Preparation method of a novel immune cell and its application

Abstract

The present application relates to the field of immune cells. The present application provides a preparation method and application of a novel immune cell, which uses a lentivirus containing a genetically modified DNA molecule to transfect cells in mammalian peripheral blood, so that the cells can be passaged multiple times and have the ability of antigen presentation and activation and expansion of natural killer cells. The cells are further genetically modified to achieve better transmembrane transfer, antigen presentation and activation of natural killer cells. After genetic modification of the cells, co-activation of the cells with different cytokines or small molecules can obtain a larger amount of natural killer cells with higher purity, and regulation of epigenetic changes in the expression amount of receptors and ligands of natural killer cells can further improve the cytotoxicity of effector cells. The present application can prepare antigen presenting cells, CTL cells and application methods for different antigens. These cells and methods have broad application prospects in the prevention and treatment of tumors and infectious diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to immune cells, in particular to engineered dendritic cells (DCs), specific CTL and NK cells produced by PBMCs, methods of preparation and uses thereof. BACKGROUND

[0002] (I) Cell immunotherapy and its types

[0003] Cell immunotherapy is to collect human immune cells, culture them in vitro after modification, increase their number by hundreds or thousands of times, enhance their targeted killing function, and then return them to the human body to kill pathogenic cells, cancer cells, mutated or virus-infected cells in the blood and tissues, break immune tolerance, activate and enhance the immune ability of the body, and achieve the dual effects of treatment and health care. Current cell immunotherapy includes cytokine-induced killer cell (CIK) therapy, dendritic cell (DC) therapy, API biological immunotherapy, DC-CIK cell therapy, natural killer cell (NK) therapy, DC-T cell therapy, chimeric antigen receptor T cell immunotherapy (CAR-T), T cell receptor T cell immunotherapy (TCR-T), etc.

[0004] DC-CIK immunotherapy is a treatment method that uses modern biotechnology to stimulate the body's own immune system to fight tumors. The basic principle is to extract immature immune cells from the body, culture them in vitro, and then return them to the patient's body. Not only can it accurately and efficiently kill tumor cells, but it can also stimulate the body's immune response to tumors, so that the immune system can kill tumor cells and start immune surveillance to prevent tumor metastasis and recurrence. DC cells recognize antigens, activate the ability of the adaptive immune system, and kill tumor cells. CIK cells kill tumor cells by exerting their cytotoxicity and secreting cytokines. The combination of the two can improve the activity of CIK cells, i.e. DC-CIK biological therapy. However, DC cells can only transmit specific signals to CIK cells to exert specific cytotoxicity after recognizing or loading antigens in vitro, which greatly limits the actual efficacy of the therapy.

[0005] NK cells are the first line of defense for human immune cells, and are used for non-specific anti-tumor and anti-viral infection, removal of necrotic cells and fat waste, and anti-aging cells. NK cells have a broad spectrum of anti-tumor effects and do not show tumor-killing specificity and MHC restriction. When other immune cells in the body (such as T and B cells) function poorly, NK cells play an important role. However, the efficiency and purity of NK cell expansion in vitro are greatly limited, which greatly limits the clinical application of NK cells.

[0006] Dendritic cells (DCs) are the body's most powerful professional antigen-presenting cells. They can efficiently absorb, process and present antigens. Immature DCs have strong migration ability, and mature DCs can effectively activate naive T cells. They are at the center of initiating, regulating and maintaining immune responses.

[0007] (II) Types of human dendritic cells (DCs) and their functions in activating T cells and presenting antigens

[0008] Human dendritic cells originate from hematopoietic stem cells. DCs can be derived from two pathways: 1. Myeloid stem cells differentiate into DCs (mDCs) under GM-CSF stimulation. These DCs share common precursors with monocytes and granulocytes, including Langerhans cells, mesothelial (or dermal) DCs, and monocyte-derived DCs. 2. They derive from lymphoid stem cells, known as lymphoid or plasmacytoid DCs (pDCs). These DCs share common precursors with T cells and NK cells. pDCs mediate anti-inflammatory responses by secreting large amounts of interferon α (IFN-α) following pathogen invasion, while mDCs play a crucial role in antigen presentation and naive T cell training. The CD11c+ subtype of mDCs is relatively abundant, while the minority CD141+ mDC subtype exhibits a robust ability to present intracellular antigens on MHC-I to CD8+ T cells, promoting their differentiation into cytotoxic T cells (CTLs). DC-mediated antigen presentation and the final differentiation of T cells into cytotoxic effector T cells are important steps in inducing antiviral and anticancer adaptive immunity.

[0009] As professional antigen-presenting cells, DCs (denaturing cytokines) not only activate T cells but also present antigens to NK cells, potentially representing another novel pathway for DCs to initiate immune responses. In the absence of inflammation, DCs enter lymphoid tissues in small numbers and slowly. While they can present antigen information, their effect on T cells is to foster tolerance rather than activation. This is related to the low expression of adhesion molecules during this period, which mediates DC adhesion and migration, and may be a key mechanism for the development of immune tolerance.

[0010] (III) The DC immune function is achieved through adhesion cascade migration

[0011] As DCs migrate, their adhesion molecule phenotype and distribution also change, regulated by a phased expression pattern. The expression of certain adhesion molecules, such as ICAM-1, gradually increases as other adhesion molecules decrease, facilitating further DC-T cell adhesion. This enhanced cell adhesion promotes the connection and binding between DCs and T lymphocytes via the antigen peptide / MHC and TCR / CD3 complexes. Furthermore, the interaction between chemokines and their receptors is a key factor in chemotactic regulation of DC migration and precise positioning.

[0012] It is generally believed that a single antigen requires dual-signal stimulation to induce T cell activation. Adhesion molecules mediate antigen presentation between DCs and T cells through a "molecular bridge" effect and synergistic signaling pathways, assisting DCs in stimulating T cell activation. As mentioned above, DCs express a variety of secondary signaling co-stimulatory molecules belonging to the adhesion molecule family, such as B7-1, B7-2, ICAM-1, ICAM-2, ICAM-3, and CD40. These components can interact with corresponding ligands expressed by T cells, such as CD28 / CTLA-4, LFA-I, and CD40L. The B7-CD28 / CTLA-4 pathway is believed to play a key role in T cell activation and is closely related to immune tolerance. The B7 family can bind to CD28 to provide a synergistic signal for T cell activation and can also bind to CTLA-4 to inhibit T cell proliferation.

[0013] DC-SIGN (DC-specific intercellular-adhesion-molecule-3 grabbing nonintegrin, also known as CD209) is a type II membrane protein with an exogenous C-type lectin. It is an adhesion receptor that is specifically and abundantly expressed on the surface of mature and immature DCs and is important for certain DC functions. For example, DC-SIGN binds to its ligand, intercellular adhesion molecule 2 (ICAM-2), which is expressed on the endothelium of vascular and lymphatic vessels. The interaction between DC-SIGN and ICAM-2 aggregates DCs and enables their transmembrane translocation, potentially crucial for DC recruitment to sites of inflammation and subsequent DC migration to lymphoid tissues. The abundant expression of DC-SIGN on DCs in peripheral tissues and lymphocytes supports this concept. The presence of DC-SIGN+ DC precursors in the blood suggests that these precursors are readily available to exit the bloodstream and reach sites of inflammation, allowing for rapid recruitment and immune surveillance at these sites. DC-SIGN can also initiate the interaction between DC and naive T cells. This function is achieved by DC-SIGN binding to another cell ligand, ICAM-3, which is expressed at high levels on the surface of naive T cells.

[0014] (IV) Deficiencies of existing DC cell therapy

[0015] DCs are rare in blood, representing less than 1% of peripheral blood mononuclear cells. Current DC therapy uses monocyte-derived DCs (MoDCs) loaded with antigen. MoDCs are mature and activated by a series of cytokines and PRR stimulators. This approach has been widely used in clinical trials of cancer vaccines, but the clinical benefit has been quite limited. This is mainly due to several apparent disadvantages of this approach: MoDCs have greater correlation with in vitro derived macrophages than primary blood-derived DCs; in addition, in vitro formed MoDCs have limited growth potential and are difficult to culture for a long time, so multiple rounds of vaccination require repeated preparation of MoDCs.

[0016] (V) Factors have important influence on the proliferation and development of immune cells

[0017] IL-15 is a cytokine that regulates the activation and proliferation of T cells and natural killer cells. IL-15 can promote the directional differentiation of CD34+ hematopoietic stem cells into NK cells. IL-15 can significantly up-regulate the proportion of NK cells, and the distribution of NK cell subsets is mainly changed from CD56dim to CD56bright cells. This may be related to the different expression states of IL15R on NK cells under different conditions. IL-15 can effectively transmit activation signals through IL-2Rβ and γ chains. IL-15 and IL-2 have a synergistic effect on stimulating the killing function of NK cells, because IL-2 cannot induce the expression of high-affinity receptors, and the addition of IL-15 promotes the expression of IL-2 high-affinity receptors, thereby promoting the killing function of NK cells.

[0018] IL15RA is a high-affinity receptor for IL-15. IL15RA, as a heterotrimer, combines with IL-2 receptor β and γ subunits to initiate signal transduction. IL-15 binds to the α subunit of IL-15R with high affinity. IL-15 also binds to the β and γ chains of the IL-2 receptor, but not to the α subunit of the IL-2 receptor. IL-15 is structurally and functionally related to IL-2. These two cytokines share some subunits of the receptor, allowing them to compete with each other and negatively regulate each other's activities.

[0019] OK432 is a freeze-dried vaccine. OK432-activated neutrophils can kill IFN-γ or TNF-α-treated cancer cells. OK432-induced neutrophils kill autologous cancer cells through the reaction between CD11b / CD18 and ICAM-1. OK432-induced monocytes can kill autologous cancer cells. After OK432 stimulates lymphocytes, LAK cell activity is shown, and even against NK cell cancer cells, the activated lymphocytes show activity.

[0020] CD52 / CDW52 is a small glycosylphosphatidylinositol (GPI)-anchored glycoprotein. It has a mature peptide consisting of only 12 amino acids and is abundantly expressed on human lymphocytes. CD52 / CDW52 may play a role in carbohydrate trafficking and targeting. It is a well-characterized target for complement-mediated cell lysis.

[0021] Among the gene modification technologies, epigenetic inheritance is an important gene modification method. Epigenetic inheritance refers to the change of gene expression without changing the DNA sequence, thereby affecting the body's phenotype. It is an important regulatory method with heritability, including DNA methylation, non-coding RNA regulation, histone modification, chromatin remodeling and other methods. Epigenetic inheritance plays an important role in the entire life process. More and more studies have shown that epigenetic regulation is not only involved in stem cell development and tissue differentiation, but is also closely related to the development of tumors, immune diseases, metabolic diseases and neurological diseases. Histone modification is one of the important epigenetic regulatory methods, including methylation, acetylation, phosphorylation and ubiquitination, etc. Cells achieve changes in chromatin conformation and regulation of gene expression through reversible modification of histones.

[0022] Histone methylation is an important histone modification mechanism that regulates various physiological and pathological processes in cells. The PRC2 complex (polycomb repressive complex 2) is a key protein complex that regulates histone methylation. It is composed of four subunits: EZH1, EZH2, Suz12, and Eed. EZH2 is the catalytic core of the PRC2 complex and can regulate the trimethylation of lysine 27 of histone H3. Studies have shown that the histone methylase EZH2 plays an important role in regulating stem cell development and immune regulation. EZH2 can regulate the development of early B cells, the plasticity of CD4+ Th1 and Th2 cell differentiation, and maintain the stability of regulatory T cells.

[0023] Therefore, EZH2 may be a new target for drug therapy, which has important scientific research value and clinical application significance.

[0024] In order to produce cell vaccines, activated cytotoxic T lymphocytes and natural killer cells with antigen presenting ability and use them for clinical treatment, it is necessary to develop new methods to prepare such cells. Summary of the Invention

[0025] The present invention aims to solve at least one of the following problems: primary blood-derived DC cells, MoDCs formed in vitro have limited growth potential and are difficult to culture for a long time, multiple rounds of vaccination require repeated preparation of MoDC cells to complete, and NK cell expansion is costly and the system is unstable.

[0026] The inventors of the present application have discovered various solutions for improving the in vitro growth potential of DC cells, enhancing the antigen presentation ability of DCs and the ability to activate CTL and NK cells.

[0027] The technical solution adopted by the present invention is:

[0028] The inventors of the present application, through creative sequence design and intellectual labor, surprisingly discovered through testing test results that by independently designing a lentiviral vector containing a target gene and transfecting PBMC cells derived from peripheral blood with the lentivirus obtained therefrom, and loading the PBMCs with multiple target influencers in stages and steps and stimulating them in combination with cytokines, engineered DCs, CTLs, and NK cells with improved lethality and antigen presentation capabilities were obtained.

[0029] The inventors first constructed lentiviral vectors carrying a self-designed recombinant NT gene and a cell-specific intercellular-adhesion-molecule-3 grabbing nonintegrin (SIGN) gene. They then transfected 293T cells with the lentiviral vector carrying the recombinant NT gene to generate lentiviral particles containing the recombinant NT and SIGN genes. These lentiviral particles were then transfected into dendritic cells (DCs) to generate a monocyte-derived dendritic cell (MoDC-NT) line, which was then loaded with antigen and stimulated with PBMCs sharing the same HLA-A as the MoDC-NT to obtain antigen-specific CTLs.

[0030] On the other hand, PBMCs were transfected with lentiviral particles carrying the recombinant NT gene to obtain engineered NT-dendritic cells (EDC-NT), and then the engineered NT-dendritic cells (EDC-NT) were infected with the aforementioned lentiviral particles carrying the SIGN gene to obtain functional NTSIGN dendritic cell (EDC-NTSIGN) strains, and PBMCs were stimulated to prepare effector cells; further, the above-mentioned EDC-NTSIGN strain was further infected with lentiviral particles carrying the HLA-A0201, HLA-A1101 and HLA-A2402 gene sequences to obtain multifunctional NT dendritic cells (E Furthermore, multifunctional NT dendritic cells (EDC-NTA) are infected with lentiviral particles carrying antigen genes to obtain antigen-specific multifunctional dendritic cells, which are then stimulated by PBMCs to obtain antigen-specific CTLs with stronger cytotoxicity. Finally, the EDC-NTSIGN strain is transfected with lentiviruses carrying cytokine and fusion protein genes to prepare a multifunctional NK trophoblast cell line (EDC-NK). The multifunctional NK trophoblast cell line (EDC-NK) is inactivated or not with a cell inactivator, and then stimulated by PBMCs. NK cells with enhanced cytotoxicity and proliferation rate are then obtained by combining cytokine culture.

[0031] The technical solutions adopted by the present invention to achieve the purpose are as follows:

[0032] In the first aspect, the present invention provides a recombinant NT gene lentiviral particle for preparing a novel immunotherapy cell, wherein the NT gene lentiviral particle is obtained by infecting a host cell with a four-plasmid virus packaging system of a lentiviral vector carrying the NT gene, and in the lentiviral vector carrying the NT gene, the recombinant NT gene consists of a nucleotide sequence fragment A, a nucleotide sequence fragment B, a nucleotide sequence fragment C and a nucleotide sequence fragment D, the nucleotide sequence of the NT gene is selected from any one of the NT1 gene and the NT2 gene, the nucleotide sequence in the NT1 gene is A, the 5′ to 3′ composition of the NT2 gene is -BCDE-, the B encodes the Tax2 gene (1-3aa), the C encodes the Tax1 gene (2-255aa), the D encodes the T2A gene, and the E encodes the Tax1 gene (227-337aa).

[0033] The nucleotide sequence of NT1 is shown in SEQ ID No. 1, the gene sequence of Tax2 (1-3aa) is shown in SEQ ID No. 2, the gene sequence of Tax1 (2-255aa) is shown in SEQ ID No. 3, and the gene sequence of Tax1 (227-337aa) is shown in SEQ ID No. 4.

[0034] In one embodiment, the structure of the NT2 gene is a 5′-3′ sequentially linked structure -Tax2(1-3aa)-Tax1(2-255aa)-T2A-Tax1(227-337aa)-, wherein the host cell is a 293T cell.

[0035] In this protocol, the Tax protein is encoded by the Tax gene in the pX gene region of the HTLV-1 genome and has a molecular weight of 40x10 3 Tax is composed of 353 amino acids, with the 48 residues at its N-terminus containing a nuclear localization signal (NLS). It is distributed in the cell nucleus. As a transactivator, Tax itself lacks DNA binding ability, but it activates viral and cellular genes through direct or indirect interactions with host cell transcription factors. These cellular transcription factors include cAMP response element binding protein / activating transcription factor (REB / ATF), nuclear factor kappa B (NFκB), and serum response factor (SRF). Through interaction with these transcription factors, Tax protein not only activates transcription of the viral long terminal repeat (LTR) itself, but also activates several immediate-early serum response genes, cytokine genes, and their receptor genes. Activation of these cellular genes leads to T cell proliferation and transformation, and, along with other factors, contributes to the development of leukemia.

