CN112980788A - Preparation method of NK (natural killer) cells with low expression of CD7 - Google Patents

Abstract

The NK cell low-expression CD7 obtained by the preparation method can avoid the suicide phenomenon of cells after being introduced into CAR targeting CD7, so that the NK cell low-expression CD7 cell preparation method has higher clinical application value.

Description

Preparation method of NK (natural killer) cells with low expression of CD7

Technical Field

The invention relates to the technical field of biology, and relates to a preparation method of NK cells of low-expression CD 7.

Background

NK cells (natural killer cells) are lymphocytes of CD56+ and CD3-, and are effector cells of the natural immune system in the organism, and can identify tumors and virus-infected cells without antigen pre-sensitization. The immune response regulator is positioned on the first defense line of a defense system of an organism, and can regulate the acquired immune response by secreting various cytokines and chemotactic factors at the early stage, so the immune response regulator is also a bridge connecting natural immunity and acquired immunity and plays an important role in the aspects of tumor immunity, cleaning non-self cells and the like.

In the current clinical NK cell therapy, an in-vitro culture method is adopted to amplify NK cells in peripheral blood of a patient, so that the number of the cells is expanded by thousands of times, the cytotoxic activity is greatly enhanced, and the cells are returned to the body of the patient. The possibility of industrial production of cells taken from the patient himself is limited above all; in addition, because the content of NK cells in peripheral blood is low and only accounts for 5% -10% of the total number of lymphocytes in the peripheral blood, the number of cells is limited, and an effective high-purity in-vitro amplification system is not available at present, the clinical application of the NK cells is limited to a great extent, and the traditional NK cell preparation method is not an ideal choice. Firstly, it is mainly used for one-to-one individual treatment method, which can only be carried out at the bedside of a hospital; second, the time for induction of natural killer cells is too long, at least 6 weeks, and in the case of patients with advanced tumors, especially patients with post-operative radiotherapy or chemotherapy, the cells may have been removed before preparation or return; thirdly, one-to-one treatment, namely, each person needs to perform a whole set of cell induction, amplification and culture process, so that the indoor quality control is difficult and the indoor quality control is more difficult in the aspect of laboratory management; fourth, many patients are deficient in their own NK cells and have limited therapeutic effect on reinfusion.

Although a great deal of research is focused on finding an optimal system for inducing differentiation from CD34+ cells of different sources to NK cells, no method is available for simply and efficiently obtaining high-purity, high-quantity and strong-killing-capacity NK cells.

Disclosure of Invention

One of the purposes of the invention is to provide an in vitro preparation method for inducing and culturing NK cells by umbilical cord blood aiming at the problems and the defects in the prior art, so as to solve the problems of insufficient NK cell sources, limited curative effect and difficult industrial production in the prior art, and have wide application prospect.

Another object of the present invention is to provide NK cells expressing a chimeric antigen receptor targeting CD7, prepared from NK cells that express CD7 at a low level.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a CD56+ NK cell induction medium, which comprises the following cytokines: IL-7, IL-15, SCF, FLt 3L.

As an alternative solution, the induction medium comprises the following cytokines: IL-7, IL-15, SCF, FLt3L, IL 3.

Preferably, the final concentration of IL-7 is 10ng/ml, the final concentration of IL-15 is 20ng/ml, the final concentration of SCF is 10ng/ml, the final concentration of FLt3L is 10ng/ml, and the final concentration of IL-3 is 5 ng/ml.

Preferably, the induction medium further comprises X-VIVO, human AB serum; preferably, the proportion of human AB serum is 5%.

Preferably, the induction medium further comprises X-VIVO, autologous plasma; preferably, the autologous plasma proportion is 5%.

The invention also provides a method for preparing CD56+ NK cells, which comprises the following steps:

1) isolating mononuclear cells from cord blood;

2) sorting to obtain CD34+ cells;

3) CD34+ cells were cultured in the induction medium described previously to obtain CD56+ NK cells.

Further, CD34+ cells were cultured in the induction medium described previouslyThe steps for obtaining CD56+ NK cells are as follows: after sorting, the cells were resuspended in the induction medium described above and plated in 24-well plates to adjust the cell density to 0.8-1.2 x 10⁵Per well.

Methods for isolating mononuclear cells from blood samples are well known in the art, such as Ficoll and Percoll layering.

Further, the step of isolating mononuclear cells from cord blood comprises: diluting the cord blood by using normal saline, adding lymphocyte separation liquid, and centrifugally collecting white membrane cells; diluting the leucocyte layer cells with normal saline, centrifuging and removing supernatant; adding erythrocyte lysate to lyse the cells, centrifuging and removing supernatant; resuspend the cell pellet.

Preferably, the ratio of 1: the cord blood was diluted at a ratio of 1.

Preferably, the ratio of 1: the buffy coat cells were diluted at a ratio of 1.

In a specific embodiment of the present invention, the step of isolating mononuclear cells from cord blood comprises: using physiological saline solution to mix at a ratio of 1: diluting the cord blood by a ratio of 1, and adding a lymphocyte separation solution with the volume of 1/2 of the diluted cord blood; 2000r/min, centrifuging for 20 min; the leucocyte layer cells were collected and then added with physiological saline at a rate of 1: 1, diluting according to a proportion; centrifuging the cells at 2000r/min for 5min, and discarding the supernatant; lysing the cells with 5ml of red blood cell lysate for 5min, then centrifuging the cells at 2000r/min for 5min, and discarding the supernatant; the cell counts were resuspended with 5-10ml of DPBS and the resulting cell numbers were calculated.

Further, the erythrocyte lysate is composed of NH₄Cl、KHCO₃EDTA and ultrapure water are prepared according to the conventional proportion in the field.

Further, the step of sorting the CD34+ cells comprises: resuspending the mononuclear cells by using a sorting buffer solution, sequentially adding an FCR (fiber channel receptor) blocking agent and CD34 magnetic beads, uniformly mixing, and incubating at a low temperature; adding a sorting buffer solution into the incubated cells, centrifuging and removing a supernatant; adding the suspension into a separation column after the suspension is resuspended by a separation buffer solution; and adding a sorting buffer solution into the sorting column, washing, adding the sorting buffer solution into the sorting column, pushing the plunger to the bottom end of the sorting column, washing the marked cells, and counting.

Preferably, low temperature incubation refers to incubation in a refrigerator at 4 ℃ for 30 min.

Preferably, the sorting buffer refers to 0.5% HSA.

