CN117604011A

CN117604011A - Fusion gene, nourishing dendritic cell expressing same, natural killer cell culture method and natural killer cell

Info

Publication number: CN117604011A
Application number: CN202311586034.9A
Authority: CN
Inventors: 卢社莲; 唐立春; 李燕荣; 李营营; 胡梦
Original assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Current assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Priority date: 2023-11-24
Filing date: 2023-11-24
Publication date: 2024-02-27

Abstract

The invention relates to the field of cell culture, in particular to a fusion gene, a nourishing dendritic cell expressing the fusion gene, a natural killer cell culture method and a natural killer cell. The dendritic cells expressing the fusion gene have the fusion genes which can express IL-21 with different transmembrane structures and are subjected to irradiation inactivation before being used as the nourishing cells for inducing and culturing natural killer cells, so that the application safety is high, and the natural killer cells provided by the invention have high ratio of the natural killer cells in the cell population obtained by culturing, high expansion times and high killing activity on tumor cells, thereby providing a new choice for developing safe and effective antitumor biological agents.

Description

Fusion gene, nourishing dendritic cell expressing same, natural killer cell culture method and natural killer cell

Technical Field

The invention belongs to the field of cell culture, and in particular relates to a fusion gene, a nourishing dendritic cell expressing the fusion gene, a natural killer cell culture method and a natural killer cell.

Background

Natural Killer (NK) cells are an important component of the human natural immune system, which are differentiated from hematopoietic stem cells, and which recognize and attack foreign pathogens and tumor cells in the body as the first line of defense, and are an important component of the natural immune system. Meanwhile, NK cells and B cells and T cells of an acquired immune system are members of lymphocyte families, and IFN gamma released after the NK cells and the T cells are activated can induce the expression of MHC molecules on the cell surface and amplify antigen presenting action, so that the NK cells can serve as a bridge to connect two major systems of innate immunity and acquired immunity.

NK cell surface does not express specific antigen recognition receptor, is not limited by MHC molecules, and can directly kill tumor cells without pre-sensitization in a variety of ways including: 1. releasing perforin and granzyme to cause tumor cell lysis; 2. releasing TNF to act on TNF receptor on the surface of tumor cells, causing apoptosis; NK cell surface ligands (FasL or TRAIL) bind to tumor cell surface receptors (Fas or TRAILR) and induce apoptosis; fc receptors on nk cell surfaces (CD 16, fcRIII gamma) bind antibodies and exert antibody-dependent cellular cytotoxicity (ADCC).

Natural Killer (NK) cells can therefore play an important role in the defense against diseases including the first line of malignancy. In contrast to cells of the acquired immune system (e.g., T cells), NK cells initiate cytotoxic activity without prior stimulation or sensitization, thereby providing an immediate natural response. NK cells have the advantage of killing tumors by both direct lysis and secretion of cytokines, which kill tumors through both perforin and FAS ligands. NK cells can produce TNF- α, IFN- γ and IL-1, which are important in NK cell anti-cancer responses and mobilizing T lymphocytes. Since NK cells have a rapid killing effect and a wide range of target cells, it is suggested that NK cells can be used for treating malignant tumors. Studies have demonstrated that adoptive transfer of activated, expanded autologous or allogeneic NK cells in vitro is effective in the treatment of a variety of leukemias and solid tumors.

NK cells can be identified by the deletion of cell surface TCRs and related CD3 molecules, as well as expression of CD 56. NK cells circulate mainly in blood, accounting for about 5 to 10% of Peripheral Blood Mononuclear Cells (PBMC), and are also present in lymphoid tissues such as bone marrow and spleen.

In tumor treatment, NK cells with a tumor killing function are prepared in vitro, and then the NK cells are returned to a patient, so that the tumor cells are killed in the patient, and the treatment effect is achieved. Therefore, the NK cells with sufficient quantity, strong killing property and stable quality are obtained, and are key to realizing clinical application of NK cell tumor treatment. However, since NK cells derived from peripheral blood are difficult to be amplified in large amounts in vitro and there is a technical difficulty in separating and purifying NK cells from peripheral blood, for example, it is difficult to avoid contamination from T lymphocytes, a large number of uniform NK cells cannot be obtained and the clinically required standard is reached, and thus this is also an obstacle that NK cells cannot be widely used in clinic; in addition, in some cases of tumor patients, NK cell mediated antitumor response is weak, which may be related to factors such as poor NK cell killing ability, poor in vivo viability or limited migration to tumor sites.

