CN111826400A

CN111826400A - Preparation method of bispecific antibody NK cell, cell and application thereof

Info

Publication number: CN111826400A
Application number: CN202010704663.7A
Authority: CN
Inventors: 张扬; 周晓宇; 李陶; 张立忠
Original assignee: Zhongkebaocheng Biomedical Technology Co ltd
Current assignee: Zhongkebaocheng Biomedical Technology Co ltd
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2020-10-27

Abstract

The application provides a preparation method of a bispecific antibody NK cell, a cell and an application thereof, wherein the method comprises the steps of culturing the NK cell, constructing a CD16A/HSP70 double-anti-lentiviral expression vector, and infecting the NK cell with the CD16A/HSP70 double-anti-lentiviral expression vector to obtain the bispecific antibody NK cell of the CD16A/HSP70 antibody. The NK cells prepared by the method have excellent cell specificity phenotype and high purity; wherein the CD3-CD56+ reaches 97.13 percent, and the CD3-CD56+ CD16+ reaches 93 percent; the NK cell carrying the CD16A/HSP70 double-anti-lentivirus expression vector can effectively eliminate the hepatoma carcinoma cell and inhibit the capacity of increasing the hepatoma carcinoma tumor volume.

Description

Preparation method of bispecific antibody NK cell, cell and application thereof

Technical Field

The application relates to the technical field of cell culture, in particular to a preparation method of a bispecific antibody NK cell, a cell and application thereof.

Background

Natural killer cells (NK) are important immune cells in the body, not only involved in anti-tumor, anti-viral infection and immune regulation, but also involved in the development of hypersensitivity and autoimmune diseases in some cases, and capable of recognizing target cells, killing mediators. Natural killer cells are derived from bone marrow lymphoid stem cells, the differentiation and development of the natural killer cells depend on bone marrow and thymus microenvironment, and the natural killer cells are mainly distributed in bone marrow, peripheral blood, liver, spleen, lung and lymph nodes. NK cells are different from T and B cells, and are lymphocytes capable of non-specifically killing tumor cells and virus-infected cells without pre-sensitization.

The source of NK is mainly supplied by self and heterogeny, the self NK cells have limited effect, cannot play a role in killing cancer cells capable of expressing HLA (human leukocyte antigen), the number of the self NK cells of a human body is limited, the NK cells are in different developmental stages, even in different tissues of the same developmental stage, and the phenotype and the function of the NK cells are greatly different. Previous allogeneic NK cells were mainly derived from peripheral blood, bone marrow, embryos and placenta, and not only were it difficult to collect donors, but also the cost of isolating other immune cells was extremely high. These all limit the clinical large-scale use of NK cells. Therefore, cord blood, which can be matched at any time, becomes the latest source of NK cells.

Because of the complexity of cord blood, obtaining high purity, high activity cord blood NK has been an obstacle to large-scale application. At present, the culture of the NK cells of the cord blood is mainly obtained by co-culturing with a feeder cell K562; or magnetic bead sorting and flow sorting are adopted; or by inducing stem cell differentiation. However, the above methods all have significant disadvantages. The use of feeder cells undoubtedly introduces exogenous cells, increasing the risk of clinical application; magnetic bead sorting and flow sorting are complicated in operation, so that the risk of cell pollution is increased undoubtedly, and the cost is high; the differentiation technology of cord blood hematopoietic stem cells is not mature yet.

A large number of researchers have conducted human NK cell culture and research on applying NK cells to cancer treatment at home and abroad, but the cultured NK cells have the problems of poor phenotype, low purity and the like, and cannot meet the requirements of practical application.

Disclosure of Invention

The application provides a preparation method of a bispecific antibody NK cell, a cell and an application thereof, which aim to solve the problems that the NK cell has poor phenotype and low purity and cannot meet the requirement of practical application in the aspect of cancer treatment.

The application provides a preparation method of a bispecific antibody NK cell, which is characterized by comprising the following steps:

culturing the NK cells;

constructing a CD16A/HSP70 double-anti-lentivirus expression vector;

and infecting the NK cells by using the CD16A/HSP70 double-anti-lentiviral expression vector to obtain the bispecific antibody NK cells of the CD16A/HSP70 antibody.

