Bispecific NK cell agonists and methods of production and use

Abstract

The present disclosure relates to the field of biomedicine, specifically to a bispecific NK cell agonist and its preparation and use. The present disclosure provides a fusion protein, which targets CD16A receptor and 4-1BB receptor to obtain a new bispecific NK cell agonist drug, namely scfv-CD16A-Trimer-4-1BBL, which has the activity of activating and expanding NK cells in vitro, enhances their anti-virus and anti-tumor abilities, and at the same time, has the potential effect of promoting the proliferation of NK cells and enhancing the function of T cells, and can be used to activate and expand NK cells in vitro, and the expanded NK cells have relatively good cytotoxicity.

Description

[Technical Field]

[0001] The present disclosure relates to the biomedicine field, and in particular to bispecific NK cell agonists and methods of making and using them. [Background technology]

[0002] NK cells are the primary effector cells of the innate immune system. Unlike T cells, NK cells do not require prior stimulation and can kill tumor cells and virus-infected cells without MHC restriction by utilizing perforin, granular enzymes, and related mechanisms. NK cells express numerous activating receptors, which enable them to effectively recognize stress ligands produced by tumor cells or infected cells and kill tumor cells. However, tumor cells also secrete and produce many immunosuppressive molecules, creating an immunosuppressive tumor microenvironment that inhibits the activity and antitumor capabilities of NK cells in this environment, resulting in NK cell exhaustion. Currently, there are many methods to restore NK cell exhaustion in the tumor microenvironment, such as immune test point blockade, activating antibodies, and activating cytokines. These methods can provide activating signals to NK cells or block inhibitory signals in the tumor microenvironment, restoring NK cells to their normal activation state and allowing them to exert antitumor or antiviral functions.

[0003] CD16A is an important activating receptor expressed on NK cells. When activated by its ligand or antibody binding, CD16A induces various cascade activation reactions, releasing granule enzymes, perforin, inflammatory cytokines, and chemokines, which potently activate NK cells and enhance their antitumor capabilities, while also temporarily increasing 4-1BB receptor expression. 4-1BB receptor is a member of the TNF receptor superfamily. 4-1BB is primarily expressed in the form of a trimer on activated NK cells and T cells. Its ligand is 4-1BBL, the only naturally occurring ligand currently known. The binding of 4-1BBL to 4-1BB activates the 4-1BB receptor, which enhances NK cell activation and ADCC.

[0004] Currently, there are still few research reports on NK cell agonists, and existing NK cell agonists have limited ability to activate NK cells, leaving room for further improvement. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure aims to solve at least part of one of the technical problems in the related art. Therefore, one objective of the present disclosure is to obtain a new bispecific NK cell agonist drug that targets CD16A receptor and 4-1BB receptor, and to provide a fusion protein that can be used to activate NK cells and enhance their antiviral and antitumor capabilities, and simultaneously has the potential effects of promoting NK cell proliferation and enhancing T cell function. [Means for solving the problem]

[0006] Thus, a first aspect of the present disclosure provides a fusion protein comprising a CD16A single-chain antibody and a 4-1BBL extracellular domain, wherein the CD16A single-chain antibody is linked to the 4-1BBL extracellular domain, and the 4-1BBL extracellular domain comprises at least one to three 4-1BBL extracellular domains.

[0007] The inventors discovered that a novel bispecific NK cell agonist drug, scfv-CD16A / Trimer-4-1BBL, was designed by targeting the CD16A receptor and the 4-1BBL extracellular segment. This NK cell agonist drug utilizes a single-chain CD16A antibody to recognize the CD16A receptor, followed by the 4-1BBL extracellular segment at the other end of the fusion protein, which recognizes the 4-1BB receptor, thereby increasing NK cell proliferation. The bispecific NK cell agonist drug, constructed by combining a single-chain anti-CD16A antibody with one to three, preferably three, 4-1BBL extracellular segments, can be used to activate NK cells and enhance their antiviral and antitumor capabilities, potentially promoting NK cell proliferation and enhancing T cell function. The scfv-CD16A / Trimer-4-1BBL fusion protein of the present disclosure can activate NK cells by binding to the CD16A receptor and the 4-1BB receptor, reverse NK cell exhaustion, and enhance the anti-tumor ability of NK cells. The scfv-CD16A / Trimer-4-1BBL fusion protein of the present disclosure can effectively expand NK cells, increasing the purity and expansion fold of NK cells.

[0008] According to the implementation means of the present disclosure, the fusion protein further comprises at least one of the following additional technical features:

[0009] According to the implementation means of the present disclosure, the 4-1BBL extracellular region comprises three 4-1BBL extracellular domains, and the three 4-1BBL extracellular domains are connected in sequence.

[0010] Three 4-1BBL extracellular domains were linked to a CD16A single-chain antibody to form scfv-CD16A-Trimer-4-1BBL, which has the ability to activate and expand NK cells in vitro, and the expanded NK cells have relatively good cytotoxicity.

[0011] According to the implementation means of the present disclosure, the CD16A single-chain antibody and the 4-1BBL extracellular domain are linked via a flexible joint.

[0012] According to the implementation means of the present disclosure, the three 4-1BBL extracellular domains are connected via flexible joints.

[0013] According to an embodiment of the present disclosure, the fusion protein is expressed by a non-mammalian cell expression system.

[0014] According to an embodiment of the present disclosure, the non-mammalian cell expression system includes at least one of a prokaryotic expression system and a lower eukaryotic expression system.

[0015] According to an embodiment of the present disclosure, the non-mammalian cell expression system includes at least one of an E. coli expression system and a yeast expression system.

[0016] According to an embodiment of the present disclosure, the yeast expression system comprises a Pichia yeast expression system.

[0017] According to the implementation of the present disclosure, the CD16A single chain antibody is V H Area and V L region, H The amino acid sequence of the VL region is shown in SEQ ID NO:1, and the amino acid sequence of the VL region is shown in SEQ ID NO:2.

[0018] According to the implementation means of the present disclosure, the V H Area and V L The regions are connected via a first joint.

