Construction method and application of membrane-bound IL-21 K562 cell strain

Abstract

The invention discloses a construction method and application of a membrane-bound IL-21 K562 cell strain, a human IL-21 mature sequence is fused with an IgG4 hinge region, a human immune globulin gamma-4 chain CH2 / CH3 region and a CD4 transmembrane structural domain through genetic engineering to construct an mbIL-21 fusion gene, and the mbIL-21 fusion gene is cloned to a PT-mnud carrier by using BamH I / Xho I double enzyme cutting sites to obtain a recombinant plasmid PCDH-mbIL-21. HEK 293T cells are transfected through a lentivirus packaging system, high-titer lentivirus particles are prepared, and the cell strain stably expressing mbIL-21 is obtained through puromycin screening after K562 cells are infected. The mbIL-21 K562 cell strain constructed by the invention has the characteristics of long acting, targeting and stable expression, and provides a powerful tool for basic research and clinical transformation of immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to a method for constructing a membrane-bound IL-21 K562 cell line and its application. Background Technology

[0002] Interleukin-21 (IL-21) is a pleiotropic cytokine primarily secreted by activated CD4+ T cells and natural killer T cells (NKT cells). It plays a central role in immune regulation by binding to the IL-21 receptor (IL-21R) and activating the JAK-STAT signaling pathway. Its functions include promoting T cell proliferation, B cell differentiation into plasma cells, enhancing the cytotoxicity of natural killer cells (NK cells), and suppressing the function of regulatory T cells (Tregs). Based on these properties, IL-21 has been extensively studied in tumor immunotherapy, antiviral immunity, and the treatment of autoimmune diseases. For example, in the tumor microenvironment, IL-21 can directly kill tumor cells by activating effector T cells and NK cells, while simultaneously inhibiting the activity of immunosuppressive cells (such as myeloid-derived suppressor cells, MDSCs), thereby remodeling the anti-tumor immune response. However, traditional soluble IL-21 has significant drawbacks in clinical applications: its half-life is extremely short (approximately 1-2 hours), requiring frequent administration to maintain effective concentrations. This not only increases treatment costs and the burden on patients but may also lead to systemic side effects such as cytokine release syndrome (CRS). Furthermore, free IL-21 is easily degraded by proteases or rapidly cleared by the kidneys, making it difficult to sustain its immunomodulatory effects in the local microenvironment.

[0003] To overcome the limitations of soluble cytokines, researchers have recently focused on developing membrane-bound cytokines. Anchoring cytokines to the cell membrane surface through genetic engineering can significantly prolong their duration of action, enhance local signal transduction efficiency, and reduce systemic toxicity. For example, the design of membrane-bound IL-2 (mbIL-2) achieves stable expression on the surface of T cells by fusing IL-2 with the Fc fragment or transmembrane domain of immunoglobulin G (IgG). This modification not only prolongs the half-life of IL-2 but also enhances the proliferation and function of anti-tumor T cells through local signal activation. Similarly, membrane-bound IL-15 (mbIL-15), by fusing the transmembrane domain of NK cell-specific receptors (such as IL-15Rα), has successfully promoted the survival and activation of NK cells and demonstrated superior efficacy compared to soluble IL-15 in the treatment of solid tumors. However, current technologies still face several challenges: First, the design of fusion proteins needs to achieve a balance between structural stability and functional activity. For example, the introduction of the IgG hinge region can enhance protein flexibility, but improper connection may lead to steric hindrance and interfere with the binding of cytokines to receptors. Secondly, the selection of transmembrane domains (such as those derived from CD28 or CD4) affects the membrane localization efficiency of proteins, and some domains may trigger non-specific signal activation or cytotoxicity. In addition, the efficiency of gene delivery systems directly affects the expression level of target proteins, and the transfection efficiency, integration site preference, and long-term expression stability of viral vectors (such as lentiviruses) still need further optimization.

[0004] The K562 cell line, as a human chronic myeloid leukemia cell model, is widely used for gene expression research, drug screening, and cell therapy vector development due to its ease of suspension culture, high proliferation rate, and good gene manipulation capabilities. For example, in chimeric antigen receptor T-cell (CAR-T) therapy, K562 cells are often engineered to express specific tumor antigens (such as CD19 or BCMA) as target cells to evaluate the killing efficacy and specificity of CAR-T cells in vitro. Furthermore, K562 cells are also used to construct engineered antigen-presenting cells (APCs) to enhance T cell activation and expansion through co-expression of co-stimulatory molecules (such as CD80 and CD86) and cytokines (such as IL-12). However, there are still technical bottlenecks in transforming K562 cells into engineered cell lines that stably express membrane-bound cytokines: (1) The design of fusion genes needs to take into account both the activity of cytokines and the anchoring efficiency of transmembrane structures. In existing methods, improper sequence ligation often leads to abnormal protein secretion or loss of function; (2) Although lentiviral vector systems can achieve efficient gene delivery, their random integration characteristics may lead to unstable gene expression levels and even the risk of insertion mutations; (3) The process of screening high-expression cell lines depends on strict antibiotic pressure (such as puromycin) and multi-level verification (such as qPCR, flow cytometry, and Western blotting), which is time-consuming and costly.

[0005] In summary, while existing technologies have made some progress in the construction and application of membrane-bound cytokines, core issues such as fusion protein design, gene delivery efficiency, and stable expression screening still need to be addressed. These challenges limit the widespread application of engineered cell lines in basic research and clinical translation, necessitating the development of more efficient methods for constructing and applying membrane-bound IL-21-containing K562 cell lines. Summary of the Invention

[0006] To address the problems of the prior art, the purpose of this invention is to provide a method for constructing an efficient membrane-bound IL-21 K562 cell line and its application.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In one aspect, this application provides a method for constructing a K562 cell line that binds to membrane-bound IL-21.

[0009] Secondly, this application provides a method for constructing a membrane-bound IL-21 K562 cell line.

[0010] Thirdly, this application provides a recombinant plasmid PCDH-mbIL-21 for constructing the K562 cell line.

[0011] Fourthly, this application provides the use of a membrane-bound IL-21 K562 cell line in the preparation of drugs that enhance immune responses.

[0012] Fifthly, this application provides a pharmaceutical composition for tumor immunotherapy.

[0013] The first aspect of this application provides a method for constructing a K562 cell line that binds IL-21 to a membrane, comprising the following steps: (1) constructing an mbIL-21 vector:

[0014] The human IL-21 sequence was sequentially linked with the IgG4 hinge region, the human immunoglobulin γ-4 chain CH2 / CH3 region, and the human CD4 transmembrane domain through gene modification to form the mbIL-21 fusion gene;

[0015] The mbIL-21 fusion gene was cloned into the PT-mnud vector by double digestion with BamHI and XhoI to obtain the recombinant plasmid PCDH-mbIL-21.

[0016] (2) Preparation of lentiviruses:

[0017] The recombinant plasmid PCDH-mbIL-21 was co-transfected with packaging plasmids PCDH-VSV-G, PCDH-Rev, and PCDH-PMDL into HEK 293T cells. The viral supernatant was collected and purified to obtain lentiviral particles carrying mbIL-21.

[0018] (3) Infecting K562 cells and screening:

[0019] K562 cells were infected with the lentivirus and screened with puromycin to obtain a stable K562 cell line expressing membrane-bound IL-21.

[0020] Further, in step (1), the construction of the mbIL-21 fusion gene includes the following sequences: the amino acid sequence encoded by the mbIL-21 fusion gene is shown in SEQ ID NO: 1, the nucleotide sequence of the mbIL-21 fusion gene is shown in SEQ ID NO: 2, the amino acid sequence encoded by mature human IL-21 is shown in SEQ ID NO: 3, the nucleotide sequence of mature human IL-21 is shown in SEQ ID NO: 4, the nucleotide sequence of the modified 12-aa IgG4 hinge region is shown in SEQ ID NO: 5, the amino acid sequence encoded by the CH2 / CH3 region of the human immunoglobulin γ-4 chain is shown in SEQ ID NO: 6, the amino acid sequence encoded by the human CD4 transmembrane domain is shown in SEQ ID NO: 7, and the nucleotide sequence of the human CD4 transmembrane domain is shown in SEQ ID NO: 8.

[0021] Further, in step (1), the primer sequences used for the double enzyme digestion reaction are as follows: the nucleotide sequence of the forward primer mbIL-21-F is shown in SEQ ID NO: 9, and the nucleotide sequence of the reverse primer mbIL-21-R is shown in SEQ ID NO: 10.

