Novel IL-15 fusion protein and application thereof

Abstract

The invention relates to the field of immunity, in particular to a novel IL-15 fusion protein and application thereof. According to the IL-15 transmembrane fusion protein provided by the invention, the structure is applied to NK cells or CAR-NK cells, so that the tumor killing function of the NK cells and the CAR-NK cells can be remarkably enhanced, and the proliferation of the NK cells and the CAR-NK cells can be effectively promoted. The IL-15 transmembrane fusion protein provides an important basis for developing NK cell products which do not depend on any tumor target and are armored by the IL-15 transmembrane fusion protein, so that the CAR-NK development period is shortened, and the CAR-NK development cost is reduced. Meanwhile, functional studies are respectively carried out in hematoma expressing CD19 and solid tumor expressing CD276, and the results indicate the broad-spectrum application prospect of the structure in tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, in particular to a novel IL-15 fusion protein and application thereof. BACKGROUND

[0002] Chimeric antigen receptor modified immune effector cell (CAR-T / NK) therapy has developed rapidly and is highly expected in recent years. NK cells are considered to have the potential to replace T cells. Compared with CAR-T cell therapy, CAR-NK cells have some significant advantages, including: (1) better safety and less side effects; (2) lower immunogenicity, suitable for developing allogeneic / off-the-shelf products; (3) multiple anti-tumor activation mechanisms, in addition to CAR-dependent activation, CAR-NK can also activate cancer cells through non-CAR-dependent cancer cell-specific activation.

[0003] Early studies found that IL-2 can stimulate the expansion of killer cells (including NK cells and T cells) in vitro and show certain anti-tumor activity after being reinfused into patients, providing a basis for subsequent cell immunotherapy. With the rapid development of CAR-T and CAR-NK therapy in recent years, in addition to IL-2, the therapeutic potential of IL-15 has also been developed. In the prior art, the effect of IL-15 on NK cells / CAR-NK cells needs to be further improved. SUMMARY

[0004] Therefore, the present application provides a novel IL-15 fusion protein and application thereof. The present application introduces a transmembrane structure to form a novel IL-15 fusion protein (tmbIL-15). The transmembrane structure can play a role in promoting proliferation of IL-15, and on the other hand, it can realize the transmission of activation signals to the NK cell interior through the transmembrane structure, thereby further activating the NK cell.

[0005] To achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] In a first aspect, the present application provides an IL-15 transmembrane fusion protein, comprising: an IL-15 coding region, a hinge region and a truncated 2B4 receptor; the truncated 2B4 receptor has an amino acid sequence as shown in SEQ ID NO. 23;

[0007] The IL-15 coding region has an amino acid sequence as shown in SEQ ID NO. 11;

[0008] The hinge region comprises a CD28 hinge; the CD28 hinge has an amino acid sequence as shown in SEQ ID NO. 13.

[0009] In some embodiments of the present application, the IL-15 transmembrane fusion protein has an amino acid sequence as set forth in SEQ ID NO. 31.

[0010] In a second aspect, the present application also provides a fusion protein comprising the IL-15 transmembrane fusion protein, further comprising one or more of a signal peptide, an antigen binding domain, a hinge & transmembrane region, a signaling region, or a cleavage peptide.

[0011] The signal peptide is selected from CD8a SP;

[0012] The antigen binding domain is selected from CD276 scFV, CD276 VHH, or FMC63;

[0013] The hinge & transmembrane region is selected from CD8a hinge & TM or CD28 hinge; and / or

[0014] The signaling region is selected from BBz;

[0015] The cleavage peptide is selected from P2A.

[0016] In some embodiments of the present application, the signal peptide CD8a SP has an amino acid sequence as set forth in SEQ ID NO. 1;

[0017] The CD276 scFV has an amino acid sequence as set forth in SEQ ID NO. 3, the CD276 VHH has an amino acid sequence as set forth in SEQ ID NO. 32, or the FMC63 has an amino acid sequence as set forth in SEQ ID NO. 34;

[0018] The hinge & transmembrane region has an amino acid sequence as set forth in SEQ ID NO. 5;

[0019] The signaling region has an amino acid sequence as set forth in SEQ ID NO. 7;

[0020] The cleavage peptide has an amino acid sequence as set forth in SEQ ID NO. 9.

[0021] In a third aspect, the present application also provides a nucleic acid molecule encoding the IL-15 transmembrane fusion protein, or encoding the fusion protein.

[0022] In a fourth aspect, the present application also provides an expression vector comprising the nucleic acid molecule.

[0023] In a fifth aspect, the present application also provides a viral particle comprising the expression vector.

[0024] In a sixth aspect, the present application also provides a host comprising any of:

[0025] (I) the IL-15 transmembrane fusion protein; or

[0026] (II) the fusion protein; or

[0027] (III) the expression vector; or

[0028] (IV) the viral particle.

[0029] In some embodiments of the present application, the host comprises an NK cell, an NKT cell, a T cell, or an induced NK cell, NKT cell, T cell.

[0030] In a seventh aspect, the present application further provides use of any of the following in the manufacture of a medicament for maintaining the activity of an immune cell, promoting the survival or proliferation of an immune cell, or enhancing the tumor-killing function of an immune cell:

[0031] (I) the IL-15 transmembrane fusion protein; or

[0032] (II) the fusion protein; or

[0033] (III) the expression vector; or

[0034] (IV) the viral particle; or

[0035] (V) the host.

[0036] In some embodiments of the present application, the tumor comprises a hematological tumor and / or a solid tumor.

[0037] As preferred, the solid tumor expresses CD276; more preferably, the solid tumor comprises one or more of ovarian cancer, prostate cancer, liver cancer, lung cancer, head and neck cancer, kidney cancer, cervical cancer, breast cancer, colorectal cancer, neuroblastoma, pituitary tumor, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, skin cancer, myeloma, bladder cancer, osteosarcoma, or glioma.

[0038] As preferred, the hematological tumor expresses CD19; more preferably, the hematological tumor comprises lymphoma or B-cell leukemia.

[0039] In some embodiments of the present application, the enhancing the tumor-killing function comprises increasing the killing power of a target cell and / or promoting the release of a killing factor;

[0040] As preferred, the killing factor comprises IFN-γ or TNF-α.

[0041] In an eighth aspect, the present application further provides use of any of the following in the manufacture of a medicament for preventing and / or treating a tumor:

[0042] (I) the IL-15 transmembrane fusion protein; or

[0043] (II) the fusion protein; or

[0044] (III) the expression vector; or

[0045] (IV) the viral particle; or

[0046] (V) the host.

[0047] In some embodiments of the present application, the tumor comprises a hematological tumor and / or a solid tumor.

[0048] As preferred, the solid tumor expresses CD276; more preferably, the solid tumor comprises one or more of ovarian cancer, prostate cancer, liver cancer, lung cancer, head and neck cancer, kidney cancer, cervical cancer, breast cancer, colorectal cancer, neuroblastoma, pituitary tumor, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, skin cancer, myeloma, bladder cancer, osteosarcoma or glioma.

[0049] As preferred, the hematological tumor expresses CD19; more preferably, the hematological tumor comprises lymphoma or B-cell leukemia.

[0050] In a ninth aspect, the present application further provides a medicine / medicine combination, characterized in that the medicine is made of any of the following items, and pharmaceutically acceptable adjuvant or auxiliary agent:

[0051] (I) the IL-15 transmembrane fusion protein; or

[0052] (II) the fusion protein; or

[0053] (III) the expression vector; or

[0054] (IV) the viral particle; or

[0055] (V) the host.

[0056] The medicine combination comprises the medicine and other any effective ingredients. BRIEF DESCRIPTION OF DRAWINGS

[0057] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows.

[0058] Figure 1 Infection efficiency of IL-15 transmembrane protein lentivirus in core plasmid CAR structure 1 group CAR-NK cells;

[0059] Figure 2 CAR+ proportion change of CAR-NK cells of the core plasmid CAR structure 1 group after stimulation culture by adding PANC-1 cells in the culture process;

[0060] Figure 3 CAR+ proportion change of CAR-NK cells of the core plasmid CAR structure 1 group after stimulation culture without adding PANC-1 cells in the culture process;

[0061] Figure 4 SDT-NK003 CAR-NK and SDT-NK045 CAR-NK cell killing (lysis) of PANC-1 cells under different effector-to-target ratio (E:T) conditions in the group without PANC-1 pretreatment;

[0062] Figure 5 SDT-NK003 CAR-NK and SDT-NK045 CAR-NK cell killing (lysis) of PANC-1 cells under different effector-to-target ratio (E:T) conditions in the PANC-1 pretreatment group;

[0063] Figure 6 Release of interferon gamma (IFN-γ) in the cell supernatant group;

[0064] Figure 7 Release of interferon gamma (IFN-γ) in the cell co-incubation group;

[0065] Figure 8 Flow cytometry detection results of IL-15 expression of the UTD-NK control group and the SDT-NK076 group;

[0066] Figure 9 Cell viability and total number of NK cells of UTD-NK and SDT-NK076 NK in the culture process;

[0067] Figure 10 Killing rate (lysis) of PANC-1 cells of SDT-NK076 NK cells obtained under the culture condition of adding IL-2 under different effector-to-target ratio (E:T) conditions;

[0068] Figure 11 Killing rate (lysis) of Huh-7 cells of SDT-NK076 NK cells obtained under the culture condition of adding IL-2 under different effector-to-target ratio (E:T) conditions;

[0069] Figure 12Figure 6 shows the lysis of HePG2-CD276 cells by SDT-NK076 NK cells obtained under culture conditions with the addition of IL-2 at different effector-to-target (E:T) ratios;