Optionally, the step of culturing NK cells comprises:

sampling umbilical cord blood and detecting viruses;

centrifuging the qualified umbilical cord blood, and inactivating the centrifuged umbilical cord blood in a water bath at 50-60 ℃ for 30-60 min;

cooling the inactivated umbilical cord blood at-20 to-10 ℃ for 10-15min, and centrifuging to obtain supernatant plasma and lower cell sap;

dividing the supernatant plasma into plasma 1 and plasma 2, and storing the plasma 1 and the plasma 2 together in a refrigerator at 4 ℃ for later use;

adding D-PBS into the lower cell sap, uniformly stirring, centrifuging and removing supernatant to obtain a cell layer;

adding 10-15ml of human lymphocyte separation liquid into the cell layer, centrifuging and shaking up, and removing supernatant to obtain a leucocyte layer;

adding a culture solution into the leucocyte layer, centrifuging, adding a culture medium into the centrifuged leucocyte layer to obtain a cell suspension, and storing the cell suspension for later use;

adding 15-20ml of D-PBS and kit factors into a culture bottle, and removing a coating solution in the culture bottle after the culture bottle is coated in an incubator at 30-40 ℃ for 2-3 h;

10ml of the plasma 1 and the cell suspension were added to the flask from which the coating solution was removed, and the flask was designated as flask 1.

Optionally, the step of culturing NK cells further comprises:

preparing an activation culture medium and a proliferation culture medium for later use;

placing the culture bottle 1 into an incubator at 37 ℃ and 5% carbon dioxide saturated humidity for culture, and recording as the first day of culture;

on the fourth day of culture, 30-40ml of the activated culture medium, 10ml of the plasma 1 and kit factors are added into the culture bottle 1, and the culture bottle 1 is placed in an incubator with 37 ℃ and 5% carbon dioxide saturated humidity for culture;

adding 60-70ml of the activated culture medium, 10ml of the blood plasma 1 and kit factors into the culture bottle 1 on the sixth day of culture, and placing the culture bottle 1 in an incubator with 37 ℃ and 5% carbon dioxide saturated humidity for culture;

adding the plasma 2 into the culture bottle 1 at the eighth day of culture to obtain cell sap, pouring the cell sap into a culture bag, and adding 500ml of the activation medium into the culture bag; placing the culture bag into an incubator at 37 ℃ and 5% carbon dioxide saturation humidity for culture;

pouring 500ml of enrichment culture medium liquid into a culture bag on the tenth day of culture, and culturing the culture bag in an incubator at 37 ℃ and 5% carbon dioxide saturation humidity;

adding 1000ml of the enrichment culture medium liquid into the culture bag on the thirteenth day of culture, and culturing the culture bag in an incubator at 37 ℃ and 5% carbon dioxide saturated humidity;

collecting NK cell suspension in the culture bag on the sixteenth day of culture, centrifuging to obtain NK cells, and counting and sealing the NK cells.

Optionally, the step of constructing the CD16A/HSP70 dual anti-lentiviral expression vector comprises:

sequentially connecting IgG leader genes, anti-CD 16A VL genes, a first linking Linker, anti-HSP70 VH genes, a second linking Linker, anti-HSP70 VL genes, a third linking Linker, anti-CD 16A VH genes, hinge sequence genes, hIgG1 CH2 genes and hIgG1 CH3 genes together to obtain fusion genes;

the fusion gene is inserted into the XbaI/DraIII site of a lentivirus overexpression plasmid vector pGreenPuroTM, and the CD16A/HSP70 double-anti-lentivirus expression vector is obtained.

Optionally, the step of infecting the NK cells with the CD16A/HSP70 double anti-lentiviral expression vector to obtain bispecific antibody NK cells of CD16A/HSP70 antibodies comprises:

respectively carrying out high-purity endotoxin-free extraction on the CD16A/HSP70 double-anti-lentivirus expression vector and the packaging plasmid;

co-transfecting the extracted CD16A/HSP70 double-anti-lentivirus expression vector and a packaging plasmid into a packaging cell;

replacing the complete medium of the transfected packaging cells and collecting the supernatant of the transfected packaging cells;

concentrating the virus and infecting NK cells to obtain the NK cells of the bispecific antibody of the CD16A/HSP70 antibody.

