Method for enhancing NK cells derived from gene-modified pluripotent stem cells and its application

Abstract

The present invention discloses a method for preparing enhanced NK cells derived from genetically modified pluripotent stem cells and its application, and provides a modified cell or its population. The cell or its population comprises: (a) F158V and S197P in the extracellular domain of CD16; (b) all or part of the extracellular domain derived from CD16; (c) the native CD16 transmembrane domain; (d) the intracellular domain with at least one of the native stimulatory domains being 2B4 or DAP10; (e) the intracellular domain of the native signal transduction domain CD3ζ, showing a mature differentiation phenotype and stronger immune killing ability, and having a more significant CD16 expression. The present invention also provides a method for preparing the cell or its population and its use. The expression levels of the maturation-related membrane protein CD94 and the killing function-related proteins NKp44 / NKG2A in the FR1-iNK cells obtained by this method are significantly increased compared with those in CTRL-iNK and FR2-iNK.

Description

Technical Field

[0001] The present invention relates to the field of immune cell products, and particularly to a method for genetically modifying pluripotent stem cell-derived enhanced NK cells and its applications. Background Art

[0002] The prior art enhances the antibody-dependent cell-mediated cytotoxicity (ADCC) of immune cells to kill tumor cells by exogenously expressing high-affinity non-cleavable hnCD16 or its variants (chimeric antigen receptor hnCD16, CAR-hnCD16). Currently, existing iPSC-derived NK cell therapy products and technologies based on CD16 engineering in immune cell therapy mainly focus on the engineering modification of iPSCs with "high-affinity non-cleavable CD16 (hnCD16) or its variants".

[0003] Regarding this technical route, the prior art has listed various transmembrane domains, stimulation domains, and signal transduction domains of membrane signaling proteins of immune cells (T, NK, macrophages, etc.) as the structural combination sources of the hnCD16 variant CAR-hnCD16. However, whether such randomly combined CAR-hnCD16 can functionally exert the ADCC effect has not been verified by the prior art. More importantly, for different immune cells, the ADCC effects of different CAR-hnCD16 structural combinations vary greatly, which will greatly affect their ability to kill tumors as cell therapy products. In addition, for the specific CAR structure of natural killer cells, especially the efficient intracellular domain and signal stimulation domain, are also the keys to affecting the efficiency of non-wild-type CD16 protein-mediated ADCC killing of tumors. For example, Chinese Patent CN113166232A discloses an immunotherapy using enhanced iPSC-derived effector cells. This patent only verified the role of exogenously expressing the hnCD16a protein (the extracellular part is a high-affinity and non-cleavable CD16a with point mutations, and the transmembrane region and intracellular region are wild-type CD16a sequences) in iPSC-differentiated NK cells, and did not confirm and optimize the CAR-hnCD16 structural combination suitable for NK cells. Summary of the Invention

[0004] The present invention provides a method for genetically modifying pluripotent stem cell-derived enhanced NK cells and its applications, aiming to solve the above problems in the background art.

[0005] The technical solution provided by the present invention is as follows:

[0006] In a first aspect, the present invention provides a modified cell or its population, wherein:

[0007] (i) The cells include: (a) induced pluripotent cells (iPSCs), (b) hematopoietic stem cells or hematopoietic progenitor cells; or (b) derivative cells obtained by differentiating the cells of (a); and

[0008] (ii) Express at least one endogenous gene encoding the following:

[0009] (1) Fc receptor FcγRIIIa (CD16a);

[0010] (2) CD94, CD56, and / or CD45;

[0011] (3) NKp46, NKG2D, and / or FasL;

[0012] (iii) Contain at least one or more exogenous nucleic acid expression constructs, which contain nucleic acid sequences encoding the following:

[0013] (1) Chimeric antigen receptor CAR;

[0014] (2) High-affinity non-cleavable hnCD16 or its engineered variant CAR-hnCD16.

[0015] In one embodiment, the hnCD16 or its engineered variant CAR-hnCD16 includes at least one of the following:

[0016] (a) F158V and S197P in the extracellular domain of CD16;

[0017] (b) All or part of the extracellular domain derived from CD16;

[0018] (c) Native CD16 transmembrane domain;

[0019] (d) Intracellular domain with at least one of the native stimulatory domains being 2B4 or DAP10;

[0020] (e) Native signal transduction domain CD3ζ intracellular domain.

[0021] In one embodiment, the cells are derivative NK cells, and the NK cells are derived from induced pluripotent stem cells or hematopoietic stem (progenitor) cells.

[0022] In one embodiment, the cells have at least one of the following characteristics compared to the natural corresponding cells differentiated from induced pluripotent stem cells:

[0023] (i) Activate immune cells;

[0024] (ii) Improve the recognition and killing of tumor cell antigens;

[0025] (iii) Enhance the ADCC effect of immune cells;

[0026] (iv) Increase cytotoxicity;

[0027] (v) Increase the types of tumor cells killed.

[0028] In a second aspect, the present invention provides a method for generating the above-mentioned modified cells or a population thereof, the method comprising:

[0029] (i) Design and produce a viral vector for transfecting iPSCs;

[0030] (ii) Perform lentiviral packaging;

[0031] (iii) Generate hnCD16-FR-iPSCs by transfecting human induced pluripotent stem cells hiPSCs expressing hnCD16-FR and GFP proteins with lentivirus, and then perform single-cell sorting and clonal selection to obtain hnCD16-FR-iPSCs positive clones.

[0032] In one embodiment, the generation of hnCD16-FR-iPSCs from the induced pluripotent stem cells includes:

[0033] (i) First differentiate the induced pluripotent stem cells into hematopoietic stem (progenitor) cells, and then differentiate the hematopoietic progenitor cells into NK cells. When CD34+ cells appear, transfer the NK cells to an NK cell differentiation medium;

[0034] (ii) Place the NK cells in the NK cell differentiation medium for 28-35 days, changing the medium weekly until it is determined that they develop into CD45+CD56+ cells.