In a particular embodiment of the invention, the step of sorting the CD34+ cells comprises: centrifuging the cells at 300g/min for 10min, discarding the supernatant, resuspending the cells with 300. mu.l of 0.5% HSA, then sequentially adding 100. mu.l of FCR blocker and CD34 magnetic beads, mixing the cells once for each addition, then placing a 50ml centrifuge tube into a 4 ℃ refrigerator for incubation for 30min, and mixing the cells once every 5 min; adding 5-10ml of 0.5% HSA into the incubated cells, centrifuging the cells at 300g/min for 10min, and discarding the supernatant; resuspend the cells with 500 μ l of 0.5% HSA, then add the cells to a sorting column that needs to be rinsed once with 3ml of 0.5% HSA in advance; 3ml of 0.5% HSA is added into the sorting column to wash the sorting column for 3 times; the column was then removed from the magnetic field, 5ml of 0.5% HSA was added to the column, the plunger was pushed hard to the bottom of the column, and the labeled cells were washed down and counted.

The invention also provides an NK cell activation medium, which comprises: IL-2, IL-15, IL-21.

As an alternative solution, the activation medium comprises OK432, IFN-gamma, IL-2, IL-15, IL-21.

Preferably, OK432 final concentration is 100ng/ml, IFN-gamma final concentration is 1000IU/ml, IL-2 final concentration is 1000IU/ml, IL-15 final concentration is 100ng/ml, and IL-21 final concentration is 100 ng/ml.

Preferably, the activation medium further comprises KBM581, human AB serum.

More preferably, the human AB serum fraction is 5%.

Preferably, the activation medium further comprises KBM581, autologous plasma; preferably, the autologous plasma proportion is 5%.

The invention provides a method for promoting conversion of CD34+ cells to CD56+ NK cells or preparing CD56+ NK cells, which comprises the following steps

1) CD56+ NK cells were obtained using the method described previously;

2) CD56+ NK cell activation.

Further, CD56+ NK cell activation includes culturing CD56+ cells obtained using the methods described previously in the activation medium described previously.

Further, CD56+ NK cell activation includes culturing cells obtained after 10-14 days of culture using the method described above in the activation medium described above; preferably, CD56+ NK cell activation includes using the method of 10-14 days after the use of the method of every 2-3 days half-change mode of the obtained cells in the activation medium; preferably, the culture is carried out in an activation medium for one week.

The invention also provides an NK cell amplification culture medium, which comprises the following cytokines: IL-2, IL-21.

Preferably, the final concentration of IL-2 is 1000 IU/ml.

Further, the amplification medium also comprises KBM581 and human AB serum.

Preferably, the human AB serum proportion is 5%.

Further, the amplification medium further comprises KBM581 and autologous plasma; preferably, the autologous plasma proportion is 5%.

The autologous plasma of the invention is preferably autologous plasma that has been inactivated.

The inactivated autologous plasma was prepared as follows: the blood was put into a sterile 50ml centrifuge tube, lifted 9 and lowered 7, and centrifuged at 750g for 15 min. Collecting supernatant plasma in 50ml centrifuge tube, incubating at 56 deg.C for 30min, standing at 4 deg.C for 30min, centrifuging at 1000g for 10min, collecting supernatant, and storing at 4 deg.C for use.

The present invention also provides a method for preparing NK cells, the method comprising:

1) obtaining cells using the method described above;

2) CD56+ NK cell activation;

3) the cells obtained in step 2) are cultured in the amplification medium described above.

Step 1) and step 2) are as defined above.

Preferably, step 3) comprises replacing the culture medium with an amplification medium for further culturing of the previously obtained cells in half-liquid changes every 2 to 3 days.

The present invention also provides NK cells prepared by the method as described above.

Preferably, the NK cells underexpress CD 7.

Preferably, the purity of the NK cells prepared by the method reaches more than 80%.

The present invention also provides a genetically modified NK cell prepared by the method as described above (low expression CD 7).

Further, the genetically modified NK cell comprises insertions/deletions of one or more of the following genes: targeting patterns, receptors, signaling molecules, transcription factors, drug target candidates, immune response modulation and regulation, or proteins that inhibit transplantation, trafficking, homing, viability, self-renewal, persistence and/or survival of the NK cells or derived cells thereof.

Further, the genetically modified NK cells include NK cells expressing a chimeric antigen receptor.

The antigen targeted by the chimeric antigen receptor referred to in the present invention includes a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is unique to tumor cells and does not occur on other cells of the body. TAA-associated antigens are not unique to tumor cells and, instead, are also expressed on normal cells in conditions where immune tolerance states to the antigen cannot be induced. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during embryonic development when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells, but which are expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-l/Melana (MART-1), gp100(Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multispectral antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p 15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and Human Papilloma Virus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, P185erbB2, P180erbB-3, C-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, Nu, K-ras, β -catenin, CDK4, Mum-1, P15, P16, 43-9F, 5T4, 791Tgp72, α -fetoprotein, β -HCG, BCA225, BTA, CA125, CA15-3\ CA27.29\ EpBCAA, CA195, CA242, CA-50, CAM43, CD68\ P1, CO-029, FGF-5, G250, Ga733\ CAM 175-175, MG-M, MOV-50, MOV-18, MOV-3970, MOV-3, MCA-3, MOV-3, MCA-3, MCA-6, MCA-11, MCB-6, TAAL6, TAG72, TLP and TPS.

In preferred embodiments, the targeting antigen includes, but is not limited to, CD7, CD19, CD20, CD22, RORl, mesothelin, CD33/IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1TCR, MAGE A3TCR, and the like.

In a specific embodiment of the invention, the chimeric antigen receptor targets CD 7.

Preferably, the amino acid sequence of the chimeric antigen receptor targeting CD7 is shown in SEQ ID NO. 1; preferably, the nucleic acid sequence of the chimeric antigen receptor targeting CD7 is shown in SEQ ID NO. 2.

The present invention also provides a method for preparing the genetically modified NK cell, the method comprising:

1) preparing the aforementioned NK cells with low expression of CD 7;

2) genetically modifying the NK cells obtained in step 1).

Further, the genetic modification comprises introducing a nucleic acid sequence encoding a chimeric antigen receptor into the NK cell obtained via step 1).

The present invention also provides a kit or a medicament comprising the NK cell prepared as described above or the genetically modified NK cell as described above.

Furthermore, the medicine also comprises a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers can be any of those conventionally used and are limited only by chemical-physical considerations (e.g., loss of solubility and reactivity with the active agent) and the manner of administration. The pharmaceutically acceptable carriers, e.g., carriers, adjuvants, excipients and diluents, described herein are well known to those skilled in the art and readily available to the public. Preferably, the pharmaceutically acceptable carrier is one that is chemically inert to the active agent and one that is not deleterious to side effects or toxicity under the conditions of use.