[0070] Figure 13 Figure 7 shows the lysis of Raji cells by SDT-NK076 NK cells obtained under culture conditions with the addition of IL-2 at different effector-to-target (E:T) ratios;

[0071] Figure 14 Figure 8 shows the lysis of HCT116 cells by SDT-NK076 NK cells obtained under culture conditions with the addition of IL-2 at different effector-to-target (E:T) ratios;

[0072] Figure 15 Figure 9 shows the lysis of HCC827 cells by SDT-NK076 NK cells obtained under culture conditions with the addition of IL-2 at different effector-to-target (E:T) ratios;

[0073] Figure 16 Figure 10 shows the lysis of SKOV-3 cells by SDT-NK076 NK cells obtained under culture conditions with the addition of IL-2 at different effector-to-target (E:T) ratios;

[0074] Figure 17 Figure 11 shows the short-term lysis of PANC-1 cells by SDT-NK076 NK cells obtained under culture conditions without the addition of IL-2 at different effector-to-target (E:T) ratios;

[0075] Figure 18 Figure 12 shows the amount of interferon gamma (IFN-γ) released by SDT-NK076 NK cells obtained under culture conditions with the addition of IL-2 after co-culture with PANC-1 cells;

[0076] Figure 19 Figure 13 shows the amount of interferon gamma (IFN-γ) released by SDT-NK076 NK cells obtained under culture conditions with the addition of IL-2 after co-culture with Huh-7 cells;

[0077] Figure 20 Figure 14 shows the amount of interferon gamma (IFN-γ) released by SDT-NK076 NK cells obtained under culture conditions with the addition of IL-2 after co-culture with Raji cells;

[0078] Figure 21 Figure 15 shows the amount of tumor necrosis factor alpha (TNF-α) released by SDT-NK076 NK cells obtained under culture conditions with the addition of IL-2 after co-culture with PANC-1 cells;

[0079] Figure 22 The amount of TNF-α released after co-culturing SDT-NK076 NK cells obtained by culturing under the condition of adding IL-2 with Huh-7 cells;

[0080] Figure 23 The amount of TNF-α released after co-culturing SDT-NK076 NK cells obtained by culturing under the condition of adding IL-2 with Raji cells;

[0081] Figure 24 The amount of IFN-γ released after co-culturing SDT-NK076 NK cells obtained by culturing under the condition of not adding IL-2 with PANC-1 cells;

[0082] Figure 25 The amount of TNF-α released after co-culturing SDT-NK076 NK cells obtained by culturing under the condition of not adding IL-2 with PANC-1 cells;

[0083] Figure 26 The proportion of CD107a expression of SDT-NK076 NK cells after stimulation by PANC-1 cells;

[0084] Figure 27 The proportion of CD107a expression of SDT-NK076 NK cells after stimulation by Raji cells;

[0085] Figure 28 The expression level of CD56 and CD16 during the culturing of SDT-NK076 NK cells;

[0086] Figure 29 The expression level of NKp30 during the culturing of SDT-NK076 NK cells;

[0087] Figure 30 The expression level of NKp44 during the culturing of SDT-NK076 NK cells;

[0088] Figure 31 The expression level of NKG2D during the culturing of SDT-NK076 NK cells;

[0089] Figure 32 The in vivo imaging result of pancreatic cancer cell PANC-1 luc of tumor-bearing mice after administration of STD-NK076;

[0090] Figure 33 The quantitative expression result of pancreatic cancer cell PANC-1 luc of tumor-bearing mice after administration of STD-NK076;

[0091] Figure 34Figure 8 shows the survival rate of NK cells in the peripheral blood of tumor-bearing mice after administration of SDT-NK076;

[0092] Figure 35 Figure 9 shows the viability of NK cells in SDT-NK078 CAR-NK and SDT-NK066 CAR-NK;

[0093] Figure 36 Figure 10 shows the number of NK cells in SDT-NK078 CAR-NK and SDT-NK066 CAR-NK;

[0094] Figure 37 Figure 11 shows the proportion of CAR+ NK cells in SDT-NK078 CAR-NK and SDT-NK066 CAR-NK;

[0095] Figure 38 Figure 12 shows the short-term lysis of PANC-1 cells by SDT-NK078 NK cells obtained under culture conditions with the addition of IL-2 at different effector-to-target (E:T) ratios;

[0096] Figure 39 Figure 13 shows the short-term lysis of Huh-7 cells by SDT-NK078 NK cells obtained under culture conditions with the addition of IL-2 at different effector-to-target (E:T) ratios;

[0097] Figure 40 Figure 14 shows the short-term lysis of HepG2-CD276 cells by SDT-NK078 NK cells obtained under culture conditions with the addition of IL-2 at different effector-to-target (E:T) ratios;

[0098] Figure 41 Figure 15 shows the killing of HCT116 cells by SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells when the culture medium contains the cytokine IL-2;

[0099] Figure 42 Figure 16 shows the killing of HCC827 cells by SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells when the culture medium contains the cytokine IL-2;

[0100] Figure 43 Figure 17 shows the killing of SKOV-3 cells by SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells when the culture medium contains the cytokine IL-2;

[0101] Figure 44SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cell killing of PANC-1 cells in the absence of IL-2 in the culture medium;

[0102] Figure 45 IFN-γ release from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells co-cultured with PANC-1 cells in the presence of IL-2 in the culture medium;

[0103] Figure 46 IFN-γ release from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells co-cultured with Huh-7 cells in the presence of IL-2 in the culture medium;

[0104] Figure 47 TNF-α release from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells co-cultured with PANC-1 cells in the presence of IL-2 in the culture medium;

[0105] Figure 48 TNF-α release from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells co-cultured with Huh-7 cells in the presence of IL-2 in the culture medium;

[0106] Figure 49 IFN-γ release from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells co-cultured with PANC-1 cells in the absence of IL-2 in the culture medium;

[0107] Figure 50 TNF-α release from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells co-cultured with PANC-1 cells in the absence of IL-2 in the culture medium;

[0108] Figure 51 CD107a release from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells co-cultured with PANC-1 cells;

[0109] Figure 52 SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cell CAR+ ratio change during PANC-1 cell stimulation;

[0110] Figure 53IFN-γ release by SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells during stimulation with PANC-1 cells;

[0111] Figure 54 TNF-α release by SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells during stimulation with PANC-1 cells;

[0112] Figure 55 CD56, CD16 expression levels during culture of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells;

[0113] Figure 56 NKp30 expression levels during culture of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells;

[0114] Figure 57 NKp44 expression levels during culture of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells;

[0115] Figure 58 NKG2D expression levels during culture of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells;

[0116] Figure 59 In vivo imaging of PANC-1 luc pancreatic cancer cells in tumor-bearing mice after administration of STD-NK066 and STD-NK078;

[0117] Figure 60 Quantitative expression of PANC-1 luc pancreatic cancer cells in tumor-bearing mice after administration of STD-NK078;

[0118] Figure 61 Survival proportion of NK cells in the peripheral blood of the orbit of tumor-bearing mice after administration of STD-NK066 and STD-NK078;

[0119] Figure 62 Killing of RAJI cells by SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells when the culture medium contains the cytokine IL-2;

[0120] Figure 63 Killing of RAJI cells by SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells when the culture medium does not contain the cytokine IL-2;

[0121] Figure 64 IFN-γ release from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells co-cultured with Raji cells in medium with IL-2 added;

[0122] Figure 65 TNF-α release from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells co-cultured with Raji cells in medium with IL-2 added;

[0123] Figure 66 IFN-γ release from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells co-cultured with Raji cells in medium without IL-2 added;

[0124] Figure 67 TNF-α release from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells co-cultured with Raji cells in medium without IL-2 added;

[0125] Figure 68 CD107a release from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells co-cultured with Raji cells;

[0126] Figure 69 CAR+ proportion of SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during Raji cell stimulation;

[0127] Figure 70 IFN-γ release from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during Raji cell stimulation;

[0128] Figure 71 TNF-α release from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during Raji cell stimulation;

[0129] Figure 72 CD56, CD16 expression levels of SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during culture;

[0130] Figure 73 NKp30 expression levels of SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during culture;

[0131] Figure 74 Figure 8 shows the NKp44 expression levels of SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during culture process;

[0132] Figure 75 Figure 9 shows the NKG2D expression levels of SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during culture process. DETAILED DESCRIPTION

[0133] The present application discloses a novel IL-15 fusion protein and its application. Those skilled in the art can refer to the content of this paper and appropriately improve the process parameters to achieve it. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiment, and the relevant personnel can obviously modify or appropriately change and combine the method and application described in this paper without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0134] Terminology:

[0135] PBMC: peripheral blood mononuclear cells

[0136] NK cells: natural killer cells

[0137] CAR-NK cells: chimeric antigen receptor NK cells

[0138] IL-15: interleukin 15

[0139] IFN-γ: interferon-γ

[0140] TNF-α: tumor necrosis factor alpha

[0141] 2B4: natural killer cell receptor 2B4

[0142] KIRS2: immune checkpoint receptor on the surface of NK cells

[0143] NKp30: natural cytotoxicity triggering receptor 3

[0144] NKp46: natural cytotoxicity triggering receptor 1

[0145] NKp44: natural cytotoxicity triggering receptor 2

[0146] DAP10: DNAX-associated protein 10

[0147] DAP12: DNAX-activated protein 12

[0148] CD16a: transmembrane molecule with unique cytoplasmic domain

[0149] The present application provides tmbIL-15 structure and its application in NK or CAR-NK, and its application in the treatment of hematological tumors and solid tumors. The present application studies NK and CAR-NK respectively, and finds that tmbIL-15 can significantly enhance the function of NK or CAR-NK, especially the enhancement of NK function is very significant, which indicates that it is possible to develop a NK cell product armored by tmbIL-15 which is independent of any tumor target, thereby reducing the cycle and cost of CAR-NK development.