[0035] In one embodiment, the medium contains DMEM / Ham F12, l-glutamine, penicillin / streptomycin, β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, interleukin-3, stem cell factor, interleukin-15, fms-like tyrosine kinase 3 ligand, and interleukin-7.

[0036] In one embodiment, the performance of lentiviral packaging includes:

[0037] (i) Prepare HEK 293T cells;

[0038] (ii) Transfect with a lentiviral packaging system;

[0039] (iii) Collect and concentrate the lentiviral solution.

[0040] In one embodiment, the design and production of a viral vector for transfecting iPSCs includes:

[0041] The CAR-hnCD16 lentiviral transfer plasmid containing a chimeric 5' long terminal repeat (LTR), packaging signal, Rev response element (RRE), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a 3' self-inactivating (SIN) LTR was placed under the control of the elongation factor 1alpha (EF1α) promoter (EF1α) and co-expressed with EGFP via an IRES.

[0042] In a third aspect, the present invention provides the use of the above-mentioned modified cell or its population in cancer immunotherapy.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] 1. The present invention provides a modified cell or its population. By designing CAR-hnCD16 with different intracellular stimulatory domain structures, the CAR-hnCD16 structure that mediates the best ADCC effect in iPSC-derived NK cells is preferably selected, so as to improve the efficacy of tumor killing by iPSC-derived NK cell therapeutic products carrying engineered CD16.

[0045] 2. The two engineered iNK cells (FR1 and FR2) expressing hnCD16FR provided by the present invention have more significant CD16 expression compared to CTRL. In particular, the expression levels of the maturation-related membrane protein CD94 and the killing function-related proteins NKp44 / NKG2A in FR1-iNK cells are significantly increased compared to CTRL-iNK and FR2-iNK.

[0046] 3. The present invention also provides a method for preparing the above-mentioned NK cells. The NK cell maturation-related membrane proteins such as CD94 and the killing function proteins such as NKp46, NKG2D, and FasL are significantly expressed in the terminal cells prepared by iPSC differentiation, demonstrating that NK cells with mature differentiation phenotypes and immune killing abilities are obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a schematic diagram of the structure of the CAR-hnCD16 lentiviral transfer plasmid in the embodiment of the present invention;

[0048] Figure 2 It is a schematic diagram of the combined domain structure of CAR-hnCD16-FR1 and CAR-hnCD16-FR2 in the embodiment of the present invention;

[0049] Figure 3 It is a schematic diagram of the results of the expression of exogenously introduced genes in iPSC positive clone cells detected by flow cytometry experiments in the embodiment of the present invention;

[0050] Figure 4 Schematic diagrams of CAR-hnCD16-FR1 and hnCD16-CAR(FR3) structures in the embodiments of the present invention;

[0051] Figure 5 Schematic diagram of the results of immunofluorescence staining with an anti-CD16 antibody after infecting HEK293 cells with lentiviruses containing FR1 and FR3 structures respectively in the embodiments of the present invention;

[0052] Figure 6 Schematic diagram of the results of flow cytometry analysis of the transmembrane structure hnCD16-CAR of NKG2D in the embodiments of the present invention, where the abscissa is CD16 and the ordinate is GFP;

[0053] Figure 7 Schematic diagram of the results of flow cytometry analysis of the expression of CD56 and CD45 in the terminal cells at the differentiation end point of CTRL-iPSC, FR1-iPSC, and FR2-iPSC in the embodiments of the present invention;

[0054] Figure 8 Flow cytometry detection results of NK cell-related function markers 28 days after differentiation in the embodiments of the present invention;

[0055] Figure 9 Schematic diagram of the results of killing Raji cells by the differentiated ink effector cells CTRL, FR1, and FR2 combined with anti-CD20 mab (Obinutuzumab) in the embodiments of the present invention;

[0056] Figure 10 Schematic diagram of the results of killing A549 cells by the differentiated ink effector cells CTRL, FR1, and FR2 combined with anti-EGFR mab (Nimotuzumab) in the embodiments of the present invention. Detailed implementation manners

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the embodiments described below are some, but not all, of the embodiments of the present application. Usually, the components of the embodiments of the present application described and illustrated in the accompanying drawings here can be arranged and designed in various different configurations.

[0058] Accordingly, the detailed description of the embodiments of the present application provided below in conjunction with the accompanying drawings is intended to represent only the selected embodiments of the present application and does not limit the scope of the present application claimed. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts belong to the scope of protection of the present application.

[0059] Natural killer (NK) cells are one of the members of the innate immune system and have two key functions: lysing target cells and providing early cytokines for immune regulation. In recent years, researchers have made great progress in studying the mechanism of action of natural killer (NK) cells in the immune efficacy against tumors and viruses. As cytotoxic innate lymphocytes, natural killer cells can produce inflammatory cytokines and chemokines, thereby limiting tumor growth and virus infection by lysing transformed or infected target cells. Studies have shown that autologous NK cells from donors can inhibit the recurrence of leukemia treatment after allogeneic hematopoietic stem cell transplantation. More importantly, the infusion of allogeneic NK cells has been shown to induce and / or maintain remission in acute myeloid leukemia (AML). More importantly, compared with T cells activated by MHC molecule recognition, NK cells express receptors that widely recognize stress-induced proteins on the surface of cancer cells. Therefore, NK cells show great potential in cancer immunotherapy, recognizing a variety of tumor antigens through their specific receptors and activating without the restriction of MHC class I molecules. Additionally, in contrast to T cells, allogeneic NK cells have the characteristic of low immunogenicity and can be used between allografts with HLA subtype mismatches without developing graft-versus-host disease. This characteristic, combined with the broad-spectrum anti-cancer activity of NK cells, makes NK cell therapy an emerging immunocytotherapy option for tumors.

