A novel il-15 fusion protein and use thereof

Abstract

The present application relates to the field of immunology, and particularly relates to a novel IL-15 fusion protein and application thereof. The present application provides an IL-15 transmembrane fusion protein, which is applied to NK cells or CAR-NK cells, can significantly enhance the tumor killing function of NK cells and CAR-NK cells, and can effectively promote the proliferation of NK cells and CAR-NK cells. It provides an important basis for developing NK cell products armored by the IL-15 transmembrane fusion protein, which are independent of any tumor target, thereby reducing the cycle and cost of CAR-NK development. Meanwhile, the present application respectively carries out function research in blood tumors expressing CD19 and solid tumors expressing CD276, and the results indicate the broad-spectrum application prospect of the structure in tumors.

Description

Technical Field

[0001] This invention relates to the field of immunology, and in particular to a novel IL-15 fusion protein and its applications. Background Technology

[0002] Chimeric antigen receptor-modified immune effector cell (CAR-T / NK) therapy has seen rapid development and high expectations 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 fewer toxic side effects; (2) lower immunogenicity, making them suitable for developing allogeneic / off-the-shelf products; and (3) multiple anti-tumor activation mechanisms, including CAR-NK cells that can be activated specifically by non-CAR-dependent cancer cells in addition to CAR-dependent activation.

[0003] Early studies found that IL-2 can stimulate the expansion of killer cells (including NK cells and T cells) in vitro and exhibit certain anti-tumor activity after infusion into patients, providing a basis for subsequent cell immunotherapy. With the rapid development of CAR-T and CAR-NK therapies in recent years, the therapeutic potential of IL-15, in addition to IL-2, has also been continuously explored. Currently, the role of IL-15 in NK cells / CAR-NK cells needs further improvement. Summary of the Invention

[0004] In view of this, the present invention provides a novel IL-15 fusion protein and its applications. The present invention introduces a transmembrane structure to form a novel IL-15 fusion protein (tmbIL-15). This transmembrane structure can, on the one hand, exert the proliferative function of IL-15, and on the other hand, facilitate the transduction of activation signals into NK cells, thereby further activating NK cells.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides an IL-15 transmembrane fusion protein comprising: an IL-15 coding region, a hinge region, and a truncated 2B4 receptor; said truncated 2B4 receptor having 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 includes a CD28 hinge; the CD28 hinge has an amino acid sequence as shown in SEQ ID NO.13.

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

[0010] In a second aspect, the present invention also provides a fusion protein, including the IL-15 transmembrane fusion protein, and further including one or more of a signal peptide, an antigen-binding domain, a hinge & transmembrane region, a signal transduction 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 and transmembrane region are selected from CD8a hinge &™ or CD28 hinge; and / or

[0014] The signal conduction region is selected from BBz;

[0015] The cleavage peptide is selected from P2A.

[0016] In some specific embodiments of the present invention, the amino acid sequence of the signal peptide CD8a SP is shown in SEQ ID NO. 1;

[0017] The amino acid sequence of CD276 scFV is shown in SEQ ID NO.3, the amino acid sequence of CD276 VHH is shown in SEQ ID NO.32, or the amino acid sequence of FMC63 is shown in SEQ ID NO.34;

[0018] The amino acid sequence of the hinge and transmembrane region is shown in SEQ ID NO.5;

[0019] The amino acid sequence of the signal transduction region is shown in SEQ ID NO.7;

[0020] The amino acid sequence of the cleaved peptide is shown in SEQ ID NO.9.

[0021] Thirdly, the present invention also provides a nucleic acid molecule encoding the IL-15 transmembrane fusion protein, or encoding the fusion protein.

[0022] Fourthly, the present invention also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0023] Fifthly, the present invention also provides viral particles, including the aforementioned expression vector.

[0024] Sixthly, the present invention also provides a host, including any of the following:

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

[0026] (II) Expressing the fusion protein; or

[0027] (III) Transfect the expression vector; or

[0028] (IV) Transducing the viral particles.

[0029] In some specific embodiments of the present invention, the host includes NK cells, NKT cells, T cells, or induced NK cells, NKT cells, and T cells.

[0030] In a seventh aspect, the present invention also provides the use of any of the following in the preparation of medicaments for maintaining the activity of immune cells, promoting the survival or proliferation of immune cells, and preparing immune cells that enhance tumor-killing function:

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

[0032] (II) The fusion protein; or

[0033] (III) The expression vector; or

[0034] (IV) The virus particles; or

[0035] (V) The host.

[0036] In some specific embodiments of the present invention, the tumor includes hematoma and / or solid tumor.

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

[0038] Preferably, the hematologic malignancy expresses CD19; more preferably, the hematologic malignancy includes lymphoma or B-cell leukemia.

[0039] In some specific embodiments of the present invention, the enhanced tumor killing function includes increasing the killing power of target cells and / or promoting the release of killing factors;

[0040] Preferably, the killing factor includes IFN-γ or TNF-α.

[0041] Eighthly, the present invention also provides the use of any of the following in the preparation of medicaments for the prevention and / or treatment of tumors:

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

[0043] (II) The fusion protein; or

[0044] (III) The aforementioned expression vector; or

[0045] (IV) The aforementioned virus particles; or

[0046] (V) The host mentioned above.

[0047] In some specific embodiments of the present invention, the tumor includes hematoma and / or solid tumor.

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

[0049] Preferably, the hematologic malignancy expresses CD19; more preferably, the hematologic malignancy includes lymphoma or B-cell leukemia.

[0050] In a ninth aspect, the present invention also provides a drug / drug combination, characterized in that the drug is made from any of the following ingredients, and pharmaceutically acceptable excipients or adjuvants:

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

[0052] (II) The fusion protein; or

[0053] (III) The expression vector; or

[0054] (IV) The virus particles; or

[0055] (V) The host;

[0056] The drug combination includes the drug and any other active ingredients. Attached Figure Description

[0057] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0058] Figure 1 This demonstrates the infection efficiency of IL-15 transmembrane protein lentivirus in CAR-NK cells with a core plasmid CAR structure 1.

[0059] Figure 2 The change in CAR+ ratio after adding PANC-1 cells to stimulate the culture of CAR-NK cells in a group of CAR-NK cells during the culture process is shown in the core plasmid CAR structure.

[0060] Figure 3 The change in CAR+ ratio in a group of CAR-NK cells after stimulating culture without the addition of PANC-1 cells during the culture process was shown to indicate the core plasmid CAR structure.

[0061] Figure 4 The study showed that SDT-NK003 CAR-NK and SDT-NK045 CAR-NK cells, without PANC-1 pretreatment, killed PANC-1 cells under different effector-to-target ratios (E:T).

[0062] Figure 5 The study showed the cytotoxicity (lysis) of PANC-1 cells by SDT-NK003 CAR-NK and SDT-NK045 CAR-NK cells under different effector-to-target ratios (E:T) in the PANC-1 pretreatment group.

[0063] Figure 6 The release of interferon-gamma (IFN-γ) in the cell supernatant group was observed.

[0064] Figure 7 The release of interferon-gamma (IFN-γ) in the co-incubation group of cells was observed.

[0065] Figure 8 The results of flow cytometry analysis of IL-15 expression in the UTD-NK control group and the SDT-NK076 group are shown.

[0066] Figure 9 The cell viability and total number of NK cells of UTD-NK and SDT-NK076 NK were shown during the culture process.

[0067] Figure 10 The killing rate (lysis) of PANC-1 cells by SDT-NK076 NK cells obtained under culture conditions with added IL-2 and different effector-to-target ratios (E:T) is shown.