The in vitro culture of NK cells mainly comprises a trophoblast cell method and a pure factor culture method. The trophoblast cell method utilizes a human cell line, and carries out co-culture with mononuclear cells after genetic engineering modification so as to activate and induce NK cells. The use of the leukemia cell line CTV-1 has a smaller effect on cell proliferation (j.immunol., 178 (1): p85-94,2007), an average increase in cell number of about 490-fold after 21 days of culture with EBV-LCL (Cytotherapy, 11 (3): p 341-355.2009), and the use of the feeder cell K562 line, which expresses 4-1BBL and membrane-bound IL-15, after 3 weeks of culture of NK cells, an average increase in NK cell number of 227-fold and exhibits high cytotoxicity in vitro and in vivo, but shows limited proliferation due to cell death (Cancer res.,69 (9): p4010-4017, 2009). After 3 weeks of NK cell culture in K562 cell line transfected with MICA, 4-1BBL and IL-15, the NK cell number was increased by an average 350-fold (Tissue anti-genes, 76 (6): p467-475, 2010), and when NK cell was cultured with K562 cell line transfected with membrane-bound IL-21 for 2 weeks while NK cell was stimulated every 7 days, the NK cell number was increased by an average 21000-fold. However, since specific cancer cells are used as feeder cells, NK cells produced have priming specificity for the specific cancer cells. In addition, NK cells cultured using tumor-derived feeder cells have potential safety hazards in clinical applications.

Therefore, searching NK cells with strong activity and establishing a culture system for in vitro large-scale allogeneic use is a problem that the NK cell adoptive immunotherapy tumor can be applied to clinic and needs to be solved. The establishment of the large-scale continuous culture and preparation process of NK cells, which is easy to operate, controllable in quality, high in yield, high in product activity and low in cost, can solve the bottleneck problem faced by the cellular immunotherapy technology, and can lead the cellular therapy products to be produced in a batched, large-scale, uniform and flow way like medicines.

Disclosure of Invention

(problem to be solved by the invention)

In view of the problems existing in the prior art, the invention aims to provide an expansion system which uses dendritic cells as trophoblasts after genetic modification and irradiation inactivation, thereby efficiently expanding NK cells, an NK cell expansion method adopting the expansion system, and NK cells obtained by the method, thereby providing a new choice for developing safe and effective antitumor biological agents.

(solution for solving the problem)

The invention provides a fusion gene, which has IL-21 expression with different transmembrane structures, and comprises a nucleotide sequence shown as SEQ ID NO. 1, wherein,

SEQ ID NO. 1 is

ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC。

Furthermore, preferably, the nucleotide sequence of the fusion gene is SEQ ID NO. 9, SEQ ID NO. 10 or SEQ ID NO. 11, wherein,

SEQ ID NO. 9 as

ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC

GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

ATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTC

AGATCCAAGAGGTCTAGGCTGCTGCACAGCGACTACATGAACATGACCCCCAGAAGGCCCGGCCCCACAAGAAAGCACTACCAGCCCTACGCTCCCCCCAGAGACTTTGCCGCTTATAGATCC

CGGTTCAGCGTCGTGAAAAGGGGGCGCAAGAAACTGCTGTACATCTTCAAGCAGCCTTTTATGCGCCCAGTGCAGACAACTCAGGAGGAAGACGGATGCTCTTGTCGGTTCCCAGAGGAGGAGGAAGGAGGCTGCGAGCTG

AGAGTGAAGTTCAGCCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAATATGACGTGCTGGATAAGCGACGAGGACGGGACCCCGAAATGGGAGGAAAACCCAGAAGGAAGAACCCTCAGGAGGGGCTGTATAATGAACTGCAGAAAGACAAGATGGCTGAGGCATACAGCGAAATTGGAATGAAAGGAGAGCGCCGACGGGGGAAGGGACACGATGGGCTGTACCAGGGACTGTCAACCGCCACTAAAGATACCTACGACGCACTGCACATGCAGGCTCTGCCCCCAAGA；