[0036] In a second aspect, the present invention provides an engineered monocyte-derived dendritic cell (MoDC-NT) strain, wherein the MoDC-NT strain is obtained by transfecting dendritic cells with the recombinant NT gene lentiviral particles described in the first aspect, and the infection is promoted by simultaneously adding a lentiviral infection-promoting agent.

[0037] In one embodiment, the agent promoting lentiviral infection is a polycationic compound polybrene. In another embodiment, the agent promoting lentiviral infection is a nanopolymer.

[0038] In one embodiment, the infection time is the 7th day of the dendritic cell culture.

[0039] In one embodiment, a culture medium containing IL-4 and GM-CSF is added to the dendritic cell culture medium every other day before and after the lentiviral infection.

[0040] In one embodiment, TNF-α and LPS are added after infection. In one embodiment, TNF-α and Poly(I:C) are added after infection. In one embodiment, the concentration of TNF-α is 5 ng / ml. In one embodiment, the concentration of TNF-α is 20 ng / ml. In one embodiment, the concentration of LPS is 5 μg / ml. In one embodiment, the concentration of LPS is 20 μg / ml. In one embodiment, the concentration of Poly(I:C) is 5 μg / ml. In one embodiment, the concentration of Poly(I:C) is 50 μg / ml.

[0041] In one embodiment, all 10 cells were successfully constructed using NT-1 lentivirus, achieving cell expansion.

[0042] In one embodiment, all 10 cells were successfully constructed using NT-2 lentivirus, achieving cell expansion.

[0043] The monocyte-derived MoDC-NT cells generated using this protocol exhibit a typical dendritic cell phenotype and can undergo multiple passages. This provides a sufficient supply of autologous DCs for antigen presentation for those who require large numbers of antigen-presenting cells but cannot afford to wait for a long time. Furthermore, the cells generated using this protocol have a higher purity and can also be used for HLA matching.

[0044] The third aspect of the present invention provides an antigen-specific CTL, the preparation steps of which include: (1) loading antigen onto a monocyte-derived dendritic cell (MoDC-NT) strain prepared by the method described in the second aspect; (2) preparing PBMC from blood with the same HLA-A as the MoDC-NT described in the second aspect; (3) stimulating the PBMC obtained in (2) with the antigen-loaded monocyte-derived dendritic cells (MoDC-NT) obtained in (1), and the addition ratio can be MoDC-NT:PBMC=1:1-1:1000; (4) the culture system used is an immune cell culture medium supplemented with IL-2; and (5) continuing the culture for a period of time.

[0045] In one embodiment, (3) the addition ratio of antigen-loaded dendritic cells (MoDC-NT) and added PBMCs can be MoDC-NT:PBMC = 1: 1. In one embodiment, (3) the addition ratio of antigen-loaded dendritic cells (MoDC-NT) and added PBMCs can be MoDC-NT:PBMC = -1:1000.

[0046] In one embodiment, the monocyte-derived dendritic cell (MoDC-NT) strain is loaded with antigen by antigen peptide loading. In one embodiment, the monocyte-derived dendritic cell (MoDC-NT) strain is loaded with antigen by transfecting the antigen gene into the dendritic cell (MoDC-NT) strain.

[0047] In one embodiment, (2) the blood is derived from peripheral blood, in one embodiment, (2) the blood is derived from umbilical cord blood. In one embodiment, the culture duration is one week, in one embodiment, the culture duration is two weeks, in one embodiment, the culture duration is three weeks or longer.

[0048] In one embodiment, the PBMCs are CD3+ T cells, and in one embodiment, CD8+ T cells.

[0049] In one embodiment, M1-specific CTLs were prepared by stimulating MHC-matched PBMCs with a certain amount of MoDC-NT loaded with M1 polypeptides, in which CD8+ positive cells accounted for 89.57% of CD3+CD56- cells, while CD3+CD56+ double positive cells accounted for 3.92%, and CD3-CD56+ double positive cells accounted for 1.70%. Among them, M1Tetramer-GILGFVFTL positive cells accounted for 20.73% of CD3+CD8+ double positive cells. In this way, while killing tumors expressing M1-specific antigens in the patient's body, it can also kill other cells in the patient's body that do not have this specific antigen but have diseased cells, thereby achieving the effect of early prevention.

[0050] The fourth aspect of the present invention provides an engineered NT dendritic cell (EDC-NT) strain, wherein the preparation method of the engineered NT dendritic cell (EDC-NT) strain comprises the steps of: using the recombinant NT gene lentiviral particles described in the first aspect of the present invention to infect pre-cultured PBMCs, continuing to culture after infection, and sorting and removing CD3+ cells from the cells, and simultaneously adding a lentiviral infection agent to promote the infection. Before the lentiviral infection, the PBMCs are cultured in a culture medium containing an immune agonist and a culture system containing IL-2, respectively. After infection, the PBMCs are cultured in a culture system containing serum and IL-2.

[0051] In one embodiment, the method for preparing the engineered NT dendritic cell (EDC-NT) strain comprises the following steps: (1) isolating mononuclear cells (PBMC) from peripheral blood containing anticoagulants; (2) culturing the cells obtained in (1) with a culture medium containing an immune cell agonist for 24 hours; (3) adding IL-2 to the culture system in (2) and continuing to culture for 4-5 days; (4) infecting the cultured cells in (3) with the recombinant NT gene lentiviral particles described in the first aspect, and adding a lentiviral infection-promoting agent at the same time; (5) culturing the cells obtained in (4) with a culture medium containing serum and IL-2 for 1-2 weeks; (6) removing CD3+ cells from the cells obtained in (5) by magnetic beads or flow cytometry; and (7) continuing to culture the cells retained in (6) under the culture conditions in (5) for 2-3 months.

[0052] In one embodiment, the immune agonist is PHA; in one embodiment, the immune agonist is PMA; in one embodiment, the immune agonist is another agonist. In one embodiment, the agent promoting lentiviral infection is a polycationic compound, polybrene; in one embodiment, the agent promoting lentiviral infection is a nanopolymer. In one embodiment, the sorting method is magnetic bead sorting; in one embodiment, the sorting method is flow cytometry screening.

[0053] In one embodiment, among 10 cells cultured with NT-1 lentivirus, one cell stopped proliferating and gradually died after about one month of culture, while the remaining 9 cells were successfully constructed, with a cell construction success rate of approximately 90%.

[0054] In one embodiment, among 10 cells using NT-2 lentivirus, two cells stopped proliferating and gradually died after about 40 days of culture, while the remaining 10 cells were successfully constructed, with a cell construction success rate of approximately 100%.

[0055] In one embodiment, the NT dendritic cell (EDC-NT) strain possesses a typical DC phenotype and can achieve long-term passage and proliferation. EDC-NT can be prepared in advance for a specific individual, stored in liquid nitrogen for a long period, and then revived (or briefly expanded) for use when needed. This provides an unlimited supply of autologous DCs for antigen presentation for individuals requiring large quantities of antigen-presenting cells, with a single preparation providing lifelong use. Furthermore, these cells can be HLA-matched for use.

[0056] The fifth aspect of the present invention provides a novel immune cell, the preparation method of which comprises the following steps: (1) the NT dendritic cell (EDC-NT) strain described in the fourth aspect is loaded or not loaded with an antigen; (2) blood with the same or different HLA-A as the EDC-NT is taken to prepare PBMC; (3) the NT dendritic cells (EDC-NT) loaded or not loaded with an antigen obtained in (1) are used to stimulate the PBMC obtained in (2), and mixed culture is performed; (4) the culture system used is an immune cell culture medium supplemented with IL-2; and (5) the culture is continued for a period of time.

[0057] In one embodiment (1), the NT dendritic cell (EDC-NT) strain is loaded with antigen by antigen peptide loading.

[0058] In one embodiment (1), the NT dendritic cell (EDC-NT) strain is loaded with antigen by transfecting the antigen gene into the NT dendritic cell (EDC-NT) strain.

[0059] In one embodiment (2), the HLA-A of the PBMC and EDC-NT is the same. In one embodiment (2), the HLA-A of the PBMC and EDC-NT is different.

[0060] In one embodiment, the blood in (2) is derived from peripheral blood. In one embodiment, the blood in (2) is derived from umbilical cord blood.

[0061] In one embodiment, the PBMCs described in (3) are selected from PBMCs and in one embodiment, the PBMCs described in (3) are CD3+T cells and in one embodiment, the PBMCs described in (3) are CD8+T cells.

[0062] In one embodiment (3), the ratio of cells in the mixed culture is cell line: PBMC = 1: 1. In one embodiment (3), the ratio of cells in the mixed culture is cell line: PBMC = 1: 1000.

[0063] The sixth aspect of the present invention provides an engineered functional NTSIGN dendritic cell (EDC-NTSIGN) strain, the preparation method of which comprises the following steps: (1) repeatedly infecting the engineered NT dendritic cell (EDC-NT) strain described in the fourth aspect of the present invention with SIGN lentiviral particles; (2) adding a reagent promoting lentiviral infection simultaneously with the viral infection in step (1); (3) sorting and collecting CD209+ cells from the cells obtained in step (2); and (4) continuing to culture the cells obtained in step (3) for a period of time.

[0064] Wherein, the sequence of the SIGN gene is selected from the nucleic acid sequence shown as SEQ ID No.5.

[0065] The method for preparing the lentiviral particles of the SIGN gene comprises obtaining the particles by infecting host cells with a four-plasmid viral packaging system containing a lentiviral vector carrying the SIGN gene.

[0066] The base sequence of the new SIGN design enables it to be efficiently transcribed and translated in human immune cells, thereby fully realizing its biological activity.

[0067] In a specific embodiment, the functional NTSIGN dendritic cell (EDC-NTSIGN) strain has a typical DC phenotype and highly expresses DC-SIGN, which can achieve better signal transduction, is more conducive to activating T cells and NK cells, and further increases the possibility of clinical application.

[0068] In one embodiment, the lentiviral infection-promoting agent is a polycationic compound, polybrene; in one embodiment, the lentiviral infection-promoting agent is a nanopolymer; in one embodiment, the sorting method is magnetic bead sorting; in one embodiment, the sorting method is flow cytometry screening.

[0069] In one embodiment, the activatable effector cells are T cells.

[0070] In one embodiment, the activatable effector cells are NK cells.

[0071] In one embodiment, EDC-NTSIGN cells can better activate effector cells (T cells, NK cells) than EDC-NT cells. After 21 days of culture, the total number of cells increased by 33.23%. Figure 14 A larger number of effector cells can more easily meet clinical application requirements, reduce the pain of patients suffering from large amounts of blood drawn, and increase the possibility of patients being cured.

[0072] The seventh aspect of the present invention provides a novel immune cell, the preparation method of which comprises the following steps: (1) the functional NTSIGN dendritic cell (EDC-NTSIGN) strain described in the sixth aspect is loaded or not loaded with an antigen; (2) blood with the same or different HLA-A as the EDC-NTSIGN is taken to prepare PBMC; (3) the functional NTSIGN dendritic cell (EDC-NTSIGN) strain loaded or not loaded with an antigen obtained in (1) is used to stimulate the PBMC obtained in (2), and mixed culture is performed; (4) the culture system used is an immune cell culture medium supplemented with IL-2; and (5) the culture is continued for a period of time.

[0073] In one embodiment (1), the functional NTSIGN dendritic cell (EDC-NTSIGN) strain is loaded with antigen by antigen peptide loading.

[0074] In one embodiment (1), the functional NTSIGN dendritic cell (EDC-NTSIGN) strain is loaded with antigen by transfecting the antigen gene into the EDC-NTSIGN cell strain.

[0075] In one embodiment (2), the HLA-A of the PBMC is the same as that of EDC-NTSIGN. In one embodiment (2), the HLA-A of the PBMC is different from that of EDC-NTSIGN.

[0076] In one embodiment, the blood in (2) is derived from peripheral blood. In one embodiment, the blood in (2) is derived from umbilical cord blood.

[0077] In one embodiment, the PBMCs described in (3) are selected from PBMCs and in one embodiment, the PBMCs described in (3) are CD3+T cells and in one embodiment, the PBMCs described in (3) are CD8+T cells.

[0078] In one embodiment (3), the ratio of cells in the mixed culture is cell line: PBMC = 1: 1. In one embodiment (3), the ratio of cells in the mixed culture is cell line: PBMC = 1: 1000.

[0079] The eighth aspect of the present invention provides an engineered multifunctional NT dendritic cell (EDC-NTA) strain, wherein the preparation method of the EDC-NTA strain comprises the following steps: (1) repeatedly infecting the EDC-NTSIGN cell line described in the sixth aspect of the present invention with lentiviral particles carrying nucleotide sequences of one or more combinations of HLA-A0201, HLA-A1101 and HLA-A2402 genes; (2) sorting and collecting HLA-A combination-positive cells from the cells obtained in step (1); and (3) continuing to culture the cells obtained in step (1) for a period of time.

[0080] The method for preparing lentiviral particles carrying the HLA-A0201, HLA-A1101 or HLA-A2402 genes respectively comprises the steps of: obtaining the obtained lentiviral particles by infecting host cells with a four-plasmid viral packaging system containing a lentiviral vector carrying the HLA-A0201, HLA-A1101 or HLA-A2402 gene respectively, wherein the host cells are 293T cells.

[0081] In one embodiment, the HLA is HLA-A0201, wherein the nucleotide sequence of the HLA-A0201 is shown as SEQ ID No. 6. In one embodiment, the HLA is HLA-A1101, wherein the nucleotide sequence of the HLA-A1101 (HA-Tag) is shown as SEQ ID No. 7. In one embodiment, the HLA is HLA-A2402, wherein the nucleotide sequence of the HLA-A2402 is shown as SEQ ID No. 8. In one embodiment, the HLA is a combination of HLA-A0201 and HLA-A1101 (HA-Tag); in one embodiment, the HLA is a combination of HLA-A0201 and HLA-A2402 (HA-Tag); in one embodiment, the HLA is a combination of HLA-A1101 (HA-Tag) and HLA-A2402 (HA-Tag); in one embodiment, the HLA is a combination of HLA-A0201, HLA-A1101 (HA-Tag), and HLA-A2402 (HA-Tag).

[0082] The resulting multifunctional NT dendritic cell (EDC-NTA, i.e., EDC-NT-A0201 / 1101 / 2402) strain simultaneously possesses HLA-A0201, HLA-A1101, and HLA-A2402, three MHC class I molecules with the highest prevalence in the Chinese population. These cells can be used to present antigens in vitro to cells bearing any of these three MHC class I molecules, enabling the large-scale production of target-specific CTLs.

[0083] The ninth aspect of the present invention provides a novel immune cell, the preparation method of which comprises the following steps: (1) taking the multifunctional NT dendritic cell (EDC-NTA) strain prepared by the method described in the eighth aspect of the present invention and mixing it with PBMC derived from anticoagulant blood; (2) using a culture system of immune cell culture medium supplemented with IL-2; and (3) continuing the culture for a period of time.

[0084] In one embodiment, the anticoagulated blood in (1) is derived from peripheral blood. In one embodiment, the anticoagulated blood in (1) is derived from umbilical cord blood.

[0085] In one embodiment, the PBMCs in (1) are selected from PBMCs and in one embodiment, the PBMCs in (3) are CD3+T cells and in one embodiment, the PBMCs in (3) are CD8+T cells.

[0086] In one embodiment (3), the ratio of cells in the mixed culture is cell line: PBMC = 1: 1. In one embodiment (3), the ratio of cells in the mixed culture is cell line: PBMC = 1: 1000.

[0087] The tenth aspect of the present invention provides an antigen-specific multifunctional dendritic cell line, the preparation steps of which include: (1) combining the antigen gene with the IκBα gene and connecting it to a lentiviral vector plasmid; (2) infecting the multifunctional NT dendritic cell (EDC-NTA) described in the eighth aspect of the present invention with a lentivirus carrying the sequence combination described in (1); (3) adding a reagent that promotes lentiviral infection during the viral infection in step (2); (4) repeating steps (2) and (3) twice; (5) screening and collecting the cells obtained that are positive for the antigen gene; and (6) continuing to culture. a period of time; wherein, (wherein, (1) the structure of the combination gene is selected from one of the general formulas IκBα(1-48aa)-antigen gene-IκBα(281-317aa), IκBα(1-317aa)-T2A-antigen gene, IκBα(1-48aa)-antigen peptide mini gene-IκBα(281-317aa) and IκBα(1-317aa)-T2A-antigen peptide mini gene; (5) the screening includes magnetic bead or flow cytometry sorting or cell monoclonal screening or resistance screening.

[0088] In one embodiment, the agent promoting lentiviral infection is a polycationic compound polybrene. In one embodiment, the agent promoting lentiviral infection is another agent promoting lentiviral infection.

[0089] In one embodiment, the screening method in (3) is magnetic bead sorting; in one embodiment, the screening method in (3) is flow cytometry sorting; in one embodiment, the screening method in (3) is cell monoclonal screening; in one embodiment, the screening method in (3) is resistance screening.