[0068] Figure 11 The killing rate (lysis) of SDT-NK076 NK cells on Huh-7 cells under different effector-to-target ratios (E:T) obtained under culture conditions with added IL-2 is shown.

[0069] Figure 12The killing rate (lysis) of SDT-NK076 NK cells obtained under culture conditions with added IL-2 on HePG2-CD276 cells under different effector-to-target ratios (E:T).

[0070] Figure 13 The killing rate (lysis) of SDT-NK076 NK cells against Raji cells under different effector-to-target ratios (E:T) obtained under IL-2-added culture conditions is shown.

[0071] Figure 14 The killing rate (lysis) of SDT-NK076 NK cells against HCT116 cells under different effector-to-target ratios (E:T) obtained under culture conditions with added IL-2 is shown.

[0072] Figure 15 The killing rate (lysis) of SDT-NK076 NK cells against HCC827 cells under different effector-to-target ratios (E:T) obtained under culture conditions with added IL-2 is shown.

[0073] Figure 16 The killing rate (lysis) of SKOV-3 cells by SDT-NK076 NK cells obtained under culture conditions with added IL-2 was shown under different effector-to-target ratios (E:T).

[0074] Figure 17 This demonstrates the short-term killing effect (lysis) of PANC-1 cells by SDT-NK076 NK cells cultured without IL-2 supplementation under different effector-to-target ratios (E:T).

[0075] Figure 18 The amount of interferon-gamma (IFN-γ) released from SDT-NK076 NK cells cultured under IL-2 supplementation and co-cultured with PANC-1 cells is shown.

[0076] Figure 19 The amount of interferon-gamma (IFN-γ) released from SDT-NK076 NK cells cultured under IL-2 supplementation and co-cultured with Huh-7 cells is shown.

[0077] Figure 20 The amount of interferon-gamma (IFN-γ) released by SDT-NK076 NK cells cultured under IL-2 supplementation and co-cultured with Raji cells is shown.

[0078] Figure 21 The amount of tumor necrosis factor α (TNF-α) released after SDT-NK076 NK cells cultured under IL-2 supplementation were co-cultured with PANC-1 cells.

[0079] Figure 22 The amount of tumor necrosis factor α (TNF-α) released after SDT-NK076 NK cells cultured under IL-2 supplementation were co-cultured with Huh-7 cells;

[0080] Figure 23 The amount of tumor necrosis factor α (TNF-α) released after co-culturing SDT-NK076 NK cells with Raji cells under IL-2 supplementation conditions is shown.

[0081] Figure 24 The figure shows the amount of interferon-gamma (IFN-γ) released by SDT-NK076 NK cells cultured without the addition of IL-2 after co-culturing with PANC-1 cells.

[0082] Figure 25 The figure shows the amount of tumor necrosis factor α (TNF-α) released after SDT-NK076 NK cells cultured without the addition of IL-2 were co-cultured with PANC-1 cells.

[0083] Figure 26 The proportion of CD107a expression in SDT-NK076 NK cells after PANC-1 cell stimulation was shown.

[0084] Figure 27 The proportion of CD107a expression in SDT-NK076 NK cells after Raji cell stimulation was shown.

[0085] Figure 28 The expression levels of CD56 and CD16 during SDT-NK076 NK cell culture were shown.

[0086] Figure 29 The expression level of NKp30 during SDT-NK076 NK cell culture was shown.

[0087] Figure 30 The expression level of NKp44 during SDT-NK076 NK cell culture was shown.

[0088] Figure 31 The expression level of NKG2D during SDT-NK076 NK cell culture was shown.

[0089] Figure 32 This shows the in vivo imaging results of PANC-1 luc pancreatic cancer cells in tumor-bearing mice after STD-NK076 administration;

[0090] Figure 33 This shows the quantitative expression results of PANC-1 luc in pancreatic cancer cells of tumor-bearing mice after STD-NK076 administration;

[0091] Figure 34This indicates the survival rate of NK cells in the peripheral blood of tumor-bearing mice after STD-NK076 administration;

[0092] Figure 35 The viability of NK cells in SDT-NK078 CAR-NK and SDT-NK066 CAR-NK is shown.

[0093] Figure 36 The number of NK cells in SDT-NK078 CAR-NK and SDT-NK066 CAR-NK is shown.

[0094] Figure 37 Showing the CAR+ ratio of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK;

[0095] Figure 38 The short-term killing effect (lysis) of PANC-1 cells by SDT-NK078 NK cells obtained under culture conditions with added IL-2 on different effector-to-target ratios (E:T) is shown.

[0096] Figure 39 The short-term killing effect (lysis) of Huh-7 cells by SDT-NK078 NK cells obtained under culture conditions with added IL-2 on different effector-to-target ratios (E:T) is shown.

[0097] Figure 40 This study demonstrates the short-term killing effect (lysis) of SDT-NK078 NK cells on HepG2-CD276 cells under different effector-to-target ratios (E:T) obtained under culture conditions with added IL-2.

[0098] Figure 41 The study showed that SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells killed HCT116 cells when the culture medium contained the cytokine IL-2.

[0099] Figure 42 The study showed that SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells killed HCC827 cells when the culture medium contained the cytokine IL-2.

[0100] Figure 43 The study showed that SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells killed SKOV-3 cells when the culture medium contained the cytokine IL-2.

[0101] Figure 44The killing effect of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells on PANC-1 cells was shown when the culture medium did not contain the cytokine IL-2.

[0102] Figure 45 The amount of IFN-γ released after co-culturing PANC-1 cells with IL-2, SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells in a culture medium supplemented with IL-2 was shown.

[0103] Figure 46 The amount of IFN-γ released after co-culturing Huh-7 cells with IL-2, SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells in a culture medium supplemented with IL-2 was shown.

[0104] Figure 47 The study showed that TNF-α was released after co-culturing NK cells with PANC-1 cells in a culture medium supplemented with IL-2, SDT-NK078 CAR-NK, and SDT-NK066 CAR-NK cells.

[0105] Figure 48 The study showed that TNF-α was released after co-culturing NK cells with Huh-7 cells in a culture medium supplemented with IL-2, SDT-NK078 CAR-NK, and SDT-NK066 CAR-NK.

[0106] Figure 49 The study showed that no IL-2 was added to the culture medium. This did not affect the release of IFN-γ from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells after co-culturing with PANC-1 cells.

[0107] Figure 50 The study showed that no IL-2 was added to the culture medium. TNF-α was released after co-culturing SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells with PANC-1 cells.

[0108] Figure 51 The release of CD107a from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells after co-culturing with PANC-1 cells was demonstrated.

[0109] Figure 52 The changes in the CAR+ ratio of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells during PANC-1 cell stimulation are shown.

[0110] Figure 53This study demonstrates the release of IFN-γ from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells during PANC-1 cell stimulation.

[0111] Figure 54 This study demonstrates the release of TNF-α from SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells during PANC-1 cell stimulation.

[0112] Figure 55 The expression levels of CD56 and CD16 during the culture of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells were shown.

[0113] Figure 56 The expression levels of NKp30 during the culture of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells were shown.

[0114] Figure 57 The expression levels of NKp44 during the culture of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells were shown.

[0115] Figure 58 The expression levels of NKG2D during the culture of SDT-NK078 CAR-NK and SDT-NK066 CAR-NK cells were shown.

