CN116240168A - Preparation and application of NK cells - Google Patents

Preparation and application of NK cells Download PDF

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CN116240168A
CN116240168A CN202310436243.9A CN202310436243A CN116240168A CN 116240168 A CN116240168 A CN 116240168A CN 202310436243 A CN202310436243 A CN 202310436243A CN 116240168 A CN116240168 A CN 116240168A
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medium
concentration
cancer
composition
cells
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王静宜
吕学燕
张晓艳
杨月峰
黄园园
王立燕
郭雷鸣
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Beijing Jingda Biotechnology Co ltd
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Abstract

The invention provides preparation of NK cells and application thereof. Specifically, the invention provides a culture medium for culturing NK cells, which comprises the following components: IL-2, IL-15, IL-12, IL-21, CD137 antibodies, CD3 antibodies, vitamins, basal medium, serum substitutes. NK cells cultured by the culture medium have high activity and good killing activity on tumor cells.

Description

Preparation and application of NK cells
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to preparation and application of NK cells.
Background
In recent years, research into cell therapies based on Chimeric Antigen Receptors (CARs) for the treatment of cancer has been quite hot. CAR-T cells have achieved great success in the treatment of blood cancers, but there are some clinical applications for which drawbacks remain to be overcome, such as severe Cytokine Release Syndrome (CRS), neurotoxicity, and high treatment costs. As important effector cells in innate immunity, NK cells are MHC-independent lymphocytes, and compared with T cells, NK cells have more mechanisms for killing target cells and have broad-spectrum anti-tumor effect. The factors secreted by NK cells are IL-3 and GM-CSF, so that cytokine storm (CRS) is almost unlikely to be caused, and in addition, allogeneic NK cells hardly cause graft versus host disease (GvHD), so that the preparation of universal CAR-NK is facilitated, and the clinical cost is reduced. Based on these advantages, NK cells are considered effector cells that have greater potential to enhance their anti-tumor ability by CAR modification and are safer and more suitable for clinical applications. According to the data on the ClinicalTrials. Gov website, a plurality of clinical trials of CAR-NK products are currently being carried out, most of the clinical trials are in the stage I and stage II, and the antitumor activity and the safety in clinical application are attracting attention.
NK cells are widely available and can be obtained from Peripheral Blood (PB), umbilical Cord Blood (UCB), human induced pluripotent stem cells (hiPSC) and NK cell lines. The NK cells from the iPSC have more advantages, including low immunogenicity, unlimited proliferation, low cost and the like, but the allogeneic iPSC may have the problems of tumorigenicity, heterogeneity and the like; NK cell lines such as NK-92 are derived from tumor tissue, which, although susceptible to genetic modification, require radiation treatment prior to their infusion into the human body, which increases safety risks and reduces the persistence and efficacy of NK cells in patients; NK cells from cord blood sources and peripheral blood sources have higher proliferation capacity, but researches show that PB-NK has stronger capability of killing tumors in vitro and is more convenient to obtain than UCB-NK, so that PB-NK has good application prospect in tumor immunotherapy compared with other NK cells from several sources.
Although the development and application of PB-NK cells has been greatly advanced in recent years, the production technology of PB-NK cells still faces many challenges. PBMCs may be obtained by direct collection of donor venous fresh blood separation, or by apheresis sources. The PBMC obtained by directly collecting fresh peripheral blood through vein separation has good activity, is beneficial to carrying out induction amplification on NK cells, but the quantity of PBMC of a single donor which can be obtained at one time is limited, the individual difference of the PBMC activation and amplification of the donor has great influence, the blood collected by the same donor at different times also has difference, if the amplification of the NK cells directly induced after collection is easy to cause failure, the stability and uniformity of cell end products are not facilitated, in addition, the open and manual blood collection step cannot meet the requirement on the safety of clinical tests, and some barriers cannot be overcome on ethical requirements. The PBMC from single blood sampling source can obtain a larger quantity at one time, can be frozen for preparing and using in a plurality of batches, is suitable for screening donors, improves the stability and uniformity of cell products in different batches, and meets the requirements of clinical tests. However, the induction of expansion of NK cells from frozen single blood-derived PBMC is a very challenging problem, namely that NK cells are extremely sensitive to the freeze thawing process, and the activity of NK cells in the recovered PBMC is reduced, so that the activation and expansion are difficult. Therefore, solving the problem of NK cell activation in PBMC after resuscitation is a great importance in achieving the preparation of high-activity NK cells derived from apheresis.