SEQ ID NO. 10

TTCGTCCCCGTGTTCCTGCCTGCCAAGCCAACAACTACCCCTGCTCCACGACCACCTACTCCAGCACCTACCATCGCAAGTCAGCCCCTGTCACTGCGACCTGAGGCTTGCCGGCCAGCAGCTGGAGGAGCAGTGCACACCCGAGGCCTGGACTTCGCATGCGATATCTACATTTGGGCACCACTGGCTGGAACCTGTGGGGTCCTGCTGCTGAGCCTGGTCATCACCCTGTATTGTAACCACAGAAAT

SEQ ID NO. 11 is

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAG

AGAGTGAAGTTCAGCCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAATATGACGTGCTGGATAAGCGACGAGGACGGGACCCCGAAATGGGAGGAAAACCCAGAAGGAAGAACCCTCAGGAGGGGCTGTATAATGAACTGCAGAAAGACAAGATGGCTGAGGCATACAGCGAAATTGGAATGAAAGGAGAGCGCCGACGGGGGAAGGGACACGATGGGCTGTACCAGGGACTGTCAACCGCCACTAAAGATACCTACGACGCACTGCACATGCAGGCTCTGCCCCCAAGA。

The present invention provides a nourishing dendritic cell, which expresses the fusion gene.

In addition, preferably, the trophoblast dendritic cells described herein have at least one selected from the group consisting of DC-mIL21-CD4 TM, DC-mIL21-CD8TM, and DC-mIL21-CD28 TM.

Furthermore, preferably, the trophoblasts described in the present invention are inactivated by irradiation.

The present invention also provides a method for culturing NK cells using the above-described feeder dendritic cells.

In addition, the present invention provides NK cells cultured using the above-described trophoblast cells.

In addition, the present invention provides a tumor therapeutic agent prepared using the above NK cells.

(effects of the invention)

The nourishing dendritic cell provided by the invention can express fusion genes (mIL-21) with different transmembrane structures and encoding IL-21, and has no amplification capability and high safety in application because the dendritic cell has strong mIL-21 presenting capability and is inactivated by irradiation before being used as a nourishing cell for inducing and culturing NK cells. In addition, the NK cells induced and cultured by using the nourishing dendritic cells provided by the invention have high ratio of the NK cells in the cell population obtained by culturing, high expansion times and improved killing activity on tumor cells.

Drawings

Fig. 1: schematic representation of fusion genes encoding IL-21 with different transmembrane structures.

Fig. 2: the flow cytometer was used to express molecules on the surface of trophoblasts.

Fig. 3: NK cell growth trend graph.

Fig. 4: four dendritic cells induced expansion fold of cultured NK cells.

Fig. 5: NK cell phenotype assay results on day 21 of induction culture.

Fig. 6: the four dendritic cells induce the killing efficiency of the cultured NK cells on the lung cancer H358 cells.

Detailed Description

Definition of the definition

Unless stated to the contrary, the terms used in the present invention have the following meanings.

In the claims and/or the specification of the present invention, the words "a" or "an" or "the" may mean "one" but may also mean "one or more", "at least one", and "one or more".

As used in the claims and specification, the words "comprise," "have," "include" or "contain" mean including or open-ended, and do not exclude additional, unrecited elements or method steps.

Throughout this application, the term "about" means: one value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters set forth herein are approximations that may vary depending upon the particular application. Any numerical value, however, inherently contains certain standard deviations found in their respective testing methods or apparatus. Accordingly, unless expressly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used herein are modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.

The term "Natural Killer (NK) cells" as used in the context of the present invention is in the form of CD3 ^- /CD56 ⁺ The lymphocyte subgroup which is the main immunophenotype characteristic, does not express T cell receptor and B cell receptor, is an important component in natural immunocytes of human body, belongs to lymphocyte family members, is differentiated from hematopoietic stem cells, and is used as a first defense line of the organism to identify and attack external pathogens and tumor cells in the body.

The term "SNK cell (stimulated natural killer cells)" refers to natural killer cells which are induced to expand, are in an activated state and have a strong killing function on various tumor target cells through in vitro culture.

The term "dendritic cells (DC cells)" is the most powerful professional antigen presenting cells in the human body, can efficiently ingest, process and present antigens, immature DC has stronger migration ability, and mature DC can effectively activate initial T cells, and is in the central link of starting, regulating and maintaining immune response.

