translocation to the nucleus. Within the nucleus, β -catenin forms a complex with TCF. When no β -catenin is present, TCF interacts with Groucho to form complexes that inhibit transcriptional activity; when β -catenin is present, it interferes with the interaction of TCF with Groucho, and together with TCF and other transcription cofactors such as CREB binding protein (CREBbindingprotein, CBP), initiates transcription of downstream target genes.
Tumors with abnormal Wnt signals all have upregulation of beta-catenin expression levels, so treatment targeting beta-catenin is of great interest. Currently, antisense oligonucleotide technology, RNA interference technology, and protein knockout technology are in the research stage. The antisense oligonucleotide aiming at the beta-catenin can reduce the expression of the tumor beta-catenin protein and effectively inhibit the growth of tumors. The adoption of beta-catenin as a target for screening to obtain a specific beta-catenin monoclonal antibody is also an important direction of research. In particular, the combined application of immune cell therapy and beta-catenin monoclonal antibody is not researched enough at present.
Disclosure of Invention
The invention overcomes the defects of the prior art, provides an improved preparation method of NK cells and uses the NK cells and monoclonal antibodies in combination for treating cancers, and has better effect.
In one aspect of the present invention, a high activity NK cell and a method for preparing the same are provided.
Specifically, the preparation method of the NK cells comprises the following steps:
the method comprises the steps of (1) anticoagulating peripheral blood of healthy people, adding a Ficoll-PaquePLUS mononuclear cell separating liquid into a centrifuge tube, slowly adding the heparin anticoagulated peripheral blood into the centrifuge tube along the tube wall to avoid destroying the water phase of the Ficoll separating liquid, centrifuging for 15min at room temperature and 3500rpm, carefully sucking upper plasma into another centrifuge tube, inactivating for 25min at 56 ℃, sucking the light yellow mononuclear cell layer into the centrifuge tube for later use, washing 3 times by using sterile PBS, discarding supernatant, precipitating to obtain peripheral blood mononuclear cell, suspending precipitated cells in a serum-free PRMI-1640 culture medium, adding the sheep anti-mouse IgG coated first into a plastic plate (adding 25 mu g of sheep anti-mouse IgG and 5ml of 0.05M Tris-HCL buffer (PH 9) into each plate, incubating for 2h at room temperature, washing 3 times by PBS, sealing for 15min at room temperature by using PBS containing 1% and washing 3 times, adding cells incubated for 2h with anti-CD3 into the coated plate, and slightly sucking cells to obtain NK cell suspension; the mononuclear cells were resuspended in NK MACS GMP medium (Miltenyi) and added with recombinant human IL-2 and IL-15, mixed well and injected into small bags, and placed in a 37℃incubator for stationary culture. On day 3, NK MACS GMP medium and recombinant human IL-2 and IL-15 were supplemented, and culture was continued after mixing. On day 4, the culture medium in the small culture bags was evenly distributed to two large culture bags for continuous culture, and NKMACS GMP culture medium and recombinant human IL-2 were supplemented to each large culture bag every 3 days for total culture for 14d, i.e., NK cells after proliferation were collected.
The invention further provides the use of NK cells in the preparation of a pharmaceutical composition for the treatment of cancer.
In addition, in another aspect, the invention also provides a monoclonal antibody specific for beta-catenin.
Further, the antibody is beta-catenin-2D 13 monoclonal antibody, and the light chain variable region sequence of the beta-catenin-2D 13 monoclonal antibody is as follows:
DIVITQRPALMAASPGEKVTITCAEYKHHAELGHVWYQQKSGISPKPWIYRSFIYKGGVPARFSGSGSGTSYSLTITSMEAEDAATYYCNTWDWFTLPFGAGTKLELK
the heavy chain variable region sequences are:
EVQLEESATELARPGASVKLSCKASGYIFSFPGFNWIKQRPGQGLEWIGPGLQRLFHHCCFDMRWGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGPDDVCQHWGLGTTLAVSS。
furthermore, the invention also provides application of the beta-catenin monoclonal antibody in preparing a pharmaceutical composition for treating cancer.