The present invention describes in detail how NK cells obtained by sorting PBMC derived from apheresis after resuscitation are activated and amplified in large quantities in vitro.
Reference is made to:
1.Martha Luevano et al,The unique profile ofcord blood natural killer cells balances incomplete maturation and effective killing function upon activation,Human Immunology 73(2012)248-257.
2.Jacob A.Myers,Jeffrey S.Miller；Exploring the NK cell platform for cancer immunotherapy,Nat Rev Clin Oncol,2021February,18(2):85-100.
3.Ying Gong et al,Chimeric antigen receptor natural killer(CAR-NK)cell design and engineering for cancer therapy,J Hematol Oncol(2021)14:73.
disclosure of Invention
The invention aims to establish a scheme which is suitable for PBMC from an apheresis source, can be effectively activated and greatly amplified after freeze thawing and finally harvest high-activity NK cells. The scheme is also suitable for fresh PB-NK amplification culture.
To achieve the above object, the present invention provides the following embodiments:
in a first aspect, the present invention provides a composition for culturing NK cells, said composition comprising an activation medium composition consisting of: IL-2, IL-15, IL-12, IL-21, CD137 antibodies, CD3 antibodies, multivitamins, basal medium, serum substitutes.
The term "natural killer cells (NK cells)" is defined as Large Granular Lymphocytes (LGL) and constitutes a third cell differentiated from common lymphoid progenitor cells that produce B and T lymphocytes. NK cells are known to differentiate and mature in bone marrow, lymph nodes, spleen, tonsils and thymus where they then enter the circulation. NK cells are phenotypically different from natural killer T cells (NKT) in terms of origin and in terms of respective effector functions; generally, NKT cell activity promotes NK cell activity by secreting ifnγ. In contrast to NKT cells, NK cells do not express the T cell antigen receptor (TCR) or the pan T marker CD3 or the surface immunoglobulin (Ig) B cell receptor, but they typically express the surface markers CD16 (FcyRIII) and CD56 in humans, NK1.1 or NK1.2 in C57BL/6 mice.
The term "culturing" as used herein includes providing the chemical and physical conditions (e.g., temperature, gas), as well as growth factors, required for NK cell maintenance. Generally culturing NK cells involves providing NK cells with conditions for expansion (proliferation). Examples of chemical conditions that may support NK cell expansion include, but are not limited to, buffers, serum, nutrients, vitamins, antibiotics, cytokines, and other growth factors, which are periodically provided (or may be manually administered) in a cell culture medium suitable for NK cell expansion.
The terms "IL-2", "IL-15", "IL-12", "IL-21" as used herein refer to the cytokines interleukin-2, interleukin-15, interleukin-12, interleukin-21 and derivatives thereof.
Herein, the term "antibody" includes glycosylated and non-glycosylated immunoglobulins relating to any isotype or subclass or antigen binding regions relating to competition for specific binding with intact antibodies, including human, humanized, chimeric, multispecific, monoclonal, polyclonal antibodies, and oligomers or antigen binding fragments thereof. Also included are proteins having antigen binding fragments or regions, such as Fab, fab ', F (ab') 2, fv, diabodies, fd, dAb, minibodies, single chain antibody molecules, complementarity Determining Region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies, and polypeptides, which contain at least a portion of an immunoglobulin sufficient to confer binding to a specific antigen of a target polypeptide. The term "antibody" includes, but is not limited to, antibodies produced, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody.
In one embodiment of the present invention, the CD137 antibody or the CD3 antibody is a CD137 monoclonal antibody or a CD3 monoclonal antibody.
The term "serum replacement" refers to an agent used in cell culture as a replacement for serum (e.g., FBS) to maintain the non-differentiated state of cells and their culture. Examples of serum substitutes include KNOKOUTTM SR (KnockOutTM serum substitute or KSR; gibco), stemSure serum substitute (SSR; wakoPure Chemical Industries, ltd.), N-2 supplement (Wako Pure Chemical Industries, ltd.), CTS TM Immune cell serum replacement. In a specific embodiment of the present invention, the serum replacement is CTS TM Immune cell serum replacement.