[0090] In one embodiment, the resistance screening is puromycin screening.

[0091] In one embodiment, the concentration of puromycin is 5 ug / ml.

[0092] In one embodiment, the concentration of puromycin is 10 ug / ml.

[0093] In one embodiment, the concentration of puromycin is 8 ug / ml.

[0094] In one embodiment, the structure of the combined gene (1) is the general formula IκBα(1-48aa)-antigen gene-IκBα(281-317aa).

[0095] In one embodiment, (1) the structure of the combined gene is the general formula IκBα(1-317aa)-T2A-antigen gene.

[0096] In one embodiment, the structure of the combined gene (1) is the general formula IκBα(1-48aa)-antigen peptide mini gene-IκBα(281-317aa).

[0097] In one embodiment, (1) the structure of the combined gene is a mini gene of the general formula IκBα(1-317aa)-T2A-antigen peptide.

[0098] The antigen gene or antigen peptide gene is selected from the full antigen gene or partial antigen gene of tumor antigen genes or viral antigen genes including but not limited to Mesothelin, CEA, NYESO-1, AFP, TPA, TPS, EGFR, VEGF, PDGFR, ALK, CA125, CA153, CA199, CA242, CA724, S100, HCG, HCH, B2-MG, MUC1 / 16, WT1, GD2, GPC3, PRAME, FOLR1, MAGEA3, Her2, Survivin, CD19, CD20, CD22, CD47, CD73, CD117, PD1, PD-L1, BCMA, etc.

[0099] IκBα is a newly engineered sequence, artificially synthesized for use. In one embodiment, the specific antigen is selected from the group consisting of mesothelin, CEA, and NYESO1. The nucleotide coding sequence of IκBα is shown in SEQ ID No. 9, and the nucleotide coding sequence of T2A is shown in SEQ ID No. 10.

[0100] In some embodiments, the antigen-specific multifunctional dendritic cells include a mesothelin-specific multifunctional dendritic cell (EDC-Mesothelin) strain, a NYESO1-specific multifunctional dendritic cell (EDC-NYESO1) strain, a CEA-specific multifunctional dendritic cell (EDC-CEA) strain, and other antigen-specific dendritic cell strains corresponding to antigen genes. The mesothelin gene sequence is shown in SEQ ID No. 11, the CEA gene sequence is shown in SEQ ID No. 12, and the NYESO1 gene sequence is shown in SEQ ID No. 13. The amino acid sequence of the antigen peptide minigene is a repeated connection sequence of multiple -antigen polypeptide-AYY-, and the nucleotide sequence of AYY is shown in SEQ ID No. 19: GCTTACTAC.

[0101] In a specific embodiment, the specific antigen is mesothelin, and the lentiviral particle is a mesothelin lentiviral particle.

[0102] The Mesothelin lentivirus particle is obtained by infecting host cells T293 cells with a mixture of a Mesothelin lentivirus vector and a viral transfection plasmid system.

[0103] In a specific embodiment, the specific antigen is Mesothelin, and the specific multifunctional dendritic cell (EDC-Mesothelin) strain, wherein the nucleotide sequence linkage mode is as shown in Figure 17 .

[0104] In a specific embodiment, the specific antigen is CEA, and the lentivirus particle is a CEA lentivirus particle.

[0105] The CEA lentivirus particle is obtained by infecting host cells T293 cells with a mixture of a CEA lentivirus vector and a viral transfection plasmid system.

[0106] In a specific embodiment, the specific antigen is CEA, and the specific multifunctional dendritic cell (EDC-CEA) strain, wherein the nucleotide sequence linkage mode is as shown in Figure 18 .

[0107] In a specific embodiment, the specific antigen is NY-ESO-1, and the lentivirus particle is a NY-ESO-1 lentivirus particle.

[0108] The NY-ESO-1 lentivirus particle is obtained by infecting host cells T293 cells with a mixture of a NY-ESO-1 lentivirus vector and a viral transfection plasmid system.

[0109] In a specific embodiment, the specific antigen is NY-ESO-1, and the specific multifunctional dendritic cell (EDC-NYESO1) strain, wherein the NY-ESO-1 nucleotide sequence linkage mode is as shown in Figure 19 .

[0110] The nucleotide coding sequence of IκBα is shown in SEQ ID No. 9, the nucleotide coding sequence of T2A is shown in SEQ ID No. 10, the nucleotide coding sequence of Mesothelin is shown in SEQ ID No. 11, the nucleotide coding sequence of CEA is shown in SEQ ID No. 12, and the nucleotide sequence of NY-ESO-1 is shown in SEQ ID No. 13.

[0111] The IκBα is a newly engineered sequence, artificially synthesized for use. IκBα promotes the rapid intracellular degradation of mesothelin, CEA, and NY-ESO-1, leading to their presentation by MHC class I molecules. This cell line can be used to generate mesothelin-, CEA-, and NY-ESO-1-specific CTLs, providing a novel treatment option for patients with mesothelin-, CEA-, and NY-ESO-1-positive tumors.

[0112] The eleventh aspect of the present invention provides a novel immune cell, the preparation method of which comprises the steps of: (1) co-culturing the antigen-specific multifunctional dendritic cells described in the tenth aspect of the present invention with PBMC prepared from blood, and (2) continuing the culture for a period of time.

[0113] In one embodiment, the blood is peripheral blood. In another embodiment, the blood is umbilical cord blood.

[0114] In one embodiment, the starting cells are PBMC cells.

[0115] In one embodiment, the starting cells are CD3+ T cells.

[0116] In one embodiment, the starting cells are CD8+ T cells.

[0117] In one embodiment, the novel immune cell is an antigen-specific CTL.

[0118] In one embodiment, the novel immune cells are antigen-specific NK cells.

[0119] In one embodiment, the blood-derived cells match the antigen-specific multifunctional dendritic cells MHC class I molecules of the tenth aspect of the present invention. In one embodiment, the blood-derived cells are HLA-A0201.

[0120] In one embodiment, the ratio of cells in the mixed culture is cell line: PBMC = 1:1. In one embodiment, the ratio of cells in the mixed culture is cell line: PBMC = 1:1000. In one embodiment, the culture system used is an immune cell culture medium supplemented with IL-2. In one embodiment, the culture duration is one week; in one embodiment, the culture duration is two weeks; in one embodiment, the culture duration is three weeks, or in one embodiment, the culture duration is other durations. In one embodiment, the PBMCs are CD3+ T cells; in one embodiment, the PBMCs are CD8+ T cells.

[0121] The antigen is selected from any one or more combinations of tumor antigens or viral antigens such as Mesothelin, CEA, NYESO-1, AFP, TPA, TPS, EGFR, VEGF, PDGFR, ALK, CA125, CA153, CA199, CA242, CA724, S100, HCG, HCH, β2-MG, MUC1 / :16, WT1, GD2, GPC3, PRAME, FOLR1, MAGEA3, Her2, Survivin, CD19, CD20, CD22, CD47, CD73, CD117, PD1, PD-L1, BCMA, etc.

[0122] In one embodiment, the target antigen is selected from Mesothelin, NYESO1 and CEA.

[0123] In a specific embodiment, the specific antigen is Mesothelin, and the CD3+CD8+ double-positive cells in the prepared CTL are as high as 89.57%, while the proportion of CD3+CD56+ double-positive cells can also reach 17.10%, and the proportion of CD3-CD56+ cells is 4.30%. In this way, while killing tumors expressing Mesothelin-specific antigens in the patient's body, it can also kill other cells in the patient's body that do not have this specific antigen but have diseased cells, thereby achieving the effect of early prevention.

[0124] In a specific embodiment, the specific antigen is CEA, and the CD3+CD8+ double-positive cells in the prepared CTL are as high as 86.86%. At the same time, the proportion of CD3+CD56+ double-positive cells can also reach 23.85%, and the proportion of CD3-CD56+ cells is 3.64%. In this way, while killing tumors expressing CEA-specific antigens in the patient's body, it can also kill other cells in the patient's body that do not have this specific antigen but have diseased cells, thereby achieving the effect of early prevention.

[0125] In a specific embodiment, the specific antigen is NY-ESO-1, and the proportion of CD3+CD8+ double-positive cells in the prepared CTL is as high as 89.74%. At the same time, the proportion of CD3+CD56+ double-positive cells can also reach 14.39%, and the proportion of CD3-CD56+ double-positive cells is 2.34%. In this way, while killing tumors expressing the NY-ESO-1 specific antigen in the patient's body, it can also kill other cells in the patient's body that do not have this specific antigen but have diseased cells, thereby achieving the effect of early prevention.

[0126] The twelfth aspect of the present application provides a multifunctional NK feeder cell (EDC-NK), and a preparation method thereof, comprising the steps of: (1) repeatedly infecting the functional NT SIGN dendritic cell (EDC-NT SIGN) strain of the fifth aspect of the present application with a plurality of lentivirus particles carrying specific cytokines in sequence, respectively; (2) adding a slow virus infection promoting agent during the virus infection of step (1); (3) screening and collecting cells positively expressing specific cytokine genes from the cells obtained in step (2); and (4) continuing to culture the cells obtained in step (3) for a period of time.

[0127] In an embodiment, the cytokines include 4-1BBL, MICA, and IL-15&CD8 fusion protein, and the lentivirus particles are obtained by sequentially infecting host cells with a four-plasmid virus packaging system containing lentivirus vectors carrying one of the genes of 4-1BBL, MICA, and IL-15&CD8 fusion protein, respectively; the nucleotide sequences of the genes of 4-1BBL, MICA, signal peptide, IL-15, and CD8 are shown in SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 18, respectively. The nucleotide composition mode of the IL-15&CD8 fusion protein is -signal peptide (87bp)-extracellular region (IL-15 Mat-peptide (342b)-Hinge (141bp))-transmembrane region (TM (72bp)). The nucleotide sequence of the signal peptide gene is shown in SEQ ID No. 16.

[0128] The structure diagram of the IL-15&CD8 fusion protein is shown in Figure 25 .

[0129] The preparation method of the lentivirus particles carrying the cytokine genes comprises the steps of:

[0130] (1) designing a cytokine-encoding nucleotide sequence and its constituent nucleotide fragments;

[0131] (2) enzyme digestion and ligation to construct a lentivirus vector of the cytokine-encoding nucleotide sequence;

[0132] (3) infecting host cells with the lentivirus vector of the cytokine-encoding nucleotide sequence to obtain lentivirus particles carrying the cytokine-encoding sequence.

[0133] 4-1BBL interacts with the NK cell-activating receptor 4-1BB to restore NK cell reactivity. MICA is a ligand for the activating receptor NKG2D, which is widely expressed on human NK cells. IL-15 can promote the directed differentiation of CD34+ hematopoietic stem cells into NK cells, significantly increasing the proportion of NK cells and shifting the NK cell subset distribution from predominantly CD56dim to predominantly CD56bright cells. The successful construction of EDC-NK provides a new option for NK cell production. This approach allows for pre-antigen stimulation of EDC-NK cells during NK cell production, generating antigen-specific cells. It may also replace K562 or other trophoblast cells currently used in clinical practice for NK cell production.

[0134] A thirteenth aspect of the present invention provides a ready-to-use multifunctional NK trophoblast (EDC-NK) cell line. The preparation method is selected from the group consisting of treating an EDC-NK cell suspension with one of Co60 irradiation, gamma irradiation, fixation with a chemical histocytological fixative, and other treatments capable of inactivating cell proliferation.

[0135] In one embodiment, the multifunctional NK trophoblast cell (EDC-NK) strain is inactivated by fixing the cells with a tissue cell fixative such as paraformaldehyde. In one embodiment, the multifunctional NK trophoblast cell (EDC-NK) strain is inactivated by treating the cells with other treatments that can cause the cells to lose their proliferation activity, but the cells can maintain their function and application safety. It can be used as a vaccine or cell preparation for clinical or scientific research use.

[0136] In one embodiment, the steps for preparing the ready-to-use multifunctional NK trophoblast cell (EDC-NK) line include: (1) preparing the multifunctional NK trophoblast cell (EDC-NK) line described in the ninth aspect of the present invention into a cell suspension; (2) irradiating and inactivating the cell suspension obtained in (1) with Co60 rays; (3) cooling the cells obtained after irradiation in (2) to -80°C using a cell freezing solution; and (4) storing the cells obtained in (3).

[0137] In one embodiment, the multifunctional NK trophoblast cell (EDC-NK) line is inactivated by Co60 radiation; in one embodiment, the irradiation dose is Dmin=19.85 kGy.

[0138] Irradiated EDC-NK cells are inactivated and cannot proliferate further, but they retain their original intracellular and surface molecular structures, thus preserving their antigen-presenting capacity and activating T and NK cells, making them safer for clinical applications. Irradiated cells can be used directly or frozen and revived.

[0139] In one embodiment, a Co60 BFT-II (2#) irradiation device is used, with circular orbital fractionated irradiation for irradiation treatment, and the cell suspension absorbed dose is: Dmin = 19.85 kGy. In one embodiment, the cell inactivation method is other radiation, or in one embodiment, the cell inactivation method is a chemical reagent.

[0140] The fourteenth aspect of the present invention provides a novel immune cell, the preparation steps of which include: (1) taking the cell line described in the fourth, sixth, eighth, tenth, twelfth or thirteenth aspect of the present invention and mixing it with PBMC derived from peripheral blood or umbilical cord blood, and the cell ratio can be cell line:PBMC=1:1-1:1000; (2) the culture system used is an immune cell culture medium supplemented with IL-2; (3) a histone methyltransferase (EZH2) inhibitor and / or cytokine can be added to the culture system described in step (2); (4) continuing the culture for a period of time.

[0141] In one embodiment, the PBMCs described in (1) are derived from peripheral blood, or in one embodiment, the PBMCs described in (1) are derived from umbilical cord blood. In one embodiment, the cell ratio in (1) is cell line:PBMC = 1:1; in one embodiment, the cell ratio in (1) is cell line:PBMC = 1:1000.

[0142] In one embodiment, the culture duration of (4) is selected from one week, two weeks, three weeks or other durations.

[0143] In one embodiment, a histone methylase (EZH2) inhibitor is added to the culture medium used to activate PBMCs for NK cell production. In one embodiment, the inhibitor is preferably UNC1999; in another embodiment, the inhibitor is preferably EPZ005687.

[0144] In one embodiment, cytokines are added to the culture medium used to activate PBMCs for NK cell production. In one embodiment, the cytokines are preferably one or more combinations of IL-15, IL-15RA, OK432, and CD52. In one embodiment, the cytokine is IL-15. In one embodiment, the cytokine is IL-15. In one embodiment, the cytokine is IL-15RA. In one embodiment, the cytokine is OK432. In one embodiment, the cytokines are IL-15 and OK432. In one embodiment, the cytokines are IL-15 and CD52. In one embodiment, the cytokines are IL-15, OK432, and CD52.

[0145] In one embodiment, NK cells are prepared by adding (irradiated or non-irradiated) EDC-NK and multi-cytokine stimulation to PBMC.

[0146] In one embodiment, the multiple cytokines are any combination of IL15, IL-15RA, OK432, and CD52.

[0147] In one embodiment, the combination cytokine is IL15, IL-15RA, OK432, and CD52.

[0148] IL-15 is a cytokine that regulates the activation and proliferation of T cells and natural killer cells. IL-15 can promote the directed differentiation of CD34+ hematopoietic stem cells into NK cells. IL-15 significantly increases the proportion of NK cells, and the distribution of NK cell subsets shifts from being predominantly CD56dim to being predominantly CD56bright cells. This may be related to the varying expression of IL15R on NK cells under different conditions. IL-15 can effectively transmit activation signals through the IL-2Rβ and γ chains. IL-15 and IL-2 synergistically stimulate the cytotoxic function of NK cells. This is because IL-2 does not induce the expression of high-affinity receptors. However, the addition of IL-15 promotes the expression of high-affinity IL-2 receptors, thereby enhancing NK cell cytotoxicity. An increase in the CD56bright subset significantly promotes cytokine secretion, while an increase in the CD56dim subset enhances NK cell cytotoxicity. However, the CD56dim and CD56bright subsets are not functionally distinct.

[0149] IL15RA is the high-affinity receptor for IL-15. IL15RA associates as a heterotrimer with the IL-2 receptor β and γ subunits to initiate signaling. IL-15 binds with high affinity to the α subunit of the IL-15R. IL-15 also binds to the β and γ chains of the IL-2 receptor, but not to the α subunit of the IL-2 receptor. IL-15 is structurally and functionally related to IL-2. These two cytokines share some receptor subunits, allowing them to compete and negatively regulate each other's activity.

[0150] OK432 is a freeze-dried vaccine. Neutrophils activated by OK432 can kill cancer cells treated with IFN-γ or TNF-α. The cytotoxicity of OK432-induced neutrophils against autologous cancer cells is achieved through the interaction between CD11b / CD18 and ICAM-1. Monocytes induced by OK432 can also kill autologous cancer cells. OK432-stimulated lymphocytes exhibit LAK cell activity, and these activated lymphocytes even show activity against cancer cells that are resistant to NK cells.

