Use of ipsc-induced ink cell population in treatment of cancer

Abstract

The present invention provides use of an iPSC-induced iNK cell population in the preparation of a drug for treating cancer, a preparation method for the iNK cell population, and the prepared iNK cell population.

Description

Application of iNK cell population induced by iPSCs in cancer treatment Technical Field

[0001] This invention relates to a method for generating an iNK cell population from induced pluripotent stem cells (iPSCs), the iNK cell population prepared by this method, and the use of said iNK cell population in the preparation of medicaments for treating cancer. This invention also relates to methods for treating cancer and iNK cell populations for treating cancer. Background Technology

[0002] Natural killer (NK) cells are a type of innate immune effector cells. Unlike acquired immune cells, NK cells have the ability to directly kill malignant tumor cells and virus-infected cells. NK cells are primarily derived from peripheral blood, bone marrow, umbilical cord blood, and induced differentiation from pluripotent stem cells (iPSCs). NK cells account for approximately 5%-20% of peripheral blood mononuclear cells, defined by the phenotype CD3- / CD56+. The limited number of NK cells in peripheral blood makes effective isolation and expansion to a sufficient therapeutic dose a major technical bottleneck in NK therapy. Using iPSCs to differentiate and produce NK (iNK) cells offers several advantages. First, iPSCs can be expanded on a large scale, facilitating the mass production and storage of NK cells and reducing production costs. Second, iPSC-derived iNK cells differ from adult NK cells in their surface receptor expression, resulting in stronger tumor-killing performance at the same dosage. Furthermore, compared to primary NK cells, iPSCs are easier to genetically engineer. Gene modification at the iPSC stage can produce multi-element genetically modified iNK cells to meet the needs of different tumor treatments. As an off-the-shelf cell therapy, iPSC-derived iNK cells have strong drug-like properties.

[0003] Summary of the Invention

[0004] iNK cell populations derived from iPSCs have shown promising efficacy in treating cancers such as ovarian cancer. This invention unexpectedly revealed a significant advantage in treating ovarian cancer compared to other tumor types, and this therapeutic effect has been confirmed through human clinical trials.

[0005] Therefore, one aspect of the present invention provides the use of iNK cell populations induced by iPSCs in the preparation of medicaments for treating cancer.

[0006] In some embodiments, the cancer is ovarian cancer, stomach cancer, colorectal cancer, or liver cancer. In a preferred embodiment, the cancer is ovarian cancer. In a preferred embodiment, the ovarian cancer is ovarian serous cystadenocarcinoma or ovarian mucinous cystadenocarcinoma.

[0007] In some embodiments, the ovarian cancer is stage II or III ovarian cancer.

[0008] In some implementations, the amount of ascites in the subject decreased by 10%-90% after treatment.

[0009] In some embodiments, the tumor marker CA125 is reduced by 25%-50% after treatment.

[0010] In some embodiments, the iNK cell population is prepared from iPSCs by a method comprising the following steps: (a) culturing iPSCs to generate embryoids and performing hematopoietic differentiation on the embryoids; (b) differentiating the hematopoietic differentiated embryoids into iNK cells; and (c) expanding the iNK cells to obtain the iNK cell population.

[0011] In some implementations, the hematopoietic differentiation of the embryoids in step (a) includes hematopoietic differentiation using different hematopoietic differentiation culture media over time.

[0012] In some implementations, four different hematopoietic differentiation culture media are used over time:

[0013] (i) First hematopoietic differentiation medium: hematopoietic differentiation basal medium supplemented with 1-20 μM CHIR99021;

[0014] (ii) Second hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of BMP4, FGF2 and VEGF, at a concentration of 25-75 ng / ml;

[0015] (iii) Third hematopoietic differentiation medium: Second hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml SCF and 1-10 μMSB431542;

[0016] (iv) Fourth hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of 25-75 ng / ml FGF2, 25-75 ng / ml VEGF and 25-75 ng / ml SCF.

[0017] In a preferred embodiment, the embryoids are cultured in the first, second, third, and fourth differentiation culture media in sequence for two days.

[0018] In some embodiments, the hematopoietic differentiation basal culture medium comprises StemPro34 medium, 0.5%-1.5% P / S, 1-5mM GlutaMAX, 25-75μg / ml ascorbic acid, 0.5%-1.5% ITS-G and 200-600μM MTG.

[0019] In some embodiments, iPSCs are dissociated into single cells prior to step (a); preferably, the single cells are cultured in a medium supplemented with 1-20 μM Y27632. In some embodiments, the single cells are seeded into 2-4 ml / well 6-well plates and cultured on a shaker.

[0020] In some implementations, IL-2 is not used in step (b).

[0021] In some implementations, step (b) is performed until the proportion of CD45+CD56+CD3- cells is >90%.

[0022] In some embodiments, step (c) includes co-culturing the iNK cells with a feeder cell line. In a preferred embodiment, the feeder cell line is the K562 myeloid leukemia cell line. In a preferred embodiment, the K562 cell line expresses membrane-bound IL-21 and 4-1BBL. In a preferred embodiment, the co-culture is performed at an iNK cell to feeder cell line ratio of 5:1 to 1:5.

[0023] In some embodiments, step (c) is performed in an NK amplification medium; preferably, the NK amplification medium contains at least one of 1%-10% human serum, 0.5%-1.5% P / S, and 25-75 U / mL IL-2. In a preferred embodiment, the NK amplification medium contains NK Xpander complete medium.

[0024] In some implementations, step (c) involves amplification for 10-15 days.

[0025] Another aspect of the present invention provides a method for inducing an iNK cell population by iPSCs, the method comprising: (a) culturing iPSCs to generate embryoids and performing hematopoietic differentiation on the embryoids; (b) differentiating the hematopoietic differentiated embryoids into iNK cells; and (c) expanding the iNK cells to obtain the iNK cell population.

[0026] In some implementations, the hematopoietic differentiation of the embryoids in step (a) includes hematopoietic differentiation using different hematopoietic differentiation culture media over time.

[0027] In some implementations, four different hematopoietic differentiation culture media are used over time:

[0028] (i) First hematopoietic differentiation medium: hematopoietic differentiation basal medium supplemented with 1-20 μM CHIR99021;

[0029] (ii) Second hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of BMP4, FGF2 and VEGF, at a concentration of 25-75 ng / ml;

[0030] (iii) Third hematopoietic differentiation medium: Second hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml SCF and 1-10 μMSB431542;

[0031] (iv) Fourth hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of 25-75 ng / ml FGF2, 25-75 ng / ml VEGF and 25-75 ng / ml SCF.

[0032] In a preferred embodiment, the embryoids are cultured in the first, second, third, and fourth differentiation culture media in sequence for two days.

[0033] In some embodiments, the hematopoietic differentiation basal culture medium comprises StemPro34 medium, 0.5%-1.5% P / S, 1-5mM GlutaMAX, 25-75μg / ml ascorbic acid, 0.5%-1.5% ITS-G and 200-600μM MTG.

[0034] In some embodiments, iPSCs are dissociated into single cells prior to step (a); preferably, the single cells are cultured in a medium supplemented with 1-20 μM Y27632. In some embodiments, the single cells are seeded into 2-4 ml / well 6-well plates and cultured on a shaker.

[0035] In some implementations, IL-2 is not used in step (b).

[0036] In some implementations, step (b) is performed until the proportion of CD45+CD56+CD3- cells is >90%.

[0037] In some embodiments, step (c) includes co-culturing the iNK cells with a feeder cell line. In a preferred embodiment, the feeder cell line is the K562 myeloid leukemia cell line. In a preferred embodiment, the K562 cell line expresses membrane-bound IL-21 and 4-1BBL. In a preferred embodiment, the co-culture is performed at an iNK cell to feeder cell line ratio of 5:1 to 1:5.

[0038] In some embodiments, step (c) is performed in an NK amplification medium; preferably, the NK amplification medium contains at least one of 1%-10% human serum, 0.5%-1.5% P / S, and 25-75 U / mL IL-2. In a preferred embodiment, the NK amplification medium contains NK Xpander complete medium.

[0039] In some implementations, step (c) involves amplification for 10-15 days.

[0040] Another aspect of the present invention provides a population of NK cells prepared by the methods described above.

[0041] The iNK cells prepared by this invention have shown good therapeutic effects in various cancers, particularly demonstrating significant advantages in the treatment of ovarian cancer. Compared with other tumor types tested (such as gastric cancer, colorectal cancer, and liver cancer), iNK cells exhibited significantly higher survival rates and tumor suppression rates in different ovarian cancer cell lines. This result was unexpected, indicating that the iNK cells provided by this invention are particularly suitable for the treatment of ovarian cancer.

