A trophoblast cell, preparation method and use thereof

Abstract

The present invention provides trophoblast cells, preparation methods, and uses thereof. Specifically, the present invention provides a fusion protein comprising the following elements fused together: (a) IL15; (b) TNFSF9 or an active fragment thereof; and (c) IL21. The present invention also provides trophoblast cells incorporating the fusion protein. The trophoblast cells of the present invention can significantly enhance the expansion multiple of natural killer cells and the expansion capacity of natural killer cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a trophoblast cell, a preparation method and use thereof. Background Art

[0002] Natural killer cells (NK) are innate immune cells in the mammalian immune system, primarily responsible for clearing virus-infected and tumor cells. NK cells recognize abnormal cells in the body using inhibitory receptors KIR and NKG2A, as well as activating receptors NKG2D, CD16, and NKp30, displayed on their cell surfaces. When target cells express low levels of inhibitory ligands and high levels of activating ligands, NK cells release perforins and granzymes, killing the target cells. NK cells also participate in acquired anti-tumor responses, playing an immunomodulatory role.

[0003] Natural killer cells are primarily distributed in peripheral blood, comprising 5% to 10% of peripheral blood mononuclear cells (PBMCs). Smaller numbers of natural killer cells are also present in lymph nodes and bone marrow, but most are in a quiescent state. In tumor immunotherapy, a high number of natural killer cells is often required for effective therapeutic effects. The key to using natural killer cells for tumor treatment lies in efficiently expanding them in vitro.

[0004] Currently, there are two main strategies for expanding natural killer cells. One is through cytokine stimulation, using cytokines such as IL2, IL12, IL15, IL18, and IL21. Cytokines are expensive, leading to high expansion costs, and their efficiency and expansion times are limited. The other approach involves stimulating natural killer cell proliferation using trophoblasts. Currently, the most commonly used trophoblasts are K562 cells. K562 cell stimulation can accelerate natural killer cell maturation and increase the expression of activating natural killer cell receptors. However, this method also has drawbacks, including a low proportion of expanded natural killer cells that are CD56+CD16+ double-positive, a low expansion time, and difficulty detecting residual natural killer cells.

[0005] Therefore, there is an urgent need in the art to develop a new trophoblast cell that enhances the expansion capacity of natural killer cells. Summary of the Invention

[0006] The object of the present invention is to provide a novel trophoblast cell for enhancing the expansion ability of natural killer cells.

[0007] In a first aspect of the present invention, a fusion protein is provided, comprising the following elements fused together: (a) IL15; (b) TNFSF9 or an active fragment thereof; and (c) IL21.

[0008] In another preferred embodiment, the fusion protein retains the biological activities of the above elements (a), (b) and (c).

[0009] In another preferred embodiment, the IL15 is derived from humans or non-human mammals, more preferably from rodents (such as mice, rats), primates and humans.

[0010] In another preferred embodiment, the IL15 includes wild-type IL15 and mutant IL15, or active fragments thereof.

[0011] In another preferred embodiment, the IL15 has the amino acid sequence of SEQ ID NO.1.

[0012] In another preferred embodiment, the amino acid sequence of IL15 is shown as SEQ ID NO.1.

[0013] In another preferred embodiment, the TNFSF9 is derived from humans or non-human mammals, more preferably from rodents (such as mice, rats), primates and humans.

[0014] In another preferred embodiment, the TNFSF9 includes wild type and mutant type.

[0015] In another preferred embodiment, the TNFSF9 includes a full-length, mature form of TNFSF9, or an active fragment thereof.

[0016] In another preferred embodiment, the TNFSF9 also includes a derivative of TNFSF9.

[0017] In another preferred embodiment, the TNFSF9 derivatives include modified TNFSF9, protein molecules whose amino acid sequences are homologous to natural TNFSF9 and have the activity of natural TNFSF9, dimers or polymers of TNFSF9, and fusion proteins containing the amino acid sequence of TNFSF9.

[0018] In another preferred embodiment, the modified TNFSF9 is PEGylated TNFSF9.

