CXCR4 high expression type iPSC-NK cell with enhanced bone marrow and tumor tissue homing ability, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and provides a CXCR4 high expression type iPSC-NK cell with enhanced bone marrow and tumor tissue homing ability, and a preparation method and application thereof. The cell takes pluripotent stem cells as starting cells, overexpresses a membrane-bound IL-15 and IL-15RA fusion protein gene and a CXCR4 receptor gene in the pluripotent stem cells; pluripotent stem cells stably expressing the target gene are obtained, and then iPSC-NK cells are obtained through induction differentiation. The membrane-bound IL-15 and IL-15RA fusion protein gene and the CXCR4 receptor gene are targetedly integrated into a safe harbor site of the induced pluripotent stem cell through a gene editing technology, so as to construct an iPSC cell strain stably expressing key proteins; the function-enhanced iNK cell is obtained through induction differentiation, and exhibits excellent bone marrow and various solid tumor tissue homing ability and persistent immune killing activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a CXCR4-overexpressing iPSC-NK cell with enhanced homing ability to bone marrow and tumor tissues, its preparation method, and its application. Background Technology

[0002] Natural killer (NK) cells are an important component of the body's innate immune system, capable of recognizing and eliminating tumor cells and virus-infected cells. NK cell immune function plays a crucial role in acute myeloid leukemia (AML) and various solid tumors. Currently, peripheral blood-based NK cell therapy suffers from limitations such as limited source, difficulty in expansion, short survival time, and insufficient homing ability, restricting its widespread clinical application.

[0003] In recent years, NK cells (iNK) derived from induced pluripotent stem cells (iPSCs) have become a hot topic in the research and development of next-generation cell therapy products due to their controllability, scalability, and functional consistency. Some literature reports that iPSC-derived NK cells have superior in vitro function and expansion potential compared to peripheral blood-derived NK cells, but they still face limitations such as limited in vivo survival and insufficient targeted homing efficiency.

[0004] CXCR4, as the receptor for CXC chemokines, mediates the chemotactic migration of NK cells to the bone marrow and tumor microenvironment, and is a key factor affecting the therapeutic efficacy of NK cell therapy.

[0005] In addition, the expression of the IL-15 / IL-15RA fusion protein can maintain the activated state of NK cells in vivo and improve their proliferation and survival, but NK cells that express IL-15 / IL-15RA alone still have the problem of insufficient homing.

[0006] Despite some progress in the preparation of NK cells derived from induced pluripotent stem cells (iPSCs) and their anti-tumor applications, the following significant shortcomings still limit their efficacy and widespread application in hematological malignancies and solid tumors.

[0007] Lack of systematically optimized homing ability regulation mechanisms: Current mainstream iPSC-NK cell products, such as FT516 and FT576 developed by FateTherapeutics, enhance cell survival by expressing IL-15, but they are not systematically designed to target the chemotactic mechanisms in the tumor microenvironment. NK cells have limited migration and infiltration efficiency in bone marrow and immune-rejecting solid tumors, resulting in insufficient killing activity in CXCL12-rich areas (such as AML lesions and pancreatic cancer).

[0008] IL-15 expression strategies have dose limitations and toxicity risks: Current methods often employ systemic exogenous IL-15 infusion or NK cell expression of free IL-15 to enhance activity. These strategies may trigger excessive activation of the immune system, leading to side effects such as cytokine release syndrome (CRS), and lack spatial limitation. Although membrane-bound IL-15 (mbIL-15) has shown some improvement, it remains difficult to maintain long-term function in the core region of solid tumors without synergistic optimization with localization mechanisms.

[0009] CXCR4 expression protocols are mostly transient expressions on peripheral blood-derived NK cells, lacking stability: some literature (e.g., Cichocki et al., Nat Commun (2020) Transient expression of CXCR4 in peripheral blood NK cells via viral transduction, but this expression is unstable and greatly affected by the cellular metabolic state, making it unsuitable for use in batch, standardized cell therapy products, especially failing to meet the GMP consistency requirements for the "off-the-shelf" use of iPSC-derived allogeneic cells.

[0010] Insufficient penetration and sustained killing ability against solid tumors: Most iPSC-NK cell products lack specific tumor tissue recognition and sustained activation mechanisms, making it difficult to establish sustained immune suppression in "cold tumors" with low immune infiltration, such as pancreatic cancer and glioma. Even if they show initial activity in solid tumor models, they are unable to maintain infiltration and survival, resulting in limited efficacy.

[0011] In summary, no existing iPSC-derived NK cell product can simultaneously meet the following key therapeutic needs: precise homing, long-lasting activation, GMP-compliant production, and adaptability expansion for solid tumors. Therefore, developing a novel NK cell product that can simultaneously meet the above key therapeutic needs has significant clinical application value. Summary of the Invention

[0012] To address the shortcomings of existing technologies, the present invention aims to provide CXCR4-overexpressing iPSC-NK cells with enhanced homing ability in bone marrow and tumor tissues, along with their preparation method and applications. This invention overcomes the deficiencies of insufficient homing ability and limited in vivo survival of existing iPSC-derived NK cells. Through gene editing technology, it constructs functionally enhanced iNK cells that simultaneously highly express CXCR4 and membrane-bound IL-15 / IL-15RA fusion proteins, achieving highly efficient homing and durable immune activity in bone marrow and CXCL12-overexpressing solid tumors, thereby improving anti-tumor efficacy and expanding the indications for cell therapy.

[0013] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides iPSC-NK cells with high CXCR4 expression that enhance the homing ability of bone marrow and tumor tissues. The cells are derived from pluripotent stem cells, which overexpress membrane-bound IL-15 and IL-15RA fusion protein genes and CXCR4 receptor genes in the pluripotent stem cells. Stable pluripotent stem cells expressing the target genes are obtained, and then iPSC-NK cells are obtained through induced differentiation.

[0014] In this invention, the cells expressing the membrane-bound IL-15 / IL-15RA fusion protein can form an autocrine / paracrine activation loop, enhancing NK cell survival, proliferation, and cytotoxicity in vivo; functional expression of the CXCR4 receptor responds to the CXCL12 gradient, enhancing the homing ability of bone marrow and tumor tissues. These cells are suitable for treating AML MRD-positive patients and various CXCL12-high expressing solid tumors (pancreatic cancer, ovarian cancer, breast cancer, non-small cell lung cancer, or glioma, etc.), possessing the dual advantages of "targeted homing + local activation." This invention also provides a method for constructing the above-mentioned cells, including designing and synthesizing expression vectors, CRISPR / Cas9-mediated safe harbor targeted insertion, screening and expanding monoclonal antibodies, and inducing differentiation into mature NK cells.

[0015] Preferably, the overexpression is performed by integrating the target gene into the safe harbor site of human pluripotent stem cells using CRISPR / Cas9 gene editing technology.

[0016] In this invention, a genomic region that can safely accommodate new exogenous genes without causing other unexpected changes to the cellular genome that may pose a risk to the patient is called a Genomic Safe Harbor (GSH), which can improve the effectiveness, durability, and predictability of gene and cell therapies.