In one embodiment of the invention, the activating medium composition has a concentration of IL-2 in the medium of 1000-10000IU/mL, IL-15 in the medium of 50-2000IU/mL, IL-12 in the medium of 50-500IU/mL, IL-21 in the medium of 0.1-10IU/mL, CD137 antibody in the medium of 5-15 μg/mL, CD3 antibody in the medium of 5-15 μg/mL, multivitamin in the medium of 0-1000ng/mL, serum replacement in the medium of 3-10%,
as a more preferred embodiment, the activation medium composition, IL-2 in the medium concentration of 1000IU/mL, IL-15 in the medium concentration of 300IU/mL, IL-12 in the medium concentration of 100IU/mL, IL-21 in the medium concentration of 5IU/mL, CD137 antibody in the medium concentration of 5u g/mL, CD3 antibody in the medium concentration of 5u g/mL, vitamin complex in the medium concentration of 100ng/mL, serum replacement in the medium concentration of 5%.
In one embodiment of the invention, the basal medium of the activation medium composition is selected from NK MACS, X-VIVO15, GT-T551H3, cellgrowSCGM, AIM-V, preferably the basal medium of the activation medium composition is X-VIVO15.
In one embodiment of the invention, the multivitamin of the activation medium composition is selected from one or more of vitamin a/retinol, vitamin B2/vitamin B6/vitamin B12, vitamin C/L-ascorbic acid, vitamin E, preferably the multivitamin of the activation medium composition comprises vitamin B2 and vitamin E.
In one embodiment of the invention, the composition of the invention further comprises a first amplification medium composition consisting of: serum replacement, IL-2, IL-15, IL-12, multivitamins, basal medium.
In one embodiment of the present invention, the concentration of the serum replacement in the medium is 3-10%, the concentration of the IL-2 in the medium is 1000-10000IU/mL, the concentration of the IL-15 in the medium is 50-2000IU/mL, the concentration of the IL-12 in the medium is 50-500IU/mL, the concentration of the multivitamin in the medium is 0-1000ng/mL,
as a more preferred embodiment, the first amplification medium composition has a serum replacement concentration of 5% in the medium, an IL-2 concentration of 1000IU/mL in the medium, an IL-15 concentration of 300IU/mL in the medium, an IL-12 concentration of 100IU/mL in the medium, and a multivitamin concentration of 100ng/mL in the medium.
In one embodiment of the invention, the composition of the invention further comprises a second amplification medium composition consisting of: serum replacement, IL-2, IL-15, vitamins, basal medium.
In one embodiment of the invention, the concentration of the serum replacement in the medium is 3-10%, the concentration of the IL-2 in the medium is 1000-10000IU/mL, the concentration of the IL-15 in the medium is 50-2000IU/mL, and the concentration of the multivitamin in the medium is 0-1000ng/mL.
As a more preferred embodiment, the second amplification medium composition has a serum replacement concentration of 5% in the medium, an IL-2 concentration of 1000IU/mL in the medium, an IL-15 concentration of 300IU/mL in the medium, and a multivitamin concentration of 100ng/mL in the medium.
In one embodiment of the invention, the basal medium of the first and second amplification medium compositions is each independently selected from NK MACS, X-VIVO15, GT-T551H3, cellgrowSCGM, AIM-V, preferably, the basal medium of the first and second amplification medium compositions is each independently X-VIVO15.
In one embodiment of the present invention, the first and second amplification medium compositions of the multivitamins are each independently selected from one or more of vitamin a/retinol, vitamin B2/vitamin B6/vitamin B12, vitamin C/L-ascorbic acid, preferably the first and second amplification medium compositions of the multivitamins each independently comprise vitamin B2 and vitamin E.
In one embodiment of the present invention, the composition of the present invention further comprises a coating liquid composition comprising a CD137 antibody, a CD28 antibody, a CD16 antibody.
In one embodiment of the present invention, the concentration of the CD137 antibody in the coating liquid is 8 μg/mL;
in one embodiment of the invention, the concentration of the CD28 antibody in the coating liquid is 8 mug/mL;
in one embodiment of the invention, the concentration of the CD16 antibody in the coating solution is 8 μg/mL.
In one embodiment of the invention, the CD137 antibody, CD28 antibody, CD16 antibody is a CD137 monoclonal antibody, a CD28 monoclonal antibody, a CD16 monoclonal antibody.
In a second aspect, the present invention provides a method of expanding NK cells, said method comprising: (1) Pretreating a cell culture vessel with a coating liquid composition according to the first aspect of the present invention;
(2) Obtaining a population of NK cells;
(3) In the cell culture vessel obtained in 1), using the activation medium composition described in the first aspect of the invention for activating the NK cell population obtained in 2) to obtain an activated NK cell population;
(4) The first and second amplification medium compositions of the first aspect of the invention are used to amplify the activated NK cell population.
