Car-ink cell, and preparation method therefor and use thereof

Abstract

The present invention relates to a CAR-iNK cell, and a preparation method therefor and the use thereof. The CAR comprises an scFv, a transmembrane domain, a costimulatory domain and a signal transduction domain, wherein the scFv comprises VH and VL of humanized FMC63, and the signal transduction domain is modified with IL15RF. By means of simultaneous transduction of the CAR structure and elements capable of increasing NK proliferation and persistence into iPSCs, the obtained CAR-iPSCs can be infinitely expanded, so that the requirements for off-the-shelf CAR-iNK cells can be met. Moreover, the high-purity CAR-iNK cells differentiated by the method show significant in-vitro specific killing function and obvious in-vivo efficacy.

Description

A CAR-iNK cell and its preparation method and application

[0001] This disclosure claims priority to the Chinese patent application filed with the Patent Office of China on March 26, 2024, with the invention name “A CAR-iNK cell and its preparation method and application” and application number 202410345720.5. The entire contents of the above application are incorporated into this disclosure by reference. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular to a CAR-iNK cell and a preparation method and application thereof. Background Art

[0003] Cell therapy is currently in full swing, with CAR-T cell therapy showing the most efficacy. However, the eight approved products all target CD-19 and BCMA, both of which are located on B cells, limiting their effectiveness to hematologic malignancies. Furthermore, since most cells in current CAR-T products are derived from patients themselves or healthy donors, they are expensive and cannot meet the demand for off-the-shelf products or maintain product homogeneity. Although NK cells can be used as a replacement for T cells in adoptive immunotherapy, existing adoptive immunotherapy approaches are often limited by the lack of antigen-specific CAR-NK cells. One approach to deriving antigen-specific NK cells is to generate CAR-expressing NK cells through induced differentiation of iPSCs.

[0004] iPSC-CAR-NK has the potential to solve all of the above problems simultaneously. First, NK cells do not have TCRs, can be used allogeneically, will not trigger an immune response in the body, and can be prepared in advance, so they can be used as universal cell therapy products. Secondly, using the potential for unlimited expansion of iPSC monoclonal stem cells, a GMP standard starting cell working library can be constructed, and then induced differentiation can be used to prepare NK products with uniform performance. Finally, NK cells have a natural broad-spectrum killing ability that is independent of specific targets and can overcome the heterogeneity of solid tumor targets.

[0005] Currently, there are four main sources for CAR-NK cell production: PBNK, CBNK, NK cell lines, and iPSC-derived NK. Most companies use the first three methods to produce CAR-NK products, and only a few companies use iPSC cells to produce CAR-NK. The CD19 antibody FMC63 is a mouse-derived monoclonal antibody, and the first CD19 CAR-T product to be marketed used this sequence. However, to reduce immune rejection, some researchers currently humanize the FMC63 sequence and then use it as an antibody to design and prepare CAR-T / CAR-NK cell products. However, traditional antibodies require humanized design and modification to obtain humanized antibodies, and their lifespan is relatively short. Therefore, the field urgently needs a CAR-iNK that is independent of exogenous IL15 factors, can be expanded indefinitely, and can remain in the body. Summary of the Invention

[0006] Problems to be solved by the invention

[0007] Based on the above problems existing in the prior art, the purpose of the present invention is to provide a universal CAR-iNK that can be prepared in advance.

[0008] Solutions for solving problems

[0009] The present invention provides a CAR-iNK cell, wherein the CAR comprises:

[0010] (1) scFv,

[0011] (2) transmembrane domain,

[0012] (3) a co-stimulatory domain, and

[0013] (4) Signaling domain,

[0014] The scFv comprises the VH and VL of humanized FMC63, and the signaling domain is modified with IL15RF.

[0015] Preferably, the amino acid sequence of the VH comprises one or more of the following sequences:

[0016] (1) the amino acid sequence shown in SEQ ID NO. 1;

[0017] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 1, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 1;

[0018] (3) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO. 1, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 1;

[0019] The amino acid sequence of the VL comprises one or more of the following sequences:

[0020] (1) the amino acid sequence shown in SEQ ID NO. 2;

[0021] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 2, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 2;

[0022] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 2.

[0023] Preferably, the scFv further comprises a connecting peptide;

[0024] The connecting peptide is located between the light chain variable region and the heavy chain variable region of the scFv, and its sequence is shown in SEQ ID NO.3.

[0025] Preferably, the scFv comprises one or more of the following sequences:

[0026] (1) the amino acid sequence shown in SEQ ID NO. 4;

[0027] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 4, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 4;

[0028] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 4, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 4.