The term "trophoblast DC cell" refers in the context of the present invention to a DC cell capable of expressing fusion genes encoding IL-21 with different transmembrane structures. In the present invention, the "trophoblast DC" is also referred to as DC-mIL-21 trophoblast. The DC-mIL-21 trophoblast of the present invention includes at least one selected from the group consisting of DC-mIL21-CD4 TM, DC-mIL21-CD8TM, and DC-mIL21-CD28 TM.

In some embodiments, the invention provides a fusion gene encoding IL-21 with different transmembrane structures, the fusion gene being artificially designed, having portions expressing different transmembrane proteins, and incorporating an IL-21 encoding gene, thereby enabling DC cells to present mIL-21 on the cell surface after transfection into the DC cells via a viral vector. A schematic diagram of the fusion gene is shown in FIG. 1. In some embodiments, the nucleotide sequences of the different segments in the fusion gene are as follows:

the nucleotide sequence encoding IL-21 is:

ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC(SEQ ID NO:1)

the nucleotide sequence encoding IgG1 is:

GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA(SEQ ID NO:2)

the nucleotide sequence encoding CD4 TM is:

ATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTC(SEQ ID NO:3)

the nucleotide sequence encoding CD8TM is:

TTCGTCCCCGTGTTCCTGCCTGCCAAGCCAACAACTACCCCTGCTCCACGACCACCTACTCCAGCACCTACCATCGCAAGTCAGCCCCTGTCACTGCGACCTGAGGCTTGCCGGCCAGCAGCTGGAGGAGCAGTGCACACCCGAGGCCTGGACTTCGCATGCGATATCTACATTTGGGCACCACTGGCTGGAACCTGTGGGGTCCTGCTGCTGAGCCTGGTCATCACCCTGTATTGTAACCACAGAAAT(SEQ ID NO:4)

the nucleotide sequence encoding CD28 TM is:

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAG(SEQ ID NO:5)

the nucleotide sequence encoding the CD28 intracellular stimulatory signal is:

AGATCCAAGAGGTCTAGGCTGCTGCACAGCGACTACATGAACATGACCCCCAGAAGGCCCGGCCCCACAAGAAAGCACTACCAGCCCTACGCTCCCCCCAGAGACTTTGCCGCTTATAGATCC(SEQ ID NO:6)

the nucleotide sequence encoding 4-1BB is:

CGGTTCAGCGTCGTGAAAAGGGGGCGCAAGAAACTGCTGTACATCTTCAAGCAGCCTTTTATGCGCCCAGTGCAGACAACTCAGGAGGAAGACGGATGCTCTTGTCGGTTCCCAGAGGAGGAGGAAGGAGGCTGCGAGCTG(SEQ ID NO:7)

the nucleotide sequence encoding CD3 ζ is:

AGAGTGAAGTTCAGCCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAATATGACGTGCTGGATAAGCGACGAGGACGGGACCCCGAAATGGGAGGAAAACCCAGAAGGAAGAACCCTCAGGAGGGGCTGTATAATGAACTGCAGAAAGACAAGATGGCTGAGGCATACAGCGAAATTGGAATGAAAGGAGAGCGCCGACGGGGGAAGGGACACGATGGGCTGTACCAGGGACTGTCAACCGCCACTAAAGATACCTACGACGCACTGCACATGCAGGCTCTGCCCCCAAGA(SEQ ID NO:8)

SEQ ID NO:9(IL21-IgG1-CD4-CD 28-4-1BB-CD3ζ)

ATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTC

AGAGTGAAGTTCAGCCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAATATGACGTGCTGGATAAGCGACGAGGACGGGACCCCGAAATGGGAGGAAAACCCAGAAGGAAGAACCCTCAGGAGGGGCTGTATAATGAACTGCAGAAAGACAAGATGGCTGAGGCATACAGCGAAATTGGAATGAAAGGAGAGCGCCGACGGGGGAAGGGACACGATGGGCTGTACCAGGGACTGTCAACCGCCACTAAAGATACCTACGACGCACTGCACATGCAGGCTCTGCCCCCAAGA

SEQ ID NO:10(IL21-IgG1-CD8-CD 28-4-1BB-CD3ζ)

SEQ ID NO:11(IL21-IgG1-CD28-CD 28-4-1BB-CD3ζ)

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGATCCAAGAGGTCTAGGCTGCTGCACAGCGACTACATGAACATGACCCCCAGAAGGCCCGGCCCCACAAGAAAGCACTACCAGCCCTACGCTCCCCCCAGAGACTTTGCCGCTTATAGATCC

in some embodiments, the present invention provides fusion genes encoding IL-21 having different transmembrane structures having the nucleotide sequence SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 11.