[0019] In another preferred embodiment, the "protein molecule whose amino acid sequence is homologous to natural TNFSF9 and has natural TNFSF9 activity" refers to a protein molecule whose amino acid sequence has ≥85% homology with TNFSF9, preferably ≥90% homology, more preferably ≥95% homology, and optimally ≥98% homology; and has TNFSF9 activity.

[0020] In another preferred embodiment, the amino acid sequence of TNFSF9 is shown as SEQ ID NO.2.

[0021] In another preferred embodiment, the IL21 is derived from humans or non-human mammals, more preferably from rodents (such as mice, rats), primates and humans.

[0022] In another preferred embodiment, the IL21 includes wild-type IL21 and mutant IL21, or active fragments thereof.

[0023] In another preferred embodiment, the IL21 has the amino acid sequence of SEQ ID NO.3.

[0024] In another preferred embodiment, the amino acid sequence of IL21 is shown as SEQ ID NO.3.

[0025] In another preferred embodiment, the fusion protein has a structure shown in the following formula I:

[0026] XYZ (I)

[0027] Where,

[0028] X is IL15;

[0029] Y is TNFSF9 or an active fragment thereof;

[0030] Z is IL21;

[0031] "-" represents a peptide bond or a peptide linker connecting the above elements.

[0032] In another preferred embodiment, any two of X, Y, and Z are connected in a head-to-head, head-to-tail, tail-to-head, or tail-to-tail manner.

[0033] In another preferred embodiment, the "head" refers to the N-terminus of a polypeptide or a fragment thereof, especially the N-terminus of a wild-type polypeptide or a fragment thereof.

[0034] In another preferred embodiment, the "tail" refers to the C-terminus of a polypeptide or a fragment thereof, especially the C-terminus of a wild-type polypeptide or a fragment thereof.

[0035] In another preferred embodiment, the length of the peptide linker is 0-20 amino acids, preferably 0-10 amino acids.

[0036] In another preferred embodiment, the fusion protein has a structure shown in Formula II:

[0037] Z0-Z1-TM-P1-Z2-P2-Z3-Z4-TM (II)

[0038] Where,

[0039] Each "-" is independently a connecting peptide or a peptide bond;

[0040] Z1 is none or a signal peptide sequence;

[0041] Z1 is IL15;

[0042] TM is the transmembrane domain;

[0043] P1 is a self-cleaving protein;

[0044] Z2 is TNFSF9 or an active fragment thereof;

[0045] P2 is a self-cleaving protein;

[0046] Z3 is none or a signal peptide sequence;

[0047] Z4 is IL21.

[0048] In another preferred embodiment, Z1 and Z3 are each independently a signal peptide of a protein selected from the following group: CD8, CD28, GMCSF, GMCSFR, IL2, IL15, and IL21.

[0049] In another preferred embodiment, Z1 and Z3 are each independently a signal peptide of a protein selected from the following group: CD8.

[0050] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD8, 41BB, and CD3.

[0051] In another preferred embodiment, the TM includes a transmembrane region derived from CD28.

[0052] In another preferred embodiment, the self-cleaving protein is selected from the group consisting of T2A, P2A, E2A, F2A, or a combination thereof.

[0053] In another preferred embodiment, the self-cleaving proteins include T2A and P2A.

[0054] In another preferred embodiment, the fusion protein is selected from the following group:

[0055] (A) a polypeptide having the amino acid sequence shown in SEQ ID NO: 4;

[0056] (B) a polypeptide having ≥80% homology (preferably ≥90% homology; more preferably ≥95% homology; most preferably ≥97% homology, such as 98% or more, 99% or more) to the amino acid sequence of SEQ ID NO: 4, and having the activity of activating NK cells and stimulating cell proliferation;

[0057] (C) A derivative polypeptide formed by substituting, deleting or adding 1-5 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4, and retaining the activity of activating NK cells and stimulating cell proliferation.

[0058] In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO.: 4.

[0059] The second aspect of the present invention provides an isolated polynucleotide encoding the fusion protein of the first aspect of the present invention.

