CN115772226A - Nourishing cell, preparation method and application thereof - Google Patents

Abstract

The invention provides a trophoblast, a preparation method and application thereof. In particular, the invention provides a fusion protein comprising the following elements fused together: (a) IL15; (b) TNFSF9 or an active fragment thereof; and (c) IL21, and the invention also provides a trophoblast integrating the fusion protein, and the trophoblast can obviously enhance the amplification multiple of the natural killer cell and enhance the amplification capacity of the natural killer cell.

Description

Nourishing cell, preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a trophoblast, a preparation method and application thereof.

Background

Natural killer cells are innate immune cells in the mammalian immune system that primarily eliminate virus-infected cells and tumor cells in vivo. The natural killer cells can utilize inhibitory receptors KIR and NKG2A displayed on the cell surface and activation receptors NKG2D, CD16 and NKp30 to identify abnormal cells in vivo, and when target cells express less inhibitory ligands and express more activating ligands, the natural killer cells can be caused to release perforin and granzyme so as to kill the target cells. The natural killer cells participate in acquired anti-tumor reaction at the same time, and play a role in immune regulation.

Natural killer cells are mainly distributed in peripheral blood, accounting for 5% to 10% of PBMC in peripheral blood, and there are also a small number of natural killer cells in lymph nodes and bone marrow, but most of them are in a resting state. In tumor immunotherapy, many natural killer cells are usually needed to achieve a good therapeutic effect. The key to the use of natural killer cells for tumor therapy is how to efficiently expand natural killer cells in vitro.

At present, there are two main expansion strategies of natural killer cells, one is stimulation by cytokines, and the expansion is stimulated by cytokines including IL2, IL12, IL15, IL18, IL21, etc. The cell factor is expensive, the amplification cost is high, the efficiency is limited, and the amplification multiple is limited. Another is the stimulation of natural killer cell proliferation by feeder cells. The most commonly used feeder cells today are the K562 cells. In the case of natural killer cells, K562 cell stimulation accelerates the maturation of natural killer cells and increases the expression of natural killer cell activating receptors. However, the method has some disadvantages, including low double positive ratio of amplified natural killer cell CD56+ CD16+, small amplification times, difficult detection of natural cell residue, and the like.

Therefore, there is an urgent need in the art to develop a novel feeder cell that enhances the expansion capability of natural killer cells.

Disclosure of Invention

The invention aims to provide a novel trophoblast for enhancing the amplification capacity of natural killer cells.

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

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

In another preferred embodiment, the IL15 is derived from a human or non-human mammal, more preferably from a rodent (e.g., mouse, rat), primate, and human.

In another preferred embodiment, the IL15 comprises wild type IL15 and mutant IL15, or active fragments thereof.

In another preferred embodiment, the IL15 has an amino acid sequence as shown in SEQ ID NO. 1.

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

In another preferred embodiment, said TNFSF9 is derived from a human or non-human mammal, more preferably from a rodent (e.g., mouse, rat), primate, and human.

In another preferred embodiment, the TNFSF9 includes a wild type and a mutant type.

In another preferred embodiment, said TNFSF9 comprises a full-length, mature form of TNFSF9, or an active fragment thereof.

In another preferred embodiment, the TNFSF9 further comprises a derivative of TNFSF9.

In another preferred embodiment, the derivative of TNFSF9 includes a modified TNFSF9, a protein molecule having an amino acid sequence homologous to native TNFSF9 and having native TNFSF9 activity, a dimer or multimer of TNFSF9, a fusion protein containing the amino acid sequence of TNFSF9.

In another preferred embodiment, the modified TNFSF9 is a pegylated TNFSF9.

In another preferred embodiment, the expression "protein molecule having an amino acid sequence homologous to native TNFSF9 and native TNFSF9 activity" means that the amino acid sequence has at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, most preferably at least 98% homology to TNFSF9; and a protein molecule having TNFSF9 activity.

In another preferred embodiment, the amino acid sequence of TNFSF9 is shown in SEQ ID NO. 2.

In another preferred embodiment, the IL21 is derived from a human or non-human mammal, more preferably from a rodent (e.g., mouse, rat), primate, and human.

In another preferred embodiment, the IL21 comprises wild-type IL21 and mutant IL21, or active fragments thereof.

In another preferred embodiment, the IL-21 has the amino acid sequence as shown in SEQ ID NO. 3.

In another preferred embodiment, the amino acid sequence of IL21 is shown in SEQ ID NO. 3.