[0019] According to an embodiment of the present disclosure, the first joint is a flexible joint.

[0020] According to an embodiment of the present disclosure, the length of the first joint is 10 to 20 amino acids.

[0021] According to an embodiment of the present disclosure, the amino acid sequence of the first joint is shown in SEQ ID NO:3.

[0022] CD16A single chain antibody V H The amino acid sequence of the region is shown in SEQ ID NO:1 below: QVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS CD16A single chain antibody V L The amino acid sequence of the region is shown in SEQ ID NO:2 below: SYVLTQPSSVVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL The amino acid sequence of the first joint is shown in SEQ ID NO:3 below: GGGGSGGGGSGGGGS.

[0023] According to the implementation means of the present disclosure, the V H Area or V L One end of the domain and the 4-1BBL extracellular domain are connected via a second joint.

[0024] According to an embodiment of the present disclosure, the second joint is a flexible joint.

[0025] According to an embodiment of the present disclosure, the length of the second joint is 10 to 20 amino acids.

[0026] According to an embodiment of the present disclosure, the amino acid sequence of the second joint is shown in SEQ ID NO:4.

[0027] The amino acid sequence of the second joint is shown in SEQ ID NO:4 below: GGGGSGGGGSGGGG.

[0028] According to an embodiment of the present disclosure, the amino acid sequence of the 4-1BBL extracellular domain is shown in SEQ ID NO:5.

[0029] The amino acid sequence of the 4-1BBL extracellular domain is shown in SEQ ID NO:5 below: REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE.

[0030] According to the implementation means of the present disclosure, the three 4-1BBL extracellular domains are connected to each other via a third joint and a fourth joint, respectively, and the third joint and the fourth joint are the same or different.

[0031] According to an embodiment of the present disclosure, the third joint and the fourth joint are both flexible joints.

[0032] According to an embodiment of the present disclosure, the length of the third joint and the fourth joint is 15 to 25 amino acids.

[0033] According to an embodiment of the present disclosure, the amino acid sequence of the third joint is shown in SEQ ID NO:6.

[0034] According to an embodiment of the present disclosure, the amino acid sequence of the fourth joint is shown in SEQ ID NO:7.

[0035] The amino acid sequence of the third joint is shown in SEQ ID NO:6 below: GGGGSGGGGSGGGGSGGGGS The amino acid sequence of the fourth joint is shown in SEQ ID NO:7 below: GGGGSGGGGSGGGGSGGGGS.

[0036] According to an embodiment of the present disclosure, the amino acid sequence of the fusion protein is shown in SEQ ID NO:8.

[0037] The amino acid sequence of the fusion protein is shown in SEQ ID NO:8 below: QVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDF ADYWGQGTLVTVSSGGGGGSGGGGSGGGGSSYVLTQPSSVSVAPQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQ AMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSEGGGGSGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPS PRSEGGGGSGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEHHHHHH

[0038] A second aspect of the present disclosure provides a nucleic acid, wherein the nucleic acid encodes a fusion protein according to the first aspect.

[0039] According to an embodiment of the present disclosure, the nucleic acid is a nucleic acid that is codon-optimized for a yeast expression system.

[0040] According to an embodiment of the present disclosure, the nucleotide sequence of the nucleic acid is set forth in SEQ ID NO:10.

[0041] The nucleotide sequence encoding the non-codon-optimized fusion protein is shown in SEQ ID NO:9 below:

[0042] The nucleotide sequence encoding the codon-optimized fusion protein is shown in SEQ ID NO: 10 below:

[0043] A third aspect of the present disclosure provides an expression vector, which comprises the nucleic acid according to the second aspect.

[0044] A fourth aspect of the present disclosure provides a recombinant cell, which, according to an embodiment of the present disclosure, harbors a nucleic acid according to the second aspect, an expression vector according to the third aspect, or expresses a fusion protein according to the first aspect.

[0045] A fifth aspect of the present disclosure provides use of the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the expression vector according to the third aspect or the recombinant cell according to the fourth aspect in the manufacture of a drug for use in the treatment or prevention of tumors or inflammatory diseases.

[0046] According to an embodiment of the present disclosure, the tumor includes at least one of ovarian cancer, lung cancer, rectal cancer, breast cancer, and leukemia.

[0047] According to an embodiment of the present disclosure, the inflammatory disease comprises a bacterial or viral infection.

[0048] According to an embodiment of the present disclosure, the bacterial infection includes at least one of pneumoniae cleber, Listeria monocytogenes, and Staphylococcus aureus infection.

[0049] According to an embodiment of the present disclosure, the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.

[0050] A sixth aspect of the present disclosure provides use of the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the expression vector according to the third aspect or the recombinant cell according to the fourth aspect in the ex vivo expansion and culture of NK cells.

[0051] According to the implementation means of the present disclosure, the NK cells are derived from peripheral blood or obtained by stem cell induction, and include at least one of NK cells derived from human peripheral blood mononuclear cells, NK cells induced from human umbilical cord blood stem cells, NK cells induced from human embryonic stem cells, and NK cells induced from human iPSCs.

[0052] A seventh aspect of the present disclosure provides a pharmaceutical composition, comprising the fusion protein of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fourth aspect.

[0053] According to the implementation means of the present disclosure, the pharmaceutical composition is used for treating or preventing tumors and inflammatory diseases.

[0054] According to an embodiment of the present disclosure, the tumor includes at least one of ovarian cancer, lung cancer, rectal cancer, breast cancer, and leukemia.

[0055] According to an embodiment of the present disclosure, the inflammatory disease comprises a bacterial or viral infection.

[0056] According to an embodiment of the present disclosure, the bacterial infection includes at least one of pneumoniae cleber, Listeria monocytogenes, and Staphylococcus aureus infection.

[0057] According to an embodiment of the present disclosure, the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.

[0058] An eighth aspect of the present disclosure provides a reagent kit, which includes the fusion protein according to the first aspect.

