CN112226407B

CN112226407B - NK cell-containing pharmaceutical composition and application thereof in treating cancer

Info

Publication number: CN112226407B
Application number: CN202011183155.5A
Authority: CN
Inventors: 刘欢; 朱小明
Original assignee: Grace Maxicell Genetic Engineering Co ltd
Current assignee: Grace maxicell genetic engineering Co.,Ltd.
Priority date: 2020-10-29
Filing date: 2020-10-29
Publication date: 2021-06-08
Anticipated expiration: 2040-10-29
Also published as: CN112226407A

Abstract

The invention provides a pharmaceutical composition containing NK cells and application thereof in treating cancer, wherein the composition comprises the NK cells optimized by the inventor and a newly prepared antibody targeting CD 38. According to the preparation method of the NK cells, the high-activity and high-purity NK cells are obtained through magnetic bead screening and specific induction optimization, and the monoclonal antibody targeting CD38 also has good binding characteristics and specificity. The composition can effectively inhibit the generation of multiple myeloma transplantation tumor, and has excellent application value.

Description

NK cell-containing pharmaceutical composition and application thereof in treating cancer

Technical Field

The invention relates to the field of biological pharmacy, in particular to a pharmaceutical composition containing NK cells and application thereof in treating cancer.

Background

Multiple Myeloma (MM) is a blood system malignant proliferative disease derived from B lymphocytes, and is characterized by abnormal proliferation of monoclonal plasma cells in bone marrow, and clinically manifests as anemia, hypercalcemia, renal insufficiency, bone destruction, and the like. In recent years, with the widespread use of drugs such as Proteasome Inhibitors (PIs) and immunomodulators (IMiDs), the prognosis of MM patients is improved significantly, but recurrence is inevitable. Statistically, median Overall Survival (OS) was only 15 months for patients with refractory/relapsed mm (rrmm).

In the last decade, the immunotherapy of MM has made a breakthrough. Treatment with allogeneic hematopoietic stem cell transplantation (allo-HSCT) and Donor Lymphocyte Infusion (DLI) may benefit a subset of MM patients due to the Graft Versus Tumor (GVT) effect of the donor lymphocytes. However, donor lymphocytes do not have the specific immune activity of myeloma, mediate non-specific immune response, and the presence of Graft Versus Host Disease (GVHD) increases the mortality of patients after receiving transplantation, limiting the wide clinical application of allo-HSCT and DLI. Thus, more effective immunotherapy is being actively sought to induce more specific, more stable and more effective anti-myeloma immune response responses while reducing treatment-related risks. With the rapid development and cross-infiltration of molecular biology and tumor immunology, a series of MM therapeutic methods with great application prospects are promoted, and immunotherapy methods such as monoclonal antibodies (mAbs), immune checkpoint inhibitors (immune checkpoint inhibitors), dendritic cell vaccines (dendritic cell vaccines) and chimeric antigen receptor T cells (CAR-T) have enjoyed favorable effects in the treatment of MM.

The monoclonal antibody induces antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), etc., by binding to a corresponding target antigen on the surface of the MM cell, thereby finally achieving the purpose of killing the tumor cell. To date, target antigens for immunotherapy have been reported: b Cell Maturation Antigen (BCMA), CD19, CD38, CD56, CD138, SLAMF7, IL-6 receptor, VEGF receptor, immunoglobulin, integrin beta, and G protein-coupled receptor family member 5D (GPRC5D), among others. For example, erlotinzumab is a humanized antibody specific for SLAMF7 that induces myeloma cell death by both directly activating NK cells and triggering ADCC. isatuximab (isa), an IgG1 monoclonal antibody developed by the company cenofibr, binds to CD38 expressed on the surface of hematopoietic cells and tumor cells [ including Multiple Myeloma (MM) cells ], and exerts an antitumor effect through various biological mechanisms, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, and the like. On day 2/3 of 2020, the united states Food and Drug Administration (FDA) approved Isa in combination with pomalidomide and dexamethasone (Isa-Pom-Dex) for the treatment of MM adult patients who have received at least 2 drugs (including lenalidomide and 1 proteasome inhibitor) therapy. Recent studies have shown that median progression-free survival of MM patients treated with Isa-Pom-Dex is 11.5 months with an objective remission rate of 60.4% (93/154), with neutropenia, thrombocytopenia, anemia and the like as common adverse effects. Monoclonal antibody therapy brings new hope for MM patients, but single antibody therapy still has various problems of unstable curative effect, drug resistance and the like, so that the effective combination of the monoclonal antibody and other immunotherapy and targeted therapy strategies is discussed, the treatment effective rate is improved to the maximum extent, the toxic and side effects are reduced, and the occurrence of drug resistance is delayed and reversed, which is the direction of traditional Chinese medicine research.