In another preferred embodiment, the fusion protein has a structure represented by formula I below:

X-Y-Z (I)

in the formula (I), the compound is shown in the specification,

x is IL15;

y is TNFSF9 or an active fragment thereof;

z is IL21;

"-" denotes a peptide bond or a peptide linker connecting the above elements.

In another preferred embodiment, any two of said X, Y, Z are joined head-to-head, head-to-tail, tail-to-head or tail-to-tail.

In another preferred embodiment, said "head" refers to the N-terminus of the polypeptide or fragment thereof, in particular of the wild-type polypeptide or fragment thereof.

In another preferred embodiment, said "tail" refers to the C-terminus of the polypeptide or fragment thereof, in particular of the wild-type polypeptide or fragment thereof.

In another preferred embodiment, the peptide linker is 0-20 amino acids, preferably 0-10 amino acids in length.

In another preferred embodiment, the fusion protein has a structure represented by formula II:

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

in the formula (I), the compound is shown in the specification,

each "-" is independently a linker peptide or a peptide bond;

z1 is a null or signal peptide sequence;

z1 is IL15;

TM is a transmembrane domain;

p1 is self-cleaving protein;

z2 is TNFSF9 or an active fragment thereof;

p2 is self-cleaving protein;

z3 is a null or signal peptide sequence;

z4 is IL21.

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

In another preferred embodiment, Z1, Z3 are each independently a signal peptide of a protein selected from the group consisting of: CD8.

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

In another preferred embodiment, the TM comprises a CD 28-derived transmembrane region.

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

In another preferred embodiment, the self-cleaving protein comprises T2A and P2A.

In another preferred embodiment, the fusion protein is selected from the group consisting of:

(A) A polypeptide having an amino acid sequence shown as SEQ ID NO. 4;

(B) A polypeptide having homology of 80% or more (preferably 90% or more; etc. preferably 95% or more; most preferably 97% or more, such as 98% or more, 99% or more) with the amino acid sequence shown in SEQ ID NO.4, and having NK activating and cell proliferation stimulating activities;

(C) The derivative polypeptide is formed by substituting, deleting or adding 1-5 amino acid residues of the amino acid sequence shown in SEQ ID NO.4, and retains the derivative polypeptide which can activate NK and stimulate cell proliferation activity.

In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID No. 4.

In a second aspect, the present invention provides an isolated polynucleotide encoding a fusion protein according to the first aspect of the invention.

In another preferred embodiment, said polynucleotide additionally comprises an auxiliary element selected from the group consisting of: a signal peptide, a secretory peptide, a tag sequence (e.g., 6 His), or a combination thereof.

In another preferred embodiment, the polynucleotide is selected from the group consisting of: a DNA sequence, an RNA sequence, or a combination thereof.

In a third aspect, the invention provides a vector comprising a polynucleotide according to the second aspect of the invention.

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

In another preferred embodiment, the vector comprises a plasmid, a viral vector.

In another preferred embodiment, the vector comprises a PiggyBac plasmid system.

In another preferred embodiment, the vector comprises an expression vector, a shuttle vector and an integration vector.

In a fourth aspect, the present invention provides a host cell comprising a vector according to the third aspect of the present invention, or having a polynucleotide according to the second aspect of the present invention integrated into its genome.

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).

In another preferred embodiment, the host cell is a feeder cell.

In another preferred embodiment, the feeder 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.

The fifth invention of the present invention provides a method for preparing an engineered trophoblast expressing the fusion protein of the first aspect of the present invention, wherein the method comprises the steps of: transducing the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention into a trophoblast, thereby obtaining the engineered trophoblast.

In another preferred embodiment, the introducing includes introducing simultaneously, sequentially or sequentially.

In another preferred embodiment, the feeder 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.

In another preferred embodiment, the method further comprises the step of performing a function and effectiveness test on the obtained engineered trophoblasts.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising 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 another preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

In another preferred embodiment, the host cell comprises a feeder cell.

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

In another preferred embodiment, the pharmaceutical composition further comprises other components for enhancing the proliferation capability of natural killer cells, such as IL2, IL7, IL12, decitabine.

In another preferred embodiment, the natural killer cells include T cells, NK cells, macrophages.

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

In a seventh aspect, the present invention provides a fusion protein according to the first aspect, a nucleic acid molecule according to the second aspect, a vector according to the third aspect, or a host cell according to the fourth aspect, or a pharmaceutical composition according to the sixth aspect, for use in preparing a medicament or a formulation for enhancing natural killer cell proliferation.