[0017] Preferably, the safe harbor site includes any one of the sites AAVS1, ROSA26, CCR5, H11 or COL1A1, with H11 being the preferred site.

[0018] Preferably, the membrane-bound IL-15 and IL-15RA fusion protein genes are expressed in tandem in the same expression vector to form an open reading frame and are driven by a single promoter, while the CXCR4 receptor gene is expressed separately in another expression vector. Alternatively, the membrane-bound IL-15 and IL-15RA fusion protein gene and the CXCR4 receptor gene are expressed in tandem in the same expression vector via the GSG-P2A self-cleaving polypeptide linker, forming an open reading frame and driven by a single promoter.

[0019] In one specific embodiment of the present invention, the membrane-bound IL-15 / IL-15RA fusion protein gene and the CXCR4 receptor gene are linked in the same expression vector via GSG-P2A and named GX11-15RFCR4.

[0020] In this invention, the promoter is selected from any one of pGK, CAG, CMV, EF1α, or UBC.

[0021] Preferably, the expression vector is co-transfected with Cas9 RNP into pluripotent stem cells.

[0022] Preferably, the Cas9 RNP includes sgRNA and Cas9 protein.

[0023] In this invention, the Cas9 RNP consists of Cas9 protein and sgRNA. The two are assembled in vitro through electrostatic interaction and structural chimerism to form an RNP complex. The RNP complex and expression vector plasmid are then delivered into the cell nucleus by electroporation to exert gene editing function.

[0024] Preferably, the expression vector comprises an upstream homologous arm, a cHS4 insulator, a pCAG promoter element, a nucleotide sequence encoding a membrane-bound IL-15 / IL-15RA fusion protein, a GSG-P2A linker, a nucleotide sequence encoding a CXCR4 receptor protein, a poly A signaling element, a CXB3 insulator, and a downstream homologous arm element.

[0025] In this invention, the membrane-bound IL-15 and IL-15RA fusion protein contains an extracellular domain fusion structure that enables autocrine / paracrine functions, thereby enhancing the survival and proliferation of NK cells in vivo.

[0026] Preferably, the amino acid sequence of the membrane-bound IL-15 and IL-15RA fusion protein gene is shown in SEQ ID NO:2.

[0027] In this invention, the nucleotide sequence of the membrane-bound IL-15 and IL-15RA fusion protein gene is shown in SEQ ID NO:1.

[0028] Preferably, the amino acid sequence of the CXCR4 receptor gene is shown in SEQ ID NO:4.

[0029] In this invention, the nucleotide sequence of the CXCR4 receptor gene is shown in SEQ ID NO:3.

[0030] In this invention, the CXCR4 receptor can respond to high concentrations of the chemokine CXCL12 in the tumor microenvironment, significantly enhancing the chemotactic homing ability of NK cells to bone marrow and solid tumor tissues.

[0031] Preferably, the step of inducing differentiation includes: (a) Days 1-7: Hematopoietic precursor induction culture, using basal medium containing cytokines BMP4, VEGF, bFGF and SCF to culture pluripotent stem cells with the target gene integrated; (b) Days 8-25: NK cells were induced to differentiate and cultured in a medium containing cytokines SCF, IL-7, FLT31, IL-15, and IL-3. (c) Days 26-39: NK cell maturation and expansion culture. Cells harvested on day 25 and K-562 feeder cells expressing mbIL-21, CD137L, and DLL-1 genes were seeded into a GREX culture system for culture, and high-purity CD56 cells were harvested. + CD16 + NK cells.

[0032] In this invention, in step (a), the basal culture medium is Stempro-34 SFM serum-free culture medium.

[0033] In this invention, in step (a), the culture conditions are as follows: continuous rotation and incubation at 70 rpm in a CO2-resistant orbital oscillator in a 37°C, 5% CO2 incubator.

[0034] Preferably, in step (a), the concentrations of the cytokines BMP4, VEGF, and bFGF in the basal culture medium are each independently 8-12 ng / mL, for example, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, or 12 ng / mL.

[0035] Preferably, in step (a), the concentration of the cytokine SCF in the basal culture medium is 18-22 ng / mL, for example, it can be 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL or 22 ng / mL, etc.

[0036] In one specific embodiment of the present invention, in step (b), the basal culture medium is DMEM medium, Ham's F12 medium and human AB serum; the proportions are 56.7%, 28.3% and 15%, respectively.

[0037] Preferably, in step (b), the concentrations of the cytokines SCF and IL-7 in the basal culture medium are each independently 18-22 ng / mL, for example, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL or 22 ng / mL, etc.

[0038] Preferably, in step (b), the concentrations of the cytokines FLT31 and IL-15 in the basal culture medium are each 8-12 ng / mL, for example, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL or 12 ng / mL.

[0039] Preferably, in step (b), the concentration of IL-3 in the basal culture medium is 4-6 ng / mL, for example, it can be 4 ng / mL, 5 ng / mL or 6 ng / mL, etc.

[0040] In one specific embodiment of the present invention, step (b) includes the following culture method: From day 8 to 14, cells are continuously incubated in a medium containing DMEM, Ham's F12, human AB serum, SCF, IL-7, FLT31, IL-15, and IL-3 on an orbital oscillator placed in a 37°C, 5% CO2 incubator at 70 rpm, with the medium being replaced every 2 days. From day 15 to 25, cells are continuously incubated in a medium containing DMEM, Ham's F12, human AB serum, SCF, IL-7, FLT31, and IL-15 on an orbital oscillator placed in a 37°C, 5% CO2 incubator at 70 rpm, with the medium being replaced every 2 days.

[0041] Preferably, in step (c), the inoculation ratio of the cells harvested on day 25 to K-562 feeder cells is 1:(1.8-2.2), for example, it can be 1:1.8, 1:2 or 1:2.2, etc.

[0042] Preferably, in step (c), the culture medium used for the culture is NK Macs medium containing 2-10% (e.g., 2%, 4%, 5%, 6%, 8% or 10%) human serum and 20-200 (e.g., 20, 50, 100, 150 or 200) IU IL-2.

[0043] In one specific embodiment of the present invention, in step (c), the culture method includes: seeding cells harvested on day 25 and K-562 feeder cells expressing genes such as mbIL-21, CD137L, and DLL-1 into the GREX culture system at a ratio of 1:2. The culture medium used is NK Macs medium containing 5% human serum and 200 IU IL-2, and the feeder cells are replenished every 7 days.

[0044] In this invention, NK cells expressing the above-mentioned membrane-bound IL-15 / IL-15RA fusion protein and CXCR4 receptor exhibit excellent migration ability of CXCL12 protein in vitro.

[0045] In this invention, a dual-function enhancing gene construct is employed to co-express the CXCR4 receptor and the membrane-bound IL-15 / IL-15RA fusion protein. This invention integrates two functional modules, CXCR4 and membrane-bound IL-15 / IL-15RA, into the same iPSC clone, respectively enhancing the cell's tumor homing ability and persistent activation function in vivo, achieving a combined effect of "directed migration + sustained activation".

