Immune cells with membrane-bound IL-21, and their preparation method and application

Abstract

We disclose immune cells containing membrane-bound IL-21, as well as methods for preparing and using them. Specifically, we disclose membrane-bound IL-21 (mbIL-21) and its application in immunotherapy. mbIL-21 enhances the proliferation and killing ability of specific immune cells, thereby enhancing the effects of NK cell therapy.

Description

[Technical Field]

[0001] The present invention relates to the field of cell therapy. Specifically, the present invention relates to immune cells having membrane-bound IL-21, and methods for preparing and applying the same. [Background technology]

[0002] Immune system homeostasis depends on two major components: the innate immune response and the adaptive immune response, both of which are regulated by a series of cytokines. The cytokine receptor gamma chain (γc) family is one of the most extensively studied cytokines and includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. This group of cytokines exhibits a wide range of pleiotropic effects, modulating both the innate and adaptive immune systems, collectively promoting the development of various immune cell populations, regulating cell differentiation, and promoting survival or inducing apoptosis depending on the cellular environment.

[0003] In cell therapy, NK cell therapy is a promising anti-cancer strategy, but the widespread clinical success of NK cell therapy has been limited in part by the challenge of producing the large doses of NK cells that may be required for clinical efficacy.

[0004] Furthermore, studies have shown that compared with T cells, NK cells have a shorter survival time in the host's body, which may affect the tumor-killing effect of NK cells. Although combined treatment with IL-2 can extend the survival of NK cells in the host's body to a certain extent, IL-2 may also induce the massive proliferation of Treg cells, which exert immunosuppressive effects.

[0005] Therefore, there is an urgent need in this field to develop NK cells with longer in vivo survival time and higher therapeutic efficacy. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide NK cells that have a longer in vivo survival time and a higher therapeutic effect, as well as methods for preparing and applying the same. [Means for solving the problem]

[0007] A first aspect of the present invention provides a recombinant protein, wherein the recombinant protein is membrane-bound IL-21 (mbIL-21), and the recombinant protein has the following characteristics: (a) the recombinant protein has the structure shown in Formula I: Z1-Z2-Z3-Z4(I) In the formula: Z1 is a signal peptide or none, Z2 is an IL-21 polypeptide, Z3 is the CD8 hinge region, Z4 is the CD8 transmembrane domain, "-" is a peptide bond or a connecting peptide. (b) the recombinant protein has a sequence as set forth in positions 1 to 249 or positions 25 to 249 of SEQ ID No:1, or a sequence as set forth in positions 1 to 246 or positions 22 to 246 of SEQ ID No:2.

[0008] In another preferred embodiment, said Z2 comprises a wild-type or mutant IL-21 element. In another preferred embodiment, said Z2 comprises IL-21 elements of human and non-human mammals.

[0009] In another preferred example, Z3 and Z4 are each independently a wild type or a mutant type. In another preferred embodiment, Z3 and Z4 are each independently derived from a human and a non-human mammal.

[0010] In another preferred embodiment, Z1 comprises the following sequence: [Table 1]

[0011] A second aspect of the present invention provides an isolated polynucleotide, which encodes a recombinant protein according to the first aspect of the invention.

[0012] A third aspect of the present invention provides a vector, said vector comprising a polynucleotide of the second aspect of the invention. In another preferred embodiment, the vector includes a plasmid or a viral vector. In another preferred embodiment, the vector is selected from the group consisting of a retroviral vector, an adenoviral vector, a lentiviral vector, and an AAV vector.

[0013] A fourth aspect of the present invention provides a genetically engineered cell, wherein the genetically engineered cell is selected from the group consisting of embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells (iPSC cells), NK cells, T cells, lymphocytes, or combinations thereof. In another preferred embodiment, the genetically engineered cells include NK cells, T cells, or lymphocytes.

[0014] The genetically engineered cells also have the following characteristics: comprising a vector according to the third aspect of the invention, or The genome incorporates a polynucleotide according to the second aspect of the invention. In another preferred embodiment, the genetically engineered cells express membrane-bound IL-21 (mbIL-21), and the mbIL-21 protein is present on the cell membrane.

[0015] A fifth aspect of the present invention provides a pharmaceutical composition comprising the genetically engineered cell according to the fourth aspect of the present invention and a pharmaceutically acceptable carrier thereof. In another preferred embodiment, the genetically engineered cells include NK cells, T cells, or lymphocytes. In another preferred embodiment, the pharmaceutical composition is used for cell therapy. In another preferred embodiment, the cell therapy is used to treat tumors.