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

[0117] Figure 60 This shows the quantitative expression results of PANC-1 luc in pancreatic cancer cells of tumor-bearing mice after STD-NK078 administration;

[0118] Figure 61 This shows the survival rate of NK cells in the peripheral blood of the orbital region of tumor-bearing mice after administration of STD-NK066 and STD-NK078.

[0119] Figure 62 The study showed that SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells killed RAJI cells when the culture medium contained the cytokine IL-2.

[0120] Figure 63 The killing effect of SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells on RAJI cells was demonstrated when the culture medium did not contain the cytokine IL-2.

[0121] Figure 64 The release of IFN-γ was observed after Raji cells were co-cultured with IL-2, SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells in a culture medium supplemented with IL-2.

[0122] Figure 65 The release of TNF-α after Raji cells were co-cultured with IL-2, SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells in a culture medium supplemented with IL-2;

[0123] Figure 66 The culture medium did not contain IL-2. The release of IFN-γ after co-culturing SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells with Raji cells was also observed.

[0124] Figure 67 The culture medium did not contain IL-2. TNF-α release was observed after co-culturing SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells with Raji cells.

[0125] Figure 68 This shows the release of CD107a from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells after co-culturing with Raji cells;

[0126] Figure 69 The changes in the CAR+ ratio of SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during Raji cell stimulation are shown.

[0127] Figure 70 This study demonstrates the release of IFN-γ from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during Raji cell stimulation.

[0128] Figure 71 This study demonstrates the release of TNF-α from SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during Raji cell stimulation.

[0129] Figure 72 The expression levels of CD56 and CD16 during the culture of SDT-NK079CAR-NK and SDT-NK004CAR-NK cells are shown.

[0130] Figure 73 The expression levels of NKp30 in SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during cell culture are shown.

[0131] Figure 74 The expression levels of NKp44 in SDT-NK079 CAR-NK and SDT-NK004 CAR-NK cells during cell culture are shown.

[0132] Figure 75 The expression levels of NKG2D during the culture of SDT-NK079CAR-NK and SDT-NK004CAR-NK cells were shown. Detailed Implementation

[0133] This invention discloses a novel IL-15 fusion protein and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0134] Terminology Explanation:

[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 α

[0141] 2B4: Natural Killer Cell Receptor 2B4

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

[0143] NKp30: Naturally occurring cytotoxicity trigger receptor 3

[0144] NKp46: Naturally occurring cytotoxicity trigger receptor 1

[0145] NKp44: Naturally occurring cytotoxicity trigger receptor 2

[0146] DAP10: DNAX-related protein 10

[0147] DAP12: DNAX activating protein 12

[0148] CD16a: A transmembrane molecule with a unique cytoplasmic domain.

[0149] This invention provides the structure of tmbIL-15 and its application in NK or CAR-NK, as well as its therapeutic applications in hematologic malignancies and solid tumors. The invention investigated NK and CAR-NK separately, finding that tmbIL-15 significantly enhances the function of NK or CAR-NK, especially NK function. This result suggests the potential to develop a tmbIL-15-armored NK cell product independent of any tumor target, thereby reducing the development cycle and cost of CAR-NK.

[0150] Meanwhile, the present invention was used to conduct functional studies in hematologic malignancies expressing CD19 and solid tumors expressing CD276, and the results indicate that this structure has broad application prospects in tumors.

[0151] Unless otherwise specified, the novel IL-15 fusion protein provided by this invention and the raw materials and reagents used in its application are all commercially available.

[0152] The present invention will be further illustrated below with reference to the embodiments:

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

[0154] 1.1 Preparation of CAR-NK cells

[0155] 1.1.1 Experimental Methods

[0156] (1) Virus preparation: 293T cells were inoculated one day before virus packaging to ensure that the confluence of 293T cells was between 80% and 90% during virus preparation. On the day of virus preparation, the core plasmid expressing different CAR structures (CAR structures are shown in Table 1), the envelope plasmid (BaEVTR-PR12, refer to CN118005808A), and the helper plasmid (psPAX2) were mixed in a ratio of 4:1:2 and 3 times PEI (YESEN, 40820ES10) conversion aid was added. After standing for 15 min, the mixture was added dropwise to 293T cells for virus production. The virus stock solution was collected at 48 h and 72 h and concentrated overnight by centrifugation at 4000 g, 4℃, and 9°C. The concentrated virus was used to infect Jurkat cells. The infection efficiency was detected after 72 h and the virus titer was calculated. The virus was frozen at -80℃ for later use.

[0157] Table 1. CAR structure of core plasmid (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 CD8a SP is shown in SEQ ID NO.2:

[0162] atggccctgcccgtgaccgccctgctgctgccactggccctgctgctgcatgccgctagacct

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

[0164] QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGTLVTVSSGG GGSGGGGSGGGGSEIVMTQSPATLSVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQNGHSFPLTFGQGTKLELKR

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

[0166] caggtgcagctggtgcagtcaggagctgaagtggtgaaacctggcgcctctgtgaagctgagttgtaagacatctggctatacattcactaattatgatattaattgggtgagacagagacctggacagggactggagtggattggctggatctttccaggagacggctctacacagtat aatgaaaagttcaagggcaaagctacactgacaaccgacaccagcaccagcaccgcctacatggagctgtccagcctgaggtccgaggataccgccgtgtacttctgcgctaggcagaccaccgccacctggttcgcctactggggccagggcaccctggtgaccgtgagcagcggcggcg gcggaagcggcggcggcggcagcggcggcgggggctccgagatcgtgatgacccagtcccccgccaccctgagcgtgagccctggcgagagggtgaccctgagctgcagagcatctcagagcatctccgactacctgtactggtaccagcagaagagccacgaaagccccagactgctgat caagtacgccagccagagcatcagcggcatccctgccaggttctccggcagcggctctggcagcgagttcaccctgaccattaatagcgtggaaccagaagatgttggagtgtattattgtcagaatggacactcttttccactgacatttggacagggcacaaaactggagctgaaaaga

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

[0168] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVIT

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

[0170] accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcacc

[0171] BBz represents the 4-BB co-stimulatory domain + CD3ζ signal transduction domain, and the amino acid sequence 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 in SEQ ID NO.9:

[0176] GSGATNFSLLKQAGDVEENPGP

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

[0178] ggctccggtgctaccaacttttcacttctgaagcaggccggcgacgtggaggagaatccaggccct

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

[0180] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

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

[0182] aactgggtcaacgtgatcagcgatctcaagaagattgaggacctgatccagagcatgcatattgacgccactctgtacacggagagtgatgtgcacccctcttgtaaagtgacggccatgaagtgcttcctgctggagttgcaagttatctcgctggagtctggggacgca tccatccatgacaccgtggagaacctgatcatcctggccaacaactccctttcgtctaatggcaacgtgactgagagcgggtgcaaagaatgtgaggagctggaagagaagaacatcaaggagttcctacagtccttcgtccacatcgtccagatgtttattaacacgtcc

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

[0184] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0185] The nucleotide sequence of 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 in SEQ ID NO.27:

[0212] CSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVP

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

[0214] tgcagcaccgtgtcccccggggttcttgctggcatcgtgatgggcgacctggtgctgaccgtcctcattgcgctggccgtgtacttcctgggtcgcttggtccct

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

[0216] FPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSST

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

[0218] ttccctcccggctaccaggtctccttctgcctggtgatggtgctgttgttcgccgttgacaccggtctctacttttctgtgaagaccaacatccgcagctcgact

[0219] (2) Preparation of CAR-NK cells: After reviving PBMCs purchased from Miaoshun Biotechnology, NK cells were sorted using CytoSinct™ CD3Nanobeads (GenScript, L00896) magnetic beads. The sorted NK cells were added to K562 feeder cells at a ratio of 1:1 and co-cultured until Day 4. CAR-NK cells were prepared by viral transduction at an MOI of 5. The culture medium was KBM581 + 10% FBS + 500 IU / mL IL-2.