[0042] This invention has discovered that while existing culture methods have achieved excellent results in vitro, the cytotoxic effects of these cells are less than expected in in vivo mouse experiments. The iNK cells of this invention incorporate numerous improvements over existing methods in their preparation process. For example, iNK cells are preferably cultured on a shaker during embryoid formation; single-cell iPSCs are preferably seeded in 2-4 ml / well of a 6-well plate and cultured on a shaker. Furthermore, this invention preferably uses culture media with different compositions at different stages of hematopoietic differentiation to better induce iPSC differentiation into hematopoietic stem cells. In addition, the inventors unexpectedly discovered that the aforementioned effects of this invention can be achieved without the use of IL-2 in the iNK cell differentiation process.

[0043] Other aspects and advantages of the invention will become apparent from the following detailed description. Attached Figure Description

[0044] Figure 1 shows a schematic diagram of the in vivo pharmacodynamics study process for peritoneal metastases using iNK cells provided by this invention.

[0045] Figure 2 shows the changes in bioluminescence intensity of tumor cells during the experiment in each group of animals in the SKOV3 xenograft model of hypospermic ovarian cancer.

[0046] Figure 3 shows the body weight change curves of each group of animals in the SKOV3 xenograft model of hypospermic ovarian cancer during the experiment.

[0047] Figure 4 shows the survival rate curves of each group of animals in the SKOV3 xenograft model of hypospermic ovarian cancer during the experiment.

[0048] Figure 5 shows the changes in bioluminescence intensity of tumor cells during the experiment in each group of animals in the SKOV3 xenograft model of hypospermic ovarian cancer.

[0049] Figure 6 shows the body weight change curves of each group of animals in the SKOV3 xenograft model of hypospermic ovarian cancer during the experiment.

[0050] Figure 7 shows the survival rate curves of each group of animals in the SKOV3 xenograft model of hypospermic ovarian cancer during the experiment.

[0051] Figure 8 shows the changes in bioluminescence intensity of tumor cells in each group of the OVCAR3 xenograft model of ovarian cancer during the animal experiments.

[0052] Figure 9 shows the weight change curves of each group of animals in the OVCAR3 xenograft model of hyperspermic ovarian cancer during the experiment.

[0053] Figure 10 shows the survival rate curves of each group of animals in the OVCAR3 xenograft model of hyperserine ovarian cancer during the experiment.

[0054] Figure 11 shows the changes in bioluminescence intensity of tumor cells in each group of animal experiments during the human gastric cancer cell line (MKN28) xenograft model.

[0055] Figure 12 shows the body weight change curves of each group of animals in the human gastric cancer cell line (MKN28) xenograft model during the experiment.

[0056] Figure 13 shows the survival rate curves of each group of animals in the human gastric cancer cell line (MKN28) xenograft model during the experiment.

[0057] Figure 14 shows the changes in bioluminescence intensity of tumor cells during the animal experiments in each group of human gastric cancer cell line (MKN28) xenograft model.

[0058] Figure 15 shows the body weight change curves of each group of animals in the human gastric cancer cell line (MKN28) xenograft model during the experiment.

[0059] Figure 16 shows the survival rate curves of each group of animals in the human gastric cancer cell line (MKN28) xenograft model during the experiment.

[0060] Figure 17 shows a schematic diagram of the iNK in vivo pharmacodynamics subcutaneous tumor model study process.

[0061] Figure 18 shows the tumor cell growth curves during the experiment in each group of animals in the human hepatocellular carcinoma cell line (HepG-2) subcutaneous tumor model.

[0062] Figure 19 shows the comparison of tumor weight at the experimental endpoint in each group of animals in the HepG-2 subcutaneous tumor model of human liver cancer cells.

[0063] Figure 20 shows some of the radiographic images of the subjects in the IIT study. Detailed Implementation

[0064] definition

[0065] As used herein, the term "differentiation" is the process by which undifferentiated ("non-specialized") or weakly specialized cells acquire the characteristics of specialized cells (such as blood cells or muscle cells). Differentiated cells, or differentiation-inducing cells, are cells that are already at a more specialized ("specialized") position within a cell lineage. When applied to the differentiation process, the term "specialization" refers to a point in the differentiation pathway where a cell has progressed to a point where, under normal circumstances, it would continue to differentiate into a specific cell type or a subpopulation of that cell type, and where, under normal circumstances, it cannot differentiate into a different cell type or revert to a weaker differentiated cell type.

[0066] As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of a body or cell body (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuous developmental efficiency ranging from incomplete or partially pluripotent cells (e.g., ectoderm stem cells or EpiSCs) that cannot produce a complete organism to more primitive, multipotent cells (e.g., embryonic stem cells) that can produce a complete organism.

[0067] Pluripotency can be determined in part by assessing the pluripotency characteristics of cells. Pluripotency characteristics include, but are not limited to: (i) pluripotent stem cell morphology; (ii) potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers, including but not limited to SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50; (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm and endoderm); (v) teratoma formation composed of the three somatic cell lineages; and (vi) embryomorphic formation composed of cells from the three somatic cell lineages.

[0068] As used in this article, the terms "permanent hematopoietic endothelial cells" (HE) or "hematopoietic differentiation" refer to the subpopulation of endothelial cells that produce hematopoietic stem cells and progenitor cells during the process of transformation from endothelial cells to hematopoietic cells.

[0069] "Embryoids (EBs)" are three-dimensional clusters that have been shown to mimic embryonic development because they generate multiple lineages within their three-dimensional regions. Through a differentiation process, typically lasting hours to days, simple EBs (e.g., aggregated pluripotent stem cells that have been induced to differentiate) continue to mature and develop into cystic EBs, at which point they are usually further treated for several days to weeks to continue differentiating. EB formation is initiated by causing pluripotent stem cells to form three-dimensional, multi-layered cell clusters that are brought close together. This is typically achieved through one of several methods, including allowing pluripotent cells to settle in droplets, allowing cells to settle in U-shaped bottom-well plates, or by mechanical agitation. To promote EB development, further differentiation cues are needed from the pluripotent stem cell aggregates because the aggregates maintained in the maintenance medium for pluripotent culture do not form suitable EBs. Therefore, the pluripotent stem cell aggregates need to be transferred to a differentiation medium that provides induction cues to the selected lineages. EB-based culture of pluripotent stem cells typically induces the production of differentiated cell populations (ectoderm, mesoderm, and endoderm germ layers) through moderate proliferation within the EB cell clusters. While proven to promote cell differentiation, EB produces heterogeneous cells with variable differentiation states because cells in the three-dimensional structure are inconsistently exposed to differentiation cues from the environment. Furthermore, EB formation and maintenance are problematic. Additionally, cell differentiation via EB is accompanied by moderate cell proliferation, which also leads to reduced differentiation efficiency.

[0070] As used in this article, the term "NK cell" or "natural killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3).

[0071] As used herein, "dissociated" cells refer to cells that have been substantially separated or purified from other cells or surfaces (e.g., culture plate surfaces). For example, cells can be dissociated from animals or tissues by mechanical or enzymatic methods. Alternatively, cells aggregated in vitro can be dissociated from each other, for example, by enzymatic or mechanical dissociation into clusters, single cells, or a suspension of a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from culture plates or other surfaces. Thus, dissociation may involve disrupting cell interactions with the extracellular matrix (ECM) and the substrate (e.g., culture surface), or disrupting the ECM between cells.

[0072] As used herein, "feeder cells" or "feeder layers" are terms used to describe a type of cell co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, proliferate, or differentiate, as the feeder cells provide stimulation, growth factors, and nutrients to support the second cell type. Feeder cells optionally originate from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts. In another instance, peripheral blood-derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells. When co-cultured with other cells, feeder cells can typically be inactivated by irradiation or treatment with an antimitotic agent (such as mitomycin) to prevent them from growing beyond the cells they support. Feeder cells can include endothelial cells, stromal cells (such as epithelial cells or fibroblasts), and leukemia cells. Not limited to the foregoing, a particular type of feeder cell can be a human feeder layer, such as human skin fibroblasts. Another type of feeder cell can be mouse embryonic fibroblasts (MEF). Generally, various feeder cells can be used in part to maintain pluripotency, direct differentiation toward a certain lineage, enhance proliferative capacity, and promote maturation toward specialized cell types (such as effector cells).

[0073] CHIR99021 is an aminopyrimidine derivative (CAS252917-06-9), a highly potent inhibitor of GSK-3α (IC50: 10 nM) and GSK-3β (IC50: 6.7 nM). CHIR-99021 exhibits more than 500 times higher selectivity for GSK-3 than its closest homologs CDC2, ERK2, and other protein kinases. CHIR-99021 activates the Wnt / β-catenin signaling pathway, inducing myocardial differentiation in embryonic stem cells.