[0060] In another preferred embodiment, the polynucleotide further contains auxiliary elements flanking the ORF of the fusion protein selected from the following group: a signal peptide, a secretory peptide, a tag sequence (such as 6His), or a combination thereof.

[0061] In another preferred embodiment, the polynucleotide is selected from the following group: a DNA sequence, an RNA sequence, or a combination thereof.

[0062] The third aspect of the present invention provides a vector comprising the polynucleotide according to the second aspect of the present invention.

[0063] In another preferred embodiment, the vector comprises one or more promoters, which are operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, replication origin, selectable marker, nucleic acid restriction site, and / or homologous recombination site.

[0064] In another preferred embodiment, the vector includes a plasmid or a viral vector.

[0065] In another preferred embodiment, the vector comprises the PiggyBac plasmid system.

[0066] In another preferred embodiment, the vector includes an expression vector, a shuttle vector, and an integration vector.

[0067] The fourth aspect of the present invention provides a host cell, which contains the vector described in the third aspect of the present invention, or the polynucleotide described in the second aspect of the present invention is integrated into its genome.

[0068] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).

[0069] In another preferred embodiment, the host cell is a trophoblast cell.

[0070] In another preferred embodiment, the trophoblast cells are selected from the group consisting of K562 cells, peripheral blood mononuclear cells, Daudi cells, THP1 cells, RPMI8226 cells, Raji cells, Jurkat cells, MOLT-4 cells, SupB15 cells, HEK293 cells, Hela cells, or a combination thereof.

[0071] The fifth invention of the present invention provides a method for preparing engineered trophoblast cells, wherein the engineered trophoblast cells express the fusion protein described in the first aspect of the present invention, wherein the method comprises the steps of: transducing the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention into the trophoblast cells, thereby obtaining the engineered trophoblast cells.

[0072] In another preferred embodiment, the introduction includes simultaneous, sequential, or order introduction.

[0073] In another preferred embodiment, the trophoblast cells are selected from the group consisting of K562 cells, peripheral blood mononuclear cells, Daudi cells, THP1 cells, RPMI8226 cells, Raji cells, Jurkat cells, MOLT-4 cells, SupB15 cells, HEK293 cells, Hela cells, or a combination thereof.

[0074] In another preferred embodiment, the method further comprises the step of performing function and effectiveness testing on the obtained engineered trophoblast cells.

[0075] The sixth aspect of the present invention provides a pharmaceutical composition, which contains the fusion protein described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0076] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0077] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0078] In another preferred embodiment, the host cells include trophoblast cells.

[0079] In another preferred embodiment, the concentration of the cells in the pharmaceutical composition is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0080] In another preferred embodiment, the pharmaceutical composition further contains other components that enhance the proliferation ability of natural killer cells, such as IL2, IL7, IL12, and decitabine.

[0081] In another preferred embodiment, the natural killer cells include T cells, NK cells, and macrophages.

[0082] In another preferred embodiment, the natural killer cells are NK cells.

[0083] The seventh aspect of the present invention provides a use of the fusion protein described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention for preparing a drug or preparation for enhancing the proliferation ability of natural killer cells.

[0084] In another preferred embodiment, the natural killer cells include T cells, NK cells, and macrophages.

[0085] In another preferred embodiment, the natural killer cells are NK cells.

[0086] In an eighth aspect, the present invention provides a kit for enhancing the proliferation ability of natural killer cells, the kit comprising a container, and the fusion protein of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, or the host cell of the fourth aspect of the present invention located in the container.

[0087] In another preferred embodiment, the kit further comprises a label or instructions for use.

[0088] A ninth aspect of the present invention provides a method for enhancing the proliferation ability of natural killer cells, comprising:

[0089] Natural killer cells are cultured in the presence of the host cells according to the fourth aspect of the present invention, thereby enhancing the proliferation capacity of the natural killer cells.

[0090] In another preferred embodiment, the method is an in vitro method.

[0091] In another preferred embodiment, the natural killer cells include T cells, NK cells, and macrophages.

[0092] In another preferred embodiment, the natural killer cells are NK cells.