[0059] According to an embodiment of the present disclosure, the reagent kit is used to detect CD16A and / or 4-1BB.

[0060] A ninth aspect of the present disclosure provides a method for treating or preventing a tumor or an inflammatory disease. According to an embodiment of the present disclosure, the method comprises administering to a subject suffering from or suspected of having a tumor or an inflammatory disease: a fusion protein according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fourth aspect, and administering at least one of the pharmaceutical compositions according to the seventh aspect.

[0061] According to an embodiment of the present disclosure, the tumor includes at least one of ovarian cancer, lung cancer, rectal cancer, breast cancer, and leukemia.

[0062] According to an embodiment of the present disclosure, the inflammatory disease comprises a bacterial or viral infection.

[0063] According to an embodiment of the present disclosure, the bacterial infection includes at least one of pneumoniae cleber, Listeria monocytogenes, and Staphylococcus aureus infection.

[0064] According to an embodiment of the present disclosure, the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.

[0065] A tenth aspect of the present disclosure provides use of the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the expression vector according to the third aspect, the recombinant cell according to the fourth aspect, or the pharmaceutical composition according to the seventh aspect in the treatment or prevention of a tumor or an inflammatory disease.

[0066] According to an embodiment of the present disclosure, the tumor includes at least one of ovarian cancer, lung cancer, rectal cancer, breast cancer, and leukemia.

[0067] According to an embodiment of the present disclosure, the inflammatory disease comprises a bacterial or viral infection.

[0068] According to an embodiment of the present disclosure, the bacterial infection includes at least one of pneumoniae cleber, Listeria monocytogenes, and Staphylococcus aureus infection.

[0069] According to an embodiment of the present disclosure, the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.

[0070] Additional aspects and advantages of the disclosure will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the disclosure. [Brief explanation of the drawings]

[0071] The above and / or additional aspects and advantages of the present disclosure will become apparent and easier to understand from the following description of the embodiments taken in conjunction with the drawings. [Figure 1] FIG. 1 is a schematic diagram of a protein expression vector and the scfv-CD16A-Trimer-4-1BBL fusion protein. [Figure 2] 1 shows the results of screening clones expressing the scfv-CD16A-Trimer-4-1BBL fusion protein. Figure (A) shows the results of dot blot analysis, and Figure (B) shows the results of Western blot analysis (anti-His tag antibody). [Figure 3] The results of fermentation expression of scfv-CD16A-Trimer-4-1BBL fusion protein are shown in Figure (A), which shows the monitoring of fermentation process parameters, and Figure (B), which shows the detection results of target protein accumulation time points. [Figure 4] Purification and physicochemical identification of scfv-CD16A-Trimer-4-1BBL fusion protein. Figure (A) shows the results of SDS-PAGE detection of the purity of scfv-CD16A-Trimer-4-1BBL fusion protein. Figure (B) shows the results of dynamic light scattering detection of scfv-CD16A-Trimer-4-1BBL fusion protein. Figure (C) shows the results of HPLC detection of scfv-CD16A-Trimer-4-1BBL fusion protein. [Figure 5]This shows the results of detecting the in vitro activated NK cell activity of scfv-CD16A-Trimer-4-1BBL fusion protein. [Figure 6] 1 shows the results of NK cell expansion and expansion effect evaluation by in vitro induction of scfv-CD16A-Trimer-4-1BBL fusion protein. Figure (A) shows the results of NK cell purity detection after NK cell expansion by in vitro induction of scfv-CD16A-Trimer-4-1BBL fusion protein. Figure (B) shows the results of NK cell expansion fold detection after in vitro induction of scfv-CD16A-Trimer-4-1BBL fusion protein. Figure (C) shows the results of total cell expansion fold detection after NK cell expansion by in vitro induction of scfv-CD16A-Trimer-4-1BBL fusion protein. Figure (D) shows the results of cell subpopulation detection after NK cell expansion by in vitro induction of scfv-CD16A-Trimer-4-1BBL fusion protein. Figure (E) shows the results of cytotoxic activity detection after NK cell expansion by in vitro induction of scfv-CD16A-Trimer-4-1BBL fusion protein. DETAILED DESCRIPTION OF THE INVENTION

[0072] Hereinafter, examples of the present disclosure will be described in detail. The examples described below are illustrative and are used only to interpret the present disclosure, and should not be understood as limitations on the present disclosure.

[0073] It should be noted that the terms "first" and "second" are for descriptive purposes only and cannot be considered to indicate or imply relative importance or the number of technical features. Thus, a feature qualified as "first" or "second" can expressly or imply the inclusion of one or more of the feature. In the description of this disclosure, unless otherwise expressly and specifically limited, the concept of "plurality" is at least two, e.g., two or three.

[0074] According to some specific embodiments of the present disclosure, the present disclosure provides a fusion protein comprising a CD16A single-chain antibody and a 4-1BBL extracellular domain, wherein the CD16A single-chain antibody is linked to the 4-1BBL extracellular domain, and the 4-1BBL extracellular domain comprises at least one to three 4-1BBL extracellular domains. For example, the 4-1BBL extracellular domain comprises three 4-1BBL extracellular domains, and the three 4-1BBL extracellular domains are linked sequentially to form an scfv-CD16A-Trimer-4-1BBL fusion protein.

[0075] The fusion protein disclosed herein targets the CD16A receptor and the 4-1BB receptor to obtain a novel bispecific NK cell agonist drug, i.e., scfv-CD16A-Trimer-4-1BBL, which has the ability to activate and expand NK cells in vitro, enhancing their antiviral and antitumor capabilities. At the same time, it has the potential to promote NK cell proliferation and enhance T cell function, and the expanded NK cells have relatively good cytotoxicity.

[0076] Here, the term "antibody" refers to an immunoglobulin protein molecule capable of binding to a specific antigen. It comprises two light chains with a low molecular weight and two heavy chains with a high molecular weight, and the heavy (H) and light (L) chains are connected by disulfide bonds to form a single tetrapeptide molecule. The amino acid sequence at the amino terminal (N-terminal) of the peptide chain varies greatly, forming the variable region (V-region), while the carboxyl terminal (C-terminal) is relatively stable and shows little variation, and is called the constant region (C-region). The V-regions of the L chain and H chain are called VL and VH, respectively, the C-region of the L chain is called CL, and the C-region of the H chain includes the CH1, CH2, and CH3 regions.