[0016] A sixth aspect of the invention provides the use of a genetically engineered cell according to the fourth aspect of the invention, wherein the genetically engineered cell is used for the preparation of a drug for immunotherapy.

[0017] A seventh aspect of the invention provides a method of cellular immunotherapy, said immunotherapy comprising administering to a patient genetically engineered cells according to the fourth aspect of the invention, wherein said genetically engineered cells comprise a vector according to the third aspect of the invention or have integrated into their genome a polynucleotide according to the second aspect of the invention.

[0018] In another preferred embodiment, the genetically engineered cells express membrane-bound IL-21 (mbIL-21), and the mbIL-21 protein is present on the cell membrane.

[0019] In another preferred embodiment, the genetically engineered cells are selected from the group consisting of embryonic stem cells, mesenchymal stem cells, iPSC cells, NK cells, T cells, lymphocytes, or a combination thereof.

[0020] In another preferred example, the patient is a tumor patient, preferably suffering from a tumor selected from the group consisting of hematological tumors, lung cancer, liver cancer, neuroblastoma, ovarian tumor, rhabdomyosarcoma, breast cancer, gastric cancer, gastrointestinal tumor, kidney cancer, and prostate cancer.

[0021] In another preferred embodiment, the patient is a tumor patient, preferably an early stage tumor patient who does not require radiotherapy or chemotherapy, or an end-stage tumor patient who cannot undergo radiotherapy or chemotherapy.

[0022] In another preferred embodiment, the immunotherapy is administered by intravenous injection or local tissue injection. [Effects of the Invention]

[0023] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief explanation of the drawings]

[0024] [Figure 1] 1 shows a schematic diagram of the structure of the recombinant protein mbIL-21. [Figure 2] NK cell isolation, culture and identification. [Figure 3] 1 shows the detection of mbIL-21 expression status on the surface of NK cells. [Figure 4] Figure 1 shows that mbIL-21 promotes NK cell division and proliferation. [Figure 5] Figure 1 shows that mbIL-21 stimulates NK cell proliferation. [Figure 6] Figure 2 shows that mbIL-21 promotes STAT3 phosphorylation in NK cells. [Figure 7] 1 shows the effect of mbIL-21 on CD226 expression. DETAILED DESCRIPTION OF THE INVENTION

[0025] As a result of extensive and thorough research, the present inventors have unexpectedly discovered a recombinant protein with a special structure, namely, membrane-bound IL-21 (mbIL-21), which, when expressed in an anchored form on the cell membrane surface of lymphocytes such as NK cells, significantly promotes the division and proliferation of NK cells, prolongs the survival time of NK cells in vivo, and significantly reduces or eliminates the potential risks of secreted IL-21 that are unfavorable to NK cell therapy, thereby enhancing the therapeutic effect of NK cell immunotherapy. Based on this, the present invention has been completed.

[0026] term To facilitate understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise specified herein, each of the following terms shall have the meaning indicated below.

[0027] The term "about" can refer to a value or configuration within an acceptable error range of a particular value or configuration, as determined by one of ordinary skill in the art, which will depend in part on how the value or configuration is measured or determined.

[0028] The term "administration" refers to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as injection or infusion.

[0029] Membrane-bound IL-21 As used herein, the terms "protein of the invention", "membrane-bound IL-21", "membrane-bound IL-21 protein", "membrane-bound IL-21 of the invention", "mbIL-21 protein" and the like are used interchangeably and refer to a recombinant protein according to the first aspect of the invention. Typically, the membrane-bound IL-21 protein of the present invention has the structure shown in Formula I:

[0030] NK cells As used herein, the terms "NK cells," "memory NK cells," "natural killer cells," "natural killer (NK) cells," "NK," and the like are used interchangeably and refer to primary immune effector cells that protect the body from viral infections and tumor cell invasion through non-antigen-specific pathways. Through engineered (genetically modified) NK cells, they may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxic effects.

[0031] vector The present invention provides a vector comprising the nucleotide of the present invention. Retrovirus-derived vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes into the cellular genome and replication through genome replication in daughter cells. Compared to vectors derived from oncogenic retroviruses, such as murine leukemia viruses, lentiviral vectors have the advantage of being able to transduce non-proliferating cells and of low immunogenicity.

[0032] Genetically engineered cells expressing membrane-bound IL-21 The present invention provides genetically engineered cells that express the membrane-bound IL-21 of the present invention, wherein the genetically engineered cells are selected from the group consisting of embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells (iPSC cells), NK cells, T cells, lymphocytes, or combinations thereof.