[0150] Meanwhile, the present application studies the function in CD19-expressing hematological tumors and CD276-expressing solid tumors respectively, and the results indicate the broad-spectrum application prospect of the structure in tumors.

[0151] Unless otherwise specified, the raw materials and reagents used in the novel IL-15 fusion protein and its application provided by the present application can be purchased from the market.

[0152] The present application will be further described below in combination with examples:

[0153] Example 1 Screening of IL-15 transmembrane fusion protein structure

[0154] 1.1 Preparation of CAR-NK cells

[0155] 1.1.1 Experimental method

[0156] (1) Virus preparation: 293T cells were inoculated one day before virus packaging to ensure that the confluence of 293T cells was between 80%-90% when the virus was prepared; the core plasmid (CAR structure, see Table 1) expressing different CAR structures, the envelope plasmid (BaEVTR-PR12, refer to CN118005808A), and the auxiliary plasmid (psPAX2) were mixed in a ratio of 4:1:2, then 3 times of PEI (YESEN, 40820ES10) was added, and the mixture was allowed to stand for 15 min before being added dropwise to 293T cells for virus production. The virus stock solution collected at 48 h and 72 h was concentrated by centrifugation at 4000 g, 4℃, 9 min, and then used to infect Jurkat cells. The infection efficiency was detected after 72 h, and the virus titer was calculated. The virus was stored at -80℃ for standby.

[0157] Table 1 Core plasmid CAR structure group 1

[0158]

[0159] The amino acid sequence of CD8a SP is shown in SEQ ID NO. 1:

[0160] MALPVTALLLPLALLLHAARP

[0161] The nucleotide sequence of the CD8a SP is shown as SEQ ID NO. 2:

[0162] atggccctgcccgtgaccgccctgctgctgccactggccctgctgctgcatgccgctagacct

[0163] The amino acid sequence of the CD276 scFV is shown as SEQ ID NO. 3:

[0164] QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQNGHSFPLTFGQGTKLELKR

[0165] The nucleotide sequence of the CD276 scFV is shown as SEQ ID NO. 4:

[0166] caggtgcagctggtgcagtcaggagctgaagtggtgaaacctggcgcctctgtgaagctgagttgtaagacatctggctatacattcactaattatgatattaattgggtgagacagagacctggacagggactggagtggattggctggatctttccaggagacggctctacacagtataatgaaaagttcaagggcaaagctacactgacaaccgacaccagcaccagcaccgcctacatggagctgtccagcctgaggtccgaggataccgccgtgtacttctgcgctaggcagaccaccgccacctggttcgcctactggggccagggcaccctggtgaccgtgagcagcggcggcggcggaagcggcggcgggggctccgagatcgtgatgacccagtcccccgccaccctgagcgtgagccctggcgagagggtgaccctgagctgcagagcatctcagagcatctccgactacctgtactggtaccagcagaagagccacgaaagccccagactgctgatcaagtacgccagccagagcatcagcggcatccctgccaggttctccggcagcggctctggcagcgagttcaccctgaccattaatagcgtggaaccagaagatgttggagtgtattattgtcagaatggacactcttttccactgacatttggacagggcacaaaactggagctgaaaaga

[0167] The amino acid sequence of the CD8a hinge & TM is shown in SEQ ID NO. 5 (the underlined part is the amino acid sequence corresponding to the CD8a hinge, and the rest is the amino acid sequence corresponding to the CD8a TM):

[0168] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVIT

[0169] The nucleotide sequence of the CD8a hinge & TM is shown in SEQ ID NO. 6:

[0170] accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcacc

[0171] BBz represents 4-BB costimulatory domain + CD3 zeta signaling domain, the amino acid sequence of which is shown in SEQ ID NO. 7:

[0172] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0173] The nucleotide sequence of BBz is shown in SEQ ID NO. 8:

[0174] aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc

[0175] The amino acid sequence of P2A is shown as SEQ ID NO. 9:

[0176] GSGATNFSLLKQAGDVEENPGP

[0177] The nucleotide sequence of P2A is shown as SEQ ID NO. 10:

[0178] ggctccggtgctaccaacttttcacttctgaagcaggccggcgacgtggaggagaatccaggccct

[0179] The amino acid sequence of IL-15 is shown as SEQ ID NO. 11:

[0180] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0181] The nucleotide sequence of IL-15 is shown as SEQ ID NO. 12:

[0182] aactgggtcaacgtgatcagcgatctcaagaagattgaggacctgatccagagcatgcatattgacgccactctgtacacggagagtgatgtgcacccctcttgtaaagtgacggccatgaagtgcttcctgctggagttgcaagttatctcgctggagtctggggacgcatccatccatgacaccgtggagaacctgatcatcctggccaacaactccctttcgtctaatggcaacgtgactgagagcgggtgcaaagaatgtgaggagctggaagagaagaacatcaaggagttcctacagtccttcgtccacatcgtccagatgtttattaacacgtcc

[0183] The amino acid sequence of CD28 hinge is shown as SEQ ID NO. 13:

[0184] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0185] The nucleotide sequence of the CD28 hinge is shown in SEQ ID NO. 14:

[0186] attgaggtgatgtaccctcccccgtacctggacaacgagaaatcgaacggcaccatcatccacgttaaaggcaagcacctgtgcccaagccctctttttcccgggccgtccaagccc

[0187] The amino acid sequence of truncated KIRS2 is shown in SEQ ID NO. 15:

[0188] NPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKK

[0189] The nucleotide sequence of truncated KIRS2 is shown in SEQ ID NO. 16:

[0190] aatccacgtcacttgcatgtcctcatcggtacttccgtggtgaagattcccttcaccatcctgctgttcttcctgctacaccgctggtgttctaacaagaag

[0191] The amino acid sequence of truncated NKp30 is shown in SEQ ID NO. 17:

[0192] EHPQLGAGTVLLLRAGFYAVSFLSVAVGSTVYYQG

[0193] The nucleotide sequence of truncated NKp30 is shown in SEQ ID NO. 18:

[0194] gagcacccccagttgggtgctggcaccgtgctgctgctccgcgccggcttttacgcggtgtccttcctgagcgtcgccgtggggtctactgtttattaccaggga

[0195] The amino acid sequence of truncated NKp46 is shown in SEQ ID NO. 19:

[0196] AQNLLRMGLAFLVLVALVWFLVEDWLSRKRTRERA

[0197] The nucleotide sequence of truncated NKp46 is shown in SEQ ID NO. 20:

[0198] gcccagaacctgctgcgtatgggcctggcgttcctggtgctggtggctcttgtctggtttttggtggaggactggctctcccgcaagcgcacccgcgagcgggcc

[0199] The amino acid sequence of truncated NKp44 is shown in SEQ ID NO. 21 :

[0200] PAAPIALVPVFCGLLVAKSLVLSALLVWWGDIWWK

[0201] The nucleotide sequence of truncated NKp44 is shown in SEQ ID NO. 22:

[0202] cctgccgctcccatcgcgctggttccggtgttctgcggtctgctggtggccaagtccttggtgctgagcgcacttctcgtctggtggggcgacatttggtggaag

[0203] The amino acid sequence of truncated 2B4 is shown in SEQ ID NO. 23:

[0204] EFRFWPFLVIIVILSALFLGTLACFCVWRRKRKEK

[0205] The nucleotide sequence of truncated 2B4 is shown in SEQ ID NO. 24:

[0206] gagttccgcttctggcccttcctggtgatcatcgtgattttgtccgctctcttcctgggcaccctggcctgcttttgcgtgtggcggcgtaagcgcaaggagaaa

[0207] The amino acid sequence of truncated DAP10 is shown in SEQ ID NO. 25:

[0208] GSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRS

[0209] The nucleotide sequence of truncated DAP10 is shown in SEQ ID NO. 26:

[0210] ggctccctttctctgcccctgctggccggtctggtggcagcggacgccgtcgctagcttgctcatcgtgggcgccgtgttcctgtgcgctcgtccgcgccgctcg

[0211] The amino acid sequence of truncated DAP12 is shown as SEQ ID NO. 27:

[0212] CSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVP

[0213] The nucleotide sequence of truncated DAP12 is shown as SEQ ID NO. 28:

[0214] tgcagcaccgtgtcccccggggttcttgctggcatcgtgatgggcgacctggtgctgaccgtcctcattgcgctggccgtgtacttcctgggtcgcttggtccct

[0215] The amino acid sequence of truncated CD16a is shown as SEQ ID NO. 29:

[0216] FPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSST

[0217] The nucleotide sequence of truncated CD16a is shown as SEQ ID NO. 30:

[0218] ttccctcccggctaccaggtctccttctgcctggtgatggtgctgttgttcgccgttgacaccggtctctacttttctgtgaagaccaacatccgcagctcgact

[0219] (2) Preparation of CAR-NK cells: After the PBMC purchased from Miaoshun Biology was resuscitated, the NK cells were sorted using CytoSinct™ CD3 Nanobeads (GenScript, L00896) magnetic beads, the sorted NK cells were added to K562 feeder layer cells at 1:1, and co-cultured to the fourth day (Day 4), then the CAR-NK cells were prepared by virus transduction at a MOI of 5, and the culture medium was KBM581+10% FBS+500 IU / mL IL-2;

[0220] (3) CAR-NK positive cell detection: On the third day after virus transduction, APC anti-human CD56 (NCAM) Antibody (Biolegend, 362504) and MonoRab™ Rabbit Anti-Humanized VHH Antibody [PE] (GenScript, A02171-200) were used to stain the cells at 4°C for 20 min, and then the cells were detected by flow cytometry. UTD-NK refers to NK cells without virus transduction.