[0151] CD52 / CDW52 is a small glycosylphosphatidylinositol (GPI)-anchored glycoprotein. It has a mature peptide consisting of only 12 amino acids and is abundantly expressed on human lymphocytes. CD52 / CDW52 may play a role in carbohydrate trafficking and targeting. It is a well-characterized target for complement-mediated cell lysis.

[0152] Among the gene modification technologies, epigenetic inheritance is an important gene modification method. Epigenetic inheritance refers to the change of gene expression without changing the DNA sequence, thereby affecting the body's phenotype. It is an important regulatory method with heritability, including DNA methylation, non-coding RNA regulation, histone modification, chromatin remodeling and other methods. Epigenetic inheritance plays an important role in the entire life process. More and more studies have shown that epigenetic regulation is not only involved in stem cell development and tissue differentiation, but is also closely related to the development of tumors, immune diseases, metabolic diseases and neurological diseases. Histone modification is one of the important epigenetic regulatory methods, including methylation, acetylation, phosphorylation and ubiquitination, etc. Cells achieve changes in chromatin conformation and regulation of gene expression through reversible modification of histones.

[0153] Histone methylation is an important histone modification mechanism that regulates various physiological and pathological processes in cells. The PRC2 complex (polycomb repressive complex 2) is a key protein complex that regulates histone methylation. It is composed of four subunits: EZH1, EZH2, Suz12, and Eed. EZH2 is the catalytic core of the PRC2 complex and can regulate the trimethylation of lysine 27 of histone H3. Studies have shown that the histone methylase EZH2 plays an important role in regulating stem cell development and immune regulation. EZH2 can regulate the development of early B cells, the plasticity of CD4+ Th1 and Th2 cell differentiation, and maintain the stability of regulatory T cells.

[0154] The use of IL15, IL-15RA, OK432 and CD52 in combination with small molecule inhibitors of EZH2 can better promote the activation and proliferation of NK cells, improve the purity of NK cells in the final product, and at the same time change the epigenetics of NK cells, thereby increasing the killing activity of target cells.

[0155] In one embodiment, NK cells are prepared by stimulating PBMCs with EDC-NK (irradiated or non-irradiated) and a histone methylase (EZH2) inhibitor.

[0156] In one embodiment, NK cells are prepared by adding (irradiated or non-irradiated) EDC-NK and histone methylase (EZH2) inhibitors and stimulating PBMC with multiple cytokines.

[0157] In one embodiment, PBMCs activated by EDC-NK stimulation can produce NK (CD3-CD56+) and NKT (CD3+CD56+), accounting for 81.11% and 4.45% respectively, and the cells expanded 253 times after 21 days of culture.

[0158] In one embodiment, PBMCs activated with EDC-NK stimulation combined with cytokines can produce NK (CD3-CD56+) and NKT (CD3+CD56+) cells accounting for 95.43% and 0.86%, respectively, with a 394-fold cell expansion after 21 days of culture, further improving NK purity and cell expansion multiples.

[0159] In one embodiment, the use of EDC-NK stimulation to activate PBMCs in combination with histone methyltransferase (EZH2) inhibitors and cytokines can better activate NK (CD3-CD56+) and increase its proportion; at the same time, the NKG2D+CD56+ cell subset also increases with the addition of EZH2 inhibitors; the killing ability of effector cells against target cells is also significantly improved after the addition of EZH2 inhibitors; the inhibitor has almost no effect on the cell expansion efficiency during the 21-day culture cycle.

[0160] In one embodiment, the PMBCs in (1) have the same HLA-A as the cell line in step (1); in one embodiment, the PMBCs in (1) have different HLA-A than the cell line in step (1).

[0161] In one embodiment, (1) the ratio of cell line addition is cell line:PBMC=1:1. In one embodiment, (1) the ratio of cell line addition is cell line:PBMC=1:1000.

[0162] In one embodiment, the cell line described in (1) is loaded with antigen before mixed culture. In one embodiment, the cell line described in (1) is not loaded with antigen before mixed culture.

[0163] In one embodiment, the antigen is loaded by antigen peptide loading; in one embodiment, the antigen is loaded by transfecting the antigen gene into a cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figure 1 .The structural diagram of the NT-2 nucleic acid sequence fragment of the present invention

[0165] Figure 2 .pCDH-EF1-MCS-T2A-copGFP plasmid map

[0166] Figure 3Gel electrophoresis results after double enzyme digestion of plasmid pUC57-NT or pCDH-EF1-MCS-T2A-copGFP

[0167] Note: ①: DNA Marker; ②: pCDH-EF1-MCS-T2A-copGFP double enzyme digestion; ③: pUC57-NT double enzyme digestion;

[0168] Figure 4 . Gel electrophoresis verification of double enzyme digestion of NT-linked vector plasmid results

[0169] Note: ①: DNA Marker; ②: pCDH-EF1-MCS-T2A-copGFP-NT double enzyme digestion;

[0170] Figure 5 .pCDH-EF1-MCS-T2A-Tdtomato plasmid map

[0171] Figure 6 Gel electrophoresis results after double enzyme digestion of plasmid pUC57-SIGN or pCDH-EF1-MCS-T2A-Tdtomato

[0172] Note: ①: DNA Marker; ②: pCDH-EF1-MCS-T2A-Tdtomato double enzyme digestion; ③: pUC57-SIGN double enzyme digestion;

[0173] Figure 7 . Gel electrophoresis verification of double enzyme digestion of SIGN connection vector plasmid results

[0174] Note: ①: DNA Marker; ②: pCDH-EF1-MCS-T2A-Tdtomato-SIGN double enzyme digestion;

[0175] Figure 8 Flow cytometry results of monocyte-derived dendritic cell (MoDC-NT) lines

[0176] Figure 9 Morphology of Monocyte-derived Dendritic Cell (MoDC-NT) Line

[0177] Figure 10 Flow cytometry results of MoDC-NT / M1 stimulating PBMC to generate M1 peptide-specific CTL cells

[0178] Figure 11 Flow cytometry results of NT dendritic cell (EDC-NT) lines carrying NT (NT-1 or NT-2) genes

[0179] Figure 12NT dendritic cell (EDC-NT) lines derived from PBMC infected with lentiviral particles carrying the NT (NT-1 or NT-2) gene

[0180] Figure 13 Flow cytometry results of functional NTSIGN dendritic cell (EDC-NTSIGN) strain

[0181] Figure 14 Proliferation curves of effector cells (T, NK) activated by EDC-NT and EDC-NTSIGN cells

[0182] Figure 15 Flow cytometry phenotype of multifunctional NT dendritic cells (EDC-NTA, i.e., EDC-NT-A0201 / 1101 / 2402)

[0183] Figure 16 .pCDH-EF1-MCS-T2A-Puro plasmid map

[0184] Figure 17 .Mesothelin nucleic acid sequence connection structure diagram

[0185] Figure 18 .CEA nucleic acid sequence connection structure diagram

[0186] Figure 19 .NY-ESO-1 nucleic acid sequence connection structure diagram

[0187] Figure 20 Flow cytometry analysis of mesothelin-specific CTLs prepared from PBMCs stimulated with EDC-Mesothelin

[0188] Figure 21 Flow cytometry analysis of CEA-specific CTL prepared from PBMC stimulated with EDC-CEA

[0189] Figure 22 Flow cytometry analysis of NY-ESO-1-specific CTLs prepared from PBMCs stimulated with EDC-NYESO1

[0190] Figure 23 Analysis of the killing ability of specific CTL cells stimulated by EDC-Mesothelin

[0191] A. Western Blot detection of mesothelin expression in FIB and FIB / A0201 / Mesothelin;

[0192] B. Flow cytometry analysis of HLA-A0201 expression;

[0193] C. Cytotoxic activity of PBMCs stimulated by EDC-Mesothelin against FIB / A0201 and FIB / A0201 / Mesothelin;

[0194] D. Add HLA-A2-blocking antibody to analyze the degree of MHC I restriction of cytotoxicity.

[0195] Figure 24 Analysis of the killing ability of specific CTL cells stimulated by EDC-NYESO1

[0196] A. Western Blot analysis of NY-ESO-1 expression in FIB, A549 / NYESO1dim, and A549 / A0201 / NYESO1;

[0197] B. Flow cytometry analysis of HLA-A0201 expression;

[0198] C. Analysis of the cytotoxic activity of cells generated by PBMCs stimulated by EDC-NYESO1 against FIB and A549 / A0201 / NYESO1.

[0199] Figure 25 .Schematic diagram of the IL-15 & CD8α fusion protein structure

[0200] Figure 26 Flow cytometry analysis of a multifunctional NK trophoblast cell line (EDC-NK) derived from NT dendritic cells (EDC-NTSIGN) infected with 4-1BBL, MICA, IL-15 & CD8 Fusion protein lentivirus.

[0201] Figure 27 Flow cytometry analysis of NK prepared from PBMC stimulated with inactivated EDC-NK for three weeks

[0202] Figure 28 .Use EDC-NK to stimulate and activate PBMC to prepare the total number of NK and NKT cell expansion curves

[0203] Figure 29 Flow cytometry analysis of NK cells prepared by PBMC stimulation with EDC-NK combined with cytokines IL-15RA, OK432, and CD52

[0204] Figure 30 .Total cell expansion curve of NK and NKT cells prepared by PBMC activated by EDC-NK combined with cytokine stimulation

[0205] Figure 31Cytotoxicity test of NK cells prepared from PBMC stimulated with EDC-NK combined with histone methylase (EZH2) inhibitor against target cells A549 after 14 days of culture

[0206] Figure 32 Total cell expansion curve after PBMC stimulation with EDC-NK combined with histone methylase (EZH2) inhibitor and cytokines DETAILED DESCRIPTION

[0207] Materials and reagents:

[0208]

[0209]

[0210]

[0211] The technical solution of the present invention is further described below in conjunction with exemplary experiments. However, the present invention is not limited to these embodiments.

[0212] Example

[0213] Example 1. Construction of NT gene lentiviral vector

[0214] Construction of NT viral vector

[0215] The newly designed NT base sequence was artificially synthesized, with Xba I and BamH I restriction sites set at both ends. NT can be NT1 or NT2. The sequence is as follows:

[0216] NT-1 (SEQ ID No. 1):

[0217]

[0218] The newly designed NT base sequence enables it to be efficiently transcribed and translated in human immune cells, thereby fully realizing its biological activity.

[0219] The structure of NT-2 is as follows Figure 1 As shown, the structure is composed of -Tax2(1-3aa)-Tax1(2-255aa)-T2A-Tax1(227-337aa)-,

[0220] Tax2(1-3aa)(SEQ ID No.2):Atggcccac

[0221] Tax1 (2-255aa) (SEQ ID No. 3):

[0222]

[0223]

[0224] T2A (SEQ ID No. 10):

[0225]

[0226] Tax1(227-337aa)(SEQ ID No.4):

[0227]

[0228] The lentiviral vector plasmid used to connect the NT gene is pCDH-EF1-MCS-T2A-copGFP, as Figure 2 shown.

[0229] pUC57-NT and pCDH-EF1-MCS-T2A-copGFP were double-digested with Xba I and BamH I, respectively. The reaction system is as follows:

[0230]

[0231] NOTE: Plasmid represents pUC57-NT or pCDH-EF1-MCS-T2A-copGFP.

[0232] Gel electrophoresis was used to detect and recover the target fragments, and UV gel imaging was performed. Figure 3 shown.

[0233] T4 Ligase was used to connect the recovered NT and pCDH-EF1-MCS-T2A-copGFP fragments. The reaction system was as follows:

[0234]

[0235] The ligation product was heat-shocked and transformed into trans 1 competent state, incubated for 1 hour, and plated for overnight culture; a single clone was picked and inoculated into LB culture medium, and plasmid was extracted after overnight culture. The plasmid size was verified by double enzyme digestion and gel electrophoresis. The UV gel imaging was shown in the figure below. Figure 4 shown.

[0236] Example 2. Construction of SIGN viral vector

[0237] The newly designed SIGN gene base sequence was artificially synthesized, and Xba I and BamH I restriction sites were set at both ends. The sequence is as follows:

[0238] SIGN (SEQ ID No. 5):

[0239]

[0240] The newly designed SIGN gene base sequence enables it to be efficiently transcribed and translated in human immune cells, thereby fully realizing its biological activity.

[0241] The lentiviral vector plasmid used to connect the SIGN gene is pCDH-EF1-MCS-T2A-tdTomato, such as Figure 5 shown.

[0242] pUC57-SIGN and pCDH-EF1-MCS-T2A-tdTomato were double-digested with BamH I and EcoR I, respectively. The reaction system is as follows:

[0243]

[0244] Note: Plasmid indicates pUC57-SIGN or pCDH-EF1-MCS-T2A-tdTomato.

[0245] Gel electrophoresis was used to detect and recover the target fragments. The UV gel imaging was shown in the figure below. Figure 6 shown.

[0246] T4 Ligase was used to ligate the recovered SIGN fragment with the pCDH-EF1-MCS-T2A-tdTomato fragment to obtain pCDH-EF1-MCS-T2A-tdTomato-SIGN. The reaction system was as follows:

[0247]

[0248]

[0249] The ligation product was heat-shocked and transformed into trans 1 competent state, incubated for 1 hour, and plated for overnight culture; a single clone was picked and inoculated into LB culture medium. After overnight culture, plasmid was extracted and double enzyme digestion was performed to verify the plasmid size. The gel electrophoresis after enzyme digestion was verified by UV imaging as shown in the figure below. Figure 7 shown.

[0250] Example 3. Lentivirus Preparation

[0251] One day before transfection, 293T cells were digested and passaged to The cell culture dish was cultured in Opti-MEM (+10% FBS) at 37°C for approximately 18-24 hours, and the cell confluence was approximately 80%.

[0252] The four plasmids pLP1 (Gag / Pol), pLP2 (Rev), pLP / VSVG (VSV-G) and pCDH-EF1-MCS-T2A-copGFP-NT were mixed evenly at a mass ratio of 1:1:1:1 and the concentration was adjusted to 4 mg / ul.

[0253] Take 920ul Opti-MEM medium and 80ul Lipofectamine3000 respectively, mix them evenly, and set them as tube 1 for later use.

[0254] Take 893ul Opti-MEM medium and 27ul plasmid mixture respectively, mix well, add 80ul P3000 reagent, mix well, and set aside as tube 2.

[0255] Slowly add the mixture in tube 1 to tube 2, mix thoroughly and let it stand at room temperature for 5 minutes.

[0256] Add the mixture to the cells to be transfected, shake well and return to the incubator. After 72 hours, harvest the supernatant, which is the virus stock solution.

[0257] The lentiviral stock solution corresponding to the pCDH-EF1-MCS-T2A-tdTomato-SIGN plasmid was prepared using the same method as above.

[0258] Example 4. Preparation of engineered monocyte-derived dendritic cell (MoDC-NT) lines

[0259] (1) Take 10 human peripheral blood aliquots, 50 ml each, containing sodium heparin;

[0260] (2) Isolation of dendritic cells (DCs) from peripheral blood using Ficoll;

[0261] (3) Resuspend the isolated cells in 1640 serum-free medium to adjust the cell density to 3E+6 cells / ml, add them to a cell culture flask, and incubate at 37°C in 5% CO2 for 1.5-2 h. Discard the suspended cells and medium, wash twice with medium, add complete medium 1640 (containing 10% FBS or 5% human AB serum or 5% autologous plasma), and add IL-4 (5 ng / ml) and GM-CSF (20 ng / ml);

[0262] (4) IL-4 (10 ng / ml) and GM-CSF (10 ng / ml) were added daily on days 1, 3, and 5;

[0263] (5) On day 7, the NT (NT-1 or NT-2) lentiviral particles prepared in Example 3 were added at an MOI of 20, and polybrene (10 μg / ml) was added. The culture was continued for one week, and IL-4 and GM-CSF were added every other day.

[0264] (6) Then, IL-2 (100 U / ml) was added to amplify the culture for a period of time.

[0265] (7) TNF-a (10 ng / ml), LPS (10 μg / ml) [or Poly(I:C), 25 μg / ml] were added and cultured for two days. Cell phenotypes were analyzed by flow cytometry. The results were as follows: Figure 8 As shown, the cell morphology Figure 9 shown.

[0266] Example 5. Preparation of engineered monocyte-derived dendritic cell (MoDC-NT) lines

[0267] The following steps were the same as those in Example 4 except that (3) IL-4 (10 ng / ml) and GM-CSF (5 ng / ml) were added, (6) and (4) IL-4 (20 ng / ml) and GM-CSF (20 ng / ml) were added every day on days 1, 3 and 5, (5) nanopolymer (5 ug / ml) was added, (6) IL-2 (200 U / ml) was added, and (7) TNF-a (5 ng / ml) and LPS (20 ug / ml) [or Poly (I:C), 10 ug / ml] were added.