The present invention also provides a method for treating cancer or an autoimmune disease in a subject, the method comprising administering to a subject in need thereof an effective amount of the aforementioned NK cell, the aforementioned genetically modified NK cell, the aforementioned medicament.

The invention also provides an application of the NK cell prepared in the previous step, wherein the application comprises any one of the following steps:

1) the application in preparing the medicine for treating cancer or autoimmune diseases;

2) the application of the chimeric antigen receptor in preparing immune cells expressing the chimeric antigen receptor;

3) use in the preparation of a kit as hereinbefore described.

The present invention also provides the use of a genetically modified NK cell as described above, said use comprising any one of:

1) the application in preparing the medicine for treating cancer or autoimmune diseases;

2) use in the preparation of a kit as hereinbefore described.

Cancer is broadly defined herein to include any neoplastic condition, whether malignant, premalignant or non-malignant. However, in general, it may be a malignant condition. Both solid tumors and non-solid tumors are included.

The cancers of the present invention encompass any type of cancer, including solid cancers and hematopoietic cancers. Representative cancers include Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), adrenocortical carcinoma, aids-related cancers (e.g., Kaposi's Sarcoma (Kaposi Sarcoma) and Lymphoma), anal cancer, appendiceal cancer, astrocytoma, atypical teratoid/rhabdoid tumors, basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bone cancer (e.g., Ewing Sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brain stem glioma, brain cancer, breast cancer, bronchial tumor, Burkitt Lymphoma (Burkitt Lymphoma), carcinoid tumors, Cardiac tumors (cardioac/Heart tumor), central nervous system cancers (including atypical teratoid/rhabdoid tumors, embryonic tumors, germ cell tumors, lymphomas), cervical cancer, chordoma, Chronic Lymphocytic Leukemia (CLL), and Lymphoma, Chronic Myelogenous Leukemia (CML), chronic myeloproliferative disorders, colon Cancer, colorectal Cancer, craniopharyngioma, cutaneous T-Cell Lymphoma, cholangiocarcinoma, extrahepatic Ductal Carcinoma In Situ (DCIS), embryonic tumors, endometrial Cancer, ependymoma, esophageal Cancer, nasal glioma, ewing's sarcoma, extracranial germ Cell tumors, extragonally germ Cell tumors, extrahepatic cholangiocarcinoma, ocular Cancer (including intraocular melanoma and retinoblastoma), osteochondral histiocytoma, gallbladder Cancer, Gastric Cancer (Gastric/Stomach Cancer), gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ Cell tumors, gestational trophoblastic disease, glioma, hairy Cell leukemia, head and neck Cancer, cardiac Cancer, hepatocellular (liver) Cancer, histiocytic polycythemia, Langerhans cells (Langerhans cells), Hodgkin Lymphoma (Hodgkin Lymphoma), Hypopharyngeal carcinoma, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, kaposi's sarcoma, kidney cancer (including kidney and Wilms tumor), langerhans ' cell histiocytosis, larynx cancer, leukemia (including Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML)), lip and oral cancer, liver cancer (primary), Lobular Carcinoma In Situ (LCIS), lung cancer, lymphoma, waldenstrom's Macroglobulinemia (macrolobuemlina), melanoma, Merkel cell carcinoma (Merkel cellcaricinoma), mesothelioma, occult primary metastatic squamous neck cancer, midline cancer involving the NUT gene, oral cancer, childhood multiple endocrine neoplasia syndrome, multiple myeloma/plasmacytoid neoplasia, Mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasms, multiple myeloma, myeloproliferative disorders, nasal and paranasal sinus cancers, nasopharyngeal cancers, neuroblastoma, non-hodgkin's lymphoma, non-small cell lung Cancer, Oral Cancer (Oral Cancer/OralCavity Cancer), oropharyngeal Cancer, osteosarcoma, ovarian Cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paragangliomas, paranasal sinuses and nasal cavity cancers, parathyroid Cancer, penile Cancer, pharyngeal Cancer, pheochromocytoma, pituitary tumor, plasmacytoma/multiple myeloma, pleuroperitoblastoma, pregnancy and breast Cancer, primary Central Nervous System (CNS) lymphoma, prostate Cancer, rectal Cancer, renal cell (kidney) Cancer, renal pelvis and ureteral transitional cell Cancer, retinoblastoma, Rhabdomyosarcoma, salivary gland carcinoma, sarcoma, sezary Syndrome, skin Cancer, small cell lung Cancer, small intestine Cancer, soft tissue sarcoma, squamous cell carcinoma, occult primary metastatic squamous neck Cancer, Gastric Cancer (Stomach/Gastric Cancer), T-cell lymphoma, testicular Cancer, laryngeal Cancer, thymoma and carcinoma of the thymus, thyroid Cancer, transitional cell carcinoma of the renal pelvis and ureter, Cancer of the urethra, uterine Cancer, endometrium, uterine sarcoma, vaginal Cancer, vulval Cancer, waldenstrom 'S macroglobulinemia, and wilms' tumor.

The subject to be treated using the methods and cells of the invention can be any mammalian species. For example, the subject may be any of the following species: a domestic pet such as a mouse, rat, gerbil, rabbit, guinea pig, hamster, cat, or dog; or livestock such as goats, sheep, pigs, cattle or horses. In another preferred embodiment of the invention, the subject may be a primate, such as a monkey, gibbon, gorilla, orang-utang, chimpanzee or bonobo. In a preferred embodiment of the invention, the subject is a human.

The term "autoimmune disease" as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves ' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxoedema, pernicious anemia, ulcerative colitis, and the like.

The term "treating" herein refers to the delivery of an effective amount of the agent for the purpose of preventing the development of any symptom or disease condition or for the purpose of preventing or delaying progression, alleviating, ameliorating or eliminating such symptom or disease condition already developed. Thus, the term "treating" is intended to include treating an individual who (i) has a partial or complete response after the first treatment, (ii) is in remission, (iii) has a detectable disease, or (iv) has a precursor of a malignant disease, to a minimal or undetectable extent.

The invention has the advantages and beneficial effects that:

the invention utilizes PBMC of umbilical cord blood to obtain CD34+ cells through CD34 sorting, forms CD56+ NK cells through induced differentiation, and the NK cells are low in CD7 expression, and has the advantages that CD7 related CAR transduction is carried out on the NK cells, and the cell suicide phenomenon can be avoided when the cells are prepared into CD 7-CAR-NK.