[0077] In the present disclosure, peripheral blood mononuclear cells (PBMCs) are cells with a single nucleus in peripheral blood, and include lymphocytes and monocytes.

[0078] According to some specific embodiments of the present disclosure, the first, second, third and fourth connecting peptides are flexible polypeptides consisting of glycine and serine.

[0079] According to some specific implementations of the present disclosure, the pharmaceutical composition of the present disclosure further comprises a pharmaceutically acceptable vector, including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavor enhancer or suspending agent, surfactant, isotonicity agent, thickener, emulsifier, preservative, solid binder, glidant, or lubricant, etc., suitable for a specific target dosage form. To the extent that any conventional additive is incompatible with the fusion protein of the present disclosure, for example, in addition to any adverse biological effects produced or interactions produced in a harmful manner with other components of the pharmaceutically acceptable composition, its use is also within the scope of the present disclosure.

[0080] For example, the fusion proteins of the present disclosure can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in a variety of forms, including, but not limited to, liquid, semi-solid, and solid dosage forms, including liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The fusion proteins can be administered by intravenous infusion or intramuscular or subcutaneous injection.

[0081] As used herein, the terms "treatment" and "prevention," and words derived therefrom, do not necessarily imply 100% or complete treatment or prevention. On the contrary, different degrees of treatment or prevention exist, and those skilled in the art will appreciate that such treatment or prevention may have potential benefits or therapeutic effects. Accordingly, treatment or prevention according to the present disclosure refers to the treatment or prevention of one or more of the diseases or symptoms of the disease being treated or prevented, such as cancer. For purposes of this specification, "prevention" also includes delaying the onset of the disease or its symptoms or disorders.

[0082] The following describes in detail the examples of the present disclosure. The examples described below are illustrative and are used only to interpret the present disclosure and should not be understood as limitations on the present disclosure. If specific techniques or conditions are not specified in the examples, they should be carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. If the manufacturer of the reagent or equipment used is not specified, it is a conventional product that can be purchased commercially.

[0083] Example 1. Construction of a fusion protein expression vector for the bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL

[0084] 1. Identification of the scfv-CD16A-Trimer-4-1BBL fusion gene Sequence information of scfv-CD16A (scfv-CD16A is V H and V L and V H and V L is connected via linker1, and V H The amino acid sequence of V is shown in SEQ ID NO:1. L The nucleotide sequence of linker 1 is shown in SEQ ID NO: 2, and the nucleotide sequence of linker 2 is shown in SEQ ID NO: 3. The 4-1BBL extracellular segment 71-254 sequence (shown in SEQ ID NO: 5) was obtained from NCBI. scfv-CD16A and the three 4-1BBL extracellular segments were connected via linker 2 (shown in SEQ ID NO: 4), and the three 4-1BBL extracellular segment domains were sequentially connected via linker 3 (shown in SEQ ID NO: 6) and linker 4 (shown in SEQ ID NO: 7). The resulting original, non-codon-optimized scfv-CD16A-Trimer-4-1BBL fusion gene is shown in SEQ ID NO: 9.

[0085] The original scfv-CD16A-Trimer-4-1BBL fusion gene sequence was modified and optimized for Pichia yeast codons, and two enzyme cleavage sites, XhoI and NotI, were added to both ends. The optimized and modified gene sequence is shown in SEQ ID NO: 10, and then the gene sequence was synthesized to obtain the scfv-CD16A-Trimer-4-1BBL fusion protein gene sequence.

[0086] 2. Construction of Pichia pastoris expression vector for scfv-CD16A-Trimer-4-1BBL fusion protein The scfv-CD16A-Trimer-4-1BBL fusion protein gene sequence obtained in the previous step and the Pichia yeast expression plasmid PGAPzα-rDNA-NTS were both double-cleaved with the restriction endonucleases Xhol and NotI. The cleaved scfv-CD16A-Trimer-4-1BBL fusion protein gene sequence product gene was recovered using a PCR product recovery kit (Axygen), and the cleaved plasmid was recovered using a DNA gel recovery kit (Axygen). Refer to the kit instructions for the procedure. The recovered cleaved fusion protein gene and cleaved vector were ligated using T4 DNA ligase. The ligated product was transformed into E. coli TOP10 recipient cells. The transformation procedure was as follows: 10 μL of the enzyme ligation product was added to 100 μL of E. coli recipient, mixed evenly, and left on ice for 15-30 minutes, flicked evenly every 5 minutes, then heat-stimulated at 42°C for 90 seconds, left on ice for 3-5 minutes, added 400 μL of antibiotic-free LB liquid medium, and resuscitated at 37°C on a rocking bed for 45 minutes, and then plated onto a Zeocin-resistant plate.

[0087] After growing clonal colonies on the plates, single colony clones were selected and placed in cell culture tubes containing LZ liquid medium (Zeocin-resistant LB medium) and cultured at 37°C for 6-8 hours. PCR identification was then performed using the bacterial liquid as a template, and the resulting positive clone was named PGAP-zα-scfv-CD16A-Trimer-4-1BBL (protein expression vector shown in Figure 1). The bacterial liquid corresponding to the positive clone was extracted using a small-volume plasmid extraction reagent kit and sent to a general-purpose biologist, Kin Weizhi, for sequencing.

[0088] The sequencing results were consistent with theoretically expected results, indicating that the clone construction was correct.

[0089] Example 2. Expression and purification of the bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL fusion protein

[0090] 1. Fusion protein expression Expand the positive clone constructed in Example 1 in 50 mL of LZ liquid medium and extract the PGAP-z α-scfv-CD16A-Trimer-4-1BBL plasmid using a medium reagent kit (Axygen). The expression plasmid PGAPz α-scfv-CD16A-Trimer-4-1BBL prepared in the previous step is linearized with the endonuclease SpeI and recovered by ethanol precipitation.