[0029] Preferably, the CAR has the structure of the following formula I:

[0030] L-scFv-H-TM-C-CD3ζ-IL15RF(I),

[0031] in,

[0032] Each "-" is independently a connecting peptide or a peptide bond;

[0033] L is an optional signal peptide sequence;

[0034] scFv is the scFv described;

[0035] H is an optional hinge region;

[0036] TM is the transmembrane domain;

[0037] C is a co-stimulatory signal molecule;

[0038] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0039] Preferably, the CAR comprises one or more of the following sequences:

[0040] (1) the amino acid sequence shown in SEQ ID NO. 5;

[0041] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 5, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 5;

[0042] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 5, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 5.

[0043] The present invention also provides a method for preparing the cell, comprising the following steps:

[0044] (a) CAR lentivirus was transduced into iPSCs to obtain CAR-iPSCs;

[0045] (b) Selecting iPSC positive monoclonal clones that simultaneously express CAR19 and IL15RF;

[0046] (c) Construction of CAR-iPSC seed bank;

[0047] (d) Inducing CAR-iPSCs to differentiate into CAR-iNK cells.

[0048] Preferably, step (a) comprises:

[0049] (a1) The chimeric antigen receptor CAR19 and IL15RF were constructed into a lentiviral vector;

[0050] (a2) The plasmid was extracted and purified, mixed with the helper plasmid, and co-transfected into 293T cells;

[0051] (a3) collecting the cell culture supernatant containing the virus particles, filtering and concentrating;

[0052] (a4) The concentrated viral particles are used to transduce iPSCs to obtain CAR-iPSCs.

[0053] The present invention also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the cells, or the cells prepared according to the method, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0054] The present invention also provides a use of the cell, the method, or the pharmaceutical composition in preparing a drug for preventing and / or treating cancer or tumor-related diseases.

[0055] Preferably, the disease is selected from B-cell lymphoma, including non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL); acute lymphoblastic leukemia (ALL), including CD19-positive B-cell ALL; chronic lymphocytic leukemia (CLL), including CD19-positive B-cell CLL; myeloma, including multiple myeloma (MM) and non-Hodgkin's lymphoma cell leukemia (LCBCL); and other CD19-positive B-cell related diseases, including CD19-positive leukemia in acute myeloid leukemia (AML).

[0056] Effects of the Invention

[0057] The present invention simultaneously transduces the CAR structure and the elements that increase NK proliferation and persistence into iPSC, and the resulting CAR-iPSC can be infinitely expanded, thus meeting the requirements of CAR-iNK spot. The high-purity CAR-iNK cells differentiated by the method of the present invention exhibit significant in vitro specific killing function and obvious in vivo efficacy. Without relying on soluble exogenous IL15 factors, they not only promote CAR-iNK cell differentiation and survival in vitro, but also promote the retention of CAR-iNK in vivo. At the same time, the CAR-iNK of the present invention establishes cytokine-driven cell autonomy, which can avoid the use of systemic high-dose related factors to promote NK cell survival in clinical practice, thereby reducing risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is a schematic diagram of the IL15RF-modified CAR structure.

[0059] Figure 2 shows the flow cytometry results of CAR19 lentivirus transduction of iPSCs.

[0060] Figure 3 shows the iPSC single clone selection criteria and the single clone selection yield. A shows the single clone selection criteria: a single clone appears in a well; B shows the single clone yield is 33.85%.

[0061] Figure 4 shows the selection of iPSC single clones and the expansion of positive clones for library construction. A shows the process of iPSC positive clone selection, expansion, and seed library construction; B shows the flow cytometry identification of positive iPSC clones; C shows the yield of double-positive iPSC clones.

[0062] Figure 5 shows the expression of CAR19 and IL15RF in iPSC-positive clones as assessed by flow cytometry. A shows the gene expression levels of iPSC monoclonal clones undergoing differentiation by flow cytometry; B shows the gene expression of CAR19 and IL15RF.

[0063] Figure 6 shows the cell morphology at different stages of iPSC iNK differentiation. Figure A shows the cell morphology at different stages of the differentiation process, measured on days 0, 2, 8, and 27. Figure B shows the cell morphology of four clones undergoing differentiation on days 0 and 8. Figure C shows the gene expression of different clones on days 0, 8, and 27. Figure D shows the expression of CAR19 and IL15RF at different stages of differentiation.

[0064] Figure 7 shows the in vitro killing function of CAR-iNK cells. A shows that compared with the control group, the three mature differentiated CAR-iNK cells exhibited significant in vitro specific killing function. B shows that the three CAR-iNK cells exhibited significant in vitro specific killing function under different E / T ratio conditions using the Incucyte real-time live cell imaging and analysis platform.