Drawings

FIG. 1 shows a graph of the results of CD56+ NK cell expansion;

FIG. 2 is a graph showing the results of the change in the proportion of CD56+ NK cells;

FIG. 3 shows the results of the percentage of CD56+ NK cells detected by flow cytometry;

fig. 4 shows a graph of NK cell identification results, in which a: CD 7; b: CD 16; c: CD 33;

figure 5 shows a graph of the results of the effect on CD7 expression following NK cell transfection with a CD 7-targeted CAR;

FIG. 6 is a graph showing the effect of IL-3 on CD34+ cells induced to produce CD56+ cells;

FIG. 7 is a graph showing the results of the effect of activation medium containing IL-2, IL-21 and IL-15 on the ratio of CD56+ NK cells.

Detailed Description

The invention is described in detail below with reference to the drawings and examples so that those skilled in the art can understand and implement the invention and further appreciate the advantages of the invention.

Unless defined otherwise in the present specification, all technical terms used herein are used in accordance with their customary definitions commonly used and understood by those of ordinary skill in the art. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1NK cell preparation

One, step

1. Sorting of CD34+ cells

Sorting of CD34+ cells was performed according to the instructions of the menini CD34+ sorting kit. Transferring the cord blood in the blood bag into a 50ml clean centrifugal tube to measure the volume of the cord blood; several additional clean 50ml centrifuge tubes were then removed, and 25ml of saline was added to each tube at a rate of 1: diluting the cord blood according to the proportion of 1, adding the cord blood into a centrifuge tube filled with normal saline by a pipette, and fully and uniformly mixing.

Taking several clean 50ml centrifuge tubes, adding 15ml of lymphocyte separation medium (Oriental Huahui, Cat: 25910) to each centrifuge tube, and adding 30ml of diluted umbilical cord blood to each centrifuge tube; centrifuge the tube at 2000r/min for 20 min.

The leucocyte layer cells were collected and then added with physiological saline at a rate of 1: 1, diluting according to a proportion; cells were centrifuged at 2000r/min for 5min and the supernatant discarded.

The cells were lysed with 5ml of erythrocyte lysate for 5min, and then centrifuged at 2000r/min for 5min and the supernatant discarded.

The cell counts were resuspended with 5-10ml of DPBS and the resulting cell numbers were calculated.

The cells were centrifuged at 300g/min for 10min, the supernatant discarded, and 300. mu.l of 0.5% HSA (1 x 10)⁸Individual cells) were resuspended, then 100 μ l of FCR blocker and CD34 magnetic beads were added in sequence, and the cells were mixed once for each addition, then 50ml centrifuge tubes were placed in a 4 ℃ freezer and incubated for 30min, and the cells were mixed every 5 min.

Adding 5-10ml of 0.5% HSA into the incubated cells, centrifuging the cells at 300g/min for 10min, and discarding the supernatant; the cells were resuspended with 500. mu.l of 0.5% HSA and then loaded onto a sorting column that required a single rinse with 3ml of 0.5% HSA.

3ml of 0.5% HSA is added into the sorting column to wash the sorting column for 3 times; the column was then removed from the magnetic field, 5ml of 0.5% HSA was added to the column, the plunger was pushed hard to the bottom of the column, and the labeled cells were washed down and counted.

After sorting, the cells were resuspended in X-VIVO medium and plated in 24-well plates to adjust the cell density to 0.8-1.2X 10⁵Per well;

X-VIVO medium: X-VIVO + IL-7(10ng/ml) + IL-15(20ng/ml) + SCF (10ng/ml) + FLt3L (10ng/ml) + 5% human AB serum (GEMINI, GemCell)^TM human serum AB，cat:100-512)；

0.5％HSA：1.25ml HSA+48.75ml DPBS。

2. Induced differentiation of NK cells

After the cells are continuously cultured for 10-14 days, the culture medium is changed to 581 activation culture medium for continuous culture for 1 week, the culture medium is changed to 581 amplification culture medium for continuous culture of the cells, and the liquid change is performed by adopting a half liquid change mode every 2-3 days.

581 activation of medium: KBM581+ OK432(100ng/ml) + IFN-gamma (1000IU/ml) + IL-2(1000IU/ml) + IL-15(100ng/ml) + IL-21(100ng/ml) + 5% human AB serum;

581 amplification medium: KBM581+ IL-2(1000IU/ml) + IL-15(100ng/ml) + IL-21(100ng/ml) + 5% human AB serum.

3. NK cell identification

NK cell surface-associated marker expression was detected by using CD56 FITC antibody, CD7 PE antibody, CD33 PE-cy7 antibody and CD16 APC-cy7 antibody.

Second, result in

1) CD56+ NK cell expansion

With the increase of cell culture days, the number of CD56+ NK cells is gradually increased, the increased cell number is converted into the expansion multiple of the cells, and finally the CD56+ NK cells can be expanded by about 25 times, and the result is shown in figure 1.

2) CD56+ NK ratio Change

With the increase of the number of days of cell culture, the proportion of CD56+ NK cells was examined by flow cytometry and plotted as a line graph, and the increase of the proportion of CD56+ NK cells was relatively slow on the first 7 days of cell culture, and then the proportion of CD56+ NK cells gradually increased, and 89% was reached on the 28 th day of culture, with the results shown in fig. 2.

3) Percentage statistics of CD56+ NK cells

With the increase of the cell culture days, the proportion of CD56+ NK cells is detected by a flow cytometer, and the flow detection results of the cell culture days of D7, D14, D21 and D28 are shown, and the proportion of CD56+ NK cells is respectively: 1.75%, 21.17%, 61.4%, 90.17%, the results are shown in FIG. 3.

4) NK cell identification

The expression of NK cell surface-associated markers, such as CD7, CD16 and CD33, was detected by flow cytometry, and the percentage of CD7 in NK cells was 16% at a low expression level, and the results are shown in FIG. 4.

Example 2CAR transduction

One, step

1. Construction of Lentiviral expression vectors expressing chimeric antigen receptor targeting CD7

1) The corresponding coding nucleic acid sequence (shown as SEQ ID NO. 2) was synthesized based on the amino acid sequence (shown as SEQ ID NO. 1) of a chimeric antigen receptor (CD7CAR) targeting CD 7. The coding nucleic acid sequence of the CD7CAR is inserted into a lentivirus expression vector in an enzyme digestion connection mode, and the linear sequence of the constructed recombinant lentivirus expression vector is shown as SEQ ID No. 3. Enzyme cutting site: pac I and SpeI. Transformation, plating, sequencing by miniprep, and confirming the success of the construction of the CD7CAR expression plasmid. The plasmid is extracted to obtain an endotoxin-free expression plasmid for packaging lentivirus.

2. Lentiviral packaging

(1)293FT cell inoculation (Day1 am)

And opening the biological safety cabinet for 30min by ultraviolet according to the use instruction of the biological safety cabinet.