NK cells are also called natural killer cells, are a class of large granular lymphocytes, are the main cells for performing non-MHC restricted killing function, and are also important components in the natural defense function in vivo. The NK cells do not need antigen stimulation, can directly kill tumor and virus infected target cells in a non-specific way, and play an important role in the immune monitoring and early anti-infection and immune processes of a body. NK cells are derived from bone marrow hematopoietic stem cells, and the developmental maturity of the NK cells depends on bone marrow and thymus microenvironment and accounts for 9% -25% of the total number of lymphocytes. Natural Killer (NK) cells are highly cytotoxic immune cells, and the goal of killing them is non-specific, and therefore, the use of NK cells for the killing of cancer cells becomes particularly powerful.

However, currently, there is no report on the use of NK cells in combination with antibodies for the treatment of multiple myeloma.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a method for effectively treating multiple myeloma.

The method comprises treating multiple myeloma with a pharmaceutical composition prepared by combining the NK cells optimized by the inventor and a newly prepared antibody targeting CD 38.

In one aspect, the invention provides a pharmaceutical composition comprising NK cells and an antibody targeting CD 38.

In another aspect of the present invention, there is provided a method for preparing high-activity NK cells, comprising the steps of collecting whole blood, separating PBMCs by Ficoll density gradient centrifugation, sucking boundary layer mononuclear cells by a Pasteur pipette, and washing twice for inducing NK cells. Buffer was prepared and 40mL of the rinse solution was decanted in advance. Filtering with 0.2um one-way filter pore membraneBuffers and washing solutions. CD1610uL + PBS2mL were plated 24 hours in advance. Counting PBMCs every 1X 10⁷40uL buffer and 10uL antibody were added to each cell. Mixing, and standing at 4 deg.C for 5 min. 30uL of buffer was added, and 20uL of magnetic beads were added per 10⁷The cells were suspended in buffer to 500uL and mixed well, containing up to 108 cells per 500uL of liquid. The mixture was allowed to stand at 4 ℃ for 10 minutes. Prepare the magnetic field, place the column in the magnetic field, and wash the column with 500uL of buffer. The liquid flow is stopped. The cell suspension was added to the column for sorting, and then the column was washed 3 times with 500uL of buffer. The washed cells were collected, and the above step was repeated 1 time with a new sorting column to purify the collected cells. The cells were added IFN-. gamma.750U/mL, IL-2800U/mL, IL-15150U/mL, 20% FBS. Standing at 37 deg.C for 5% CO₂Cultured in an incubator for 3 days. After 3 days fresh medium and 800U/mL IL-2,250U/mL IL-15, and 20% FCS were added to adjust the cell concentration to 2X10⁶Per mL, shake the cell culture flask daily to place the cells in dispersed suspension, count every 3 days, and replenish every 3 days with new 800U/mL IL-2,250U/mL IL-15, and 20% FCS medium. The high activity NK cells are harvested after 18 days of culture.