[0220] (3) Detection of CAR-NK positive cells: On the third day after viral transduction, 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 and then detected. UTD-NK cells were NK cells that had not undergone viral transduction.

[0221] 1.1.2 Experimental Results

[0222] Figure 1 The results showed that the viruses in each experimental group could infect NK cells, producing the corresponding CAR-NK cells. Due to differences in structural design, the infection efficiency of each group on NK cells varied.

[0223] 1.2 Changes in CAR+ ratio during target cell stimulation

[0224] 1.2.1 Experimental Methods

[0225] Various CAR-NK cells were prepared according to the experimental method described in 1.1.1. Based on the counting results and flow cytometry results, the total number of CAR+ cells and the total number of cells were adjusted to be consistent, and the CAR-NK cells were divided into two groups, with 2.0 × 10⁶ cells in each group. 6 Cell volume was determined by culturing in KBM581 medium with 10% FBS. One group was supplemented with 5.0 × 10⁶ cells every two days. 5 The target cells were stimulated with the human pancreatic cancer cell line PANC-1, while the other group grew normally without the addition of target cell stimulation. The CAR+ ratio and cell count were measured every 2 days, and treatment was stopped when CAR+ <20%.

[0226] 1.2.2 Experimental Results

[0227] Figure 2 , Figure 3 The results showed that, with the CAR and cell quantity adjusted to be consistent, the CAR+ ratio of SDT-NK003 and SDT-NK045 gradually increased with the increase of culture time, reaching >50% on Day 12, while the CAR+ ratio of the other groups continued to decrease until the CAR+ ratio was <20% and then the treatment was stopped. Figure 3The results showed that, without the addition of PANC-1 cells for stimulation during culture, the CAR+ ratio in the SDT-NK045 group was higher than that in the SDT-NK003 group. This indicates that both SDT-NK045 and SDT-NK003 cells exhibit strong sustained proliferation capabilities for CAR+ cells, with SDT-NK045 cells demonstrating the strongest sustained proliferation ability. Further functional validation was performed using SDT-NK045 and SDT-NK003 cells.

[0228] 1.3 In vitro short-term lethality verification

[0229] 1.3.1 Experimental Methods

[0230] Human pancreatic cancer cell line PANC-1 was selected as the target cell.

[0231] SDT-NK045 CAR-NK cells and SDT-NK003 CAR-NK cells were selected as effector cells and subjected to two different treatments. NK cells (UTD-NK) were used as a control.

[0232] PANC-1 pretreatment group: PANC-1 cells were co-cultured with SDT-NK045 CAR-NK cells and SDT-NK003 CAR-NK cells for 12 days, and then SDT-NK045 CAR-NK cells and SDT-NK003 CAR-NK cells were collected as effector cells.

[0233] Untreated group without PANC-1: SDT-NK045 CAR-NK cells and SDT-NK003 CAR-NK cells were cultured for 12 days, and then collected as effector cells.

[0234] ① Add target cells at a rate of 10,000 cells / 100 μL per well to a 96-well white plate;

[0235] ② Calculate the amount of effector cells 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 dilute the effector cells to the corresponding concentrations in clear 96-well plates;

[0236] ③ Take 100 μL of diluted effector cells and seed them into the target cells of step ①;

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

[0238] ⑤ After 20 hours, ONE-Glo substrate (Promega, #E6120) was added, and the results were detected using a microplate reader;

[0239] ⑥ Calculate the kill efficiency using the following formula:

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

[0241] The target cell reading represents the value detected by the microplate reader after seeding only 10,000 target cells per well.

[0242] 1.3.2 Experimental Results

[0243] Figure 4 , Figure 5 The results showed that both SDT-NK003 CAR-NK and SDT-NK045 CAR-NK cells significantly killed PANC-1 target cells. Figure 4 The results showed that, without pretreatment with PANC-1, both SDT-NK003 and SDT-NK045 exhibited strong lethality with no difference. Figure 5 The results showed that when SDT-NK045 was pretreated with PANC-1 and repeatedly stimulated, its killing ability was stronger than that of SDT-NK003.

[0244] 1.4 Cytokine Release Detection

[0245] 1.4.1 Experimental Methods

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

[0247] Cell supernatant group: SDT-NK045, SDT-NK003, and UTD-NK cells were divided into two groups. One group of cells was cultured normally, and the other group was supplemented with PANC-1 cells at a ratio of 4:1. The same number of PANC-1 cells were added every other day. After culturing for 12 days, the cell supernatant was collected for IFN-γ cytokine detection. For specific detection methods, please refer to the instructions of the IFN-γ (ACRO CRS-A017) factor detection kit.

[0248] Cell co-incubation group: SDT-NK045, SDT-NK003, and UTD-NK cells were randomly divided into two groups. One group was cultured normally, while the other group was supplemented with PANC-1 cells at a 4:1 ratio. The same number of PANC-1 cells were added every other day. After 12 days of co-culture, cells from each group were collected, and 1.0 × 10⁶ cells were used. 5 CAR-NK cells with 1.0 × 10 5 After co-incubating PANC-1 target cells for 18 hours, the supernatant was collected by centrifugation for IFN-γ cytokine detection. The specific detection method is described in the instructions of the IFN-γ (ACRO CRS-A017) factor detection kit.

[0249] 1.4.2 Experimental Results

[0250] Figure 6 The results showed that after 12 days of pretreatment with PANC-1 target cells, the release of IFN-γ from SDT-NK045 in the supernatant was higher than that from SDT-NK003; the release of IFN-γ in the supernatant of the PANC-1 target cell treatment group was significantly higher than that of the group not treated with PANC-1 target cells.

[0251] Figure 7 The results showed that after co-incubation of SDT-NK045 and SDT-NK003 cells with PANC-1 target cells, the IFN-γ release in the supernatant of SDT-NK045 cells was significantly higher than that of SDT-NK003 cells. CAR-NK cells pretreated with PANC-1 for 6 days showed higher IFN-γ release in the untreated group than in the pretreated group due to decreased sensitivity to PANC-1 target cells under long-term stimulation.

[0252] The above experimental results show that the SDT-NK045 structure 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 this structure is abbreviated as tmbIL-15, and its amino acid sequence is shown in SEQ ID NO.31:

[0253] MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPEFRFWPFLVIIVILSALFLGTLACFCVWRRKRKEK

[0254] Example 2: The impact of the tmbIL-15 structure on NK function

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

[0256] 2.1.1 Experimental Methods:

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

[0258] Table 2 Structure of core plasmid tmbIL-15

[0259]

[0260] IL-15 detection: 3 days after preparation with SDT-NK076, a count of 1.0 × 10⁻⁶ was collected. 6 Cells were stained with Biotin anti-human IL-15 Antibody (BioLegend, 515104) and APC anti-human CD56 (NCAM) Antibody (Biolegend, 362504) at 4°C for 20 min and then detected by flow cytometry; NK cells that were not transduced (UTD-NK) were used as the control group.