[0046] In this invention, a stable expression system is constructed at a safe harbor site. Gene editing technologies such as CRISPR / Cas9 are used to integrate the above-mentioned functional modules into the H11 safe harbor gene site of iPSC, ensuring the stability of expression, genetic safety, and scalable GMP production.

[0047] In this invention, a standardized engineered iPSC master cell bank (MCB) is constructed. The master cell bank is established based on monoclonal, highly characterized human iPSCs, which enables the development of cell therapy products with unified and traceable sources, thus distinguishing it from the batch-to-batch variation problem of traditional allogeneic NK cells.

[0048] In this invention, the induction and differentiation system of iPSCs into NK cells is optimized, and a specific stage induction system is established, including hematopoietic progenitor cell induction, NK cell induction differentiation and maturation expansion process, so as to achieve high-efficiency, high-purity and high-activity functional iNK cells.

[0049] In this invention, NK cells expressing the IL-15 / IL-15RA fusion protein can survive and proliferate on their own. The iNK cells obtained by this invention construct a stable autocrine / paracrine activation signal by binding IL-15 and its receptor α chain to the membrane, without relying on exogenous IL-2 / IL-15 supplementation, and have better proliferation and persistence in vivo.

[0050] In this invention, high expression of CXCR4 significantly improves the homing efficiency of tumor tissue and bone marrow tissue. The CXCR4 functional expression structure designed in this invention can respond efficiently to the CXCL12 concentration gradient and achieve a significant localization enrichment effect in AML and solid tumors with high expression of CXCL12 (such as pancreatic cancer, ovarian cancer, and glioblastoma).

[0051] In this invention, the cells are suitable for use in a universal iNK product for both hematologic and solid tumor applications. This invention overcomes the limitations of traditional NK products, enabling the use of iNK cells in the hematologic system (such as AML MRD) and CXCL12. + Its dual-indication application in solid tumors demonstrates broad-spectrum anti-tumor potential.

[0052] In this invention, the "off-the-shelf" immunotherapy product form of the cell-based CAR-T therapy, LB-09 iNK cells, has advantages such as cryopreservation, ready-to-use, and low toxicity, making it suitable for large-scale allogeneic applications and avoiding the problems of long manufacturing cycles, high toxicity, and high costs associated with autologous CAR-T.

[0053] In this invention, a non-viral gene editing system (such as CRISPR / Cas9 RNP) is used to mediate the targeted integration of the target gene into a safe harbor site on the genome of pluripotent stem cells, thereby improving clinical safety and reducing potential tumorigenicity and off-target risks.

[0054] In a second aspect, the present invention provides a method for preparing CXCR4-overexpressing iPSC-NK cells with enhanced homing ability to bone marrow and tumor tissues as described in the first aspect, the preparation method comprising: (1) Construct an expression vector plasmid containing the membrane-bound IL-15 and IL-15RA fusion protein gene and the CXCR4 receptor gene; (2) Co-transfect pluripotent stem cells with the expression vector plasmid constructed in step (1) and Cas9 RNP to screen pluripotent stem cells that stably express the target gene. (3) Inducing differentiation of the pluripotent stem cells screened in step (2) to obtain iPSC-NK cells.

[0055] Preferably, in step (1), the membrane-bound IL-15 and IL-15RA fusion protein genes are expressed in tandem in the same expression vector to form an open reading frame and are driven by a single promoter, while the CXCR4 receptor gene is expressed separately in another expression vector; Alternatively, the membrane-bound IL-15 and IL-15RA fusion protein gene and the CXCR4 receptor gene are expressed in tandem in the same expression vector via the GSG-P2A self-cleaving polypeptide linker, forming an open reading frame and driven by a single promoter.

[0056] Preferably, the promoter is selected from any one of pGK, CAG, CMV, EF1α, or UBC.

[0057] Preferably, in step (1), the expression vector of the membrane-bound IL-15 / IL-15RA fusion protein gene includes an upstream homologous arm, a cHS4 insulator, a pCAG promoter element, a nucleotide sequence encoding the membrane-bound IL-15 / IL-15RA fusion protein, a GSG-P2A linker, a nucleotide sequence encoding the CXCR4 receptor protein, a poly A signaling element, a CXB3 insulator, and a downstream homologous arm element.

[0058] Preferably, in step (1), the amino acid sequence of the membrane-bound IL-15 and IL-15RA fusion protein gene is shown in SEQ ID NO:2.

[0059] Preferably, in step (1), the amino acid sequence of the CXCR4 receptor gene is as shown in SEQ ID NO:4.

[0060] Preferably, in step (1), the Cas9 RNP includes sgRNA and Cas9 protein.

[0061] Preferably, in step (1), the Cas9 RNP targets the safe harbor site of human pluripotent stem cells.

[0062] Preferably, the safe harbor site includes any one of AAVS1, ROSA26, CCR5, H11 or COL1A1 sites, with H11 site being the preferred one.

[0063] Preferably, in step (2), the co-transfection conditions are as follows: Cas9 protein and sgRNA are assembled in vitro through electrostatic interaction and structural chimerism to form an RNP complex, and then the RNP complex and expression vector plasmid are delivered into the cell nucleus together by electroporation to exert the gene editing effect.

[0064] Preferably, in step (2), the screening method includes: flow cytometry sorting, PCR and ddPCR identification, flow cytometry detection of exogenous gene expression, flow cytometry detection of cell totipotency, cell passage stability detection, and WGS and WES detection of pathogenicity assessment.

[0065] Preferably, in step (3), the step of inducing differentiation includes: (a) Days 1-7: Hematopoietic precursor induction culture, using basal medium containing cytokines BMP4, VEGF, bFGF and SCF to culture pluripotent stem cells with the target gene integrated; (b) Days 8-25: NK cells were induced to differentiate and cultured in a medium containing cytokines SCF, IL-7, FLT31, IL-15, and IL-3. (c) Days 26-39: NK cell maturation and expansion culture. Cells harvested on day 25 and K-562 feeder cells expressing mbIL-21, CD137L, and DLL-1 genes were seeded into a GREX culture system for culture, and high-purity CD56 cells were harvested. + CD16 + NK cells.

[0066] Thirdly, the present invention provides the application of CXCR4-overexpressing iPSC-NK cells with enhanced homing ability to bone marrow and tumor tissues as described in the first aspect in the preparation of antitumor drugs, wherein the tumors include bone marrow hematologic malignancies or solid tumors expressing high levels of the chemokine CXCL12.

[0067] Preferably, the bone marrow hematologic malignancies include acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, myeloproliferative neoplasm, or myelodysplastic syndrome; preferably, acute myeloid leukemia.

[0068] Preferably, the solid tumors expressing high levels of the chemokine CXCL12 include pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, non-small cell lung cancer, glioma, or melanoma.