[0091] Ethanol precipitation step: 1) Add 2 volumes of absolute ethanol and 0.1 volumes of 3M sodium acetate to the enzyme cleavage reaction system and precipitate at -20°C for at least 2 hours. 2) Centrifuge at 12,000g for 10 minutes and discard the supernatant. 3) Resuspend and precipitate in 300μL of 70% ethanol, centrifuge at 12,000g for 10 minutes, and discard the supernatant. 4) Dry at 37°C, resuspend in deionized water, measure the concentration, and adjust to 0.5-1.0μg / μL.

[0092] Preparation of Yeast Receptor: Preparation Steps: 1) A well-preserved strain of Bastepickia yeast X33 was taken, streaked onto a YPD plate, and incubated at 30°C in a yeast culture tank. 2) When the clones grew to approximately 1 mm in diameter, they were transferred to 4 mL of liquid YPD medium and incubated at 30°C on a rocking bed. 3) Cultured for 24-48 hours. 1 mL of the resulting culture was inoculated into 50 mL of fresh YPD medium. 4) When the OD600 reached 1-1.5, the culture was transferred to a 50 mL centrifuge tube and centrifuged at 1500 g for 5 minutes at 4°C. The supernatant was discarded. 5) The culture was resuspended in 50 mL of ice water and centrifuged at 1500 g for 5 minutes at 4°C. The supernatant was discarded. This process was repeated once. 6) Resuspend in 50 mL of 1 M cold sorbitol, centrifuge at 1500 g for 5 minutes at 4°C, discard the supernatant, and repeat once. 7) Resuspend in 500 μL of 1 M cold sorbitol.

[0093] Electrotransformation: Place 100 μL of the recipient cells into a sterile, pre-cooled rotating cup, add 10 μL of linearized plasmid (5-10 μg), and mix thoroughly. Set the rotator parameters to 2000 V, 200 Ω, and 25 μF. Immediately after rotation, add 1 mL of cold sorbitol to the rotating cup and incubate on ice for 5-10 minutes. Transfer the entire bacterial suspension to a 50 ml centrifuge tube containing 1 ml of YPD and incubate at 30°C, 225 rpm, and 2 hours. After incubation, 100-1000 μL of the bacterial suspension was applied to a YPDZ (Zeocin-resistant) plate and placed in a 30°C yeast incubation tank for incubation.

[0094] Screening for positive expression clones: After growing the yeast clones on the YPDZ plates, select some clones and inoculate them into 2 mL of BMGY medium. Incubate the yeast on a rocking bed at 28°C until the culture becomes milky white. One mL of the resulting culture was frozen and stored for seed storage. The remaining 1 mL of culture was centrifuged at 12,000 g for 5 minutes and the supernatant was removed. An initial screening by Dot blot analysis using an anti-His tag antibody was performed (results shown in Figure 2) to select yeast strains with relatively high expression levels. The yeast strains with relatively high expression levels (e1, c7, c11, d1, and f13) were then plated on YPD plates containing zeocin. After clone growth, several clones were selected from the cultures and shaken to obtain the supernatant. Western blot analysis using an anti-His tag antibody was performed to screen for high-expressing strains. The remaining 1 mL of the corresponding culture was frozen and stored for seed storage. This high-expressing strain (f13-3) is a Pichia yeast strain capable of expressing the scfv-CD16A-Trimer-4-1BBL complex protein.

[0095] 2. Fermentation, purification and identification of fusion protein Expression strain cultivation: Fermentation of the recombinant strain was carried out according to Invitrogen's fermentation instruction manual. The screened high-expression strain was inoculated into a cell culture tube containing 4 mL of YPD medium and cultured at 30°C on a rocking bed for 24-48 hours until the OD600 reached 2-6, which resulted in the first fermented seed liquid. 1 mL of the seed liquid was transferred to a corn bottle containing 200 mL of BMGY medium and cultured at 30°C on a rocking bed for 12-24 hours, which resulted in the second fermented seed liquid. The 200 mL of second fermented seed liquid was then inoculated into a fermentation tank containing 6 L of BMGY medium. Setting parameters: culture temperature 30°C, pH 6.0, dissolved oxygen, rotation speed, etc. were monitored, and fermentation culture was initiated. During the bacterial growth stage, BMGY medium was used. When the dissolved oxygen concentration increased rapidly, the basal glycerol in the medium was consumed, and glycerol addition was started at a rate of 70 ml / h. The fermentation temperature was adjusted to 25°C, and the fermentation continued until completion.

[0096] Clarification of culture broth: After the fermentation is completed, the broth is centrifuged at 10,000 g for 30 minutes, the supernatant is collected, and filtered through 0.22 μm and 500 kDa microfilters for clarification, and then the pH is adjusted to 7.4.

[0097] Protein capture: A nickel affinity column is used to capture recombinant fusion proteins. The specific steps are as follows: 1) Rinse the column with deionized water for 5 column volumes. 2) Rinse the column with PBS for 3 column volumes. 3) Add the sample. 4) After sample addition, rinse the column with PBS for 3 column volumes. 5) Impurity wash: Rinse the column stepwise with PBS solutions containing 10 mM, 20 mM, 30 mM, and 40 mM imidazole, for 1 to 1.5 column volumes per concentration. 6) Elute the target protein. The eluate is a PBS solution containing 200 mM imidazole. The collected eluate is concentrated and used for subsequent purification.

[0098] Fine purification: Using AKTA PURE 25, equilibrate the Superdex 200 molecular sieve with PBS buffer. After equilibration, add the sample to the neutral eluate, rinse with PBS buffer, and collect the eluted sample. The sample was then concentrated and passed through a Superdex 75 molecular sieve in the same manner to obtain the scfv-CD16A-Trimer-4-1BBL recombinant protein.

[0099] Identification of recombinant proteins: Purified proteins are identified by SDS-PAGE, HPLC, and dynamic light scattering.