The invention does not limit the source individuals used for amplifying the activated NK cells, and only needs to screen the transfusion infectious diseases to be qualified. According to one embodiment of the invention, the HANK cells are obtained by ex vivo activation of at least one of the following ex vivo NK cells: the patient's NK cells, the patient's semi-compatible NK cells, and unrelated allogeneic NK cells. The term semi-compatible NK cells of a patient refers to NK cells from the patient's relatives. For example, the patient's own PBMCs are collected to prepare NK cells, preferably prior to routine treatment; or collecting patient relatives, namely semi-compatible PBMC to prepare NK cells; or the NK cells produced by PBMC or the NK cells produced by umbilical cord blood of an unrelated individual, is not limited to this.

In another aspect of the invention, a monoclonal antibody that specifically binds to CD38 is provided, wherein the antibody is obtained by performing multiple rounds of screening on a library. Wherein the heavy chain variable region of the antibody is SEQ ID NO: 1, the light chain variable region is SEQ ID NO: 2, respectively.

The antibody drug is monoclonal antibody or polyclonal antibody. The two antigen binding sites on an antibody molecule can be the same or different, i.e., bispecific antibodies, called antigen-binding fragments (Fab) at the ends of the two arms. According to one embodiment of the invention, the antibody drug is an intact mab that includes an Fc fragment.

According to a preferred embodiment of the present invention, the ratio of the NK cell and the antibody drug in the NK cell in the pharmaceutical composition is 1X 10⁶To 5X 10⁷The method comprises the following steps: 10 mg/kg). When the antibody drug is administered, a doctor or a medical instruction gives the usage amount according to the condition of a patient, and in vitro experiments and animal experiments show that the specific proportion of NK cells are jointly administered during administration, so that the effect of treating cancer can be obviously enhanced.

Advantageous effects

The invention provides a high-activity pharmaceutical composition for treating multiple myeloma, wherein the composition comprises NK cells optimized by the inventor and a newly prepared antibody targeting CD 38. According to the preparation method of the NK cells, the high-activity and high-purity NK cells are obtained through magnetic bead screening and specific induction optimization, and the monoclonal antibody targeting CD38 also has good binding characteristics and specificity. The composition can effectively inhibit the generation of multiple myeloma transplantation tumor, and has excellent application value.

Drawings

FIG. 1 is a graph showing the results of amplification of NK cells in culture for several days

FIG. 2 is a graph showing the results of the antibody-inhibited cell assay

FIG. 3 is a graph showing the results of the antibody + NK cell suppressor cell assay

FIG. 4 is a graph showing the volume change of mouse graft tumor inhibited by the composition

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 preparation of high-Activity NK cells

Diluting heparinized fresh blood by one time with sterile normal saline, adding 4mL of lymphocyte separation solution into a 15mL centrifugal tube, inclining the centrifugal tube, slowly injecting 8mL of diluted whole blood along the tube wall, and enabling the blood to be uniformly distributed on the upper layer and form an obvious interface with the separation solution. Separating PBMCs by using a Ficoll density gradient centrifugation method, centrifuging at 20 ℃ for 1800 rpm for 20min, taking PBMCs as a separation liquid and serum interface layer, sucking interface layer mononuclear cells by using a Pasteur pipette, and washing twice for later use to induce NK cells.