[0060] Among human immune cells, NK cells are distributed in the bone marrow, peripheral blood, liver, spleen, lungs and lymph nodes, but the content is low, accounting for 8-20% of circulating lymphocytes, and the phenotype is CD56 positive and CD3 negative. In contrast, T cells can account for 90% in the thoracic duct lymph fluid, about 75% in the lymph nodes, and 60% to 80% in the peripheral blood. The number of NK cells obtained by extracting PBMC from human peripheral blood is limited, and the stability of products from different donors is poor. In 2006, Japanese scientist Shinya Yamanaka took the lead in publishing a technical method for the successful preparation of induced pluripotent stem cells (iPS cells) in the world-renowned academic journal Cell, and won the Nobel Prize in Physiology or Medicine in 2012 for this technology. Specifically, iPSC technology uses specific transcription factors to reverse terminally differentiated somatic cells (such as peripheral blood cells, urine cells, skin cells, etc.) into stem cells with sub-totipotency, that is, induced pluripotent stem cells. This reversal process is called cell reprogramming. Since iPS cells have similar sub-totipotency to embryonic stem cells, they can differentiate into various tissues and organ cells in the human body, such as neurons, glial cells, cardiomyocytes, liver cells, pancreatic islet cells, immune cells, etc., which enables the large-scale preparation of these difficult-to-obtain and difficult-to-amplify cells, providing a new platform for the study of the mechanism of related diseases. Therefore, the preparation of mature NK cells differentiated from iPS cells provides a possible way to achieve the clinical transformation of NK cell therapy.

[0061] CD16 protein binds to the Fc segment of antibody IgG. Through the ADCC effect, it activates immune cells, enabling them to kill tumor cells recognized by the antibody, so CD16 can be paired with different antibodies to kill tumor cells. Currently, CD16 has been identified as two isomers: Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells, which is the receptor for the Fc segment of antibody IgG, binds to target cells recognized by the antibody, and activates the ADCC effect of NK cells. CD16b is only expressed by human neutrophils and does not mediate the ADCC killing of tumors by NK cells. The expression level of CD16 in mature natural killer cells is very low, and the protein will be cleaved and inactivated after binding to the antibody. Therefore, the ADCC effect of wild-type CD16 is limited under natural conditions. The expression of CD16a in mature NK cells prepared by iPSC differentiation is very low, and generally about 10% of iNK cells are CD16 positive. In addition, wild-type CD16a has a low affinity for the Fc segment of IgG, and a specific region of the extracellular domain will be cleaved and shed by the protease ADAM17, thereby regulating the surface density on cells such as NK. Therefore, wild-type CD16 will be cleaved and shed after binding to the antibody on the surface of the target cell, reducing its mediated tumor killing effect.

[0062] hnCD16 is introduced with extracellular F158V mutation into wild-type CD16 to make it have high affinity, and extracellular S197P mutation, which is not cleaved and shed by ADAM17 protease. The variants of hnCD16 are mainly fusion proteins of the extracellular region of hnCD16 and transmembrane domains, signal transduction domains, and stimulatory domains that are not derived from CD16 and are derived from the same or different polypeptides. Multiple cell and animal experiments have verified that the combination of hnCD16-iPSC-NK cells with corresponding antibodies can greatly enhance the killing efficacy of NK cells against various tumors.

[0063] The exogenous expression of non-cleavable hnCD16 enhances the ADCC effect and the conjugation of bispecific, trispecific, or multispecific conjugates to a certain extent. However, its intracellular signal transduction pathway in NK is still the intracellular signal domain of wild-type CD16. Therefore, in order to further enhance the ADCC killing effect mediated by CD16, especially the enhancement of its killing ability against tumor cells with multiple targets in combination with multispecific antibodies, hnCD16 is modified into hnCD16-CAR (hnCD16-chimera antigen receptor), replacing the transmembrane domain and / or intracellular domain of native CD16, generating a chimeric hnCD16 containing a non-wild-type transmembrane domain, non-wild-type stimulatory domain, and / or non-wild-type signal transduction domain.

[0064] For different types of immune cells from different sources, such as T cells or NK cells derived from PBMC or iPSC, the effects of hnCD16 variants with different CAR structures on activating the ADCC effect of immune cells vary greatly, and ultimately will lead to significant differences in the therapeutic effects of CAR-hnCD16-immune cell products on eliminating tumor cells. The present invention preferably selects the CAR-hnCD16 structural combination suitable for NK cells derived from iPSC, designs immune cell membrane signal proteins from different sources, and combines them into the hnCD16 variant structure of extracellular, transmembrane, and intracellular stimulation domains. We propose and prove that the CAR-hnCD16 structure of "signal peptide-hnCD16 extracellular region-transmembrane domain-signaling domain-stimulatory domain" mediates better tumor killing efficacy. Therefore, compared with the prior art, the present invention preferably selects the CAR-hnCD16 structure and sequence that can more efficiently mediate the ADCC effect to kill tumor cells in NK cells derived from iPSC, providing important support for the application of engineered CD16 in NK cell therapy products derived from iPSC.

[0065] References throughout this specification to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "certain embodiments", "another embodiment" or "other embodiments", or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the foregoing phrases appearing throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0066] The term "in vivo" generally refers to activities carried out inside a living organism.

[0067] As used herein, the term "differentiation" is the process by which unspecialized ("not specialized") or weakly specialized cells acquire the characteristics of specialized cells (such as blood cells or muscle cells). Differentiated cells or cells induced to differentiate are cells that have reached a more specialized ("specialized") position within a cell lineage. The term "specialized", when applied to the process of differentiation, refers to a cell that has progressed in the differentiation pathway to the point where, under normal circumstances, it will continue to differentiate into a specific cell type or a subset of cell types and, under normal circumstances, it cannot differentiate into different cell types or revert to a more weakly differentiated cell type. As used herein, the term "pluripotent" refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potency that ranges from incompletely or partially pluripotent cells (such as epiblast stem cells or EpiSCs) that cannot give rise to a complete organism to more primitive, pluripotent cells (such as embryonic stem cells) that can give rise to a complete organism.

[0068] As used herein, the term "induced pluripotent stem cell" or iPSC means a stem cell generated from a differentiated adult, neonatal, or fetal cell that has been induced or altered, i.e., reprogrammed, to a cell capable of differentiating into tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. The resulting iPSC does not refer to a cell as it is found in nature.