[0074] "SB431542" is a Src family kinase inhibitor (CAS 301836-41-9), an inhibitor of the TGF-β signaling pathway, and a potent ALK5 inhibitor (with an IC50 value of 94 nM). It can inhibit TGF-β-mediated SMAD protein activation, cell proliferation, and cell motility, and can effectively inhibit the migration and invasion of cells induced by growth factor β1 in primary and secondary cancer cells.

[0075] “Y27632” is a Rho-associated kinase inhibitor (CAS 331752-47-7) that can prevent cell death and improve the survival rate and proliferation efficiency of isolated human stem cells without affecting the pluripotency of stem cells.

[0076] BMP4 refers to bone morphogenetic protein 4. Studies have found that it has a significant promoting effect on maintaining the pluripotency of mouse embryonic stem cells. Adding BMP4 to a serum-free culture system can appropriately activate the BMP signaling pathway, thereby upregulating the expression of damaged downstream target genes Ube2s and Chmp4b in the serum-free system, so as to ensure the chromosomal integrity and proliferation capacity of mouse embryonic stem cells, and further improve and maintain the in vivo developmental potential of mouse embryonic stem cells in the long term.

[0077] "FGF2" refers to fibroblast growth factor 2, also known as basic fibroblast growth factor (bFGF) and FGF basic. It belongs to the FGF family and is a globular protein composed of a single polypeptide with a molecular weight of 18 kDa. FGF-2 is an important component of embryonic stem cell culture media, enabling cells to remain undifferentiated in serum-free medium. FGF-2 may also play an important role in the differentiation and function of the nervous system, as well as in the regeneration of the eyes and bones.

[0078] VEGF, or Vascular Endothelial Growth Factor, also known as Vascular Permeability Factor (VPF) or Angiotensin, is a novel growth factor initially discovered in various cultured tumor cell lines. It increases the permeability of microvessels and small veins. Later, it was found that this factor specifically acts on vascular endothelial cells and promotes their proliferation, hence the name vascular endothelial growth factor. The structure of VEGF is highly conserved.

[0079] Stem cell factor (SCF) is a widely expressed type I transmembrane glycoprotein that promotes the survival, differentiation, and mobilization of various cell types, including bone marrow cells, erythroid progenitor cells, and megakaryocytes. In bone marrow culture, SCF stimulates the proliferation of myeloid progenitor cells, erythroid hematopoietic progenitor cells, and lymphoid progenitor cells, and has a synergistic effect with colony-stimulating factor. Mature SCF consists of an extracellular region containing 189 amino acids, a transmembrane region containing 23 amino acids, and an intracellular tail containing 36 amino acids. The extracellular region is cleaved by proteases to form free SCF.

[0080] GlutaMAX additive is an alternative to L-glutamine with improved stability, enhancing cell health. Suitable for both adherent and suspension cultures of mammalian cells, GlutaMAX requires no acclimatization. GlutaMAX supplement is supplied as a 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl. GlutaMAX is also included in various culture medium formulations. Compared to L-glutamine, GlutaMAX significantly reduces the accumulation of toxic ammonia, improves cell viability and growth, and remains stable over a wide temperature range. Unlike L-glutamine, GlutaMAX does not spontaneously decompose, thus preventing ammonia formation. Instead, cells cleave the dipeptide bonds as needed to release L-glutamine. This system prevents waste accumulation and maintains a fresh supply of L-glutamine during long-term culture. Improved cell viability and growth are observed; cultures with lower ammonia levels exhibit higher cell viability.

[0081] "P / S" or "PS" refers to penicillin and streptomycin.

[0082] "ITS-G" refers to an insulin-transferrin-selenium supplement. Insulin (INS) is a double-chain (α,β) polypeptide hormone secreted by pancreatic β cells. It is currently the only known hormone in the body that lowers blood glucose and is also the only hormone that simultaneously promotes the synthesis of glycogen, fat, and protein. Transferrin is an essential component of body fluids, participating not only in iron transport and metabolism, but also in the regulation of respiration, cell proliferation, and the immune system. It also regulates iron ion balance and energy balance, and has antibacterial protective functions. Selenium, provided as sodium selenite, is a cofactor for glutathione peroxidase and other proteins, and acts as an antioxidant in culture media. ITS-G solution was initially used in combination with RPMI 1640 and minimum essential medium (MEM), but is now commonly used in combination with other basal media to support various cell types in the presence of 2–4% FBS.

[0083] "MTG" refers to thioglycerol, which is an essential reducing agent for culturing ES and iPS cells and is equivalent to 2ME.

[0084] Application of iNK cell population induced by iPSCs in cancer treatment

[0085] A first aspect of the invention provides the use of an iNK cell population induced by iPSCs in the preparation of a medicament for treating cancer in a subject. Accordingly, the invention also provides a method for treating cancer in a subject, the method comprising administering a therapeutically effective amount of the iNK cell population of the invention to the subject. Accordingly, the invention also provides the iNK cell population of the invention for treating cancer in a subject.

[0086] In some embodiments of the present invention, the cancer is ovarian cancer, stomach cancer, colorectal cancer, or liver cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is liver cancer.

[0087] In embodiments where the cancer is ovarian cancer, the iNK cell population of the present invention can be applied to any stage and / or any histological type of ovarian cancer.

[0088] Ovarian cancer is classified into four stages based on its malignancy: Stage I, Stage II, Stage III, and Stage IV. This invention is intended to be applicable to any stage of ovarian cancer. In some embodiments, the NK cell population of this invention can be used to treat Stage II or III ovarian cancer. According to the classification method established by the World Health Organization (WHO), ovarian cancer histologically falls into four main categories: epithelial tumors, germ cell malignancies, sex cord-stromal tumors, and metastatic tumors. This invention is intended to be applicable to any histological type of ovarian cancer. Epithelial tumors include serous tumors, mucinous tumors, endometrioid tumors, clear cell tumors, and mixed epithelial carcinomas. This invention is intended to be applicable to any epithelial tumor of the ovary. In some embodiments, the NK cell population of this invention can be used to treat ovarian serous cystadenocarcinoma or ovarian mucinous cystadenocarcinoma.

[0089] In some embodiments, the iNK cell population of the present invention can be used to treat stage II or III ovarian serous cystadenocarcinoma or ovarian mucinous cystadenocarcinoma.

[0090] In some embodiments, the amount of ascites in the subject is reduced after treatment with the iNK cell population of the present invention. In some embodiments, the amount of ascites in the subject is reduced by 10%-90%, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0091] In some embodiments, the tumor marker CA125 is reduced after treatment with the iNK cell population of the present invention. In some embodiments, the tumor marker CA125 is reduced by 25%-50%, for example, 25%, 30%, 35%, 40%, 45%, or 50%.

[0092] In some embodiments, after treatment with the iNK cell population of the present invention, the amount of ascites in the subject decreases, and the subject's tumor marker CA125 decreases. In some embodiments, after treatment with the iNK cell population of the present invention, the amount of ascites in the subject decreases by 10%-90%, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and the subject's tumor marker CA125 decreases by 25%-50%, for example, 25%, 30%, 35%, 40%, 45%, or 50%.

[0093] In some embodiments, the object is a human being. In such embodiments, the therapeutically effective dose of the present invention can be as low as 3 x 10⁻⁶. 8 per cell / individual. This low dose is one-third of the safe dose reported for similar products. Human trials have demonstrated that this low dose can achieve safe and effective treatment for ovarian cancer. In this embodiment, the maximum dose of iNK cell population can be 6 x 10-1. 8 This is one-tenth of the highest dose reported for similar products per cell / individual.

[0094] In some embodiments, the iNK cell population is prepared from iPSCs by a method comprising the following steps: (a) culturing iPSCs to generate embryoids and performing hematopoietic differentiation on the embryoids; (b) differentiating the hematopoietic differentiated embryoids into iNK cells; and (c) expanding the iNK cells to obtain the iNK cell population.

[0095] In some implementations, the hematopoietic differentiation of the embryoids in step (a) includes hematopoietic differentiation using different hematopoietic differentiation culture media over time.

[0096] In some implementations, in step (a), four different hematopoietic differentiation media are used over time:

[0097] (i) First hematopoietic differentiation medium: hematopoietic differentiation basal medium supplemented with 1-20 μM CHIR99021;

[0098] (ii) Second hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of BMP4, FGF2 and VEGF, at a concentration of 25-75 ng / ml;

[0099] (iii) Third hematopoietic differentiation medium: Second hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml SCF and 1-10 μM SB431542;

[0100] (iv) Fourth hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of 25-75 ng / ml FGF2, 25-75 ng / ml VEGF and 25-75 ng / ml SCF.