[0022] Furthermore, in step (2), the amount of packaging plasmids used is: PCDH-VSV-G 3 μg; PCDH-Rev 2 μg; PCDH-PMDL 5 μg; PCDH-mbIL-21 15 μg.

[0023] Further, in step (3), the specific steps of the puromycin screening include:

[0024] The puromycin-sensitive concentration for K562 cells was determined to be 1 μg / ml;

[0025] Infected K562 cells were cultured in a medium containing 1 μg / ml puromycin for 48 hours, and surviving cells were screened.

[0026] Further, step (3) includes a verification step: the mRNA expression level of mbIL-21 is detected by qPCR, the nucleotide sequence of the forward primer WY-pcdh-IL21qPCR1F is shown in SEQ ID NO:11, the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR1R is shown in SEQ ID NO:12, the nucleotide sequence of the forward primer WY-pcdh-IL21qPCR2F is shown in SEQ ID NO:13, and the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR2R is shown in SEQ ID NO:14, the IL-21 protein expression level is detected by Western Blot, and a specific IL-21 antibody is used.

[0027] The second aspect of this application provides a method for constructing a membrane-bound IL-21-containing K562 cell line, wherein the K562 cell line stably expresses a fusion protein comprising human IL-21, the IgG4 hinge region, the CH2 / CH3 region of the human immunoglobulin γ-4 chain, and the human CD4 transmembrane domain.

[0028] The third aspect of this application provides a recombinant plasmid PCDH-mbIL-21 for constructing the K562 cell line. The recombinant plasmid PCDH-mbIL-21 contains the mbIL-21 fusion gene and is inserted into the PT-mnud vector via double restriction sites of BamHI and XhoI.

[0029] The fourth aspect of this application provides the use of a membrane-bound IL-21 K562 cell line in the preparation of a drug that enhances the immune response. The drug exerts its immunomodulatory effect by means of the following: the mbIL-21 fusion gene expressed on the surface of the K562 cell line binds to the IL-21 receptor of neighboring immune cells, activating the JAK-STAT signaling pathway and promoting T cell proliferation, NK cell toxicity, or B cell differentiation.

[0030] The fifth aspect of this application provides a pharmaceutical composition for tumor immunotherapy, comprising a membrane-bound IL-21 K562 cell line and a pharmaceutically acceptable carrier or excipient.

[0031] Beneficial Effects: The mbIL-21 K562 cell line constructed in this invention exhibits long-lasting, targeted, and stable expression, providing a powerful tool for basic research and clinical translation in immunotherapy. Through rational design of fusion proteins, efficient gene delivery systems, and rigorous validation procedures, key issues in existing membrane-bound cytokine technologies, such as unstable expression, insufficient activity, and long screening cycles, have been successfully addressed.

[0032] Compared with the prior art, the present invention has the following advantages: (1) Long-lasting effect and enhanced local effect: By fusing IL-21 with the hinge region of IgG4, the CH2 / CH3 region of human immunoglobulin γ-4 chain and the transmembrane domain of CD4, the present invention successfully constructed a membrane-bound IL-21 (mbIL-21) fusion protein. This design enables IL-21 to be stably anchored on the cell membrane surface, significantly prolonging its action time and avoiding the problem of short half-life (only 1-2 hours) caused by rapid degradation or clearance of soluble IL-21. Experiments show that mbIL-21 is continuously expressed on the K562 cell membrane surface and can maintain a high concentration in the local microenvironment (such as tumor tissue), thereby enhancing the activation effect on adjacent immune cells (such as T cells and NK cells) and reducing the risk of side effects such as systemic cytokine release syndrome (CRS).

[0033] (2) Structural optimization and preservation of functional activity: In the fusion protein design of this invention, the IgG4 hinge region endows the protein with flexibility, ensuring that the binding space of IL-21 to the receptor is unrestricted; the CH2 / CH3 region enhances signal transduction efficiency by promoting dimerization, mimicking the binding mode of the natural receptor complex; and the CD4 transmembrane domain achieves efficient membrane localization. Western blot analysis confirmed that mbIL-21 was stably expressed in K562 cells, and its molecular weight was consistent with expectations, indicating that the fusion protein did not undergo abnormal folding or degradation, and the biological activity of IL-21 was fully preserved. Furthermore, qPCR analysis showed that the mRNA expression level of mbIL-21 was significantly higher than that of the untransfected group, further demonstrating the stability of gene integration and expression.

[0034] (3) High-efficiency gene delivery and screening system: This invention uses the PT-mnud vector based on the transposon system, combined with BamHI and XhoI double restriction sites, to achieve high-efficiency cloning and stable integration of the mbIL-21 gene. The lentiviral packaging system (VSV-G, Rev, and PMDL plasmids co-transfected into HEK 293T cells) produces high viral titers. After infecting K562 cells, positive cell lines can be efficiently enriched in just 48 hours through pressure screening with puromycin (1 μg / ml), significantly shortening the screening cycle of several weeks required by the traditional limiting dilution method. Restriction enzyme digestion identification and sequencing results confirm that the recombinant plasmid construction is accurate and without sequence bias, ensuring the reproducibility of the experiment.

[0035] (4) Multifunctional Validation and Reliability Assurance: This invention verifies the functionality of cell lines through multi-level technical means: qPCR detection: Specific primers (WY-pcdh-IL21qPCR1F / R, WY-pcdh-IL21qPCR2F / R) are used to quantify the mRNA expression level of mbIL-21, and melting curve analysis is combined to ensure primer specificity. Figure 5 Western Blot analysis: Protein expression was confirmed using an IL-21-specific antibody. The results showed that the experimental group (mbIL-21 K562) had a clear band at the expected molecular weight position, while the control group (untransfected group) showed no expression. Functional verification: Co-culture experiments confirmed that mbIL-21 K562 cells could significantly enhance the proliferation and cytotoxicity of effector T cells (not detailed in the disclosure, but a potential application direction).

[0036] (5) Broad application prospects: The mbIL-21 K562 cell line of this invention not only provides an ideal model for studying the local immune regulation mechanism of IL-21, but can also be used as engineered antigen-presenting cells (APCs) for tumor immunotherapy. For example, co-culturing it with CAR-T cells can evaluate the enhancing effect of membrane-bound IL-21 on T cell persistence and killing efficacy; or combining it with chemotherapy / radiotherapy can explore its synergistic effect of reversing immunosuppression in the tumor microenvironment. In addition, this technical route can be extended to the construction of other membrane-bound cytokines (such as IL-12 and IL-15), providing a universal platform for the development of personalized immunotherapy regimens. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 The amino acid sequence and schematic diagram of mbIL-21 of the present invention are shown below.

[0039] Figure 2 This is an agarose gel electrophoresis image of the PCDH vector of the present invention after double digestion (enzyme sites: BamHI and XhoI).

[0040] Figure 3 This image shows the enzyme digestion identification of the plasmid extracted after the mbIL-21 recombinant and transformed product of the present invention.

[0041] Figure 4 This is a schematic diagram of the sequencing results comparison of the present invention.

[0042] Figure 5 The diagram shows the mRNA level of mbIL-21 in K562 cells detected by RT-PCR experiment according to the present invention.

[0043] Figure 6 This is a diagram showing the WB detection of IL-21 protein levels in the K562 cell line mbIL-21 according to the present invention.

[0044] Figure 7 This is a schematic diagram of the co-culture process of PBMCs and irradiated mbIL-21 K562 cells according to the present invention.

[0045] Figure 8 The purity of NK (CD3-CD16+CD56+) cells in the in vitro expansion culture of NK cells according to the present invention; Figure 8 A is a line graph showing the percentage of NK (CD3-CD16+CD56+) cells in the co-culture system monitored by flow cytometry according to the present invention. Figure 8 B is a flow cytometry representation of the percentage of NK (CD3-CD16+CD56+) cells in the co-culture system monitored by flow cytometry according to the present invention. Note: On days 0, 3, 6, 10, 12, and 14, the percentage of NK cells in PBMC+ irradiated mbIL-21 K562 cells was 10.30%, 33.40%, 46.50%, 60.30%, 80.80%, and 90.40%, respectively; the percentage of NK cells in the co-culture system of PBMC+ irradiated untransfected mbIL-21 K562 cells was 10.80%, 11.90%, 19.80%, 27.30%, and 40.10%, respectively.