[0065] Figure 8 shows the in vitro killing function of CAR-iNK cells. A shows the phenotypic identification of CAR-iNK cells; B shows that CAR-iNK cells exhibit significant specific killing function under different E / T ratios; C shows that CAR-iNK cells still exhibit significant killing effects under multiple rounds of stimulation; D shows that during incubation with Nalm6 tumor cells, CAR-iNK cells exhibited high secretion of CD107a and IFNγ cytokines; E shows that CAR-iNK cells continued to expand 7 days after feeder cell removal.

[0066] FIG9 is a flow chart of the in vivo efficacy experiment, showing the in vivo efficacy experiment protocol.

[0067] Figure 10 shows the persistence of CAR-iNK cells. A shows the persistence of CAR-iNK cells in vivo (100-200 cells / ul in peripheral blood); B shows IVIS images of each mouse at different time points after cell injection.

[0068] Figure 11 shows the preclinical anti-tumor toxicity of CAR-iNK. A and B show the in vivo efficacy and survival results of CAR-iNK. CAR-iNK significantly inhibited tumor growth and prolonged mouse survival. C shows the weight changes of mice after CAR-iNK injection. DETAILED DESCRIPTION

[0069] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0070] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0071] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0072] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0073] As used herein, the term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0074] As used herein, the term "and / or" refers to any one of the options or a combination of any two or more of the options.

[0075] As used herein, the term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In the present invention, when the term "comprising" or "including" is used, unless otherwise indicated, it also covers the situation consisting of the elements, integers or steps described.

[0076] As used herein, the term "iPSC" refers to induced pluripotent stem cells, which are cells generated from somatic cells by expressing or inducing expression of cytokines, with the potential to differentiate into various germ layers. Various reprogramming methods known in the art can be used to reprogram somatic cells into induced pluripotent stem cells. See, for example, disclosed U.S. Patent Application No. 20090246875, U.S. Patent Application No. 2010 / 0210014, U.S. Patent Application No. 20120276636, U.S. Patent No. 8,058,065, U.S. Patent No. 8,129,187, U.S. Patent No. 8,268,620, PCT Publication No. WO 2007 / 069666A1 and U.S. Patent No. 8,268,620, which are incorporated herein by reference. Typically, somatic cells are reprogrammed to dedifferentiation or pluripotent state, relating to the expression of reprogramming factors (including transcription factors). The relevant transcription factors may include one or more of the following, or be composed of or essentially consist of one or more of the following factors: OCT4, SOX2, KLF4 and MYC (OSKM); SOX2, KLF4 and OCT4 (SKO); OCT4, SOX2, KLF4 and GLIS1 (OSKG); OCT4, SOX2, NANOG and LIN28 (OSNL); or OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28 (OKSMNL). Methods for introducing reprogramming factors or nucleic acids encoding these reprogramming factors into somatic cells are known in the art, see, for example, U.S. Patent Nos. 8,268,620, 8,691,574, 8,741,648, 8,546,140, ​​and U.S. Patent Nos. 8,900,871 and 8,071,369. iPSCs can also be generated using somatic cell nuclear transplantation (SCNT).

[0077] As used herein, the term "differentiation" refers to one or more steps of converting a less differentiated cell into a more differentiated cell, particularly a post-mitotic tissue-specific cell type, such as differentiating iPSCs into NK cells. iPSCs can be induced to differentiate into NK cells, for example, by adding differentiation factors to the cell culture medium.

[0078] As used herein, the term "iNK" refers to iPSC-derived NK cells.

[0079] As used herein, the term "IL15RF" refers to IL-15 / IL-15 receptor fusion protein.

[0080] As used herein, the term "domain" refers to a region of a polypeptide that folds into a specific structure independently of other regions.

[0081] As used herein, the term "single-chain variable region fragment" or "scFv" refers to a single-chain polypeptide derived from an antibody that retains the ability to bind to an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA technology, and wherein the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for modifying ScFv are known to those skilled in the art.

[0082] As used herein, the term "tumor antigen" refers to a biological molecule with antigenicity, the expression of which leads to cancer.

[0083] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric antigen receptor" is sometimes also referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide that can bind to a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits signals to cause activation or inhibition of a biological process.

[0084] As used herein, the term "hinge region" generally refers to the region between the CH1 and CH2 domains of an immunoglobulin heavy chain. The hinge region is located between the extracellular antigen-binding domain (e.g., scFv) and the NK cell membrane. The hinge region is typically derived from the IgG family, for example, IgG1 and IgG4, but can also be derived from IgD and CD8.