[0069] In this invention, the iPSC-NK cells can be used to prepare therapeutic drugs for treating residual molecular disease in acute myeloid leukemia (AML), and also for treating various solid tumors with high CXCL12 expression, such as pancreatic cancer, ovarian cancer, glioblastoma, breast cancer, or prostate cancer. They can also be used to improve the homing ability of NK cells in tumor tissues and their survival and anti-tumor persistence in the tumor microenvironment. Furthermore, they can be used as "off-the-shelf" allogeneic immunotherapy cell products to replace autologous CAR-T cells and reduce the risk of immunotoxicity.

[0070] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0071] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a functionally enhanced NK cell product with significant advantages by stably expressing the CXCR4 and membrane-bound IL-15 / IL-15RA fusion protein in iPSC-derived NK cells. The main effects and advantages are as follows: (1) Significant improvement in targeted homing ability.

[0072] Compared with iNK cells that express only IL-15, the CXCR4-overexpressing iNK cells constructed in this invention showed a significantly improved migration rate in the in vitro CXCL12 chemotaxis assay.

[0073] In the NOG mouse model, after intravenous injection of luciferase-labeled iNK cells, the cumulative signal in the bone marrow was 52 times and 47 times that of the control group on day 10 and day 28, respectively, indicating a significant enhancement in their sustained homing ability.

[0074] (2) Improvement in immune activation and survival ability.

[0075] The construction of membrane-bound IL-15 / IL-15RA enabled LB-09 iNK cells to maintain a survival rate of over 80% after continuous in vitro culture for more than 14 days without exogenous cytokines.

[0076] Compared with wild-type iNK cells, it can maintain significant CD107a expression, IFN-γ secretion level and tumor killing ability under IL-2-free conditions.

[0077] At a 1:1 effector-target ratio, the specific lysis rate of Thrp-1 cells was increased by approximately 60% (p<0.01).

[0078] (3) Expansion in the treatment of solid tumors.

[0079] CXCR4 overexpression enables iNK cells to efficiently enter CXCL12-enriched regions, such as "immune rejection" solid tumors like pancreatic cancer and ovarian cancer.

[0080] In a xenograft pancreatic cancer model, the cells of this invention significantly inhibited tumor growth, with a tumor volume difference of more than 70% between the treatment group and the control group on day 21 (p<0.01).

[0081] (4) Improvement in process consistency and product stability.

[0082] By integrating dual-function modules at safe harbor sites such as AAVS1 and H11, the risk of insertional mutations from viral vectors is avoided, ensuring consistent product genetic background and supporting large-scale clinical-grade GMP production.

[0083] The finished LB-09 iNK cells have a batch-to-batch coefficient of variation (CV) of <10%, a viability of >90%, and target gene expression fluctuations within ±15%.

[0084] (5) Safety and efficacy in preliminary clinical trials.

[0085] In the IIT (Investigator Initiated Trial), among 10 patients with acute myeloid leukemia who were MRD-positive, 78% achieved MRD negativity after using LB-09 (including q-PCR and flow cytometry double positivity conversion), with the effect being particularly significant in the pediatric group.

[0086] No serious cytokine release syndrome (CRS), ICANS, or graft-versus-host disease (GvHD) events occurred in any of the subjects, indicating a favorable safety profile.

[0087] (6) Other process and usage advantages.

[0088] This product is ready to use after cryopreservation and is packaged in 30 mL cryopreservation bags with standardized packaging for easy transportation and rapid application. It possesses all the characteristics of "off-the-shelf" allogeneic immunotherapy products: large-scale production, standardization, consistency, high safety, and ease of use. It reduces dependence on exogenous cytokines, which helps reduce drug compatibility complexities and the risk of immunotoxicity. It can be applied to patients with immunodeficiency and those experiencing bone marrow suppression after chemotherapy, with a wide range of indications. Attached Figure Description

[0089] Figure 1 To detect the site-directed integration of the KI gene in LB monoclonal strains using PCR as a preliminary screening method.

[0090] Figure 2 To detect the site-directed integration of the KI gene in LB monoclonal strains using PCR and ddPCR.

[0091] Figure 3 The results of random insertion of LB monoclonal vector fragments were used for PCR detection.

[0092] Figure 4 The expression of transgenes (IL-15, CXCR4) in LB monoclonal strains.

[0093] Figure 5 The results show the functional activity of iNK and K562 from different iPSC clone sources under an effector-target ratio of 1:1.

[0094] Figure 6 The results show the functional activity of iNK and K562 from different iPSC clone sources under an effector-target ratio of 3:1.

[0095] Figure 7 The results show the functional activity of iNK and THP1 from different iPSC clone sources under an effector-target ratio of 1:1.

[0096] Figure 8 The results show the functional activity of iNK and THP1 from different iPSC clone sources under an effector-target ratio of 3:1.

[0097] Figure 9 The graph shows the results of the in vitro migration ability of LB-09 into the lower chamber containing different concentrations (0, 2, 10, 50 or 250 ng / mL) of CXCL12 by the Transwell assay.

[0098] Figure 10 The production of IFN-γ after iNK cells were co-cultured with K562 and THP1 cells for 24 hours.

[0099] Figure 11 The production of TNF-α was observed after iNK cells were co-cultured with K562 and THP1 cells for 24 hours.

[0100] Figure 12 Results show the in vitro survival ability of LB-09 under cytokine-free conditions.

[0101] Figure 13 Bioluminescence imaging results of mice loaded with THP-1 tumors after treatment with vector control, wild-type iNK, iNK expressing IL-15, or dual-modified iNK co-expressing IL-15 and CXCR4.

[0102] Figure 14 The tumor burden curves for each group in the Thrp-1-luc-GFP xenograft model at different time points are shown.

[0103] Figure 15 Bioluminescence imaging of isolated femur was used to assess intramedullary tumor burden in each group.

[0104] Figure 16 The distribution of iNK cells in peripheral blood as detected on day 10.

[0105] Figure 17 The distribution of iNK cells in (femoral-derived) bone marrow as detected on day 10.

[0106] Figure 18 The distribution of iNK cells in peripheral blood as detected on day 28.

[0107] Figure 19 The distribution of iNK cells in (femoral-derived) bone marrow as detected on day 28. Detailed Implementation

[0108] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0109] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0110] Example 1 A human iPSC cell line was constructed that highly expresses CXCR4 and the membrane-bound IL-15 / IL-15RA fusion protein.

[0111] 1. Materials and Methods.

[0112] (1) Cell line: Human CD34 with normal karyotype and strict quality control. +The umbilical cord blood-derived iPSC cell line (named WT-iPSC).

[0113] (2) Gene editing tools: The CRISPR / Cas9 system was used to design an sgRNA sequence (SEQ ID NO:5) targeting the H11 safe harbor site. The corresponding sequence of the sgRNA is acaaggctatgctatctata.

[0114] (3) Carrier construction.