[0261] 2.1.2 Experimental Results

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

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

[0264] 2.2.1 Experimental Methods:

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

[0266] 2.2.2 Experimental Results:

[0267] Figure 9 The results showed that the initial cell count was the same in both the experimental and control groups (1.0 × 10⁻⁶). 7 Under conditions close to cell viability, SDT-NK076 maintained a stable viability of over 80% during culture, and the cell number continued to increase until Day 5 when the cell number reached 6.0 × 10⁻⁶. 7 The above results indicate that, in contrast, the control group UTD-NK showed a continuous decrease in cell viability and a negative growth trend in cell number during culture. This suggests that tmbIL-15 promotes the maintenance of NK cell viability and proliferation.

[0268] 2.3 Short-term killing effect of tmbIL-15 NK cells on various target cells under IL-2-added culture conditions.

[0269] 2.3.1 Experimental Methods

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

[0271] ② Seven types of cancer cells were selected as target cells: 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). Target cells were added to 96-well white plates at a rate of 10,000 cells / 100 μL per well.

[0272] ③ Calculate the amount of effector cells 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 dilute the effector cells to the corresponding concentrations in clear 96-well plates;

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

[0274] ⑤ After seeding target cells and effector cells, the 96-well white plate was placed in a CO2 incubator for incubation; after 20 hours, ONE-Glo substrate (Promega, #E6120) was added, and the results were detected using a microplate reader;

[0275] 2.3.2 Experimental Results:

[0276] Table 3. Kill rate statistics of SDT-NK076 and UTD-NK against PANC-1 (with IL-2 added)

[0277]

[0278] Table 4. Kill Rate Statistics of SDT-NK076 and UTD-NK against Huh-7 (with IL-2 added)

[0279]

[0280] Table 5. Kill rate statistics of SDT-NK076 and UTD-NK against HePG2-CD276 (with IL-2 added).

[0281]

[0282] Table 6. Kill Rate Statistics of SDT-NK076 and UTD-NK against RAJI (with IL-2 added)

[0283]

[0284] Table 7. Kill rate statistics of SDT-NK076 and UTD-NK against HCT116 (with IL-2 added)

[0285]

[0286] Table 8. Kill rate statistics of SDT-NK076 and UTD-NK against HCC827 (with IL-2 added)

[0287]

[0288] Table 9. Kill Rate Statistics of SDT-NK076 and UTD-NK against SKOV-3 (with IL-2 added)

[0289]

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

[0291] 2.4 Short-term killing effect of tmbIL-15 NK cells cultured without IL-2 supplementation on target cells

[0292] 2.4.1 Experimental Methods

[0293] The procedure was carried out according to the method in Example 2.3.1, except that the cells were cultured in KBM581 medium with 10% FBS and the target cells were PANC-1.

[0294] 2.4.2 Test Results

[0295] Table 10. Kill rate statistics of SDT-NK076 and UTD-NK against PANC-1 (without IL-2 added).

[0296]

[0297] Figure 17 The results in Table 10 show that the SDT-NK076 in the experimental group had significantly stronger killing power than the UTD-NK in the control group. Compared with the results in Table 3, the killing ability of SDT-NK076 against PANC-1 target cells was not weakened, while UTD-NK significantly reduced its killing ability against PANC-1 target cells after IL-2 culture was removed, indicating that even without IL-2 culture, tmbIL-15 can still maintain a high in vitro killing function of NK cells.

[0298] 2.5 Detection of cytokine release from cultured cells cultured under IL-2 supplementation conditions

[0299] 2.5.1 Experimental Methods

[0300] tmbIL-15 NK cells were cultured in KBM581 + 10% FBS + 500 IU / mL IL-2 medium. Three types of cancer cells, namely human pancreatic cancer (PANC-1), human liver cancer (Huh-7), and human lymphoma (Raji), were selected as target cells.

[0301] 1.0×10 5 tmbIL-15 NK cells with 1.0×10 5 Target cells were co-incubated for 18 h, and the supernatant was collected by centrifugation for the detection of IFN-γ and TNF-α. Specific experimental procedures were performed according to the instructions for the IFN-γ (ACRO CRS-A017) and TNF-α (ACRO CRS-A002) factor detection kits.

[0302] Experimental results

[0303] Table 11 IFN-γ Release Statistics (with IL-2 added)

[0304]

[0305] Table 12 TNF-α Release Statistics (with IL-2 added)

[0306]

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

[0308] 2.6 Detection of cytokine release from cultures cultured without IL-2 supplementation

[0309] 2.6.1 Experimental Methods

[0310] The procedure was carried out according to the method in Example 2.5.1, except that the cells were cultured in KBM581 medium with 10% FBS and the target cells were PANC-1.

[0311] 2.6.2 Experimental Results

[0312] Table 13 IFN-γ Release Statistics (without IL-2 addition)

[0313]

[0314] Table 14 TNF-α Release Statistics (without IL-2)

[0315]

[0316] Figure 24 , Figure 25 The results in Tables 13 and 14 show that tmbIL-15 NK cells (SDT-NK076) obtained under culture conditions without IL-2 supplementation, after co-culturing with PANC-1 target cells at a 1:1 effector-target ratio for 18 h, exhibited high levels of IFN-γ and TNF-α release, while the release of IFN-γ and TNF-α in the control group UTD-NK was almost undetectable. Compared with the results in Tables 11 and 12, the release levels were significantly increased, indicating that tmbIL-15 enhances the release of NK cell cytotoxic factors and can achieve high cytotoxic factor release without the addition of IL-2 during culture.

[0317] 2.7 CD107a detection

[0318] 2.7.1 Experimental Methods

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

[0320] 100 μL (1.0 × 10⁻⁶) of effector cells 5 ), 100 μL of target cells (1.0 × 10⁻⁶) 5 Add a 96-well plate;

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

[0322] Add 2 μL of GolgiStop™ Protein Transport Inhibitor (BD, 554724) to 3 mL of complete culture medium, add 20 μL to the cells and mix well. After incubating for 2.5 h–3 h, add 0.5 μL of APC anti-human CD56(NCAM) Antibody (Biolegend, 362504), incubate at 37 °C for 30 min, wash once with FACS buffer, and then perform instrumental analysis to detect the expression of CD107a molecules.

[0323] 2.7.2 Experimental Results

[0324] Upregulation of CD107a expression is consistent with perforin secretion, and its expression level is significantly correlated with the cytotoxic activity of NK cells. NK cells with positive expression of CD107a can represent cytotoxic NK cells. Figure 26 , Figure 27 The results showed that, compared with the control group UTD-NK, tmbIL-15 NK (SDT-NK076) released higher levels of CD107a after stimulation with different types of target cells.

[0325] 2.8 Detection of NK cell killing phenotype

[0326] 2.8.1 Experimental Methods

[0327] ① 1.0 × 10⁻⁶ mg / L was collected on Day 0, Day 5, and Day 7 of CAR-NK culture. 6 tmbIL-15 NK (SDT-NK076) and UTD-NK cells were centrifuged and the supernatant was removed.

[0328] ② Wash once with FACS buffer, then add 1 μL of CD56 (Biolenged, 362508), CD16 (Biolenged, 360716), NKp30 (Biolenged, 325234), NKp44 (Biolenged, 325112), and NKG2D (Biolenged, 320824) antibody to each sample and stain at 4℃ for 20 min;

[0329] ③ After washing with FACS buffer once, resuspend in 200 μL of FACS buffer and then perform flow cytometry to detect the expression levels of CD56, CD16, NKp30, NKp44, and NKG2D.