[0046] Figure 9This is a graph showing the change in the number of NK cells during the in vitro expansion culture of NK cells according to the present invention. Note: The graph shows the total number of NK cells in the co-culture system of PBMC+ irradiated mbIL-21 K562 cells and PBMC+ irradiated untransfected mbIL-21 K562 cells after trypan blue staining, and the total number of NK cells calculated by combining the proportion of CD56+CD16+ cells by flow cytometry at days 0, 3, 6, 10, 12, and 14. * indicates a significant difference. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0049] In this application, "-one less" means one or more, and "more than" means two or more. "-one less item (item) below" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "-one less item (item) in a, b, or c", or "-one less item (item) in a, b, and c", can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0050] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0051] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0052] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0053] The first aspect of this application provides a method for constructing a K562 cell line that binds IL-21 to a membrane, comprising the following steps: (1) constructing an mbIL-21 vector:

[0054] The human IL-21 sequence was sequentially linked with the IgG4 hinge region, the human immunoglobulin γ-4 chain CH2 / CH3 region, and the human CD4 transmembrane domain through gene modification to form the mbIL-21 fusion gene;

[0055] The mbIL-21 fusion gene was cloned into the PT-mnud vector by double digestion with BamHI and XhoI to obtain the recombinant plasmid PCDH-mbIL-21.

[0056] (2) Preparation of lentiviruses:

[0057] The recombinant plasmid PCDH-mbIL-21 was co-transfected with packaging plasmids PCDH-VSV-G, PCDH-Rev, and PCDH-PMDL into HEK 293T cells. The viral supernatant was collected and purified to obtain lentiviral particles carrying mbIL-21.

[0058] (3) Infecting K562 cells and screening:

[0059] K562 cells were infected with the lentivirus and screened with puromycin to obtain a stable K562 cell line expressing membrane-bound IL-21.

[0060] In some embodiments, in step (1), the construction of the mbIL-21 fusion gene includes the following sequences: the amino acid sequence encoded by the mbIL-21 fusion gene is shown in SEQ ID NO: 1, the nucleotide sequence of the mbIL-21 fusion gene is shown in SEQ ID NO: 2, the amino acid sequence encoded by mature human IL-21 is shown in SEQ ID NO: 3, the nucleotide sequence of mature human IL-21 is shown in SEQ ID NO: 4, the nucleotide sequence of the modified 12-aa IgG4 hinge region is shown in SEQ ID NO: 5, the amino acid sequence encoded by the CH2 / CH3 region of the human immunoglobulin γ-4 chain is shown in SEQ ID NO: 6, the amino acid sequence encoded by the human CD4 transmembrane domain is shown in SEQ ID NO: 7, and the nucleotide sequence of the human CD4 transmembrane domain is shown in SEQ ID NO: 8.

[0061] In some embodiments, in step (1), the primer sequences used in the double enzyme digestion reaction are as follows: the nucleotide sequence of the forward primer mbIL-21-F is shown in SEQ ID NO: 9, and the nucleotide sequence of the reverse primer mbIL-21-R is shown in SEQ ID NO: 10.

[0062] In some embodiments, in step (2), the amount of packaging plasmid used is: PCDH-VSV-G 3 μg; PCDH-Rev 2 μg; PCDH-PMDL 5 μg; PCDH-mbIL-21 15 μg.

[0063] In some embodiments, the specific steps of puromycin screening in step (3) include:

[0064] The puromycin-sensitive concentration for K562 cells was determined to be 1 μg / ml;

[0065] Infected K562 cells were cultured in a medium containing 1 μg / ml puromycin for 48 hours, and surviving cells were screened.

[0066] In some embodiments, step (3) is followed by a verification step: the mRNA expression level of mbIL-21 is detected by qPCR, the nucleotide sequence of the forward primer WY-pcdh-IL21qPCR1F is shown in SEQ ID NO:11, the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR1R is shown in SEQ ID NO:12, the nucleotide sequence of the forward primer WY-pcdh-IL21qPCR2F is shown in SEQ ID NO:13, and the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR2R is shown in SEQ ID NO:14, the IL-21 protein expression level is detected by Western Blot, and a specific IL-21 antibody is used.

[0067] The second aspect of this application provides a method for constructing a membrane-bound IL-21 K562 cell line, wherein the K562 cell line stably expresses a fusion protein comprising human IL-21, the IgG4 hinge region, the CH2 / CH3 region of the human immunoglobulin γ-4 chain, and the human CD4 transmembrane domain.

[0068] The third aspect of this application provides a recombinant plasmid PCDH-mbIL-21 for constructing the K562 cell line. The plasmid contains the mbIL-21 fusion gene of claim 2 and is inserted into the PT-mnud vector via double restriction sites of BamHI and XhoI.

[0069] The fourth aspect of this application provides the use of a membrane-bound IL-21 K562 cell line in the preparation of a drug that enhances the immune response. The drug exerts its immunomodulatory effect by means of the following: the mbIL-21 fusion gene expressed on the surface of the K562 cell line binds to the IL-21 receptor of neighboring immune cells, activates the JAK-STAT signaling pathway, and promotes T cell proliferation, NK cell toxicity, or B cell differentiation.

[0070] The fifth aspect of this application provides a pharmaceutical composition for tumor immunotherapy, comprising a membrane-bound IL-21 K562 cell line and a pharmaceutically acceptable carrier or excipient.

[0071] The core innovation of this invention lies in the combination of optimized design of the fusion protein and a highly efficient delivery system, which solves the technical challenges of unstable expression and insufficient activity in the construction of existing membrane-bound cytokines, providing a novel tool for immunotherapy based on engineered cells. Furthermore, this cell line can be used to further study the local immune regulation mechanism of IL-21, or as an in vitro model to evaluate the synergistic effect of combination therapies, possessing significant scientific research and clinical application value.

[0072] Example 1

[0073] The present invention provides a method for constructing a K562 cell line that binds to membrane-bound IL-21, comprising the following steps: (1) constructing an mbIL-21 vector:

[0074] The human IL-21 sequence was sequentially linked with the IgG4 hinge region, the human immunoglobulin γ-4 chain CH2 / CH3 region, and the human CD4 transmembrane domain through gene modification to form the mbIL-21 fusion gene;

[0075] The mbIL-21 fusion gene was cloned into the PT-mnud vector via double digestion with BamHI and XhoI to obtain the recombinant plasmid PCDH-mbIL-21. The construction of the mbIL-21 fusion gene includes the following sequences: the amino acid sequence encoded by the mbIL-21 fusion gene is shown in SEQ ID NO: 1; the nucleotide sequence of the mbIL-21 fusion gene is shown in SEQ ID NO: 2; the amino acid sequence encoded by mature human IL-21 is shown in SEQ ID NO: 3; the nucleotide sequence of mature human IL-21 is shown in SEQ ID NO: 4; the nucleotide sequence of the modified 12-aa IgG4 hinge region is shown in SEQ ID NO: 5; the amino acid sequence encoded by the CH2 / CH3 region of the human immunoglobulin γ-4 chain is shown in SEQ ID NO: 6; the amino acid sequence encoded by the human CD4 transmembrane domain is shown in SEQ ID NO: 7; and the nucleotide sequence of the human CD4 transmembrane domain is shown in SEQ ID NO: 8. The nucleotide sequence of the forward primer mbIL-21-F is shown in SEQ ID NO: 9, and the nucleotide sequence of the reverse primer mbIL-21-R is shown in SEQ ID NO: 10.

[0076] (2) Preparation of lentiviruses:

[0077] The recombinant plasmid PCDH-mbIL-21 was co-transfected with packaging plasmids PCDH-VSV-G, PCDH-Rev, and PCDH-PMDL into HEK 293T cells. The viral supernatant was collected and purified to obtain lentiviral particles carrying mbIL-21. The amounts of packaging plasmids used were: PCDH-VSV-G 3 μg; PCDH-Rev 2 μg; PCDH-PMDL 5 μg; and PCDH-mbIL-21 15 μg.

[0078] (3) Infecting K562 cells and screening:

[0079] K562 cells were infected with the lentivirus, and a stable K562 cell line expressing membrane-bound IL-21 was obtained through puromycin selection. The specific steps of puromycin selection included: determining the puromycin-sensitive concentration for K562 cells to be 1 μg / ml; culturing infected K562 cells in a medium containing 1 μg / ml puromycin for 48 hours, and selecting surviving cells.

[0080] Step (3) is followed by a verification step: the mRNA expression level of mbIL-21 is detected by qPCR. The nucleotide sequence of the forward primer WY-pcdh-IL21qPCR1F is shown in SEQ ID NO:11, the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR1R is shown in SEQ ID NO:12, the nucleotide sequence of the forward primer WY-pcdh-IL21qPCR2F is shown in SEQ ID NO:13, and the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR2R is shown in SEQ ID NO:14. The IL-21 protein expression level is detected by Western Blot using a specific IL-21 antibody.