[0085] As used herein, the term "transmembrane region" generally refers to the transmembrane segment connecting the extracellular antigen binding domain and the intracellular signaling domain, generally derived from dimeric membrane proteins, mainly including CD3ζ, CD4, CD8, CD28, etc., which can anchor the CAR structure to the NK cell membrane. Different designs of the transmembrane region can affect the expression of the introduced CAR gene.

[0086] As used herein, the term "signaling domain" generally refers to the functional signaling domains from CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ (FCER1G), FcRβ (Fc Epsilon R1b), CD79a, CD79b, FcγRIIa, DAP10, and DAP12 proteins.

[0087] As used herein, the term "costimulatory domain" generally refers to a functional signaling domain from one or more of the following proteins: CD27, CD28, 41BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, NKG2C, B7-H3, a ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGA L, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TR ANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96, CEACAM1, CRTAM, Ly9(CD229), CD1 60(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), B LAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46 and NKG2D.

[0088] As used herein, the term "CAR-NK cell" refers to NK cells expressing CAR, which are usually obtained by transducing NK cells with an expression vector encoding CAR. Commonly used expression vectors are viral vectors, such as lentiviral expression vectors. NK cells modified with chimeric antigen receptors (CAR-NK) are not restricted by the major histocompatibility complex and have specific targeted killing activity and the ability to proliferate persistently. In addition to NK cells, other lymphocytes such as T cells can also be transformed with expression vectors encoding CAR to obtain targeted killer cells expressing the CAR.

[0089] As used herein, the terms "sequence identity", "sequence identity" and "sequence identity" are used interchangeably and refer to the amount of consistency between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of consistency between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If the amino acid residues or bases in the two sequences are the same at a certain alignment position, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence consistency. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment and calculate the sequence consistency between two amino acid or nucleotide sequences using the default settings.

[0090] As used herein, the term "vector" refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (e.g., a nucleic acid, a plasmid, or a virus, etc.). A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of the polynucleotide of interest (such as a promoter and / or enhancer), and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric analysis, such as β-galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0091] As used herein, the term "host cell" refers to mammalian immune effector cells, especially human cells, such as T cells or NK cells, that can express CAR provided herein. Host cells include the offspring of a single host cell, and the offspring may not necessarily be completely consistent with the original mother cell (in terms of morphology or genomic DNA complementation) due to natural, accidental or intentional mutations. Host cells are also included in cells transfected with nucleic acid molecules or expression vectors provided herein in vivo.

[0092] As used herein, the term "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that can be safely administered to humans and / or animals without excessive adverse side effects and is suitable for maintaining the activity of the drug or active agent contained therein. Depending on the route of administration, various carriers well known in the art can be used, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer, emulsifiers, isotonic saline, and / or pyrogen-free water. The pharmaceutical compositions provided herein can be prepared into clinically acceptable dosage forms such as powders and injections. The pharmaceutical compositions of the present invention can be administered to a subject using any appropriate route, for example, orally, by intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneally, rectally, sublingually, or by inhalation, transdermally, or other routes of administration.

[0093] As used herein, the terms "cancer" and "tumor" are well known in the art and refer to the presence of cells that have unregulated cell growth and morphological characteristics that differ from normal cell types of similar origin, also known as dysregulated cells. Malignant refers to those cancerous cells that are capable of causing morbidity and / or mortality. As used herein, "cancer" and "tumor" include both precancerous and malignant types.

[0094] The present invention provides a CAR-iNK cell, wherein the CAR comprises:

[0095] (1) scFv,

[0096] (2) transmembrane domain,

[0097] (3) a co-stimulatory domain, and

[0098] (4) Signaling domain,

[0099] The scFv comprises the VH and VL of humanized FMC63, and the signaling domain is modified with IL15RF.

[0100] In certain embodiments, the amino acid sequence of the VH comprises one or more of the following sequences:

[0101] (1) the amino acid sequence shown in SEQ ID NO. 1;

[0102] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 1, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 1;

[0103] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.1, and which retains the activity of the amino acid sequence shown in SEQ ID NO.1.

[0104] SEQ ID NO.1:

[0105] In certain embodiments, the amino acid sequence of the VL comprises one or more of the following sequences:

[0106] (1) the amino acid sequence shown in SEQ ID NO. 2;

[0107] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 2, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 2;

[0108] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 2.

[0109] SEQ ID NO.2:

[0110] In certain embodiments, the scFv further comprises a connecting peptide.

[0111] In certain embodiments, the connecting peptide is between the light chain variable region and the heavy chain variable region of the scFv, and the sequence is shown in SEQ ID NO.3.