[0330] 2.8.2 Experimental Results

[0331] Figures 28 to 31 The results showed that, compared with the control group UTD-NK, the expression of NK activation receptors such as NKp30, NKp44, and NKG2D in tmbIL-15 NK cells (SDT-NK076) gradually increased with prolonged culture time; at the same time, the trend of increasing CD56+CD16+ double-positive NK phenotype was more obvious, indicating increased NK cell maturity. This suggests that tmbIL-15 can promote the expression of NK killing receptors, thereby promoting NK cell activation and maturation.

[0332] The above experiments demonstrate that tmbIL-15 can significantly promote the survival and proliferation of NK cells, and exhibits significant in vitro killing effect and release of cytotoxic factors against multiple tumor cells. Under long-term treatment with IL-2-free culture medium, tmbIL-15 can significantly maintain the function and proliferation of NK cells, unaffected by IL-2.

[0333] 2.9 Effects on tumors in animals

[0334] 2.9.1 Experimental Methods

[0335] The tumor animal model used was the severely immunodeficient mouse NCG model. Pancreatic cancer cells (PANC-1luc) were selected as the tumor cells, and tumor grafting was performed via tail vein at a dose of 1.0 × 10⁻⁶. 6 Cells / animal, administered at 1.0 × 10⁻⁶ on days 28 and 42 post-tumor bearing. 7 The mice were administered the dose twice, with a control group (DPBS group) and a UTD-NK cell group. All experimental samples were obtained from PBMCs purchased from Miaoshun Biotechnology, where NK cells were obtained through magnetic bead sorting. CAR-NK samples were obtained after NK cell virus infection. Tumor burden changes were periodically monitored using in vivo imaging after drug administration, and mouse body weight changes were also observed. Peripheral blood was collected from the orbits of mice on days 49, 56, and 63, and the proportion of NK cells was detected by flow cytometry.

[0336] 2. Experimental Results:

[0337] (1) Results of live imaging

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

[0339]

[0340] Figure 32 , Figure 33 The results in Table 15 show that, compared with the control group DPBS and the NK group, the tmbIL-15 armored NK cell group SDT-NK076 had a significant tumor-suppressing effect.

[0341] (2) Observation of the persistence of NK cells in mice.

[0342] Table 16. Observation of the persistence of CAR-NK cells in mice.

[0343]

[0344] "-" indicates that the mouse died;

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

[0346]

[0347] Tables 16 and 17 Figure 34 The results showed that in peripheral blood samples collected from the orbital fossa of mice at D49, D56 and D63, the tmbIL-15-NK group still had a high proportion of NK cell survival, while the NK group had almost no detectable NK cells, indicating that tmbIL-15-NK has significant long-term survival ability in vivo.

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

[0349] 3.1 Effects of tmbIl-15 on CAR+ survival and proliferation

[0350] 3.1.1 Experimental Methods

[0351] (1) Preparation of CAR-NK cells: SDT-NK066 and SDT-NK078 cells were prepared according to the method in 1.1. The CAR structure on the core plasmid is shown in Table 3. Untransduced NK cells (UTD-NK) were used as the control group.

[0352] Table 18 Two sets of CAR core plasmid structures

[0353]

[0354] CD276 VHH is a CD276 single-domain antibody. The amino acid sequence of the antibody is shown in SEQ ID NO.32.

[0355] QVQLVESGGGLVQPGGSLRLSCSASGFTPSIYTMGWYRQAPGKGREFVASIVNEGIPGYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAFATYYGLGDPRYWGQGTLVTVSS

[0356] The nucleotide sequence of CD276 VHH is shown in 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 showed that, comparing the CAR changes between SDT-NK078 and SDT-NK066, it was found that with the change of culture time, the CAR+ of (SDT-NK078) with tmbIL-15 gradually increased in the later stage of culture, while the CAR+ of (SDT-NK066) without tmbIL-15 structure continued to decrease during the culture process; indicating that tmbIL-15 promotes the maintenance and proliferation of CAR-NK, and further affects CAR expression.

[0363] 3.2 Short-term lethality experiment of tmbIL-15-CD276 VHH-CAR-NK

[0364] 3.2.1 Experimental Methods

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

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

[0367] Short-term lethality experiments were conducted according to method 1.3.

[0368] 3.2.2 Experimental Results

[0369] Table 19. Kill rate statistics of SDT-NK066, SDT-NK078 and UTD-NK against PANC-1 (with IL-2 added).

[0370]

[0371] Table 20. Kill rate statistics of SDT-NK066, SDT-NK078 and UTD-NK against Huh-7 (with IL-2 added).

[0372]

[0373] Table 21 Kill Rate Statistics of SDT-NK066, SDT-NK078 and UTD-NK against HepG2-CD276 (with IL-2 added)

[0374]

[0375] Table 22 Kill Rate Statistics of SDT-NK066, SDT-NK078 and UTD-NK against HCC827 (with IL-2 added)

[0376]

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

[0378]

[0379] Table 24. Kill Rate Statistics of SDT-NK066, SDT-NK078, and UTD-NK against SKOV-3 (with IL-2 added).

[0380]

[0381] Table 25. Kill rate statistics of SDT-NK066, SDT-NK078 and UTD-NK against PANC-1 (without IL-2).

[0382]

[0383] Figures 38 to 43 The results in Tables 19 to 24 show that when the culture medium for CAR-NK cells contains the cytokine IL-2, at the same effector-to-target ratio, SDT-NK078 containing the tmbIL-15 structure has a significantly higher killing ability against six types of cancer cells than SDT-NK066 without the tmbIL-15 structure. This indicates that the tmbIL-15 structure can significantly enhance the killing ability of CAR-NK cells against solid tumor target cells.

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

[0385] 3.3 Cytokine release detection:

[0386] 3.3.1 Experimental Methods

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

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

[0389] 1.0×10 5 Effector cells and 1.0 × 10 5 Target cells were co-incubated for 18 h, and the supernatant was collected by centrifugation for cytokine detection. For specific experimental procedures, please refer to the instructions for the IFN-γ (ACRO CRS-A017) / TNF-α (ACRO CRS-A002) factor detection kit.

[0390] 3.3.2 Experimental Results

[0391] Table 26 IFN-γ Release Statistics (with IL-2 added)

[0392]

[0393] Table 27 TNF-α Release Statistics (with IL-2 added)

[0394]

[0395] Table 28 IFN-γ Release Statistics (without IL-2 addition)

[0396]

[0397] Table 29 TNF-α Release Statistics (without IL-2 added)

[0398]

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

[0400] Figure 49 , Figure 50The results in Tables 28 and 29 show that, in the absence of IL-2 in the culture medium, SDT-NK078 cells containing the tmbIL-15 structure released significantly higher levels of IFN-γ and TNF-α than the control group SDT-NK066 cells without the tmbIL-15 structure after co-culturing with target cells. This indicates that the tmbIL-15 structure can still enhance the release of CAR-NK cell killing factors even without IL-2 stimulation.

[0401] 3.4 CD107a detection

[0402] 3.4.1 Experimental Methods

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

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

[0405] 100 μL (1.0 × 10⁻⁶) of effector cells 5 ), 100 μL of target cells (1.0 × 10⁻⁶) 5 Add a 96-well plate;

[0406] Add 2 μL of PE anti-human CD107a (LAMP-1) Antibody (BioLenged, 328608) and incubate at 37°C for 1 h;

[0407] Add 2 μL of GolgiStop™ Protein Transport Inhibitor (BD, 554724) to 3 mL of complete culture medium, add 20 μL to the cells and mix well. After incubating for 2.5 h–3 h, add 0.5 μL of APC anti-human CD56 (NCAM) Antibody (Biolegend, 362504), incubate at 37 °C for 30 min, wash once with FACS buffer, and then perform CD107a expression detection.