[0069] As used herein, the term "multipotent stem cell" refers to a cell that has the developmental potential to differentiate into one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three germ layers. Thus, multipotent cells can also be referred to as "partially differentiated cells". Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Multipotent" indicates that a cell can form many types of cells within a given lineage, but not cells of other lineages. For example, multipotent hematopoietic cells can form many different types of blood cells (red blood cells, white blood cells, platelets, etc.), but they cannot form neurons. Thus, the term "multipotency" refers to a cell state that has a lower degree of developmental potential than totipotency and pluripotency.

[0070] Pluripotency can be determined, in part, by assessing the pluripotent characteristics of cells. Pluripotent characteristics include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) the expression of pluripotent stem cell markers, which include, but are 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 lineages (ectoderm, mesoderm, and endoderm); (v) teratoma formation consisting of the three somatic lineages; and (vi) the formation of embryoid bodies composed of cells from the three somatic lineages.

[0071] The term "culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture medium", "medium" (in the singular "medium" in all cases), "supplement", and "culture medium supplement" refer to the nutritional compositions for culturing cell cultures.

[0072] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursor cells" refer to cells committed to the hematopoietic lineage but capable of further hematopoietic differentiation, and include multipotent hematopoietic stem cells (hemocytoblasts), myeloid progenitors, megakaryocyte progenitors, erythroid progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). As used herein, the term "long-term hematopoietic stem cells" refers to CD34+ hematopoietic cells capable of giving rise to both mature myeloid cell types and lymphocyte types, including T cells, NK cells, and B cells. Hematopoietic cells also include multiple subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0073] As used herein, the term "exogenous" is intended to mean a reference molecule or activity introduced into a host cell or not native to the host cell. The molecule can be introduced, for example, by introducing a coding nucleic acid into the host genetic material, such as by integration into the host chromosome or as extrachromosomal genetic material (e.g., a plasmid). Thus, the term when used in reference to the expression of a coding nucleic acid refers to the introduction of the coding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a reference molecule or activity present in the host cell. Similarly, the term when used in reference to the expression of a coding nucleic acid refers to the expression of a coding nucleic acid contained within the cell and not introduced exogenously.

[0074] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotide sequences are composed of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) (when the polynucleotide is RNA, uracil replaces thymine). Polynucleotides can include genes or gene fragments (e.g., probes, primers, ESTs or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides also refer to double-stranded and single-stranded molecules.

[0075] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a molecule having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, the terms refer to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (which are commonly referred to in the art as polypeptides or proteins). "Polypeptide" includes, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides or combinations thereof.

[0076] The term "Fc receptor" (abbreviated as FcR) is classified based on the type of antibody it recognizes. For example, the receptor that binds the most common class of antibody IgG is called Fc - gamma receptor (FcγR), the receptor that binds IgA is called Fc - alpha receptor (FcαR) and the receptor that binds IgE is called Fc - epsilon receptor (FcεR). The classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells and B cells) and the signal transduction properties of each receptor. Fc- The γ receptors (FcγRs) include several members: FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), and their antibody affinities vary due to differences in their molecular structures.

[0077] The term "long terminal repeat (LTR)" refers to a long terminal repeat sequence (5'-LTR and 3'-LTR) at each end of the genome of retroviruses. It does not encode proteins but contains regulatory elements such as promoters and enhancers. The LTR within the viral genome can be transferred adjacent to cellular proto-oncogenes, activating these proto-oncogenes under the action of the strong LTR promoters and enhancers, and transforming normal cells into cancer cells.

[0078] The term "lentivirus" is the main gene delivery platform currently used in cell and gene therapy research, especially in CAR-T cell therapy. Lentiviruses are single-stranded RNA viruses, mostly developed based on human immunodeficiency virus type 1 (HIV-1). They can be used for gene delivery into the vast majority of mammalian cells, including non-dividing cells such as hematopoietic stem cells and nerve cells, which are difficult to achieve using retroviruses. The target genes introduced using recombinant lentiviruses can also be integrated into the genome of host cells, enabling long-term and stable gene expression. Lentiviral vectors (LVs) are viral vector systems modified based on the human immunodeficiency virus (HIV-1 virus). They can efficiently introduce target genes (or RNAi) into primary cells or cell lines of animals and humans, and have the ability to infect both dividing and non-dividing cells. The genome of the lentiviral vector is single-stranded positive-sense RNA. After its genome enters the cell, it is reverse-transcribed into DNA by the reverse transcriptase carried by itself in the cytoplasm, forming a pre-integration DNA complex. After entering the nucleus, the DNA is integrated into the cell genome. The integrated DNA is transcribed into mRNA, which returns to the cytoplasm to express the target gene or fragment. The gene expression or RNAi interference mediated by lentiviral vectors is continuous and stable. The target gene is integrated into the genome of the host cell and divides with the division of the cell genome. In addition, lentiviral vectors can effectively infect and integrate into non-dividing cells. These characteristics make lentiviral vectors have distinct features compared with other viral vectors (such as non-integrating adenoviral vectors, adeno-associated viral vectors with low integration rates, and traditional retroviral vectors that only integrate into dividing cells). A large number of literature studies have shown that tissues or cells with long-term expression of target genes mediated by lentiviral vectors include the brain, liver, muscle, retina, hematopoietic stem cells, bone marrow mesenchymal stem cells, macrophages, etc.

[0079] The lentiviral expression vector has deleted most of the genes of the HIV virus, and only retains the LTR sequence, packaging signal, Rev response element, etc. of the HIV virus. Therefore, it has low immunogenicity, no cellular immune response at the injection site, and a relatively low humoral immune response, without affecting the second injection of the viral vector.