[0101] In some embodiments, embryoids are cultured using first, second, third, and fourth hematopoietic differentiation media, respectively, for 1-3 days, for example, 2 days. In a preferred embodiment, embryoids are cultured using first, second, third, and fourth hematopoietic differentiation media, respectively, for 2 days. In a further preferred embodiment, embryoids are cultured using first, second, third, and fourth hematopoietic differentiation media in sequence for 2 days.

[0102] In some embodiments, the hematopoietic differentiation basal culture medium comprises StemPro34 medium, 0.5%-1.5% P / S, 1-5mM GlutaMAX, 25-75μg / ml ascorbic acid, 0.5%-1.5% ITS-G and 200-600μM MTG.

[0103] In some embodiments, the hematopoietic differentiation basal culture medium comprises StemPro34 medium, 1% P / S, 2mM GlutaMAX, 50μg / ml ascorbic acid, 1% ITS-G and 400μM MTG.

[0104] In some embodiments, the first hematopoietic differentiation medium is a hematopoietic differentiation basal medium supplemented with 1-20 μM CHIR99021, for example, CHIR99021 supplemented with 1, 5, 10, 15 or 20 μM.

[0105] In some embodiments, the second hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with at least one of BMP4, FGF2, and VEGF. In some embodiments, the second hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with 25-75 ng / ml BMP4, FGF2, and VEGF. In some embodiments, the second hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with 50 ng / ml BMP4, 50 ng / ml FGF2, and 50 ng / ml VEGF, wherein the basal hematopoietic differentiation medium comprises StemPro34 medium, 1% P / S, 2 mM GlutaMAX, 50 μg / ml ascorbic acid, 1% ITS-G, and 400 μM MTG.

[0106] In some embodiments, the third hematopoietic differentiation medium is the second hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml SCF and 1-10 μM SB431542. In some embodiments, the third hematopoietic differentiation medium is the second hematopoietic differentiation medium supplemented with 25-75 ng / ml SCF and 1-10 μM SB431542. In some embodiments, the third hematopoietic differentiation medium is the second hematopoietic differentiation medium supplemented with 50 ng / ml SCF and 6 μM SB431542, wherein the second hematopoietic differentiation medium is a basal hematopoietic differentiation medium supplemented with 50 ng / ml BMP4, 50 ng / ml FGF2, and 50 ng / ml VEGF, and the basal hematopoietic differentiation medium comprises StemPro34 medium, 1% P / S, 2 mM GlutaMAX, 50 μg / ml ascorbic acid, 1% ITS-G, and 400 μM MTG.

[0107] In some embodiments, the fourth hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml FGF2, 25-75 ng / ml VEGF, and 25-75 ng / ml SCF. In some embodiments, the fourth hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with 25-75 ng / ml FGF2, 25-75 ng / ml VEGF, and 25-75 ng / ml SCF. In some embodiments, the fourth hematopoietic differentiation medium is a basal hematopoietic differentiation medium supplemented with 50 ng / ml FGF2, 50 ng / ml VEGF, and 50 ng / ml SCF, wherein the basal hematopoietic differentiation medium comprises StemPro34 medium, 1% P / S, 2 mM GlutaMAX, 50 μg / ml ascorbic acid, 1% ITS-G, and 400 μM MTG.

[0108] In some implementations, iPSCs are dissociated into single cells prior to step (a). For example, human iPSCs are routinely cultured in commercially available Essential 8 medium. Before initiating iNK cell differentiation, iPSCs are ensured to be morphologically normal, confluenced to or below 70%, and without significant spontaneous differentiation, and are dissociated into single cells using Accutase.

[0109] In some embodiments, prior to step (a), iPSCs are dissociated into single cells, and the single cells are cultured in medium supplemented with 1-20 μM (e.g., 1, 5, 10, 15, or 20 μM) Y27632. For example, single cells are seeded in 6-well plates at 2-4 ml / well and cultured on a shaker. For example, collected cells are counted and seeded at a density of 1.5E5 cells / ml in 6-well plates at 2-4 ml / well, and cultured in StemScale PSC suspension complete medium supplemented with 10 μM Y27632 at a volume of 2 mL / well. The cell-seeded 6-well plates are then placed in a CO2 cell culture incubator with a CO2 shaker (70 rpm) and incubated at 37°C in 5% CO2 to generate embryoids (EB).

[0110] In some implementations, IL-2 is not used in step (b).

[0111] In some embodiments, step (b) uses 1-10 ng / mL of IL-3 in the first phase and does not use IL-3 in the second phase. In some embodiments, the second phase is 5-10 days after the first phase, for example, 8 days.

[0112] In some implementations, step (b) uses 10-30 ng / mL (e.g., 20 ng / mL) SCF, 10-30 ng / mL (e.g., 20 ng / mL) IL-7, 5-15 ng / mL (e.g., 10 ng / mL) IL-15 and 5-15 ng / mL (e.g., 10 ng / mL) FLT3L in both the first and second stages.

[0113] In some embodiments, step (b) in the first stage uses an NK differentiation medium containing the following components: 56.6% DMEM / F12+GlutaMAX™-I, 28.3% F12+GlutaMAX-I, 15% heat-inactivated human AB serum, 1% P / S, 2mM L-glutamine, 1μM β-mercaptoethanol, 5ng / mL sodium selenite, 50μM ethanolamine, 20ug / mL ascorbic acid, 5ng / mL IL-3, 20ng / mL SCF, 20ng / mL IL-7, 10ng / mL IL-15, and 10ng / mL FLT3 ligand (FLT3L).

[0114] In some embodiments, step (b) in the second stage uses an NK differentiation medium containing the following components: 56.6% DMEM / F12+GlutaMAX™-I, 28.3% F12+GlutaMAX-I, 15% heat-inactivated human AB serum, 1% P / S, 2mM L-glutamine, 1μM β-mercaptoethanol, 5ng / mL sodium selenite, 50μM ethanolamine, 20ug / mL ascorbic acid, 20ng / mL SCF, 20ng / mL IL-7, 10ng / mL IL-15, 10ng / mL FLT3 ligand (FLT3L), and IL-3-free.

[0115] In some implementations, step (b) is performed until the proportion of CD45+CD56+CD3- cells is >90%, for example, as determined by flow cytometry. Step (c) is then performed.

[0116] In some embodiments, step (c) includes co-culturing the iNK cells with a feeder cell line. In a preferred embodiment, the feeder cell line is the K562 myeloid leukemia cell line. In a preferred embodiment, the K562 cell line expresses membrane-bound IL-21 and 4-1BBL.

[0117] In some embodiments, the co-culture in step (c) is performed at a ratio of 5:1 to 1:5 of iNK cells to feeder cell lines. In some embodiments, the co-culture in step (c) is performed at a ratio of 1:1 of iNK cells to feeder cell lines. In some embodiments, the co-culture in step (c) is performed at a ratio of 1:1 of iNK cells to K562 cell lines.

[0118] In some implementations, step (c) involves amplification for 10-15 days, e.g., 12 days. In some implementations, the amplification is performed at 5x10⁻⁶. 5 cells / cm 2 The density was co-cultured statically in NK amplification medium (e.g., containing NK Xpander complete medium, 5% human serum, 1% P / S and 50 U / mL IL-2).

[0119] NK cell population and its preparation method

[0120] Another aspect of the present invention provides a method for inducing an iNK cell population by iPSCs, the method comprising: (a) culturing iPSCs to generate embryoids and performing hematopoietic differentiation on the embryoids; (b) differentiating the hematopoietic differentiated embryoids into NK cells; and (c) expanding the NK cells to obtain the NK cell population.

[0121] In some implementations, the hematopoietic differentiation of the embryoids in step (a) includes hematopoietic differentiation using different hematopoietic differentiation culture media over time.

[0122] In some implementations, in step (a), four different hematopoietic differentiation media are used over time:

[0123] (i) First hematopoietic differentiation medium: hematopoietic differentiation basal medium supplemented with 1-20 μM CHIR99021;

[0124] (ii) Second hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of BMP4, FGF2 and VEGF, at a concentration of 25-75 ng / ml;

[0125] (iii) Third hematopoietic differentiation medium: Second hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml SCF and 1-10 μMSB431542;

[0126] (iv) Fourth hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of 25-75 ng / ml FGF2, 25-75 ng / ml VEGF and 25-75 ng / ml SCF.

[0127] In some embodiments, embryoids are cultured using first, second, third, and fourth hematopoietic differentiation media, respectively, for 1-3 days, for example, 2 days. In a preferred embodiment, embryoids are cultured using first, second, third, and fourth hematopoietic differentiation media, respectively, for 2 days. In a further preferred embodiment, embryoids are cultured using first, second, third, and fourth hematopoietic differentiation media in sequence for 2 days.