[0033] Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising the engineered immune cells of the present invention, as well as pharmaceutically acceptable carriers, diluents or excipients thereof. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation.

[0034] In one embodiment, the formulation may include a buffer solution such as neutral buffered saline or sulfate buffered saline, a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol, a protein, a polypeptide or an amino acid such as glycine, an antioxidant, a chelating agent such as EDTA or glutathione, and a preservative. The formulation of the present invention is preferably formulated for intravenous administration or local tissue administration.

[0035] Therapeutic Applications The present invention includes therapeutic applications of cells (e.g., NK cells) transduced with vectors (e.g., lentiviral vectors) containing the recombinant proteins of the present invention. NK cells can target surface markers on tumor cells, significantly improving the proliferation efficiency of the transduced NK cells and their killing ability against tumor cells.

[0036] The main advantages of the present invention are: (a) The mbIL-21 of the present invention significantly enhances the proliferation and killing ability of NK cells in vivo and in vitro, thereby enhancing the effects of NK cell therapy. (b) The mbIL-21 nucleotide fragment of the present invention can be inserted into an expressible site of pluripotent stem cells (iPSCs, ESCs, etc.) through gene editing, and the differentiated immune cells, represented by NK cells, have stronger proliferation and killing abilities.

[0037] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions are generally in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by manufacturers. Unless otherwise specified, percentages and parts are calculated as weight percentages and weight parts.

[0038] Example 1. Design of the recombinant protein mb-IL21 The main structure of the recombinant protein mb-IL21 in this example is as shown in Figure 1, in which the signal peptide sequence of IL21, the hinge region sequence of CD8 (hinge), and the transmembrane sequence (TM) of CD8 are used.

[0039] The amino acid sequence of mbIL-21 in this example is as shown in SEQ ID No: 1 or 2, [ka] [ka]

[0040] The sequence of the recombinant protein mb-IL21 in this example is an artificially synthesized nucleotide sequence corresponding to SEQ ID No: 1 or 2. Furthermore, we constructed mb-IL21 with a P2A sequence, and mbIL-21 is linked to the mCherry reporter gene via P2A, allowing for easier and faster monitoring of the stability of the entire expression system during the initial evaluation and screening of design schemes for these molecules.

[0041] Example 2. Isolation, culture, expansion and identification of NK cells NK cells are isolated from human peripheral blood mononuclear cells (PBMCs). After culture and expansion, the acquired NK cells are identified. The identification results are shown in Figure 2. The results showed that CD3 + CD56 + demonstrates that the NK cell population is greater than 90%.

[0042] Example 3. Preparation of genetically engineered NK cells expressing mbIL-21 To verify the function of mbIL-21, the coding sequence of mbIL-21 was cloned into a retroviral vector, packaged, and used to generate retroviruses that were then used to infect NK cells. Flow cytometry was then used to detect IL-21 expression on the NK cell membrane. The expression rates of IL-21 on NK cells from different donors were found to range from 25% to 40%. In this example, the mbIL-21 used was IL21SP-IL21-GGGGS-CD8 Hinge-CD8™ (SEQ ID No: 1). result As shown in Figure 3, NK cells expressing mbIL-21 on the cell surface are obtained.

[0043] Example 4. Effect of mbIL-21 on NK cell proliferation To examine the effect of mbIL-21 on NK cell proliferation, in this example, NK-Ctrl and NK-mbIL-21 cells are labeled with CFSE and cultured in the absence of IL-2 (with or without IL-2) and at low concentrations of IL-2 (20 U / ml IL-2), respectively. In this example, the mbIL-21 used is IL21SP-IL21-GGGGS-CD8 hinge-CD8™ (SEQ ID No: 1).

[0044] The procedure is as follows: 1. Remove 6E6 NK-Ctrl or NK-mbIL-21 cells and centrifuge at 300g for 5 minutes at room temperature. Discard the supernatant. 2. Add 10mL of room temperature PBS, gently blow off the cells, and centrifuge at 300g for 5 minutes at room temperature. Discard the supernatant. 3. Dilute 6µL of CellTrace™ CFSE dye in 6mL of PBS to prepare a mixture. 4. Resuspend the cells in the mixture from the previous step, corresponding to 1mL of mixture per 1E6 cells. 5. Place in a 37°C incubator in the dark and stain for 20 minutes. 6. Add 5x the volume of complete medium, and then place in a 37°C incubator in the dark for 5 minutes to stop staining. 7. Centrifuge at 300g for 5 minutes at room temperature and discard the supernatant. 7. Wash the cells 2-3 times with 10mL of room temperature PBS. Resuspend the cells in 8.12 mL of NK medium to adjust the cell density to 5E5 / mL, add to a 12-well plate, and place in a 37 °C incubator to culture for 3-5 days before detection.