[0080] The earliest used exogenous expression vector contained all the cis-acting elements of the lentiviral vector (packaging signal, reverse transcription and integration elements), exogenous insertion fragments, and related regulatory elements (promoter and insertion fragment). Specifically, the N-terminus of the vector contains a replication initiation binding site, a splice donor site, a packaging signal, an RRE sequence, and a splice acceptor site; the heterologous promoter and insertion fragment are in the middle; the C-terminus contains a PolyA tailing signal and the 3' LTR. To increase safety, reduce the possibility of generating RCR by recombination, increase expression efficiency, and enrich regulatory expression patterns, researchers have carried out a series of modifications on the exogenous expression vector:

[0081] 1), Construction of SIN vector: Since the U3 region of the 3' LTR of retrovirus will be used as a template during reverse transcription and viral DNA replication and finally generate the U3 region corresponding to the 5' LTR, if we want to remove the viral own transcriptional initiation element to make the exogenous fragment undergo Tat-independent transcription, we must replace the U3 region of the 5' LTR with a heterologous promoter, and at the same time remove the corresponding sequence of 133 - 400 bp of the U3 region of the 3' LTR containing the TATA box and transcriptional binding sites such as Sp1, NF-kB, and other enhancers. The modified U3 region without this sequence will be replicated and transferred to form the new U3 region of the 5' LTR in the transcriptional product, so the transcriptional product no longer contains the viral own transcriptional initiation element, and thus no longer depends on the Tat protein factor for transcription, enabling the removal of the Tat coding gene in other plasmid vectors. At the same time, after integrating into the host cell genome, it can also reduce the transcriptional interference with genes near the integration site, such as avoiding activating downstream genes (common in early retroviral vectors and a major factor inducing tumorigenesis when using retroviral vectors for gene therapy).

[0082] 2), The post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE, woodchuck hepatitis virus posttranscriptional regulatory element) and the cPPT (central polypurine tract) sequence can greatly increase the expression efficiency of exogenous fragments.

[0083] The packaging factor expression vector expresses all the trans - acting factors necessary for packaging and infection except for the envelope proteins. To avoid packaging such sequences into pseudovirus particles (lentiviral vectors), the packaging factor expression vector lacks cis - packaging signals and LTR sequences. However, the Rev response element (RRE) sequence and the splice donor site are still retained. Using other promoters, such as the CMV or RSV promoter, and the polyA tail signal sequence of the insulin gene to replace the corresponding sequences of the LTR can make the transcription products more stable and have higher expression efficiency.

[0084] The term "promoter" is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for specific binding of RNA polymerase and transcription initiation. Most are located upstream of the transcription start point of the structural gene, and the promoter itself is not transcribed. However, some promoters (such as tRNA promoters) are located downstream of the transcription start point, and these DNA sequences can be transcribed. The characteristics of promoters were initially identified through mutations that increase or decrease the gene transcription rate. Promoters are generally located upstream of the transcription start site.

[0085] The term "promoter (EF1a) or EF1α promoter" is a strong mammalian expression promoter derived from the elongation factor 1alpha (EF1A) gene, which can stably drive the constitutive expression of its downstream genes in a variety of cells and can also be used for stem cells, primary cells, hematopoietic cells, etc.

[0086] The term "Internal ribosome entry site sequence (IRES)" is a non - translated RNA that can recruit ribosomes to translate mRNA. By fusing IRES with foreign cDNA, it was found that IRES can independently initiate translation. Generally, the translation of eukaryotic mRNA requires a 5' cap to mediate ribosome binding, but there are also some exceptions in eukaryotes and viruses. For example, some genes have a short RNA sequence (about 150 - 250 BP) at the 5' end. This type of RNA sequence can fold into a structure similar to the initiator tRNA, thus mediating the binding of ribosomes to RNA and initiating protein translation.

[0087] The term "Enhanced Green Fluorescent Protein", EGFP is a mutant line of GFP. A more widely used mutant of GFP is the enhanced green fluorescent protein (EGFP), whose emitted fluorescence intensity is more than 6 times greater than that of GFP. Therefore, it is more suitable than GFP as a reporter gene to study gene expression, regulation, cell differentiation, and the localization and transport of proteins in organisms.

[0088] The term "HEK293T cell", abbreviated as 293T cell, is a cell line derived from 293 cells by genetic engineering. It has been transfected with the adenovirus E1A gene, can express SV40 large T antigen, and contains the SV40 replication origin and promoter region. Many eukaryotic expression vectors such as pcDNA3.1 contain the replication origin of SV40 virus and can replicate in cell lines expressing SV40 virus T antigen, thereby increasing the expression level of foreign genes. Therefore, 293T cells are widely used in virus packaging. The transfection efficiency with calcium phosphate can be as high as 50%. The protein expression level is high, and the expressed protein can be easily detected by alkaline phosphatase analysis 2-3 days after transfection. Transient transfection of 293T cells is a convenient way to overexpress proteins and obtain intracellular and extracellular (secreted or membrane) proteins. 293 cells are a human renal epithelial cell line with multiple derivative strains, such as HEK293, 293T / 17, etc. They are all derived from human embryonic kidney cells, rarely express endogenous receptors required for extracellular ligands, and are relatively easy to transfect, making them a very commonly used cell line for expressing foreign genes in research. The 293T cells used in the present invention are the cell line numbered "ATCC, CRL-3216".

[0089] The term "ddH2O" refers to ultrapure water that has been distilled twice.

[0090] The term "Opti-MEM medium" is a medium that can be used for various suspension and adherent mammalian cells, including Sp2, AE-1, CHO, BHK-21, HEK, and primary fibroblasts.

[0091] The term "ExFect TM Transfection Reagent" is a transfection reagent based on biodegradable polymer dendrimers and is suitable for DNA transfection of various cells. Due to the uniform size and definite shape of the activated dendrimers used, highly reproducible transfection results can be achieved. After ExFect TM Transfection Reagent carries DNA into cells, it can quickly release the DNA and be rapidly degraded, greatly reducing the toxicity to cells. The transfection reagent-DNA complex can be directly added to the complete medium, and serum and antibiotics do not affect its transfection effect. There is no need to change the medium before and after transfection, so the operation is very simple.