As used herein, the term "expansion" or "proliferation" refers to cell growth and an increase in cell number. Amplification or proliferation as used herein relates to an increased number of NK cells that occur during culture, such as disclosed herein.
The cell culture vessel used for the culture of NK cells is not particularly limited as long as it can culture NK cells, but includes a bottle, a tissue culture bottle, a dish, a plate, a tissue culture dish, a multi-dish, a microplate, a multi-dish, a multi-well plate, a microscope slide, a chamber slide, a culture dish, a tube, a tray, a culture bag, and a roller bottle.
The cell culture apparatus may be cell-adhesive or cell-non-adhesive, and is appropriately selected according to the purpose. The cell-adhesive incubator may be coated with any cell-supporting matrix such as extracellular matrix (ECM) for the purpose of enhancing the adhesion between the surface of the incubator and cells. The cell support matrix may be any substance for the purpose of adhesion of NK cells.
Other culture conditions may be appropriately set. For example, the culture temperature is not particularly limited, and may be about 30 to 40℃and preferably about 37 ℃. CO 2 The concentration may be about 1 to 10%, preferably about 2 to 5%. The oxygen partial pressure may be 1 to 10%.
In one embodiment of the invention, the NK cell population is obtained from peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, stem cells or bone marrow, preferably the NK cell population is obtained from peripheral blood mononuclear cells, more preferably the peripheral blood mononuclear cells are peripheral blood mononuclear cells after cryopreservation resuscitation.
In one embodiment of the invention, the method of obtaining the NK cell population comprises subjecting the peripheral blood mononuclear cells to magnetic bead sorting. Preferably, the magnetic bead sorting comprises magnetic bead negative sorting or magnetic bead positive sorting.
More preferably, the magnetic beads used for the magnetic bead anion selection comprise CD3 magnetic beads, CD14 magnetic beads and CD19 magnetic beads, and most preferably, the magnetic beads are used in an amount of 20 mu L/10 7 And (3) cells.
More preferably, the magnetic beads used in the magnetic bead cation comprise CD56 magnetic beads.
In one embodiment of the present invention, the method of using the first and second amplification medium compositions of the first aspect of the present invention for amplifying the activated NK cell population comprises:
a. day3-Day5, culturing the population of activated NK cells obtained in step 3) of the second aspect with the first amplification medium composition of the invention;
b. day6-Day24, culturing the NK cell population as described in a using the second expansion medium composition of the invention;
c. day25 harvest NK cells.
In a third aspect, the invention provides an NK cell product obtained by culturing by the method of the second aspect of the invention.
In one embodiment of the present invention, the purity of the NK cells in the NK cell product is up to 99%.
In a fourth aspect, the invention provides a pharmaceutical composition comprising an NK cell product according to the third aspect of the invention and a pharmaceutically acceptable carrier.
The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when properly administered to an animal, such as a human. The preparation of pharmaceutical compositions comprising antibodies or additional active ingredients is known to those skilled in the art in view of the present disclosure. Furthermore, for animal (e.g., human) administration, it should be understood that the preparation should meet sterility, pyrogenicity, general safety and purity standards as required by the biological standard FDA office (FDAOffice ofBiologicalStandards).
As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol/water solutions, saline solutions, parenteral vehicles such as sodium chloride, ringer's dextrose, and the like), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelators, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, pharmaceutical stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids, and nutritional supplements, and combinations thereof, as will be known to those of ordinary skill in the art. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well known parameters.
In a fifth aspect, the invention provides the use of any one of the following:
(1) The use of a product according to the third aspect of the invention or a pharmaceutical composition according to the fourth aspect of the invention for the preparation of a reagent having a killing effect on tumour cells;
(2) Use of a product according to the third aspect of the invention or a pharmaceutical composition according to the fourth aspect of the invention for the manufacture of a medicament for the treatment of autoimmune diseases, diseases of the blood system and/or cancer.
In a specific embodiment of the invention, the tumor cells comprise K562 and Raji.
"treatment" refers to an execution regimen that may include administering one or more drugs to a patient in an effort to alleviate the signs or symptoms of the disease. Desirable therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and moderating or improving prognosis. Remission may occur before the signs or symptoms of the disease or disorder appear and after they appear. Thus, "treating" may include "preventing" a disease or undesired condition. In addition, "treatment" does not require complete relief of signs or symptoms, does not require cure, and specifically includes regimens that have only marginal effects on the patient.