[0128] In some embodiments, the hematopoietic differentiation basal culture medium comprises StemPro34 medium, 0.5%-1.5% P / S, 1-5mM GlutaMAX, 25-75μg / ml ascorbic acid, 0.5%-1.5% ITS-G and 200-600μM MTG.

[0129] In some embodiments, the hematopoietic differentiation basal culture medium comprises StemPro34 medium, 1% P / S, 2mM GlutaMAX, 50μg / ml ascorbic acid, 1% ITS-G and 400μM MTG.

[0130] In some embodiments, the first hematopoietic differentiation medium is a hematopoietic differentiation basal medium supplemented with 1-20 μM CHIR99021, for example, CHIR99021 supplemented with 1, 5, 10, 15 or 20 μM.

[0131] In some embodiments, the second hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with at least one of BMP4, FGF2, and VEGF. In some embodiments, the second hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with 25-75 ng / ml BMP4, FGF2, and VEGF. In some embodiments, the second hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with 50 ng / ml BMP4, 50 ng / ml FGF2, and 50 ng / ml VEGF, wherein the basal hematopoietic differentiation medium comprises StemPro34 medium, 1% P / S, 2 mM GlutaMAX, 50 μg / ml ascorbic acid, 1% ITS-G, and 400 μM MTG.

[0132] In some embodiments, the third hematopoietic differentiation medium is the second hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml SCF and 1-10 μM SB431542. In some embodiments, the third hematopoietic differentiation medium is the second hematopoietic differentiation medium supplemented with 25-75 ng / ml SCF and 1-10 μM SB431542. In some embodiments, the third hematopoietic differentiation medium is the second hematopoietic differentiation medium supplemented with 50 ng / ml SCF and 6 μM SB431542, wherein the second hematopoietic differentiation medium is a basal hematopoietic differentiation medium supplemented with 50 ng / ml BMP4, 50 ng / ml FGF2, and 50 ng / ml VEGF, and the basal hematopoietic differentiation medium comprises StemPro34 medium, 1% P / S, 2 mM GlutaMAX, 50 μg / ml ascorbic acid, 1% ITS-G, and 400 μM MTG.

[0133] In some embodiments, the fourth hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml FGF2, 25-75 ng / ml VEGF, and 25-75 ng / ml SCF. In some embodiments, the fourth hematopoietic differentiation medium is the basal hematopoietic differentiation medium supplemented with 25-75 ng / ml FGF2, 25-75 ng / ml VEGF, and 25-75 ng / ml SCF. In some embodiments, the fourth hematopoietic differentiation medium is a basal hematopoietic differentiation medium supplemented with 50 ng / ml FGF2, 50 ng / ml VEGF, and 50 ng / ml SCF, wherein the basal hematopoietic differentiation medium comprises StemPro34 medium, 1% P / S, 2 mM GlutaMAX, 50 μg / ml ascorbic acid, 1% ITS-G, and 400 μM MTG.

[0134] In some implementations, iPSCs are dissociated into single cells prior to step (a). For example, human iPSCs are routinely cultured in commercially available Essential 8 medium. Before initiating iNK cell differentiation, iPSCs are ensured to be morphologically normal, confluenced to or below 70%, and without significant spontaneous differentiation, and are dissociated into single cells using Accutase.

[0135] In some embodiments, prior to step (a), iPSCs are dissociated into single cells, and the single cells are cultured in medium supplemented with 1-20 μM (e.g., 1, 5, 10, 15, or 20 μM) Y27632. For example, single cells are seeded in 6-well plates at 2-4 ml / well and cultured on a shaker. For example, collected cells are counted and seeded at a density of 1.5E5 cells / ml in 6-well plates at 2-4 ml / well, and cultured in StemScale PSC suspension complete medium supplemented with 10 μM Y27632 at a volume of 2 mL / well. The cell-seeded 6-well plates are then placed in a CO2 cell culture incubator with a CO2 shaker (70 rpm) and incubated at 37°C in 5% CO2 to generate embryoids (EB).

[0136] In some implementations, IL-2 is not used in step (b).

[0137] In some embodiments, step (b) uses 1-10 ng / mL of IL-3 in the first phase and does not use IL-3 in the second phase. In some embodiments, the second phase is 5-10 days after the first phase, for example, 8 days.

[0138] In some implementations, step (b) uses 10-30 ng / mL (e.g., 20 ng / mL) SCF, 10-30 ng / mL (e.g., 20 ng / mL) IL-7, 5-15 ng / mL (e.g., 10 ng / mL) IL-15 and 5-15 ng / mL (e.g., 10 ng / mL) FLT3L in both the first and second stages.

[0139] In some embodiments, step (b) in the first stage uses an NK differentiation medium containing the following components: 56.6% DMEM / F12+GlutaMAX™-I, 28.3% F12+GlutaMAX-I, 15% heat-inactivated human AB serum, 1% P / S, 2mM L-glutamine, 1μM β-mercaptoethanol, 5ng / mL sodium selenite, 50μM ethanolamine, 20ug / mL ascorbic acid, 5ng / mL IL-3, 20ng / mL SCF, 20ng / mL IL-7, 10ng / mL IL-15, and 10ng / mL FLT3 ligand (FLT3L).

[0140] In some embodiments, step (b) in the second stage uses an NK differentiation medium containing the following components: 56.6% DMEM / F12+GlutaMAX™-I, 28.3% F12+GlutaMAX-I, 15% heat-inactivated human AB serum, 1% P / S, 2mM L-glutamine, 1μM β-mercaptoethanol, 5ng / mL sodium selenite, 50μM ethanolamine, 20ug / mL ascorbic acid, 20ng / mL SCF, 20ng / mL IL-7, 10ng / mL IL-15, 10ng / mL FLT3 ligand (FLT3L), and IL-3-free.

[0141] In some implementations, step (b) is performed until the proportion of CD45+CD56+CD3- cells is >90%, for example, as determined by flow cytometry. Step (c) is then performed.

[0142] In some embodiments, step (c) includes co-culturing the iNK cells with a feeder cell line. In a preferred embodiment, the feeder cell line is the K562 myeloid leukemia cell line. In a preferred embodiment, the K562 cell line expresses membrane-bound IL-21 and 4-1BBL.

[0143] In some embodiments, the co-culture in step (c) is performed at a ratio of 5:1 to 1:5 of iNK cells to feeder cell lines. In some embodiments, the co-culture in step (c) is performed at a ratio of 1:1 of iNK cells to feeder cell lines. In some embodiments, the co-culture in step (c) is performed at a ratio of 1:1 of iNK cells to K562 cell lines.

[0144] In some implementations, step (c) involves amplification for 10-15 days, e.g., 12 days. In some implementations, the amplification is performed at 5x10⁻⁶. 5 cells / cm 2 The density was co-cultured statically in NK amplification medium (e.g., containing NK Xpander complete medium, 5% human serum, 1% P / S and 50 U / mL IL-2).

[0145] Another aspect of the invention provides an iNK cell population prepared by any of the embodiments described above. The iNK cell population has a CD45+CD56+CD3- phenotypic profile.

[0146] In some embodiments of the present invention, the cells in the iNK cell population may be further genetically engineered to form engineered iNK cells, such as CAR-iNK. Methods for forming CAR-iNK are known in the art, and the present invention does not impose any limitations. The iNK cells provided by the present invention are intended to be applicable to any suitable CAR formation process and CAR targeting any suitable target.

[0147] Example

[0148] Example 1. Differentiation and expansion of iNK cell population

[0149] Human iPSCs were routinely cultured in commercially available Essential 8 medium. Before initiating NK cell differentiation, iPSCs were ensured to be morphologically normal, confluence close to or below 70%, and without significant spontaneous differentiation. Cells were dissociated into single cells using Accutase, and the collected cells were counted and seeded at a density of 1.5E5 cells / ml in 6-well plates. The plates were then cultured in StemScale PSC suspension complete medium supplemented with 10 μM Y27632 at a volume of 2 mL / well. The seeded 6-well plates were then placed in a CO2 cell culture incubator with a CO2 shaker (70 rpm) and incubated at 37°C in 5% CO2 to generate embryoids (EBs). 24 hours later (D1), the medium was changed, and the embryoids were collected in 15 ml centrifuge tubes and collected by sedimentation. The EBs were then resuspended in the original wells with hematopoietic differentiation basal medium (StemPro34 medium, 1% PS, 2 mM GlutaMAX, 50 μg / ml ascorbic acid, 1% ITS-G, 400 μM MTG) supplemented with 10 μM CHIR99021 and cultured with shaking. On D2 and D4, the medium change was repeated, and the EBs were resuspended in the original wells with hematopoietic differentiation medium 1 (hematopoietic differentiation basal medium, 50 ng / ml BMP4, 50 ng / ml FGF2, 50 ng / ml VEGF) and hematopoietic differentiation medium 2 (hematopoietic differentiation basal medium 1, 50 ng / ml SCF, 6 μM SB431542) and cultured with shaking, respectively. Starting from day 6, free suspended cells appear in the culture medium supernatant. When changing the medium thereafter, embryonic bodies and suspended cells need to be collected by sedimentation and centrifugation respectively, and then resuspended in the original wells with hematopoietic differentiation medium 3 (basal hematopoietic differentiation medium, 50 ng / ml FGF2, 50 ng / ml VEGF, 50 ng / ml SCF) and shaken.