In another preferred embodiment, the natural killer cells include T cells, NK cells, macrophages.

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

In an eighth aspect, the present invention provides a kit for enhancing natural killer cell proliferation ability, the kit comprising a container, and 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, in the container.

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

The ninth aspect of the present invention provides a method for enhancing natural killer cell proliferation potency, comprising:

culturing a natural killer cell in the presence of the host cell according to the fourth aspect of the present invention, thereby enhancing the proliferative capacity of the natural killer cell.

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

In another preferred embodiment, the natural killer cells include T cells, NK cells, macrophages.

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

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

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

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

FIG. 1 shows a schematic diagram of the gene sequence;

FIG. 2 shows a flow chart of the surface expression of different cytokine proteins by trophoblasts;

FIG. 3 shows the signaling pathways by trophoblasts to activate different cytokine receptors;

FIG. 4 shows that trophoblasts activate natural killer cell expansion.

FIG. 5 shows the number of cells and the fold of proliferation of trophoblasts-activated natural killer cells.

FIG. 6 shows the fold-expansion of different cytokine combination protocols to activate natural killer cells.

Detailed Description

The inventor of the present invention has extensively and deeply studied, and unexpectedly found for the first time that a novel trophoblast, which expresses a fusion protein containing IL15, IL21 and TNFSF9, can significantly enhance the amplification factor of natural killer cells and enhance the amplification capacity of natural killer cells. On the basis of this, the present inventors have completed the present invention.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

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.

IL15

Human interleukin 15 (interleukin-15, IL-15) IL-15 is a pleiotropic cytokine with the function of activating T cells, B cells and NK cells and mediating the proliferation and survival of these cells.

TNFSF9

Is a transmembrane protein which 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.

IL21

Human interleukin 21 (interleukin-21, IL-21) IL-21 is a pleiotropic cytokine involved in regulating B cell proliferation, in conjunction with IL-15, promoting bone marrow precursor cell proliferation and NK cell proliferation, differentiation and cytotoxic activity.

Fusion proteins

As used herein, "fusion protein of the invention", or "polypeptide" both refer to a fusion protein according to the first aspect of the invention.

In a preferred embodiment, the fusion protein of the 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, a linking sequence may or may not be included between the elements (e.g., between element a and element b, or element c). The linker sequence is generally a sequence that does not affect both proteins.

In another preferred embodiment, the fusion protein has the structure shown as X-Y-Z (I), wherein X is IL15; y is TNFSF9 or an active fragment thereof; z is IL21.

In some embodiments, the fusion protein has the structure of Z0-Z1-TM-P1-Z2-P2-Z3-Z4-TM (II), wherein Z1 is a null or signal peptide sequence; z1 is IL15; TM is a transmembrane domain; p1 is self-cleaving protein; z2 is TNFSF9 or an active fragment thereof; p2 is self-cleaving protein; z3 is a null or signal peptide sequence; z4 is IL21.

In another preferred embodiment, the fusion protein has an amino acid sequence as shown in SEQ ID No. 4.

The term "fusion protein" as used herein also includes variants of the fusion protein (sequence shown in SEQ ID No.: 4) having the above-described activity. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or several (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of a fusion protein of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which an antigenic peptide is fused to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (a fusion protein in which a leader sequence, a secretory sequence or a tag sequence such as 6 × His is fused). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 3, preferably up to 2, more preferably up to 1 amino acid with an amino acid of similar or analogous nature as compared to the amino acid sequence of formula I or formula II. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

The invention also provides analogs of the fusion proteins of the invention. The analogs may differ from the polypeptide of SEQ ID No.4 by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylated or carboxylated, in vivo or in vitro. Modifications also include glycosylation, such as those that result from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that effects glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

Feeder cell

Trophoblast cells are a class of trophoblast cells that do not divide and proliferate, but remain metabolically active. The cell is modified by a genetic engineering technology, and then is irradiated by rays, so that various cell factors are stably expressed on the surface of a cell membrane, and directional activation and proliferation of other cells can be directionally stimulated under the synergistic action of the various cell factors.