[0115] The expression vector contains an upstream homologous arm, a cHS4 insulator, a pCAG promoter element, a nucleotide sequence encoding a membrane-bound IL-15 / IL-15RA fusion protein, a GSG-P2A linker, a nucleotide sequence encoding a CXCR4 receptor protein, a polyA signaling element, a CXB3 insulator, and a downstream homologous arm element.

[0116] The nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO:23.

[0117] The nucleotide sequence of the cHS4 insulator is shown in SEQ ID NO:24.

[0118] The nucleotide sequence of the pCAG promoter element is shown in SEQ ID NO:25.

[0119] The amino acid sequence of the GSG-P2A linker is shown in SEQ ID NO:26.

[0120] The nucleotide sequence of the GSG-P2A linker is shown in SEQ ID NO:30.

[0121] The nucleotide sequence of the poly A signal element is shown in SEQ ID NO:27.

[0122] The nucleotide sequence of the CXB3 insulator is shown in SEQ ID NO:28.

[0123] The nucleotide sequence of the downstream homologous arm element is shown in SEQ ID NO:29.

[0124] The coding sequences of the membrane-bound IL-15 and IL-15RA fusion protein and the full-length coding sequence of CXCR4 were artificially synthesized and cloned into an expression vector with a strong promoter, serving as donor plasmids for gene editing.

[0125] The gene coding sequence of the membrane-bound IL-15 and IL-15RA fusion protein is shown in SEQ ID NO:1.

[0126] The amino acid sequence of the membrane-bound IL-15 and IL-15RA fusion protein gene is shown in SEQ ID NO:2.

[0127] The full-length CXCR4 coding sequence is shown in SEQ ID NO:3.

[0128] The amino acid sequence of the CXCR4 receptor gene is shown in SEQ ID NO:4.

[0129] (4) Transfection method: The Cas9-sgRNA complex and expression vector plasmid were simultaneously transfected into WT-iPSC using electroporation. The specific steps are as follows.

[0130] (a) The molar ratio of sgRNA to Cas9 (GenCRISPR™ Cas9 v1.1) was 4:1, and the amounts used were 60 pmol and 15 pmol, respectively. After mixing, the mixture was incubated at room temperature for 15 min.

[0131] (b) Add 1 μg of expression vector plasmid GX11-15RFCR4 and electroporate it into 8×10⁸ cells using a Lonza 4D MAX electroporation system and Lonza P3 Primary reagent. 5 WT-iPSC cells; 20 μL electroporation system, electroporation parameters (CA-137).

[0132] (c) After 8 days of electroporation, single clones were sorted using a flow cytometer.

[0133] (5) Single clone screening: After flow cytometry sorting, single clones were cultured and amplified, and then PCR and droplet digital PCR (ddPCR) were used to detect the site-specific integration of exogenous genes.

[0134] (a) PCR screening was performed using primers GX2-H11-outF / pCAG-JDR (primer information is shown in Table 1 below) across the 5' end homologous arm.

[0135] Table 1 (b) The single clones that are positive in the initial screening are cultured and the clones are selected according to their cell status. DNA is extracted using the PuroLink GenomicDNA Mini Kit for PCR identification and droplet digital PCR (ddPCR) detection.

[0136] (c) PCR identification of KI type of each clone.

[0137] PCR identification of the KI type of each clone was performed using three primer pairs: GX2-H11-outF / pCAG-JDR (primer pair across the 5' homologous arm), GX9-bGH-pF1 / C2332_3'-1152_R1 (primer pair across the 3' homologous arm), and H11-fragF201 / H11-fragR943 (primer information is shown in Table 2 below).

[0138] Table 2 If both the 5' and 3' homologous arms are positive and the WT test is negative, the KI type is determined to be + / +, i.e., homozygous knock-in. If both the 5' and 3' homologous arms are positive and the WT test is positive, the KI type is determined to be + / -, i.e., heterozygous knock-in. If no such result is obtained, the relevant clone is discarded.

[0139] (d) Detection of IL15RF copy number using droplet digital PCR (ddPCR) technology.

[0140] Droplet digital PCR (ddPCR) technology was used to quantitatively analyze the transgenes integrated on each monoclonal genome: the IL15RF transgene sequence was detected by targeting specific TaqMan probes (primers and probes are shown in Table 3 below), and the endogenous gene RNase P with two copies in the human diploid genome was used as the reference standard (TaqMan™ Copy Number Reference Assay, human, RNase P). The transgene copy number was calculated according to the formula (IL15RF concentration (copies / µL) / RNase P concentration (copies / µL)) × 2.

[0141] Table 3 (e) PCR identification of the residual status of each cloning vector backbone.

[0142] To identify residual vector backbones in each clone, PCR was performed using three primer pairs: 18T-ori-pF1-1 / 18T-ori-pR1 (for detecting the Ori sequence in the vector backbone), Bb-K-qPF2 / Bb-K-qPR2 (for detecting the Kan sequence in the vector backbone), and Kan-pF / Kan-pR (for detecting the Kan sequence in the vector backbone). All three primer pairs were negative to preliminarily determine the absence of residual backbones; otherwise, residual backbones were considered to be present.

[0143] Table 4 2. Experimental results.

[0144] (1) Results of monoclonal screening.

[0145] I. Initial screening results for monoclonal antibodies.

[0146] Forty-five monoclonal antibodies were detected using the primer pair GX2-H11-outF / pCAG-JDR, which identifies the 5' homologous arm. The expected fragment size was 1584 bp. The results were obtained based on the agarose gel electrophoresis image of the product. Figure 1 The 26 single clones showing positive bands (LB02, LB04, LB09, LB10, LB14, LB15, LB16, LB17, LB18, LB19, LB22, LB23, LB24, LB25, LB26, LB28, LB32, LB33, LB34, LB35, LB36, LB37, LB38, LB39, LB44, and LB45) were selected for further culture.

[0147] II. Continue to identify monoclonal antibodies.

[0148] The initially positive single clones were cultured and 12 clones, namely LB02, LB04, LB09, LB10, LB22, LB23, LB24, LB25, LB28, LB35, LB36, and LB37, were selected for further testing based on cell status.

[0149] (a) PCR identification results of KI type of each clone.

[0150] Based on the PCR test results and judgment criteria, the KI type of clones LB02, LB04, and LB09 is initially determined to be + / +, while the KI type of clones LB22, LB23, LB24, LB25, LB28, LB35, LB36, and LB37 is determined to be + / -. A comprehensive judgment needs to be made in conjunction with the ddPCR test results.

[0151] Figure 2 To detect the site-directed integration of the KI gene in LB monoclonal strains using PCR and ddPCR, the genotypes shown are presented in Table 5 below.

[0152] Table 5 (b) Results of IL15RF copy number detection using droplet digital PCR (ddPCR) technology.

[0153] First, probe 15RF-P2 was used to detect 12 clones, namely LB02, LB04, LB09, LB10, LB22, LB23, LB24, LB25, LB28, LB35, LB36, and LB37, and the copy number was calculated based on the detection results. Then, probe 15RF-P4 was used to detect 5 clones, namely LB04, LB09, LB22, LB24, LB28, and LB37, and the copy number was calculated based on the detection results.