[0112] SEQ ID NO.3:

[0113] In certain embodiments, the scFv comprises one or more of the following sequences:

[0114] (1) the amino acid sequence shown in SEQ ID NO. 4;

[0115] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 4, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 4;

[0116] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 4, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 4.

[0117] SEQ ID NO.4:

[0118] In certain embodiments, the CAR has the structure of Formula I below:

[0119] L-scFv-H-TM-C-CD3ζ-IL15RF(I),

[0120] in,

[0121] Each "-" is independently a connecting peptide or a peptide bond;

[0122] L is an optional signal peptide sequence;

[0123] scFv is the scFv described;

[0124] H is an optional hinge region;

[0125] TM is the transmembrane domain;

[0126] C is a co-stimulatory signal molecule;

[0127] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0128] In certain embodiments, the CAR comprises one or more of the following sequences:

[0129] (1) the amino acid sequence shown in SEQ ID NO. 5;

[0130] (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 5, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 5;

[0131] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 5, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 5.

[0132] SEQ ID NO.5:

[0133] In certain embodiments, the nucleic acid sequence of the CAR is shown as SEQ ID NO.6.

[0134] SEQ ID NO.6:

[0135] The present invention also provides a method for preparing the cell, comprising the following steps:

[0136] (a) CAR lentivirus was transduced into iPSCs to obtain CAR-iPSCs;

[0137] (b) Selecting iPSC positive monoclonal clones that simultaneously express CAR19 and IL15RF;

[0138] (c) Construction of CAR-iPSC seed bank;

[0139] (d) Inducing CAR-iPSCs to differentiate into CAR-iNK cells.

[0140] In certain embodiments, step (a) comprises:

[0141] (a1) The chimeric antigen receptor CAR19 and IL15RF were constructed into a lentiviral vector;

[0142] (a2) The plasmid was extracted and purified, mixed with the helper plasmid, and co-transfected into 293T cells;

[0143] (a3) collecting the cell culture supernatant containing the virus particles, filtering and concentrating;

[0144] (a4) The concentrated viral particles are used to transduce iPSCs to obtain CAR-iPSCs.

[0145] In certain embodiments, the step (a1) comprises connecting the chimeric antigen receptor CAR19 and IL15RF in series, adding a CAG promoter element upstream, and constructing them into the same lentiviral vector.

[0146] In certain embodiments, the nucleic acid sequence of the CAG promoter element is shown as SEQ ID NO.7.

[0147] SEQ ID NO.7:

[0148] In certain embodiments, the helper plasmid in step (a2) is psPAX2 and / or pMD2.G.

[0149] In certain embodiments, the co-transfection in step (a2) is co-transfection of 293T cells.

[0150] In certain embodiments, the co-transfection in step (a2) is performed for 48 hours.

[0151] In certain embodiments, the filtration in step (a3) ​​comprises filtering using a filter after centrifugation.

[0152] In certain embodiments, the centrifugation is performed at 4°C and 500 g for 10 min.

[0153] In certain embodiments, the filter is a 0.45 μm filter.

[0154] In certain embodiments, the concentration in step (a3) ​​is performed by using a lentivirus concentration kit to concentrate the virus.

[0155] In certain embodiments, the kit is Lenti-X TM .

[0156] The present invention also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the cells, or the cells prepared according to the method, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0157] The present invention also provides a use of the cell, the method, or the pharmaceutical composition in preparing a drug for preventing and / or treating cancer or tumor-related diseases.

[0158] In certain embodiments, the disease is selected from B-cell lymphoma, including non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL); acute lymphoblastic leukemia (ALL), including CD19-positive B-cell ALL; chronic lymphocytic leukemia (CLL), including CD19-positive B-cell CLL; myeloma, including multiple myeloma (MM) and non-Hodgkin lymphoma cell leukemia (LCBCL); and other CD19-positive B-cell related diseases, including CD19-positive leukemia in acute myeloid leukemia (AML).

[0159] Example 1: Preparation of iPSCs

[0160] 1. iPSC Digestion Process

[0161] After aspirating the culture medium supernatant, wash the cells with DPBS first, then add 0.5mM EDTA (diluted with DPBS) to digest the cells, leave them at room temperature for 8 minutes, and then observe the cell status under a microscope to see if they have shrunk; after confirming the digestion status, discard the EDTA digestion solution and terminate the digestion with E8 complete medium supplemented with ROCKi.

[0162] 2. iPSC Passaging Process

[0163] The digested cells were passaged at a certain ratio using E8 complete medium supplemented with ROCKi as the culture medium. The next day, the culture medium was replaced with E8 complete medium without ROCKi. The medium was changed every day and the cells were passaged when they reached 70% confluence.