DMEM containing 10% fetal calf serum, PBS and Trypsin without EDTA-0.25% are placed at 37 ℃ in 5% CO₂The cell culture box is preheated for 30 min.

293FT cell culture flasks (T175) grown to 80% -90% were grown from 37 ℃ with 5% CO₂The cell culture flask was taken out of the flask, sprayed with 75% ethanol onto the surface of the flask, and then the flask was placed in a safety cabinet, the medium therein was pumped down with a sterile vacuum pump, and 6mL of pre-warmed PBS was added with a 10mL pipette and washed once, and the PBS was aspirated.

To a T175 cell culture flask was added 2mL TrypLE^TMEXPRESS, which was allowed to spread well on the cell surface, the cap was closed, and it was observed under a microscope until the cells became round, 8mL of DMEM medium containing 10% fetal bovine serum was added to stop the digestion, the cells on the wall of the flask were blown down with a pipette, and the cell fluid was transferred to a15 mL sterile centrifuge tube and centrifuged at 1500rpm for 5 min.

The supernatant was discarded, 1mL of DMEM medium was added, the cells were resuspended and dispersed by gentle aspiration with a pipette, a certain amount of cells were taken, diluted n times, and counted with a hemocytometer.

Part of the cells were treated at 1.3X 10⁷Inoculating every 150mm plate, and adding 20mL of DMEM medium into every 150mm plate for virus packaging; the remaining cells were counted at 0.75X 10⁷Each T175 flask was plated with 25mL of DMEM medium for passaging. Shaking the above cells gently, adding 5% CO at 37 deg.C₂Culturing in an incubator.

(2) Virus packing (Day2 morning)

When the cells are well packed on a 150mm plate paved with Day1 under a microscope, the density reaches 70-80%, and then the virus can be packed.

The biological safety cabinet is opened for 30min by ultraviolet according to the use instruction of the biological safety cabinet.

Will be provided with

buffer、

The CD7CAR expression plasmid and the two packaging plasmids (psPAX2 and pmd2.g) were returned to room temperature.

1.25mL per 150mm dish

buffer, 15. mu.g of CD7CAR expression plasmid, 7.5. mu.g of psPAX2, and 3.75. mu.g of pMD2. G. Adding into the mixed solution

62.5. mu.L/150 mm dish, mixed well again, and left to stand at room temperature for 10 min.

293FT cells for packaging virus 5% CO from 37 ℃₂Taking out the cell culture box, spraying 75% alcohol on the surface of a cell culture dish, placing the cell culture dish into a safety cabinet, evenly distributing the mixed solution into a culture medium of a 150mm flat dish, gently shaking, and placing the culture medium into a container with 37 ℃ and 5% CO₂An incubator.

Packaging for 4h, discarding old culture medium, adding 5mL preheated PBS to wash cells, adding 20mL fresh preheated DMEM culture medium containing 10% fetal calf serum, and adding 5% CO at 37 deg.C₂An incubator.

(3) Virus collection and concentration (Day4)

And replacing the solution for 48-72 h to collect virus stock solution. The stock solution was collected in 50ml centrifuge tubes and centrifuged at 2000rpm for 5 min.

The supernatant was left 1mL for virus-related detection. The remainder was filtered through a 0.45 μm filter into a new centrifuge tube and centrifuged at 18500g at 4 ℃ for 2 h.

The supernatant was discarded as clean as possible and serum-free medium was added to resuspend the virus particles. Volume of medium added: the volume of the virus stock solution was 1: 500. This is the virus concentrate.

The virus concentrate was dispensed at 120. mu.l/tube, and 10. mu.l of the virus concentrate was retained for transfection efficiency measurement. Storing the concentrated solution in a refrigerator at-80 deg.C.

3. Lentiviral transfection and detection

Cells will be transfected with lentiviral senl-7-re packaged using adherent 293FT cells. Cell counts were re-seeded into 24-well plates, 5 x 10 per well⁵The cells are centrifuged for 2h at 35 ℃ at 2000r/min by using PGE2 as transfection assisting agent, and after centrifugation, the cells are placed in CO₂The culture was continued in the incubator, and 500. mu.l of medium was replenished per well the next day, and CAR expression was detected by flow cytometry the fourth day after transfection.

Second, result in

NK cells could significantly reduce CD7 expression after transfection with CD7CAR, with the results shown in figure 5.

Example 3 Effect of IL-3 cytokines on CD56+ NK cell production

1. Step (ii) of

Changing the culture medium of X-VIVO: X-VIVO + IL-7(10ng/ml) + IL-15(20ng/ml) + SCF (10ng/ml) + FLt3L (10ng/ml) + IL-3(5ng/ml) + 5% human AB serum. NK cells were prepared under the same conditions as in example 1.

2. Results

Addition of cytokines IL7, IL15, SCF, FLt3L, IL3 to X-VIVO medium also promoted the conversion of CD34+ cells to CD56+ NK cells, but at a slower rate than addition of cytokines IL7, IL15, SCF, FLt3L to X-VIVO medium, as shown in fig. 6.

Example 4 Effect of OK432 and IFN-. gamma.on CD56+ NK cell production

1. Step (ii) of

Change 581 activation medium: KBM581+ IL-2(1000IU/ml) + IL-15(100ng/ml) + IL-21(100ng/ml) + 5% human AB serum.

2. Results

Replacement of the 581-activated cytokines in the medium with IL-2, IL-21, IL-15 also promoted the conversion of CD34+ cells to CD56+ NK cells, but at a slower rate than the addition of cytokines OK432, IFN-. gamma., IL-2, IL-21, IL-15, as shown in FIG. 7.

Sequence listing

<110> Hebei Senlang Biotech Co., Ltd

<120> preparation method of NK cell with low expression of CD7

<141> 2021-03-08

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2585

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttaattaagc caccatgctg ctgctggtga ccagcctgct gctgtgcgag ctgccccacc 60