[0092] The term "Package Plasmid Mix" is a mixture of the lentiviral packaging plasmid psPAX2 derived from the human immunodeficiency virus and the pMD2.G plasmid containing VSV-G. These plasmids provide all the necessary structural, regulatory, and replication proteins required for packaging pseudotyped lentiviral expression. Co-transfection of 293T producer cells (such as ATCC, catalog number CRL-11268) with a lentiviral expression construct (such as the Cellecta PRSI shRNA expression construct) produces pseudotyped packaged lentiviruses. Lentiviral particles pseudotyped with the VSV-G envelope protein can infect a variety of mammalian or non-mammalian, dividing or non-dividing cells.

[0093] The term "induced pluripotent stem cells (iPSCs)" is obtained by artificially inducing non-pluripotent cells to express certain specific genes. iPSCs and natural pluripotent stem cells are similar in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, the formation of different chimeras, and differentiation potential.

[0094] The term "human induced pluripotent stem cells (hiPSCs)" is induced from human cells by non-integrating methods, suitable for clinical-grade stem cell research and applications, with highly similar clone morphology and gene expression to human ES cells, maintaining a normal karyotype for a long time, and having the potential to differentiate into the three germ layers in vivo and in vitro.

[0095] Based on the above detailed description, these and other changes can be made to the embodiments. Generally, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments together with the full scope of equivalent rights enjoyed by these claims. Therefore, the claims are not limited by this disclosure.

[0096] Example 1

[0097] Experiment on lentiviral transfection to construct CAR-hnCD16a-iPSC

[0098] 1) Design and production of viral vectors for transfecting iPSCs: The CAR-hnCD16 lentiviral transfer plasmid contains a chimeric 5' long terminal repeat (LTR), a packaging signal, a Rev response element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a 3' self-inactivating (SIN) LTR. The CAR-hnCD16 lentiviral transfer plasmid is placed under the control of the EF1α promoter and co-expressed with EGFP via an IRES. The virus is produced in 293T cells (ATCC, CRL-3216) by co-transfecting the transfer plasmid, the packaging plasmid, and the envelope plasmid.

[0099] Figure 1 This is a schematic diagram of the structure of the CAR-hnCD16 lentiviral transfer plasmid in the examples of the present invention. The overall size of the CAR-hnCD16 lentiviral transfer plasmid used in this example is 10536 bp.

[0100] 2) Lentiviral packaging process:

[0101] A. Preparation of HEK 293T cells: 3 - 5×10 6 HEK 293T cells were passaged and inoculated into a 100 mm cell culture dish, placed in an incubator at 37°C and 5% CO2, and cultured for 16 h - 24 h. During the passage process, the cells need to be fully digested into a single cell suspension to obtain a better packaging effect.

[0102] B. Transfection of the lentiviral packaging system: Dilute the packaging plasmid mixture (PackagePlasmid Mix) and the lentiviral expression plasmid in the lentiviral packaging kit with ddH2O to a plasmid solution with a final concentration of 1.0 μg / μL; take a 1.5 mL centrifuge tube (labeled as tube A), add 300 μL of Opti-MEM medium and 40 μL of ExFect TM Transfection Reagent, mix well and let stand at room temperature for 5 min; take a 1.5 mL centrifuge tube (labeled as tube B), add 2.5 μL of the lentiviral expression plasmid solution with a final concentration of 1.0 μg / μL and 7.5 μL of Package Plasmid Mix, mix well; add the solution in tube A to tube B, mix well, and let stand at room temperature for 15 - 30 min; slowly and evenly add the mixed solution in tube B dropwise to the culture dish inoculated with HEK 293T cells, gently shake the culture dish horizontally to mix, place the culture dish in an incubator at 37°C and 5% CO2 for 6 h, and replace the medium with fresh complete medium pre-warmed in a 37°C water bath.

[0103] C. Virus collection and concentration: 48 hours after transfection, collect the supernatant containing lentivirus, and supplement 10 - 15 mL of fresh complete medium into the culture dish; continue culturing for 24 hours, and conduct the second collection of virus supernatant; mix the virus supernatants collected twice, filter through a 0.45 μm filter, and the filtered virus solution can be concentrated or directly used to infect target cells; aliquot the virus concentrate and store it at -80 °C for immediate use.

[0104] 3) Methods for constructing multiple hnCD16 - CAR - iPSCs:

[0105] Generate hnCD16 - FR - iPSCs by transducing hiPSCs with lentiviral particles expressing hnCD16 - FR and GFP, and then perform single - cell sorting and clonal selection to obtain positive hnCD16 - FR - iPSC clones.

[0106] Refer to Figure 2 , hnCD16 - FR - iPSCs include CAR - hnCD16 - FR1 and CAR - hnCD16 - FR2. The nucleotide sequences of the two CAR - hnCD16s are shown in the sequence listing.

[0107] Refer to Figure 3 , detect the expression of exogenously introduced genes in the established iPSC positive clone cells through flow cytometry experiments. Unstained is the iPSC control group without added antibody; CTRL is the iPSC control group transfected with GFP virus; FR1 and FR2 are the experimental groups of positive clone cells transfected with CAR - hnCD16 - FR1 and CAR - hnCD16 - FR2. The first row shows the flow cytometry detection results of GFP antibody, and the second row shows the flow cytometry detection results of CD16 antibody. Through Figure 3 It shows that both the FR1 and FR2 clones normally express membrane - localized CD16a protein.

[0108] Construct chimeric antigen receptors (CARs) with the combination (FR1) of type I transmembrane proteins and the transmembrane domain combination (FR3) of type II transmembrane protein NKG2D. Infect the immortalized cell line YT-NK cells with lentivirus to compare their cellular localization. The extracellular regions of both structures are high-affinity non-cleavable CD16 (hnCD16). The transmembrane regions use the transmembrane region sequence of CD16 (FR1) or the transmembrane region sequence of NKG2D (FR3) respectively, and are co-expressed with green fluorescent protein (GFP) through IRES (internal ribosome entry site). Infect HEK293 cells with lentiviruses containing FR1 and FR3 structures respectively, then perform immunofluorescence staining with anti-CD16 antibody and observe with a laser confocal microscope. From the immunofluorescence results, it can be seen that for the combination FR1 of type I transmembrane proteins, the fluorescence signal of CD16 is significantly localized on the cell membrane, and there are also some signals inside the cell; while for the transmembrane region of type II transmembrane protein NKG2D and the combination FR3 of type I transmembrane proteins, CD16 does not form the unique membrane structure signal of the cell membrane and is mainly localized inside the cell (cytoplasm).