[0164] Example 2: Lentiviral transduction of iPSCs and preparation of CAR-iPSCs

[0165] 1. Construction of CAR19-IL15RF lentiviral expression vector

[0166] The construction and synthesis of the plasmid vectors involved in the present invention were completed by a CRO company (Yunzhou Biotechnology Co., Ltd.). After the chimeric antigen receptor CAR19 and IL15RF were connected in series, a CAG promoter element was added upstream, and the same lentiviral vector was constructed, and the plasmid was extracted and purified. The extracted expression plasmid was mixed with psPAX2 and pMD2.G auxiliary plasmids in a certain proportion and co-transfected into 293T cells. After 48 hours of transfection, the cell culture supernatant containing viral particles was collected, centrifuged at 4°C and 500g for 10 minutes, and the supernatant was filtered through a 0.45um filter and concentrated using a lentiviral concentration kit Lenti-X. TM The virus was concentrated by Takara, and the concentrated virus was aliquoted and stored at -80°C until use.

[0167] 2. Lentiviral Transduction of iPSC Cells

[0168] (1) After iPSCs were digested, the cells were collected and counted, and then plated in 12-well plates (coated with vitronectin VTN one day in advance), with 6E4 cells per well. The culture medium used was complete E8 medium supplemented with 10 μM ROCKi, and the cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0169] (2) On the next day, cells from two wells were taken for digestion and counting, and then the medium from the planned transduction wells was aspirated and replaced with 500uL E8 medium + 6ug / ml polybrene (diluted in E8 medium).

[0170] (3) Count the cells as in step 2, add lentiviral particles containing CAR structure and elements that increase NK proliferation and persistence at an MOI (3-5), infect iPSC cells to prepare iPSC cells expressing chimeric antigen receptor CAR, and carefully shake the well plate to evenly distribute the virus in the culture medium.

[0171] (4) The transduced cells were cultured in a 37°C, 5% CO2 incubator and the medium was replaced with complete E8 medium without polybrene the next day.

[0172] (5) The medium was changed every day. During cell passage, the expression of the transduced gene in the virally transduced iPSC cells was identified by flow cytometry. 1e5 cells / sample after cell culture were flow stained, and appropriate amount of antibodies (CD19 protein; anti-IL15RF; dead / live dye) were added and incubated in a refrigerator at 4°C for 30 minutes. After 30 minutes, the cells were washed twice with FACS buffer (DPBS containing 1% FBS) at 1500 rpm for 3 minutes. The supernatant was discarded and the cells were resuspended in 200 μl FACS buffer and detected by the instrument.

[0173] (6) After confirming that the virally transduced iPSC cells successfully express CAR19 and IL15RF, the next step is to plate and select single clones.

[0174] Example 3: Selection of iPSC monoclones

[0175] When the cell confluence reaches about 70%, observe the cell morphology and confirm that the cells have no self-differentiation phenomenon, then proceed with cell processing and digestion; wash the cells with DPBS once, then use TryPLE Express or TryPLE Select to digest the iPSC cells, place them in a 37℃ incubator for 5 minutes, observe whether the cells shrink under a microscope, blow the cells into single cells with a pipette, add E8 medium in a 1:1 ratio to terminate the digestion, transfer the cell solution to a 15ml centrifuge tube and centrifuge at 400g for 5 minutes; after the centrifugation, discard the supernatant and use 10% CloneR TM Resuspend the cells in complete E8 medium, count the cells, and add 10% CloneR TM The cell density was adjusted to 10 cells / ml with complete E8 medium. 100 μl of cell suspension was plated into 96-well plates (coated with vitronectin one day in advance) using a multichannel pipette. 48 h later (day 2), 100 μl of culture medium (supplemented with 10% CloneR) was added to each well. TM 2 complete E8 medium); after 48 h (day 4), the medium was changed to 150 μl per well with 10% CloneR TM 2 complete E8 medium. The medium was changed daily with complete E8 medium. Colony formation could be observed under a microscope on days 7-10. Individual clones were observed and labeled under a microscope. When the iPSC single clone reached 70% confluence, cell digestion was performed as described in Example 1. All digested cells were transferred to a 24-well plate for further culture. When the cells reached 70% confluence, they were digested and passaged, and gene expression analysis of the selected single clones was performed using flow cytometry.

[0176] The monoclonal selection criteria are shown in FIG3A . The iPSC monoclones obtained were in good growth condition, with normal clonal proliferation and no self-differentiation. The statistical yield of the selected monoclones was 33.85% ( FIG3B ).