[0045] result As shown in Figures 4 and 5, on day 0, the CFSE signal intensities of NK-Ctrl and NK-mbIL-21 are consistent. On days 3 and 4, under culture conditions without IL-2, mbIL-21 can significantly promote the division and proliferation of NK cells. In the presence of IL-2, mbIL-21 cannot play an additional role, since IL-2 itself can significantly stimulate NK cell proliferation. After modification with mbIL-21 designed according to the present invention, NK cells can proliferate independently of exogenous cytokines.

[0046] Example 5. Effect of mbIL-21 on STAT3 phosphorylation STAT3 plays a key role in the signal transduction and proliferative effects of IL-21. In this example, to detect the effect of mbIL-21 on STAT3 phosphorylation, the expression of phosphorylated STAT3 (pSTAT3) in NK-Ctrl and NK-mbIL-21 was detected by flow cytometry, and the mean fluorescence intensity (MFI) of pSTAT3 was calculated. In this example, the mbIL-21 used is IL21SP-IL21-GGGGS-CD8 hinge-CD8™.

[0047] The procedure is as follows: 1. Wash the cells twice with 10 mL of BD Pharmingen™ Stain Buffer (Cat. No. 554656). 2. Dilute the required amount of BD Phosflow™ Lyse / Fix Buffer (5x concentrate) 1:5 with deionized or distilled water, then preheat the solution to 37°C. 3. Homogenize the cells by pipetting with 500 μL of prewarmed 1x BD Phosflow™ Lyse / Fix Buffer (Cat. No. 558049), then incubate the cells at 37°C for 10 minutes. 4. Wash the cells twice with 1 mL of BD Pharmingen™ Stain Buffer (Cat. No. 554656). 5. Cool BD Phosflow™ Perm Buffer III to -20°C or 4°C before use. 6. Gently resuspend the cells in 500 μL of chilled BD Phosflow™ Perm Buffer III (Cat. No. 558050) and incubate on ice for 30 minutes. 7. Wash the cells twice with 1 mL of BD Pharmingen™ Stain Buffer (Cat. No. 554656). 8. Resuspend the cells in 100 μL of BD Pharmingen™ Stain Buffer (FBS) and stain with Alexa Fluor® 647 Mouse IgG2a Isotype Control (Cat. No. 558053) and Alexa Fluor® 647 Mouse Anti-Stat3 (pY705) Antibody (Cat. No. 557815) for 30 minutes in the dark at room temperature. 9. Wash the cells once with BD Pharmingen™ Stain Buffer and resuspend the cells in 200 μL of BD Pharmingen™ Stain Buffer for flow cytometry analysis.

[0048] The results are shown in FIG. 6, and demonstrate that expression of mbIL-21 significantly promotes the phosphorylation of STAT3.

[0049] Example 6. Effect of mbIL-21 on CD226 expression CD226, also known as DNAM-1, belongs to the immunoglobulin superfamily and is primarily expressed on NK cells, T cells, and some B cell subsets. Binding of CD226 on the surface of NK cells to ligands such as CD155 and CD112 on tumor cells enhances NK cell cytokine secretion and cytotoxicity. Studies have shown that CD226 expression on the surface of NK cells gradually decreases during aging and the development of cancer, suggesting that CD226 may mediate the immune surveillance function of NK cells. Furthermore, CD226 also plays an important role in maintaining the function of memory NK cells.

[0050] In this example, we investigated whether mbIL-21 expressed by NK cells affects CD226 expression. The procedure was as follows: 1. Collect 1E5-2E5 test cells and centrifuge at 200-400g for 5 minutes at 4°C. Discard the supernatant. 2. Wash once with 200ul of FACS buffer (1% FBS dissolved in PBS), then centrifuge at 200-400g for 5 minutes at 4°C and discard the supernatant. 3. Add 100ul of CD226 directly labeled antibody (1:100), mix thoroughly by pipetting, and incubate at 4°C for 30-60 minutes. 4. After incubation, add 200ul / well of FACS buffer, centrifuge at 200-400g for 5 minutes at 4°C, and discard the supernatant. 5. Repeat step 4 twice. 6. After washing, add 200 ul / well of FACS buffer to resuspend the cells and detect them by flow cytometry.