Buffer (containing PBS900uL, FBS100uL, MACSRS 19mL) was prepared and 40mL of the rinse solution was decanted. The buffer and wash were filtered through a 0.2um single direction pore membrane. CD1610uL + PBS2mL were plated 24 hours in advance. 1 part of PBMCs was counted for each 1X 10⁷40uL buffer and 10uL antibody were added to each cell. Mixing, and standing at 4 deg.C for 5 min. 30uL of buffer was added, and 20uL of magnetic beads were added per 10⁷And (4) cells. Preparing cell suspension with buffer solution to 500uL, mixing well, each 500uL liquid contains 10 at most⁸And (4) cells. The mixture was allowed to stand at 4 ℃ for 10 minutes. Prepare the magnetic field, place the column in the magnetic field, and wash the column with 500uL of buffer. The liquid flow is stopped. The cell suspension was added to the column for sorting, and then the column was washed 3 times with 500uL of buffer. The washed cells were collected, and the above step was repeated 1 time with a new sorting column to purify the collected cells. The cells were added IFN-. gamma.750U/mL, IL-2800U/mL, IL-15150U/mL, 20% FBS. Standing at 37 deg.C for 5% CO₂Cultured in an incubator for 3 days. 3 days later, fresh culture medium and 800U/mLIL-2,250U/mLIL-15, and 20% FCS were added to adjust the cell concentration to 2X10⁶The cells were kept in suspension by shaking the cell culture flask daily, counted every 3 days, and supplemented with new 800U/mLIL-2,250U/mLIL-15, and 20% FCS medium every 3 days. The culture was carried out for 18 days. Flow cytometry analysis was performed using direct immunofluorescent labeling. Adjusting the density of the NK cell suspension to 1X 107/mL, 50uL of cell suspension is taken, 20ULCD3-FITC and 20ULPE-CD56 are added, vortex for 2 seconds and protected from light and stored for 30 minutes at 4 ℃. Hanks buffer solution washed twice, immediately tested on machine, and counted for each sample>5×10⁴And (4) respectively. And acquiring positive expression rates of cell surface determinants CD3 and CD56 in the sample, and using FITC labeled homotypic mouse anti-human IgG as a corresponding negative control. Detecting and culturing the NK cells for 18 days, and determining the proportion of the cells.

The results are shown in FIG. 1, the cell volume is small at 0 th day of culture, the cells are scattered under the microscope and are round and bright, the NK cells are purer through the previous screening of the magnetic beads, and the cell number reaches 1.15 multiplied by 10 by inducing and culturing the cells through a specific inducing method to 18 th day¹⁰And the cell number is much higher than that obtained by the culture method in the prior art.

The flow cytometry detection result shows that the positive rate of NK cells CD3-CD56+ on day 0 is (12.33 +/-2.41)%, while the positive rate of NK cells CD3-CD56+ cultured for 18d is (98.79 +/-4.52)%, and the difference is statistically significant (P is less than 0.01). The purity is already quite high and can be used for corresponding clinical trials.

Example 2 killing experiment of NK cells

Inoculating U266 into RPMI1640 culture medium containing 10% fetal calf serum, and culturing at 37 deg.C with 5% CO₂Culturing in a constant-temperature incubator with saturated humidity, carrying out passage for 1 time every 1-2 days, and taking cells in logarithmic growth phase as an experimental object.

Measurement of cell killing Activity by LDH Release method, U266 cells in logarithmic growth phase were taken and the cell density was adjusted to 1X 10 by RPMI1640 containing 10% FCS⁴Per ml, 100. mu. l U266 cells (target cells) per well in 96-well culture plates, where the target cells natural release wells were free of effector NK cells and 100. mu.l of culture medium; maximum release pore 100 μ l of 1% NP 40; the experimental holes are respectively 1: 1. 5: 1. 10: 1. 20: 1 volume ratio of NK cells added with equal concentration of 100ul, each of which is made into 3 multiple wells, 37 ℃, 5% CO₂Culturing for 4-6 h, adding chromogenic reaction system, measuring absorbance A value at 490nm wavelength (reference wavelength 650nm), and calculating killing activity according to LDH release method, wherein the killing activity (percent) is [ (experimental group A value-total self value)Then release A value)/(maximum release group A value-Total Natural release A value)]X 100%. The results are shown in table 1 below.

TABLE 1NK cytotoxic Activity assay results

NK cell/U266 cell ratio	Killing activity (%)
		1:1	(43.8±2.1)％
5∶1	(55.2±3.7)％
		10：1	(66.8±4.5)％
20：1	(73.1±5.2)％

As can be seen from table 1, the NK cytotoxic activity assay effective target ratio was 1: 1. 5: 1. 10: 1. 20: 1, the killing rate of NK cells to U266 cells is respectively (43.8 +/-2.1)%, (55.2 +/-3.7)%, (66.8 +/-4.5)%, and (73.1 +/-5.2)%, and the killing activity is enhanced with the increase of the effective target ratio (P is less than 0.05). From the perspective of optimal use conditions, it is advantageous to select NK cells with a target-to-effect ratio of 10:1 for killing experiments.