[0093] In another preferred embodiment, the cells are cells cultured in vitro.

[0094] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0095] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 A schematic diagram of the gene sequence is shown;

[0097] Figure 2Flow cytometry plots of different cytokine proteins expressed on the surface of trophoblasts are shown;

[0098] Figure 3 The signaling pathways by which trophoblasts activate different cytokine receptors are shown;

[0099] Figure 4 Trophoblasts were shown to activate natural killer cell expansion.

[0100] Figure 5 The cell number and proliferation fold of NK cells activated by trophoblasts are shown.

[0101] Figure 6 The fold-expansion of natural killer cells activated by different cytokine combinations is shown. DETAILED DESCRIPTION

[0102] After extensive and in-depth research, the inventors unexpectedly discovered a novel trophoblast cell line that expresses a fusion protein containing IL15, IL21, and TNFSF9. These trophoblast cells can significantly enhance the expansion multiple and the expansion capacity of natural killer cells. Based on this, the inventors completed the present invention.

[0103] the term

[0104] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0105] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0106] IL15

[0107] Human interleukin-15 (IL-15) IL-15 is a pleiotropic cytokine that activates T cells, B cells, and NK cells and mediates the proliferation and survival of these cells.

[0108] TNFSF9

[0109] It is a transmembrane protein that can activate the co-stimulatory factor CD137 and activate a variety of immune cells, such as DC cells, monocytes, B cells, mast cells, NK cells and neutrophils.

[0110] IL21

[0111] Human interleukin-21 (IL-21) IL-21 is a pleiotropic cytokine that participates in regulating B cell proliferation and synergizes with IL-15 to promote the proliferation of bone marrow precursor cells and the proliferation, differentiation and cytotoxic activity of NK cells.

[0112] Fusion protein

[0113] As used herein, "the fusion protein of the present invention" or "polypeptide" refers to the fusion protein described in the first aspect of the present invention.

[0114] In a preferred embodiment, the fusion protein of the present invention comprises the following elements: (a) IL15; (b) TNFSF9 or an active fragment thereof; and (c) IL21. In the fusion protein of the present invention, linker sequences may or may not be present between the elements (e.g., between element a and element b, or between element b and element c). The linker sequence typically does not affect the interaction between the two proteins.

[0115] In another preferred embodiment, the structure of the fusion protein is shown as XYZ (I), wherein X is IL15; Y is TNFSF9 or an active fragment thereof; and Z is IL21.

[0116] In some embodiments, the structure of the fusion protein is as shown in Z0-Z1-TM-P1-Z2-P2-Z3-Z4-TM(II), wherein Z1 is none or a signal peptide sequence; Z1 is IL15; TM is a transmembrane domain; P1 is a self-cleaving protein; Z2 is TNFSF9 or an active fragment thereof; P2 is a self-cleaving protein; Z3 is none or a signal peptide sequence; and Z4 is IL21.

[0117] In another preferred embodiment, the fusion protein has an amino acid sequence as shown in SEQ ID NO.: 4.

[0118] As used herein, the term "fusion protein" also includes variant forms of the fusion protein (such as the sequence shown in SEQ ID NO.: 4) having the above-mentioned activity. These variant forms include (but are not limited to): deletion, insertion and / or substitution of 1-3 (usually 1-2, more preferably 1) amino acids, and addition or deletion of one or several (usually within 3, preferably within 2, more preferably within 1) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not change the function of the protein. For another example, addition or deletion of one or several amino acids at the C-terminus and / or N-terminus generally does not change the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the present invention. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0119] The present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion proteins. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the fusion protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of an antigenic peptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusion with a leader sequence, secretory sequence or tag sequence such as 6×His). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0120] A preferred class of active derivatives are polypeptides in which no more than three, preferably no more than two, and more preferably no more than one amino acid sequence is replaced with an amino acid sequence having similar or similar properties, compared to the amino acid sequence of Formula I or Formula II. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0121] Table A

[0122]

[0123]

[0124] The present invention also provides analogs of the fusion proteins of the present invention. These analogs may differ from the polypeptide set forth in SEQ ID NO.: 4 in terms of amino acid sequence, modifications that do not affect the sequence, or a combination of these. Analogs also include those with residues other than naturally occurring L-amino acids (e.g., D-amino acids), as well as those with non-naturally occurring or synthetic amino acids (e.g., β- and γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0125] Modifications (generally without altering the primary structure) include chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from polypeptide synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the polypeptide to glycosylation enzymes (e.g., mammalian glycosylases or deglycosylases). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or optimize their solubility.