ccgcctttct gctgatcccc ggggcccagc cggccatggc ggcctacaaa gatatccaga 120

tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc atcagttgca 180

gtgcaagtca gggcattagc aattatttaa actggtatca gcagaaacca gatggaactg 240

ttaaactcct gatctattac acatcaagtt tacactcagg agtcccatca aggttcagtg 300

gcagtgggtc tgggacagat tattctctca ccatcagcaa cctggaacct gaagatattg 360

ccacttatta ttgtcagcag tatagcaagc ttccgtacac gttcggaggg gggaccaagc 420

tggaaataaa acgtggtggt ggtggttctg gtggtggtgg ttctggcggc ggcggctccg 480

gtggtggtgg atccgaggtg caactggtgg agtctggggg aggcttagtg aagcctgggg 540

ggtccctgaa actctcctgt gcagcctctg gactcacttt cagtagctat gccatgtctt 600

gggttcgcca gactccagag aagaggctgg agtgggtcgc atccattagt agtggtggtt 660

tcacctacta tccagacagt gtgaagggcc gattcaccat ctccagagat aatgccagga 720

acatcctgta tctgcaaatg agcagtctga ggtctgagga cacggccatg tattactgtg 780

caagagacga ggtacggggg tacctcgatg tctggggcgc agggaccacg gtcaccgttt 840

cctcggcctc gggggccgaa tctaagtacg gaccgccctg ccccccttgc cctatgttct 900

gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc accgtggcct 960

tcatcatctt ttgggtgaaa cggggcagaa agaaactcct gtatatattc aaacaaccat 1020

ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga tttccagaag 1080

aagaagaagg aggatgtgaa ctgcgggtga agttcagcag aagcgccgac gcccctgcct 1140

accagcaggg ccagaatcag ctgtacaacg agctgaacct gggcagaagg gaagagtacg 1200

acgtcctgga taagcggaga ggccgggacc ctgagatggg cggcaagcct cggcggaaga 1260

acccccagga aggcctgtat aacgaactgc agaaagacaa gatggccgag gcctacagcg 1320

agatcggcat gaagggcgag cggaggcggg gcaagggcca cgacggcctg tatcagggcc 1380

tgtccaccgc caccaaggat acctacgacg ccctgcacat gcaggccctg cccccaaggc 1440

tcgagggcgg cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc 1500

ccggccctag gatgcttctc ctggtgacaa gccttctgct ctgtgagtta ccacacccag 1560

cattcctcct gatcccacgc aaagtgtgta acggaatagg tattggtgaa tttaaagact 1620

cactctccat aaatgctacg aatattaaac acttcaaaaa ctgcacctcc atcagtggcg 1680

atctccacat cctgccggtg gcatttaggg gtgactcctt cacacatact cctcctctgg 1740

atccacagga actggatatt ctgaaaaccg taaaggaaat cacagggttt ttgctgattc 1800

aggcttggcc tgaaaacagg acggacctcc atgcctttga gaacctagaa atcatacgcg 1860

gcaggaccaa gcaacatggt cagttttctc ttgcagtcgt cagcctgaac ataacatcct 1920

tgggattacg ctccctcaag gagataagtg atggagatgt gataatttca ggaaacaaaa 1980

atttgtgcta tgcaaataca ataaactgga aaaaactgtt tgggacctcc ggtcagaaaa 2040

ccaaaattat aagcaacaga ggtgaaaaca gctgcaaggc cacaggccag gtctgccatg 2100

ccttgtgctc ccccgagggc tgctggggcc cggagcccag ggactgcgtc tcttgccgga 2160

atgtcagccg aggcagggaa tgcgtggaca agtgcaacct tctggagggt gagccaaggg 2220

agtttgtgga gaactctgag tgcatacagt gccacccaga gtgcctgcct caggccatga 2280

acatcacctg cacaggacgg ggaccagaca actgtatcca gtgtgcccac tacattgacg 2340

gcccccactg cgtcaagacc tgcccggcag gagtcatggg agaaaacaac accctggtct 2400

ggaagtacgc agacgccggc catgtgtgcc acctgtgcca tccaaactgc acctacggat 2460

gcactgggcc aggtcttgaa ggctgtccaa cgaatgggcc taagatcccg tccatcgcca 2520

ctgggatggt gggggccctc ctcttgctgc tggtggtggc cctggggatc ggcctcttca 2580

tgtga 2585

<210> 2

<211> 856

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gly Ala Gln Pro Ala Met Ala Ala Tyr Lys