Expression vectors and host cells

The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells transformed with the vectors of the invention or the coding sequences of the fusion proteins of the invention, and methods for producing the polypeptides of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant fusion proteins by conventional recombinant DNA techniques. Generally, the following steps are provided:

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

(2) A host cell cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present invention, the polynucleotide sequence encoding the fusion protein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a fusion protein of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters which can control the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits 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 E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell (e.g., E.coli), or 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 are: 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 (e.g., pichia pastoris, kluyveromyces lactis, or a combination thereof; preferably, the yeast cell comprises Kluyveromyces lactis, more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis) is selected as the host cell.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer, which is 100 to 270 bp on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers, among others.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl ₂ Methods, the steps used are well known in the art. Another method is to use MgCl ₂ . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the physical, chemical and other properties of the recombinant protein can be utilized for isolation and purification of the recombinant protein by various separation methods. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.

Peptide linker

The invention provides a fusion protein, which optionally contains a peptide linker. The size and complexity of the peptide linker may affect the activity of the protein. In general, the peptide linker should be of sufficient length and flexibility to ensure that the two proteins being linked have sufficient degrees of freedom in space to function. Meanwhile, the influence of alpha helix or beta sheet formation in the peptide joint on the stability of the fusion protein is avoided.

The length of the linker peptide is generally 0 to 20 amino acids, preferably 0 to 10 amino acids.

Preparation

The invention provides a fusion protein according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of said cells in said preparation is 1X 10 ³ -1×10 ⁸ One cell/Kg body weight, more preferably 1X 10 ⁴ -1×10 ⁷ One cell/Kg body weight.

In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the invention are preferably formulated for in vitro cell culture (e.g., culture expansion of natural killer cells such as NK cells).

The main advantages of the invention include:

(1) The invention discovers a new trophoblast for the first time, which expresses a fusion protein containing IL15, IL21 and TNFSF9, and the trophoblast can obviously enhance the amplification multiple of natural killer cells and enhance the amplification capacity of the natural killer cells.

(2) The invention designs a novel trophoblast for the first time, and in order to enhance the amplification capacity of K562 to natural killer cells, the invention modifies natural soluble IL15 and IL21 cytokine sequences, so that the sequences can be expressed on the surface of a cell membrane, and simultaneously, TNFSF9 is expressed, thus the amplification capacity of K562 is enhanced together. The gene sequence of the expressed protein is optimized, so that no homologous human gene interference exists, and the residue of the trophoblasts after culture can be monitored by high-sensitivity quantitative PCR detection.

(3) The invention designs a new trophoblast for the first time, and the amplification multiple of the natural killer cell is higher by simultaneously expressing IL15, IL21 and CD37L displayed on the surface of the membrane.

(4) According to the invention, by optimizing the sequence of the overexpressed gene, high sensitivity, high specificity and rapid detection of residual trophoblast cells can be realized by utilizing qPCR.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, reagents and materials used in examples are commercially available products.

In the invention: PCR amplification kit purchased from Novowed; nucleic acid fragment synthesis was performed by Jinwei Zhi, suzhou.

General procedure

The invention firstly constructs an expression vector of fusion protein through molecular cloning, extracts plasmids after successful construction and sends the plasmids to DNA sequencing, after the sequencing is correct, the plasmids are transferred into target cells by using an electric transfer system, stably transfected cells are screened and amplified, gamma-ray irradiation is used after the amplification, the irradiated cells are trophoblast cells, and the trophoblast cells are finally stored in liquid nitrogen for later use. When in use, the cells are recovered and co-cultured with peripheral blood mononuclear cells, and the expansion of NK cells in the cells is stimulated.

Example 1 design of PicgyBac non-viral vector-based mIL15-TNFSF9-mIL21

According to the characteristics of the PiggyBac vector, a mIL15-TNFSF9-mIL21 gene fragment sequence is searched, and an intermediate fragment consists of a CD 8-derived connecting and transmembrane fragment.

The fragment design synthesis method comprises the following steps: through DNA sequence optimization, high homologous sequences do not exist in the human gene, and subsequent gene detection is facilitated. The synthetic mIL15-TNFSF9-mIL21 gene fragment (King of King Kogyo Co.) was designed. The fragment mainly comprises an upper membrane signal peptide (CD 8), an active structural domain of IL15, a CD28 transmembrane structural domain, a T2A sequence, a TNFSF9 sequence, a P2A sequence, an upper membrane signal peptide (CD 8), an IL21 active structural domain and a CD28 transmembrane structural domain, and the sequence is connected to a Piggybab vector after synthesis. The specific sequence design is shown in FIG. 1.