The present application provides bispecific antibody NK cells prepared according to the methods described above.

The application also provides application of the bispecific antibody NK cell prepared by the method in preparation of a medicine for treating liver cancer.

The present application provides for continuous secretion of bispecific antibodies in vivo by genetically modified NK cells, inherits the advantages of CAR-T (chimeric antigen receptor T cells) and bispecific antibodies and overcomes the disadvantages thereof, being a cell that fuses autoimmune cells, genetic modification and bispecific antibodies.

Compared with the prior art, the technical scheme provided by the application has the following beneficial effects: the NK cell provided by the invention has excellent cell phenotype and high purity, wherein the cell phenotype of the NK cell is 97.13% for CD3-CD56+, and 93% for CD3-CD56+ CD16 +; the cell carrying the CD16A/HSP70 double-anti-lentivirus expression vector can effectively eliminate the liver cancer cell and inhibit the capacity of increasing the tumor volume of the liver cancer.

Drawings

In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow-through phenotypic assay of NK cell sample 1 prepared in an example of the present application;

fig. 2 is a flow-through phenotypic assay of NK cell sample 2 prepared in the examples of the present application.

Detailed Description

Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following examples do not represent all embodiments consistent with the present application. But merely as exemplifications of systems and methods consistent with certain aspects of the application, as recited in the claims.

Adoptive immunotherapy mainly includes specific TIL (tumor infiltrating lymphocytes), TCR (T cell antigen receptor), CAR-T (chimeric antigen receptor T cells), CAR-NK (chimeric antigen receptor NK cells), nonspecific CIK (cytokine-induced killer cells), DCs (dendritic cells), NK, and the like. The NK can be used as an important immune effector cell to directly kill target cells infected or mutated by viruses without pre-sensitization, and has the following characteristics: no specific antigen recognition is required; killing target cells directly; not restricted by MHC (histocompatibility complex); has wide antitumor spectrum and basically no adverse reaction. Plays a very important role in controlling the occurrence and development of cancer. It can destroy viruses, bacteria and cancer cells in the human body by releasing perforin/granzyme, ADCC effect (antibody-dependent cell-mediated cytotoxicity), Fas/FasL system (apoptosis system) and NK cytotoxic. Due to the unique characteristics of the NK, the application of the NK in tumor immunotherapy is more and more emphasized. In Japan, NK cells are widely used not only for the treatment of cancer patients but also for sub-health people to prevent the occurrence of cancer.

The application provides a method for preparing bispecific antibody NK cells, which comprises the following steps:

s1: culturing the NK cells;

s2: constructing a CD16A/HSP70 double-anti-lentivirus expression vector;

s3: and infecting the NK cells by using the CD16A/HSP70 double-anti-lentiviral expression vector to obtain the bispecific antibody NK cells of the CD16A/HSP70 antibody.

The specificity of NK cell surface markers is relative compared to T cells, B cells. Human NK cells mIg-, some as well as NK cells CD2, CD3 and CD8 are positive, express IL-2 receptor beta chain (P75, CD122), CD11b/CD18 are positive. Labeled antibodies commonly used for detection of NK cells are CD16, CD56, CD57, CD59, CD11b, CD94 and LAK-1.

The reagent kit factors in the application are all purchased from Tianjin ocean biological product science and technology responsibility limited company.

S1: the step of culturing the NK cells comprises:

sampling umbilical cord blood and detecting viruses;

adding 15-20ml of D-PBS, one JV-1 kit factor and one JV-2 kit factor into a culture bottle, coating the culture bottle in an incubator at 30-40 ℃ or a refrigerator at 4 ℃ for 2-3h, and removing a coating solution in the culture bottle;

adding 10ml of plasma 1, the cell suspension, five JV-3 kit factors and one JV-R-Plus kit factor into the culture bottle without the coating solution, and marking the culture bottle as a culture bottle 1;

adding 30-40ml of the activation medium, 10ml of the plasma 1, JV-R-plus kit factors and JV-6 kit factors into the culture bottle 1 on the fourth day of culture, and culturing the culture bottle 1 in an incubator at 37 ℃ and 5% carbon dioxide saturation humidity;

adding 60-70ml of the activated culture medium, 10ml of the plasma 1 and JV-7 kit factors into the culture bottle 1 on the sixth day of culture, and placing the culture bottle 1 in an incubator at 37 ℃ and 5% carbon dioxide saturated humidity for culture;

In practical application, the prepared NK cell has the function of tumor targeted migration, can induce tumor cell apoptosis and has the effect of keeping tumor cell growth all the time, and can be used as a liver cancer tumor targeted therapy carrier.