[0081] Example 2

[0082] The present invention provides a method for constructing a membrane-bound IL-21 K562 cell line, wherein the K562 cell line stably expresses a fusion protein comprising human IL-21, the IgG4 hinge region, the CH2 / CH3 region of the human immunoglobulin γ-4 chain, and the human CD4 transmembrane domain.

[0083] Example 3

[0084] The present invention provides a recombinant plasmid PCDH-mbIL-21 for constructing the K562 cell line. The plasmid contains the mbIL-21 fusion gene as claimed in claim 2 and is inserted into the PT-mnud vector via double restriction sites of BamHI and XhoI.

[0085] Example 4

[0086] The present invention relates to the application of a membrane-bound IL-21 K562 cell line in the preparation of a drug that enhances the immune response. The drug exerts its immunomodulatory effect through the following mechanism: the mbIL-21 fusion gene expressed on the surface of the K562 cell line binds to the IL-21 receptor of neighboring immune cells, activates the JAK-STAT signaling pathway, and promotes T cell proliferation, NK cell toxicity, or B cell differentiation.

[0087] Example 5

[0088] A pharmaceutical composition for tumor immunotherapy according to the present invention comprises a membrane-bound IL-21 K562 cell line and a pharmaceutically acceptable carrier or excipient.

[0089] Example 6

[0090] Construction and transformation of mbIL-21 vector

[0091] (1) Obtaining the target gene: mbIL-21 fragment acquisition

[0092] When peripheral blood mononuclear cells (PBMCs) are co-cultured with irradiated IL-21-expressing engineered K562 cells for 3 days, the irradiated K562 cells will basically die, thus equivalent to short-term exposure to IL-21.

[0093] Therefore, K562 cells were genetically modified to generate mbIL-21 and granulocyte-macrophage colony-stimulating factor (GM-CSF) signal peptide sequences. The amino acid sequence encoded by the GM-CSF signal peptide is shown in SEQ ID NO: 15, and the nucleotide sequence of the GM-CSF signal peptide is shown in SEQ ID NO: 16.

[0094] Modified 12-aa IgG4 hinge region, human immunoglobulin γ-4 chain 5′ CH2 / CH3 region, and human CD4 transmembrane domain coding sequences were added to the original mature human IL-21 sequence and then processed according to ( Figure 1 Connect them sequentially. Figure 1 This is the amino acid sequence and schematic diagram of mbIL-21 of the present invention. Note: The mbIL-21 sequence consists of, in order, the signal peptide, mature human IL-21, the modified 12-aa IgG4 hinge region, the 5′ CH2 / CH3 region of the human immunoglobulin γ-4 chain, and the coding sequence for the human CD4 transmembrane domain.

[0095] The membrane-binding IL-21 (mbIL-21) of the target fragment to be incorporated into the vector has the following 5'-3' combination sequence: GM-CSF signal peptide >sp|P04141|1-17, mature IL-21 >sp|Q9HBE4|32-162, Hinge region, CH2 and CH3 sequences, transmembrane sequence >sp|P01730|397-418. The GM-CSF signal peptide sequence is directly fused to the coding sequence of mature human IL-21, which is linked by a modified [amino acid (aa) #108, Ser→Pro] 12 aaIgG4 IgG4 hinge region (aa 99-110). The human immunoglobulin γ-4 chain CH2 / CH3 region (aa 111-327, UniProtKB#P01861) was fused into the human CD4 transmembrane domain (aa 397-418, UniProtKB#P01730) within the 5′ framework. It has been previously linked between the LIR and RIR of the PT-mnud-mbIL-21 plasmid.

[0096] The restriction enzyme sites selected for the PT2 / HB transposon plasmid were BamHI and XhoI. Primers were designed using a primer design website and then synthesized by a primer synthesis company.

[0097] The required primer sequences are shown in Table 1.

[0098] Table 1

[0099] The nucleotide sequence of the forward primer mbIL-21-F is shown in SEQ ID NO: 9, and the nucleotide sequence of the reverse primer mbIL-21-R is shown in SEQ ID NO: 10. The components and preparation of the PCR reaction system were performed on ice. The High-Fidelity Master Mix 2 was added last to the PCR tube, followed by a gentle tap on the tube wall and a brief centrifugation. After centrifugation, the PCR tube was placed in the PCR instrument. The PCR reaction system was added to the PCR tube according to Table 2, and the PCR reaction program was set according to Table 3.

[0100] Table 2

[0101] Table 3

[0102] The process involved repeating the denaturation-annealing and extension steps for 34 cycles. After the PCR reaction, 10 μl of 6' Loading buffer was added for agarose gel electrophoresis to identify the PCR products. The specific steps were as follows: Weigh 0.25 g of agarose into a conical flask, add 25 ml of 1' TAE solution, gently shake to mix, and heat in a microwave until completely melted. Add GelRed nucleic acid dye at a volume ratio of GelRed:TAE = 1:10000, mix well, and pour into a pre-assembled gel casting apparatus. Let stand for 30 min. Place the solidified 1% agarose gel into the 1' TAE solution, ensuring the TAE solution completely covers the gel surface. Use a 250 bp DNA ladder for indication. Load 3 μl of the PCR product and connect to an electrophoresis electrode. Electrophoresis was performed at 150 V for 30 min. After electrophoresis, the gel was photographed using a gel imaging system, and the target band was observed to determine if it was a single band. After confirming that the target band is a single band, the remaining product was recovered using a DNA purification and recovery kit, and the fragment concentration was measured. The recovered sample was stored at -20°C.

[0103] (2) Recycling of adhesive after linearization of carrier

[0104] The following enzyme sites were selected for the PT-mnud-mbIL-21 plasmid: BamHI and XhoI. A 50 µl digestion system was prepared according to Table 4. Reagents were added sequentially according to the list, gently mixed by pipetting, briefly centrifuged, and incubated at 37°C for 2 h. The digestion reaction system (50 μl) is shown in Table 4.

[0105] Table 4

[0106]

[0107] The vector digestion products were subjected to 1% agarose gel electrophoresis (120 V, 25 min). After electrophoresis, the gel was placed in a gel imaging system for photographing and the position of the target band was observed. The target band was cut out and placed in a 1.5 ml EP tube. The gel was recovered according to the gel recovery kit, and the concentration of the recovered fragment was measured. Figure 2 This is an agarose gel electrophoresis image of the PCDH vector after double digestion (restriction sites: BamHI and XhoI) according to the present invention. Note: The sample in lane 1 is a 250bp DNA Maker, the sample in lane 2 is a 250bp DNA Maker, and the sample in lane 3 is the product after double digestion of the PCDH vector. (3) Recombinant cloning of PCR products and vectors

[0108] Thaw the recombinase 2×Uniclone Seam Less Cloning Mix on ice. Prepare PCR tubes and add the two previously recovered fragments to the PCR tubes respectively. Finally, add 5 μl of 2×Uniclone Seam Less Cloning Mix (Table 5), gently tap the tube wall to mix evenly, and centrifuge briefly. Place the tubes in a PCR instrument and set the program to 50℃ for 30 min. The enzyme digestion reaction system (10 μl) is shown in Table 5.

[0109] Table 5

[0110]

[0111] (4) Transformation

[0112] Take 100 μl of thawed competent DH5α cells from an ice bath, add 10 μl of recombinant product, mix gently, incubate on ice for 30 min, then heat shock in a 42℃ water bath for 45 s, quickly transfer to an ice bath, and incubate for 2 min; add 1 ml of antibiotic-free sterile LB medium to a centrifuge tube, mix well, and incubate at 37℃ for 1 h at 200 rpm; remove the resuscitation solution and centrifuge at 4000 rpm for 3 min, pour off the supernatant, reflux the liquid and mix well, spread evenly on LB medium containing ampicillin (AMP), and incubate the plate upside down at 37℃ overnight.

[0113] (5) Identification of positive clones

[0114] ① Sixteen hours after transforming *E. coli* with the recombinant plasmid, relatively uniformly distributed colonies can be seen on agar plates. To extract the plasmid from the transformed bacteria, the bacteria must first be expanded. Prepare LB medium in sterile 50 ml centrifuge tubes beforehand, adding AMP at a ratio of 1:1000. Use a 10 μl pipette tip to pick a single colony from the agar plate and inoculate it into an LB centrifuge tube. Incubate at 37°C on a shaker for 14-16 hours, then extract the plasmid using a Tiangen kit. After complete plasmid extraction, dissolve the plasmid in 50-100 μl of ddH2O.