[0221] 1.1.2 Experimental results

[0222] Figure 1 The results show that the viruses in each experimental group can infect NK cells and prepare corresponding CAR-NK cells. Due to the different structural designs, the infection efficiency of each group on NK cells is different.

[0223] 1.2, Change of CAR+ proportion during target cell stimulation

[0224] 1.2.1 Experimental method

[0225] According to the experimental method of 1.1.1, various CAR-NK cells were prepared. According to the counting results and flow detection results, the CAR+ and cell amounts were adjusted to be consistent, and the CAR-NK cells were divided into two groups, 2.0x10 6 cells, and cultured in the culture medium KBM581+10%FBS. One group was added with 5.0x10 5 target cells, human pancreatic cancer cell line PANC-1, for stimulation, and the other group grew normally without target cell stimulation. The CAR+ proportion and cell counting were detected every 2 days, and the treatment was stopped when the CAR+ proportion was less than 20%.

[0226] 1.2.2 Experimental results

[0227] Figure 2 , Figure 3 The results show that under the condition of adjusting the CAR and cell amounts to be consistent, with the increase of the culture time, the CAR+ proportion of SDT-NK003 and SDT-NK045 gradually increases, and the CAR+ proportion increases to more than 50% on Day 12. The CAR+ proportion of the other groups continues to decrease until the CAR+ proportion is less than 20% and the treatment is stopped. Figure 3It is shown that the proportion of CAR+ in SDT-NK045 group is higher than that in SDT-NK003 after stimulation culture without adding PANC-1 cells in the culture process. It indicates that the CAR+ cells of SDT-NK045 and SDT-NK003 have strong ability of continuous proliferation, and the CAR+ cells of SDT-NK045 have the strongest ability of continuous proliferation. SDT-NK045 and SDT-NK003 are selected for further functional verification.

[0228] 1.3 Short-term killing verification in vitro

[0229] 1.3.1 Experimental method

[0230] PANC-1 cells of human pancreatic cancer cell line are selected as target cells.

[0231] SDT-NK045 CAR-NK cells and SDT-NK003 CAR-NK cells are selected as effector cells, and two different treatments are performed, and NK cells (UTD-NK) are selected as controls.

[0232] PANC-1 pretreatment group: after co-culturing PANC-1 cells with SDT-NK045 CAR-NK cells and SDT-NK003 CAR-NK cells for 12 days, respectively, SDT-NK045 CAR-NK cells and SDT-NK003 CAR-NK cells are collected as effector cells.

[0233] No PANC-1 pretreatment group: after culturing SDT-NK045 CAR-NK cells and SDT-NK003 CAR-NK cells for 12 days, respectively, SDT-NK045 CAR-NK cells and SDT-NK003 CAR-NK cells are collected as effector cells.

[0234] ① 10000 cells / 100 μL of target cells are added to each well of a 96-well white plate;

[0235] ② The amount of effector cells is calculated according to the set 6 effector-to-target ratios (2:1, 1:1, 0.5:1, 0.25:1, 0.125:1, 0.0625:1), and the effector cells are diluted to the corresponding concentration in a transparent 96-well plate;

[0236] ③ 100 μL of diluted effector cells are inoculated into the target cells of step ①;

[0237] ④ After inoculating the target cells and effector cells, they are placed in a carbon dioxide incubator for culture;

[0238] ⑤ After 20 hours, ONE-Glo substrate (Promega, #E6120) is added, and the enzyme marker is detected;

[0239] vi. Calculate the killing efficiency according to the following formula:

[0240] Effector cell killing rate% = (1 - sample group reading / target cell reading)%

[0241] Target cell reading refers to the value detected by the microplate reader after only inoculating 10000 cells / well of target cells.

[0242] 1.3.2 Experimental results

[0243] Figure 4 、 Figure 5 The results show that SDT-NK003 CAR-NK and SDT-NK045 CAR-NK cells have significant killing effect on PANC-1 target cells. Figure 4 It is shown that when not pretreated with PANC-1, SDT-NK003 and SDT-NK045 have strong killing effect and no difference; Figure 5 It is shown that when repeatedly stimulated by pretreatment with PANC-1, SDT-NK045 has stronger killing ability than SDT-NK003.

[0244] 1.4 Cytokine release detection

[0245] 1.4.1 Experimental method

[0246] SDT-NK045, SDT-NK003 and UTD-NK cells were prepared respectively, and the cells were treated in two groups:

[0247] Cell supernatant group: SDT-NK045, SDT-NK003 and UTD-NK cells were evenly divided into two groups, one group of cells was normally cultured, and the other group was added with PANC-1 cells at a ratio of 4:1, and the same number of PANC-1 cells was added every other day, and the cell supernatant was collected after 12 days of co-culture for IFN-γ cytokine detection, and the specific detection method was referred to the IFN-γ (ACRO CRS-A017) factor detection kit instruction manual.

[0248] Cell co-incubation group: SDT-NK045, SDT-NK003 and UTD-NK cells were evenly divided into two groups, one group of cells was normally cultured, and the other group was added with PANC-1 cells at a ratio of 4:1, and the same number of PANC-1 cells was added every other day, and the cells in each group were collected after 12 days of co-culture, and 1.0×10 5 CAR-NK cells were co-incubated with 1.0×10 5 PANC-1 target cells for 18h, and the supernatant was collected by centrifugation for IFN-γ cytokine detection, and the specific detection method was referred to the IFN-γ (ACRO CRS-A017) factor detection kit instruction manual.

[0249] 1.4.2 Experimental results

[0250] Figure 6 The results show that the release amount of IFN-γ in the supernatant of SDT-NK045 is higher than that of SDT-NK003 after PANC-1 target cells are pretreated for 12 days; the release amount of IFN-γ in the supernatant of PANC-1 target cell treatment group is significantly higher than that of the group without PANC-1 target cell treatment.

[0251] Figure 7 The results show that the release amount of IFN-γ in the supernatant of SDT-NK045 is significantly higher than that of SDT-NK003 after SDT-NK045 and SDT-NK003 cells are co-incubated with PANC-1 target cells; the release amount of IFN-γ in the group without PANC-1 treatment is higher than that in the group pretreated with PANC-1 for 6 days because the sensitivity of CAR-NK cells to PANC-1 target cells is reduced under long-term stimulation of target cells.

[0252] The above experimental results show that the structure of SDT-NK045 is significantly superior to other structures in terms of CAR+ expression, killing ability and IFN-γ release. The transmembrane fusion protein CD8a SP+IL-15+CD28 hinge+truncated2B4 in the structure is referred to as tmbIL-15, and the amino acid sequence is shown in SEQ ID NO. 31:

[0253] MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPEFRFWPFLVIIVILSALFLGTLACFCVWRRKRKEK

[0254] Example 2 Influence of tmbIL-15 structure on NK function

[0255] 2.1 Expression of tmbIL-15 in NK cells

[0256] 2.1.1 Experimental method:

[0257] Prepare tmbIL-15 NK (SDT-NK076) cells as the experimental group according to the method of 1.1, wherein the tmbIL-15 structure on the core plasmid is shown in Table 2.

[0258] Table 2. Core plasmid tmbIL-15 structure

[0259]

[0260] IL-15 detection: 3 days after the preparation of SDT-NK076, 1.0 x 10 6 Cells were detected by flow cytometry after staining with Biotin anti-human IL-15 Antibody (BioLegend, 515104) and APC anti-human CD56 (NCAM) Antibody (Biolegend, 362504) at 4°C for 20 min; NK cells without transduction (UTD-NK) were used as a control group.

[0261] 2.1.2 Experimental results

[0262] Figure 8 The results show that, compared with the control group UTD-NK, the experimental group SDT-NK076 successfully overexpresses IL-15 in NK cells.

[0263] 2.2 Effect of tmbIL-15 on NK cell activity and proliferation

[0264] 2.2.1 Experimental method

[0265] CAR-NK positive cell detection: CAR-NK cell number ≥ 2 x 10 7 SDT-NK076 cells were cultured in KBM581 + 10% FBS basic medium without the addition of IL-2, a cytokine that promotes NK proliferation, and the NK activity and proliferation changes were detected on the 2nd day and the 5th day of culture.

[0266] 2.2.2 Experimental results

[0267] Figure 9 The results show that, under the condition of consistent initial amount of cells (1.0 x 10 7 ) and close cell viability between the experimental and control groups, the viability of SDT-NK076 is stably maintained at more than 80% during the culture process, and the cell number continues to grow, reaching 6.0 x 10 7 above on Day 5; while the cell viability of the control group UTD-NK continuously decreases during the culture process, and the cell number shows a negative growth trend. This indicates that tmbIL-15 promotes the maintenance of NK viability and proliferation.

[0268] 2.3 Short-term killing of tmbIL-15 NK obtained under the culture condition with the addition of IL-2 to various target cells

[0269] 2.3.1 Experimental method

[0270] ① tmbIL-15 NK cells were prepared according to the method of Example 2.1 and cultured in culture medium (KBM581 + 10% FBS + 500 IU / mL IL-2).

[0271] ② Seven kinds of cancer cells, namely human pancreatic cancer (PANC-1), human liver cancer (Huh-7 / HepG2-CD276), human lymphoma (Raji), human colon cancer (HCT116), human non-small cell lung cancer (HCC827), and human ovarian cancer (SKOV-3), were selected as target cells. The target cells were added to the 96-well white plate at 10000 cells / 100 μL per well.