Further, a specific type 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 application of the beta-catenin monoclonal antibody combined with NK cells in preparing a medicine box for treating cancers.
Further, the beta-catenin monoclonal antibody in the kit is in the form of a pharmaceutical composition, and further comprises a pharmaceutically acceptable carrier.
Drug carriers are known to those skilled in the art. These are most typically standard carriers for administration of drugs to humans, including solutions at physiological pH, such as sterile water, saline and buffer solutions. The composition can be administered intramuscularly or subcutaneously. The other compounds will be administered according to standard methods used by those skilled in the art.
In addition to the selected molecules, the pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surfactants and the like. The pharmaceutical composition may also include one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, and the like.
Certain compositions may 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,
furthermore, NK cells of the present invention are used at a concentration of 1X 10 cells density 7 /mL-1×10 9 Preferably, the concentration of the catalyst is 2X 10 7 /mL。
Further, the NK cells and monoclonal antibodies of the present 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 may be by any suitable route including oral, topical, intravenous, subcutaneous, transdermal, intramuscular, intra-articular, parenteral, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrastem, intrathecal, intraperitoneal, intrahepatic, enteral and intracranial injection or infusion techniques), etc., intraarterial, intradermal, intraventricular, intracranial, intraperitoneal, intranasal, rectal, intravaginal. As used herein, "simultaneous administration", "combination administration", "simultaneous administration" or "substantially simultaneous administration" refers to administration at the same point in time or substantially immediately following each other. In the latter case, the two compounds are administered in close enough time that the observed results are indistinguishable from those obtained when administered at the same point in time.
The frequency of administration (e.g., NK cells) of the compositions disclosed herein, comprising: 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 a month. Or at least once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week or once a 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 about 6,5,4,3,2 or any of 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 per day. 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 hours. In some embodiments, the interval between each administration is constant. For example, the administration may be daily, every two days, every three days, every four days, every five days, 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 x 10 8 NK cells/kg (e.g., at least about 1X 104,1X 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 NK cells obtained by the method, and verifies that the NK cells have better tumor killing capability, and after the NK cells are combined with a beta-catenin monoclonal antibody, the NK cells can remarkably inhibit the growth of tumors in mice and have excellent tumor treatment effect.
Drawings
FIG. 1 is a diagram of the result of specific western blot identification of beta-catenin monoclonal antibody
FIG. 2 is a graph showing the result of inhibition of cancer cells by beta-catenin monoclonal antibody
FIG. 3 shows the effect of mab on Wnt/beta-catenin pathway activity
FIG. 4 graphs showing the results of NK cell and/or mab 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 that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
EXAMPLE 1 preparation of beta-catenin immunogen
According to the human beta-catenin gene sequenceCounting primers, namely 5'-AGGATCCAACTTGATTAACTATCAA-3' of an upstream primer PF; the downstream primer PR 5'-ACTCGAGCAGGTCAGTATCAAACCA-3'. Human blood DNA is used as a template to amplify the beta-catenin fragment. The PCR reaction conditions were denaturation at 94℃for 5min, annealing at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 60s, and total extension at 72℃for 10min after 30 cycles. The PCR amplification result was detected by 1.0% agarose gel electrophoresis to obtain a specific fragment of about 1932 bp. And (3) connecting the fragment with a T vector, performing sequencing and identification to be correct, performing double digestion with a pET-30a (+) vector, and connecting the fragment with a T4 DNA ligase at 4 ℃ overnight to construct a recombinant plasmid pET-30a (+) -beta-catenin. The recombinant plasmid is transformed into E.coli DH5 alpha competent cells, the identified plasmid is extracted after kanamycin resistance screening, the recombinant plasmid is transformed into E.coli Rosetta (DE 3) competent cells, LB solid medium is coated, and the culture is carried out at 37 ℃ overnight. Single colonies positive in identification were picked up and inoculated into 5ml LB liquid medium (containing 50ug/ml kanamycin), beta-catenin expression was induced (1 mmol/L IPTG, induction at 25 ℃ C. For 10 hours), the supernatant was taken out, precipitated with 25% saturated ammonium sulfate solution, and diluted with an appropriate amount of equilibration solution (25 mmol/L Tris, 0.5mol/L NaCl, 50mmol/L imidazole pH 7.8) to prepare a crude extract. HisTrap is used TM The column was equilibrated by washing with 10 volumes of equilibration liquid and loaded at a flow rate of 0.5 ml/min. Finally, the target protein was collected by gradient elution with eluent (25 mmol/L Tris, 0.5mol/LNaCl, 0.5mol/L imidazole pH 7.8). Purified beta-catenin protein was analyzed by SDS-PAGE, and the eluate containing the target protein was dialyzed against TBS (25 mmol/L Tris, 0.15mol/L NaClpH8.0) and then purified by BCA method at a concentration of 1.4mg/ml.