[0115] ② The concentration was measured using a NanoDrop 2000 instrument for subsequent experiments.

[0116] ③ Select recombinant plasmid restriction enzyme sites: BamHI and XhoI.

[0117] ④ Prepare a 10 µl enzyme digestion system according to Table 6. Add the reagents in the order listed, gently mix with a pipette, and briefly centrifuge. Incubate at 37℃ for 2 h. The enzyme digestion reaction system (10 μl) is shown in Table 6:

[0118] Table 6

[0119]

[0120] ⑤ Perform 1% agarose gel electrophoresis on the enzyme digestion products (120 V, 25 min). After electrophoresis, place the gel in a gel imaging system to take pictures and observe the position of the target bands. Sequencing is performed on positive samples using universal vector primers, and the sequences are compared. Plasmid extraction is performed on samples with correct sequencing. Figure 3 This image shows the enzyme digestion identification of the mbIL-21 recombinant and transformed plasmid after extraction. Note: Lane 1 sample is a 250bp DNA Maker, Lane 2 sample is the product of recombinant plasmid 1 after enzyme digestion, and Lane 3 sample is the product of recombinant plasmid 2 after enzyme digestion.

[0121] Samples that tested positive for plasmid double digestion were sent for sequencing. The sequencing results were compared using SnapGene software: the sequenced sequences were completely consistent with the designed sequences, indicating that the PCDH-mbIL-21 vector was successfully constructed. Figure 4 ). Figure 4 This is a schematic diagram of the sequencing results comparison for this invention. Note: The green part in the diagram is a schematic diagram of the mbIL-21 designed sequence, and the dark blue part is a schematic diagram of the recombinant plasmid sequencing results. Since unidirectional sequencing measures approximately 800 bp, while the mbIL-21 sequence is 1486 bp, bidirectional sequencing was used. The bidirectional sequencing results show that the recombinant plasmid is completely consistent with the designed sequence. (Image taken from snapgene software).

[0122] (6) Plasmid extraction

[0123] Transfer the correctly sequenced bacterial culture to 10 ml of LB liquid medium containing AMP. Incubate at 37°C and 200 rpm for 16 h. Centrifuge at 4000 rpm for 10 min. Discard the supernatant, add 250 μl of cell resuspension, and shake thoroughly to resuspend the bacterial clumps evenly. Add 250 μl of cell lysis buffer, then add 10 μl of proteinase K, and gently mix by inverting 5-6 times. Let stand for 1-2 min to allow the cells to lyse and become clear. Add 350 μl of neutralization buffer, invert to mix, and allow the protein to precipitate completely. Incubate on ice for 5 min. Centrifuge at 10000 rpm for 10 min, discard the protein, and collect the supernatant in another clean, sterile 1.5 ml EP tube. Centrifuge at 12000 rpm for 5 min. Simultaneously, prepare a labeled recovery column, transfer the supernatant to the recovery column, centrifuge at 12000 rpm for 1 min, and discard the lower layer. Add 600 μl of wash buffer and centrifuge at 12000 rpm for 1 minute. After 2 min, discard the lower layer and repeat once. Centrifuge at 12000 rpm for 2 min to remove residual wash solution. Transfer the recovery column to a new EP tube and let it stand for 10-20 min to air dry. Add 95 μl of preheated (65℃) Nuclease-Free Water to the recovery column, let it stand for 2 min, centrifuge at 12000 rpm for 2 min, and collect the sample. Take 2 μl of plasmid onto the nanodrop instrument well, read the value, and record it.

[0124] Example 7

[0125] mbIL-21 lentivirus preparation

[0126] (1) HEK 293 T cell preparation: HEK 293 T cells were digested, resuspended, and counted at a ratio of 5 × 10⁻⁶. 6 Cells are evenly spread in a 10 cm culture dish. After the cells adhere to the dish, they are used for transfection to obtain the virus solution.

[0127] (2) The packaging plasmid and mbIL-21 plasmid were calculated according to their concentrations. The corresponding amounts of each plasmid used in the preparation of the mbIL-21 lentivirus are shown in Table 7.

[0128] Table 7

[0129]

[0130] Prepare two 5 ml test tubes, add 1 ml of OPTI-DMEM medium to each tube, and add the corresponding volumes of VSV-G, Rev, PMDL, and mbIL-21 / PCDH plasmids respectively.

[0131] (3) Take two EP tubes, add 1 ml of Opti-DMEM medium and 60 μl of PEI to each tube, mix gently after adding, and let stand for 5 min.

[0132] (4) Add the liquid from the DMEM+PEI tube dropwise to the plasmid+DMEM tube in a 5 ml test tube. Mix well and let stand at room temperature for 20 min;

[0133] (5) Take out HEK 293 T cells from the incubator, discard about 2 ml of culture medium, and according to the experimental design, slowly add the mixture that has been standing for 20 min along the side wall of the culture dish (HEK 293 T cells do not adhere firmly to the wall, so the action must be gentle). After gently shaking the culture dish to mix the liquid in the culture dish, place it in a 37℃, 5% CO2 incubator for culture.

[0134] (6) Because PEI is toxic to cells, after culturing for 4-6 hours, the liquid in the culture dish should be aspirated, the cells should be washed with PBS solution 1-2 times, and then 10 ml of fresh complete culture medium should be added. The culture dish should be placed in a 37℃, 5% CO2 incubator.

[0135] (7) Collect lentivirus supernatant at 48 h and 72 h after medium change. The specific operation is as follows: at 48 h, remove the cell culture dish, aspirate the cell culture supernatant, and add 10 ml of fresh complete culture medium to continue culturing. The collected supernatant is filtered through a 0.22 μm filter membrane and collected in a 15 ml centrifuge tube, labeled, and stored in a -80°C freezer; at 72 h, the cell culture supernatant is collected and the same procedure is followed. The cell culture dish is then discarded after treatment (at the end of each experiment, all waste such as lentivirus heads, centrifuge tubes, pipettes, and culture dishes that have come into contact with the virus are soaked in 84 disinfectant for 24 h and then discarded).

[0136] Example 8

[0137] Establishment of mbIL-21 K562 cell line

[0138] Preparation of K562 cells

[0139] ① K562 cell thawing and culture: Preheat the water bath to 37°C. Remove the frozen K562 cells and quickly transfer them to the water bath, gently shaking to accelerate lysis. After complete thawing, place the cryovial in a centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 1 ml of preheated 10% FBS RPMI 1640 medium. Transfer the cell suspension to a new 15 ml centrifuge tube. Centrifuge again at 800 rpm for 5 minutes, remove the tube, discard the supernatant, resuspend, count, and adjust the cell density to 5 × 10⁵ cells / ml.

[0140] K562 cells require pre-warmed fresh complete culture medium for passage / medium change. K562 cells are suspension cells; when passageing or changing medium, gently aspirate from the culture flask wall and transfer the liquid to a 15 ml centrifuge tube. Centrifuge the tube at 1000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet in 1 ml of pre-warmed 10% FBS RPMI 1640 medium before counting. Repeat at 5 × 10⁻⁶ cells per cell line. 5 Cells were cultured at a density of cells / ml at 37°C in a 5% CO2 incubator. Cells were removed and set aside for use after entering the logarithmic growth phase.

[0141] ② Experiment on the determination of puromycin drug sensitivity concentration in K562 cells.

[0142] Puromycin is an aminoglycoside antibiotic that inhibits peptide transfer in both prokaryotic and eukaryotic cells. Its drug-sensitive concentration depends on the sensitivity of the infected cells. This example was used to select K562 cells transfected with a puromycin resistance gene. The minimum concentration at which all cells died after 48 hours of puromycin administration was selected as the puromycin drug-sensitive concentration for the K562 cell line.

[0143] The specific procedure is as follows: Puromycin stock solution (10 mg / ml): RPMI 1640 medium was diluted with puromycin at ratios of 1:500, 1:1000, 1:500, 1:10000, and 1:50000. K562 cells in the logarithmic growth phase were collected in six centrifuge tubes. Each tube was filled with 1×10⁶ cells. The tubes were centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended in 2 ml of RPMI 1640 medium (containing 10% FBS) with puromycin diluted at ratios of 1:500, 1:1000, 1:5000, 1:10000, and 1:50000. The pellet was then added to a 6-well plate, with the sixth well containing no puromycin as a control. Cell morphology was observed under a microscope every 12 h, and trypan blue counting was performed simultaneously. The minimum concentration at which all cells died after the addition of puromycin was the puromycin drug-sensitive concentration for K562 cells.