[0154] Clones with a KI copy number close to 1 and a PCR result of + / - are considered single-copy (heterozygous). Clones with a KI copy number close to 2 and a PCR result of + / - are considered single-copy but contain random integration. Clones with a KI copy number close to 2 and a PCR result of + / + are considered double-copy (homozygous). Clones with a KI copy number close to any other positive integer are considered clones with random integration, regardless of the PCR result. Based on this, LB02, LB04, and LB09 are double-copy clones; LB22, LB24, and LB28 are single-copy clones; and LB10, LB23, LB25, LB35, LB36, and LB37 are single-copy clones containing random integration. Table 6 shows the results of the KI gene copy number identification analysis for LB single-clone strains.

[0155] Table 6 (c) PCR identification of the residual status of each cloning vector backbone.

[0156] Based on the PCR test results and judgment criteria, it was initially determined that LB02 and LB04 clones contained backbone integration, but a comprehensive judgment needs to be made in conjunction with the ddPCR test results.

[0157] Figure 3 The results of random insertion of LB monoclonal vector fragments were used for PCR detection.

[0158] In summary, we successfully electroporated the GX11-15RFCR4 vector plasmid into iPSC-ASE cells to obtain LB Pool cells. Flow cytometry sorted the LB Pool cells to obtain 190 LB monoclonal cell lines. Subsequent cell culture and screening identified 12 LB monoclonal cell lines (02, 04, 09, 10, 22, 23, 24, 25, 28, 35, 36, and 37). Among these, 6 lines (10, 23, 25, 35, 36, and 37) showed random transgene integration; 2 lines (02 and 04) showed plasmid backbone integration; and 4 lines (09, 22, 24, and 28) showed successful site-directed transgene integration. LB09 was a double copy, while LB22, LB24, and LB28 were single copies.

[0159] (2) Flow cytometry detection of transgenic expression.

[0160] In the cell phenotype analysis, nine monoclonal cell lines (LB-02, LB-04, LB-09, LB-22, LB-24, LB-28, LB-35, LB-36, and LB-37) were cultured to passage 28. The expression of transgenic CXCR4 and IL15 was detected by flow cytometry. Isotype controls were used as the phylogenetic basis, and the proportion of cells that were both CXCR4-positive and IL15RF-positive (i.e., the double-positive rate) was defined accordingly. Figure 4 The flow cytometry results showed that the double positivity rate of monoclonal cell lines LB-09, LB-22 and LB-28 was higher than 90%.

[0161] Example 2 This embodiment describes the functional differentiation and function of the above-mentioned gene-edited iPSCs into functional iNK cells.

[0162] 1. LB-09 / LB-22 / LB-28 iPSC-NK cell differentiation and expansion process.

[0163] (1) Culture of LB-09 / LB-22 / LB-28 monoclonal iPSCs.

[0164] Three types of monoclonal iPSCs were cultured statically in six-well plates at 37°C in an incubator with 5% CO2 and 5% O2. Cells were passaged every four days starting from the day of revival. Cells on the day of revival were designated as P+0 generation, and each subsequent passage was designated as P+1, P+2, P+3, and so on.

[0165] (2) Inducing LB-09 / LB-22 / LB-28 monoclonal iPSCs to form hematopoietic progenitor cells (HPCs).

[0166] On day 0, three monoclonal iPSCs cultured to passage P+2 were digested into single cells using accutase and then seeded into ultra-low adsorption six-well plates, with one well for each monoclonal cell line and 3e5 cells per well. From day 0 to 7, hematopoietic progenitor cells (HPCs) were induced by culturing them in serum-free Stempro-34 SFM medium containing cytokines BMP4, VEGF, bFGF, and SCF on a shaker at 70 rpm in a 5% CO2, 5% O2, 37°C incubator.

[0167] (3) Inducing LB-09 / LB-22 / LB28 to differentiate into NK cells.

[0168] From day 8 to 14, cells were continuously incubated in a medium containing DMEM, Ham's F12 medium, human AB serum, SCF, IL-7, FLT31, IL-15, and IL-3 on an orbital shaker placed in a 37°C, 5% CO2 incubator at 70 rpm, with the medium being replaced every 2 days. From day 15 to 25, cells were continuously incubated in a medium containing DMEM, Ham's F12 medium, human AB serum, SCF, IL-7, FLT31, and IL-15 on an orbital shaker placed in a 37°C, 5% CO2 incubator at 70 rpm, with the medium being replaced every 2 days.

[0169] (4) Maturation and expansion of iNK cells (NK cells formed by iPSCs) differentiated from LB-09, LB-22, and LB-28.

[0170] Cells harvested on day 25 were seeded into the GREX culture system at a ratio of 1:2 with K-562 feeder cells expressing genes such as mbIL-21, CD137L, and DLL-1. The culture medium used was NK Macs medium containing 5% human serum and 200 IU IL-2. Feeder cells were replenished every 7 days for a total expansion period of 14 days.

[0171] (5) Control group induction procedure: Induction was performed using iPSC clones without gene editing as described above.

[0172] 2. Phenotypic and functional characterization of LB-09 / LB-22 / LB-28 iPSC-NK cells.

[0173] (1) LB-09 / LB-22 / LB-28 cells were harvested at days 0, 14, 21, 25, and 39 of iPSC-NK differentiation. The expression of transgenes CXCR4 and IL15RF was detected by flow cytometry. The results are shown in Table 7. The expression of CD56 was detected in cells at days 25 and 39 of differentiation to characterize the purity of NK cells. The results are shown in Table 8. Table 7 shows the transgene expression at each stage of LB-09 / LB-22 / LB-28 differentiated NK cells and NK cell expansion.

[0174] Table 7 Table 8 shows the CD56 expression in LB-09 / LB-22 / LB-28 differentiated NK cells.

[0175] Table 8 (2) The in vitro functional activity of iNK amplified by LB-09 / LB-22 / LB-28 was detected by continuous killing experiment.

[0176] iNK cells differentiated from LB-09 / LB-22 / LB-28 cells, expanded for 14 days, were co-incubated with GFP-expressing K562 and GFP-expressing THP1 cells at effector-target ratios of 1:1 and 3:1, respectively, in the absence of IL-2. Green fluorescence during incubation was detected using an INCUCYTE instrument to indicate target cell death, further determining the killing capacity of each iNK cell line, which was then defined as an indicator of in vitro iNK functional activity. In this experiment, iNK cells derived from unedited iPSC clones were used as a control to evaluate the in vitro functional activity of CXCR4+IL15RF-edited iNK cells.

[0177] Figure 5 The results show the functional activity of iNK and K562 from different iPSC clone sources under an effector-target ratio of 1:1.

[0178] Figure 6 The results show the functional activity of iNK and K562 from different iPSC clone sources under an effector-target ratio of 3:1.