EXAMPLE 2 preparation of beta-catenin monoclonal antibodies
The beta-catenin protein prepared in example 1 is used as an immunogen, and BALB/c female mice with the age of 6 weeks are taken, and the subcutaneous multipoint immunization method is adopted, wherein the immunization dose is 50 mug/mouse. And finally screening out 2 cell strains with strongest positive values, namely beta-catenin-1F 2 and beta-catenin-2D 13, by adopting a conventional cell fusion and screening method. The two hybridoma cells are respectively adopted to prepare the abdominal water type antibody by a mouse in-vivo induction method, protein G affinity chromatography columns are adopted to purify, and the concentration is adjusted to be 1mg/ml for standby. And performing Westernblot detection on the two monoclonal antibodies beta-catenin-1F 2 and beta-catenin-2D 13 and the beta-catenin protein respectively. Specifically, PC9 human lung cancer cells (autumn-transmitted organisms, product number: H086) are lysed, total proteins are extracted, the proteins are transferred to PVDF membranes after SDS-PAGE electrophoresis, diluted 2 purified antibodies (1:4000) are respectively added after 5% of skimmed milk powder seals the membranes, the membranes are taken out after overnight reaction at 4 ℃ for washing 3 times, HRP enzyme-labeled secondary antibodies (1:8000) are added for reaction for 1.5 hours at room temperature, washing 6 times, chemiluminescent liquid is added for reaction, and imaging is carried out on a gel imager. The results are shown in FIG. 1.
From the results shown in FIG. 1, all 2 monoclonal antibodies prepared by the invention have better binding to the beta-catenin protein in tumor cells.
The screened antibody Ig class and subclass was assayed using the SBAClonotyping System-HRP kit, all procedures strictly followed the kit instructions. The results showed that both beta-catenin-1F 2 and beta-catenin-2D 13 were IgG1.
Example 3 characterization and Performance analysis of beta-catenin monoclonal antibody beta-catenin-2D 13
The affinity constant (Affinity constant), ka value, was determined by non-competitive enzyme immunoassay. According to the method of Method for determining the affinity of monoclonal antibody using non-competitive ELISA: a computer program, the method is described as formula Ka= (n-1)/2 (n [ Ab ]']t－[Ab]t) calculating the affinity constant Ka value. The results showed that the Ka value of beta-catenin-2D 13 was 8.71X 10 9 L/mol, has better affinity property.
Amplification of V by PCR L And V H The gene and PCR method amplification kit is purchased from Takara company, and the amplification product is sequenced and identified, and the light chain variable region sequence of the beta-catenin-2D 13 monoclonal antibody is as follows:
the light chain variable region sequences are:
DIVITQRPALMAASPGEKVTITCAEYKHHAELGHVWYQQKSGISPKPWIYRSFIYKGGVPARFSGSGSGTSYSLTITSMEAEDAATYYCNTWDWFTLPFGAGTKLELK
the heavy chain variable region sequences are:
EVQLEESATELARPGASVKLSCKASGYIFSFPGFNWIKQRPGQGLEWIGPGLQRLFHHCCFDMRWGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGPDDVCQHWGLGTTLAVSS。
EXAMPLE 4 Activity analysis of beta-catenin monoclonal antibody beta-catenin-2D 13
PC9 human lung cancer cells are placed in RPMI1640 culture solution containing 10% of fetal bovine serum, 5% CO 2 Incubating the incubator for 48 hours, detecting the cell state before the experiment, and performing EDTA digestion on the cells in the logarithmic growth phase with good state for later use.