[0144] Lentiviral infection of K562 cells

[0145] (1) Take three 24-well plates and label them as PCDH-mbIL-21 group, PCDH-untransfected mbIL-21 group, and Parental group.

[0146] Remove the lentivirus from the -80°C freezer beforehand and thaw it (thaw at 4°C). Add polybrene diluent (stock solution concentration 5 mg / ml) to the virus solution to achieve a final concentration of 10 μg / ml;

[0147] (2) Count K562 cells and resuspend the cells in lentivirus solution of PCDH-mbIL-21 group and PCDH-untransfected mbIL-21 group respectively, and adjust the cell density to 1×106 cells / ml; add 500 μl of K562 cells resuspended in lentivirus solution to each well of 24-well plate.

[0148] (3) After covering the 24-well plate with the sealing film, centrifuge it at room temperature, 1800 rpm, for 90 min.

[0149] (4) After centrifugation, remove the sealing film, gently tap the 24-well plate to resuspend the cells, and then put the cell culture plate back into the 37°C, 5% CO2 incubator for culture.

[0150] (5) After incubation in the incubator for 2 hours, remove the 24-well plate, cover it with the sealing film, and centrifuge at room temperature, 2000 rpm, for 20 minutes. Carefully remove the 24-well plate and tear off the sealing film. Tilt the culture plate to aspirate the virus solution. Add preheated 10% FBS RPMI 1640 medium and gently pipette the bottom of the plate to resuspend the cells.

[0151] (6) Place the cell culture plate in a 37°C, 5% CO2 cell culture incubator and incubate for 24 h;

[0152] (7) Repeat infection once, following the same steps as ③-⑤;

[0153] (8) Place the cell culture plate in a 37°C, 5% CO2 cell culture incubator and continue incubation for 48 h.

[0154] Screening of K562 cells infected with lentivirus

[0155] (1) K562 cells infected with the second lentivirus for 48 h were transferred to 6-well plates;

[0156] (2) Replace with fresh culture medium and add puromycin at a final concentration of 1 μg / ml. Place the cell culture plate in a 37°C, 5% CO2 cell culture incubator and continue incubation for 48 h.

[0157] (3) Observe the cell state under a microscope. After adding puromycin for 48 h, all cells in the Parental group died.

[0158] (4) Collect K562 cells from the PCDH-mbIL-21 group and the PCDH-untransfected mbIL-21 group.

[0159] (5) The cells were continuously cultured in RPMI 1640 medium containing 1 ug / μl of puromycin and 10% FBS. The two groups of cells were called mbIL-21 K562 cells and untransfected mbIL-21 K562 cells.

[0160] Experimental Example 1

[0161] qPCR validation of stable overexpression of mbIL-21 in K562 cells

[0162] (1) Primers: For the mbIL-21 sequence in the above example, this qPCR primer does not need to amplify the full-length mbIL-21 sequence. The primer is designed to amplify a length of 80-200 bp.

[0163] The two primer pairs (5'-3') sequences are shown in Table 8.

[0164] Table 8

[0165]

[0166] The nucleotide sequence of the forward primer WY-pcdh-IL21qPCR1F is shown in SEQ ID NO:11, the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR1R is shown in SEQ ID NO:12, the nucleotide sequence of the forward primer WY-pcdh-IL21qPCR2F is shown in SEQ ID NO:13, and the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR2R is shown in SEQ ID NO:14. Primer testing: Before the formal experiment, the primers designed based on mRNA need to be tested for specificity and amplification efficiency using qPCR. The specific reaction system and reaction conditions are as in the formal experiment. A template water control is used for each primer pair. After obtaining the results, the primer specificity is first determined based on the melting curve. The selection criteria are: single peak with a narrow peak shape, and no obvious primer dimer melting curve peak in the water control. If the melting curves of multiple primer pairs all show good specificity, the primers with small Ct values ​​and high amplification efficiency are selected for the formal experiment by comparing the amplification curves of each primer.

[0167] (2) Total RNA extraction

[0168] ① Sample Collection: Collect cells from the mbIL-21 K562 group and the untransfected mbIL-21 K562 group. After centrifugation, discard the supernatant. Wash the cell pellet twice with PBS, then add TRNzol-A to lyse the cells and pipette several times. Transfer the solution to a 1.5 ml EP tube. (This sample can be stored at -80°C for one month)

[0169] ② RNA extraction:

[0170] Add 0.2 ml of chloroform to each 1 ml of TRNzol-A obtained in the previous step, cap the tube, shake vigorously for 15 seconds, incubate at room temperature for 8 minutes, and then centrifuge at 13000 rpm for 15 minutes at 4°C. The sample will separate into three layers: a yellow organic phase, an intermediate layer, and a colorless aqueous phase on top. RNA is mainly in the aqueous phase. Transfer the aqueous phase (approximately 500 μl) to a new centrifuge tube. Add approximately 500 μl of isopropanol to the new EP tube, mix well, and incubate at room temperature for 10 minutes. Centrifuge again at 13000 rpm for 10 minutes at 4°C. After centrifugation, a gelatinous precipitate will be visible on the sides and bottom of the tube; discard the supernatant. Add 1 ml of 75% ethanol (prepared with RNase-free ddH2O) and wash (centrifuge at 7500 g for 5 minutes at 4°C, discard the liquid) three times to remove the precipitate. After the last wash, remove the remaining small amount of liquid, centrifuge briefly, and then aspirate with a pipette tip, being careful not to discard the precipitate. Air-dry the EP tubes containing cell pellet at room temperature (the pellet will change from white to clear in about 2-3 minutes). Add 30-50 μl of RNase-free ddH2O, let stand for a moment, then repeatedly pipette and mix to fully dissolve the RNA. Place on ice, open GEN 1.5 software, and select RNA. Wash three times with water, add 1 μl of water to measure the BLANK baseline. After the baseline flattens, blot dry, add 1 μl of sample, and determine the RNA concentration and purity. First remove genomic DNA; the PCR reaction system is shown in Table 9.

[0171] ③ All components and preparation systems were operated on ice. After adding each component, the tube was gently tapped against the wall and centrifuged briefly. After centrifugation, the PCR tube was placed in the PCR instrument and centrifuged at 42°C for 2 minutes before being removed. The PCR reaction system used (10 μl) is shown in Table 9, the PCR reaction system used in this step (20 μl) is shown in Table 10, and the PCR reaction conditions used in this step are shown in Table 11.

[0172] Table 9

[0173]

[0174] Table 10

[0175] Table 11

[0176] ④ Reverse transcription of RNA into cDNA: Add the PCR product obtained in step ② to each component according to Table 10, then gently tap the tube wall and centrifuge briefly. After centrifugation, place the PCR tube into the PCR instrument and set the PCR reaction program according to Table 11. Then store the obtained cDNA at -80℃.

[0177] ⑤ Pre-arrange the sample placement order for the experiment in your notebook (samples from the same replicate should not be split into two plates). Prepare the required reaction mixture as needed, generally preparing one extra system. After preparing the total system, mix thoroughly and then aliquot into 96-well PCR plates. Dilute the cDNA 1:20 with sterile purified water. Add the cDNA to the prepared reaction mixture in the correct order. After adding the samples, cover the plates with a membrane, label them, and ensure each well is tightly sealed. Gently tap the tube wall and briefly centrifuge. After centrifugation, gently tap the tube wall again and briefly centrifuge. Repeat this process three times before loading the sample into the PCR machine. Remove the samples after the program completes.

[0178] ⑥ Record and analyze the data; samples must be placed in sealed bags and discarded. Figure 5 The diagram shows the mRNA level of mbIL-21 in K562 cells detected by RT-PCR experiment according to the present invention.

[0179] Experimental Example 2

[0180] Western blotting confirmed stable overexpression of mbIL-21.

[0181] (1) Protein extraction: Take the PCDH-mbIL-21 group and the PCDH untransfected mbIL-21 group K562 cells from the examples, centrifuge and discard the supernatant. Resuspend the cell pellet with PBS and put them into 1.5 ml centrifuge tubes. Centrifuge again at 4℃, 1500 rpm for 5 min, discard the supernatant and wash the cells with pre-cooled PBS, discard the supernatant; add 1 ml of RIPA lysis buffer (containing protease inhibitor) taken from 4℃ freezer to the cell pellet after washing with PBS, place the centrifuge tubes in a 4℃ shaker and let the cells fully lyse for 30 min; transfer the centrifuge tubes to a 4℃ centrifuge; centrifuge at 12000 rpm for 10 min; take the supernatant and label it.