The present invention provides a trophoblast DC cell that expresses the fusion gene of the present invention encoding IL-21 having different transmembrane structures. In some embodiments, the trophoblast DC cell expresses the fusion gene set forth in SEQ ID NO. 9, SEQ ID NO. 10, or SEQ ID NO. 11. In some embodiments, the trophoblast DC cells are inactivated by irradiation.

The present invention provides a culture method for culturing NK cells, which comprises using the trophoblast DC cell of the present invention as a trophoblast for inducing culture of NK cells. In some embodiments, the trophoblast DC cells used in the method express the fusion gene of the invention encoding IL-21 having a different transmembrane structure. In some embodiments, the trophoblast DC used in the method expresses the fusion gene set forth in SEQ ID NO 9, SEQ ID NO 10, or SEQ ID NO 11.

The present invention provides NK cells which are cultured by induction using the feeder DC cells of the present invention as feeder cells. In some embodiments, the NK cells are NK cells cultured using the culture methods of the invention for culturing NK cells.

The invention also provides the use of the NK cells of the invention in the preparation of a formulation for tumor treatment.

Examples

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

The experimental techniques and experimental methods used in this example are conventional techniques unless otherwise specified, or according to conditions, operating methods, etc. suggested by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.

EXAMPLE 1 preparation of trophoblast DC cells expressing transmembrane IL-21 fusion Gene

1.1 preparation of recombinant DC cells

Fusion genes encoding IL-21 (schematic diagram shown in figure 1) with different transmembrane structures and the TAX2 gene were synthesized by Jin Wei inc. The synthesized gene was ligated into lentiviral vector pCDH, viral supernatants were harvested by lentiviral packaging, and the supernatants were concentrated and transfected into DC cells. The specific steps of transfection of DC cells are:

a) 3 flasks of 293T cells were seeded using T75 flasks such that the cell density in each flask was 3X 10 ⁶ Shaking up individual cells/10 ml, placing at 37deg.C and CO ₂ Culturing overnight in an incubator; the next day, slow virus packaging is carried out when the cell density reaches 80%;

b) Taking A, B two 15mL centrifuge tubes, respectively adding 1.5mL opti-MEM, adding 60 mug plasmid into the A tube and uniformly mixing, adding 300ng liposome 3000 into the B tube and uniformly mixing, and standing at room temperature for 5min;

c) Adding the liquid in the pipe B into the pipe A after 5min, uniformly mixing (the strength is lighter than that of the first time), and standing for 30min at room temperature;

d) The supernatant was discarded from 293T cells having a density of 80%, and 5mL of opti-MEM was added thereto, followed by washing once and then 4mL of opti-MEM;

e) After 30min, the mixed solution obtained by mixing the tube A and the tube B is gently dripped into the 293T cells obtained in the step d), 1mL of the mixed solution is gently shaken uniformly for each bottle, and the mixture is placed at 37 ℃ and CO ₂ Incubating for 4 hours in an incubator;

f) After the incubation was completed, 10mL of fresh opti-MEM medium was replaced, gently shaken, and placed at 37℃in CO ₂ Culturing overnight in an incubator;

g) Culturing for 48 hr, collecting supernatant containing virus, and storing in refrigerator at 4deg.C; adding 10mL opti-MEM culture medium into 293T cells for further culture;

h) After 72h of incubation, the virus supernatant was harvested, combined with the virus supernatant harvested after 48h of incubation, centrifuged at 2000rmp at 4℃for 10min, the supernatant was filtered through a 0.45 μm needle filter and concentrated by centrifugation through a virus concentration column (60 mL of virus supernatant), centrifuged at 3000rmp for 30min, finally resuspended in 300. Mu.L of sterile PBS and the obtained virus supernatant was stored at-80℃for further use.

i) Lentivirus infects DC cells: thawing frozen 300 μl of TAX 2-transferred lentiviral concentrate, and adding to the isolated cells with a number of about 1×10 ⁶ cells were cultured in RPMI1640 medium supplemented with 10% FBS and 200IU/mL IL-2 for 14 days;

j) Sorting IL-21 in a flow cytometer using anti-IL-21 antibodies ⁺ The cells that are sorted are DC cells that express transmembrane IL-21, i.e., trophoblast-type DC cells described herein (as shown in FIG. 2), also referred to herein as DC-mIL-21 trophoblasts, including, for example, DC-mIL21-CD4, DC-mIL21-CD8, and DC-mIL21-CD28, as shown in FIG. 2;

k) Culturing DC-mIL-21 trophoblast in RPMI1640 medium supplemented with 10% FBS and 200IU/mL IL-2 for 10 days, and collecting appropriate amount of cells at 1×10 ⁷ After 100Gy irradiation at a cell/mL density, the cells were used as highly safe and non-amplifying trophoblast cells for the induction of PBMC.