[0100] Figure 2 shows the isolation of yeast expression strains for fusion proteins by screening on YPDZ-resistant culture plates. Initial screening was performed using dot blot analysis to identify clones with relatively high expression levels. These were then confirmed and compared by Western blot analysis to screen for yeast expression clones with high expression levels, which were then used for subsequent protein expression and purification.

[0101] Figure 3 shows the monitoring of each parameter during the fermentation process. The entire fermentation process lasted for 50 hours, and the fermentation process was stable and easy to repeat. Panel A in Figure 3 shows that glycerol was added at the end of the basal glycerol culture stage to induce protein expression (20 hours). Protein continued to accumulate throughout the process, and there was little difference in the amount of protein accumulated between 43 and 50 hours. This allowed us to determine the end point of fermentation (Panel B in Figure 3 shows the amount of scfv-CD16A-Trimer-4-1BBL recombinant protein in the fermentation supernatant after 20 hours, 28 hours, 35 hours, 43 hours, and 50 hours of fermentation, respectively). After two-step purification using centrifugation and filtration, the protein purity reached over 95% as determined by SDS-PAGE and HPLC (Figures A and C in Figure 4). The molecular weight of the scfv-CD16A-Trimer-4-1BBL protein was approximately 95 kDa, as expected. Dynamic light scattering identification revealed that the molecular diameter of scfv-CD16A-Trimer-4-1BBL was approximately 10 nm, with good intermolecular uniformity (Figure 4B).

[0102] Example 3: Examination of the in vitro activation function of bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL fusion protein

[0103] Human PBMCs were isolated using Ficoll density gradient centrifugation. The isolated PBMCs were diluted to 1.0 x 10 in RPMI 1640 containing 10% FBS. 6 Dilute the cells to 100 cells / mL. Add 10 mL of diluted cells to a T25 culture bottle. After 12 hours of culture, add scfv-CD16A-Trimer-4-1BBL fusion protein to the culture bottle at a final concentration of 20 nM. After 24 hours of culture, detect phenotypic changes in NK cells using a flow cytometer.

[0104] The results are shown in Figure 5. Compared with the control group (PBS), the experimental group containing scfv-CD16A-Trimer-4-1BBL fusion protein showed significantly increased expression of major activating molecules on the surface of NK cells, such as CD69, NKp30, the killing molecules 4-1BB, TRAIL, Granzyme B, and the chemotactic molecule CX3CR1. This indicates that NK cells are effectively activated by the scfv-CD16A-Trimer-4-1BBL fusion protein.

[0105] Example 4: Evaluation of the in vitro induced NK cell expansion and expansion effect of the fusion protein of bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL

[0106] 1. Detection of purity and number of fusion protein-amplified NK cells: 1) T75 culture bottles were coated with the fusion protein 24 hours prior to the incubation. 7.5 ml of the coating solution was used per bottle to cover the bottom of the bottle. The bottles were then placed in a refrigerator at 4°C overnight. 2) After isolating PBMC cells from the blood sample, they are counted (Meyer counter) and the percentage of each cell subpopulation among the PBMC cells is recorded. 6 / ml density (20ml culture volume). The total number of cells is 30*10 6 This is day 0. At the same time, 1% IL2 (1000 IU / ml) and 5% autologous plasma of the corresponding PBMCs were added to KBM581 medium without scfv-CD16A-Trimer-4-1BBL. 3) On day 3, supplement with IL2 factor at 1% of the corresponding culture volume. 4) On day 5, observe the color of the medium and the state of cell adhesion to the bottom of the T75 bottle. If necessary, replenish with 5-10 ml of KBM581 medium containing scfv-CD16A-Trimer-4-1BBL and add IL-2 (1000 IU / ml) factor at 1% by volume of the supplemented medium. 5) On day 6, sample and count the cells for subculture expansion (1.3*10 6The cells were stained (CD3 / CD45 / CD56) and the percentages of the NK cell subpopulation and CD56+ cell subpopulation were detected using a flow cytometer. The total cell count was calculated, and the NK cell count was calculated from the flow purity results. From day 5 onwards, the cells were subcultured and expanded in a basal medium containing scfv-CD16A-Trimer-4-1BBL, supplemented with 1% IL-2 and 2% autologous plasma. 6) On the seventh day, observe the color of the medium and the state of cell growth on the wall of the bottle. If necessary, replenish the KBM581 medium and add 1% IL2 factor by volume of the supplemental liquid. 7) On day 8, sample and count. If necessary, expand the cells (T75 or T175 bolt, 1.4*10 6 / ml) and add 1% IL2 factor and 2% autologous plasma to the medium. 8) On the 9th day, observe the color of the medium and the state of cell growth on the wall of the bottle, and replenish the fluid if necessary (add IL2 factor at 1% of the volume of the replenishment fluid). 9) On day 10, sample and count the cells for subculture expansion (1.5*10 6 The cells were stained (CD3 / CD45 / CD56) and the percentages of the NK cell subpopulation and CD56+ cell subpopulation were detected using a flow cytometer, and the total cell number was calculated. The number of NK cells was also calculated from the flow purity detection results. 10) On the 12th day, observe the color of the medium and the state of cell growth on the wall of the bottle, and replenish the fluid if necessary (add IL2 factor at 1% of the volume of the replenishment fluid). 11) On day 13, samples were taken and counted, and the cells were subcultured and expanded (1.5*10 6 The cells were stained (CD3 / CD45 / CD56) and the percentages of the NK cell subpopulation and CD56+ cell subpopulation were detected using a flow cytometer, and the total cell number was calculated. The number of NK cells was also calculated from the flow purity detection results. 12) On day 15, sample and count. If necessary, expand the cells (T175 bolt or culture bag, 1.5*10 6 / ml) and add 1% of the medium IL2 factor. 13) On day 17, samples were taken and counted, and stained (CD3 / CD45 / CD56) to detect the percentage of NK cell subpopulations and the percentage of CD56+ cell subpopulations using a flow cytometer. The total cell number was calculated, and the NK cell count was calculated from the flow purity detection results. Graphs were created showing the time course of the percentage and number of NK cells according to the NK purity and cell count on days 0, 6, 10, 13, and 17.