[0272] ③ The amount of effector cells was calculated according to the set 6 effector-to-target ratios (2:1, 1:1, 0.5:1, 0.25:1, 0.125:1, 0.0625:1), and the effector cells were diluted to the corresponding concentrations in a transparent 96-well plate.

[0273] ④ 100 μL of the diluted effector cells were transferred to the target cells (96-well white plate);

[0274] ⑤ After inoculation of the target cells and effector cells, the 96-well white plate was placed in a carbon dioxide incubator for culture. After 20 hours, ONE-Glo substrate (Promega, #E6120) was added, and the enzyme-labeled instrument was detected.

[0275] 2.3.2 Experimental results:

[0276] Table 3 SDT-NK076 and UTD-NK killing rate statistics table for PANC-1 (with IL-2)

[0277]

[0278] Table 4 SDT-NK076 and UTD-NK killing rate statistics table for Huh-7 (with IL-2)

[0279]

[0280] Table 5 SDT-NK076 and UTD-NK killing rate statistics table for HePG2-CD276 (with IL-2)

[0281]

[0282] Table 6 SDT-NK076 and UTD-NK killing rate statistics table for RAJI (with IL-2)

[0283]

[0284] Table 7 Statistics of killing rate of SDT-NK076 and UTD-NK on HCT116 (with IL-2 added)

[0285]

[0286] Table 8 Statistics of killing rate of SDT-NK076 and UTD-NK on HCC827 (with IL-2 added)

[0287]

[0288] Table 9 Statistics of killing rate of SDT-NK076 and UTD-NK on SKOV-3 (with IL-2 added)

[0289]

[0290] Figure 10 to Figure 16 The results of Tables 3 to 9 show that under different effector-target ratio conditions, the killing ability of SDT-NK076 on the seven target cells is significantly higher than that of the control group UTD-NK, indicating that the tmbIL-15 structure enhances the killing ability of NK cells on target cells.

[0291] 2.4 Short-term killing of target cells by tmbIL-15 NK cells cultured without IL-2 added

[0292] 2.4.1 Experimental method

[0293] The method of Example 2.3.1 was followed, except that the culture medium was KBM581 + 10% FBS, and the target cells were PANC-1.

[0294] 2.4.2 Test results

[0295] Table 10 Statistics of killing rate of SDT-NK076 and UTD-NK on PANC-1 (without IL-2 added)

[0296]

[0297] Figure 17 The results of Table 10 show that the experimental group SDT-NK076 is significantly stronger than the control group UTD-NK. Compared with the results of Table 3, the killing ability of SDT-NK076 on PANC-1 target cells does not weaken, while UTD-NK significantly reduces the killing ability on PANC-1 target cells under the condition of removing IL-2 culture, indicating that even under the condition of no IL-2 culture, tmbIL-15 can still maintain the high in vitro killing function of NK cells.

[0298] 2.5 Cytokine release assay of cells cultured under conditions with IL-2 added

[0299] 2.5.1 Experimental method

[0300] The tmbIL-15 NK cells were cultured in the culture medium KBM581+10%FBS+500 IU / mL IL-2, and three kinds of cancer cells, i.e., human pancreatic cancer (PANC-1), human hepatocarcinoma (Huh-7), and human lymphoma (Raji), were selected as target cells.

[0301] 1.0×10 5 tmbIL-15 NK cells were incubated with 1.0×10 5 target cells for 18 h, centrifuged to collect the supernatant for detection of IFN-γ and TNF-α; for specific experimental procedures, refer to the IFN-γ (ACRO CRS-A017) and TNF-α (ACRO CRS-A002) factor detection kit instructions:

[0302] Experimental results

[0303] Table 11. IFN-γ release amount statistics table (with IL-2 added)

[0304]

[0305] Table 12. TNF-α release amount statistics table (with IL-2 added)

[0306]

[0307] Figure 18 to Figure 23 The results of Tables 11, 12 show that, compared with the control group UTD-NK, the experimental group tmbIL-15 NK (SDT-NK076) releases higher levels of IFN-γ and TNF-α under the condition of the culture medium with IL-2.

[0308] 2.6 Cytokine release assay of cells cultured under conditions without IL-2 added

[0309] 2.6.1 Experimental method

[0310] According to the method of Example 2.5.1, the difference is that the culture medium KBM581+10%FBS is used for culture, and the target cell is PANC-1.

[0311] 2.6.2 Experimental results

[0312] Table 13. IFN-γ release amount statistics table (without IL-2 added)

[0313]

[0314] Table 14 TNF-α release statistics table (no IL-2 added)

[0315]

[0316] Figure 24 、 Figure 25 The results of Table 13 and Table 14 show that tmbIL-15 NK cells (SDT-NK076) obtained under culture conditions without the addition of IL-2 have a higher release level of IFN-γ and TNF-α after co-culturing with PANC-1 target cells at a 1:1 effector-target ratio for 18 h, and the release of IFN-γ and TNF-α of the control group UTD-NK is almost undetectable. Compared with the results of Table 11 and Table 12, the release level is significantly increased, indicating that tmbIL-15 enhances the release of NK cell killing factors, and higher release of killing factors can be achieved without adding IL-2 during culture.

[0317] 2.7 CD107a detection

[0318] 2.7.1 Experimental method

[0319] The tmbIL-15 NK cells were cultured in KBM581+10%FBS medium, and two cancer cells, human pancreatic cancer (PANC-1) and human lymphoma (Raji), were selected as target cells.

[0320] The effector cells were 100 μL (1.0×10 5 ), and the target cells were 100 μL (1.0×10 5 ) added to a 96-well plate.

[0321] 2 μL PE anti-human CD107a (LAMP-1) Antibody (BioLenged, 328608) was added, and incubated at 37°C for 1 h;

[0322] 2 μL GolgiStop™ Protein Transport Inhibitor (BD, 554724) was added to 3 mL complete medium, and 20 μL was added to the cells and mixed, and after incubation for 2.5-3 h, 0.5 μL APC anti-human CD56 (NCAM) Antibody (Biolegend, 362504) was added, and incubated at 37°C for 30 min, and washed once with FACS buffer, and the expression of CD107a molecules was detected by FACS.

[0323] 2.7.2 Experimental results

[0324] The up-regulation of expression of CD 108a molecules is consistent with the secretion of perforin, and the expression level is significantly correlated with the killing activity of NK cells. NK cells with positive expression of CD 108a molecules can represent NK cells with killing activity. Figure 26 、 Figure 27 The results of Table 2 show that, compared with the control group UTD-NK, tmbIL-15 NK (SDT-NK076) releases higher levels of CD 108a after stimulation by different types of target cells.

[0325] 2.8 Detection of NK cell killing phenotype

[0326] 2.8.1 Experimental method

[0327] ① At Day0, Day5, and Day7 of CAR-NK culture, 1.0x10 6 tmbIL-15 NK (SDT-NK076) and UTD-NK cells were centrifuged to remove the supernatant;

[0328] ② FACS buffer was washed once, and then 1 μL of CD56 (Biolenged, 362508), CD16 (Biolenged, 360716), NKp30 (Biolenged, 325234), NKp44 (Biolenged, 325112), and NKG2D (Biolenged, 320824) antibodies were added to each sample for 4°C staining for 20 min;

[0329] ③ FACS buffer was washed once, and then 200 μL of FACS buffer was resuspended for flow detection of CD56, CD16, NKp30, NKp44, and NKG2D expression levels;

[0330] 2.8.2 Experimental results

[0331] Figure 28 to Figure 31 The results of Table 2 show that, compared with the control group UTD-NK, tmbIL-15 NK (SDT-NK076) gradually increases the expression of NK activating receptors such as NKp30, NKp44, and NKG2D with the extension of culture time; at the same time, the trend of increase in CD56+CD16+ double positive NK phenotype is more obvious, indicating the increase in NK maturity. It is shown that tmbIL-15 can promote the expression of NK killing receptors, thereby promoting the activation and maturation of NK cells.

[0332] The above experiments show that tmbIL-15 can significantly promote the survival and proliferation of NK cells, and has significant in vitro killing and killing factor release on multiple tumor cells. Under the condition of long-term treatment of culture medium without IL-2, tmbIL-15 can significantly maintain the function and proliferation of NK cells, which is not affected by IL-2.

[0333] 2.9 Effect on tumor in vivo of animals

[0334] 2.9.1 Experimental method

[0335] The tumor animal model is the severe immunodeficient mouse NCG model, and the tumor cell is the pancreatic cancer cell PANC-1 luc. The tumor is loaded through the tail vein, and the tumor loading dose is 1.0 x 10 6 cells per mouse, and the mice are administered twice at a dose of 1.0 x 10 7 cells per mouse on day D28 and day D42 after tumor loading, and a control group DPBS group and UTD-NK cell group are set. All experimental samples are NK cells obtained by magnetic bead sorting from PBMCs purchased from Miaoshun Biology, and CAR-NK samples are obtained after viral infection of NK cells. After administration, the change of tumor load is detected by in vivo imaging regularly, and the change of mouse weight is observed. On D49, D56 and D63, the peripheral blood of mouse orbit is collected, and the proportion of NK cells in it is detected by flow cytometry.

[0336] 2. Experimental results:

[0337] (1) In vivo imaging results

[0338] Table 15 Statistical analysis of the inhibitory effect of CAR-NK on pancreatic cancer cell PANC-1 luc

[0339]

[0340] Figure 32 、 Figure 33 The results of Table 15 show that the tmbIL-15 armored NK cell group SDT-NK076 has a significant tumor inhibition effect compared with the control group DPBS and the NK group.