The cancers described herein encompass any type of cancer, including both solid and non-solid cancers. In particular, the cancer comprises cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, stomach cancer, esophageal cancer, thyroid cancer, transitional bladder epithelial cancer, leukemia, brain tumor, stomach cancer, peritoneal cancer, head and neck cancer, endometrial cancer, renal cancer, female genital tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma.
Autoimmune diseases as used herein refer to diseases that result from an inappropriate immune response in a subject against substances and tissues normally found in the body. In other words, the immune system mistreats a part of the body as a pathogen and attacks its own cells. This may be limited to certain tissues (e.g., in autoimmune thyroiditis) or to specific tissues including different sites (e.g., godpasture disease, which may affect the basal membrane of both the lung and kidney). Immunosuppressants, e.g., drugs that reduce the immune response, are commonly used to treat autoimmune diseases. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, goldPaschel's syndrome, necrotizing vasculitis, lymphadenitis, perinodular vasculitis, systemic lupus erythematosus, rheumatoid disease, arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-related vasculitis (e.g., wegener granulomatosis, microscopic polyangiitis), uveitis, shoegren's syndrome, crohn's disease, lorenter's syndrome, ankylosing spondylitis, lyme arthritis, golda-Bares syndrome, hashimoto's thyroiditis, and cardiomyopathy.
Compared with the prior art, the invention has the advantages and beneficial effects that:
NK cells have limited proliferation potential and in vitro expansion is a challenge. In recent years, research on NK cell expansion culture generally adopts two schemes of a trophoblast cell culture method and a pure factor culture method. The addition of the trophoblast cells can enable NK cells to be activated well and expand in a large quantity, but the added trophoblast cells still have great risks even if irradiated, and are not suitable for clinical application; the pure factor method is used for inducing and amplifying NK cells in PBMC, so that the method is safer and suitable for clinic, but the amplification factor is generally not ideal, and in addition, the reinfusion of allogeneic NK cells needs to completely remove T cells so as to avoid GVHD; in contrast, for the recovered PBMC, it is clearly an extremely difficult task to amplify and obtain high-activity NK cells with purity of more than 99% in large quantities by the pure factor method.
Compared with the prior art, the invention provides a method for culturing NK cells, and the amplification factor of the NK cells obtained by the method can reach 1000-10000 times finally, and the purity is more than 99%. The invention can effectively solve the problem that high-purity NK cells are difficult to obtain, and provides possibility for the allogenic use of NK cells separated from peripheral blood of an apheresis source.
Drawings
FIG. 1 is a cell flow result before and after magnetic bead sorting on day0, wherein panels A and B are NK cell flow results before and after magnetic bead sorting on day 0; panels C and D are the ratios of CD14 and CD19 in total cell cells after day0 bead sorting;
FIG. 2 is a graph showing the results of detection of the expression ratio of CD16 in NK cells, wherein FIG. A is a graph showing the results of the expression ratio of CD16 in NK cells on day0 and FIG. B is a graph showing the results of the expression ratio of CD16 in NK cells on day25 in culture;
FIG. 3 is a graph showing NK cell flow results after 25 days of culture;
FIG. 4 is a graph of the killing capacity of NK cells against tumors at 15 and 25 days of culture, wherein graph A is a graph of the killing capacity of NK cells against tumors at 15 days of culture and graph B is a graph of the killing capacity of NK cells against tumors at 25 days;
FIG. 5 is a proliferation curve of cells cultured for 25 days;
FIG. 6 is a graph comparing NK cell flow results after 25 days of culture;
FIG. 7 is a graph of the results of comparison of proliferation curves of cells cultured for 25 days.
Detailed Description
The present invention will be described in detail below with reference to examples and drawings to facilitate understanding and practice of the invention and to further realize advantages thereof by those skilled in the art.
Unless otherwise defined in the specification of the present invention, all technical terms herein are used according to conventional 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, unless otherwise specified, are commercially available.
EXAMPLE 1NK cell culture method
1. Experimental procedure
1. Pretreatment of cell culture flasks
Phosphate buffer containing 8 μg/mL of CD137 monoclonal antibody, 8 μg/mL of CD28 monoclonal antibody and 8 μg/mL of CD16 monoclonal antibody was added to a 6-well cell culture plate, 850 μl/well, and the liquid was allowed to disperse in the well to cover the whole bottom, and was kept flat overnight at 4 ℃.