[0150] After the first 8 days of hematopoietic differentiation, on day 8, all EB and suspension cells from two 6-well plates were collected and seeded into a T75 culture flask. The cells were then cultured in 15 mL of NK differentiation medium 1 (56.6% DMEM / F12+GlutaMAX™-I, 28.3% F12+GlutaMAX-I, 15% heat-inactivated human AB serum, 1% P / S, 2 mM L-glutamine, 1 μM β-mercaptoethanol, 5 ng / mL sodium selenite, 50 μM ethanolamine, 20 μg / mL ascorbic acid, 5 ng / mL IL-3, 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL IL-15, 10 ng / mL...). NK cell differentiation was induced by static culture in FLT3 ligand (FLT3L) at 377°C and 5% CO2. On the fourth day of NK differentiation, an equal volume (15 ml / T75) of NK differentiation medium 1 was added to the culture flasks, and static culture continued. On the eighth day of NK differentiation, all suspension cells in each T75 flask were collected (if any non-adherent EB cells were collected after sedimentation), centrifuged, and resuspended in 20 mL / T75 flask of NK differentiation medium 2 (NK differentiation medium 1 without IL-3) and static cultured. On the 12th day of NK differentiation, 20 mL of NK differentiation medium 2 was added to each T75 flask and static culture was continued. Subsequently, every 4 days, the suspension cells were centrifuged and the medium was partially replaced using NK differentiation medium 2. On day 28 of NK differentiation, all suspended spindle-shaped NK cells were collected by centrifugation (avoiding EB collection), and samples were taken for counting and flow cytometry analysis (at this time, the proportion of CD45+CD56+CD3- cells was >90%) for initial iNK amplification.

[0151] For the first round of expansion after differentiation, iNK cells were co-cultured with irradiated K562 myeloid leukemia cell lines expressing mbIL-21 and 4-1BBL at a 1:1 ratio and a density of 5 x 10⁵ cells / cm² in NK expansion medium (NK Xpander complete medium, 5% human serum, 1% P / S, and 50 U / mL IL-2) in a G-Rex 6M Well with a culture volume of 100 mL. Fresh IL-2 (50 U / mL) was added to the supernatant every 4 days. The cells at the bottom of the G-Rex were kept undisturbed throughout the expansion culture. After 12 days of expansion, the iNK cells at the bottom of the G-Rex were harvested by centrifugation, counted, and cryopreserved using CS10.

[0152] Example 2. In vivo efficacy test of human ovarian cancer cell line-1 (hypoglycemic ovarian cancer SKOV3) xenograft model

[0153] Objective: To investigate the in vivo efficacy of the drug in a human ovarian cancer xenograft model, we examined the inhibitory effect of the iNK cell population (hereinafter referred to as iNK) prepared in Example 1 on the growth of xenografts in the peritoneum of mice after multiple intraperitoneal injections.

[0154] Methods: The in vivo efficacy experiment procedure is shown in Figure 1. Six- to eight-week-old female immunodeficient mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were intraperitoneally inoculated with 2 × 10⁶ mmol / L of the drug. 5 Human ovarian cancer cell line -1-luc cells were grouped by fluorescence signal detection using IVIS 4 days later. After 1.4 Gy irradiation, the mice were intraperitoneally injected with in vitro expanded iNK cells (1×10⁻⁶ cells) 24 hours later. 7 Cells / session, once a week, for a total of 3 times) or PBNK cells (1×10⁻⁶ cells / session, once a week, for a total of 3 times) 7 Cells / time, once a week, for a total of 3 times), and administered to mice via intraperitoneal injection 1×10 5 IU hIL-2 (twice a week for 3 weeks, for a total of 6 times), and D-luciferin substrate injected intraperitoneally twice a week, followed by administration via IV fluid 5 minutes later. The Lumina III in vivo imaging system monitors tumor growth rates.

[0155] Tumor cell proliferation (tumor cell bioluminescence intensity): As shown in Figure 2, the bioluminescence imaging results detected by IVIS showed that on day 45 after the start of the experiment, the iNK cells after three administrations achieved a tumor inhibition rate of 99.88% against human ovarian cancer cell line-1, which was statistically significant (p < 0.0001****), exhibiting similar antitumor activity to PBNK cells after the same dose and number of administrations (TGI = 99.98%, p < 0.0001****). Therefore, the iNK cell population of the present invention exhibited a strong inhibitory effect on human ovarian cancer cell line-1 in vivo under intraperitoneal administration conditions.

[0156] Body weight: As shown in Figure 3, the mice maintained normal body weight after multiple iNK administrations, demonstrating good safety.

[0157] General clinical observation: As shown in Figure 4, by day 90 of the experiment, all mice in the tumor-bearing control group (8 / 8) and the PBNK group (4 / 4) had died, while 37.5% (3 / 8) of the mice in the iNK cell group survived, which was statistically significant compared with the tumor-bearing control group (p < 0.0001). Animal mortality in the tumor-bearing control group began on day 12, which is presumably related to excessive tumor burden.

[0158] Example 3: In vivo dose-effect test of human ovarian cancer cell line-1 (hypoplasmic ovarian cancer SKOV3) xenograft model

[0159] Objective: To investigate the dose-response effect of different doses of iNK administered via multiple intraperitoneal injections in a human ovarian cancer xenograft model, we examined the inhibitory effect of iNK on the growth of xenografts in the peritoneal cavity of mice.

[0160] Methods: The in vivo efficacy experiment procedure is shown in Figure 1. Six- to eight-week-old female immunodeficient mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were intraperitoneally inoculated with 2 × 10⁶ mmol / L of the drug. 5 Human ovarian cancer cell line -1-luc cells were grouped by fluorescence signal detection using IVIS 4 days later. After 1.4 Gy irradiation, the mice were intraperitoneally injected with in vitro expanded iNK cells (1×10⁻⁶ cells) 24 hours later. 7 Cells / time or 3×10 6 Cells / time or 1×10 6 Cells / time, once a week, for a total of 3 times), and administered to mice via intraperitoneal injection 1×10 5 IU hIL-2 (twice a week for 3 weeks, for a total of 6 times), and monitor tumor growth rate twice a week via IVIS.

[0161] Tumor cell proliferation (tumor cell bioluminescence intensity): As shown in Figure 5, the bioluminescence imaging results detected by IVIS showed that on day 45 after the start of the experiment, the inhibition rates of iNK against human ovarian cancer cell line-1 after three doses were: 1E7 iNK TGI = 85.21% (P = 0.0021**), 3E6 iNK TGI = 72.03% (p = 0.0093**), and 1E6 iNK TGI = 22.03% (p = 0.6523). ns Therefore, under intraperitoneal administration, iNK cells 1E6, 3E6, and 1E7 all showed certain inhibitory effects on human ovarian cancer cell line-1 in vivo, exhibiting a good dose-response relationship. Among them, the inhibitory effects of iNK cells 3E6 and 1E7 on human ovarian cancer cell line-1 were statistically different.

[0162] Body weight: As shown in Figure 6, the mice maintained normal body weight after multiple administrations of high, medium, and low doses of iNK, demonstrating good safety. The tumor-bearing control group mice experienced a significant decrease in body weight, presumably due to a higher tumor burden.

[0163] General clinical observation: As shown in Figure 7, up to day 90 after the start of the experiment, the median survival of the tumor-bearing control group was 51.5 days, and the median survival of the high-dose iNK group was 66 days, which was statistically significant compared with the tumor-bearing control group (p = 0.0005***); the median survival of the medium-dose iNK group was 61.5 days, which was statistically significant compared with the tumor-bearing control group (p = 0.0107*); the median survival of mice in the low-dose iNK group was 60 days, which was statistically significant compared with the tumor-bearing control group (p = 0.0270*).

[0164] Example 4: In vivo efficacy test of human ovarian cancer cell line-2 (hyperserum ovarian cancer OVCAR3) xenograft model

[0165] Objective: To verify the in vivo efficacy of iNK in an ovarian cancer xenograft model, we examined the inhibitory effect of iNK on tumor growth after multiple intraperitoneal injections in a human ovarian cancer cell line-2 xenograft model.