[0268] Example 6. Preparation of Engineered Monocyte-Derived Dendritic Cell (MoDC-NT) Strain

[0269] The following procedures were the same as those in Example 4 except that (3) IL-4 (20 ng / ml) and GM-CSF (40 ng / ml) were added, (6) and (4) IL-4 (5 ng / ml) and GM-CSF (40 ng / ml) were added every day on days 1, 3, and 5, (5) nanopolymer (20 ug / ml) was added, (6) IL-2 (200 U / ml) was added, and (7) TNF-a (5 ng / ml) and LPS (20 ug / ml) [or Poly (I:C), 10 ug / ml] were added.

[0270] Flow cytometry analysis results showed that all 10 cells using NT-1 and NT-2 lentiviruses were successfully constructed and cell expansion was achieved.

[0271] The use of NT-1 and NT-2 lentiviruses enabled the large-scale expansion of MoDC-NT cells. All 10 constructed cells were successfully constructed, with a cell construction success rate of approximately 100%, providing sufficient cells for clinical and scientific research use.

[0272] The monocyte-derived MoDC-NT cells prepared by this protocol have a typical dendritic cell phenotype and can be proliferated multiple times through passages. The cells prepared by this protocol have higher purity and can also be used for HLA matching. They can provide sufficient autologous DC cells for antigen presentation for some people who need a large number of antigen-presenting cells but cannot wait for a long time.

[0273] Example 7. Preparation of M1 polypeptide-specific CTL cells

[0274] The preparation method of monocyte-derived dendritic cell (MoDC-NT) strain is as described in Example 4.

[0275] (1) Prepare a 2E+6 cell / ml suspension of a well-grown monocyte-derived dendritic cell (MoDC-NT) strain, add M1 polypeptide to a final concentration of 50 μg / ml, and continue culturing for 48 h;

[0276] (2) Obtain 50 ml of peripheral blood from a human (with the same HLA-A as MoDC-NT, HLA-A0201) containing sodium heparin;

[0277] (3) PBMCs were separated using Ficoll, a portion of the cells were used for CTL preparation, and the remaining cells were frozen for future use;

[0278] (4) PBMCs were diluted to a suspension of 2E+6 cells / ml and inoculated into a cell culture flask. MoDC-NT / M1:PBMCs were added to the flask at a ratio of 1:10. IL-2 was added to a final concentration of 200 U / ml (IL-2 needs to be added to this concentration during the entire culture process). After mixing, the cells were placed in a cell culture incubator for culture.

[0279] (7) Continue culturing for two weeks and take a small amount of cell suspension for flow cytometry and other quality control analysis. Figure 10 shown.

[0280] Example 8. Preparation of M1 polypeptide-specific CTL cells

[0281] (1) M1 polypeptide was added to a final concentration of 15 μg / ml. (2) 50 ml of human (with the same HLA-A as MoDC-NT, HLA-A0201) sodium heparinized umbilical cord blood was collected. (3) CD8+ T cells were separated using Ficoll, with the ratio of antigen-MoDC-NT to CD8+ T cells being 1:500. IL-2 was added to a final concentration of 50 U / ml. (7) Culture was continued for one week. The rest of the process was the same as in Example 5.

[0282] Example 9. Preparation of M1 polypeptide-specific CTL cells

[0283] (1) M1 polypeptide was added to a final concentration of 25 μg / ml. (2) 50 ml of human umbilical cord blood (with the same HLA-A as MoDC-NT, HLA-A0201) containing sodium heparin was collected. (3) CD3+ T cells were separated using Ficoll, with the ratio of antigen-MoDC-NT to CD3+ T cells being 1:1000. IL-2 was added to a final concentration of 800 U / ml. (7) Culture was continued for three weeks. The rest of the process was the same as in Example 5.

[0284] The flow cytometry results showed that the M1-specific CTLs prepared by stimulating MHC-matched PBMCs with a certain amount of MoDC-NT loaded with M1 polypeptide had CD8+ positive cells accounting for 89.57% of the CD3+CD56- cells, while CD3+CD56+ double-positive cells accounted for 3.92%, and CD3-CD56+ double-positive cells accounted for 1.70%. Among them, M1Tetramer-GILGFVFTL positive cells accounted for 20.73% of the CD3+CD8+ double-positive cells, indicating that this scheme can achieve specific antigen presentation, so that while it can kill tumors expressing M1-specific antigens in the patient's body, it can also kill other cells in the patient's body that do not have this specific antigen but have diseased cells, thereby achieving the effect of early prevention.

[0285] Example 10. Preparation of NT dendritic cell (EDC-NT) strain

[0286] (1) Take 10 human peripheral blood aliquots, 50 ml each, containing sodium heparin;

[0287] (2) PBMCs were isolated using Ficoll;

[0288] (3) PBMCs were resuspended in 1640 medium (containing 10% FBS or 5% human AB serum or 5% autologous plasma) and the cell density was adjusted to 2E+6 cells / ml. The cells were added to a cell culture flask and the immune cell agonist PHA was added to a final concentration of 5 μg / ml. The cells were cultured at 37°C under 5% CO2 for 24 h.

[0289] (4) On day 3, IL-2 was added to the culture supernatant to a final concentration of 100 U / ml and culture was continued for 4–5 days;

[0290] (5) On day 7, NT (NT-1 or NT-2) lentiviral particles were added at an MOI of 20, along with polybrene (10 μg / ml), and culture was continued for 1–2 weeks.

[0291] (6) Collect cells, wash twice with PBS, add anti-CD3 antibody magnetic beads, incubate for 2 h, and perform magnetic bead sorting to remove CD3+ cells;

[0292] (7) The remaining cells were adjusted to a cell density of 2E+6 cells / ml and added to a cell culture flask. The cells were cultured for 2-3 months and the cell phenotypes were analyzed by flow cytometry. Figure 11 The cell morphology is shown in Figure 12 shown.

[0293] Example 11. Preparation of NT dendritic cell (EDC-NT) strain

[0294] The following procedures were the same as in Example 6, except that in (3), the immune cell agonist was changed to PMA to a final concentration of 10 μg / ml, IL-2 was added to a final concentration of 500 U / ml in (4), the MOI in (5) was 10, and polybrene (20 μg / ml) was added at the same time, and in (6), the cells were collected and washed twice with PBS and then screened by flow cytometry to remove CD3+ cells.

[0295] Example 12. Preparation of NT dendritic cell (EDC-NT) strain

[0296] The following procedures were the same as in Example 6, except that in (3), the immune cell agonist was changed to PMA to a final concentration of 20 μg / ml, in (4), IL-2 was added to a final concentration of 200 U / ml, the MOI in (5) was 15, and nanopolymers (10 μg / ml) were added at the same time, and in (6), the cells were collected and washed twice with PBS and then screened by flow cytometry to remove CD3+ cells.

[0297] The results show that

[0298] The NT-1 base sequence enables better protein expression in cells, bringing the success rate of constructing the EDC-NT cell line to 90%. Among the 10 cells cultured with the NT-1 lentivirus, one cell stopped proliferating and gradually died after about a month of culture, while the remaining 9 cells were successfully constructed, with a cell construction success rate of approximately 90%.

[0299] The NT-2 amino acid sequence can enable EDC-NT cells to be expanded multiple times through passages, and the success rate of cell line construction is very high, reaching 100%; all 10 cells using NT-2 lentivirus were successfully constructed, with a cell construction success rate of 100%.

[0300] The NT dendritic cell (EDC-NT) strain prepared by this method has a typical dendritic cell phenotype and can achieve long-term passage and proliferation. The EDC-NT of the individual in need can be prepared in advance, stored in liquid nitrogen for a long time, and then revived (or briefly expanded and cultured) for use when needed. This method also provides an unlimited supply of autologous DC cells for antigen presentation for those who require large quantities of antigen-presenting cells, and the cells can be prepared once for lifelong use. The cells can also be used for HLA matching.

[0301] Example 13. Preparation of functional NT SIGN dendritic cell (EDC-NT SIGN) strain

[0302] (1) Take EDC-NT cells in good growth state, add SIGN lentivirus particles prepared by the method of Examples 2 and 3, MOI is 20, and simultaneously add polybrene (10 ug / ml), continue to culture for 1 week;

[0303] (2) Take part of the cells infected with SIGN virus, repeat infection with SIGN lentivirus twice, MOI is 20, and simultaneously add polybrene (10 ug / ml), then continue to culture for 1 week;

[0304] (3) Collect the cells, and wash with PBS for 2 times, add Anti-CD209 antibody magnetic beads, incubate thoroughly, then perform magnetic bead sorting, and collect CD209+ cells;

[0305] (4) Adjust the density of the collected cells to 2E+6 cells / ml, add to a cell culture flask, continue to culture for 1-2 weeks, and analyze the produced EDC-NT SIGN by flow cytometry, the results are shown in Figure 13 .

[0306] Example 14. Preparation of functional NT SIGN dendritic cell (EDC-NT SIGN) strain

[0307] Except that polybrene (10 ug / ml) in (1) and (2) is changed to nano-polymer (20 ug / ml), and (3) Anti-CD209 antibody magnetic bead incubation screening is changed to flow cytometry screening to collect CD209+ cells, the rest is the same as Example 13.

[0308] The flow cytometry results show that,

[0309] The EDC-NT SIGN cell strain prepared by transferring SIGN gene into EDC-NT cells has a typical DC phenotype, and can initiate the interaction of DC with naive T cells, and this function is realized through the combination of DC-SIGN with another cell ligand ICAM-3, which is highly expressed on the surface of naive T cells; the cell strain highly expresses DC-SIGN, which can realize better signal transduction, is conducive to the activation of T cells and NK cells, so that the expansion efficiency of DC-CTL is improved, the antigen-specific CTL is also improved, a larger number of effector cells are obtained, which is more conducive to clinical application, reduces the pain of patients caused by a large amount of blood sampling, increases the possibility of curing patients, and further improves the possibility of clinical application.

[0310] Example 15. Activation of effector cells by NT SIGN dendritic cell (EDC-NT SIGN) strain

[0311] (1) Obtain 50 ml of human peripheral blood containing sodium heparin;

[0312] (2) PBMCs were separated using Ficoll, a portion of which was used for effector cell preparation, and the remaining cells were frozen for future use;

[0313] (3) PBMCs were diluted to a suspension of 2E+6 cells / ml and inoculated into a cell culture flask. EDC-NTSIGN was added to the flask at a ratio of 1:100, and IL-2 was added to a final concentration of 500 U / ml (IL-2 needs to be added to this concentration during the entire culture process). After mixing, the cells were cultured in a cell culture incubator with a basal medium of X VIVO-15 + 10% FBS.

[0314] (4) Culture the cells continuously for three weeks and take a small amount of cell suspension for quality control analysis such as flow cytometry. Figure 14 shown.

[0315] The results showed that compared with EDC-NT cells, EDC-NTSIGN cells can better activate effector cells (T cells, NK cells). After a 21-day culture period, the total number of cells increased by 33.23%. A larger number of effector cells can more easily meet clinical application requirements, reduce the pain caused by large amounts of blood drawn from patients, and increase the possibility of cure.

[0316] Example 16. Preparation of multifunctional NT dendritic cell (EDC-NTA, i.e., EDC-NT-A0201 / 1101 / 2402) strain

[0317] (1) Take a suspension of EDC-NTSIGN cells in good growth condition;

[0318] (2) Lentiviral particles carrying the gene sequences of HLA-A0201 (as shown in SEQ ID No. 6), HLA-A1101 (as shown in SEQ ID No. 7), and HLA-A2402 (as shown in SEQ ID No. 8) were added simultaneously at an MOI of 20, and polybrene (10 μg / ml) was added, and the culture was continued for 1 week;

[0319] (3) Repeat step (2) twice;

[0320] (4) The cells were diluted and divided into 96-well plates for monoclonal cell screening. After culture, DCs were measured by flow cytometry. The results were as follows: Figure 15 shown.

[0321] HLA-A0201 (SEQ ID No. 6):

[0322]

[0323] HLA-A1101(HA-Tag)(SEQ ID No.7):

[0324] HLA-A2402 (SEQ ID No. 8):

[0325]

[0326]

[0327] The results showed that the multifunctional NT dendritic cell (EDC-NTA, i.e., DC-A0201 / 1101 / 2402) strain simultaneously possesses HLA-A0201, HLA-A1101, and HLA-A2402, three MHC class I molecules with the highest prevalence among Chinese people, and can also be other HLA subtypes. This cell can be used to present antigens in vitro to cells containing one of the corresponding MHC I molecules, making large-scale target-specific CTL production possible.

[0328] Example 17. Preparation of functional NT dendritic cell (EDC-NTA, i.e., EDC-NT-A0201) strain

[0329] The same procedures as in Example 16 were used except that (2) lentiviral particles carrying the HLA-A0201 (as shown in SEQ ID No. 6) gene sequence were added at an MOI of 10, and nanopolymers (20 μg / ml) were added, and the cells were cultured for 2 weeks. (4) The screening method was to wash the cells twice with PBS, add anti-HLA-A02 antibody magnetic beads, and after sufficient incubation, perform magnetic bead sorting to collect the EDC-NTA0201 cells.

[0330] Example 18. Preparation of multifunctional NT dendritic cell (EDC-NTA, i.e., EDC-NT-A1101) strain

[0331] The same procedures as in Example 16 were used except that (2) lentiviral particles carrying the HLA-A1101 (as shown in SEQ ID No. 7) gene sequence were added at an MOI of 10, polybrene (10 μg / ml) was added, and the cells were cultured for 3 weeks; (4) the screening method was to wash the cells twice with PBS, add anti-tag antibody flow cytometry antibodies, incubate the cells sufficiently, and then sort the cells on a flow cytometer to collect the EDC-NTA1101 cells.

[0332] Example 19. Preparation of multifunctional NT dendritic cell (EDC-NTA, i.e., EDC-NT-A0201 / 1101) strain

[0333] The method was the same as Example 16 except that (2) lentiviral particles carrying the gene sequences of HLA-A0201 (as shown in SEQ ID No. 6) and HLA-A1101 (as shown in SEQ ID No. 7) were added.

[0334] Example 20. Preparation of multifunctional NT dendritic cell (EDC-NTA, i.e., EDC-NT-A0201 / 2402) strain

[0335] The method was the same as Example 16 except that (2) lentiviral particles carrying the gene sequences of HLA-A0201 (as shown in SEQ ID No. 6) and HLA-A2402 (as shown in SEQ ID No. 8) were added.

[0336] Example 21. Preparation of multifunctional NT dendritic cell (EDC-NTA, i.e., EDC-NT-A1101 / 2402) strain

[0337] The method was the same as Example 16 except that (2) lentiviral particles carrying the gene sequences of HLA-A1101 (as shown in SEQ ID No. 7) and HLA-A2402 (as shown in SEQ ID No. 8) were added.

[0338] Example 22. Preparation of Mesothelin Lentiviral Particles, CEA Lentiviral Particles, and NY-ESO-1 Lentiviral Particles

[0339] As shown in Examples 2 and 3, the newly designed Mesothelin, CEA, and NY-ESO-1 base sequences were artificially synthesized. By designing a new sequence of IκBα, the new sequence and the antigen gene were artificially synthesized into a recombinant viral vector, with Xba I and BamH I restriction sites set at both ends, respectively. The lentiviral vector plasmid used to connect the three antigen genes is pCDH-EF1-MCS-T2A-Puro, as shown in FIG. Figure 16 Lentivirus was prepared using a four-plasmid system.

[0340] The procedures were the same as in Example 3 except that pCDH-EF1-MCS-T2A-copGFP-NT was replaced by plasmids pCDH-EF1-MCS-T2A-Puro-Mesothelin target antigen, pCDH-EF1-MCS-T2A-Puro-CEA target antigen, and pCDH-EF1-MCS-T2A-Puro-NY-ESO-1 target antigen, respectively.

[0341] (1) The target antigen nucleic acid sequence structure is IκBα(1-48aa)-Mesothelin-IκBα(281-317aa), except that pCDH-EF1-MCS-T2A-copGFP-NT is replaced by plasmid pCDH-EF1-MCS-T2A-Puro-IκBα(1-48aa)-Mesothelin-IκBα(281-317aa), and the rest is the same as Example 3.

[0342] (2) The target antigen nucleic acid sequence structure is IκBα(1-317aa)-T2A-CEA, except that pCDH-EF1-MCS-T2A-copGFP-NT is replaced by plasmid pCDH-EF1-MCS-T2A-Puro-IκBα(1-317aa)-T2A-CEA, and the rest is the same as Example 3.

[0343] (3) The target antigen nucleic acid sequence structure is IκBα(1-48aa)-NYESO1 mini gene-IκBα(281-317aa), except that pCDH-EF1-MCS-T2A-copGFP-NT is replaced with plasmid pCDH-EF1-MCS-T2A-Puro-IκBα(1-48aa)-NYESO1-mini gene-IκBα(281-317aa), and the rest is the same as Example 3.