Taking 5×10 4 The PC9 human lung cancer cells are inoculated into a 96-well plate, and the culture medium is sucked out after 12h of adherent culture. 100.0 mug/mL of cetuximab is added into DMEM culture medium (positive control group), 1, 10, 50 and 100.0 mug/mL of beta-catenin-2D 13 monoclonal antibody is added into DMEM culture medium (corresponding to experimental groups 1-4), pure DMEM culture medium is used as blank group, after 48 hours of drug treatment, 8 mul of MTT solution is dripped into each hole, the solution is incubated for 8 hours at room temperature, the solution in the hole is sucked out, 80 mul of dimethyl maple is dripped into each hole, shaking is carried out for 30 seconds, purple crystals are dissolved, a full-automatic enzyme-labeled analyzer is selected for measuring light absorption value, and the cell proliferation inhibition rate is calculated, wherein the calculation formula is as follows: cell inhibition (%) =1- (treatment OD/blank OD) ×100%. The results are shown in FIG. 2.
As can be seen from fig. 2, after the monoclonal antibody is treated and cultured for 48 hours, the inhibition rate of the monoclonal antibody group for treating PC9 human lung cancer cells is significantly lower than that of a blank group (P < 0.05); under the same concentration, the beta-catenin-2D 13 monoclonal antibody has better effect of inhibiting the proliferation of PC9 human lung cancer cells compared with a positive control cetuximab group, and has better inhibition effect, wherein the cell inhibition rate reaches (95.9+/-2.1%).
Placing a grouping sample into a 96-hole white fluorescent detection plate, dripping 30 mu L of LAR II working solution into each hole, dripping 8ul of cell lysate into each group, absorbing for 10min, mixing uniformly, immediately placing into a mulus micro-pore plate multifunctional photometer for detection, recording the original value of fluorescence excited by firefly luciferase, taking out the detection plate, dripping 30 mu LStop & Glo working solution into each hole, mixing uniformly, placing into the mulus micro-pore plate multifunctional photometer for detection, recording the original value of fluorescence excited by Renilla luciferase, and performing data processing according to the following formula, wherein relative luciferase activity = the luciferase activity of each hole sample/the Renilla luciferase activity of the same hole sample; activity of Wnt/β -catenin pathway of each group = TOPFlash relative luciferase activity/FOPFlash relative luciferase activity. The results are shown in FIG. 3.
As can be seen from fig. 3, the activity TOP/FOP values of Wnt/β -catenin pathway of each mab group were significantly lower than that of the blank group (P < 0.05) after 48h incubation of mab. Under the same concentration, the beta-catenin-2D 13 monoclonal antibody has better activity effect of reducing Wnt/beta-catenin channels than the positive control cetuximab for PC9 human lung cancer cells, and has better inhibition effect at TOP/FOP value position (4.21+/-0.3)% under the concentration of 100.0 mug/mL.
EXAMPLE 5 isolation and culture of immune cells NK cells
Peripheral blood of healthy people is anticoagulated by heparin, 15mL of heparin-PaquePLUS mononuclear cell separation liquid is added into a 50mL centrifuge tube, 30mL of heparin-anticoagulated peripheral blood is slowly added to the centrifuge tube wall to avoid destroying the water phase of the Ficoll separation liquid, the mixture is centrifuged for 15min at room temperature and 3500rpm, upper plasma is carefully sucked into another 50mL centrifuge tube, the mixture is inactivated for 25min at 56 ℃, the faint yellow mononuclear cell layer is sucked into a 15mL centrifuge tube, the mixture is washed for 3 times by sterile PBS, the supernatant is discarded, and the sediment is peripheral blood mononuclear cells.