In practice, the following steps are required after obtaining NK cells:

firstly, injecting HSP70 antigen into an animal to obtain an animal cell, secondly, fusing the animal cell with an immune human HSP70 antigen cell, culturing the obtained fused cell, obtaining a hybridoma cell after screening, and culturing and subcloning the hybridoma cell to obtain the hybridoma cell secreting the monoclonal antibody.

And finally, carrying out expanded culture on the hybridoma cells, centrifuging to collect cell supernatant, and purifying the antibody in the cell supernatant by using a protein A affinity chromatography method.

In the actual process, total RNA is extracted, reverse transcription is carried out to form cDNA, VH and VL gene segments are amplified by using VH and VL primers, and a carrier is connected with a PCR product, transformed, screened, identified and sequenced to obtain the variable gene sequence of the anti-human HSP70 monoclonal antibody. After obtaining the variable gene sequence of the anti-human HSP70 monoclonal antibody, a CD16A/HSP70 double-anti-lentiviral expression vector is constructed.

S2: the steps for constructing the CD16A/HSP70 double anti-lentivirus expression vector comprise the following steps:

In the actual process, PCR technology (polymerase chain reaction) can be used for in vitro amplification to obtain a target gene segment, and the recombinant technology is used for transferring the coded gene segment into a lentiviral vector to construct a CD16A/HSP70 double-resistant lentiviral expression vector.

In practice, the bispecific antibody is also called BsAb, so the bispecific antibody NK cell is also called BsAb-NK cell, so the bispecific antibody NK cell is called BsAb-NK cell in the present application.

S3: the step of infecting said NK cells with a CD16A/HSP70 dual anti-lentiviral expression vector to give BsAb-NK cells of CD16A/HSP70 antibody (bispecific antibody NK cells) includes:

concentrating the virus and infecting NK cells to obtain BsAb-NK cells of CD16A/HSP70 antibody.

In the actual process, the CD16A/HSP70 double-anti-lentivirus expression vector obtained by the construction is used for transfecting NK cells, and the replication of the double-anti-lentivirus vector in human umbilical cord blood cells is utilized to ensure that the human umbilical cord blood cells are continuously and stably expressed.

In practical applications, the properties of BsAb-NK cells for detecting the CD16A/HSP70 antibody include:

taking a BsAb-NK cancer cell line in a logarithmic growth phase, and culturing for 4-8 hours at a concentration of an effective target ratio of 5:1 to obtain a supernatant;

the supernatant was assayed for IFN-. gamma. (serum cytokine anti-interferon), TNF-. alpha.and IL-2 (interleukin) secretion by BsAb-NK cells using an ELISA kit.

In practical application, BsAb-NK cells of the autocrine anti-CD 16A/HSP70 antibody which are highly expressed are subjected to in vivo and in vitro validity verification, and the antitumor activities of unmodified NK cells and the BsAb-NK cells are examined when the same effect target ratio is achieved. Unmodified NK cells and BsAb-NK cells were examined for secretion of cytokines IFN-. gamma.levels, TNF-. alpha.levels and IL-2 levels in a high-expression HSP70 liver cancer cell line and a low-expression HSP70 liver cancer cell line. And (3) analyzing the in vivo effectiveness: taking HSP70 mouse model as an example, the tumor volume of mice was examined with respect to the growth rate and weight loss rate of the control group by NK cell injection therapy and BsAb-NK cell injection therapy. Median survival was compared for the BsAb-NK cell group, NK cell group and control group.

The following are examples provided by the present application:

first, NK cells were prepared:

the experimental equipment is firstly disinfected by ultraviolet.