[0107] 2. Subpopulation analysis of NK cells derived from fusion protein-amplified PBMCs 1) Intracellular molecular labeling cells Monensin induction: 4 x 10 cultured cells 6 The cells (2 ml) were placed in a 6-well plate and induced with 10 μl of monensin (0.5 μg / μl) at 37° C. in a 5% CO 2 incubator for 4 hours. 2) Preparation of single-cell suspension: 9.0 x 10 cells cultured from surface molecule-labeled cells 6 Cells were collected by centrifugation at 400 g for 8 minutes, washed twice with 10 ml of 1x PBS, and finally resuspended in 0.81 ml of 1x PBS to form a single-cell suspension. After the induction of intracellular molecular labeling (intracellular monensin), the cells were washed twice with 5 ml of 1x PBS and finally resuspended in 180 μl of 1x PBS to form a single-cell suspension. 3) Closure: 90 μl of mouse serum was added to the surface molecule-labeled cells and mixed evenly. 20 μl of mouse serum was added to the intracellular molecule-labeled cells and mixed evenly. The cells were then left to stand at room temperature for 15 to 30 minutes. 4) Antibody labeling: After closure, surface molecule-labeled cells were dispensed into nine flow tubes at 0.09 ml per tube. For simultaneous detection of multiple batches of cells, cells from tubes 1-8 could be mixed with cells from multiple batches in equal amounts for labeling. The cell usage in each tube was 0.8-1 × 10 cells. 6 1 x 10 cells, and sample tube 1 is surface molecular labeled. The cell usage is 0.8-1 x 10 cells. 6 Sample tube 2 is for intracellular molecular labeling, and the cell count is 3-4 × 10 6 The corresponding fluorescently labeled antibody is added, mixed uniformly, and then allowed to stand at 4°C in the dark for 30 minutes (mixing once every 15 minutes during this period).

[0108] [Table 1]

[0109] 5) Washing: Add 1 ml of 1x PBS to each tube of surface molecule-labeled cells and collect them by centrifugation at 400 g for 8 minutes at 4°C. Wash twice with 1 ml of 1x PBS. Finally, add 200 μl of PBS to each tube to resuspend the labeled cells. Add 5 μl of DAPI (50 μg / ml, 40x) to the experimental group, transfer to a tube, and detect on the microscope. 6) Detection: Calibrate and adjust the flow cytometer according to the instrument manual. A blank control tube sample is used to adjust the forward and side scatter voltages. A single-target control sample is used to adjust the fluorescence compensation for each channel. After drawing a cell gate during detection, 1 x 10 cells are collected within each sample gate. 4 Collect cells.

[0110] 3. Detection of the killing activity of fusion protein-amplified NK cells

[0111] Preparation of target cell (K562) suspension 1) Cell counting: Cultured K562 cells were resuspended in 1640 medium containing 0.5% FBS (the medium used for target cells) and counted to determine a cell density of 1 × 10 6 / mL, 2) CFSE staining: Add CFSE (working concentration 5 μM) to the cell suspension, immediately mix by spraying with a 1 mL pipette, vortex thoroughly, and incubate in a 37 °C culture tank under light shielding for 15 min, remove the vortex every 5 min and mix once. 3) Termination of staining: Add 5 times the volume of pre-chilled complete medium (the medium used for target cells) at 4°C to terminate staining, and place in an ice bath for 5 minutes. Centrifuge at 140g, 4°C, for 5 minutes. 4) Cell washing: Resuspend the cells in complete medium (the medium used for target cells) pre-cooled at 4°C, and then centrifuge the cells at 140g for 5 minutes at 4°C. Repeat this process twice to wash the cells. 5) Counting: Resuspend and count the cells to obtain a cell density of 2 x 10 5 / mL, 6) Plate: 100 μl of K562 suspension was added to each well of a 96-well round-bottom plate, and the final cell number per well was 20,000 cells / well.

[0112] NK cell preparation and addition 7) Prepare the appropriate NK cell density. 8) Take out the E-Plate 16 and place it on the ultra-clean table. 9) 100 μl of NK cell suspension was added to the culture plate at different CD56+:K562 effector target ratios (1:1, 2:1, 4:1, 8:1). Three sets of controls were also set up: target cells stained with CFSE only (to detect the natural death of target cells), effector cells only (to verify that effector cells do not contain CFSE non-specific staining), and target cells + Tween-20 (as a positive control for target cell apoptosis). 10) After co-incubation, add NK cells to each well in the predetermined order. Centrifuge at 120g for 2 minutes at room temperature to ensure sufficient contact with the effective target cells. Return to the culture tank at 37°C and incubate for 4 hours. 11) After the incubation is completed, add 5 μl of PI, mix evenly, incubate for 5 minutes in the dark, and then detect with a microscope. 12) Result analysis: Kill percentage = [(Cell mortality rate (%) of experimental group target cells - Mortality rate (%) of target cells) / (100% - Natural mortality rate (%) of target cells)] x 100%)

[0113] 4, Result analysis As shown in Figures A to C of Figure 6, when NK cells derived from PBMCs were expanded using scfv-CD16A-Trimer-4-1BBL, NK cells were effectively expanded, improving the purity and expansion fold of NK cells, and the percentage of NK cells reached the highest level of about 60% during the entire culture process. As shown in Figure 6, Panel D, the subpopulation analysis of the expanded cells revealed that the PBMC cells after treatment and expansion with scfv-CD16A-Trimer-4-1BBL had a NK cell ratio of 55%, NKT ratio of approximately 20%, and total CD56-positive cells of approximately 75%. The remaining cell subpopulations had low ratios among the expanded PBMC cells. As shown in Figure 6E, the cytotoxicity of the expanded cells was evaluated, and the expanded cells were found to be able to effectively kill K562 cells.