Monocytes were resuspended in 90mL NK MACS GMP medium (Miltenyi) and 5 ten thousand units of recombinant human IL-2 and IL-15 were added, mixed well, injected into a small culture bag, and placed in a incubator at 37℃for stationary culture. On day 3, 50mL NK MACS GMP medium and 2.5 ten thousand units of recombinant human IL-2 and IL-15 were supplemented, and culture was continued after mixing. On day 4, the culture solution in the small culture bags is evenly distributed to two large culture bags for continuous culture, 100mL of NK MACS GMP culture solution and 15 ten thousand units of recombinant human IL-2 are supplemented to each large culture bag every 3 days, and total culture is carried out for 14 days, namely the amplified NK cells are collected. The amplified NK cells were smeared, dried and anti-CD56 and anti-CD3 labeled by Envision two-step method, and the working concentration of the antibody was 1:20. The results showed 89.7% cd56+ and 3.72% cd3+ indicating higher purity of the NK cells prepared.
EXAMPLE 6 characterization of immune cells NK cells
The PC9 human lung cancer cell suspension is placed at 37 ℃ and 5 percent CO 2 Culturing in incubator, collecting PC9 human lung cancer cells in logarithmic growth phase, and adjusting cell number to 2.0X10 5 mL －1 A96-well U-shaped plate, 100. Mu.L/well, was added. NK cells prepared in example 5 are grouped according to different effect target ratios, namely, NK group, NK+PC9 human lung cancer cells 10:1 and 50:1), PC9 human lung cancer cells and blank group are cultured in 96-hole U-shaped plates at 37 ℃ and 5% CO2 for 12 hours, 20ul MTT 5mg/ml is added to each hole, incubation is carried out at 37 ℃ for 4 hours, 100ul DMSO is added to each hole, the mixture is fully blown and uniformly mixed, and the OD value of the wavelength of 570nm is detected by an enzyme label instrument, and the method is calculated according to the following formula: killing (%) =1- (experimental well a value-effector cell well a value)/target cell well a value x 100%. The results are shown in Table 1.
Table 1 killing Activity of groups against cancer cells
| Group of | Killing rate (%) |
| Group 10:1 | 83.46±2.02 |
| 50:1 group | 90.31±3.47 |
From the results in Table 1, it is found that NK has a killing effect on PC9 human lung cancer cells, and the killing rate is (90.31.+ -. 3.47)% when the effective target ratio is 50:1.
Example 7 Combined therapy experiment with NK cells and beta-catenin-2D 13 mab
Grouping nude mice: placing BALB/c nude mice in a sterile environment for breeding, and randomly grouping 10 nude mice in each group; a is a sterile nude mouse blank control group, a PC9 human lung cancer cell group is transplanted in the B body, a beta-catenin-2D 13 monoclonal antibody group is injected in the C body, an NK cell group is injected in the D body, a beta-catenin-2D 13 monoclonal antibody and an NK cell group are injected in the E body, and a cetuximab control group is injected in the F body.
Establishing a mouse model: the cultured PC9 human lung cancer cells are digested by trypsin, centrifuged, serum-free DMEM/F12 culture medium is used for preparing glioma cell suspension, and the cell suspension is injected into the back subcutaneous of BALB/c nude mice subcutaneously by a 0.2mL syringe with the injection amount of 2X 10 6 (0.2 ml). In each experimental group, NK cell therapy was: the NK cell suspension prepared in example 5 was taken and injected into the body (cell density was 2X 10) by the tail vein of the mouse using a 0.2mL syringe 7 /mL), interval 7d was dosed once more. The monoclonal antibody treatment group is: the 0.2mL syringe is taken to respectively take beta-catenin-2D 13 monoclonal antibody or cetuximab, and the beta-catenin-2D 13 monoclonal antibody or cetuximab is injected into a mouse body in the same way, the injection quantity is 150.0 mug/mouse, and the administration is carried out for 1 time in 3 days, and the total administration is carried out for 3 times. All of the above operations are completed in the same time period and the injection of the cancer cells is completed first. The NK cells and the monoclonal antibodies were injected at 30min intervals during the combination therapy. Nude mice were sacrificed at 14d post-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 for each of 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 NK cells are treated in a combined mode, the tumor weight is only (0.24+/-0.05) g, and the tumor weight of the PC9 human lung cancer cell group is (2.70+/-0.12) g, so that the combined treatment group has obvious inhibition effect compared with the control.