Example 3 preparation of monoclonal antibody targeting CD38

Natural synthetic yeast libraries were generated according to the method of WO2012009568 or synthetic methods conventional in the art for artificial yeast libraries, and yeast cell lines expressing monoclonal antibodies were propagated and subjected to high throughput screening and selection. The screening was performed with a commercially available biotinylated human CD38 antigen protein. Yeast cells (about 1010 cells/library) were incubated with 3ml of 100nM biotinylated monomeric human CD38 antigen protein in FACS wash buffer PBS containing 0.1% BSA for 15 minutes at room temperature. After washing once with 50mL ice-cold wash buffer, the cell pellet was resuspended in 40mL wash buffer and 500 μ l streptavidin microbeads were added to the yeast cells and incubated for 15 min at 4 ℃. Next, the yeast cells were pelleted, resuspended in 5mL of wash buffer, and loaded onto a MACS LS column. After loading 5mL, the column was washed 3 times with 3mL FACS wash buffer. The column was removed from the magnetic field and the yeast cells were eluted with 5mL of growth medium and then allowed to grow overnight. After two rounds of MACS, five rounds of sorting were performed using flow cytometry (FACS). For the first round of FACS selection, approximately 4 × 107 yeast cells were pelleted, washed three times with wash buffer, and incubated with 100nM biotinylated monomeric human CD38 antigen protein for 10 minutes at room temperature. Next, the yeast cells were washed twice and stained with goat anti-human F (ab')2 κ -FITC diluted 1:100 and a secondary antibody, streptavidin-Alexa Fluor 633 diluted 1:500, at 4 ℃ for 15 minutes. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.4mL of wash buffer and transferred to a sieve-capped sorting tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and the sort gate (sort gate) was determined to select only CD38 binding. The cynomolgus monkey CD38 protein was used as a countersieve. These pools were then sorted for human CD38 binding to identify cross-reactive binders in a second round of FACS, thereby reducing multispecific binders of reagents. The fourth round of FACS consisted primarily of positive selection using 100nM biotinylated monomer CD38 as antigen. The heavy chains from the fourth round of FACS sort output were used to prepare light chain diversity libraries for additional rounds of selection. The first round of selection involved the conjugation of MiltenyiMACs beads with either 100nM biotinylated monomeric human CD38 or 200nM biotinylated monomeric murine CD38 as antigen. Following MAC bead selection, three rounds of FACS sorting were performed. The first round of FACS involved 100nM or 10nM of human CD 38. Competitive selection was performed with 75-100nM competitor IgG, simultaneously with the second round of FACS described above. After selection, a third positive sorting was performed with 1 or 10nM of human CD38 antigen protein and then plated. Yeast clones were grown to saturation and then induced at 30 ℃ for 48 hours with shaking. After induction, yeast cells were pelleted and the supernatant collected for purification. IgG was purified using a protein a column and eluted with acetic acid at pH 2.0. The purified antibodies obtained from the sorting were subjected to affinity (affinity) measurement of anti-CD 38 antibody. The affinity of these antibodies was determined by measuring the KD of the CD38 antibody with Forte Bio. The data for the first 7 with stronger binding capacity are shown in table 2 below.

TABLE 2 Kd values of mAb antibodies

As can be seen from Table 2, the present invention obtained 7 monoclonal antibodies with better binding properties by screening. Among them, 2B4 and 4D6 showed the best binding properties. In addition, through detection, 2B4 and 4D6 are only combined with human CD38 protein, are not combined with CD38 extracellular protein of recombinant cynomolgus macaques and mice, and have better specificity.