[0344] The structural formula of the target antigen nucleic acid sequence is: (1) The structure of the combined gene is selected from the general formula IκBα(1-48aa)-antigen gene-IκBα(281-317aa), IκBα(1-317aa)-T2A-antigen gene, IκBα(1-48aa)-antigen peptide mini gene-IκBα(281-317aa) and IκBα(1-317aa)-T2A-antigen peptide mini gene.

[0345] The sequence structure of Mesothelin, CEA and NY-ESO-1 is as follows Figure 17 、 Figure 18 and Figure 19 The antigen sequences of IκBα, Mesothelin, CEA and NY-ESO-1 are shown in SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No. 13, respectively.

[0346] IκBα (SEQ ID No. 9):

[0347]

[0348] T2A (SEQ ID No. 10):

[0349]

[0350] Mesothelin (SEQ ID No. 11):

[0351]

[0352] CEA (SEQ ID No. 12):

[0353]

[0354]

[0355] NY-ESO-1 (SEQ ID No. 13):

[0356]

[0357] The amino acid sequence of the antigen peptide mini gene is the repeated connection sequence of multiple-antigen polypeptide-AYY, and the nucleotide sequence of AYY is shown in SEQ ID No. 19: GCTTACTAC.

[0358] By designing a new sequence of IκBα, the new sequence is artificially synthesized with the antigen gene to prepare a lentivirus, and the gene is transferred into MoDC-NT or EDC-NT or EDC-NT SIGN, so that the antigen presentation after intracellular degradation of the antigen can be realized.

[0359] Example 23. Preparation of mesothelin-specific multifunctional dendritic cell (EDC-Mesothelin) strain

[0360] (1) Take a suspension of well-grown multifunctional NT dendritic cells (EDC-NTA);

[0361] (2) At the same time, add the mesothelin lentivirus particles obtained in Example 22, and the MOI is 20, and add polybrene (10 ug / ml), and continue to culture for 1 week;

[0362] (3) Continue to repeat step (2) twice;

[0363] (4) Add puromycin to the cell culture solution to a final concentration of 5 ug / ml, and continuously pressurize for one week.

[0364] (5) After removing the puromycin, continue to expand the culture for standby use.

[0365] Results show that by using IκBα can promote Mesothelin in the cell rapid degradation, and MHC I molecule presentation, the construction of this cell line can be used to prepare Mesothelin specific CTL, for Mesothelin positive tumor patients provide a brand new choice.

[0366] Example 24. Preparation of CEA specific multifunctional dendritic cell (EDC-CEA) strain

[0367] (1) Take the suspension of the well growing multifunctional NT dendritic cells (EDC-NTA);

[0368] (2) At the same time, add the CEA lentivirus particles obtained in Example 22, and the MOI is 20, and add the nano polymer (20ug / ml), continue to culture for 2 weeks;

[0369] (3) Continue to repeat step (2) twice;

[0370] (4) Add puromycin to the cell culture solution to a final concentration of 8ug / ml, and continuously pressurize for one week.

[0371] (5) After removing the puromycin, continue to expand the culture, and reserve.

[0372] Results show that by using IκBα can promote CEA in the cell rapid degradation, and MHC I molecule presentation, the construction of this cell line can be used to prepare CEA specific CTL, for CEA positive tumor patients provide a brand new choice.

[0373] Example 25. Preparation of NY-ESO-1 specific multifunctional dendritic cell (EDC-NYESO1) strain

[0374] (1) Take the suspension of the well growing multifunctional NT dendritic cells (EDC-NTA);

[0375] (2) At the same time, add the NY-ESO-1 lentivirus particles obtained in Example 22, and the MOI is 20, and add the polybrene (20ug / ml), continue to culture for 1 week;

[0376] (3) Continue to repeat step (2) twice;

[0377] (4) Add puromycin to the cell culture solution to a final concentration of 10ug / ml, and continuously pressurize for one week.

[0378] (5) After removing the puromycin, continue to expand the culture, and reserve.

[0379] The results showed that the use of IκBα can promote the rapid degradation of NY-ESO-1 in cells and its presentation by MHC class I molecules. The construction of this cell line can be used to prepare NY-ESO-1-specific CTLs, providing a new option for the treatment of patients with NY-ESO-1-positive tumors.

[0380] Example 26. Preparation of EGFR-specific multifunctional dendritic cell (EDC-EGFR) strain

[0381] Except that (2) NY-ESO-1 lentiviral particles were changed to EGFR lentiviral particles, the rest were the same as Example 25.

[0382] Example 27. Preparation of CA153-specific multifunctional dendritic cell (EDC-CA153) strain

[0383] Except that (2) NY-ESO-1 lentiviral particles were changed to CA153 lentiviral particles, the rest were the same as Example 25.

[0384] Example 28. Preparation of MAGEA3-specific multifunctional dendritic cell (EDC-MAGEA3) strain

[0385] Except that (2) NY-ESO-1 lentiviral particles were changed to β2-MG lentiviral particles, the rest were the same as Example 25.

[0386] Example 29. Preparation of Mesothelin-specific CTL cells

[0387] (1) Obtain 50 ml of peripheral blood from a human (HLA-A0201) containing sodium heparin;

[0388] (2) Using Ficoll-separated CD3+ T cells, a portion of the cells are used for CTL preparation, and the remaining cells are frozen for future use;

[0389] (3) Dilute CD3+ T cells to a suspension of 2E+6 cells / ml and inoculate them into a cell culture flask. Add EDC-Mesothelin to the flask at a ratio of 1:1000 and add IL-2 to a final concentration of 200 U / ml (IL-2 needs to be added daily to this concentration during the entire culture process). Mix well and place in a cell culture incubator for culture.

[0390] (4) On the 7th day of cell culture, EDC-Mesothelin was added to the culture flask at a ratio of 1:200 and cultured for two weeks. A small amount of cell suspension was taken for flow cytometry and other quality control analysis. Figure 20 shown.

[0391] The results showed that by using a certain amount of EDC-Mesothelin to stimulate MHC class I molecule-matched PBMCs, the mesothelin-specific CTLs prepared had a CD3+CD8+ double-positive cell ratio as high as 89.57%, while the proportion of CD3+CD56+ double-positive cells could reach 17.10%, and the proportion of CD3-CD56+ cells was 4.30%. In this way, while killing tumors expressing mesothelin-specific antigens in the patient's body, it can also kill other cells in the patient's body that do not have this specific antigen but have diseased cells, thereby achieving the effect of early prevention.

[0392] Example 30. Preparation of CEA-specific CTL cells

[0393] (1) Obtain 50 ml of peripheral blood from a human (HLA-A0201) containing sodium heparin;

[0394] (2) Using Ficoll-separated CD8+ T cells, a portion of the cells are used for CTL preparation, and the remaining cells are frozen for future use;

[0395] (3) Dilute CD8+ T cells to a suspension of 2E+6 cells / ml and inoculate them into a cell culture flask. Add EDC-CEA to the flask at a ratio of 1:100, and add IL-2 to a final concentration of 200 U / ml (IL-2 needs to be added daily to this concentration during the entire culture process). Mix well and place in a cell culture incubator for culture.

[0396] (4) On the 7th day of cell culture, EDC-CEA was added to the culture flask at a ratio of 1:200 and cultured for another week. A small amount of cell suspension was taken for quality control analysis such as flow cytometry. Figure 21 shown.

[0397] The results showed that by using a certain amount of EDC-CEA to stimulate MHC class I molecule-matched PBMCs, the CEA-specific CTLs prepared had a CD3+CD8+ double-positive cell ratio of 86.86%, while the CD3+CD56+ double-positive cell ratio could reach 23.85%, and the CD3-CD56+ cell ratio was 3.64%. In this way, while killing tumors expressing CEA-specific antigens in the patient's body, it can also kill other cells in the patient's body that do not have this specific antigen but have diseased cells, thereby achieving the effect of early prevention.

[0398] Example 31. Preparation of NY-ESO-1 specific CTL cells

[0399] (1) Obtain 50 ml of human (HLA-A0201) umbilical cord blood containing sodium heparin;

[0400] (2) PBMCs were separated using Ficoll, a portion of which was used for CTL preparation, and the remaining cells were frozen for future use;

[0401] (3) PBMCs were diluted to a 2E+6 cells / ml suspension and inoculated into a cell culture flask. EDC-NYESO1 was added to the flask at a ratio of 1:10. IL-2 was also added to a final concentration of 200 U / ml (IL-2 should be added daily to this concentration during the entire culture process). After mixing, the cells were placed in a cell culture incubator for culture.

[0402] (4) Continue culturing for three weeks and take a small amount of cell suspension for flow cytometry and other quality control analysis. Figure 22 shown.

[0403] The results showed that by using a certain amount of EDC-NYESO1 to stimulate MHC class I molecule-matched PBMCs, the NY-ESO-1-specific CTLs prepared had a CD3+CD8+ double-positive cell ratio as high as 89.74%, while the CD3+CD56+ double-positive cell ratio could also reach 14.39%, and the CD3-CD56+ cell ratio was 2.34%. This can not only kill tumors expressing NY-ESO-1 specific antigens in the patient's body, but also kill other cells in the patient's body that do not have this specific antigen but have diseased cells, thereby achieving the effect of early prevention.

[0404] It can start with PBMC, CD3+T or CD8+T.

[0405] Example 32. Analysis of the killing ability of specific CTL cells stimulated by EDC-Mesothelin

[0406] Human fibroblasts (FIB) (HLA-A01:01 A33:03) provided by Jinmeitai Biotechnology Hebei Co., Ltd. were selected as negative controls, and HLA-A0201 was transfected into FIB via lentivirus to obtain FIB / A0201. Mesothelin was further transfected into FIB / A0201 via lentivirus to obtain FIB / A0201 / Mesothelin. Western Blot analysis of mesothelin expression in FIB and FIB / A0201 / Mesothelin was performed. Figure 23 As shown in A; Flow cytometry analysis of HLA-A0201 expression Figure 23 As shown in B; and the killing activity of cells generated by PBMC stimulated by EDC-Mesothelin against FIB / A0201 and FIB / A0201 / Mesothelin was analyzed Figure 23C; at the same time, the degree of cytotoxicity restricted by MHC I was analyzed by adding HLA-A2-blocking antibody. The results are shown in Figure 23 As shown in D.

[0407] Western blot results showed that mesothelin transfected into FIB cells via lentivirus was successfully expressed in the cells, while FIB cells without mesothelin transfection did not express it. Flow cytometry results showed that HLA-A0201 transfected into FIB cells via lentivirus was successfully expressed in the cells and could be transported to the cell membrane surface. Cell killing results showed that CTL cells generated by PBMCs stimulated by EDC-AFPMesothelin could specifically kill target cells expressing mesothelin antigen. Anti-HLA A2 antibody blocking experiments showed that the killing of target cells expressing mesothelin antigen by CTL cells generated by PBMCs stimulated by EDC-Mesothelin was restricted by MHC class I molecules.

[0408] Example 33. Analysis of the killing ability of specific CTL cells stimulated by EDC-NYESO1

[0409] Human fibroblasts (FIB) (HLA-A01:01 A33:03) provided by Jinmeitai Biotechnology Hebei Co., Ltd. were selected as negative controls, and HLA-A0201 and NY-ESO-1 were sequentially transfected into HLA-A0201-negative cells A549 / NYESO1 by lentivirus. dim Western Blot detection of NY-ESO-1 in FIB, A549 / NYESO1 dim The expression results in A549 / A0201 / NYESO1 are as follows Figure 24 As shown in A; flow cytometry analysis of HLA-A0201 expression is shown in 24B; and analysis of the cytotoxic activity of cells generated by EDC-NYESO1-stimulated PBMC against FIB and A549 / A0201 / NYESO1 is shown in 24C.

[0410] Western blot analysis showed that A549 / NYESO1 dim The cells expressed low levels of NY-ESO-1, and after lentiviral transfection, A549 / NYESO1 dimThe expression of NY-ESO-1 in the cells was significantly enhanced, while the control group FIB cells did not express NY-ESO-1. Flow cytometry results showed that HLA-A0201 transfected into A549 cells by lentivirus was successfully expressed in the cells and could be transported to the cell membrane surface. Cell killing results showed that the cells generated by PBMC stimulated by EDC-NYESO1 could inhibit A459 / NYESO1 without being restricted by MHC class I molecules. dim The cells can be effectively killed, which is mainly due to the role of NK and NKT; while the MHC class I molecule restriction of CTL and the enhancement of A549 / NYESO1 dim When NY-ESO-1 specific antigen is expressed, CTL cells generated by PBMC stimulated by EDC-NYESO1 can further enhance the killing effect on target cells, showing stronger killing activity against A549 / A0201 / NYESO1.

[0411] Example 34. Preparation of multifunctional NK trophoblast cell line (EDC-NK)

[0412] (1) Obtain a suspension of NT dendritic cells (EDC-NTSIGN) in good growth condition;

[0413] (2) 4-1BBL (as shown in SEQ ID No. 14), MICA (as shown in SEQ ID No. 15), IL-15Mat-peptide (as shown in SEQ ID No. 17) & Hinge / TM CD8 (as shown in SEQ ID No. 18) fusion protein lentiviral particles were added simultaneously (IL-15 & CD8α fusion protein structure as shown in Figure 25 As shown), the MOI was 20, and polybrene (10 μg / ml) was added and cultured for 1 week;

[0414] (3) Repeat step (2) twice;

[0415] (4) The cells were diluted and divided into 96-well plates for monoclonal cell screening. After culture, DCs were measured by flow cytometry. The results were as follows: Figure 26 shown.

[0416] 4-1BBL (SEQ ID No. 14):

[0417]

[0418]

[0419] MICA (SEQ ID No. 15):

[0420]

[0421] IL-15&CD8αFusion protein:

[0422] Signal peptide (SEQ ID No. 16):

[0423]

[0424] IL-15Mat-peptide (SEQ ID No.17):

[0425]

[0426] Hinge / TM CD8 (SEQ ID No. 18):

[0427]

[0428] Results showed that EDC-NTSIGN cells could be transfected with 4-1BBL, MICA, and IL-15 & CD8 Fusion protein genes to successfully generate EDC-NK cells. 4-1BBL interacts with the NK cell-activating receptor 4-1BB to restore NK cell reactivity. MICA is a ligand for the activating receptor NKG2D, which is widely expressed on human NK cells. IL-15 promotes the directed differentiation of CD34+ hematopoietic stem cells into NK cells, significantly increasing the proportion of NK cells and shifting the NK cell subset distribution from predominantly CD56dim to predominantly CD56bright cells. The successful construction of EDC-NK cells provides a new option for NK cell production. This approach allows for pre-antigen stimulation of EDC-NK cells during NK cell production, generating antigen-specific cells. It may also serve as an alternative to the currently used K562 trophoblast cells for NK cell production.

[0429] Example 35. Inactivation of multifunctional NK trophoblast cell (EDC-NK) strain

[0430] (1) Take EDC-NK cells in good growth condition, adjust the cell density to 2E+6 cells / ml, and place the cell suspension in a cell culture flask;

[0431] (2) The cell suspension was sent to the laboratory of Beijing Hongyi Sifang Radiation Technology Co., Ltd. for irradiation treatment. A Co60 BFT-II (2#) irradiation device was used, and the irradiation treatment was carried out by circular orbital fractionated irradiation. The absorbed dose of the cell suspension was: Dmin = 19.85 kGy.

[0432] (3) The test method is: GB / T 15053-2008 "Standard method for measuring absorbents using radiochromic film and polymethyl methacrylate dose measurement system".

[0433] (4) Retrieve the cell suspension after irradiation, collect the cells by centrifugation, and then freeze the cells. The freezing density is 1E+7 cells / ml. The freezing medium is GMP-grade cell freezing medium, and programmed cooling is used for freezing.

[0434] (5) Irradiated cells can be used directly, or frozen cells can be revived and used.

[0435] Analysis shows that irradiated EDC-NK cells are inactivated and cannot proliferate further, but they retain their original intracellular and surface molecular structures. This preserves their antigen-presenting capacity, does not affect their ability to activate T and NK cells, and is more conducive to the safety of clinical applications. Alternatively, the cells can be fixed with paraformaldehyde or other tissue cell fixatives or treated with other methods that can inactivate cell proliferation, but the cells can still maintain their function and application safety. They can be used as vaccines or cell preparations for clinical or scientific research purposes.

[0436] Example 36. Preparation of NK cells

[0437] (1) Obtain 50 ml of human peripheral blood containing sodium heparin;

[0438] (2) PBMCs were separated using Ficoll, a portion of which was used for NK cell preparation, and the remaining cells were frozen for future use;

[0439] (3) PBMCs were diluted to a suspension of 2E+6 cells / ml and inoculated into a cell culture flask. EDC-NK (irradiated or non-irradiated) was added to the flask at a ratio of 1:50. IL-2 was also added to a final concentration of 500 U / ml (IL-2 was added to this concentration each time the fluid was replenished during the entire culture process). After mixing, the cells were cultured in a cell culture incubator. The basal culture medium was X VIVO-15 + 10% FBS.