[0109] For the FR1 and FR3 structures, please refer to Figure 4 。

[0110] Refer to Figure 5 , the cellular localization analysis of the transmembrane region structures of CD16 and NKG2D. HEK293 cells were infected with lentiviruses containing FR1 and FR3 structures respectively. In the figure, blue is the nuclear DNA, green is GFP, red is CD16, and the scale bar is 10 μm.

[0111] Meanwhile, infect YT-NK cells with the lentivirus of the hnCD16-CAR (FR3) structure FR3 with the transmembrane region of NKG2D, and perform flow cytometry analysis. Using the absence of anti-CD16 antibody as the negative control, the results are shown in Figure 6 。If the cell membrane is not disrupted with 0.05% Triton and anti-CD16 antibody is added, 21.8% of the cells are positive. After deducting the control, only about 16% of the CD16-positive cells; if the cell membrane is disrupted with Triton, 66.9% of the cells are CD16-positive, and the difference between the two is 45.1%. This shows that translated CD16 exists in the cytoplasm of more cells, but cannot be presented or localized on the cell membrane.

[0112] The localization of the encoded protein to the cell membrane is the basis for the CAR of lymphocytes to recognize and kill tumor cells. If a structure that is stably expressed on the cell membrane cannot be formed, according to the principles of cell biology and the action of CAR, the function of specifically recognizing and killing tumor cells cannot be exercised. Therefore, combining the type I transmembrane region of the 2B4 protein with the domains of other type I transmembrane proteins in CAR-hnCD16a can effectively avoid the confusion of intracellular and extracellular signals, thereby efficiently exercising its function and enhancing the ability of lymphocytes (such as NK cells) to kill tumor cells.

[0113] Example 2

[0114] Experimental method for the differentiation of hnCD16-CAR-iPSC into iNK

[0115] In the first step, hiPSCs are first differentiated into hematopoietic progenitor cells and then into NK cells. When CD34+ cells appear, the cell population is transferred to the NK cell differentiation medium. The hematopoietic progenitor cells are transferred to the NK cell differentiation medium, which contains DMEM / Ham F12 (Thermo Fisher Scientific, Waltham, MA, 11965092, 11765054), 2 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA, 25030081), 1% penicillin / streptomycin (Thermo Fisher Scientific, Waltham, MA, 15140122), 25 μM β-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, 21985023), 5 ng / mL sodium selenite (Merck Millipore, Burlington, MA, S5261), 50 μM ethanolamine (MP Biomedicals, ICN19384590), 20 mg / mL ascorbic acid (Merck Millipore, Burlington, MA, A4544), interleukin-3; stem cell factor (SCF), interleukin-15, fms-like tyrosine kinase 3 ligand (FLT3L), and interleukin-7. The cells are placed under these conditions for 28 - 35 days, and the medium is changed weekly until flow cytometry determines that they have developed into CD45+CD56+ cells. As Figure 7 shown, the cells at the differentiation endpoint significantly express the NK cell maturation marker CD56 and the hematopoietic cell marker CD45 (blue peak) (all antibodies are from biolegend).

[0116] Refer to Figure 8, by analyzing the expression of a series of NK cell-related markers, it is demonstrated that: 1. Membrane proteins related to NK cell maturation such as CD94 and killing functional proteins such as NKp46, NKG2D, FasL, etc. are all significantly expressed in the terminal cells prepared by iPSC differentiation, indicating that NK cells with mature differentiation phenotypes and immune killing abilities are obtained through our differentiation method. 2. Two engineered iNK cells expressing hnCD16FR (FR1 and FR2) have more significant CD16 expression compared to CTRL. In particular, the expression levels of the membrane protein CD94 related to maturation and the proteins NKp44 / NKG2A related to killing function in FR1-iNK cells are significantly increased compared to CTRL-iNK and FR2-iNK.

[0117] Example 3

[0118] 1. hnCD16-CAR-iNK cell killing experiment:

[0119] 1) Experimental method and process: The differentiated iNK effector cells CTRL, FR1, and FR2 were combined with antibodies against the highly expressed targets of cancer cells, and co-incubated with the luciferase-expressing target cells Raji or A549 at different effector-to-target ratios at 37°C and 5% CO2 for 24 h. Then, the bioluminescence substrate was added and detected by a microplate reader. The cytotoxicity against target cells was evaluated using a luciferase-based standard bioluminescence method. The experimental results of killing tumor cells are as follows:

[0120] 1) Killing Raji cells in combination with anti-CD20 mab (Obinutuzumab), the results are shown in Figure 9 .

[0121] 2) Killing A549 cells in combination with anti-EGFR mab (Nimotuzumab), the results are shown in Figure 10 .

[0122] Through Figure 9 , Figure 10 It is shown that NK cells engineered to express the FR structure can significantly enhance the killing effect on tumor cells from multiple sources through combination with therapeutic monoclonal antibodies. The killing function of CTRL cells that do not express CD16 on tumor cells does not show a large difference after the addition of antibodies, suggesting that its ADCC effect is not significantly activated. Comparing FR1 with FR2, it can be found that even at a lower effector-to-target ratio, a more significant killing enhancement effect can be achieved, reflecting the high tumor killing efficiency of the CAR-hnCD16a structure.

[0123] Through the above examples, genetically modified NK cells or their populations can be obtained, wherein the NK cells or their populations are derived from induced pluripotent stem cells or hematopoietic progenitor cells:

[0124] (i) The NK cells include: (a) induced pluripotent cells (iPSCs), (b) hematopoietic stem cells or hematopoietic progenitor cells; or (b) derivative cells obtained by differentiating the cells of (a); and

[0125] (ii) express at least one endogenous gene encoding the following:

[0126] (1) The Fc receptor FcγRIIIa (CD16a);

[0127] (2) CD94, CD56, and / or CD45;

[0128] (3) NKp46, NKG2D, and / or FasL;

[0129] (iii) contain at least one or more exogenous nucleic acid expression constructs, which contain nucleic acid sequences encoding the following:

[0130] (1) The chimeric antigen receptor CAR;

[0131] (2) F158V and S197P in the extracellular domain of CD16;

[0132] (3) All or part of the extracellular domain derived from CD16;

[0133] (4) The native CD16 transmembrane domain;

[0134] (5) The intracellular domain with at least one of the native stimulatory domains 2B4 and DAP10;

[0135] (6) The intracellular domain of the native signaling domain CD3ζ.