The virus detection of the cord blood sampling comprises the following detection contents: hepatitis b five, HCV (hepatitis c virus), HTLV (human T-cell virus), HIV (human immunodeficiency virus), EBV (lymphotropic virus of the herpesviridae), CMV (cytomegalovirus) and syphilis helix, detection criteria: the five tests of hepatitis B only can make the surface antibody be positive, and the two and three antibody positivity do not meet the requirements.

Adding 20ml of D-PBS, one JV-1 kit factor and one JV-2 kit factor into a culture bottle, sucking the D-PBS in the culture bottle by a pipette to wash the kit for 2 times, sealing by a sealing film, and coating in an incubator for 3 hours at 30 ℃.

Preparing an activation medium and a proliferation medium:

adding a kit factor JV-5 into 1000ml of basal medium to obtain a proliferation medium;

adding one JV-4 strain into 500ml proliferation culture medium to obtain activation culture medium. The activation culture medium and the proliferation culture medium are reserved;

centrifuging qualified umbilical cord blood for 15min, wherein the rotation speed is 1000g, inactivating the centrifuged umbilical cord blood in a water bath at 60 ℃ for 60min, cooling the inactivated umbilical cord blood at-20 ℃ for 15min, centrifuging for 20min, wherein the rotation speed is 1000g to obtain supernatant plasma and lower cell sap, dividing the supernatant plasma into plasma 1 and plasma 2, and storing the plasma in a refrigerator at 4 ℃ for later use.

D-PBS was added to the lower cell sap to 50ml and stirred uniformly, and the supernatant was removed by centrifugation to obtain a cell layer.

15ml of human lymphocyte separation liquid is added into the cell layer, the cell layer is centrifuged (the rotating speed is 200g, the centrifugation is 5min) and shaken up, and the supernatant is removed to obtain the leucocyte layer.

Adding a basic culture medium to the leucocyte layer to 50ml, then centrifuging (rotating speed 500g, centrifuging for 15min), and adding the centrifuged leucocyte layer into a culture medium without any factors to obtain a cell suspension.

Sucking out the coating solution in the culture bottle coated in the step 1 by using a suction pipe, wherein the coating surface does not touch in the sucking process, adding 10ml of blood plasma 1, 40ml of the cell suspension (the coating surface of the culture bottle is kept in a wet state in the whole process), five JV-3 kit factors and one JV-R-Plus kit factor into the culture bottle after sucking out, and placing the culture bottle in an incubator at 37 ℃ and 5% carbon dioxide saturated humidity for culture, wherein the culture is recorded as the first day of culture.

On the fourth day of culture, 40ml of the activated medium was added to the flask, 10ml of plasma 1 was added, and one JV-R-plus kit factor and one JV-6 kit factor were added, and the flask 1 was incubated at 37 ℃ in a 5% carbon dioxide saturated humidity incubator.

On the sixth day of culture, 60ml of the activation medium was added to the flask, 10ml of plasma 1 and one JV-7 kit factor were added, and the flask 1 was incubated at 37 ℃ in a 5% carbon dioxide saturated humidity incubator.

On the eighth day of culture, the flask 1 was taken out of the incubator, and the state and number of cells were observed under a microscope. Adding plasma 2 into the culture bottle 1, sucking 10ml of activation culture medium into the culture bottle by using a pipette, continuously blowing and washing the inner wall of the bottle for 10-20 times, repeating the steps for multiple times until most of cells on the inner wall of the bottle are blown off to obtain cell sap, pouring the cell sap into a culture bag, and adding 500ml of activation culture medium into the culture bag; the culture bag is placed into an incubator at 37 ℃ and 5% carbon dioxide saturation humidity for culture.

In practice, if the number of cells is large and the culture medium turns yellow before the eighth day, the liquid in the culture flask can be expanded into another culture flask, and the two culture flasks can be simultaneously bagged at the eighth day.

In practice, during the pouring of the cell sap into the culture bag, it is necessary to first detect the leakage of the culture bag, and 100ml of the activation medium may be added first, and if the bag does not have the leakage, the activation medium may be continuously added to the culture bag, and a total of 500ml of the activation medium (including the activation medium that was originally detected as the leakage of the bag) may be added.

The culture medium turns yellow obviously on the tenth day of culture, 500ml of enrichment culture medium liquid is poured into a culture bag, and a culture bottle is placed in an incubator at 37 ℃ and 5% carbon dioxide saturation humidity for culture.