[0440] (4) After culturing the cells for three weeks, a small amount of cell suspension was taken for flow cytometry analysis. The results were as follows: Figure 27 shown.

[0441] The cell expansion curves of NK (CD3-CD56+) and NKT (CD3+CD56+) obtained by activating PBMC with EDC-NK stimulation for 21 days are shown in Figure 2. Figure 28 shown.

[0442] The results showed that EDC-NK could stimulate and activate PBMC to specifically expand NK cell subsets. The resulting NK (CD3-CD56+) and NKT (CD3+CD56+) accounted for 81.11% and 4.45%, respectively. After 21 days of culture, the cells expanded 253 times. This method is used as a new solution for NK cell preparation.

[0443] Example 37. Preparation of NK cells using EDC-NK combined with cytokines

[0444] (1) Obtain 50 ml of peripheral blood from a person with the same HLA-A as EDC-NK, containing sodium heparin;

[0445] (2) PBMCs were separated using Ficoll, a portion of which was used for NK cell preparation, and the remaining cells were frozen for future use;

[0446] (3) PBMC were diluted to a suspension of 2E+6 cells / ml and inoculated into a cell culture flask. EDC-NK (irradiated or non-irradiated) was added to the culture flask at a ratio of 1:50. IL-2 was added to a final concentration of 500 U / ml (IL-2 was added to this concentration each time the fluid was replenished during the entire culture process). IL-15RA was added to a final concentration of 20 ng / ml, OK432 to a final concentration of 0.2 KE / ml, and CD52 to a final concentration of 10 μg / ml. After mixing, the cells were cultured in a cell culture incubator. After 7 days of culture, the basal culture medium containing only IL-2 was continued to be supplemented. The basal culture medium was X VIVO-15 + 10% FBS.

[0447] (4) After culturing the cells for three weeks, a small amount of cell suspension was taken for flow cytometry analysis. The results were as follows: Figure 29 shown.

[0448] Combining cytokines with EDC-NK stimulation to activate PBMCs can produce NK (CD3-CD56+) and NKT (CD3+CD56+). The cell expansion curve after 21 days of culture is shown in the figure below. Figure 30 shown.

[0449] Results showed that EDC-NK combined with cytokines IL-15RA, OK432, CD52, and IL-2 stimulated PBMC to specifically expand NK cell subsets, resulting in NK (CD3-CD56+) and NKT (CD3+CD56+) cell subsets accounting for 95.43% and 0.86%, respectively. After 21 days of culture, the cells expanded 394-fold. This further improved NK purity and cell expansion multiples can be used as a new protocol for NK cell preparation.

[0450] Example 38. Preparation of NK cells in combination with histone methylase (EZH2) inhibitors

[0451] (1) Take and EDC-NK with different HLA-A human umbilical cord blood 50 ml, containing heparin sodium;

[0452] (2) Use Ficoll to separate PBMC, part of the cells are used for NK preparation, and the remaining cells are frozen for standby;

[0453] (3) Dilute the PBMC into a suspension of 2E+6 cells / ml, inoculate into a cell culture bottle, and add (irradiated or non-irradiated) EDC-NK into the culture bottle at a ratio of 1:50, while adding IL-2 to a final concentration of 500 U / ml (IL-2 needs to be added to this concentration every time the liquid is supplemented during the entire culture process), adding IL-15RA to a final concentration of 20 ng / ml, OK432 to a final concentration of 0.2 KE / ml, and CD52 to a final concentration of 10 ug / ml;

[0454] (4) Divide the cells into three groups, the first group without inhibitor; the second group with EPZ005687 to a final concentration of 2.5 uM; and the third group with UNC1999 to a final concentration of 2.5 uM, mix well, and place in a cell culture incubator for culture. After 7 days of culture, continue to supplement the basic culture medium with only IL-2, and the basic culture medium is X VIVO-15 + 10% FBS;

[0455] (5) Continuously culture the cells for two weeks, take a small amount of cell suspension for flow detection, and sample and detect the killing ability of the cells against target cells A549 (E:T = 10:1) at 14 days of culture. The results are shown in Figure 31 . The flow results of using EDC-NK to stimulate PBMC for NK cell preparation combined with histone methyltransferase (EZH2) inhibitor are shown in Table 1. The cell expansion curve chart of using EDC-NK to stimulate and activate PBMC combined with histone methyltransferase (EZH2) inhibitor and cytokines for 21 days of culture cycle is shown in Figure 32 .

[0456]

[0457] Table 1. Percentage of individual cell subpopulation in parent cell population

[0458] The results show that using EDC-NK to stimulate and activate PBMC combined with histone methyltransferase (EZH2) inhibitor and cytokines can better activate NK (CD3-CD56+), increase its proportion; at the same time, the proportion of NKG2D+CD56+ double positive cell subpopulation also increases with the addition of EZH2 inhibitor; after the addition of EZH2 inhibitor, the killing ability of effector cells on target cells also significantly increases; the inhibitor has almost no effect on cell expansion efficiency in 21 days of culture cycle.

[0459] The results of the above examples show that by designing a new gene sequence, preparing a lentivirus, infecting immune cells to obtain a cell line, and further stimulating PBMC with a cell line or a combined cytokine, the monocyte-derived dendritic cell method (MoDC-NT) strain, engineered NT dendritic cell (EDC-NT), functional NTSIGN dendritic cell (EDC-NTSIGN) strain, multifunctional NT dendritic cell (EDC-NTA cell), target antigen-specific multifunctional dendritic cell, target antigen-specific CTL, engineered multifunctional NK trophoblast and NK cells were obtained in sequence. The novel immune cell preparation method of the present invention successfully prepared a variety of engineered functional cell lines and immune cells.

[0460] The present invention provides a preparation method and application of a novel immune cell, relating to the field of immune cells, and in particular to the preparation of DNA molecules, viral vectors and cells containing the DNA molecules, the modification and application of cells with antigen presenting ability, and the modification and application of cells with NK stimulation ability.

[0461] The present invention genetically modifies cells in mammalian peripheral blood using lentivirus, enabling them to possess antigen-presenting capabilities and the ability to activate and expand natural killer cells. Further genetic modification of these cells enables enhanced transmembrane transfer, antigen presentation, and natural killer cell activation. The genetically modified cells can present antigen information through antigen loading or antigen gene transfection. The genetically modified cells can present antigen information to mononuclear cells of a wider range of HLA types. The genetically modified cells can induce and stimulate mononuclear cells to expand and differentiate into natural killer cells. This function is retained even after cells have lost their proliferative activity following irradiation or treatment with tissue cell fixatives such as paraformaldehyde or cell inactivators. After genetic modification, these cells can stimulate mononuclear cells to expand and differentiate into cytotoxic T lymphocytes with specific killing capabilities against cells expressing specific antigen information. These cells can also activate and expand natural killer cells after genetic modification. Co-activation and expansion of mononuclear cells with different cytokines after genetic modification can yield larger numbers of natural killer cells. Co-activation and expansion of mononuclear cells with cytokines in the presence of a histone methyltransferase (EZH2) inhibitor can increase the proportion of natural killer cells in effector cells, regulate epigenetic changes in natural killer cell receptor and ligand expression, and enhance the cytotoxicity of effector cells. The cells prepared by the present invention, which have antigen-presenting and natural killer cell activation and expansion capabilities, have the ability to expand through multiple generations and can undergo multiple genetic modifications. The present invention can be used to prepare antigen-presenting cells, CTL cells, and application methods specific for mesothelin, CEA, NY-ESO-1, or other antigens. The present invention can also be used to prepare high-purity, highly cytotoxic natural killer cells on a large scale and for application methods. These cells and methods of use have broad application prospects in the prevention and treatment of tumors and infectious diseases.