The following experiments were conducted on 2B4 monoclonal antibody. The sequence of the 2B4 single-antibody heavy chain variable region is obtained by analyzing the sequence of the light chain variable region and the heavy chain variable region and is SEQ ID NO: 1; the light chain variable region sequence is SEQ ID NO: 2.

example 42B4 antibody inhibition of cell assay

Cells were collected at logarithmic growth phase at 5X 10³One/well was inoculated in a 96-well plate, 4 replicate wells were set, and 2B4 monoclonal antibody was added to each group to final mass concentration0, 6.25, 12.5, 25, 50, 100. mu.g/mL respectively, 5% CO at 37 ℃₂Incubate in incubator for 24 h. mu.L of MTT solution was added to each well and incubation was continued in the cell incubator for 4 h. Add 100. mu.L of Formazan lysis solution to each well, mix well, and continue incubation in cell culture box. Until formazan was completely dissolved by observation under a normal light microscope, absorbance was measured at 570nm, and cell survival rate and inhibition rate were calculated. The wells without cells and with only nutrient solution and MTT reaction reagent on the 96-well plate are blank groups, the wells with U266 cells and without monoclonal antibody are negative control groups, and the cell survival rate and inhibition rate are calculated. Cell viability was 100% (a experiment-a blank)/(a control-a blank). The results are shown in FIG. 2.

As can be seen from the results in FIG. 2, the 2B4 monoclonal antibody has a significant inhibitory effect on the in vitro proliferation of myeloma U266 cells. The survival rate of myeloma U266 cells decreased gradually with the increase of antibody concentration, and the survival rate of cells was only (20.2. + -. 1.1)%, at 100. mu.g/mL, as compared with the control group.

Example 52B4 antibody in combination with NK cell suppressor cell assay

Inoculating U266 in RPMI1640 culture solution containing 10% fetal calf serum, culturing in a constant temperature incubator with 37 ℃, 5% CO2 and saturated humidity, carrying out passage for 1 time every 1-2 days, and taking cells in logarithmic growth phase as an experimental object.

Cells were collected at 1X 10 log phase growth⁴One cell/well is inoculated on a 96-well plate, 4 repeated wells are arranged, 50 mu g/mL of 2B4 monoclonal antibody is added in each group, and the NK cell is 1 multiplied by 10 respectively³/ml、5×10³/ml、1×10⁴/ml、5×10⁴/ml、1×10⁵Per ml, 5% CO at 37 ℃₂Incubate in incubator for 24 h. mu.L of MTT solution was added to each well and incubation was continued in the cell incubator for 4 h. Add 100. mu.L of Formazan lysis solution to each well, mix well, and continue incubation in cell culture box. Until formazan was completely dissolved by observation under a normal light microscope, absorbance was measured at 570nm, and the cell survival rate was calculated. Wells without cells and with only nutrient solution and MTT reaction reagent on 96-well plates were blank, and the cell viability was 100% (a experiment-a blank)/(a control-a blank). The results are shown in FIG. 3.

As can be seen from FIG. 3, the combined use of the antibody and NK cells of the present invention has significant systemic synergistic therapeutic effect, and can significantly inhibit the proliferation of cancer cells at a concentration of 1 × 10⁵Under the treatment of/mlNK +2B450 micrograms/mL, the survival rate of cancer cells is only 5%, and the effect is better.

Example 6 treatment experiments with multiple myeloma mouse model

Random equal numbers divided 25 nude mice into five groups: group A: non-transplanted group; group B: u266 transplant group; group C: monoclonal antibody + NK cell transplantation group; group D: a NK cell transplantation group; group E: and (5) monoclonal antibody transplanting groups.

U266 at the logarithmic growth phase was digested with trypsin, centrifuged, counted and formulated to contain 1X 10 per 1mL⁷U266 serum-free DMEM/F12 suspension.

Group A injected with an equal amount of physiological saline subcutaneously on the back of nude mice as a control;

group B injected with 0.1mL of U266 alone (1X 10)⁶Individual cells) suspension was injected subcutaneously into the back of BALB/c nude mice.

Group C: a therapeutic dose of 10mg/kg of 2B4 mab and 0.2mL of NK cell suspension (5X 10)⁷Individual cell) +0.1mL U266 (1X 10)⁶Individual cells) suspension was injected subcutaneously into the back of nude mice via tail vein.