[0408] (2) Experimental results

[0409] Figure 51 The results showed that after co-culturing with target cells, SDT-NK078 containing the tmbIL-15 structure was able to release a large amount of CD107a compared with the control group SDT-NK06 which did not contain the tmbIL-15 structure.

[0410] 3.5 tmbIL-15-CD276 VHH-CAR-NK Long-Term Lethality Experiment

[0411] 3.5.1 Experimental Methods:

[0412] 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.

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

[0414] The seeding density of target cells was 1.0 × 10⁶. 5 At an effector-to-target ratio of 1:5, effector cells and target cells were co-cultured for 2 days, followed by a half-medium change, and the cells were replenished with target cells (2.0 × 10⁻⁶). 5 Continue co-culturing for 2-3 days. CAR+ changes are detected on day 0, day 4, and day 6 after co-culturing. On day 6, cell culture supernatant is collected to detect the release of cytokines IFN-γ and TNF-α.

[0415] 3.5.2 Experimental Results:

[0416] Table 30 Statistical table of CAR+ ratio after co-culture with PANC-1

[0417]

[0418] Table 31 Statistical Table of IFN-γ Release

[0419]

[0420] Table 32 Statistical Table of TNF-α Release Amount

[0421]

[0422] Figure 52 The results in Table 30 show that with repeated stimulation of target cells, the CAR+ ratio of SDT-NK078 cells containing the tmbIL-15 structure continuously increased, while the CAR+ ratio of the control group SDT-NK066 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.

[0423] Figure 53 , Figure 54 The results in Tables 31 and 32 show that, after repeated stimulation by target cells, the amounts of IFN-γ and TNF-α released by SDT-NK078 containing the tmbIL-15 structure were significantly higher than those released by the control group SDT-NK066 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.

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

[0425] 3.6.1 Experimental Methods

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

[0427] 3.6.2 Experimental Results

[0428] Figures 55 to 58 The results showed that, compared with the control group SDT-NK066 (containing no tmbIL-15), SDT-NK078 containing the tmbIL-15 structure exhibited a gradual increase in the expression of NK-activating receptors such as NKp30, NKp44, and NKG2D with prolonged culture time; simultaneously, the trend of increasing CD56+CD16+ double-positive NK phenotype was more pronounced, indicating increased NK cell maturity. This suggests that, under continuous stimulation by 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 Effects of tmbIL-15-CD276 VHH-CAR-NK on tumors in animals

[0430] 1. Experimental Methods:

[0431] The tumor animal model used was the severely immunodeficient mouse NCG model. Pancreatic cancer cells (PANC-1luc) were selected as the tumor cells, and tumor grafting was performed via tail vein at a dose of 1.0 × 10⁻⁶. 6 Cells / animal, single CAR-NK administration on day 14 after tumor bearing, tmbIL15 CD276-CAR-NK (SDT-NK078) was administered in two dose groups, 1.0 × 10 6 CAR-NK / only 2.0×10 6 CAR-NK / animal, CD276 CAR-NK (SDT-NK066) sample at 2.0×10 6 CAR-NK cells were administered at a single dose per mouse, with a control group consisting of UTD-NK cells. All experimental samples were obtained from PBMCs purchased from Miaoshun Biotechnology, where NK cells were obtained through magnetic bead sorting. CAR-NK samples were then obtained after NK cell virus infection. Tumor burden changes were periodically monitored using in vivo imaging after drug administration, and mouse body weight changes were also observed. Peripheral blood was collected from the orbital region of mice on days 25, 32, 39, and 46, and the proportion of NK cells in the blood was analyzed by flow cytometry.

[0432] 2. Experimental Results:

[0433] (1) Results of live imaging

[0434] Table 33 Statistical analysis of the inhibitory effect of tmbIL-15-CD276 VHH-CAR-NK on pancreatic cancer cells PANC-1 luc

[0435]

[0436] Table 33 Figure 59 , Figure 60 The results showed that, compared with the control group NK group, both dose groups of SDT-NK078 cells had significant tumor-suppressing effects.

[0437] (2) Observation of the persistence of NK cells in mice.

[0438] Table 34. Observation of the persistence of CD276 VHH-CAR-NK cells in mice.

[0439]

[0440] "-" indicates that the mouse died;

[0441] Table 35 Statistical analysis of the persistence of CD276 VHH-CAR-NK cells in mice.

[0442]

[0443] Tables 34 and 35 Figure 61 The results showed that both dose groups of SDT-NK078 had high survival rates, while the NK and SDT-NK066 groups were almost undetectable, indicating that the CD276 VHH-CAR-NK of the tmbIL-15 armor has significant long-term survival capability in vivo.

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

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

[0446] 4.1.1 Experimental Methods

[0447] Preparation of CAR-NK cells: SDT-NK079 and SDT-NK004 cells were prepared according to method 1.1, and the CAR structure on the core plasmid is shown in Table 3. Untransduced NK cells (UTD-NK) were used as the control group.

[0448] Table 36 CAR structures of core plasmids (3 groups)

[0449]

[0450] FMC63 is an anti-CD19 single-chain antibody, and its amino acid sequence is shown in SEQ ID NO.34:

[0451] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGG SEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

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

[0453] gacatccagatgacccagacgaccagctccctctcggcatccctgggggatcgcgtgactatttcatgccgggcttcccaggacatctctaagtacctcaactggtaccagcagaagcccgacggcaccgtgaaactgctgatctaccataccagccgtcttcacagcggtgtcccttcca ggttttcaggatcggggtcgggcaccgactactctctgaccatctccaacctggagcaggaggacatcgcgacctatttctgtcaacagggcaacacgttgccctacaccttcggcggcgggaccaagctggagatcaccggcggaggcggttcgggaggcggaggctctggcggtggcggc tctgaggtgaagctgcaggagagcgggcctgggctggtggctccctctcagagcctgtccgtcacctgcacagtgtctggcgtgtctctgccggactacggcgttagttggattcgccagccaccgcgcaagggcctggaatggctaggtgtaatctggggctccgagaccacctactaca actccgccctcaaatcgcgccttactatcatcaaggacaactccaaatcacaggtgttcctgaagatgaatagcttgcagactgatgacaccgccatttactactgtgccaagcactactattacggtggtagctacgcgatggattattggggccagggtactagtgtcacagtgtcctcc

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

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

[0456] Short-term lethality experiments were conducted according to method 1.3.

[0457] 4.1.2 Experimental Results

[0458] Table 37. Kill Rate Statistics of SDT-NK004, SDT-NK079, and UTD-NK against RAJI (with IL-2 added)

[0459]

[0460] Table 38. Kill rate statistics of SDT-NK004, SDT-NK079 and UTD-NK against RAJI (IL-2 not added).

[0461]

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

[0463] Figure 63 The results in Table 38 show that when the culture medium for CAR-NK cells did not contain the cytokine IL-2, SDT-NK079 containing the tmbIL-15 structure still exhibited significantly higher killing ability against human pancreatic cancer cells (PANC-1) than the control group SDT-NK004 which did not contain the tmbIL-15 structure. This indicates that CAR-NK cells containing the tmbIL-15 structure can maintain a very strong in vitro killing function even in the absence of IL-2.