In practice, after 500ml of culture medium was poured, a total of 1L of culture medium, 500ml of activated culture medium and 500ml of propagation culture medium, was added to the bag.

And adding 1000ml of proliferation culture medium liquid into each culture bag in the thirteenth day of culture, uniformly mixing, and taking out 30ml of culture liquid for detection of bacteria, fungi, mycoplasma and endotoxin.

And collecting NK cell suspension in the culture bag on the sixteenth day of culture, centrifuging to obtain NK cells, and counting and sealing the NK cells. Two NK cell samples, namely NK cell sample 1 and NK cell sample 2, were co-prepared in this example.

FIG. 1 is a flow-type phenotypic assay chart of NK cell sample 1 prepared in the example of the present application, from which it can be seen that CD3-CD 56-is 1.84%, CD3-CD56+ is 97.13%, CD3+ CD 56-is 0.07%, CD3+ CD56+ is 0.96%, and CD3- (CD56+ CD16+) is 93.00%. FIG. 2 is a flow-type phenotypic assay of NK cell sample 2 prepared in the example of the present application, showing that CD3-CD56+ is 98.34% and CD3-CD56+ CD16+ is 96.06%.

Obtaining NK cells requires the preparation of hybridoma cells secreting monoclonal antibodies.

Mu.g of human HSP70 antigen was mixed with the same dose of IFA kit for emulsion reaction to immunize BALB/c mice at multiple points on the back, under the armpits and in the groin. On both day 14 and day 28, immunization was performed again in the same manner.

On day 38, tail serum was taken from the mice and the serum titer was measured by indirect ELISA. 3 days before fusion, 20. mu.g of human HSP70 antigen was taken for abdominal booster immunization.

Mixing mouse myeloma Sp2/0 cell and spleen cell of mouse immunized with human HSP70 antigen at a ratio of 1:10, adding cell fusion agent PEG, stopping PEG effect in serum-free culture medium within 1min, centrifuging to remove supernatant to obtain fusion cell, and suspending the fusion cell in HAT selective culture medium.

Adding into 96-well cell culture plate, and standing at 37 deg.C with 5% CO₂Culturing for 15 days in the culture box, centrifuging to remove the supernatant, performing high-throughput screening by using indirect ELISA (enzyme-linked immunosorbent assay), performing culture medium conversion on the hybridoma cells determined to be positive, changing the hybridoma cells from HAT selective culture medium to HT selective culture medium, changing the hybridoma cells to 10% serum-containing culture medium, and finally changing the hybridoma cells to serum-free culture medium.

The obtained hybridoma cells are subcloned to obtain the hybridoma cells secreting the monoclonal antibody.

Then, a CD16A/HSP70 double anti-lentivirus expression vector is constructed according to the hybridoma cells.

Subcloned cells were treated at 10⁵Adding into a cell culture flask containing serum-free medium with volume of 1L, and standing at 37 deg.C under 5% CO₂The culture was expanded in an incubator for 20 days, and the cell supernatant was collected by centrifugation.

The anti-HSP70 in the cell supernatant was purified by protein A affinity chromatography. And (3) carrying out purity identification, titer identification and subtype identification on the anti-HSP70 monoclonal antibody. Wherein purity identification is detected using SDS-PAGE gel electrophoresis (polyacrylamide gel electrophoresis); performing titer identification by using ELISA; subtype identification the screened antibodies were subjected to subtype identification using a kit.

Total RNA from the hybridoma was extracted, reverse transcribed to form cDNA, and VH and VL fragments were amplified using VH and VL primers. Connecting the pGEM-T vector with a PCR product (VH or VL), transforming, screening, identifying and sequencing to obtain the variable region gene sequence of the anti-human HSP70 monoclonal antibody.

Sequentially connecting IgG leader gene, CD16A VL resisting gene, a first linking linker, HSP70 VH resisting gene, a second linking linker, HSP70 VL resisting gene, a third linking linker, CD16A VH resisting gene, hinge sequence gene, hIgG1 CH2 gene and hIgG1 CH3 gene together, and then filling the mixture into XbaI/DraIII site of a lentiviral over-expression plasmid vector pGreenPuroTM to obtain lentiviral expression vector plasmid.