20 25 30

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

35 40 45

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

50 55 60

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

65 70 75 80

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

85 90 95

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro

100 105 110

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr

115 120 125

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

165 170 175

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr

180 185 190

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

195 200 205

Ala Ser Ile Ser Ser Gly Gly Phe Thr Tyr Tyr Pro Asp Ser Val Lys

210 215 220

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu

225 230 235 240

Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

245 250 255

Arg Asp Glu Val Arg Gly Tyr Leu Asp Val Trp Gly Ala Gly Thr Thr

260 265 270

Val Thr Val Ser Ser Ala Ser Gly Ala Glu Ser Lys Tyr Gly Pro Pro

275 280 285

Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val Val Gly Gly Val

290 295 300

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

305 310 315 320

Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

325 330 335

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

340 345 350

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

355 360 365

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

370 375 380

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

385 390 395 400

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

405 410 415

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

420 425 430

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

435 440 445

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

450 455 460

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu Glu Gly Gly Gly

465 470 475 480

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

485 490 495

Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu

500 505 510

Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile

515 520 525

Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile

530 535 540

Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu

545 550 555 560

Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp

565 570 575

Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe

580 585 590

Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe

595 600 605

Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe

610 615 620

Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser

625 630 635 640

Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn

645 650 655

Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser

660 665 670

Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys

675 680 685

Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp

690 695 700

Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly

705 710 715 720

Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu

725 730 735

Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro

740 745 750

Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile

755 760 765

Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro

770 775 780

Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp

785 790 795 800

Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys

805 810 815

Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro

820 825 830

Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val

835 840 845

Ala Leu Gly Ile Gly Leu Phe Met

850 855

<210> 3

<211> 9947

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

actagtgtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 60

aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 120

attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 180

tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 240

gcaaccccca ctggttgggg cattgccacc aactgtcagc tcctttccgg gactttcgct 300

ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 360

ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt 420

ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 480

ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 540

cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 600

cctggaattc gagctcggta cctttaagac caatgactta caaggcagct gtagatctta 660

gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag 720

atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct 780

ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag 840

tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt 900

cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac 960

ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt 1020

acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 1080

gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc 1140

cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg 1200

ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca 1260

gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag 1320

tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc 1380

gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa 1440

gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg 1500

ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 1560

cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 1620

gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 1680

ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg 1740

ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 1800

ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg 1860

attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 1920

aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg 1980

gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 2040

aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 2100

gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 2160

cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 2220

tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 2280

tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 2340

agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 2400

cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 2460

tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 2520

ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 2580

tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 2640

cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 2700

actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 2760

ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 2820

tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 2880

ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 2940

aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 3000

ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 3060

agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 3120

ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 3180

tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 3240

cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 3300

ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 3360

gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 3420

ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 3480

aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 3540

cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 3600

ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 3660

gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 3720

ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 3780

ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 3840

gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 3900

cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 3960

ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 4020

aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 4080

ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa 4140

gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg 4200

caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg 4260

ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct 4320

gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct 4380

agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg 4440

ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt 4500

gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt 4560

gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag 4620

gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg 4680

cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc 4740

gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg 4800

ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa 4860

cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga 4920

cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta 4980

gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac 5040

aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt 5100

cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt 5160

agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag 5220

agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag 5280

cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat 5340

agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact 5400

cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa 5460

ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt 5520

gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg 5580

gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga 5640

atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag 5700

tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat 5760

agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt 5820

taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga 5880

caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt 5940

agtgaacgga tctcgacggt atcggttaac ttttaaaaga aaagggggga ttggggggta 6000

cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca 6060

aaaacaaatt acaaaaattc aaaattttat cgatgtcgac gataagcttt gcaaagatgg 6120

ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga 6180

gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag 6240

aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac 6300

tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat 6360

ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag 6420

gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg 6480

ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcggggttgg 6540

aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt 6600

tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg 6660

tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct 6720

ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt 6780

ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg 6840

gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc 6900

tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg 6960

gtcggcacca gtagcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc 7020

aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag 7080

ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag 7140

gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt 7200

ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca 7260

cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa 7320

gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgat taattaagcc 7380

accatgctgc tgctggtgac cagcctgctg ctgtgcgagc tgccccaccc cgcctttctg 7440

ctgatccccg gggcccagcc ggccatggcg gcctacaaag atatccagat gacacagact 7500

acatcctccc tgtctgcctc tctgggagac agagtcacca tcagttgcag tgcaagtcag 7560

ggcattagca attatttaaa ctggtatcag cagaaaccag atggaactgt taaactcctg 7620

atctattaca catcaagttt acactcagga gtcccatcaa ggttcagtgg cagtgggtct 7680

gggacagatt attctctcac catcagcaac ctggaacctg aagatattgc cacttattat 7740

tgtcagcagt atagcaagct tccgtacacg ttcggagggg ggaccaagct ggaaataaaa 7800

cgtggtggtg gtggttctgg tggtggtggt tctggcggcg gcggctccgg tggtggtgga 7860

tccgaggtgc aactggtgga gtctggggga ggcttagtga agcctggggg gtccctgaaa 7920

ctctcctgtg cagcctctgg actcactttc agtagctatg ccatgtcttg ggttcgccag 7980

actccagaga agaggctgga gtgggtcgca tccattagta gtggtggttt cacctactat 8040

ccagacagtg tgaagggccg attcaccatc tccagagata atgccaggaa catcctgtat 8100

ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc aagagacgag 8160

gtacgggggt acctcgatgt ctggggcgca gggaccacgg tcaccgtttc ctcggcctcg 8220

ggggccgaat ctaagtaccc tgcccccctt gccctatgtt ctgggtgctg gtggtggtcg 8280

gaggcgtgct ggcctgctac agcctgctgg tcaccgtggc cttcatcatc ttttgggtga 8340

aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga ccagtacaaa 8400

ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa ggaggatgtg 8460

aactgcgggt gaagttcagc agaagcgccg acgcccctgc ctaccagcag ggccagaatc 8520

agctgtacaa cgagctgaac ctgggcagaa gggaagagta cgacgtcctg gataagcgga 8580

gaggccggga ccctgagatg ggcggcaagc ctcggcggaa gaacccccag gaaggcctgt 8640

ataacgaact gcagaaagac aagatggccg aggcctacag cgagatcggc atgaagggcg 8700

agcggaggcg gggcaagggc cacgacggcc tgtatcaggg cctgtccacc gccaccaagg 8760

atacctacga cgccctgcac atgcaggccc tgcccccaag gctcgagggc ggcggagagg 8820

gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct aggatgcttc 8880

tcctggtgac aagccttctg ctctgtgagt taccacaccc agcattcctc ctgatcccac 8940

gcaaagtgtg taacggaata ggtattggtg aatttaaaga ctcactctcc ataaatgcta 9000

cgaatattaa acacttcaaa aactgcacct ccatcagtgg cgatctccac atcctgccgg 9060

tggcatttag gggtgactcc ttcacacata ctcctcctct ggatccacag gaactggata 9120

ttctgaaaac cgtaaaggaa atcacagggt ttttgctgat tcaggcttgg cctgaaaaca 9180

ggacggacct ccatgccttt gagaacctag aaatcatacg cggcaggacc aagcaacatg 9240

gtcagttttc tcttgcagtc gtcagcctga acataacatc cttgggatta cgctccctca 9300

aggagataag tgatggagat gtgataattt caggaaacaa aaatttgtgc tatgcaaata 9360

caataaactg gaaaaaactg tttgggacct ccggtcagaa aaccaaaatt ataagcaaca 9420

gaggtgaaaa cagctgcaag gccacaggcc aggtctgcca tgccttgtgc tcccccgagg 9480

gctgctgggg cccggagccc agggactgcg tctcttgccg gaatgtcagc cgaggcaggg 9540

aatgcgtgga caagtgcaac cttctggagg gtgagccaag ggagtttgtg gagaactctg 9600

agtgcataca gtgccaccca gagtgcctgc ctcaggccat gaacatcacc tgcacaggac 9660

ggggaccaga caactgtatc cagtgtgccc actacattga cggcccccac tgcgtcaaga 9720

cctgcccggc aggagtcatg ggagaaaaca acaccctggt ctggaagtac gcagacgccg 9780

gccatgtgtg ccacctgtgc catccaaact gcacctacgg atgcactggg ccaggtcttg 9840

aaggctgtcc aacgaatggg cctaagatcc cgtccatcgc cactgggatg gtgggggccc 9900

tcctcttgct gctggtggtg gccctgggga tcggcctctt catgtga 9947

Claims (10)

1. A CD56+ NK cell induction medium, wherein the induction medium comprises the following cytokines: IL-7, IL-15, SCF, FLt 3L; preferably, the final concentration of IL-7 is 10ng/ml, the final concentration of IL-15 is 20ng/ml, the final concentration of SCF is 10ng/ml, and the final concentration of FLt3L is 10 ng/ml; preferably, the induction medium further comprises IL-3; preferably, the final concentration of IL-3 is 5 ng/ml; preferably, the culture medium further comprises X-VIVO, human AB serum; preferably, the human AB serum ratio is 5%; preferably, the culture medium further comprises X-VIVO, autologous plasma; preferably, the autologous plasma proportion is 5%.