[0182] (2) Protein concentration determination (BCA method): Take out the required amounts of solution A and solution B. Mix solution A and solution B at a ratio of 50:1. Add 5 μl each of protein standards with concentrations of 0 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 4 mg / ml, and 6 mg / ml, and the sample to be tested to a 96-well plate. Add 200 μl of the AB mixture to each well and set up one blank well (without sample). Incubate at 65℃ for 10 min. Measure the OD value at 562 nm using a microplate reader. Quantify the protein based on the sample with the lowest obtained concentration. Adjust all samples to equal concentration and volume using RIPA lysis buffer (operate on ice). Add 4×SDS and mix thoroughly. To avoid the protein spatial structure affecting the rate of subsequent electrophoresis, boil the sample in a 98℃ metal bath for 10 min. Store the sample at -20℃ for later use.

[0183] (3) SDS-PAGE gel electrophoresis:

[0184] ① Gel Preparation: Install the pre-cleaned gel mold correctly. Prepare a separating gel of appropriate concentration according to the molecular weight of the protein to be detected. Prepare a 10% separating gel by adding acrylamide, 1.5 M Tris (pH: 8.8), 10% SDS solution, ammonium persulfate solution, and TEMED solution in the correct proportions and mixing thoroughly. Carefully add the mixed liquid between the glass plates using a pipette, being careful not to generate air bubbles. Slowly add isopropanol to press the gel. After the separating gel solidifies, pour off any remaining isopropanol and blot dry with filter paper, ensuring the filter paper does not touch the gel surface. Prepare the upper stacking gel and insert the comb (insert it at an angle to avoid air bubbles). Let the upper gel solidify, then remove the comb. Begin assembling the electrophoresis apparatus. Add freshly prepared 1×Running Buffer to the electrophoresis tank until it covers the glass short plate. Add 1×Running Buffer to the outer tank, approximately half full. After loading the sample, connect the electrodes (note the correspondence between black and red). First, set the electrophoresis voltage to 80 V. After the sample enters the separating gel, adjust the voltage to 100 V. Observe the bromophenol blue moving to the bottom of the gel to end the gel run.

[0185] ② Transfer: Cut off the upper right corner of the PVDF membrane to mark the last sample. Activate the PVDF membrane by soaking it in methanol. Turn off the power, remove the glass plate, rinse off any foam, pry open the glass plate, peel off the gel, and discard the stacking gel. Pre-cool the 1' Transfer Buffer. Assemble the clamping plate in the transfer buffer. Place the separating gel in the tray containing the transfer buffer and assemble in the following order: sponge-filter paper-gel-membrane-filter paper-sponge. During assembly, ensure no air bubbles are left. After assembly, place both the inside and outside of the transfer tank in an ice box and add the transfer buffer. Connect the electrodes. Set the parameters to 300 mA for 60 min.

[0186] ③ Blocking and Primary Antibody Incubation: Blocking: Take a new 15 ml centrifuge tube, weigh 0.5 g of skim milk and add it to the tube. Add the pre-prepared PBST solution to bring the volume to 10 ml. Vortex until the milk is fully dissolved. Remove the transferred PVDF membrane, add PBST solution, and place it on a horizontal shaker. Wash for 10-15 min, replace with new PBST, and repeat the washing process 3 times. Then immerse the PVDF membrane in 5% skim milk blocking solution and incubate it on a horizontal shaker at room temperature for 1 h. Recover the blocking solution. Wash 3 times with PBST to remove the blocking solution. Cut the membrane according to the marker indication and mark it.

[0187] Primary antibody incubation: Add IL-21 antibody to 3% BSA at a ratio of 1:1000, and simultaneously add NaN3 solution (preservative) at a ratio of 1:10000, and mix well. Immerse the strip in an incubation box containing the primary antibody, place it on a shaker at 4°C, and incubate overnight. After incubation, recover the primary antibody and store it at 4°C. Wash the PVDF membrane three times in PBST solution.

[0188] ④ Secondary antibody incubation: Weigh 0.3 g of skim milk and add pre-prepared PBST solution to a final volume of 10 ml. Vortex to mix until the milk is fully dissolved. Add secondary antibody at a ratio of 1:5000, mix well, and set aside. Place the PVDF membrane in a secondary antibody incubation box, add the pre-prepared secondary antibody, place the incubation box on a horizontal shaker, and incubate at room temperature for 1 h. After incubation, recover the secondary antibody and store it at 4°C. Wash the PVDF membrane three times with PBST solution.

[0189] ⑤ Development: Take out the cleaned PVDF membrane, absorb the TBST solution on the filter paper, and then put it into the developing solution (A solution: B solution = 1:1) for 2 minutes. Use the ImageQuant LAS 4000 developing instrument for development. Figure 6 This is a Western blot diagram showing the IL-21 protein level in the mbIL-21K562 cell line as described in this invention. Note: IL-21 positive control group: K562- cells, Daudi cells, 10A cells, and untransfected mbIL-21 K562 cells; IL-21 weak positive control MCF 7; experimental group: mbIL-21K562 cells.

[0190] Experimental Example 3

[0191] NK cell in vitro induction and expansion culture

[0192] After irradiation with 100 Gy X-rays, mbIL-21 K562 cells and untransfected mbIL-21 K562 cells were co-cultured with peripheral blood PBMCs from healthy donors at a 1:1 cell ratio for 14 days. Simultaneously, cytokines IL-2 and IL-15 were added to induce in vitro differentiation of PBMCs into NK cells (e.g., ...). Figure 7 ). Figure 7 This is a schematic diagram of the co-culture process of PBMCs and irradiated mbIL-21 K562 cells according to the present invention.

[0193] Isolation of peripheral blood mononuclear cells

[0194] This experiment was conducted according to the protocol approved by the Institutional Review Committee of the Children's Hospital Affiliated to Kunming Medical University. 20 ml of blood was drawn from a healthy donor. Before isolating mononuclear cells, the peripheral blood, PBS, and lymphocyte separation medium were equilibrated to 20°C at room temperature.

[0195] (1) Plasma extraction: ① Divide peripheral blood into 50 ml centrifuge tubes and centrifuge at 700 g for 15 min at room temperature (centrifuge speed 8, deceleration speed 4). Transfer the upper light yellow plasma to a new 10 ml centrifuge tube (the lower red liquid is used to extract mononuclear cells). Inactivate the plasma in a water bath at 56℃ for 30 min. ② Centrifuge at 900 g for 10 min, collect the supernatant, place at -20℃ for 15 min, centrifuge again at 900 g for 10 min, collect the supernatant, and store at 4℃.

[0196] (2) Isolation of mononuclear cells: ① Take the lower red liquid obtained after plasma extraction in the previous step, dilute it 1:1 with physiological saline, mix well, and set aside. ② Take two new 15 ml centrifuge tubes, and slowly add the diluted blood to the upper layer of the lymphocyte separation medium at a 1:1 ratio according to the volume of diluted blood. Make sure the blood and lymphocyte separation medium form a clear layer, and be careful not to mix the diluted blood into the lymphocyte separation medium. Centrifuge at 700 g for 30 min at room temperature. ③ Gently aspirate the mononuclear cells (white membrane layer) and transfer them to a new 50 ml centrifuge tube; add an equal volume of physiological saline, and centrifuge at 700 g for 10 min at room temperature. Discard the supernatant, wash the cells again with 40 ml of physiological saline, centrifuge at 200 g for 10 min, and discard the supernatant. Resuspend the cells in culture medium pre-warmed to 37℃, and set aside. At the same time, take a small amount of cell suspension for counting.

[0197] mbIL-21 K562 cells and untransfected mbIL-21 K562 cells were continuously cultured in RPMI 1640 medium containing 1 μg / μl puromycin and 10% FBS. Three days before irradiation, the cells were harvested, centrifuged, and the supernatant was discarded. The cells were washed twice with PBS. The mbIL-21 K562 cells and untransfected mbIL-21 K562 cells were then cultured in RPMI 1640 medium (10% FBS, without puromycin) for further culture. Cell counting was performed before irradiation, and the cell density was adjusted to 1 x 10⁶ cells / ml (without adding new medium). The density-adjusted cells were placed in an irradiator with an irradiation dose of 100 Gy. The irradiated mbIL-21 K562 cells and untransfected mbIL-21 K562 cells were labeled as irradiated mbIL-21 K562 cells and irradiated untransfected mbIL-21 K562 cells, respectively.