2. Thawing recovery of cryopreserved PBMC
RPMI 1640 medium containing 10% fbs was preheated in 37 ℃ water bath, frozen PBMC cells were removed from liquid nitrogen, the tube cap of the frozen tube was unscrewed for 1/4 of the pressure release, then immediately screwed, the frozen tube was placed in 37 ℃ water bath for about 1.5 minutes, rotated continuously during which time the frozen tube was removed when small ice crystals remained in the tube in trace amounts, and the exterior of the frozen tube was wiped with 75% ethanol and placed in a biosafety cabinet. Sucking 9mL to 15mL of the preheated RPMI 1640 culture medium into a centrifuge tube, transferring the cell suspension in the freezing tube into the centrifuge tube by using a 1mL pipettor, lightly flushing the freezing tube by using 1mL of the culture medium, transferring flushing liquid into the centrifuge tube, placing the centrifuge tube into 400g/min of the centrifuge, centrifuging for 8 min, removing the supernatant after centrifugation, lightly beating the bottom of the tube to loosen cell precipitation, immediately adding 10mL of the preheated culture medium to resuspend the cells, and thawing the PBMC.
3. Magnetic bead sorting
Filtering the thawed PBMC suspension with 70 μm cell sieve to remove cell mass, sampling and counting, calculating MACS-Buffer and magnetic bead dosage according to counting result, performing magnetic bead negative selection of CD3, CD14 and CD19, and magnetic bead dosage is 20 μL/10 7 Cells, collecting the falling CD56 + NK cells, centrifuged and the supernatant was discarded, resuspended in 2mL of X-VIVO15 basal medium, sampled and counted, and a small amount of cell suspension was further taken for flow detection, NK cells (CD 3) - CD56 + ) The proportion is 99.17%.
Day0 vaccination
After the NK cells obtained in the step 1.3 were washed again by centrifugation, they were resuspended in X-VIVO15 medium containing IL-21000IU/mL, IL-15300IU/mL, IL-12100IU/mL, IL-215IU/mL, CD 1375. Mu.g/mL, CD 35. Mu.g/mL, vitamin complex 100ng/mL (vitamin B2: vitamin E=1:1) and counted at a density of 1.26×10 6 Inoculating into 6-well plate coated in 1.1, 1.8 mL/well, adding 5% serum replacement (CTS) TM Immune cell serum replacement), rehydration to 2mL, in an incubator (37 ℃, CO) 2 The concentration is 5%, the humidity is 45% -55%) and culturing.
5. Fluid infusion on day3
The fluid replacement was performed on day3 after cell inoculation, with a fluid replacement volume of 2mL. At this time, the medium was supplemented with 5% serum replacement (CTS) TM Immune cell serum replacement), IL-21000IU/mL, IL-15300IU/mL, IL-12100IU/mL, multivitamin 100ng/mL (vitamin B2: vitamin e=1: 1) Amplification medium 1 (basal medium X-VIVO 15).
6. Fluid infusion on day6
The second fluid replacement was performed on day6 of culture, with a fluid replacement volume of 4mL. At this time, the medium was supplemented with 5% serum replacement (CTS) TM Immune cell serum replacement), IL-21000IU/mL, IL-15300IU/mL, multivitamin 100ng/mL (vitamin B2: vitamin e=1: 1) Is amplified medium 2 (basal medium is X-VIVO 15).
7. Fluid replacement on day7
Cell clusters were blown off at 1X 10 on day7 of culture 6 The density of individual/mL was supplemented with amplification medium 2.
8.Day9-Day23
After Day7 is filled, cells are counted and filled every other Day, i.e. cells are counted and filled at 1×10 in Day9, day11, day13, day15, day17, day19, day21, day23, respectively 6 The amplification medium 2 was supplemented with a density of/mL and sampled and fed-stream to determine NK purity.
9. End of culture and detection on day25
NK cell purity, cell killing ability against tumor, cell expression of activated receptor and inhibitory receptor were examined by Day25 sampling, and the culture was ended at Day 25.
2. Experimental results
The cell suspension is harvested on the 25 th DAY of culture, the whole cell number is amplified by more than 5000 times compared with the DAY0 inoculation, and meanwhile, the flow detection and the killing function detection of tumor cells (K562 and Raji) are carried out on the amplified cells.