[0341] (2) Persistence observation of NK cells in vivo of mice.

[0342] Table 16 Persistence observation of CAR-NK cells in vivo of mice

[0343]

[0344] “-” indicates that the mouse died;

[0345] Table 17 Statistical analysis of persistence observation of CAR-NK cells in vivo of mice

[0346]

[0347] Table 16, Table 17, Figure 34 The results of Table 16, Table 17,

[0348] Example 3 Influence of tmbIL-15 structure on the function of CD276 VHH-CAR-NK

[0349] 3.1 Influence of tmbIL-15 on CAR+ viability and proliferation

[0350] 3.1.1 Experimental method

[0351] (1) Preparation of CAR-NK cells: SDT-NK066 and SDT-NK078 cells were prepared according to the method of 1.1, wherein the CAR structure on the core plasmid is shown in Table 3. NK cells without transduction (UTD-NK) were used as a control group.

[0352] Table 18 Core plasmid CAR structure 2 group

[0353]

[0354] CD276 VHH is a CD276 single domain antibody, and the amino acid sequence of the antibody is shown as SEQ ID NO. 32:

[0355] QVQLVESGGGLVQPGGSLRLSCSASGFTPSIYTMGWYRQAPGKGREFVASIVNEGIPGYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAFATYYGLGDPRYWGQGTLVTVSS

[0356] The nucleotide sequence of CD276 VHH is shown as SEQ ID NO. 33:

[0357] caggtgcaactggtggagagcggaggtggattggtgcagccagggggttccctacgcctgtcatgctccgcctccggctttacaccctctatctacaccatgggctggtaccgccaggcccccgggaagggccgagagttcgtcgcttccattgtcaacgagggcatccctggctacg cggggtccgtaaaaggccgcttcaccatctctcgggacaactccaagaacacgctttacctgcagatgaactccctgcgcgccgaagacaccgccgtgtactactgtgccttcgccacttattacggcctcggtgatccgcgttattggggccagggcaccctggttactgtgagctcg

[0358] CAR testing: 3 days after CAR-NK preparation, a count of 1.0 × 10⁻⁶ cells was collected. 6 Cells were stained with APC anti-human CD56 (NCAM) Antibody (Biolegend, 362504) and MonoRab™ Rabbit Anti-Humanized VHH Antibody [PE] (GenScript, A02171-200) at 4 ℃ for 20 min on the third day after viral transduction, and then analyzed by instrument.

[0359] Adjust the initial total number of CAR-NK cells and ensure consistency in CAR characterization. Prepare various CAR-NK cell lines at a ratio of 1.0 × 10⁻⁶. 7 Cells were inoculated into KBM581 basal medium with 10% FBS and cultured without the commonly used cytokine IL-2, which promotes NK cell proliferation. NK cell viability and proliferation changes were recorded using a cell counter during culture.

[0360] 3.1.2 Experimental Results

[0361] Figure 35 , Figure 36 The results showed that the viability of SDT-NK078 remained stable without decline during culture, and the number of cells continued to increase. In contrast, the viability of the control group UTD-NK and CAR-NK (SDT-NK066) continued to decrease during culture, and the number of cells showed a negative growth trend.

[0362] Figure 37The results show that, compared the CAR change between SDT-NK078 and SDT-NK066, it is found that the CAR+ with tmbIL-15 (SDT-NK078) gradually increased in the late culture period, while the CAR+ without tmbIL-15 structure (SDT-NK066) continued to decline during the culture process; indicating that tmbIL-15 promotes the survival and proliferation of CAR-NK, further affecting CAR expression.

[0363] 3.2 tmbIL-15-CD276 VHH-CAR-NK short-term killing experiment

[0364] 3.2.1 Experimental method

[0365] Six kinds of cancer cells expressing CD276, namely human pancreatic cancer (PANC-1), human hepatocarcinoma (Huh-7), human hepatocarcinoma (HepG2-CD276), human colon cancer (HCT116), human non-small cell lung cancer (HCC827) and human ovarian cancer (SK-OV-3) were selected as target cells.

[0366] The prepared SDT-NK066, SDT-NK078 cells and UTD-NK cells were respectively cultured in KBM581+10%FBS+500 IU / mL IL-2 or KBM581+10%FBS for 7 days as effector cells.

[0367] The short-term killing experiment was carried out according to the method of 1.3.

[0368] 3.2.2 Experimental results

[0369] Table 19 SDT-NK066, SDT-NK078 and UTD-NK killing rate statistics table of PANC-1 (add IL-2)

[0370]

[0371] Table 20 SDT-NK066, SDT-NK078 and UTD-NK killing rate statistics table of Huh-7 (add IL-2)

[0372]

[0373] Table 21 SDT-NK066, SDT-NK078 and UTD-NK killing rate statistics table of HepG2-CD276 (add IL-2)

[0374]

[0375] Table 22 Summary of killing rate of SDT-NK066, SDT-NK078 and UTD-NK against HCC827 (with IL-2 added)

[0376]

[0377] Table 23 Summary of killing rate of SDT-NK066, SDT-NK078 and UTD-NK against HCT116 (with IL-2 added)

[0378]

[0379] Table 24 Summary of killing rate of SDT-NK066, SDT-NK078 and UTD-NK against SKOV-3 (with IL-2 added)

[0380]

[0381] Table 25 Summary of killing rate of SDT-NK066, SDT-NK078 and UTD-NK against PANC-1 (without IL-2 added)

[0382]

[0383] Figure 38 to Figure 43 The results of Tables 19 to 24 show that when the culture medium of CAR-NK cells contains the cytokine IL-2, the killing ability of SDT-NK078 containing the tmbIL-15 structure against 6 kinds of cancer cells is significantly higher than that of SDT-NK066 not containing the tmbIL-15 structure at the same effector target ratio. It is shown that the tmbIL-15 structure can significantly improve the killing ability of CAR-NK against solid tumor target cells.

[0384] Figure 44 The results of Table 25 show that when the culture medium of CAR-NK cells does not contain the cytokine IL-2, the killing ability of SDT-NK078 containing the tmbIL-15 structure against human pancreatic cancer cells (PANC-1) is still significantly higher than that of the control group SDT-NK066 not containing the tmbIL-15 structure. It is shown that CAR-NK cells containing the tmbIL-15 structure can still maintain very strong in vitro killing function without IL-2.

[0385] 3.3 Cytokine release detection:

[0386] 3.3.1 Experimental method

[0387] The prepared SDT-NK066, SDT-NK078 cells, and UTD-NK cells were respectively cultured in a culture medium of KBM581+10% FBS+500 IU / mL IL-2 or KBM581+10% FBS for 7 days as effector cells.

[0388] Two cancer cells, human pancreatic cancer cells (PANC-1) and human liver cancer cells (Huh-7), were respectively used as target cells.

[0389] 1.0×10 5 The effector cells were co-incubated with 1.0×10 5 The target cells were co-incubated for 18 h, centrifuged to collect the supernatant for cytokine detection; for specific experimental steps, refer to the IFN-γ (ACRO CRS-A017) / TNF-α (ACRO CRS-A002) factor detection kit instruction manual:

[0390] 3.3.2 Experimental results

[0391] Table 26: IFN-γ release amount statistics (with IL-2)

[0392]

[0393] Table 27: TNF-α release amount statistics (with IL-2)

[0394]

[0395] Table 28: IFN-γ release amount statistics (without IL-2)

[0396]

[0397] Table 29: TNF-α release amount statistics (without IL-2)

[0398]

[0399] Figure 45 to Figure 48 The results of Tables 26, 27 show that the amount of IFN-γ and TNF-α released by SDT-NK078 containing tmbIL-15 structure is significantly higher than that of the control group SDT-NK066 without tmbIL-15 structure after co-culturing with target cells in the culture medium containing IL-2. It is shown that tmbIL-15 structure can enhance the release of CAR-NK cell killing factors.

[0400] Figure 49 、 Figure 50The results of Table 28, Table 29 show that the amount of IFN-γ and TNF-α released by SDT-NK078 containing tmbIL-15 structure is significantly higher than that of control group SDT-NK066 without tmbIL-15 structure after co-cultured with target cells without adding IL-2 in the culture medium. It shows that tmbIL-15 structure can still enhance the release of CAR-NK cell killing factors without IL-2 stimulation.

[0401] 3.4 CD107a detection

[0402] 3.4.1 Experimental method

[0403] The prepared SDT-NK066, SDT-NK078 cells and UTD-NK cells were used as effector cells.

[0404] Human pancreatic cancer cells (PANC-1) were used as target cells.

[0405] The effector cells 100 μL (1.0×10 5 ), target cells 100 μL (1.0×10 5 ) were added to a 96-well plate.

[0406] 2 μL PE anti-human CD107a (LAMP-1) Antibody (BioLenged, 328608) was added and incubated at 37°C for 1 h.

[0407] 2 μL GolgiStop™ Protein Transport Inhibitor (BD, 554724) was added to 3 mL complete medium, 20 μL was taken and added to the cells and mixed, and incubated for 2.5-3 h, then 0.5 μL APC anti-human CD56 (NCAM) Antibody (Biolegend, 362504) was added and incubated at 37°C for 30 min, washed once with FACS buffer, and detected by FACS for CD107a expression.

[0408] (2) Experimental results

[0409] Figure 51 The results show that SDT-NK078 containing tmbIL-15 structure can release a large amount of CD107a relative to control group SDT-NK06 without tmbIL-15 structure after co-cultured with target cells.