The amino acid sequence of the IL15 is shown in SEQ ID NO. 1: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLSGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEELEKNIKEFLIKEFVHIVQMFINTS

The amino acid sequence of TNFSF9 is shown in SEQ ID NO. 2: MEYASDASLDPEAPWPAPPRARACRAPRVLPWALGLLLLLLLLLLAACVFLACPWAVSGARASPGSAASPRLREGPLEGPRPLDDPLLDLRQLVAQLNGDLGPLSWYSGLGAGVSLTGGLSYKEDTKELVVAKAGVGVFQLELRRVVAGSGAGVSLAQPLQPSAAGAALALTLPPASSARGSAFQGRVLGRVLGRVLTHVLTHVLTHVLTHALGALVHARLQLTLGLFPMAPSPRSE

The amino acid sequence of IL21 is shown as the amino acid sequence of SEQ ID NO. 3:

HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

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

MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGYPYDVPDYAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEARPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFI IFWVRSKRSRPEGRGSLLTCGDVEENPGPMEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRP

example 2 construction of K562 cells expressing mIL15-TNFSF9-mIL21

Connecting the synthesized target gene to a pMD 18-T simple vector, connecting the target gene to a PiggyBac vector by adopting a method of enzyme digestion, connection and transformation, detecting whether the sequence of the synthesized target gene is correct by using a vector specific primer to carry out PCR amplification and sequencing, and then transferring a connection product into escherichia coli competent TOP10 to ensure that the PiggyBac vector containing a target fragment is amplified in escherichia coli in a large quantity. And (3) sequencing the constructed vector after the construction is successful, and extracting the plasmid by adopting an endotoxin-free plasmid large-sampling kit after the sequencing is correct.

Transfection of the co-transfected plasmids Piggyback-mIL 15-TNFSF9-mIL21 and Piggyback Transposase into K562 cells (cell number 1E 7) using the Nucleofector cell transfection system, the medium was RPMI 1640 medium containing 10% FBS, after 48 hours, cells were collected, HA (Cat.901523, biolegend), TNFSF9 (Cat.130-125-112, miltenyi) and IL21 (Cat.12-7213-82, thermo) fluorescent antibody markers, cells positive for expression of IL15, TNFSF9 and mIL21 were sorted using a flow cytometer, and then repeated three times to obtain stable cell lines for amplification culture.

Irradiation was carried out for 10 minutes with 100Gy of gamma ray, and the branches were frozen in liquid nitrogen after irradiation.

And (4) conclusion: the designed vector can stably express three proteins on the upper surface of a target cell at the same time, wherein IL15 and IL21 are naturally secreted proteins and cannot be expressed on the surface of a cell membrane, and the three proteins can be stably expressed on the surface of the cell membrane after being modified. The results are shown in FIG. 2

Example 3 functional assay of trophoblasts

1ug of receptors for IL-15, IL-21 and TNFSF9 were transiently transfected into 1E6 293T cells in 24-well plates, respectively, and downstream reporter plasmids including pGL4.47 (JAK-STAT 3 pathway, promega), pGL4.52 (JAK-STAT 5 pathway, promega), pGL4.32 (NF-. Kappa.B pathway, promega) were transfected, respectively. Then, the cells K562, K562-mIL15-TNFSF9-mIL21, K562-TNFSF9-mIL21 and K562-TNFSF9 were added, respectively, to detect the expression level of Luciferase in the cells, and the results are shown in FIG. 3.

In conclusion, K562-mIL15-TNFSF9-mIL21 can efficiently activate receptors for IL15, TNFSF9 and IL21.

Example 4 Natural killer cell expansion

Obtaining peripheral blood mononuclear cells, and adding 15 ml of lymphocyte separation solution into a 50 ml centrifuge tube; slowly adding 30 ml of fresh blood into the centrifugal tube; centrifuging at 2000 rpm for 20 min; transferring the leucocyte into a new centrifuge tube, supplementing 50 ml of physiological saline at 1800 rpm, quickly rising and quickly falling, and centrifuging for 8 minutes; the supernatant was discarded, and the cells were suspended with 1ml of physiological saline, supplemented with physiological saline to 50 ml, and centrifuged at 1200 rpm for 8 minutes in a fast-rising and fast-falling manner to obtain buffy coat cells.