[0462] The present invention improves the in vitro growth potential of DC cells, enhances the DC antigen presentation ability and the ability to activate CTL and NK cells, greatly promotes the progress of cellular immunotherapy medical practice, and has broad industrialization prospects and commercial space. Sequence Listing <110> Hou Li <120> Preparation method of a novel immune cell and its application <130> 210811 <160> 19 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1009 <212> DNA <213> designed <400> 1 gctctagaat ggcccacttc cccggcttcg gccagagcct gctgtacggc taccccgtgt 60 acgtgttcgg caactgcgtg caggccgact ggtgccccgt gagcggcggc ctgtgcagca 120 cccgcctgca ccgccacgcc ctgctggcca cctgccccga gcaccagctg acctgggacc 180 ccatcgacgg ccgcgtggtg agcagccccc tgcagtacct gatcccccgc ctgcccagct 240 tccccaccca gcgcaccagc cgcaccctga aggtgctgac cccccccacc acccccgtga 300 gccccaaggt gccccccgcc ttcttccaga gcatgcgcaa gcacaccccc taccgcaacg 360 gctgcctgga gcccaccctg ggcgaccagc tgcccagcct ggccttcccc gagcccggcc 420 tgcgccccca gaacatctac accacctggg gcaagaccgt ggtgtgcctg tacctgtacc 480 agctgagccc ccccatgacc tggcccctga tcccccacgt gatcttctgc cacccccgcc 540 agctgggcgc cttcctgacc aaggtgcccc tgaagcgcct ggaggagctg ctgtacaaga 600 tgttcctgca caccggcgcc gtgatcgtgc tgcccgagga cgacctgccc accaccatgt 660 tccagcccgt gcgcgccccc 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ggggcggcag cgtggtgtgc atgtacctgt accagctgag cccccccatc 480 acctggcccc tgctgcccca cgtggtgttc tgccaccccg gccagctggg cgccttcctg 540 accaacgtgc cctacaagcg catcgaggag ctgctgtaca agatcagcct gaccaccggc 600 gccctgatca tcctgcccga ggactgcctg cccaccaccc tgttccagcc cgtgcgcgcc 660 cccgtgaccc tgaccgcctg gcagcacggc ctgctgccct tccacagcac cctgaccacc 720 cccggcctga tctggacctt caccgacggc acccccatga tc 762 <210> 4 <211> 333 <212> DNA <213> designed <400> 4 gcctggcagc acggcctgct gcccttccac agcaccctga ccacccccgg cctgatctgg 60 accttcaccg acggcacccc catgatcagc ggcccctgcc ccaaggacgg ccagcccagc 120 ctggtgctgc agagcagcag cttcatcttc cacaagttcc agaccaaggc ctaccacccc 180 agcttcctgc tgagccacgg cctgatccag tacagcagct tccacaacct gcacctgctg 240 ttcgaggagt acaccaacat ccccatcagc ctgctgttca acgagaagga ggccgacgac 300 aacgaccacg agccccagat cagccccggc gag 333 <210> 5 <211> 1231 <212> DNA <213> designed <400> 5 cgggatccat gagcgacagc aagggagcccc gcctgcagca gctggggcctg ctggaggagg 60 agcagctgcg cggcctgggc ttccgccaga cccgcggcta caagagcctg gccggctgcc 120 tgggccacgg ccccctggtg ctgcagctgc tgagcttcac cctgctggcc ggcctgctgg 180 tgcaggtgag caaggtgccc agcagcatca gccaggagca gagccgccag gacgccatct 240 accagaacct gacccagctg aaggccgccg tgggcgagct gagcgagaag agcaagctgc 300 aggagatcta ccaggagctg acccagctga aggccgccgt gggcgagctg cccgagaaga 360 gcaagctgca ggagatctac caggagctga cccgcctgaa ggccgccgtg ggcgagctgc 420 ccgagaagag caagctgcag gagatctacc aggagctgac ctggctgaag gccgccgtgg 480 gcgagctgcc cgagaagagc aagatgcagg agatctacca ggagctgacc cgcctgaagg 540 600 gcctgaaggc cgccgtgggc gagctgcccg agaagagcaa gcagcaggag atctaccagg 660 720 tctaccagga gctgacccag ctgaaggccg ccgtggagcg cctgtgccac ccctgcccct 780 gggagtggac cttcttccag ggcaactgct acttcatgag caacgccag cgcaactggc 840 acgacagcat caccgcctgc aaggaggtgg gcgcccagct ggtggtgatc aagagcgccg 900 aggaggaa cttcctgcag ctgcagagca gccgcagcaa ccgcttcacc tggatgggcc 960 tgagcgacct gaaccaggag ggcacctggc agtgggtgga cggcagcccc ctgctgccca 1020 gcttcaagca gtactggaac cgcggcgagc ccaacaacgt gggcgaggag gactgcgccg 1080 agttcagcgg caacggctgg aacgacgaca agtgcaacct ggccaagttc tggatctgca 1140 agaagagcgc cgccagctgc agccgcgacg aggagcagtt cctgagcccc gccccgcca 1200 cccccaaccc cccccccgcc taagaattcc g 1231 <210> 6 <211> 1095 <212> DNA <213> designed <400> 6 atggccgtca tggcgccccg aaccctcgtc ctgctactct cgggggctct ggccctgacc 60 cagacctggg cgggctctca ctccatgagg tatttcttca catccgtgtc ccggcccggc 120 cgcggggagc cccgcttcat cgcagtgggc tacgtggacg acacgcagtt cgtgcggttc 180 gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggt 240 ccggagtatt gggacgggga gacacggaaa gtgaaggccc actcacagac tcaccgagtg 300 gacctgggga ccctgcgcgg ctactacaac cagagcgagg ccggttctca caccgtccag 360 aggatgtatg gctgcgacgt ggggtcggac tggcgcttc tccgcgggta ccaccagtac 420 gcctacgacg gcaaggatta catcgccctg aaagaggacc tgcgctcttg gaccgcggcg 480 gacatggcag ctcagaccac caagcacaag tgggaggcgg cccatgtggc ggagcagttg 540 agagcctacc tggagggcac gtgcgtggag tggctccgca gatacctgga gaacgggaag 600 gagacgctgc agcgcacgga cgcccccaaa acgcatatga ctcaccacgc tgtctctgac 660 catgaagcca ccctgaggtg ctgggccctg agcttctacc ctgcggagat cacactgacc 720 tggcagcggg atggggagga ccagacccag gacacggagc tcgtggagac caggcctgca 780 ggggatggaa ccttccagaa gtgggcggct gtggtggtgc cttctggaca ggagcagaga 840 tacacctgcc atgtgcagca tgagggtttg cccaagcccc tcaccctgag atgggagccg 900 tcttcccagc ccaccatccc catcgtgggc atcattgctg gcctggttct ctttggagct 960 gtgatcactg gagctgtggt cgctgctgtg atgtggagga ggaagagctc agatagaaaa 1020 ggagggagct actctcaggc tgcaagcagt gacagtgccc agggctctga tgtgtctctc 1080 acagcttgta aagtg 1095 <210> 7 <211> 1128 <212> DNA <213> designed <400> 7 atggccgtca tggcgccccg aaccctcctc ctgctactct cgggggccct ggccctgacc 60 cagacctggg cgggctccca ctccatgagg tatttctaca cctccgtgtc ccggcccggc 120 cgcggggagc cccgcttcat cgccgtgggc tacgtggacg acacgcagtt cgtgcggttc 180 gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggg 240 ccggagtatt gggaccagga gacacggaat gtgaaggccc agtcacagac tgaccgagtg 300 gacctgggga ccctgcgcgg ctactacaac cagagcgagg acggttctca caccatccag 360 ataatgtatg gctgcgacgt ggggccggac gggcgcttcc tccgcgggta ccggcaggac 420 gcctacgacg gcaaggatta catcgccctg aacgaggacc tgcgctcttg gaccgcggcg 480 gacatggcag ctcagatcac caagcgcaag tgggaggcgg cccatgcggc ggagcagcag 540 agagcctacc tggagggccg gtgcgtggag tggctccgca gatacctgga gaacgggaag 600 gagacgctgc agcgcacgga cccccccaag acacatatga cccaccaccc catctctgac 660 catgaggcca ccctgaggtg ctgggccctg ggcttctacc ctgcggagat cacactgacc 720 tggcagcggg atggggagga ccagacccag gacacggagc tcgtggagac caggcctgca 780 ggggatggaa ccttccagaa gtgggcggct gtggtggtgc cttctggaga ggagcagaga 840 tacacctgcc atgtgcagca tgagggtctg cccaagcccc tcaccctgag atgggagctg 900 tctcccagc ccaccatccc catcgtgggc atcattgctg gcctggttct cttggagct 960 gtgatcactg gagctgtggt cgctgccgtg atgtggagga ggaagagctc agatagaaaaa 1020 ggagggagtt acactcaggc tgcaagcagt gacagtgccc agggctctga tgtgtctctc 1080 acagcttgta aagtgggatc ctatccatat gatgttccag attacgct 1128 <210> 8 <211> 1095 <212> DNA <213> designed <400> 8 atggccgtca tggcgccccg aaccctcgtc ctgctactct cgggggccct ggccctgacc 60 cagacctggg caggctccca ctccatgagg tatttctcca catccgtgtc ccggcccggc 120 cgcggggagc cccgcttcat cgccgtgggc tacgtggacg acacgcagtt cgtgcggttc 180 gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggg 240 ccggagtatt gggacgagga gacagggaaa gtgaaggccc actcacagac tgaccgagag 300 aacctgcgga tcgcgctccg ctactacaac cagagcgagg ccggttctca caccctccag 360 atgatgtttg gctgcgacgt ggggtcggac gggcgcttcc tccgcgggta ccaccagtac 420 gcctacgacg gcaaggatta catcgccctg aaagaggacc tgcgctcttg gaccgcggcg 480 gacatggcgg ctcagatcac caagcgcaag tgggaggcgg cccatgtggc ggagcagcag 540 agagcctacc tggagggcac gtgcgtggac gggctccgca gatacctgga gaacgggaag 600 gagacgctgc agcgcacgga cccccccaag acacatatga cccaccaccc catctctgac 660 catgaggcca ctctgagatg ctgggccctg ggcttctacc ctgcggagat cacactgacc 720 tggcagcggg atggggagga ccagacccag gacacggagc ttgtggagac caggcctgca 780 ggggatggaa ccttccagaa gtgggcagct gtggtggtac cttctggaga ggagcagaga 840 tacacctgcc atgtgcagca tgagggtctg cccaagcccc tcaccctgag atgggagcca 900 tcttcccagc ccaccgtccc catcgtgggc atcattgctg gcctggttct ccttggagct 960 gtgatcactg gagctgtggt cgctgctgtg atgtggagga ggaacagctc agatagaaaa 1020 ggagggagct actctcaggc tgcaagcagt gagtgccc agggctctga tgtgtctctc 1080 acagcttgta aagtg 1095 <210> 9 <211> 951 <212> DNA <213> designed <400> 9 atgttccagg ccgccgagcg cccccaggag tgggccatgg agggccccg cgacggcctg 60 aagaaggagc gcctgctgga cgaccgccac ngagcggcc tggacaccat gaggacgag 120 gagtacgagc agatggtgaa ggagctgcag gagatccgcc tggagcccca ggaggtgccc 180 cgcggcagcg agccctggaa gcagcagctg accgaggacg gcgacagctt cctgcacctg 240 gccatcatcc acgaggagaa ggccctgacc atggaggtga tccgccaggt gaagggcgac 300 ctggccttcc tgaacttcca gaacaacctg cagcagaccc ccctgcacct ggccgtgatc 360 accaaccagc ccgagatcgc cgaggccctg ctggccgccg gctgcgaccc cgagctgcgc 420 gacttccgcg gcaacccc cctgcacctg gcctgcgagc agggctgcct ggccagcgtg 480 ggcgtgctga cccagagctg caccaccccc cacctgcaca gcatcctgaa ggccaccaac 540 tacaacggcc acacctgcct gcacctggcc agcatccacg gctacctggg catcgtggag 600 ctgctggtga gcctgggcgc cgacgtgaac gcccaggagc cctgcaacgg ccgcaccgcc 660 ctgcacctgg ccgtggacct gcagaacccc gacctggtga gcctgctgct gaagtgcggc 720 gccgacgtga accgcgtgac ctaccagggc tacagcccct accagctgac ctggggccgc 780 cccagcaccc gcatccagca gcagctgggc cagctgaccc tggagaacct gcagatgctg 840 cccgagagcg aggacgagga gagctacgac accgagagcg agttcaccga gttcaccgag 900 gacgagctgc cctacgacga ctgcgtgttc ggcggccagc gcctgaccct g 951 <210> 10 <211> 54 <212> DNA <213> designed <400> 10 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg ccca 54 <210> 11 <211> 1761 <212> DNA <213> designed <400> 11 atgctggctg gagagacagg gcaggaggct gcgcccctgg acggagtcct ggccaaccca 60 cctaacattt ccagcctctc ccctcgccaa ctccttggct tcccgtgtgc ggaggtgtcc 120 ggcctgagca cggagcgtgt ccgggagctg gctgtggcct tggcacagaa gaatgtcaag 180 ctctcaacag agcagctgcg ctgtctggct caccggctct ctgagccccc cgaggacctg 240 gacgccctcc cattggacct gctgctattc ctcaacccag atgcgttctc ggggccccag 300 gcctgcaccc gtttcttctc ccgcatcacg aaggccaatg tggacctgct cccgaggggg 360 gctcccgagc gacagcggct gctgcctgcg gctctggcct gctggggtgt gcgggggtct 420 ctgctgagcg aggctgatgt gcgggctctg ggaggcctgg cttgcgacct gcctgggcgc 480 tttgtggccg agtcggccga agtgctgcta ccccggctgg tgagctgccc gggacccctg 540 gaccaggacc agcaggaggc agccagggcg gctctgcagg gcgggggacc cccctacggc 600 cccccgtcga catggtctgt ctccacgatg gacgctctgc ggggcctgct gcccgtgctg 660 ggccagccca tcatccgcag catcccgcag ggcatcgtgg ccgcgtggcg gcaacgctcc 720 tctcgggacc catcctggcg gcagcctgaa cggaccatcc tccggccgcg gttccggcgg 780 gaagtggaga agacagcctg tccttcaggc aagaaggctc ccgagataga cgagagcctc 840 atcttctaca agaagtggga gctggaagcc tgcgtggatg cggccctgct ggccacccag 900 atggaccgcg tgaacgccat ccccttcacc tacgagcagc tggacgtcct aaagcataaa 960 ctggatgagc tctacccaca aggttacccc gagtctgtga tccagcacct gggctacctc 1020 ttcctcaaga tgagccctga ggacattcgc aagtggaatg tgacgtccct ggagaccctg 1080 aaggctttgc ttgaagtcaa caaagggcac gaaatgagtc ctcaggtggc caccctgatc 1140 gaccgctttg tgaagggaag gggccagcta gacaaagaca ccctagacac cctgaccgcc 1200 ttctaccctg ggtacctgtg ctccctcagc cccgaggagc tgagctccgt gccccccagc 1260 agcatctggg cggtcaggcc ccaggacctg gacacgtgtg acccaaggca gctggacgtc 1320 ctctatccca aggcccgcct tgctttccag aacatgaacg ggtccgaata cttcgtgaag 1380 atccagtcct tcctgggtgg ggcccccacg gaggatttga aggcgctcag tcagcagaat 1440 gtgagcatgg acttggccac gttcatgaag ctgcggacgg atgcggtgct gccgttgact 1500 gtggctgagg tgcagaact tctgggaccc cacgtggagg gcctgaggc ggaggagcgg 1560 caccgcccgg tgcgggactg gatcctacgg cagcggcagg acgacctgga cacgctgggg 1620 ctggggctac agggcggcat ccccaacggc tacctggtcc tegctcag catgcaagg 1680 gccctctcgg gggacccctg cctcctagga cctggacctg ttccaccct cctggcactg 1740 CTCCTAGCCT CACCCTGCC C 1761 <210> 12 <211> 2004 <212> DNA <213> designed <400> 12 aagctcacta ttgaatccac gccgttcaat gtcgcagagg ggaaggaggt gcttctactt 60 gtccacaatc tgcccagca tctttttggc tacagctggt acaaggtga aagagtgat 120 ggcaccgtc aaattatagg atatgtaata ggaactcac aagctacccc agggcccgca 180 tacagtggtc gagagataat ataccccaat gcatccctgc tgatccagaa catcatccag 240 aatgacacag gattctacac cctacacgtc ataaagtcag atcttgtgaa tgagaagca 300 actggccagt tccgggtata cccggagctg cccaagccct ccatctccag caacaactcc 360 aaacccgtgg aggacaagga tgctgtggcc ttcacctgtg aacctgagac tcaggacgca 420 acctacctgt ggtgggtaaa caatcagagc ctcccggtca gtcccaggct gcagctgtcc 480 aatggcaaca ggaccctcac tctattcaat gtcacaagaa atgacacagc aagctacaaa 540 tgtgaaaccc agaacccagt gagtgccagg cgcagtgatt cagtcatcct gaatgtcctc 600 tatggcccgg atgcccccac catttcccct ctaaacacat cttacagatc aggggaaaat 660 ctgaacctct cctgccacgc agcctctaac ccacctgcac agtactcttg gtttgtcaat 720 gggactttcc agcaatccac ccaagagctc tttatcccca acatcactgt gaataatagt 780 ggatcctata cgtgccaagc ccataactca gacactggcc tcaataggac cacagtcacg 840 acgatcacag tctatgcaga gccacccaaa cccttcatca ccagcaacaa ctccaacccc 900 gtggaggatg aggatgctgt agccttaacc tgtgaacctg agattcagaa cacaacctac 960 ctgtggtggg taaataatca gagcctcccg gtcagtccca ggctgcagct gtccaatgac 1020 aacaggaccc tcactctact cagtgtcaca aggaatgatg taggacccta tgagtgtgga 1080 atccagaaca aattaagtgt tgaccacagc gacccagtca tcctgaatgt cctctatggc 1140 ccagacgacc ccaccatttc cccctcatac acctattacc gtccaggggt gaacctcagc 1200 ctctcctgcc atgcagcctc taacccacct gcacagtatt cttggctgat tgatgggaac 1260 atccagcaac acacacaaga gctcttatc tccaacatca ctgagaagaa cagcggactc 1320 tatacctgcc aggccaataa ctcagccagt ggccacagca ggactacagt caagacaatc 1380 acagtctctg cggagctgcc caagccctcc atctccagca acaactccaa acccgtggag 1440 gacaaggatg ctgtggcctt cacctgtgaa cctgaggctc agaacacaac ctacctgtgg 1500 tgggtaaatg gtcagagcct cccagtcagt cccaggctgc agctgtccaa tggcaacagg 1560 accctcactc tattcaatgt cacaagaaat gacgcaagag cctatgtatg tggaatccag 1620 aactcagtga gtgcaaaccg cagtgaccca gtcaccctgg atgtcctcta tgggccggac 1680 acccccatca tttccccccc agactcgtct tacctttcgg gagcgaacct caacctctcc 1740 tgccactcgg cctctaaccc atccccgcag tattctttggc gtatcaatgg gataccgcag 1800 caacacacac aagttctct tatcgccaaa atcacgccaa ataataacgg gacctatgcc 1860 tgttttgtct ctaacttggc tactggccgc aataattcca tagtcaagag catcacagtc 1920 tctgcatctg gaacttctcc tggtctctca gctggggcca ctgtcggcat catgattgga 1980 gggctggttg gggttgctct gata 2004 <210> 13 <211> 540 <212> DNA <213> designed <400> 13 atgcaggccg aaggccgggg cacagggggt tcgacgggcg atgctgatgg cccaggaggc 60 cctggcattc ctgatggccc agggggcaat gctggcggcc caggagaggc gggtgccacg 120 ggcggcagag gtccccgggg cgcaggggca gcaagggcct cggggccggg aggaggcgcc 180 ccgcggggtc cgcatggcgg cgcggcttca gggctgaatg gatgctgcag atgcggggcc 240 agggggccgg agagccgcct gcttgagtc tacctcgcca tgcctttcgc gacacccatg 300 gaagcagagc tggcccgcag gagcctggcc caggatgccc caccgcttcc cgtgccaggg 360 gtgcttctga aggagttcac tgtgtccggc aacatactga ctatccgact gactgctgca 420 gaccaccgcc aactgcagct ctccatcagc tcctgtctc agcagctttc cctgttgatg 480 tggatcacgc agtgctttct gcccgtgttt ttggctcagc ctccctcagg gcagaggcgc 540 <210> 14 <211> 762 <212> DNA <213> designed <400> 14 atggaatacg cctctgacgc ttcactggac cccgaagccc cgtggcctc cgcgcccgc 60 gctcgcgcct gccgcgtact gccttgggcc ctggtcgcgg ggctgctgct gctgctgctg 120 ctcgctgccg cctgcgcgt cttcctcgcc tgcccctggg ccgtgtcgg ggctcgcgcc 180 tcgccggct ccgcggccag ccgagactc cgcgagggtc ccgagctttc gccgacgat 240 cccgccggcc tcttggacct gcggcagggc atgtttgcgc agctggtggc ccaaaatgtt 300 ctgctgatcg atgggcccct gagctggtac agtgacccag gcctggcagg cgtgtccctg 360 acggggggcc tgagctacaa agaggacacg aaggagctgg tggtggccaa ggctggagtc 420 tactatgtct tctttcaact agagctgcgg cgcgtggtgg ccggcgaggg ctcaggctcc 480 gtttcacttg cgctgcacct gcagccactg cgctctgctg ctggggccgc cgccctggct 540 ttgaccgtgg acctgccacc cgcctcctcc gaggctcgga actcggcctt cggtttccag 600 ggccgcttgc tgcacctgag tgccggccag cgcctgggcg tccatcttca cactgaggcc 660 agggcacgcc atgcctggca gcttacccag ggcgccacag tcttgggact cttccggggtg 720 acccccgaa tcccagccgg actcccttca ccgaggtcgg aa <210> 15 <211> 1164 <212> DNA <213> designed <400> 15 atggggctgg gcccggtctt cctgcttctg gctggcatct tcccttttgc acctccgggga gctgctgctg agccccacag tcttcgttat aacctcacgg tgctgtccgg ggatggatct gtgcagtcag ggtttctcgc tgaggtacat ctggatggtc agcccttcct gcgctgtgac 180 aggcagaat gcagggcaaa gccccaggga cagtgggcag aagatgtcct gggaataag acatgggaca gagagaccag ggacttgaca gggacgga aggacctcag gatgaccctg gctcatatca aggaccaga agaaggcttg cattccctcc aggattag ggtctgtgag 360 atccatgaag acaacagcac caggagctcc cagcatttct actacgatgg ggagctcttc 420 ctctcccaaa acctggagac tgaggaatg acaatgcccc agtcctccag agctcagacc 480 ttggccatga acgtcaggaa tttcttgaag gaagatgcca tgaagaccaa gacacactat 540 cacgctatgc atgcagactg cctgcaggaa ctacggcgat atctaaaatc cggcgtagtc 600 ctgaggagaa cagtgccccc catggtgaat gtcacccgca gcgaggcctc agagggcaac 660 attaccgtga catgcagggc ttctggcttc tatccctgga atatcacact gagctggcgt 720 caggatgggg tatctttgag ccacgacacc cagcagtggg gggatgtcct gcctgatggg 780 aatggaacct accagacctg ggtggccacc agatttgcc aaggagagga gcagaggttc 840 acctgctaca tggaacacag cgggaatcac agcactcacc ctgtgccctc tgggaaagtg 900 ctggtgcttc agagtcattg gcagacattc catgtttctg ctgttgctgc tgctgctgct 960 gctgctgctg ctatttttgt tattattatt ttctacgtct gttgttgtaa gaagaaaaca 1020 tcagctgcag agggtccaga gctcgtgagc ctgcaggtcc tggatcaaca cccagttggg 1080 acgagtgacc acagggatc cacacagctc ggatttcagc ctctgatgtc agatcttggg tccactggct ccctgaggg cgcc1164 <210> 16 <211> 87 <212> DNA <213> designed <400> 16 60. atgagaattt cgaaaccaca tttgagaagt atttccatcc agtgctactt gtgtttactt ctaaacagtc attttctac tgaagct <210> 17 <211> 342 <212> DNA <213> designed <400> 17 aactgggtga atgtaataag tgatttgaaa aaaattgaag atcttattca atctatgcat attgatgcta ctttatatac ggaagtgat gttcacccca gttgcaaagt aacagcaatg aagtgctttc tcttggagtt acaagttatt tcacttgagt ccggagatgc aagtattcat 180 240. sightseeing aaaatctgat catcctagca aacaacagtt tgtcttctaa tgggaatgta acagaatctg gatgcaaaga atgtgagga ctggagga aaaatattaa agaatttttg cagagttttg tacatattgt ccaaatgttc atcaacactt ct <210> 18 <211> 213 <212> DNA <213> designed <400> 18 gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 60 cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 120 agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 180 gtccttctcc tgtcactggt tatcaccctt tac 213 <210> 19 <211> 9 <212> DNA <213> designed <400> 19 gcttactac 9

Claims

1. A lentiviral particle for preparing a recombinant NT gene of a novel immune cell, characterized in that: The lentiviral particles of the recombinant NT gene are obtained by infecting host cells with a four-plasmid viral packaging system containing a lentiviral vector carrying the recombinant NT gene, wherein the recombinant NT gene consists of a nucleotide sequence fragment A, a nucleotide sequence fragment B, a nucleotide sequence fragment C and a nucleotide sequence fragment D, and the sequence of the recombinant NT gene is selected from NT1 as shown in SEQ ID No. 1 or the NT2 gene whose 5′ to 3′ sequence is -BCDE-, wherein the nucleotide sequence of A is shown in SEQ ID No. 1, the nucleotide sequence of B is shown in SEQ ID No. 2, the nucleotide sequence of C is shown in SEQ ID No. 3, the nucleotide sequence of D is shown in SEQ ID No. 10, and the nucleotide sequence of E is shown in SEQ ID No. 4, wherein the host cell is a 293T cell.

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