[0126] Trophoblasts

[0127] Trophoblasts are a type of cell that does not divide or proliferate but remains metabolically active. These cells are genetically engineered and then irradiated with radiation, resulting in the stable expression of multiple cytokines on their cell membranes. These cytokines, acting in concert, can stimulate the targeted activation and proliferation of other cells.

[0128] Expression vectors and host cells

[0129] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the coding sequence of the fusion protein of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0130] The polynucleotide sequences of the present invention can be used to express or produce recombinant fusion proteins using conventional recombinant DNA techniques. Generally, the following steps are involved:

[0131] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the fusion protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0132] (2) Host cells cultured in a suitable culture medium;

[0133] (3) Isolate and purify proteins from culture medium or cells.

[0134] In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it generally contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0135] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operatively linked to an appropriate promoter within the expression vector to direct mRNA synthesis. Representative examples of such promoters include the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoter, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0136] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0137] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0138] The host cell can be a prokaryotic cell (such as Escherichia coli), a lower eukaryotic cell, or a higher eukaryotic cell, such as a yeast cell, a plant cell, or a mammalian cell (including human and non-human mammals). Representative examples include Escherichia coli, wheat germ cells, insect cells, SF9, HeLa, HEK293, CHO, yeast cells, etc. In a preferred embodiment of the present invention, a yeast cell (such as Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell includes Kluyveromyces, more preferably Kluyveromyces marxianus, and / or Kluyveromyces lactis) is selected as the host cell.

[0139] When the polynucleotides of the present invention are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting DNA factors, typically about 10 to 300 base pairs in length, that act on promoters to increase gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs on the late replication origin side), the polyoma enhancer on the late replication origin side, and adenovirus enhancers.

[0140] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0141] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0142] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0143] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0144] Peptide linker

[0145] The present invention provides a fusion protein that may optionally contain a peptide linker. The size and complexity of the peptide linker may affect the activity of the protein. Generally, the peptide linker should be of sufficient length and flexibility to ensure that the two linked proteins have sufficient spatial freedom to perform their functions. At the same time, the peptide linker should avoid the formation of α-helices or β-sheets that may affect the stability of the fusion protein.

[0146] The length of the connecting peptide is generally 0-20 amino acids, preferably 0-10 amino acids.

[0147] preparation

[0148] The present invention provides a fusion protein according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, or a host cell according to the fourth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the cells in the preparation is 1×103 -1×10 8 cells / kg body weight, preferably 1×10 4 -1×10 7 cells / Kg body weight.

[0149] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for in vitro cell culture (such as culture expansion of natural killer cells such as NK cells).

[0150] The main advantages of the present invention include:

[0151] (1) The present invention discovered for the first time a new trophoblast cell that expresses a fusion protein containing IL15, IL21 and TNFSF9. The trophoblast cell of the present invention can significantly enhance the expansion multiple of natural killer cells and enhance the expansion ability of natural killer cells.

[0152] (2) This invention is the first to design a new type of trophoblast cell. To enhance the ability of K562 to expand natural killer cells, the invention modifies the natural soluble IL15 and IL21 cytokine sequences so that these sequences can be expressed on the cell membrane surface, and simultaneously express TNFSF9, thereby enhancing the expansion ability of K562. The gene sequence of the expressed protein is optimized to ensure that there is no interference from homologous human genes. Subsequently, quantitative PCR can be used with high sensitivity to monitor the residual trophoblast cells after culture.

[0153] (3) The present invention designs a new trophoblast cell for the first time, which can increase the expansion multiple of natural killer cells by simultaneously expressing IL15, IL21 and CD37L displayed on the membrane surface.