[0177] Example 4: Identification of iPSC positive clones

[0178] Flow cytometry was used to identify positive iPSC clones. According to Example 1, iPSC monoclones were digested and 1e5 cells / sample of each clone were flow stained. Appropriate antibodies (CD19 protein; anti-IL15RF; dead / live dye) were added and incubated in a refrigerator at 4°C for 30 minutes. After 30 minutes, the cells were washed twice with FACS buffer (DPBS containing 1% FBS) at 1500 rpm for 3 minutes. The supernatant was discarded and the cells were resuspended in 200 μl of FACS buffer for detection. After data analysis, monoclonal clones expressing both CAR19 and IL15RF were selected for further passage and the next step of iNK differentiation.

[0179] As shown in Figure 5, the expression of CAR19 and IL15RF in iPSC-positive monoclonal clones was close to 100%. Identification and statistical analysis of all monoclonal positive clones revealed a yield of 61.54%. All identified positive clones were expanded and cultured. Four positive clones (021, 029, 035, and 057; gene expression is shown in Figure 5B) were selected for iNK differentiation.

[0180] Example 5: Construction of iPSC Seed Bank

[0181] According to the method of Example 4, the iPSC positive clones were identified, and the positive clones with high gene expression were selected for CAR-iPSC seed bank construction. The process is shown in Figure 4A. The positive clones were selected for cell passage treatment. When the positive monoclonal clones selected in the 24-well plate reached 70% confluence, they were continued to be passaged. The cells were passaged to 6-well plates, and the positive clones were re-identified by flow cytometry, as shown in Figure 5B. When the cell confluence reached 70%, the culture was continued to be expanded. The yield was shown in Figure 5C. All cells were passaged to T25 cell culture flasks, and the cells were frozen when they reached 70% confluence in T25. The frozen monoclonal cells were finally stored in a liquid nitrogen tank.

[0182] Example 6: Cryopreservation of iPSCs

[0183] When the confluence of iPSC clones reached 70%, the cells were observed for autodifferentiation. The cells were digested according to the method of Example 1, harvested, and centrifuged at 400 g for 3 minutes. The supernatant was discarded, and the cell pellet was placed on ice. iPSC cells were cryopreserved according to the following steps: CS10 freezing solution was placed on ice in advance. The number of cells to be frozen in each cryovial was calculated based on the ratio (500 μl CS10 / tube). The cell pellet was resuspended in the corresponding volume of CS10 freezing solution. The cell suspension was aliquoted into each cryovial (with pre-printed labels). The cryovials were placed in a freezing box and quickly placed in a -80°C freezer. After 24 hours, the cells were transferred to a liquid nitrogen tank for long-term storage.

[0184] Example 7: Recovery of iPSCs

[0185] Remove the cryovial from liquid nitrogen and quickly thaw in a 37°C water bath. Once thawed, quickly remove the cryovial from the 37°C water bath (approximately 1 minute). Add the cells containing the cryovial to a 15ml centrifuge tube (add 4.5ml of pre-warmed complete E8 medium). Centrifuge at 400g for 3 minutes. Discard the supernatant and resuspend the cell pellet in complete E8 medium supplemented with ROCKi. Place the cell suspension in a culture flask or well plate (coated with vitronectin one day in advance) and continue culturing. The next day, replace the medium with complete E8 medium without ROCKi. Repeat the medium change daily, and passage the cells when they reach 70% confluence.

[0186] Example 8: Inducing iPSCs to differentiate into iNK cells

[0187] Using TrypLE TM iPSC cells were dissociated into single cells by Express and filtered through a 40 μm cell sieve to remove any undissociated cell aggregates. The collected cells were counted and seeded at a density of 8,000 cells / well in an ultra-low attachment round-bottom 96-well plate in STEMdiff supplemented with 40 ng / mL SCF, 20 ng / mL BMP4, 20 ng / mL VEGF, and 10% CloneR. TM APEL TM 2 medium, the final volume is 100 μL. The plate is then centrifuged at 300x g for 5 minutes and incubated at 37 ° C, 5% CO2 for 6 days to generate hematopoietic embryoid bodies (HE EB). The HE EB formed on the 6th day is then pooled into a 15 ml centrifuge tube and collected by sedimentation. The collected HE EB plates are plated onto 2% gelatin-coated or uncoated 6-well plates in NK cell differentiation medium, which is composed of 56.6% DMEM + GlutaMAX TM -I, 28.3% F12+GlutaMAXTM -I, 15% heat-inactivated human AB serum, 1% P / S, 2mM L-glutamine, 1μM β-mercaptoethanol, 5ng / mL sodium selenite, 50μM ethanolamine, 20mg / L ascorbic acid, 5ng / mL IL-3, 20ng / mL SCF, 20ng / mL IL-7, 10ng / mL IL-15, 10ng / mL FLT3 ligand (FLT3L) and 50U / mL IL-2. For each well of a 6-well plate, approximately 16 EB plates were inoculated. The culture medium was replaced every 6 days for the first 14 days of differentiation, and every 3 days after 14 days of differentiation. From the 7th day of differentiation, IL-3 was no longer supplemented in the culture medium. Floating NK cells with a spindle-shaped morphology gradually appeared around day 21-35 of differentiation and were collected for subsequent analysis and verification. The cell morphology at different stages of the differentiation process is shown in Figure 6. For expansion, NK cells were incubated with K562 myeloid leukemia cell line expressing mbIL-21 at a 1:1 ratio and 2.5x10 5 The cells were co-cultured at a minimum density of 10 cells / mL in NK expansion medium (RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% P / S, and 50 U / mL IL-2).