Example 2 comparison of effects of different culture protocols
1. Experimental protocol
After the single nucleated cells of the frozen single blood sampling are recovered, the following schemes are adopted for culture respectively:
control 1: NK cell sorting is carried out after the cell Day0 is recovered, and then culture is carried out by using a culture medium (IL-21000 IU/mL, IL-15300IU/mL and 5% serum substitute) only containing the combination of two factors of IL-2 and IL-15 and the serum substitute;
p19 (experimental group): NK cells were sorted after recovery of Day0 cells, and cultured in the same manner as in the examples (medium composition: IL-21000IU/mL, IL-15300IU/mL, IL-12100IU/mL, IL-215IU/mL, CD1375ug/mL, CD35ug/mL, vitamin complex 100ng/mL (vitamin B2: vitamin E=1:1), and 5% CTS was added TM Immune cell serum substitutes);
control2 group: cells Day0 were recovered and cultured directly (unsorted) using the same protocol as the experimental group.
2.Experimental results
A) As can be seen from FIG. 6, the NK cell purity was low (< 60%) after 25 days of culture in Control1 and Control2 groups, and the experimental group purity reached 99% or more.
B) As can be seen from fig. 7, the NK cell expansion ability of the experimental group was significantly stronger than that of the control group, and thus, the experimental group was considered to be superior to the control group.
While the invention has been described with reference to the drawings and preferred embodiments, various modifications and changes will occur to those skilled in the art. Various modifications, variations, and equivalents of the present invention are intended to be covered by the contents of the appended claims.

Claims (10)

1. A composition for culturing NK cells, said composition comprising an activation medium composition consisting of: IL-2, IL-15, IL-12, IL-21, CD137 antibodies, CD3 antibodies, multivitamins, basal medium, serum substitutes;
preferably, in the activating medium composition, the concentration of IL-2 in the medium is 1000-10000IU/mL, the concentration of IL-15 in the medium is 50-2000IU/mL, the concentration of IL-12 in the medium is 50-500IU/mL, the concentration of IL-21 in the medium is 0.1-10IU/mL, the concentration of CD137 antibody in the medium is 5-15 mug/mL, the concentration of CD3 antibody in the medium is 5-15 mug/mL, the concentration of vitamin complex in the medium is 0-1000ng/mL, the concentration of serum replacement in the medium is 3-10%,
more preferably, the activating medium composition has a concentration of IL-2 in the medium of 1000IU/mL, IL-15 in the medium of 300IU/mL, IL-12 in the medium of 100IU/mL, IL-21 in the medium of 5IU/mL, CD137 antibody in the medium of 5 mug/mL, CD3 antibody in the medium of 5 mug/mL, multivitamin in the medium of 100ng/mL, serum replacement in the medium of 5%;
preferably, the basal medium of the activation medium composition is selected from NK MACS, X-VIVO15, GT-T551H3 and cellgrowSCGM, AIM-V, and more preferably, the basal medium of the activation medium composition is X-VIVO15;
preferably, the multivitamins of the activation medium composition comprise one or more of vitamin a/retinol, vitamin B2/vitamin B6/vitamin B12, vitamin C/L-ascorbic acid, vitamin E, preferably, the multivitamins of the activation medium composition comprise vitamin B2 and vitamin E.
2. The composition of claim 1, further comprising any one or more of the following:
1) A first amplification medium composition, said first amplification medium composition consisting of: serum replacement, IL-2, IL-15, IL-12, multivitamins, basal medium;
preferably, in the first amplification medium composition, the concentration of the serum replacement in the medium is 3-10%, the concentration of the IL-2 in the medium is 1000-10000IU/mL, the concentration of the IL-15 in the medium is 50-2000IU/mL, the concentration of the IL-12 in the medium is 50-500IU/mL, the concentration of the compound vitamin in the medium is 0-1000ng/mL,
more preferably, in the first amplification medium composition, the concentration of the serum replacement in the medium is 5%, the concentration of the IL-2 in the medium is 1000IU/mL, the concentration of the IL-15 in the medium is 300IU/mL, the concentration of the IL-12 in the medium is 100IU/mL, and the concentration of the multivitamin in the medium is 100ng/mL;
2) A second amplification medium composition, said second amplification medium composition consisting of: serum replacement, IL-2, IL-15, multivitamins, basal medium;
preferably, in the second amplification medium composition, the concentration of the serum replacement in the medium is 3-10%, the concentration of the IL-2 in the medium is 1000-10000IU/mL, the concentration of the IL-15 in the medium is 50-2000IU/mL, the concentration of the compound vitamin in the medium is 0-1000ng/mL,
more preferably, in the second amplification medium composition, the concentration of the serum replacement in the medium is 5%, the concentration of the IL-2 in the medium is 1000IU/mL, the concentration of the IL-15 in the medium is 300IU/mL, and the concentration of the multivitamin in the medium is 100ng/mL;
preferably, the basal medium of the first and second amplification medium compositions is each independently selected from NKMACS, X-VIVO15, GT-T551H3, cellgrowSCGM, AIM-V,
more preferably, the basal medium of the first and second amplification medium compositions is X-VIVO15;
preferably, the first and second amplification medium compositions are each independently selected from one or more of vitamin A/retinol, vitamin B2/vitamin B6/vitamin B12, vitamin C/L-ascorbic acid, and vitamin E, and preferably, the first and second amplification medium compositions are each independently selected from vitamin B2 and vitamin E.