[0114] These results demonstrate that scfv-CD16A-Trimer-4-1BBL has the ability to activate and expand NK cells in vitro, and the expanded NK cells have relatively good cytotoxicity.

[0115] In the description herein, references such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" mean that the specific features, structures, materials, or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In the description herein, the terms "exemplary" and "specific examples" do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, if not in conflict with each other, those skilled in the art may combine and combine features of different embodiments or examples described herein.

[0116] Although the embodiments of the present disclosure have been presented and described, the above embodiments are illustrative and cannot be understood as limiting the present disclosure, and it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present disclosure.

[0117] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefits from a patent application with patent application number 202111566165.1, filed with the State Intellectual Property Office of China on December 20, 2021, the entire contents of which are incorporated herein by reference.

Claims

1. A fusion protein comprising a CD16A single chain antibody and a 4-1BBL extracellular domain, wherein the CD16A single chain antibody is linked to the 4-1BBL extracellular domain; The fusion protein, wherein the 4-1BBL extracellular region comprises three 4-1BBL extracellular domains, and the three 4-1BBL extracellular domains are connected in sequence.

2. the CD16A single-chain antibody and the 4-1BBL extracellular domain are linked via a flexible joint; and The fusion protein according to claim 1, wherein the three 4-1BBL extracellular domains are linked via flexible joints.

3. the fusion protein is expressed by a non-mammalian cell expression system; the non-mammalian cell expression system comprises at least one of a prokaryotic expression system and a lower eukaryotic expression system; the non-mammalian cell expression system comprises at least one of an E. coli expression system and a yeast expression system; The fusion protein of claim 1 , wherein the yeast expression system comprises a Pichia yeast expression system.

4. The CD16A single chain antibody is V Ｈ Area and V Ｌ region, and the V Ｈ The amino acid sequence of the V region is shown in SEQ ID NO:

1. Ｌ The amino acid sequence of the region is shown in SEQ ID NO:2, The V Ｈ Area and V Ｌ The regions are connected via a first joint; the first joint is a flexible joint; the length of said first joint is 10 to 20 amino acids; and The fusion protein of claim 1, wherein the amino acid sequence of the first joint is set forth in SEQ ID NO:

3.

5. The V Ｈ Region or V Ｌ one end of the domain and the 4-1BBL extracellular domain are connected via a second joint; the second joint is a flexible joint; the length of said second joint is 10 to 20 amino acids; and The fusion protein of claim 4, wherein the amino acid sequence of the second joint is set forth in SEQ ID NO:

4.

6. The fusion protein of claim 1, wherein the amino acid sequence of the 4-1BBL extracellular domain is set forth in SEQ ID NO:

5.

7. The three 4-1BBL extracellular domains are connected to each other via a third joint and a fourth joint, respectively, and the third joint and the fourth joint are the same or different; the third joint and the fourth joint are both flexible joints; the length of the third and fourth joints is 15 to 25 amino acids; The amino acid sequence of the third joint is shown in SEQ ID NO: 6, and The fusion protein of claim 1, wherein the amino acid sequence of the fourth joint is shown in SEQ ID NO:

7.

8. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is set forth in SEQ ID NO:

8.

9. A nucleic acid encoding the fusion protein of claim 1.

10. the nucleic acid is a nucleic acid that has been codon-optimized for yeast expression systems; The nucleic acid of claim 9, wherein the nucleotide sequence of the nucleic acid is set forth in SEQ ID NO:

10.

11. An expression vector comprising the nucleic acid of claim 9.

12. A recombinant cell expressing a fusion protein carrying the nucleic acid of claim 9.

13. Use of the fusion protein according to any one of claims 1 to 8, the nucleic acid according to claim 9 or 10, the expression vector according to claim 11, or the recombinant cell according to claim 12 in the manufacture of a drug used in the treatment or prevention of tumors or inflammatory diseases.

14. the tumor comprises at least one of ovarian cancer, lung cancer, rectal cancer, breast cancer, and leukemia; the inflammatory disease comprises a bacterial or viral infection; The bacterial infection may include pneumonia, Listeria monocytogenes, or Staphylococcus aureus infections. The use according to claim 13, wherein the viral infection comprises at least one of HPV, HBV, influenza virus, and coronavirus infection.

15. Use of the fusion protein according to any one of claims 1 to 8, the nucleic acid according to claim 9 or 10, the expression vector according to claim 11, or the recombinant cell according to claim 12 in the ex vivo culture and expansion of NK cells.

16. The NK cells are derived from peripheral blood or obtained by stem cell derivation; and The use according to claim 15, wherein the NK cells comprise at least one of NK cells derived from human peripheral blood mononuclear cells, NK cells derived from human umbilical cord blood stem cells, NK cells derived from human embryonic stem cells, and NK cells derived from human iPSCs.

17. A pharmaceutical composition comprising the fusion protein of claim 1.

18. The pharmaceutical composition is used for treating or preventing tumors or inflammatory diseases, the tumor comprises at least one of ovarian cancer, lung cancer, rectal cancer, breast cancer, and leukemia; the inflammatory disease comprises a bacterial or viral infection; The bacterial infection may include pneumonia, Listeria monocytogenes, or Staphylococcus aureus infections. The pharmaceutical composition of claim 17, wherein the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.

19. A reagent kit comprising the fusion protein according to any one of claims 1 to 8.

20. The reagent kit according to claim 19, which is used to detect CD16A and / or 4-1BB.

21. A therapeutic agent for treating or preventing a tumor or an inflammatory disease, comprising the fusion protein according to any one of claims 1 to 8, the nucleic acid according to claim 9 or 10, the expression vector according to claim 11, the recombinant cell according to claim 12, or the pharmaceutical composition according to claim 17 or 18.

22. the tumor comprises at least one of ovarian cancer, lung cancer, rectal cancer, breast cancer, and leukemia; the inflammatory disease comprises a bacterial or viral infection; The bacterial infection includes at least one of pneumoniae, Listeria monocytogenes, and Staphylococcus aureus infection; and The agent according to claim 21, wherein the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.

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