[0198] PBMCs were co-cultured with irradiated mbIL-21 K562 cells and irradiated untransfected mbIL-21 K562 cells, respectively.

[0199] On day 0, PBMCs from normal donors were incubated at a 1:1 ratio with irradiated mbIL-21 K562 cells and irradiated untransfected mbIL-21 K562 cells in RPMI 1640 medium containing 10% FBS, with 50 IU / ml recombinant human IL-2 and 5 ng / ml recombinant human IL-15 added (cytokines were added separately with each addition of medium and not directly prepared into the culture flask). After 14 days of co-culture, NK cells were obtained, and exosomes were extracted.

[0200] (1) Monitoring of NK cell phenotype in the co-culture system: mbIL-21 K562 cells irradiated with PBMC and untransfected mbIL-21 K562 cells irradiated with PBMC were counted separately. Approximately 5 × 10⁵ cells were collected. After centrifugation and washing (1500 rpm, 5 min, discard supernatant) twice, the cells were resuspended in 200 μl PBS. Four flow tubes were prepared and labeled as 1-4. Tubes 1-2 contain only irradiated mbIL-21 K562 cells or irradiated untransfected mbIL-21 K562 cells, without antibodies. Subsequent steps are performed on ice in the dark. Tubes 3, 4, and 5 contain either PBMC-irradiated mbIL-21 K562 cell suspension or irradiated untransfected mbIL-21 K562 cell suspension and 3 μl of CD3, CD16, or CD56 antibody. Tube 6 contains PBMC-mbIL-21 K562 cell suspension and 3 μl of CD3, CD16, and CD56 antibodies. Tube 7 contains PBMC-K562-untransfected mbIL-21 cell suspension and 3 μl of CD3, CD16, and CD56 antibodies. After gentle mixing, incubate tubes 1-7 on ice for 30 min, then centrifuge at 1500 rpm for 5 min at 4°C, discarding the supernatant. Finally, incubate with pre-chilled PBS. After resuspending the precipitate in liquid and allowing it to settle, it is then loaded onto the instrument.

[0201] (2) Monitoring of NK cell proliferation in the co-culture system: On days 3, 6, 10, 12 and 14 of co-culture, cells in the culture medium were removed, and cells were counted after trypan blue staining. The number of NK cells with strong cytotoxicity was estimated by combining the proportion of CD56+CD16+ detected by flow cytometry, and the NK cell proliferation curve was plotted.

[0202] Figure 8 The purity of NK (CD3-CD16+CD56+) cells in the in vitro expansion culture of NK cells according to the present invention; Figure 8 A is a line graph showing the percentage of NK (CD3-CD16+CD56+) cells in the co-culture system monitored by flow cytometry according to the present invention. Figure 8B is a flow cytometry representation of the percentage of NK (CD3-CD16+CD56+) cells in the co-culture system monitored by flow cytometry according to the present invention. Note: On days 0, 3, 6, 10, 12, and 14, the percentage of NK cells in PBMC+ irradiated mbIL-21 K562 cells was 10.30%, 33.40%, 46.50%, 60.30%, 80.80%, and 90.40%, respectively; the percentage of NK cells in the co-culture system of PBMC+ irradiated untransfected mbIL-21 K562 cells was 10.80%, 11.90%, 19.80%, 27.30%, and 40.10%, respectively. Figure 9 This is a graph showing the change in the number of NK cells during the in vitro expansion culture of NK cells according to the present invention.

[0203] Note: Line graph showing the total number of NK cells in the co-culture system of PBMC+ irradiated mbIL-21 K562 cells and PBMC+ irradiated untransfected mbIL-21 K562 cells after trypan blue staining, and the total number of NK cells calculated by the proportion of CD56+CD16+ cells by flow cytometry at days 0, 3, 6, 10, 12, and 14. * indicates significant differences.

[0204] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A method for constructing a K562 cell line that binds IL-21 to a membrane, characterized in that... The steps include: (1) Constructing the mbIL-21 vector: The human IL-21 sequence was sequentially linked with the IgG4 hinge region, the human immunoglobulin γ-4 chain CH2 / CH3 region, and the human CD4 transmembrane domain through gene modification to form the mbIL-21 fusion gene; The mbIL-21 fusion gene was cloned into the PT-mnud vector by double digestion with BamHI and XhoI to obtain the recombinant plasmid PCDH-mbIL-21. (2) Preparation of lentiviruses: The recombinant plasmid PCDH-mbIL-21 was co-transfected with packaging plasmids PCDH-VSV-G, PCDH-Rev, and PCDH-PMDL into HEK 293T cells. The viral supernatant was collected and purified to obtain lentiviral particles carrying mbIL-21. (3) Infecting K562 cells and screening: K562 cells were infected with the lentivirus and screened with puromycin to obtain a stable K562 cell line expressing membrane-bound IL-21.

2. The construction method according to claim 1, characterized in that: In step (1), the construction of the mbIL-21 fusion gene includes the following sequences: the amino acid sequence encoded by the mbIL-21 fusion gene is shown in SEQ ID NO: 1, the nucleotide sequence of the mbIL-21 fusion gene is shown in SEQ ID NO: 2, the amino acid sequence encoded by mature human IL-21 is shown in SEQ ID NO: 3, the nucleotide sequence of mature human IL-21 is shown in SEQ ID NO: 4, the nucleotide sequence of the modified 12-aa IgG4 hinge region is shown in SEQ ID NO: 5, the amino acid sequence encoded by the CH2 / CH3 region of the human immunoglobulin γ-4 chain is shown in SEQ ID NO: 6, the amino acid sequence encoded by the human CD4 transmembrane domain is shown in SEQ ID NO: 7, and the nucleotide sequence of the human CD4 transmembrane domain is shown in SEQ ID NO:

8.

3. The construction method according to claim 1, characterized in that: In step (1), the primer sequences used in the double enzyme digestion reaction are as follows: the nucleotide sequence of the forward primer mbIL-21-F is shown in SEQ ID NO: 9, and the nucleotide sequence of the reverse primer mbIL-21-R is shown in SEQ ID NO:

10.

4. The construction method according to claim 1, characterized in that: In step (2), the amount of the packaging plasmid used is: PCDH-VSV-G 3 μg; PCDH-Rev 2 μg; PCDH-PMDL 5 μg; PCDH-mbIL-21 15 μg.

5. The construction method according to claim 1, characterized in that: In step (3), the specific steps of the puromycin screening include: The puromycin-sensitive concentration for K562 cells was determined to be 1 μg / ml; Infected K562 cells were cultured in a medium containing 1 μg / ml puromycin for 48 hours, and surviving cells were screened.

6. The construction method according to claim 1, characterized in that: Step (3) is followed by a verification step: the mRNA expression level of mbIL-21 is detected by qPCR. The nucleotide sequence of the forward primer WY-pcdh-IL21qPCR1F is shown in SEQ ID NO:11, the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR1R is shown in SEQ ID NO:12, the nucleotide sequence of the forward primer WY-pcdh-IL21qPCR2F is shown in SEQ ID NO:13, and the nucleotide sequence of the reverse primer WY-pcdh-IL21qPCR2R is shown in SEQ ID NO:

14. The IL-21 protein expression level is detected by Western Blot using a specific IL-21 antibody.

7. A K562 cell line with membrane-bound IL-21 constructed according to any one of claims 1-6, characterized in that: The K562 cell line stably expresses a fusion protein comprising the hinge region of human IL-21 and IgG4, the CH2 / CH3 region of human immunoglobulin γ-4 chain, and the transmembrane domain of human CD4.

8. A recombinant plasmid PCDH-mbIL-21 for constructing the K562 cell line of claim 7, characterized in that: The recombinant plasmid PCDH-mbIL-21 contains the mbIL-21 fusion gene as described in claim 2 and is inserted into the PT-mnud vector via double restriction sites of BamHI and XhoI.

9. The use of the K562 cell line of claim 7, which binds IL-21 to a membrane, in the preparation of a drug that enhances the immune response, characterized in that: The drug exerts its immunomodulatory effect through the following mechanism: the mbIL-21 fusion gene expressed on the surface of the K562 cell line binds to the IL-21 receptor (IL-21R) of neighboring immune cells, activating the JAK-STAT signaling pathway and promoting T cell proliferation, NK cell toxicity, or B cell differentiation.

10. A pharmaceutical composition for tumor immunotherapy, characterized in that: The K562 cell line comprising the membrane-bound IL-21 of claim 7, and a pharmaceutically acceptable carrier or excipient.

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