[0154] (4) By optimizing the sequence of overexpressed genes, the present invention can achieve high sensitivity, high specificity and rapid detection of residual trophoblast cells using qPCR.

[0155] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0156] Unless otherwise specified, the reagents and materials used in the examples are all commercially available products.

[0157] In the present invention, the PCR amplification kit was purchased from Novozymes; and the nucleic acid fragment synthesis was completed by Suzhou Genewise Co., Ltd.

[0158] General Methods

[0159] The present invention first constructs an expression vector for the fusion protein through molecular cloning. After successful construction, the plasmid is extracted and sent for DNA sequencing. Once the sequencing is correct, the plasmid is transferred into the target cells using an electroporation system. Stably transfected cells are screened for amplification and then irradiated with gamma rays. The irradiated cells are trophoblasts and finally stored in liquid nitrogen for future use. When used, they are resuscitated and co-cultured with peripheral blood mononuclear cells to stimulate the expansion of NK cells therein.

[0160] Example 1. Design of mIL15-TNFSF9-mIL21 based on PiggyBac non-viral vector

[0161] According to the characteristics of the PiggyBac vector, the sequence of the mIL15-TNFSF9-mIL21 gene fragment was searched. The middle fragment consisted of a CD8-derived linker and a transmembrane fragment.

[0162] The fragment design and synthesis method is: through DNA sequence optimization, it is ensured that there are no high homologous sequences in the human gene, which is convenient for subsequent gene detection. The mIL15-TNFSF9-mIL21 gene fragment was designed and synthesized (Golden Wisdom Company). The fragment mainly includes the upper membrane signal peptide (CD8), the active domain of IL15, the CD28 transmembrane domain, the T2A sequence, the TNFSF9 sequence, the P2A sequence, the upper membrane signal peptide (CD8), the IL21 active domain, and the CD28 transmembrane domain. After synthesis, the sequence is connected to the Piggybac vector. The specific sequence design is as follows Figure 1 shown.

[0163] The amino acid sequence of IL15 is shown in SEQ ID NO. 1: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0164] The amino acid sequence of TNFSF9 is as SEQ ID Shown in NO.2: MEYASDASLDPEAPWPPARARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGG LSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE

[0165] The amino acid sequence of IL21 is as shown in SEQ ID NO.3:

[0166] HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0167] The amino acid sequence of the mIL15-TNFSF9-mIL21 fusion protein is shown in SEQ ID NO.4:

[0168] MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGYPYDVPDYAALSNSIMYFSHFVPVFLAPKPTTTPAPRPPTPAPTIASQPLSLRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLTVAFI IFWVRSKRSRPEGRGSLTCDGDVEENPGPMEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLAAACAVFLACWPAWVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYS DPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPR SEGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPHKSSSQGQDRHMIRMQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHR LTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSALSNSIMYFSHFVPVFLAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRP

[0169] Example 2. Construction of K562 cells expressing mIL15-TNFSF9-mIL21

[0170] The synthesized target gene was ligated to the pMD 18-T simple vector. The target gene was then linked to the PiggyBac vector using enzyme digestion, ligation, and transformation. The correct sequence of the synthesized target gene was verified by PCR amplification using vector-specific primers and sequencing. The ligation product was then transformed into competent E. coli TOP10 cells to allow the PiggyBac vector containing the target fragment to be amplified in large quantities in E. coli. After successful construction, the constructed vector was sequenced and, if confirmed, the plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0171] The co-transfection plasmid Piggybac-mIL15-TNFSF9-mIL21 and Piggybac Transposase were transfected into K562 cells (cell number 1E7) using the Nucleofector cell transfection system. The culture medium was RPMI1640 medium containing 10% FBS. After 48 hours, the cells were collected and labeled with HA (Cat.901523, biolegend), TNFSF9 (Cat.130-125-112, Miltenyi) and IL21 (Cat.12-7213-82, Thermo) fluorescent antibodies. Cells positive for IL15, TNFSF9 and mIL21 expression were sorted by flow cytometry. The process was repeated three times to obtain stably transfected cell lines for expansion culture.