[0410] 3.5 tmbIL-15-CD276 VHH-CAR-NK long-term killing experiment

[0411] 3.5.1 Experimental method:

[0412] The prepared SDT-NK066, SDT-NK078 cells, UTD-NK cells were cultured in KBM581 + 10% FBS for 6 days as effector cells.

[0413] PANC-1 cells were used as target cells.

[0414] The inoculation amount of target cells was 1.0 x 10 5 According to the effector to target ratio of 1:5, the effector cells were co-cultured with the target cells for 2 days, half of the medium was replaced, and the target cells (2.0 x 10 5 The CAR+ change was detected on day 0, day 4, and day 6 after co-culture, and the release of cytokines IFN-γ and TNF-α was detected on day 6.

[0415] 3.5.2 Experimental results:

[0416] Table 30 CAR+ ratio statistics after co-culture with PANC-1

[0417]

[0418] Table 31 IFN-γ release amount statistics

[0419]

[0420] Table 32 TNF-α release amount statistics

[0421]

[0422] Figure 52 The results of Table 30 show that with repeated stimulation of target cells, the CAR+ ratio of SDT-NK078 containing tmbIL-15 structure continues to rise, and the CAR+ ratio of SDT-NK066 control group without tmbIL-15 structure continues to decline. It shows that in the process of NK killing tumor cells, tmbIL-15 structure can long-term promote the clonal proliferation of CAR-NK cells.

[0423] Figure 53 、 Figure 54 The results of Tables 31 and 32 show that after repeated stimulation of target cells, the amount of IFN-γ and TNF-α released by SDT-NK078 containing tmbIL-15 structure is significantly higher than that of SDT-NK066 without tmbIL-15 structure. It shows that tmbIL-15 structure can long-term enhance the release of CAR-NK cell killing factors.

[0424] 3.6 Detection of the killing phenotype of tmbIL-15-CD276 VHH-CAR-NK cells

[0425] 3.6.1 Experimental method

[0426] The prepared SDT-NK066, SDT-NK078 cells and UTD-NK cells were detected for the expression of NKp30, NKp44, NKG2D, CD56 and CD16 according to the method of 2.8.

[0427] 3.6.2 Experimental results

[0428] Figure 55 to Figure 58 The results show that the SDT-NK078 containing the tmbIL-15 structure gradually increases the expression of NK activating receptors such as NKp30, NKp44 and NKG2D with the extension of the culture time, compared with the control group SDT-NK066 without the tmbIL-15 structure; at the same time, the trend of increase of the NK phenotype of CD56+CD16+ double positive is more obvious, indicating that the maturity of NK is increased. It is shown that in the process of continuous stimulation of tumor cells, the tmbIL-15 structure can promote the expression of CAR-NK cell killing receptors, thereby promoting the activation and maturation of CAR-NK cells.

[0429] 3.7 Effect of tmbIL-15-CD276 VHH-CAR-NK on tumor in vivo

[0430] 1. Experimental method:

[0431] The severe immunodeficient mouse NCG model was selected as the tumor animal model, and the pancreatic cancer cell PANC-1luc was selected as the tumor cell. The tumor was loaded through the tail vein, and the tumor loading dose was 1.0×10 6 cells per mouse. After tumor loading, the CAR-NK was administered once on D14, and the tmbIL15 CD276-CAR-NK (SDT-NK078) was set in two dose groups, 1.0×10 6 CAR-NK per mouse and 2.0×10 6 CAR-NK per mouse. The CD276 CAR-NK (SDT-NK066) sample was administered at a dose of 2.0×10 6 CAR-NK per mouse, and a control group of UTD-NK cells was set. All experimental samples were obtained from PBMC purchased from Miaoshun Biology, and the NK cells were obtained by magnetic bead sorting. The CAR-NK sample was obtained after viral infection of the NK cells. After administration, the change of tumor load was detected by in vivo imaging regularly, and the change of mouse body weight was observed. The peripheral blood of mouse orbit was collected on D25, D32, D39 and D46, and the change of the proportion of NK cells in the peripheral blood was detected by flow cytometry

[0432] 2. Experimental results:

[0433] (1) In vivo imaging results

[0434] Statistical analysis of the inhibitory effect of tmbIL-15-CD276 VHH-CAR-NK on pancreatic cancer cell PANC-1 luc

[0435]

[0436] The results of Table 33, Figure 59 , Figure 60 show that the two dose groups of SDT-NK078 cell group have a significant tumor inhibition effect compared with the control group NK group.

[0437] (2) Persistence of NK cells in vivo.

[0438] Table 34 Persistence of CD276 VHH-CAR-NK cells in vivo in mice

[0439]

[0440] “-” means the mouse died;

[0441] Statistical analysis of the persistence of CD276 VHH-CAR-NK cells in vivo in mice

[0442]

[0443] The results of Table 34, Table 35, Figure 61 show that the two dose groups of SDT-NK078 group have a higher survival rate, while the NK group and the SDT-NK066 group are almost undetectable, indicating that tmbIL-15 armored CD276 VHH-CAR-NK has a significant persistent survival ability in vivo.

[0444] Example 4 Effect of tmbIL-15 structure on the function of CD19-CAR-NK

[0445] 4.1 Short-term killing experiment of tmbIL-15-CD19 VHH-CAR-NK

[0446] 4.1.1 Experimental method

[0447] Preparation of CAR-NK cells: SDT-NK079 and SDT-NK004 cells were prepared according to the method of 1.1, and the CAR structure on the core plasmid is shown in Table 3. NK cells without transduction (UTD-NK) were used as the control group.

[0448] Table 36 Core plasmid CAR structure Group 3

[0449]

[0450] FMC63 is an anti-CD19 single chain antibody, the amino acid sequence of the antibody is shown as SEQ ID NO. 34:

[0451] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0452] The nucleotide sequence of FMC63 is shown as SEQ ID NO. 35:

[0453] gacatccagatgacccagacgaccagctccctctcggcatccctgggggatcgcgtgactatttcatgccgggcttcccaggacatctctaagtacctcaactggtaccagcagaagcccgacggcaccgtgaaactgctgatctaccataccagccgtcttcacagcggtgtcccttccaggttttcaggatcggggtcgggcaccgactactctctgaccatctccaacctggagcaggaggacatcgcgacctatttctgtcaacagggcaacacgttgccctacaccttcggcggcgggaccaagctggagatcaccggcggaggcggttcgggaggcggaggctctggcggtggcggctctgaggtgaagctgcaggagagcgggcctgggctggtggctccctctcagagcctgtccgtcacctgcacagtgtctggcgtgtctctgccggactacggcgttagttggattcgccagccaccgcgcaagggcctggaatggctaggtgtaatctggggctccgagaccacctactacaactccgccctcaaatcgcgccttactatcatcaaggacaactccaaatcacaggtgttcctgaagatgaatagcttgcagactgatgacaccgccatttactactgtgccaagcactactattacggtggtagctacgcgatggattattggggccagggtactagtgtcacagtgtcctcc

[0454] The human lymphoma cell line (Raji) expressing CD19 was selected as the target cell.

[0455] The prepared SDT-NK079, SDT-NK004 cells and UTD-NK cells were cultured in the culture medium KBM581+10%FBS+500 IU / mL IL-2 or KBM581+10%FBS for 7 days as effector cells.

[0456] The short-term killing experiment was performed according to the method of 1.3.

[0457] 4.1.2 Experimental results

[0458] Table 37 SDT-NK004, SDT-NK079 and UTD-NK killing rate statistics of RAJI (add IL-2)

[0459]

[0460] Table 38 SDT-NK004, SDT-NK079 and UTD-NK killing rate statistics of RAJI (without adding IL-2)

[0461]

[0462] Figure 62 The results of Table 37 show that when the culture medium of CAR-NK cells contains the cytokine IL-2, the killing ability of SDT-NK079 containing the tmbIL-15 structure to Raji cells is significantly higher than that of SDT-NK004 without the tmbIL-15 structure at the same effector target ratio. It is shown that the tmbIL-15 structure can significantly improve the killing ability of CAR-NK to solid tumor target cells.

[0463] Figure 63 The results of Table 38 show that when the culture medium of CAR-NK cells does not contain the cytokine IL-2, the killing ability of SDT-NK079 containing the tmbIL-15 structure to human pancreatic cancer cells (PANC-1) is still significantly higher than that of the control group SDT-NK004 without the tmbIL-15 structure. It is shown that CAR-NK cells containing the tmbIL-15 structure can still maintain very strong in vitro killing function without IL-2.

[0464] 4.2 Cytokine release detection:

[0465] 4.2.1 Experimental method

[0466] The prepared SDT-NK079, SDT-NK004 cells and UTD-NK cells were cultured in KBM581+10%FBS+500 IU / mL IL-2 or KBM581+10%FBS for 7 days as effector cells.

[0467] The human lymphoma cell line (Raji) was used as target cells.

[0468] 1.0×10 5 The effector cells were co-incubated with 1.0×10 5 The supernatant was collected by centrifugation and subjected to cytokine detection; the specific experimental steps refer to the IFN-γ (ACRO CRS-A017) / TNF-α (ACRO CRS-A002) factor detection kit instructions:

[0469] 4.2.2 Experimental results

[0470] Table 39 Statistics of IFN-γ release (with IL-2 added)

[0471]

[0472] Table 40 Statistics of TNF-α release (with IL-2 added)

[0473]

[0474] Table 41 Statistics of IFN-γ release (without IL-2 added)

[0475]

[0476] Table 42 Statistics of TNF-α release (without IL-2 added)

[0477]

[0478] Figure 64 、 Figure 65 The results of Table 39 and Table 40 show that the amount of IFN-γ and TNF-α released by SDT-NK079 containing tmbIL-15 structure is significantly higher than that of control group SDT-NK004 without tmbIL-15 structure after co-culturing with target cells in the presence of IL-2 in the culture medium. This indicates that tmbIL-15 structure can enhance the release of CAR-NK cell killing factors.