[0179] Figure 7 The results show the functional activity of iNK and THP1 from different iPSC clone sources under an effector-target ratio of 1:1.

[0180] Figure 8 The results show the functional activity of iNK and THP1 from different iPSC clone sources under an effector-target ratio of 3:1.

[0181] (3) Cell migration ability was determined by CXCL12 chemotaxis assay.

[0182] To evaluate the chemotactic migration ability of LB-09 expressing CXCR4, 4 × 10⁻⁶ cells were used. 5 One WT iNK cell or LB-09 cell was seeded in the upper chamber of a 5 μm pore size Transwell chamber. The lower chamber contained serum-free medium supplemented with 0, 2, 10, 50, or 250 ng / mL recombinant human CXCL12 (Biolegend, 581208). After incubation at 37°C and 5% CO2 for 3 hours, migrating cells in the lower chamber were collected and quantified by flow cytometry using counting microspheres (CountBright™, Thermo Fisher). Specific migration rate (%) was calculated as: (Number of migrating cells in the experimental group - Number of spontaneously migrating cells) / (Maximum number of cells (4 × 10⁻⁶)). 5 (Number of spontaneously migrating cells) × 100.

[0183] The results showed that in the presence of CXCL12, LB-09's migration from the upper chamber to the lower chamber was significantly enhanced, while WT iNK only showed background-level migration. This indicates that LB-09 has the potential to migrate to tissues rich in CXCL12.

[0184] Figure 9 The graph shows the results of the in vitro migration ability of LB-09 into the lower chamber containing different concentrations (0, 2, 10, 50 or 250 ng / mL) of CXCL12 by the Transwell assay.

[0185] (4) Cytokine production.

[0186] To assess cytokine production after co-culturing LB-09 cells with K-562 and THP-1, LB-09 or WT iNK cells were cultured at 3 × 10⁶ cells per well. 4 Cells were seeded at a density of 50 μL and co-cultured with K-562 or THP-1 tumor cells at a 1:1 effector-target ratio. After 24 hours, the culture plates were centrifuged (300 g, 5 min) and the supernatant was collected. Using the Linko Biotech Human IFN-γ ELISA Kit and the Dacoway Human TNF-α Pre-coated ELISA Kit, following the manufacturer's instructions, the pro-inflammatory cytokine IFN-γ in the collected supernatant was analyzed. Figure 10 ) and TNF-α ( Figure 11 Quantitative analysis was performed. Data showed that under stimulation by K-562 or THP-1 cells, all three batches of LB-09 (batch numbers: 20250401, 20250403, 20250501) were able to secrete a certain amount of IFN-γ and TNF-α, and their secretion levels were higher than those of WT iNK cells.

[0187] Figure 10 The production of IFN-γ after iNK cells were co-cultured with K562 and THP1 cells for 24 hours.

[0188] Figure 11 The production of TNF-α was observed after iNK cells were co-cultured with K562 and THP1 cells for 24 hours.

[0189] (5) The in vitro survival ability of LB-09.

[0190] This experiment aimed to evaluate the effect of enhanced IL-15 / IL-15Ra expression on the sustained survival of LB-09 cells under cytokine-free conditions. NK cells were resuspended in NKMACKS + 10% AB serum at a concentration of 2 × 10⁻⁶. 7Cells / mL, seeded in duplicate in 96-well plates (200 μL per well), with no added IL-2 or other cytokines in the culture system. At specified time points, add 1 μL Annexin V-Brilliant Violet™ 421 and 5 μL counting microspheres to each well. Analyze the samples using flow cytometry (Beckman Coulter, CytoFLEX S), and calculate the absolute cell count using the formula (number of cells detected × total number of microspheres) / number of microspheres detected.

[0191] The results showed that WT iNK cells were undetectable by day 14, while three independent batches of LB-09 (batch numbers: 20250401, 20250403, 20250501) remained detectable until day 31. These findings indicate that IL-15 / IL-15Ra expression significantly enhances the in vitro survival of NK cells.

[0192] Figure 12 Results show the in vitro survival ability of LB-09 under cytokine-free conditions.

[0193] Example 3 This embodiment evaluates the bone marrow homing and antitumor activity of LB-09 iNK cells in an animal model.

[0194] This embodiment aims to evaluate the antitumor efficacy and bone marrow homing ability of LB-09 iNK cells in a THP-1 xenograft mouse model.

[0195] In a NOG mouse model of acute myeloid leukemia (THML) using the Thp-1-luc-GFP protocol, animals were divided into three groups: a wild-type iNK group, an IL15-iNK group (genetically engineered iPSC-derived NK cells expressing IL-15), and an LB-09 iNK group. Test samples were administered intravenously at a dose of 1 × 10⁻⁶. 7 Cells / dose, administered for two consecutive days (once every 24 hours, for a total of two doses). Tumor growth was monitored using bioluminescence imaging, and data acquisition and analysis were performed using Tanon Image software. Antitumor activity was assessed by tumor growth rate (expressed as %T / C), calculated as: %T / C = (mean BLI signal in the treatment group / mean BLI signal in the control group) × 100%. Peripheral blood and femoral bone marrow samples were collected from euthanized mice on days 10 and 28 post-treatment, and the biodistribution of iNK cells was analyzed by detecting CD56 using flow cytometry.

[0196] In the THP-1 tumor model (n=4), engineered LB-09 iNK cells co-expressing IL-15 and CXCR4 exhibited potent systemic antitumor efficacy. Compared with the wild-type iNK group, both the IL15-iNK group and the LB-09 iNK group showed significant inhibition of systemic tumor progression, with %T / C values ​​of 18.6% and 9.3% respectively at day 28. Figure 13 , Figure 14 This effect was associated with the persistence of IL-15 enhancement: although all iNK groups were detectable in the blood at day 28, the LB-09 and IL-15-iNK groups showed higher retention rates (although IL-15-iNK did not reach statistical significance compared to wild-type iNK). Figure 16 , Figure 17 , Figure 18 , Figure 19 Crucially, the LB-09 iNK group showed significantly stronger inhibitory effects on intramedullary tumors of the femoral bone marrow than the IL15-iNK group alone. Figure 15 Flow cytometry analysis of femoral bone marrow showed that on day 10 (n=2) and day 28 (n=4), the accumulation of LB-09 iNK was 50 times higher than that of IL15-iNK. Figure 16 , Figure 17 , Figure 18 , Figure 19 This confirms its ability to migrate CXCR4-dependent components.

[0197] Figure 13 Bioluminescence imaging results of mice loaded with THP-1 tumors after treatment with vector control, wild-type iNK, iNK expressing IL-15, or dual-modified iNK co-expressing IL-15 and CXCR4.

[0198] Figure 14 Tumor burden curves at different time points for each group in the Thrp-1-luc-GFP xenograft model. Values ​​are expressed as mean ± standard error. Statistical significance was determined by two-way ANOVA. ;ns, no statistical significance.