[0172] The cells were then irradiated with 100 Gy of gamma rays for 10 minutes, and the branches were frozen in liquid nitrogen after irradiation.

[0173] Conclusion: The designed vector can stably express three proteins on the surface of target cells. IL15 and IL21 are naturally secreted proteins and are not expressed on the cell membrane surface. After modification, they can be stably expressed on the cell membrane surface. Figure 2 shown

[0174] Example 3. Functional detection of trophoblast cells

[0175] 1ug of IL-15, IL-21, and TNFSF9 receptors were transiently transfected into 1E6 293T cells in a 24-well plate. Downstream reporter plasmids, including pGL4.47 (JAK-STAT3 pathway, Promega), pGL4.52 (JAK-STAT5 pathway, Promega), and pGL4.32 (NF-κB pathway, Promega), were also transfected. K562, K562-mIL15-TNFSF9-mIL21, K562-TNFSF9-mIL21, and K562-TNFSF9 cells were then added to detect the expression of luciferase in the cells. The results are shown in Figure 2. Figure 3 shown.

[0176] Conclusion: K562-mIL15-TNFSF9-mIL21 can effectively activate the receptors of IL15, TNFSF9 and IL21.

[0177] Example 4. Natural Killer Cell Expansion

[0178] To obtain peripheral blood mononuclear cells, add 15 ml of lymphocyte separation solution to a 50 ml centrifuge tube; slowly add 30 ml of fresh blood to the centrifuge tube; centrifuge at 2000 rpm for 20 minutes at room temperature; transfer the buffy coat cells to a new centrifuge tube, add physiological saline to 50 ml, and centrifuge at 1800 rpm for 8 minutes; discard the supernatant, suspend the cells with 1 ml of physiological saline, add physiological saline to 50 ml, and centrifuge at 1200 rpm for 8 minutes to obtain the buffy coat cells.

[0179] The cell density was adjusted to 1E6 / ml using X-VIVO serum-free medium containing 200 IU / ml IL2, and then K562-mIL15-TNFSF9-mIL21 trophoblasts irradiated with 100 Gy of γ-rays were added for co-culture in T75 or T150 culture flasks. The cell number ratio of K562 and PBMC cells was 2:1. When the cell density was high, X-VIVO serum-free medium containing IL2 was added at twice the original volume. On the 7th day of co-culture, natural killer cells were given After cell count, 100 Gy gamma-irradiated trophoblasts were added for a second round of co-culture expansion (37°C, 5% (v / v) CO2). The K562 and natural killer cell ratio was 2:1. The cells were then transferred to T175 culture flasks and cultured (37°C, 5% (v / v) CO2). During this period, X-VIVO medium containing IL-2 (at one-fold the original volume) was added depending on cell growth. Culture was continued (37°C, 5% (v / v) CO2) for 7 days. The total culture period was 15 days.

[0180] Conclusion: If Figure 4 As shown in Figure 5, K562-mIL15-TNFSF9-mIL21 can effectively activate the proliferation of natural killer cells, and the purity of natural killer cells after culture reaches more than 85%, which is better than the proliferation of cells with only TNFSF9 or TNFSF9 and IL21 added.

[0181] like Figure 6 As shown, the three components were split into different combinations, set as experimental scheme 1, expressing the fusion protein mIL15-TNFSF9-mIL21; experimental scheme 2, expressing mIL15 and TNFSF9-mIL21 respectively; experimental scheme 3, TNFSF9 and mIL15-mIL21; experimental scheme 4, expressing mIL21 and mIL15-TNFSF9 respectively; then the proliferation of natural killer cells was activated. The results showed that the cumulative effect of the separate expression of experimental schemes 2, 3, and 4 was not as good as the effect of expressing the three at the same time in experimental scheme 1.