[0051] In this example, the mbIL-21 used is IL21SP-IL21-GGGGS-CD8 hinge-CD8™. The results are shown in FIG. 7, which demonstrate that mbIL-21 expressed on the surface of NK cells unexpectedly significantly promotes the expression of CD226 on the surface of NK cells.

[0052] discussion Cytokines such as IL-2 and IL-15 have many effects on immune cells such as NK cells. IL-2 is used to prolong the survival of NK cells in the host, but it can also induce the proliferation of Treg cells, which exert immunosuppressive effects. However, in clinical applications, intravenous administration of IL-2 has been found to cause symptoms such as fever and vomiting, as well as water and salt metabolism disorders and functional abnormalities of the kidneys, liver, heart, and lungs. The most common and serious complication is capillary leak syndrome, which requires patients to discontinue treatment.

[0053] IL-15 also has several functions similar to IL-2, including stimulating the proliferation of activated T cells, generating cytotoxic effector T cells, and activating and maintaining NK cells. Unlike IL-2, IL-15 is generally not thought to induce the massive proliferation of Treg cells. Furthermore, IL-15 also plays an important role in the generation and maintenance of memory NK cells. However, studies have shown that the prolonged presence of IL-15 under prolonged in vitro expansion conditions can cause NK cells to exhibit an exhausted phenotype.

[0054] IL-21 activates the JAK-STAT, PI3K, and MAPK pathways. STAT3 plays a major role in the biological effects of IL-21, but STAT1 also contributes to the expression of IL-21-regulated genes. IL-21 plays an important role in regulating NK cell proliferation, cytotoxicity, and memory. For example, IL-21 can promote the secretion of IFN-γ by NK cells. The use of IL-21-expressing K562 cells as trophoblast cells allows for the expansion of large numbers of highly activated NK cells in vitro and ameliorate the exhaustion of NK cells that often occurs during the culture process.

[0055] Through research, the inventors have unexpectedly discovered that by modifying IL-21 to express it fixedly on the surface of specific cells to form membrane-bound IL-21 (mbIL-21), the in vitro proliferation ability of NK cells is significantly enhanced, NK cell exhaustion is reduced, and the lifespan of NK cells within the host is extended, thereby enhancing the killing ability of NK cells against tumor cells. In particular, the optimized mbIL-21 protein can significantly promote NK cell proliferation and the phosphorylation of intracellular signals such as STAT3, and has the prospect of very broad clinical application.

[0056] Furthermore, when used in combination with factors such as IL-12, IL-15, and IL-18, mbIL-21 of the present invention can synergistically promote the generation of memory NK cells.

[0057] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application.

Claims

1. A recombinant protein comprising: The recombinant protein is characterized in that it is membrane-bound IL-21 (mbIL-21).

2. The membrane-bound IL-21 has the structure shown in Formula I: Z1-Z2-Z3-Z4(I) In the formula: Z1 is a signal peptide or none, Z2 is an IL-21 polypeptide; Z3 is the CD8 hinge region, Z4 is the CD8 transmembrane domain, "-" is a peptide bond or a connecting peptide The recombinant protein of claim 1.

3. The membrane-bound IL-21 has a sequence shown in positions 1 to 249 or 25 to 249 of SEQ ID No: 1, or a sequence shown in positions 1 to 246 or 22 to 246 of SEQ ID No:

2. The fusion protein of claim 2.

4. 1. An isolated polynucleotide comprising:

2. The isolated polynucleotide, characterized in that it encodes the recombinant protein of claim 1.

5. A vector comprising: The vector, characterized in that it comprises the polynucleotide of claim 4.

6. A genetically engineered cell, A genetically engineered cell, characterized in that the genetically engineered cell contains the vector described in claim 5 or has the polynucleotide described in claim 4 integrated into its genome.

7. The genetically engineered cells are selected from the group consisting of embryonic stem cells, mesenchymal stem cells, iPSC cells (induced pluripotent stem cells), NK cells, T cells, lymphocytes, or a combination thereof. The genetically engineered cell of claim 6.

8. 1. A pharmaceutical composition comprising:

10. The pharmaceutical composition, comprising the genetically engineered cells of claim 6 and a pharmaceutically acceptable carrier.

9. 7. Use of the genetically engineered cells of claim 6, 7. Use of the genetically engineered cells according to claim 6, characterized in that the genetically engineered cells are used for the preparation of drugs for use in immunotherapy.

10. 2. A cellular immunotherapy comprising: The cellular immunotherapy method comprises administering to a patient genetically engineered cells, wherein the genetically engineered cells contain the vector of claim 5 or have the polynucleotide of claim 4 integrated into their genome.

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