The nucleic acid sequence of the TAX2 gene is:

ATGGCCCATTTCCCAGGATTTGGACAGAGCCTCCTATATGGATACCCCGTCTACGTGTTTGGCGATTGTGTACAGGCCGATTGGTGTCCCGTCTCAGGTGGTCTATGTTCCACCCGCCTACATCGACATGCCCTCCTGGCCACCTGTCCAGAGCACCAACTCACCTGGGACCCCATCGATGGACGCGTTGTCAGCTCTCCTCTCCAATACCTTATCCCTCGCCTCCCCTCCTTCCCCACCCAGAGAACCTCAAGGACCCTCAAGGTCCTTACCCCTCCCACCACTCCTGTCTCCCCCAAGGTTCCACCTGCCTTCTTTCAATCAATGCGAAAGCACACCCCCTACCGAAATGGATGCCTGGAACCAACCCTCGGGGATCAGCTCCCCTCCCTCGCCTTCCCTGAACCTGGCCTCCGTCCCCAAAACATCTACACCACCTGGGGAAAAACCGTAGTATGCCTATACCTATACCAGCTCTCCCCACCCATGACATGGCCACTTATACCCCATGTCATATTCTGCCACCCCAGACAATTAGGAGCCTTCCTCACCAAGGTGCCTCTAAAACGATTAGAAGAACTTCTATACAAAATGTTCCTACACACAGGGGCAGTCGTAGTCCTCCCGGAGGACGACCTACCCACCACAATGTTCCAACCCGTGAGGGCTCCCTGTATCCAGACTGCCTGGTGTACAGGACTTCTCCCCTATCACTCCATCTTAACAACCCCAGGTCTAATATGGACCTTCAATGACGGCTCACCAATGATTTCCGGCCCTTGCCCCAAGGCAGGGCAGCCATCTTTAGTAGTTCAGTCCTCCCTATTAATCTTCGAAAAATTCCAAACCAAAGCCTTCCATCCCTCCTATCTACTCTCTCATCAGCTTATACAATACTCCTCCTTCCATAACCTTCACCTTCTATTCGATGAATACACCAACATCCCTGTCTCTATTTTATTTAATAAAGAAGAGGCGGATGACAATGGCGACCAGCCTCCCGAGCCAGCCACCCAGGGCGAGTCATCGACCCAAAAGGTCAGACCGTCTCACACAAACAATCCCAAGTAT

1.2 cultivation and inactivation of trophoblast DC cells

The trophoblast DC cells were cultured using RPMI1640 basal medium supplemented with 10% FBS such that the cell density was maintained at 1X 10 ⁶ cells/mL～3×10 ⁶ cells/mL. Subsequently, the cells were inactivated using an irradiation dose of 100 Gy.

EXAMPLE 2 Induction culture of NK cells

In PBMC (1X 10) ⁶ cells/mL～1.5×10 ⁶ cells/mL) cells were cultured on day 0, and inactivated trophoblast DC cells (PBMC: trophoblast DC = 1: 1) IL-2 was then fed daily according to the cell culture volume so that the concentration of IL-2 was maintained at about 200IU/mL. On day 7 of culture, inactivated trophoblast DC cells (total cells in culture: trophoblast DC cells=20:1) were added again and culture continued until day 25. Wherein the cell number per bottle of cells was recorded on day 0, day 3, day 7, day 10, day 13, day 16, day 19, day 22 (as shown in fig. 3).

a) Human peripheral blood mononuclear cells were isolated, centrifuged and counted for resuspension in RPMI1640 medium supplemented with 10% fbs and 200IU/mL IL-2;

b) In PBMC (1X 10) ⁶ cells/mL～1.5×10 ⁶ cells/mL) cells were cultured on day 0, 100Gy of inactivated different trophoblast DC cells (PBMCs: trophoblast DC = 1: 1) The method comprises the steps of carrying out a first treatment on the surface of the Placing at 37deg.C, CO ₂ Culturing in an incubator;

c) Supplementing 50 IU/mL-500 IU/mL of IL-2 every day according to the actual volume, and changing the culture medium according to the volume which is about half of the volume of the culture medium in a culture bottle at the moment when the culture medium turns yellow according to the growth condition of cells;

d) On days 18 to 28 (depending on the days when the medium turns yellow), NK cells, that is, SNK cells after the culture, were obtained.