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 or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or of being 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 cis-yuan Dai biological products Co., ltd
Preparation method and application of <120> clinical blood immune cells
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<213> Artificial sequence (Artificial Sequence)
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Gly His Val Trp Tyr Gln Gln Lys Ser Gly Ile Ser Pro Lys Pro Trp
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Ile Tyr Arg Ser Phe Ile Tyr Lys Gly Gly Val Pro Ala Arg Phe Ser
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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
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Leu Pro Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
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Glu Val Gln Leu Glu Glu Ser Ala Thr Glu Leu Ala Arg Pro Gly Ala
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Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Phe Pro
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Gly Phe Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
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Gly Pro Gly Leu Gln Arg Leu Phe His His Cys Cys Phe Asp Met Arg
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Trp Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Claims (7)
1. A 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:
DIVITQRPALMAASPGEKVTITCAEYKHHAELGHVWYQQKSGISPKPWIYRSFIYKGGVPARFSGSGSGTSYSLTITSMEAEDAATYYCNTWDWFTLPFGAGTKLELK
the heavy chain variable region sequences are:
EVQLEESATELARPGASVKLSCKASGYIFSFPGFNWIKQRPGQGLEWIGPGLQRLFHHCCFDMRWGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGPDDVCQHWGLGTTLAVSS。
2. use of a β -catenin monoclonal antibody according to claim 1 for the preparation of a pharmaceutical composition for the treatment of lung cancer.
3. The use according to claim 2, wherein the composition further comprises a pharmaceutically acceptable carrier.
4. The use according to claim 3, wherein the carrier further comprises a preservative.
Use of nk cells in combination with a β -catenin monoclonal antibody for the preparation of a kit for the treatment of cancer lung cancer; the beta-catenin monoclonal antibody is beta-catenin-2D 13, and the light chain variable region sequence of the monoclonal antibody is as follows:
DIVITQRPALMAASPGEKVTITCAEYKHHAELGHVWYQQKSGISPKPWIYRSFIYKGGVPARFSGSGSGTSYSLTITSMEAEDAATYYCNTWDWFTLPFGAGTKLELK
the heavy chain variable region sequences are:
EVQLEESATELARPGASVKLSCKASGYIFSFPGFNWIKQRPGQGLEWIGPGLQRLFHHCCFDMRWGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGPDDVCQHWGLGTTLAVSS；
the preparation method of the NK cells comprises the following steps: separating peripheral blood mononuclear cells, adding serum-free PRMI-1640 culture medium suspension precipitated cells into a plastic plate, coating sheep anti-mouse IgG firstly, sealing for 15 minutes at room temperature by using PBS containing 1% FCS, washing for 3 times by using PBS, adding cells incubated with anti-CD3 for 2 hours into the coated plate, 4 ℃ for 2 hours, gently sucking cell suspension, re-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 a 37 ℃ incubator for standing culture; on day 3, half of NK MACS GMP culture medium and half of recombinant human IL-2 and IL-15 are supplemented, and culture is continued after uniform mixing; on day 4, the culture medium in the small culture bags was evenly distributed to two large culture bags for continuous culture, and NK MACS GMP culture medium and recombinant human IL-2 were supplemented to each large culture bag every 3 days for total culture for 14d, i.e., NK cells after proliferation were collected.
6. The use according to claim 5, characterized in that the β -catenin monoclonal antibody itself is in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
7. The use according to claim 6, wherein the carrier further comprises a preservative.
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