And (3) after respectively carrying out high-purity endotoxin-free extraction on the lentivirus expression vector plasmid, the lentivirus shuttle plasmid and the auxiliary plasmid thereof, co-transfecting the NK cell, replacing the transfected NK cell with a complete culture medium for 6h, and infecting the transfected NK cell after concentrating the virus to obtain the BsAb-NK cell.

BsAb-NK cells are used as effector cells, and a liver cancer cell line with low expression of HSP70 is used as target cells, so that the killing effect of the BsAb-NK cells is detected.

The cancer cell lines in the logarithmic growth phase (the liver cancer cell line with low expression HSP70 and the liver cancer cell line with high expression HSP 70) are cultured together for 4 hours at the concentration of the effective target ratio of 5:1, the supernatant is collected, and the IFN-gamma, TNF-alpha and IL-2 secretion level of BsAb-NK cells is detected by an ELISA kit.

30 female nude mice of 6 weeks old are taken, and each nude mouse is injected with 1X 10 subcutaneous injections in the right armpit⁷The human HSP70 positive tumor cell line of (a), was randomized into 3 groups for tail vein injection treatment: saline control group, NK cell group and BsAb-NK cell group.

Tumor volume was calculated and mice were weighed. And counting the death number per day, and drawing a survival rate curve of the transplanted tumor model mouse.

The in vivo and in vitro effectiveness of BsAb-NK cells of the high-efficiency expression autocrine anti-CD 16A/HSP70 antibody is verified, and the antitumor activity of unmodified NK cells is lower than that of the BsAb-NK cells at the same effective target ratio. The liver cancer cell line of high expression HSP70 and the liver cancer cell line of low expression HSP70, the level of secreted cytokine IFN-gamma, the level of TNF-alpha and the level of IL-2 of BsAb-NK cells are all higher than those of unmodified NK cells. The in vivo effectiveness analysis result shows that: in HSP70 mouse model, with NK and BsAb-NK cell injection treatment, the tumor volume of mice increased slowly relative to the control group, the body weight decreased slowly, and BsAb-NK cell group > control group. BsAb-NK cell group median survival > control group median survival. Therefore, the bispecific antibody NK cell prepared by the method for preparing the bispecific antibody NK cell can be applied to the preparation of medicines for treating liver cancer.

Compared with the prior art, the technical scheme provided by the application has the following beneficial effects: the NK cell provided by the invention has excellent cell phenotype and high purity, wherein the cell phenotype of the NK cell is 97.13% for CD3-CD56+, and 93% for CD3-CD56+ CD16 +; the cell carrying the CD16A/HSP70 double-anti-lentivirus expression vector can effectively eliminate the liver cancer cell and inhibit the capacity of increasing the tumor volume of the liver cancer. Therefore, the bispecific antibody NK cell prepared by the preparation method of the bispecific antibody NK cell can be applied to the preparation of medicines for treating liver cancer.

The embodiments provided in the present application are only a few examples of the general concept of the present application, and do not limit the scope of the present application. Any other embodiments extended according to the scheme of the present application without inventive efforts will be within the scope of protection of the present application for a person skilled in the art.

Claims

1. A method for preparing a bispecific antibody NK cell, comprising the steps of:

culturing the NK cells;

constructing a CD16A/HSP70 double-anti-lentivirus expression vector;

2. The method of claim 1, wherein the step of culturing NK cells comprises:

sampling umbilical cord blood and detecting viruses;

3. The method of claim 2, wherein the step of culturing NK cells further comprises:

4. The method of claim 1, wherein the step of constructing the CD16A/HSP70 dual anti-lentiviral expression vector comprises:

inserting the fusion gene into a lentivirus overexpression plasmid vector pGreenPuro^TMThe XbaI/DraIII site is the CD16A/HSP70 double anti-lentiviral expression vector.

5. The method of claim 1, wherein the step of infecting the NK cells with a CD16A/HSP70 dual anti-lentiviral expression vector to obtain bispecific antibody NK cells of CD16A/HSP70 antibodies comprises:

6. A bispecific antibody NK cell produced by the method of claim 1.

7. Use of bispecific antibody NK cells prepared by the method of claim 1 in the preparation of a medicament for the treatment of liver cancer.