As can be seen from FIG. 3, the trophoblast DC cells (DC-mIL 21-CD 4. TM., DC-mIL21-CD 8. TM., and DC-mIL21-CD 28. TM.) that express IL-21 in the membrane were able to induce a significant increase in the number of NK cells cultured, compared to the DC cells in the figure (no mIL-21 expression), with the SNK cells cultured in the DC-mIL21-CD 28. TM. Induction being the best in growth.

EXAMPLE 3 detection of cell phenotype in SNK cells obtained by culture

On day 22 of cell culture, expression of CD3, CD56, CD16 of the cells was examined using a flow cytometer, thereby analyzing the phenotype of the SNK cells after culture. The method comprises the following specific steps:

a) SNK cells on day 21 of culture were collected, centrifuged at 1000rpm for 5min, and the supernatant was discarded, washed once with 10mL of PBS, and the wash was discarded.

b) Cells were resuspended in 6mL pbs each, 5 flow tubes each, centrifuged (1000 rpm,5 min) at 1mL per tube, the supernatant discarded after centrifugation, and then resuspended in 50 μl bs per tube.

c) Antibody incubation: each group of cells was divided into 6 tubes, each: (1) negative control (no antibody added), (2) single-stained tube added 10. Mu.L anti-CD3 antibody, (3) single-stained tube added 10. Mu.L anti-CD4 antibody, (4) single-stained tube added 10. Mu.L anti-CD8 antibody, (5) single-stained tube added 10. Mu.L anti-CD56 antibody, (6) single-stained tube added 10. Mu.L anti-CD16 antibody, incubated at room temperature in the dark for 30min.

d) After incubation of the antibodies, each tube was washed with 2mL of PBS, centrifuged at 1000rpm for 5min, and the supernatant was discarded, and then resuspended in 1mL of PBS, respectively, and the expression of CD3, CD56, CD16 was detected using a flow cytometer.

As can be seen from FIG. 5, the SNK cells induced by the DC-mIL-21 trophoblast had an increased ratio compared to the NK cells induced by the DC-mIL-21 trophoblast alone, and the SNK cells induced by the DC-mIL-21-CD28 were higher than the SNK cells induced by the other two DC-mIL-21 trophoblasts (i.e., DC-mIL-21-CD 4. TM. And DC-mIL-21-CD 8. TM.).

Example 4 detection of the killing Capacity of induced cultured SNK cells against cancer target cells

On the 21 st day of cell culture, the killing efficiency of the cultured SNK cells on lung cancer cells NCI-H358 is detected, and the specific operation steps are as follows:

a) Different target cells (i.e. lung cancer cells NCI-H358) were taken, digested with pancreatin, stopped by adding DMEM medium when the cells became round or were about to fall off the walls, gently pipetting the cells on the walls, transferring the cells to a 50mL centrifuge tube, centrifuging at 1000rpm for 5min, and discarding the supernatant. Washing with 10ml PBS, collecting in centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, re-suspending with 10ml RPMI1640 medium supplemented with 2% FBS, sampling, counting, and adjusting cell density to 8×10 according to cell number ⁴ cells/mL；

b) Inoculation of target cells: target cells after cell density adjustment were inoculated into 96-well cell culture plates, 50 μl per well, wells without target cells were set as control groups, and 50 μl of RPMI1640 medium with 2% fbs was added per well;

c) Preparation and dilution of SNK cells: collecting SNK cells on day 21 of culture, centrifuging, re-suspending with 30mLPBS, sampling, counting, centrifuging, washing, discarding supernatant, re-suspending cells with RPMI1640 medium supplemented with 2% FBS according to counting result, and adjusting cell density to 1.6X10 ⁶ cells/mL；