Group D: 0.2mL NK cell suspension (5X 10)⁷Individual cell) +0.1mL U266 (1X 10)⁶Individual cells) suspension was injected subcutaneously into the back of nude mice via tail vein.

Group E: therapeutic dose of 10mg/kg 2B4 mAb +0.1mL U266 (1X 10)⁶Individual cells) suspension was injected subcutaneously into the back of nude mice via tail vein.

Monitoring the growth of the tumor: from the day of transplantation, the growth of tumor cells in nude mice was observed every day at regular time, after the tumor appeared, the maximum values of length and width (vertical to length and width) of the tumor were measured with a vernier caliper and recorded as a and b, respectively, and the tumor volume was calculated. Nude mice were sacrificed 3 weeks after tumor appearance, tumors were removed in whole blocks and volume was measured. The results are shown in FIG. 4 below.

In nude mice, whether the transplanted NK cell or 2B4 monoclonal antibody can effectively inhibit the transplantationThe growth of the U266 graft tumor of (a). The growth curve shows that at 21d, the combined treatment with 2B4 mab and NK cells can significantly reduce the volume growth of the transplanted tumor, and at 21d, the volume is only (211 +/-3.5) mm³Significantly less than (3005. + -. 3.5) mm of control³。

Sequence listing

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Claims

1. Use of high activity NK cells in the manufacture of a medicament for the treatment of multiple myeloma, characterized in that: the medicine also contains a monoclonal antibody targeting CD 38; the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and the light chain variable region is shown as SEQ ID NO: 2 is shown in the specification; what is needed isThe preparation method of the high-activity NK cell comprises the following steps: diluting heparinized fresh blood by one time with sterile normal saline, adding 4mL of lymphocyte separation solution into a 15mL centrifugal tube, inclining the centrifugal tube, slowly injecting 8mL of diluted whole blood along the tube wall, and enabling the blood to be uniformly distributed on the upper layer and form an obvious interface with the separation solution; separating PBMCs by using a Ficoll density gradient centrifugation method, centrifuging at 20 ℃ for 1800 rpm for 20min at a centrifugal force, taking PBMCs as a separation liquid and serum interface layer, sucking interface layer mononuclear cells by using a Pasteur pipette, and washing twice for later use to induce NK cells; preparation of PBS900uL, FBS100uL, MACS RS solution 19mL buffer, in advance of pouring the washing solution 40mL using 0.2um single filter hole membrane filtration buffer, using CD1610uL + PBS2mL in advance of 24 hours culture dish, 1 PBMCs count, each 1 x10⁷Adding 40uL buffer solution and 10uL antibody into each cell, uniformly mixing, and standing at 4 ℃ for 5 minutes; 30uL of buffer was added, and 20uL of magnetic beads were added per 10⁷(ii) individual cells; preparing cell suspension with buffer solution to 500uL, mixing well, each 500uL liquid contains 10 at most⁸(ii) individual cells; standing for 10 minutes at 4 ℃; preparing a magnetic field, placing the separation column in the magnetic field, and washing the separation column by using 500uL buffer solution; stopping the liquid flow; adding the cell suspension into a separation column, sorting, and then adding 500uL buffer solution to wash the separation column for 3 times; collecting the washed cells, repeating the above step 1 times with a new sorting column, and purifying the collected cells; adding IFN-gamma 750U/mL, IL-2800U/mL, IL-15150U/mL, 20% FBS to the cells; standing at 37 deg.C for 5% CO₂The culture box is used for culturing for 3 days; after 3 days fresh medium and 800U/mL IL-2,250U/mL IL-15, and 20% FCS were added to adjust the cell concentration to 2X10⁶Shaking the cell culture flask every day to make the cells in a dispersed suspension state, counting every 3 days, supplementing new 800U/mL IL-2,250U/mL IL-15 every 3 days, and culturing in 20% FCS medium for 18 days, and harvesting the high-activity NK cells; the blood is from autologous or allogeneic sources.

2. A monoclonal antibody targeting CD38, characterized by: the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and the light chain variable region is shown as SEQ ID NO: 2, respectively.