[0136] For clinical use as an immunotherapeutic agent, for example, in the context of immuno-oncology applications, genetically modified enhanced NK cells are generated. By combining therapeutic monoclonal antibodies, the killing effect on tumor cells from multiple sources can be significantly enhanced. The killing function of CTRL cells that do not express CD16 on tumor cells does not show a large difference after adding the antibody, suggesting that its ADCC effect is not significantly activated. Comparing FR1 with FR2, it can be found that even at a lower effector-to-target ratio, a more significant killing enhancement effect can be achieved, reflecting the high tumor killing efficiency of the CAR-hnCD16a structure.

[0137] The present invention designs different structural combinations of chimeric antigen receptor hnCD16 (CAR-hnCD16). Two structures of CAR-hnCD16 are designed with the extracellular region of high-affinity non-cleavable hnCD16 and different transmembrane regions (2B4 and DAP10) and intracellular co-stimulatory regions (CD3ζ). By comparing the CAR-CD16 combination with the NKG2D transmembrane domain as the key in the CD16 variant proposed in the patent CN 113166232 A, it is found that the CAR protein expressed by it is significantly lower than the CAR-hnCD16 combination provided by the present invention in terms of membrane localization ability. The reason may be that as a type I transmembrane protein, the transmembrane domain / signal transduction domain / stimulatory domain combined with CD16 and its signal peptide (the extracellular region of hnCD16) needs to also be derived from a type I transmembrane protein in order to make the variant CAR-hnCD16 structure conduct signals without chaos and mediate better tumor killing efficacy. Combining the extracellular signal domain of a type I transmembrane protein (the extracellular domain of CD16) with the transmembrane domain of a type II transmembrane protein (the NKG2D transmembrane domain) may cause signal chaos in the resulting chimeric antigen receptor combined protein. The two designed CAR-hnCD16 structures and sequences (CAR-hnCD16-FR1 and CAR-hnCD16-FR2) prove that two of them (FR1, FR2) have an obvious effect of enhancing the killing of tumor cells when used in combination with human antibodies in iPSC-derived NK cells (significantly higher than the effect of wild-type hnCD16), and the killing effect of FR1 is better.

[0138] As described above, it is only the optimal specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions within the technical scope disclosed in this application should be covered within the protection scope of this application. Therefore, the protection scope of this application should be subject to the protection scope of the claimed rights.

Claims

1. A modified cell or population thereof, characterized in that, The cell is a derived NK cell, and the NK cell is derived from induced pluripotent stem cells differentiated into hematopoietic progenitor cells, wherein: (i) The cell expresses endogenous genes encoding the following: (1) Fc receptor FcγRIIIa (CD16a); (2) CD94, CD56, and CD45; (3) NKp46, NKG2D, and FasL; (ii) It contains an exogenous nucleic acid expression construct, which contains a nucleic acid sequence encoding an engineered variant CAR-hnCD16 of hnCD16; The nucleotide sequence of the CAR-hnCD16 is shown in SEQ ID No.

1.

2. The cell or population thereof according to claim 1, characterized in that, The cell has at least one of the following characteristics compared to the natural corresponding cells differentiated from induced pluripotent stem cells, including the following: (i) Activating immune cells; (ii) Improving the recognition and killing of tumor cell antigens; (iii) Enhancing the ADCC effect of immune cells; (iv) Increasing cytotoxicity; (v) Increasing the types of tumor cells killed.

3. A method for generating the cell or population thereof as described in claim 1 or 2, characterized in that, The method includes: (i) Designing and producing a viral vector for transfecting iPSCs; (ii) Performing lentiviral packaging; (iii) Generating hnCD16-FR-iPSCs by lentiviral transfection of human induced pluripotent stem cells hiPSCs expressing hnCD16-FR and GFP proteins, and then performing single-cell sorting and clone selection to obtain a positive clone of hnCD16-FR-iPSCs; The nucleotide sequence of the hnCD16-FR is shown in SEQ ID No.

1.

4. The method of a cell or a population thereof according to claim 3, wherein the induced pluripotent stem cells generate hnCD16-FR-iPSCs, characterized in that, Including: (i) First differentiating induced pluripotent stem cells into hematopoietic progenitor cells, and then differentiating the hematopoietic progenitor cells into NK cells. When CD34+ cells appear, transferring the NK cells into an NK cell differentiation medium; (ii) Placing the NK cells in the NK cell differentiation medium for 28 - 35 days, changing the medium weekly until it is determined that they develop into CD45+CD56+ cells.

5. The method according to claim 4 for the cell or its population, characterized in that: The medium contains DMEM / Ham F12, l-glutamine, penicillin / streptomycin, β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, interleukin-3, stem cell factor, interleukin-15, fms-like tyrosine kinase 3 ligand, and interleukin-7.

6. The method according to claim 3 for a cell or a population thereof, wherein lentivirus packaging is carried out, characterized in that Including: (i) Preparing HEK 293T cells; (ii) Transfecting with a lentiviral packaging system; (iii) Collecting and concentrating the lentiviral solution.

7. The method of a cell or a population thereof according to claim 3, wherein the viral vector for transfecting iPSCs is designed and produced, characterized in that, Including: Placing the CAR-hnCD16 lentiviral transfer plasmid containing a chimeric 5' long terminal repeat sequence (LTR), packaging signal, Rev response element (RRE), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and 3' self-inactivating (SIN) LTR under the control of a constitutive promoter (ef1a) and co-expressing it with EGFP through IRES.

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