d) Inoculation of SNK cells: adding diluted SNK cells with good density into a corresponding 96-well plate containing target cells, wherein 50 mu L/well is used for making the effective target ratio be 20:1, and simultaneously setting a T high group and a T low group, wherein 50 mu L of RPMI1640 culture medium added with 2% FBS is added;

e) Co-cultivation: after cell inoculation is finished, the cell is placed at 37 ℃ and CO ₂ Co-culturing in an incubator for 4 hours;

f) Cell lysis: after 4h of cell CO-culture, 10 mu L of cell lysate is added to each well of the Tgao group, and the cells are placed at 37 ℃ and CO ₂ The incubator is fully cracked for 1h;

g) After cell lysis, taking out the cells, adding 100 mu L Working Solution into each hole, and reacting for 30min in a light-shielding environment at room temperature;

h) Immediately after 50. Mu.L of Stop Solution was added to each well, the absorbance at 490nm was measured by an enzyme-labeled instrument, and the cell killing efficiency was calculated according to the formula, and the result is shown in FIG. 6.

Cell killing ratio (%) = [ (a-B-E)/(C-D-E) ]x100

A: absorbance of E+T well-absorbance of background Blank

B: absorbance of E Low control wells-absorbance of background Blank

C: absorbance of T high control wells-absorbance of background Blank

D: absorbance of T high control Blank-absorbance of background Blank

E: absorbance of T low control-absorbance of background Blank

As shown in fig. 6, the effective target ratio is 20:1, the killing efficiency of the SNK cells induced and cultured by the DC cells, the DC-mIL-21-CD4 TM, the DC-mIL-21-CD 8TM and the DC-mIL-21-CD28 TM serving as the trophoblasts to the target cells is 58%, 61%, 71% and 73%, respectively, so that the SNK cells induced and cultured by the DC-mIL-21 trophoblasts have strong tumor cell killing efficiency.

Example 5 modification of example 2

When the induction culture of NK cells as described in example 2 was performed:

the time for adding the nourishing DC cells for the second time is 0 day and 5-8 days respectively, wherein the optimal time for adding the nourishing DC cells for the second time is 7 days;

when feeder DC cells were added on day 0, the cell number ratio of feeder DC cells to PBMC was (0.8-1.2): 1, the optimal ratio is 1:1.

when the trophoblast DC cells were added on day 7, the ratio of trophoblast DC cells to total cell population in culture was 1: (15-25), the optimal ratio is 1:20.

in the case of the cell culture performed in step a) of example 2, the IL-2 may be added at a concentration of 150 to 250IU/mL.

Suitable harvest time for induction of cultured SNK cells may be days 18-28.

From the above examples, the present invention has high safety in application by using DC cells with high IL-21 presenting ability to irradiate so that they lose amplifying ability. And, through expressing the fusion genes with different transmembrane structures and encoding IL-21 on the surface of DC cells, and using the DC cells as trophoblasts for culturing NK cells after irradiation inactivation, the NK cells induced to culture can occupy a relatively high proportion in a cell population obtained by culture, have high expansion times and have strong killing capacity on tumor cells.

The above examples of the present invention are only examples for clearly illustrating the present invention, and are not limiting of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims

1. A fusion gene for expressing IL-21 with different transmembrane structures, which comprises a nucleotide sequence shown in SEQ ID NO. 1,

SEQ ID NO. 1 is ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC.

2. The fusion gene according to claim 1, wherein the nucleotide sequence of the fusion gene is SEQ ID NO. 9, SEQ ID NO. 10 or SEQ ID NO. 11,

SEQ ID NO. 9 is ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC

ATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTC

SEQ ID NO. 10 is ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC

SEQ ID NO. 11 is ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC

3. A trophoblast dendritic cell expressing the fusion gene of claim 1.

4. The trophoblast-type dendritic cell of claim 3, having at least one selected from the group consisting of DC-mIL21-CD4 TM, DC-mIL21-CD8TM, and DC-mIL21-CD28 TM.

5. The trophoblast of claim 3, wherein said trophoblast is inactivated by irradiation.

6. A natural killer cell culture method, characterized in that the method uses the trophoblast dendritic cell of any one of claims 3 to 5.

7. A natural killer cell cultured using the feeder dendritic cell of any one of claims 3 to 5.

8. A tumor therapeutic agent prepared using the natural killer cell of claim 7.