[0199] Figure 15 Intramedullary tumor burden in each group was assessed using bioluminescence imaging of isolated femurs. Values ​​are expressed as mean ± standard error. Statistical significance was determined by one-way ANOVA. ;ns, no statistical significance.

[0200] Figure 16 The distribution of iNK cells in peripheral blood as detected on day 10.

[0201] Figure 17The distribution of iNK cells in (femoral-derived) bone marrow as detected on day 10.

[0202] Figure 18 The distribution of iNK cells in peripheral blood as detected on day 28.

[0203] Figure 19 The distribution of iNK cells in (femoral-derived) bone marrow as detected on day 28.

[0204] Figures 16-19 In this study, numerical values ​​are expressed as mean ± standard error. Statistical significance was determined using one-way ANOVA. There was no statistical significance.

[0205] In summary, this invention presents a complete process and advantages, from gene editing and cell differentiation induction to functional evaluation and animal model validation. The data is clear and reproducible, fully demonstrating the effectiveness of the technical solution. The cell products obtained by this invention are suitable for immunotherapy of acute myeloid leukemia, overcoming the shortcomings of traditional NK cell therapy such as poor homing and low survival, and possess excellent clinical translational potential and broad application prospects.

[0206] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A CXCR4-overexpressing iPSC-NK cell with enhanced homing ability to bone marrow and tumor tissues, characterized in that, The cells are pluripotent stem cells as the starting cells. The membrane-bound IL-15 and IL-15RA fusion protein genes and CXCR4 receptor genes are overexpressed in the pluripotent stem cells to obtain pluripotent stem cells that stably express the target genes. Then, iPSC-NK cells are obtained through induced differentiation.

2. The CXCR4-overexpressing iPSC-NK cells with enhanced homing ability to bone marrow and tumor tissues according to claim 1, characterized in that, The overexpression method is as follows: the target gene is integrated into the genomic safe harbor site of human pluripotent stem cells using CRISPR / Cas9 gene editing technology; The safe harbor sites include any one of AAVS1, ROSA26, CCR5, H11, or COL1A1 sites.

3. The CXCR4-overexpressing iPSC-NK cells with enhanced homing ability to bone marrow and tumor tissues according to claim 1, characterized in that, The membrane-bound IL-15 and IL-15RA fusion protein genes are expressed in tandem in the same expression vector, forming an open reading frame and driven by a single promoter, while the CXCR4 receptor gene is expressed separately in another expression vector. Alternatively, the membrane-bound IL-15 and IL-15RA fusion protein gene and the CXCR4 receptor gene are expressed in tandem in the same expression vector via the GSG-P2A self-cleaving polypeptide linker, forming an open reading frame and driven by a single promoter; The promoter is selected from any one of pPGK, CAG, CMV, EF1α or UBC; The expression vector was co-transfected with Cas9 RNP into pluripotent stem cells; The Cas9 RNP includes sgRNA and Cas9 protein.

4. The CXCR4-overexpressing iPSC-NK cells with enhanced homing ability to bone marrow and tumor tissues according to claim 1, characterized in that, The expression vector comprises an upstream homologous arm, a cHS4 insulator, a pCAG promoter element, a nucleotide sequence encoding a membrane-bound IL-15 / IL-15RA fusion protein, a GSG-P2A linker, a nucleotide sequence encoding a CXCR4 receptor protein, a poly A signaling element, a CXB3 insulator, and a downstream homologous arm element. The amino acid sequence of the membrane-bound IL-15 and IL-15RA fusion protein gene is shown in SEQ ID NO:2; The amino acid sequence of the CXCR4 receptor gene is shown in SEQ ID NO:

4.

5. The CXCR4-overexpressing iPSC-NK cells with enhanced homing ability to bone marrow and tumor tissues according to claim 1, characterized in that, The steps of inducing differentiation include: (a) Days 1-7: Hematopoietic precursor induction culture, using basal medium containing cytokines BMP4, VEGF, bFGF and SCF to culture pluripotent stem cells with the target gene integrated; (b) Days 8-25: NK cells were induced to differentiate and cultured in a basal medium containing cytokines SCF, IL-7, FLT31, IL-15, and IL-3. (c) Days 26-39: NK cell maturation and expansion culture. Cells harvested on day 25 and K-562 feeder cells expressing mbIL-21, CD137L, and DLL-1 genes were seeded into a GREX culture system for culture, and high-purity CD56 cells were harvested. + CD16 + NK cells.

6. The CXCR4-overexpressing iPSC-NK cells with enhanced homing ability to bone marrow and tumor tissues according to claim 5, characterized in that, In step (a), the concentrations of the cytokines BMP4, VEGF, and bFGF in the basal culture medium are each 8-12 ng / mL. In step (a), the concentration of the cytokine SCF in the basal culture medium is 18-22 ng / mL; In step (b), the concentrations of the cytokines SCF and IL-7 in the basal culture medium are each 18-22 ng / mL independently; In step (b), the concentrations of the cytokines FLT31 and IL-15 in the basal culture medium are each 8-12 ng / mL independently; In step (b), the concentration of IL-3 in the basal culture medium is 4-6 ng / mL; In step (c), the inoculation ratio of the cells harvested on day 25 to K-562 feeder cells is 1:(1-2); In step (c), the culture medium used is NK Macs medium containing 2-10% human serum and 20-200 IU IL-2.

7. The method for preparing CXCR4-overexpressing iPSC-NK cells with enhanced homing ability to bone marrow and tumor tissues as described in any one of claims 1-6, characterized in that, The preparation method includes: (1) Construct an expression vector plasmid containing the membrane-bound IL-15 and IL-15RA fusion protein gene and the CXCR4 receptor gene; (2) Co-transfect pluripotent stem cells with the expression vector plasmid constructed in step (1) and Cas9 RNP to screen pluripotent stem cells that stably express the target gene. (3) Inducing differentiation of the pluripotent stem cells screened in step (2) to obtain iPSC-NK cells.

8. The preparation method according to claim 7, characterized in that, In step (2), the co-transfection conditions are as follows: Cas9 protein and sgRNA are assembled in vitro through electrostatic interaction and structural chimerism to form an RNP complex, and then the RNP complex and expression vector plasmid are delivered into the cell nucleus together by electroporation to exert the gene editing effect. In step (2), the screening methods include: flow cytometry sorting, PCR and ddPCR identification, flow cytometry detection of exogenous gene expression, flow cytometry detection of cell totipotency, cell passage stability detection, and WGS and WES detection of pathogenicity assessment.

9. The use of CXCR4-overexpressing iPSC-NK cells with enhanced homing ability to bone marrow and tumor tissues as described in any one of claims 1-6 in the preparation of antitumor drugs, characterized in that, The tumors include bone marrow hematologic malignancies or solid tumors expressing high levels of the chemokine CXCL12.

10. The application according to claim 9, characterized in that, The bone marrow hematologic malignancies include acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, myeloproliferative neoplasm, or myelodysplastic syndrome. Solid tumors expressing high levels of the chemokine CXCL12 include pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, non-small cell lung cancer, glioma, or melanoma.

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