[0182] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto. Sequence Listing <110> Shanghai Huaiyue Biotechnology Co., Ltd. <120> A trophoblast cell, preparation method and use thereof <130> P2021-2182 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 114 <212> PRT <213> artificial sequence <400> 1 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60<00004​​​​​​​​​​​​​​​​​​​​<213> artificial sequence <400> 2 Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro 1 5 10 15 Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val 20 25 30 Ala Gly Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe 35 40 45 Leo Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser 50 55 60 Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp 65 70 75 80 Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val 85 90 95 Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp 100 105 110 Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu 115 120 125 Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe 130 135 140 Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser 145 150 155 160 Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala 165 170 175 Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala 180 185 190 Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala 195 200 205 Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His 210 215 220 Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val 225 230 235 240 Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 245 250 <210> 3 <211> 138 <212> PRT <213> artificial sequence <400> 3 His Lys Ser Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg 1 5 10 15 Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu 20 25 30 Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu 35 40 45 Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn 50 55 60 Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys 65 70 75 80 Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu 85 90 95 Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe 100 105 110 Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu 115 120 125 Ser Ser Arg Thr His Gly Ser Glu Asp Ser 130 135 <210> 4 <211> 793 <212> PRT <213> artificial sequence <400> 4 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys 20 25 30 Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr 35 40 45 Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe 50 55 60 Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile 65 70 75 80 His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser 85 90 95 Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu 100 105 110 Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val 115 120 125 Gln Met Phe Ile Asn Thr Ser Gly Tyr Pro Tyr Asp Val Pro Asp Tyr 130 135 140 Ala Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val 145 150 155 160 Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 165 170 175 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 180 185 190 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Lys Pro 195 200 205 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 210 215 220 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 225 230 235 240 Arg Pro Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu 245 250 255 Asn Pro Gly Pro Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu 260 265 270 Ala Pro Trp Pro Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro 275 280 285 Trp Ala Leu Val Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala 290 295 300 Cys Ala Val Phe Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala 305 310 315 320 Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu 325 330 335 Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe 340 345 350 Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser 355 360 365 Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu 370 375 380 Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val 385 390 395 400 Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu 405 410 415 Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser 420 425 430 Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala 435 440 445 Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu 450 455 460 His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala 465 470 475 480 Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly 485 490 495 Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg 500 505 510 Ser Glu Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly 515 520 525 Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu 530 535 540 Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro His Lys Ser 545 550 555 560 Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile 565 570 575 Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu 580 585 590 Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala 595 600 605 Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn 610 615 620 Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro 625 630 635 640 Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro 645 650 655 Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg 660 665 670 Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg 675 680 685 Thr His Gly Ser Glu Asp Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe 690 695 700 Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro 705 710 715 720 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 725 730 735 Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His 740 745 750 Thr Arg Gly Leu Asp Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 755 760 765 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 770 775 780 Trp Val Arg Ser Lys Arg Ser Arg Pro 785 790

Claims

1. A fusion protein, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO:

4.

2. An isolated polynucleotide, characterized in that The polynucleotide encodes the fusion protein according to claim 1.

3. A carrier, characterized in that It contains the polynucleotide according to claim 2.

4. A host cell, characterized in that The host cell contains the vector according to claim 3, or the polynucleotide according to claim 2 is integrated into its genome.

5. A method for preparing engineered trophoblast cells, characterized in that: The engineered trophoblast expresses the fusion protein of claim 1, wherein the method comprises the steps of: transducing the polynucleotide of claim 2 or the vector of claim 3 into the trophoblast, thereby obtaining the engineered trophoblast.

6. A composition for non-therapeutic purposes, characterized in that The composition contains the fusion protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, or the host cell of claim 4.

7. A use of the fusion protein according to claim 1, the polynucleotide according to claim 2, the vector according to claim 3, the host cell according to claim 4, or the composition according to claim 6, characterized in that: Used for preparing non-therapeutic preparations for enhancing the proliferation ability of natural killer cells.

8. A kit for enhancing the proliferation ability of natural killer cells, characterized in that: The kit contains a container, and the fusion protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, or the host cell of claim 4 located in the container.

9. A method for enhancing the proliferation ability of natural killer cells for non-therapeutic purposes, characterized in that: include: Natural killer cells are cultured in the presence of the host cell according to claim 4, thereby enhancing the proliferation ability of the natural killer cells.

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