2. A method of producing CD56+ NK cells, said method comprising the steps of:

1) isolating mononuclear cells from cord blood;

2) sorting to obtain CD34+ cells;

3) culturing CD34+ cells using the induction medium of claim 1;

preferably, the step of isolating mononuclear cells from cord blood comprises: diluting the cord blood by using normal saline, adding lymphocyte separation liquid, and centrifugally collecting white membrane cells; diluting the leucocyte layer cells with normal saline, centrifuging and removing supernatant; adding erythrocyte lysate to lyse the cells, centrifuging and removing supernatant; resuspending the cell pellet;

preferably, the ratio of 1: diluting the cord blood in a proportion of 1;

preferably, the ratio of 1: 1, diluting white membrane layer cells in proportion;

preferably, the step of isolating mononuclear cells from cord blood comprises: using physiological saline solution to mix at a ratio of 1: diluting the cord blood by a ratio of 1, and adding a lymphocyte separation solution with the volume of 1/2 of the diluted cord blood; 2000r/min, centrifuging for 20 min; the leucocyte layer cells were collected and then added with physiological saline at a rate of 1: 1, diluting according to a proportion; centrifuging the cells at 2000r/min for 5min, and discarding the supernatant; lysing the cells with 5ml of red blood cell lysate for 5min, then centrifuging the cells at 2000r/min for 5min, and discarding the supernatant; resuspending the cell count with 5-10ml of DPBS, and calculating the number of the obtained cells;

preferably, the step of sorting the CD34+ cells comprises: resuspending the mononuclear cells by using a sorting buffer solution, sequentially adding an FCR (fiber channel receptor) blocking agent and CD34 magnetic beads, uniformly mixing, and incubating at a low temperature; adding a sorting buffer solution into the incubated cells, centrifuging and removing a supernatant; adding the suspension into a separation column after the suspension is resuspended by a separation buffer solution; adding a sorting buffer solution into the sorting column, washing, adding the sorting buffer solution into the sorting column, pushing the plunger to the bottom end of the sorting column, washing the marked cells, and counting;

preferably, low temperature incubation means incubation in a refrigerator at 4 ℃ for 30 min;

preferably, the sorting buffer refers to 0.5% HSA;

preferably, the step of sorting the CD34+ cells comprises: centrifuging the cells at 300g/min for 10min, discarding the supernatant, resuspending the cells with 300. mu.l of 0.5% HSA, then sequentially adding 100. mu.l of FCR blocker and CD34 magnetic beads, mixing the cells once for each addition, then placing a 50ml centrifuge tube into a 4 ℃ refrigerator for incubation for 30min, and mixing the cells once every 5 min; adding 5-10ml of 0.5% HSA into the incubated cells, centrifuging the cells at 300g/min for 10min, and discarding the supernatant; resuspend the cells with 500 μ l of 0.5% HSA, then add the cells to a sorting column that needs to be rinsed once with 3ml of 0.5% HSA in advance; 3ml of 0.5% HSA is added into the sorting column to wash the sorting column for 3 times; then removing the sorting column from the magnetic field, adding 5ml of 0.5% HSA into the sorting column, forcibly pushing the plunger to the bottom end of the sorting column, washing the marked cells, and counting;

preferably, the steps of culturing CD34+ cells using the medium of claim 1 are as follows: after sorting the cells, resuspending the cells in the medium of claim 1, and plating the cells in a 24-well plate to adjust the cell density to 0.8-1.2 x 10⁵Per well.

3. An NK cell activation medium, comprising: IL-2, IL-15, IL-21; preferably, the final concentration of IL-2 is 1000IU/ml, the final concentration of IL-15 is 100ng/ml, and the final concentration of IL-21 is 100 ng/ml; preferably, the medium further comprises: OK432, IFN-gamma; preferably, the final concentration of OK432 is 100ng/ml, and the final concentration of IFN-gamma is 1000 IU/ml; preferably, the medium further comprises KBM581, human AB serum; preferably, the human AB serum ratio is 5%; preferably, the medium further comprises KBM581, autologous plasma; preferably, the autologous plasma proportion is 5%.

4. A method of making CD56+ NK cells, the method comprising:

1) obtaining cells using the method of claim 2;

2) CD56+ NK cell activation;

preferably, CD56+ NK cell activation comprises culturing the cells obtained in step 1) in the medium of claim 3; preferably, CD56+ NK cell activation comprises culturing cells obtained after 10-14 days of culture using the method of claim 2 in the medium of claim 3; preferably, CD56+ NK cell activation includes using claim 2 the method of 10-14 days after the use of every 2-3 days half liquid change way will be obtained in the culture medium of claim 3 culture; preferably, the culture is carried out in the medium of claim 3 for one week.

5. An NK cell expansion medium, comprising the following cytokines: IL-2, IL-21; preferably, the final concentration of IL-2 is 1000IU/ml, and the final concentration of IL-21 is 100 ng/ml; preferably, the medium further comprises KBM581, human AB serum; preferably, the human AB serum proportion is 5%.

6. A method of producing NK cells, comprising

1) Culturing by the method of claim 4;

2) culturing the cells obtained in step 1) in the amplification medium of claim 5;

preferably, the step 2) comprises the step 1) of half-changing the culture medium for every 2-3 days by replacing the culture medium with the amplification medium of claim 5.

7. An NK cell produced by the method of claim 2, claim 4 or claim 6; preferably, the NK cells under-express CD 7; preferably, the purity of the NK cells reaches more than 80%.

8. A genetically modified NK cell, or a method of making the same, which is the NK cell of claim 7; preferably, the genetically modified NK cells comprise NK cells expressing a chimeric antigen receptor; preferably, the chimeric antigen receptor targets CD 7; preferably, the amino acid sequence of the chimeric antigen receptor targeting CD7 is shown in SEQ ID NO. 1; preferably, the nucleic acid sequence of the chimeric antigen receptor targeting CD7 is shown in SEQ ID NO. 2;

preferably, the method for preparing the genetically modified NK cell comprises the steps of:

1) preparing the NK cell of claim 7;

2) performing gene modification on the NK cells obtained in the step 1);

preferably, the genetic modification comprises introducing a nucleic acid sequence encoding a chimeric antigen receptor into the NK cells obtained via step 1).

9. A kit or medicament comprising the NK cell of claim 7 or the genetically modified NK cell of claim 8.

10. An application, the application comprising any one of:

1) use of the NK cell of claim 7 in the preparation of a medicament for the treatment of cancer or an autoimmune disease;

2) use of the NK cell of claim 7 for the preparation of an immune cell expressing a chimeric antigen receptor;

3) use of the NK cell of claim 7 for the preparation of a kit according to claim 9;

4) use of the genetically modified NK cell of claim 8 for the preparation of a medicament for the treatment of cancer or an autoimmune disease;

5) use of the genetically modified NK cell of claim 8 for the preparation of a kit according to claim 9.

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