[0166] Methods: As shown in Figure 1, 6-8 week old female severely immunodeficient mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were intraperitoneally inoculated with 2×10⁶ mice. 5 Human ovarian cancer cell line -2-luc cells were grouped by IVIS detection of fluorescence signal values ​​after 7 days. Mice enrolled were irradiated with 1.4 Gy for 24 hours, and then intraperitoneally injected with in vitro expanded PBNK cells (1×10⁻⁶). 7 Cells / time, once a week, for a total of 3 times) and iNK cells (1×10 7 Cells / time or 3×10 6 Cells / time or 1×10 6 Cells / time, once a week, for a total of 3 times), and administered to mice via intraperitoneal injection 1×10 5 IU hIL-2 (twice a week for 3 weeks, for a total of 6 times), and monitor tumor growth rate twice a week via IVIS.

[0167] Tumor cell proliferation (tumor cell bioluminescence intensity): As shown in Figure 8, the bioluminescence imaging results detected by IVIS showed that on day 54 after the start of the experiment, the inhibition rate of iNK against human ovarian cancer cell line-2 after 3 administrations was similar to that of PBNK after the same dose and number of administrations (TGI = 97.45%, p < 0.0001****), all of which were statistically significant (p < 0.0001****). Specifically, the TGI of 1E7 iNK was 98.32%, the TGI of 3E6 iNK was 88.72%, and the TGI of 1E6 iNK was 59.40%. Therefore, iNK, under intraperitoneal administration conditions, showed a strong inhibitory effect on human ovarian cancer cell line-2 in vivo, exhibiting a good dose-response relationship.

[0168] Body weight: As shown in Figure 9, the mice maintained normal body weight after multiple administrations of high and medium doses of iNK, demonstrating good safety. The tumor-bearing control group and the low-dose iNK group showed significant weight loss, presumably due to higher tumor burden.

[0169] General clinical observation: As shown in Figure 10, by day 68 after the start of the experiment, all mice in the tumor-bearing control group (6 / 6) were found to have died. In the same dose PBNK and high-dose iNK groups, 100% (6 / 6) of the mice survived, which was statistically significant compared to the tumor-bearing control group (p = 0.0012**). In the medium-dose iNK group, 83.33% (5 / 6) of the mice survived, which was not statistically significant compared to the tumor-bearing control group (p = 0.0138*). In the low-dose iNK group, 50% (3 / 6) of the mice survived, which was not statistically significant compared to the tumor-bearing control group (p = 0.1486). ns Animals in the tumor-bearing control group began to die from day 15 onwards, which is presumably related to excessive tumor burden and rapid tumor development.

[0170] Example 5: In vivo efficacy test of human gastric cancer cell line (MKN28) xenograft model

[0171] Objective: To verify the in vivo efficacy of iNK in a gastric cancer xenograft model, we examined the inhibitory effect of iNK on tumor growth after multiple intraperitoneal injections in a human gastric cancer cell line xenograft model.

[0172] Methods: As shown in Figure 1, 6-8 week old female severely immunodeficient mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were intraperitoneally inoculated with 2×10⁶ mice. 5 Human gastric cancer cell line -luc cells were grouped by fluorescence signal detection by IVIS 7 days later. After 1.4 Gy irradiation, mice were intraperitoneally injected 24 hours later with in vitro expanded PBNK cells and iNK cells (1×10⁻⁶). 7 Cells / time, once a week, for a total of 3 times), and administered to mice via intraperitoneal injection 1×10 5 IU hIL-2 (twice a week for 3 weeks, for a total of 6 times), and monitor tumor growth rate twice a week via IVIS.

[0173] Tumor cell proliferation (tumor cell bioluminescence intensity): As shown in Figure 11, the bioluminescence imaging results detected by IVIS showed that on day 50 after the start of the experiment, iNK after 3 doses showed a certain anti-tumor activity against human gastric cancer cell lines (TGI = 57.28%, p = 0.0247*); PBNK after 3 doses showed a tumor inhibition rate against human gastric cancer cell lines (TGI = -53.86%, p > 0.05). ns The iNK cells did not exhibit antitumor activity. Therefore, iNK cells showed some inhibitory effect on human gastric cancer cell lines under intraperitoneal administration conditions, but the inhibitory effect was not as good as that on ovarian cancer cell lines.

[0174] Body weight: As shown in Figure 12, the body weight of mice gradually remained normal after multiple administrations of iNK and PBNK, demonstrating good safety.

[0175] General clinical observation: As shown in Figure 13, by day 108 of the experiment, 50% (3 / 6) of the tumor-bearing control group mice survived, and 83.33% (5 / 6) of the iNK group mice survived. There was no statistically significant difference between the tumor-bearing control group and the iNK group (p = 0.4138). ns Therefore, iNK administration via intraperitoneal administration had no significant effect on the survival of human gastric cancer cell line-bearing mice.

[0176] Example 6: In vivo efficacy test of human colorectal cancer cell line (RKO) xenograft model

[0177] Objective: To verify the in vivo efficacy of iNK in a colorectal cancer xenograft model, we examined the inhibitory effect of iNK on tumor growth after multiple intraperitoneal injections in a human colorectal cancer cell line xenograft model.

[0178] Methods: As shown in Figure 1, 6-8 week old female severely immunodeficient mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were intraperitoneally inoculated with 2×10⁶ mice. 5 Human colorectal cancer cell line -luc cells were grouped by fluorescence signal detection by IVIS 7 days later. After 1.4 Gy irradiation, mice were intraperitoneally injected 24 hours later with in vitro expanded PBNK cells and iNK cells (1×10⁻⁶). 7 Cells / time, once a week, for a total of 3 times), and administered to mice via intraperitoneal injection 1×10 5 IU hIL-2 (twice a week for 3 weeks, for a total of 6 times), and monitor tumor growth rate twice a week via IVIS.

[0179] Tumor cell proliferation (tumor cell bioluminescence intensity): As shown in Figure 14, the bioluminescence imaging results detected by IVIS showed that on day 50 after the start of the experiment, the tumor inhibition rates of iNK and PBNK against human colorectal cancer cell lines after three administrations were 94.86% (p = 0.0148*) and 80.48% (p = 0.0461*), respectively, both showing statistically significant differences. Therefore, iNK showed a certain inhibitory effect on human colorectal cancer cell lines under intraperitoneal administration conditions in vivo, but the inhibitory effect was not as good as that of ovarian cancer cell lines.

[0180] Body weight: As shown in Figure 15, the mice maintained normal body weight after multiple administrations of iNK and PBNK, demonstrating good safety.

[0181] General clinical observation: As shown in Figure 16, by day 40 after the start of the experiment, no mice survived in either the tumor-bearing control group or the iNK group, which was not statistically significant (p = 0.4138). ns Therefore, iNK administration via intraperitoneal administration had no significant effect on the survival of mice bearing human colorectal cancer cell lines.

[0182] Example 7: In vivo efficacy test of a subcutaneous tumor model of human hepatocellular carcinoma cell line (HepG-2)

[0183] Objective: To verify the in vivo efficacy of iNK in a hepatocellular carcinoma xenograft model, we examined the inhibitory effect of iNK on tumor growth after multiple tail vein injections in a human hepatocellular carcinoma subcutaneous tumor model.

[0184] Methods: As shown in Figure 16, 6-8 week old female severely immunodeficient mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were subcutaneously inoculated with 5×10⁶ mice. 6 Human liver cancer cell line, after 14 days, was measured by tumor volume (~50 mm). 3 Mice were divided into groups. Twenty-four hours after irradiation with 1.4 Gy, the mice were injected via tail vein with in vitro expanded iNK cells (1 × 10⁻⁶). 7 Cells / time, twice a week, for a total of 6 times), and administered to mice via tail vein injection 1×10 5 IU hIL-2 (twice a week for 3 weeks, for a total of 6 times), and tumor growth rate measured twice a week using calipers.

[0185] Tumor cell proliferation: As shown in Figure 18, the bioluminescence imaging results obtained by IVIS showed that on day 32 after the start of the experiment, the tumor inhibition rate of iNK against human hepatocellular carcinoma cell lines after 6 administrations was 54.14%, which was statistically different from the tumor-bearing control group (p < 0.05*). Therefore, iNK showed a certain inhibitory effect on human hepatocellular carcinoma cell lines in vivo under tail vein administration, but the inhibitory effect was not as good as that of ovarian cancer cell lines.

[0186] Tumor volume: As shown in Figure 19, the tumor volume of mice decreased after multiple administrations of iNK, demonstrating a certain tumor inhibition effect (p < 0.01**).