Adjusting the cell density to 1E6/ml by using X-VIVO serum-free culture medium containing 200IU/ml IL2, adding K562-mIL15-TNFSF9-mIL21 feeder cells irradiated by 100Gy dose gamma rays, and co-culturing in a T75 or T150 culture bottle, wherein the cell number ratio of K562 to PBMC cells is 2:1; when the cell density is higher, supplementing the X-VIVO serum-free culture medium containing IL2, wherein the volume of the X-VIVO serum-free culture medium is one time of that of the original culture medium; on the 7 th day of CO-culture, the number of natural killer cells was counted, and then feeder cells irradiated with 100Gy gamma radiation were added for the 2 nd round of CO-culture amplification (37 ℃,5% (v/v) CO 2), the ratio of the number of K562 cells to the number of natural killer cells was 2:1, cells were transferred to a T175 flask and cultured (37 ℃ C., 5% (v/v) CO 2), during which time X-VIVO medium containing IL2 (added in an amount of one time the volume of the original medium) was added depending on the growth of the cells, and the culture was continued (37 ℃ C., 5% (v/v) CO 2) for 7 days. The whole process is cultured for 15 days.

And (4) conclusion: as shown in FIG. 4 and FIG. 5, K562-mIL15-TNFSF9-mIL21 can effectively activate the proliferation of natural killer cells, and the purity of the cultured natural killer cells reaches more than 85%, which is better than the proliferation of cells added with TNFSF9, TNFSF9 and IL21.

As shown in FIG. 6, the three components were split into different combinations, set as protocol 1, expressing the fusion protein mIL15-TNFSF9-mIL21; experimental scheme 2, mIL15 and TNFSF9-mIL21 were expressed separately; experimental protocol 3, TNFSF9 and mIL15-mIL21; experimental scheme 4, mIL21 and mIL15-TNFSF9 are expressed separately; then the proliferation of natural killer cells is activated, and the result shows that the accumulation effect of two separate expressions in the experimental schemes 2,3 and 4 is not as good as the effect of simultaneously expressing three in the experimental scheme 1.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Sequence listing

<110> Shanghai Huayue Biotechnology Co., ltd

<120> a trophoblast, preparation method and application thereof

<130> P2021-2182

<160> 4

<170> PatentIn version 3.5

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Thr Ser

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Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe

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Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val

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Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp

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Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu

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Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe

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Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser

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Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala

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Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala

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Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala

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Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His

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Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val

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Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

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Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn

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Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys

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Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu

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Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe

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Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu

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Ser Ser Arg Thr His Gly Ser Glu Asp Ser

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His Ala Ala Arg Pro Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys

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Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr

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Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe

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Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile

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Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

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Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

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Arg Pro Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu

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Asn Pro Gly Pro Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu

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Ala Pro Trp Pro Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro

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Trp Ala Leu Val Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala

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Cys Ala Val Phe Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala

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Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu

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Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe

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Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser

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Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu

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Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val

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Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu

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Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser

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Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala

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Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu

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His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala

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Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly

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Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg

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Ser Glu Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly

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Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu

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Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro His Lys Ser

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Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile

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Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu

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Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala

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Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro

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Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro

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Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg

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Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His

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Claims (10)

1. A fusion protein comprising the following elements fused together: (a) IL15; (b) TNFSF9 or an active fragment thereof; and (c) IL21.

2. The fusion protein of claim 1, having the structure of formula I:

X-Y-Z (I)

in the formula (I), the compound is shown in the specification,

x is IL15;

y is TNFSF9 or an active fragment thereof;

z is IL21;

"-" denotes a peptide bond or a peptide linker connecting the above elements.

3. An isolated polynucleotide encoding the fusion protein of claim 1.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell comprising the vector of claim 4, or having the polynucleotide of claim 3 integrated into its genome.

6. A method of preparing an engineered trophoblast cell expressing the fusion protein of claim 1, wherein the method comprises the steps of: transducing the nucleic acid molecule of claim 3 or the vector of claim 4 into a trophoblast, thereby obtaining the engineered trophoblast.

7. A pharmaceutical composition comprising the fusion protein of claim 1, the nucleic acid molecule of claim 3, the vector of claim 4, or the host cell of claim 5, and a pharmaceutically acceptable carrier, diluent, or excipient.

8. Use of the fusion protein of claim 1, the nucleic acid molecule of claim 3, the vector of claim 4, or the host cell of claim 5, or the pharmaceutical composition of claim 7, for the preparation of a medicament or formulation for enhancing natural killer cell proliferation.

9. A kit for enhancing natural killer cell proliferation capability, comprising a container, and the fusion protein of claim 1, the nucleic acid molecule of claim 3, the vector of claim 4, or the host cell of claim 5 disposed within the container.

10. A method of enhancing natural killer cell proliferation potency comprising:

culturing a natural killer cell in the presence of the host cell of claim 5, thereby enhancing natural killer cell proliferation potency.

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