[0464] 4.2 Cytokine release detection:

[0465] 4.2.1 Experimental Methods

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

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

[0468] 1.0×10 5 Effector cells and 1.0 × 10 5 Target cells were co-incubated for 18 hours, and the supernatant was collected by centrifugation for cytokine detection. For specific experimental procedures, please refer to the instructions for the IFN-γ (ACRO CRS-A017) / TNF-α (ACRO CRS-A002) factor detection kit.

[0469] 4.2.2 Experimental Results

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

[0471]

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

[0473]

[0474] Table 41 IFN-γ Release Statistics (without IL-2 addition)

[0475]

[0476] Table 42 TNF-α Release Statistics (without IL-2)

[0477]

[0478] Figure 64 , Figure 65 The results in Tables 39 and 40 show that, after co-culturing with target cells in the culture medium supplemented with IL-2, the amounts of IFN-γ and TNF-α released by SDT-NK079 containing the tmbIL-15 structure were significantly higher than those of 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.

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

[0480] 4.3 CD107a detection

[0481] 4.3.1 Experimental Methods

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

[0483] CD107a expression was detected according to the method in 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 experimental method described in 3.5.4 was followed to study changes in CAR+ and the release of cytokines IFN-γ and TNF-α.

[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 expression of NKp30, NKp44, NKG2D, CD56, and CD16 in the prepared SDT-NK079, SDT-NK004, and UTD-NK cells was detected according to method 2.8.

[0497] 3.5.2 Experimental Results

[0498] Figures 72 to 75 The results showed that, compared with the control group SDT-NK004 which did not contain the tmbIL-15 structure, the expression of NK-activating receptors such as NKp30, NKp44, and NKG2D in SDT-NK079 containing the tmbIL-15 structure gradually increased with prolonged culture time; at the same time, the trend of increasing CD56+CD16+ double-positive NK phenotype was more obvious, indicating increased NK cell maturity. This suggests that during continuous stimulation by 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 effect and release of cytotoxic factors against various solid tumor cells and hematological malignancies. Furthermore, under long-term treatment with IL-2-free culture medium, tmbIL-15 can significantly maintain the function and proliferation of NK cells, unaffected by IL-2. In long-term killing experiments with repeated stimulation of tumor cells, tmbIL-15-armored CAR-NK cells still maintain high CAR+ maintenance and clonal proliferation, and exhibit high release of cytotoxic factors and expression of cytotoxic receptors.

[0500] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An IL-15 transmembrane fusion protein, characterized in that, The amino acid sequence of the IL-15 transmembrane fusion protein is shown in SEQ ID NO.

31.

2. A fusion protein, characterized in that, Includes the IL-15 transmembrane fusion protein as described in claim 1, wherein the fusion protein is composed of a signal peptide, an antigen-binding domain, a hinge & transmembrane region, a signal transduction region, a cleavage peptide, and the IL-15 transmembrane fusion protein as described in claim 1 in sequence; The signal peptide is selected from CD8a SP; The antigen-binding domain is selected from anti-CD276 scFV, anti-CD276 VHH, or anti-CD19 antibody FMC63; The hinge and transmembrane region are selected from CD8a hinge™. The signal conduction region is selected from BBz; The cleavage peptide is selected from P2A.

3. The fusion protein as described in claim 2, characterized in that, The amino acid sequence of the signal peptide CD8a SP is shown in SEQ ID NO.1; The amino acid sequence of CD276 scFV is shown in SEQ ID NO.3, the amino acid sequence of CD276 VHH is shown in SEQ ID NO.32, or the amino acid sequence of FMC63 is shown in SEQ ID NO.34; The amino acid sequence of the hinge and transmembrane region is shown in SEQ ID NO.5; The amino acid sequence of the signal transduction region is shown in SEQ ID NO.7; The amino acid sequence of the cleaved peptide is shown in SEQ ID NO.

9.

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

5. An expression vector, characterized in that, Includes the nucleic acid molecules as described in claim 4.

6. Virus particles, characterized in that, Including the expression vector as described in claim 5.

7. The host, characterized in that, Includes any of the following: (I) Expressing the IL-15 transmembrane fusion protein as described in claim 1; or (II) Expressing the fusion protein as described in claim 2 or 3; or (III) Transfection with the expression vector as described in claim 5; or (IV) Transduction of viral particles as described in claim 6.

8. The host as described in claim 7, characterized in that, The host is selected from NK cells, NKT cells, and T cells.

9. The host as described in claim 8, characterized in that, The host is selected from induced NK cells, NKT cells, or T cells.

10. Any of the following applications in maintaining the activity of immune cells, promoting their survival, or their proliferation: The application is for purposes other than disease diagnosis or treatment; (I) The IL-15 transmembrane fusion protein as described in claim 1; or (II) The fusion protein as described in claim 2 or 3; or (III) The expression vector as described in claim 5; or (IV) Virus particles as described in claim 6; or The immune cells mentioned are NK cells.

11. The application as described in claim 10, characterized in that, The NK cells mentioned are induced NK cells.

12. Any of the following applications in the preparation of immune cells that enhance tumor-killing function: (I) The fusion protein as described in claim 2 or 3; or (II) An expression vector containing a nucleic acid molecule encoding a fusion protein as shown in (I); or (III) Viral particles containing the expression vector shown in (II); When the antigen-binding domain is selected from anti-CD276 scFV or anti-CD276 VHH, the tumor is a solid tumor; The solid tumor expresses CD276; the solid tumor is selected from one or more of the following: ovarian cancer, prostate cancer, liver cancer, lung cancer, head and neck cancer, kidney cancer, cervical cancer, breast cancer, colorectal cancer, neuroblastoma, pituitary adenoma, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, skin cancer, myeloma, bladder cancer, osteosarcoma, or glioma. When the antigen-binding domain is selected from anti-CD19 antibody FMC63, the tumor is a hematologic malignancy; The hematologic malignancy expresses CD19; the hematologic malignancy is either lymphoma or B-cell leukemia.

13. The application as described in claim 12, characterized in that, The enhanced tumor-killing function is to improve the killing power of target cells and / or promote the release of killing factors; The killing factor is IFN-γ or TNF-α.

14. The use of any of the following in the preparation of drugs for the prevention and / or treatment of tumors: (I) The fusion protein as described in claim 2 or 3; or (II) An expression vector containing a nucleic acid molecule encoding a fusion protein as shown in (I); or (III) Viral particles containing the expression vector shown in (II); or (IV) A host containing an expression vector as shown in (II) or viral particles as shown in (III); When the antigen-binding domain is selected from anti-CD276 scFV or anti-CD276 VHH, the tumor is a solid tumor; The solid tumor expresses CD276; the solid tumor is one or more of the following: ovarian cancer, prostate cancer, liver cancer, lung cancer, head and neck cancer, kidney cancer, cervical cancer, breast cancer, colorectal cancer, neuroblastoma, pituitary adenoma, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, skin cancer, myeloma, bladder cancer, osteosarcoma, or glioma. When the antigen-binding domain is selected from anti-CD19 antibody FMC63, the tumor is a hematologic malignancy; The hematologic malignancy expresses CD19; the hematologic malignancy is either lymphoma or B-cell leukemia.

15. A drug / drug combination, characterized in that, The drug is made from any of the following raw materials, and pharmaceutically acceptable excipients or adjuvants: (I) The IL-15 transmembrane fusion protein as described in claim 1; or (II) The fusion protein as described in claim 2 or 3; or (III) The expression vector as described in claim 5; or (IV) Virus particles as described in claim 6; or (V) The host as described in claim 7 or 8; The drug combination includes the drug.

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