[0187] Example 8. Investigator-Initiated Clinical Trial (IIT) of NK Cells and its Results

[0188] IIT was performed using the NK cell population (iNK) prepared according to Example 1. The dose level was 3 × 10⁻⁶. 8 6×10 8 Cells / individuals, administered weekly for three consecutive weeks, with each cycle lasting 28 days, for a total of two treatment courses. The clinical trial was conducted at the First Affiliated Hospital of Bengbu Medical Center. The subject was a 63-year-old female of Han ethnicity, who first signed informed consent on May 26, 2023. On February 9, 2017, she underwent radical oophorectomy for ovarian cancer (abdominal total hysterectomy + bilateral salpingo-oophorectomy + pelvic lymph node dissection + omentectomy + appendectomy). Postoperative pathological diagnosis was stage IIIA bilateral mucinous cystadenocarcinoma of the ovaries, with extensive omental metastasis and metastatic nodules in the mesentery. As of May 31, 2023, the subject was confirmed to have TxN0M0, stage III ovarian cancer.

[0189] Summary after two cycles of dosing: The actual AEs were mild (grade 1-2), with fever, peritoneal fibrin formation, and hypoalbuminemia all within the predicted range. After symptomatic treatment, all AEs were controllable and reversible. No serious adverse events (SAEs) occurred. Therefore, the subjects tolerated the 6 doses of the iNK cell population prepared in Example 1 of this invention well. No AEs related to the iNK cell population of this invention occurred during the subsequent follow-up period. Five consecutive imaging assessments after dosing showed stable disease (SD); the latest imaging assessment on February 18, 2024, showed disease progression (PD). The subjects were enrolled for 9 months, and telephone survival follow-up is ongoing.

[0190] Following administration, the subjects' quality of life improved. The tumor marker carbohydrate antigen 125 (CA125) decreased (baseline level 295.5 IU / mL, most recent follow-up level 214.10 IU / mL, with a minimum level of 141.1 IU / mL). After administration, the improvement in ascites volume was maintained for 5 months. Changes in ascites volume and serum CA125 levels are shown in Tables 1 and 2 below. Imaging results from follow-up are shown in Figure 20, and the results of the five follow-up visits are summarized in Table 3.

[0191] Table 1. Changes in serum CA125 levels in the subjects

[0192] Table 2. Changes in ascites volume in the subjects

[0193] Table 3. Summary of some imaging results of the subjects

Claims

1. The use of an iNK cell population induced by iPSCs in the preparation of a drug for treating cancer.

2. The application according to claim 1, wherein the cancer is ovarian cancer, gastric cancer, colorectal cancer or liver cancer; preferably, the cancer is ovarian cancer.

3. The application according to claim 2, wherein the ovarian cancer is ovarian serous cystadenocarcinoma or ovarian mucinous cystadenocarcinoma.

4. The application according to claim 2, wherein the ovarian cancer is stage III ovarian cancer.

5. In the application according to claim 2, the amount of ascites in the subject is reduced by 10%-90% after treatment; and / or, the tumor marker CA125 in the subject is reduced by 25%-50% after treatment.

6. The application according to claim 1, wherein the iNK cell population is prepared from iPSCs by a method comprising the following steps: (a) Culture iPSCs to generate embryoids and perform hematopoietic differentiation on the embryoids; (b) Differentiating hematopoietic embryoids into iNK cells; and (c) Expand the iNK cells to obtain the iNK cell population.

7. The application according to claim 6, wherein, Step (a) involves hematopoietic differentiation of the embryoids using different hematopoietic differentiation media over time; preferably, four different hematopoietic differentiation media are used over time: (i) First hematopoietic differentiation medium: hematopoietic differentiation basal medium supplemented with 1-20 μM CHIR99021; (ii) Second hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of BMP4, FGF2 and VEGF, at a concentration of 25-75 ng / ml; (iii) Third hematopoietic differentiation medium: Second hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml SCF and 1-10 μM SB431542; (iv) Fourth hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of FGF2, VEGF and SCF at 25-75 ng / ml; More preferably, the embryoids are cultured in the first, second, third and fourth differentiation media in sequence for two days.

8. The application according to claim 7, wherein the hematopoietic differentiation basal culture medium comprises StemPro34 medium, 0.5%-1.5% PS, 1-5mM GlutaMAX, 25-75μg / ml ascorbic acid, 0.5%-1.5% ITS-G and 200-600μM MTG.

9. The application according to claim 6, wherein the iPSCs are dissociated into single cells prior to step (a); preferably, the single cells are cultured in a medium supplemented with 1-20 μM Y27632; and / or, preferably, the single cells are seeded in 2-4 ml / well 6-well plates and cultured on a shaker.

10. The application according to claim 6, wherein IL-2 is not used in step (b); and / or, wherein NK differentiation medium is used in step (b), the NK differentiation medium comprising at least one of 1-15 ng / mL IL-3, 1-30 ng / mL FLT3L and 1-30 ng / mL IL-15.

11. The application according to claim 10, wherein step (b) is performed after 5-10 days in NK differentiation medium without IL-3.

12. The application according to claim 6, wherein step (b) is performed until the proportion of CD45+CD56+CD3- cells is >90%.

13. The application according to claim 6, wherein step (c) comprises co-culturing the iNK cells with a feeder cell line; preferably, the feeder cell line is the K562 myeloid leukemia cell line; preferably, the K562 cell line expresses membrane-bound IL-21 and 4-1BBL; preferably, the co-culturing is performed at a ratio of iNK cells to feeder cell line of 5:1 to 1:

5.

14. The application according to claim 6, wherein step (c) is performed in NK amplification medium; preferably, the NK amplification medium contains at least one of 1%-10% human serum, 0.5%-1.5% P / S and 25-75 U / mL IL-2; preferably, the NK amplification medium contains NK Xpander complete medium.

15. The method of claim 14, wherein step (c) involves amplification for 10-15 days.

16. A method for inducing iNK cell populations by iPSCs, the method comprising: (a) Culture iPSCs to generate embryoids and perform hematopoietic differentiation on the embryoids; (b) Differentiating hematopoietic embryoids into iNK cells; and (c) Expand the iNK cells to obtain the iNK cell population.

17. The method of claim 16, wherein the hematopoietic differentiation of the embryoids in step (a) comprises performing hematopoietic differentiation using different hematopoietic differentiation media over time; preferably, wherein four different hematopoietic differentiation media are used over time: (i) Primary hematopoietic differentiation medium: basic hematopoietic differentiation culture supplemented with 1-20 μM CHIR99021 base; (ii) Second hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of BMP4, FGF2 and VEGF, at a concentration of 25-75 ng / ml; (iii) Third hematopoietic differentiation medium: Second hematopoietic differentiation medium supplemented with at least one of 25-75 ng / ml SCF and 1-10 μM SB431542; (iv) Fourth hematopoietic differentiation medium: the hematopoietic differentiation basal medium supplemented with at least one of FGF2, VEGF and SCF at 25-75 ng / ml; More preferably, the embryoids are cultured in the first, second, third and fourth differentiation media in sequence for two days.

18. The method according to claim 16, wherein the hematopoietic differentiation basal culture medium comprises StemPro34 medium, 0.5%-1.5% PS, 1-5mM GlutaMAX, 25-75μg / ml ascorbic acid, 0.5%-1.5% ITS-G and 200-600μM MTG.

19. The method of claim 16, wherein the iPSCs are dissociated into single cells prior to step (a); preferably, the single cells are cultured in a medium supplemented with 1-20 μM Y27632; and / or, preferably, the single cells are seeded in 2-4 ml / well 6-well plates and cultured on a shaker.

20. The method of claim 16, wherein IL-2 is not used in step (b); and / or, wherein NK differentiation medium is used in step (b), the NK differentiation medium comprising at least one of 1-15 ng / mL IL-3, 1-30 ng / mL FLT3L and 1-30 ng / mL IL-15.

21. The method of claim 20, wherein step (b) is performed after 5-10 days in NK differentiation medium without IL-3.

22. The method of claim 16, wherein step (b) is performed until the proportion of CD45+CD56+CD3- cells is >90%.

23. The method of claim 16, wherein step (c) comprises co-culturing the iNK cells with a feeder cell line; preferably, the feeder cell line is the K562 myeloid leukemia cell line; preferably, the K562 cell line expresses membrane-bound IL-21 and 4-1BBL; preferably, the co-culturing is performed at a ratio of iNK cells to feeder cell line of 5:1 to 1:

5.

24. The method of claim 16, wherein step (c) is performed in an NK amplification medium; preferably, the NK amplification medium comprises at least one of 1%-10% human serum, 0.5%-1.5% P / S and 25-75 U / mL IL-2; preferably, the NK amplification medium comprises NK Xpander complete medium.

25. The method of claim 24, wherein step (c) involves amplification for 10-15 days.

26. iNK cell population, which is prepared by the method according to any one of claims 16 to 25.

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