3. The composition of claim 1 or 2, wherein the composition further comprises a coating solution composition, wherein the coating solution composition comprises a CD137 antibody, a CD28 antibody, a CD16 antibody,
preferably, in the coating liquid composition, the concentration of the CD137 antibody in the coating liquid is 8 mug/mL;
preferably, in the coating liquid composition, the concentration of the CD28 antibody in the coating liquid is 8 mug/mL;
preferably, the concentration of the CD16 antibody in the coating liquid composition is 8 mug/mL.
4. A method of expanding NK cells, said method comprising:
(1) Pretreating a cell culture vessel with the coating liquid composition of claim 3;
(2) Obtaining a population of NK cells;
(3) In the cell culture vessel obtained in 1), using the activation medium composition of claim 1 to activate the NK cell population obtained in 2) to obtain an activated NK cell population;
(4) Use of the first amplification medium composition, the second amplification medium composition of claim 2 or 3 for amplifying the activated NK cell population.
5. The method of claim 4, wherein the NK cell population is obtained from peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, stem cells or bone marrow,
preferably, the NK cell population is obtained from peripheral blood mononuclear cells,
preferably, the peripheral blood mononuclear cells are peripheral blood mononuclear cells after cryopreservation and resuscitation.
6. The method of claim 5, wherein said method of obtaining a population of NK cells comprises subjecting said peripheral blood mononuclear cells to magnetic bead sorting,
preferably, the magnetic bead sorting comprises magnetic bead negative sorting or magnetic bead positive sorting;
preferably, the magnetic beads used for the magnetic bead anion selection comprise CD3 magnetic beads, CD14 magnetic beads and CD19 magnetic beads, and more preferably, the magnetic beads are used in an amount of 20 mu L/10 7 A cell;
preferably, the magnetic beads used in the magnetic bead cation selection comprise CD56 magnetic beads.
7. An NK cell product, characterized in that said NK cell product is cultured by the method of any one of claims 4-6,
preferably, the purity of NK cells in the NK cell product reaches more than 99%.
8. A pharmaceutical composition comprising the NK cell product of claim 7 and a pharmaceutically acceptable carrier.
9. The use of any one of the following:
(1) Use of the product of claim 7 or the pharmaceutical composition of claim 8 for the preparation of an agent having a killing effect on tumor cells;
(2) Use of the product of claim 7 or the pharmaceutical composition of claim 8 for the manufacture of a medicament for the treatment and/or prophylaxis of autoimmune disorders, diseases of the blood system and/or cancer;
preferably, the cancer comprises a solid cancer and a non-solid cancer;
preferably, the cancer comprises cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, transitional-bladder carcinoma, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, renal cancer, female genital tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hematopoietic tumor, multiple myeloma, non-hodgkin's lymphoma, acute myelogenous leukemia, B-cell lymphoma, T-cell lymphoma;
preferably, the autoimmune disease comprises glomerulonephritis, goldPascher's syndrome, necrotizing vasculitis, lymphadenitis, perinodular periarteritis, systemic lupus erythematosus, rheumatoid disease, arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-related vasculitis, uveitis, schoenlen syndrome, crohn's disease, lyter's syndrome, ankylosing spondylitis, lyme's arthritis, goldan-barre syndrome, hashimoto's thyroiditis and cardiomyopathy.
10. The use of claim 9, wherein said tumor cells comprise K562, raji.
CN202310436243.9A 2023-04-21 2023-04-21 Preparation and application of NK cells Pending CN116240168A (en)

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