[0479] Figure 66 、 Figure 67 The results of Table 41 and Table 42 show that the amount of IFN-γ and TNF-α released by SDT-NK079 containing tmbIL-15 structure is significantly higher than that of control group SDT-NK004 without tmbIL-15 structure after co-culturing with target cells in the absence of IL-2 in the culture medium. This indicates that tmbIL-15 structure can still enhance the release of CAR-NK cell killing factors without the stimulation of IL-2.

[0480] 4.3 CD107a detection

[0481] 4.3.1 Experimental method

[0482] The prepared SDT-NK079, SDT-NK004 cells and UTD-NK cells were used as effector cells, and human lymphoma cells (Raji) were used as target cells.

[0483] The expression of CD107a was detected according to the method of 3.4.1.

[0484] 4.3.2 Experimental Results

[0485] Figure 68 The results showed that after co-culturing with target cells, SDT-NK079 containing the tmbIL-15 structure was able to release a large amount of CD107a compared with the control group SDT-NK004 which did not contain the tmbIL-15 structure.

[0486] 4.4 Long-term lethality experiment of tmbIL-15-CD276 VHH-CAR-NK

[0487] 4.4.1 Experimental Methods:

[0488] The prepared SDT-NK066, SDT-NK078 and UTD-NK cells were cultured in KBM581 + 10% FBS for 6 days and then used as effector cells.

[0489] Raji cells were used as target cells.

[0490] The changes in CAR+ and the release of cytokines IFN-γ and TNF-α were studied according to the experimental method in 3.5.4.

[0491] 4.4.2 Experimental Results:

[0492] Figure 69 The results showed that with repeated stimulation of target cells, the CAR+ ratio of SDT-NK079 cells containing the tmbIL-15 structure continuously increased, while the CAR+ ratio of the control group SDT-NK004 CAR-NK cells without the tmbIL-15 structure continuously decreased. This indicates that the tmbIL-15 structure can promote the clonal proliferation of CAR-NK cells in the long term during NK cell killing of tumor cells.

[0493] Figure 70 , Figure 71 The results showed that, after repeated stimulation by target cells, SDT-NK079 containing the tmbIL-15 structure released significantly higher levels of IFN-γ and TNF-α than the control group SDT-NK004 without the tmbIL-15 structure. This indicates that the tmbIL-15 structure can enhance the release of CAR-NK cell killing factors in the long term.

[0494] 3.5 Detection of tmbIL-15-CD276 VHH-CAR-NK cell killing phenotype

[0495] 3.5.1 Experimental Methods

[0496] The prepared SDT-NK079, SDT-NK004 cells and UTD-NK cells were detected for the expression of NKp30, NKp44, NKG2D, CD56 and CD16 according to the method 2.8.

[0497] 3.5.2 Experimental results

[0498] Figure 72 to Figure 75 The results show that the SDT-NK079 containing the tmbIL-15 structure has gradually increased expression of NK activating receptors such as NKp30, NKp44 and NKG2D with the extension of culture time, compared with the control group SDT-NK004 without the tmbIL-15 structure; at the same time, the trend of increase of CD56+CD16+ double positive NK phenotype is more obvious, indicating the increase of NK maturity. It is shown that in the process of continuous stimulation of tumor cells, the tmbIL-15 structure can promote the expression of CAR-NK cell killing receptors, thereby promoting the activation and maturation of CAR-NK cells.

[0499] The results of the above examples show that the tmbIL-15 structure can significantly promote the survival and proliferation of CAR-NK cells, and has significant in vitro killing and killing factor release to various solid tumor cells and hematological tumor cells. In addition, under the long-term treatment condition of IL-2-free medium, tmbIL-15 can significantly maintain the function and proliferation of NK cells, which is not affected by IL-2. In the long-term killing experiment of repeated stimulation of tumor cells, the tmbIL-15 armored CAR-NK can still maintain a high CAR+ maintenance and clonal proliferation, and show a high killing factor release and killing receptor expression.

[0500] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. An IL-15 transmembrane fusion protein characterized in that, Comprising: an IL-15 coding region, a hinge region, and a truncated 2B4 receptor; the truncated 2B4 receptor has an amino acid sequence as set forth in SEQ ID NO. 23; the IL-15 coding region has an amino acid sequence as set forth in SEQ ID NO. 11; the hinge region comprises a CD28 hinge; the CD28 hinge has an amino acid sequence as set forth in SEQ ID NO.

13.

2. The IL-15 transmembrane fusion protein of claim 1, wherein, the IL-15 transmembrane fusion protein has an amino acid sequence as set forth in SEQ ID NO.

31.

3. A fusion protein, characterized in that, Comprising the IL-15 transmembrane fusion protein as set forth in claim 1 or 2, further comprising one or more of a signal peptide, an antigen binding domain, a hinge & transmembrane region, a signaling region, or a cleavage peptide; the signal peptide is selected from a CD8a SP; the antigen binding domain is selected from a CD276 scFV, a CD276 VHH, or a FMC63; the hinge & transmembrane region is selected from a CD8a hinge & TM; the signaling region is selected from a BBz; the cleavage peptide is selected from a P2A.

4. The fusion protein of claim 3, wherein, the amino acid sequence of the signal peptide CD8a SP is as set forth in SEQ ID NO. 1; the amino acid sequence of the CD276 scFV is as set forth in SEQ ID NO. 3, the amino acid sequence of the CD276 VHH is as set forth in SEQ ID NO. 32, or the amino acid sequence of the FMC63 is as set forth in SEQ ID NO. 34; the amino acid sequence of the hinge & transmembrane region is as set forth in SEQ ID NO. 5; the amino acid sequence of the signaling region is as set forth in SEQ ID NO. 7; the amino acid sequence of the cleavage peptide is as set forth in SEQ ID NO.

9.

5. A nucleic acid molecule encoding the IL-15 transmembrane fusion protein as set forth in claim 1 or 2, or encoding the fusion protein as set forth in claim 3 or 4.

6. An expression vector, characterized in that, Comprising the nucleic acid molecule as set forth in claim 5.

7. A viral particle, characterized in that, Comprising the expression vector as set forth in claim 6.

8. A host, characterized in that, Comprising any of: (I) expressing the IL-15 transmembrane fusion protein as set forth in claim 1 or 2; or (II) expressing the fusion protein as set forth in claim 3 or 4; or (III) transfecting the expression vector as set forth in claim 6; or (IV) transducing the viral particle as set forth in claim 7.

9. The host of claim 8, wherein, The host comprises an NK cell, an NKT cell, a T cell, or an induced NK cell, NKT cell, T cell.

10. Use of any of: (I) the IL-15 transmembrane fusion protein as set forth in claim 1 or 2; or (II) the fusion protein as set forth in claim 3 or 4; or (III) the expression vector as set forth in claim 6; or (IV) the viral particle as set forth in claim 7; or (V) the host as set forth in claim 8 or 9, 11. Use according to claim 10, wherein the compound is ###0002### in the manufacture of a medicament for maintaining the activity of an immune cell, for promoting the survival or proliferation of an immune cell, for preparing an immune cell with enhanced tumor-killing function. The tumor comprises a hematological tumor and / or a solid tumor.

12. The use according to claim 11, wherein the compound is ###0002### The solid tumor expresses CD276; the solid tumor comprises one or more of ovarian cancer, prostate cancer, liver cancer, lung cancer, head and neck cancer, kidney cancer, cervical cancer, breast cancer, colorectal cancer, neuroblastoma, pituitary tumor, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, skin cancer, myeloma, bladder cancer, osteosarcoma or glioma.

13. The use according to claim 11, wherein the compound is ###00006### 11 The hematological tumor expresses CD19; the hematological tumor comprises lymphoma or B-cell leukemia.

14. The use according to claim 10, wherein the compound is ###00010### 10 The enhanced tumor-killing function comprises improved killing of target cells and / or promoted release of killing factors; The killing factor comprises IFN-γ or TNF-α.

15. Use of any of the following in the preparation of a medicament for preventing and / or treating a tumor: (I) the IL-15 transmembrane fusion protein according to claim 1 or 2; or (II) the fusion protein according to claim 3 or 4; or (III) the expression vector according to claim 6; or (IV) the viral particle according to claim 7; or (V) the host according to claim 8 or 9.

16. The use of claim 15, wherein, The tumor comprises a hematological tumor and / or a solid tumor.

17. The use according to claim 16, wherein The solid tumor expresses CD276; the solid tumor comprises one or more of ovarian cancer, prostate cancer, liver cancer, lung cancer, head and neck cancer, kidney cancer, cervical cancer, breast cancer, colorectal cancer, neuroblastoma, pituitary tumor, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, skin cancer, myeloma, bladder cancer, osteosarcoma or glioma.

18. The use of claim 16, wherein, The hematological tumor expresses CD19; the hematological tumor comprises lymphoma or B-cell leukemia.

19. Medicament / Pharmaceutical combination, characterized in that, The medicament comprises any of the following as a raw material, as well as a pharmaceutically acceptable excipient or adjuvant: (I) the IL-15 transmembrane fusion protein according to claim 1 or 2; or (II) the fusion protein according to claim 3 or 4; or (III) the expression vector according to claim 6; or (IV) the viral particle according to claim 7; or (V) the host according to claim 8 or 9. The medicament combination comprises the medicament and other any effective ingredients.

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