[0188] Example 9: In vitro phenotype identification of CAR-iNK

[0189] Phenotypic identification of differentiated and mature CAR-iNK cells was performed by flow cytometry, including CD45, CD56, CD16, CAR19, and IL15RF. The results are shown in Figure 7. 1e5 cultured CAR-iNK cells / sample were collected for flow cytometry staining, and appropriate amounts of detection antibodies were added. The cells were incubated in a refrigerator at 4°C for 30 minutes. After 30 minutes, the cells were washed twice with FACS buffer (DPBS containing 1% FBS) at 1500 rpm for 3 minutes. The supernatant was discarded and the cells were resuspended in 200 μl of FACS buffer before detection.

[0190] The results showed that the expression of the CAR gene and IL15RF varied across iPSC clones during differentiation, with some showing downregulation followed by recovery. However, some positive clones achieved normal expression during the final maturation stage of CAR-iNK cells. Further functional validation of the differentiated and mature CAR-iNK cells will be performed.

[0191] Example 10: Identification of CAR-iNK killing function in vitro

[0192] The luciferase reporter gene assay was used to detect the cytotoxicity of CAR-iNK cells. For target cell plating, count Nalm6-luc cells, calculate the number of cells required for plating, aspirate the desired cell suspension, centrifuge at 250g for 4 minutes, discard the supernatant, resuspend the cell pellet in complete RPMI 1640 medium, adjust the cell density to 2E5 cells / ml, and plate 50 μl of cell suspension per well in a 96-well plate using a multichannel pipette. For effector cell plating, count CAR-iNK cells and control cells, aspirate the desired cell number, centrifuge at 300g for 5 minutes, discard the supernatant, and resuspend the cells in complete RPMI 1640 medium. Adjust the cell density by isocratic dilution, and add effector cells at E / T ratios of 4, 2, 1, 0.5, and 0.25. The 96-well plate was placed at 37°C and 5% CO2 for incubation for 24 hours. After 24 hours, the 96-well plate was removed and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in 50 μl of DPBS. 50 μl of luciferase reporter gene reagent was added to each well and reacted in the dark for 5 minutes. 80 μl of the mixture was aspirated from each reaction well to a black-walled transparent plate for detection.

[0193] The results are shown in FIG7 , and the differentiated and mature CAR-iNK cells have significant in vitro specific killing activity compared with the iNK control group.

[0194] Example 11: In vitro recovery and culture of CAR-iNK

[0195] The CAR-iNK cells and feeder cells were removed from liquid nitrogen and quickly placed in a 37°C water bath for thawing. After the cells thawed, they were quickly removed from the 37°C water bath (approximately 1 minute). The CAR-iNK cells were added to a 15ml centrifuge tube (with 9ml of pre-warmed complete RPMI 1640 medium), and the feeder cells were added to a 50ml centrifuge tube (with 45ml of pre-warmed complete RPMI 1640 medium). The cells were centrifuged at 300g for 5 minutes, and the supernatant was discarded. The CAR-iNK cells and feeder cells were resuspended in complete RPMI 1640 medium supplemented with 50IU IL-2 and counted. The feeder cells were added to the CAR-iNK cells at a ratio of 1:1 and cultured in a 37°C, 5% CO2 incubator. The cells were cultured every 3 days. The feeder cells were observed on the 7th day to see if they were completely removed. If so, the next experiment could be carried out.

[0196] Example 12: CAR-iNK in vitro cytokine secretion

[0197] According to Example 11, on the seventh day, the CAR-iNK cells and the control group cells that had been cleared of feeder cells were taken to detect cytokine secretion during the killing process. Tumor cells Nalm6 and effector cells were co-incubated and plated at an E / T ratio of 5:1, and CD107a monoclonal antibody (1 ul / well) was added. The cells were incubated in an incubator for 1 hour, and Golgi Stop was added according to the ratio after 1 hour. TM (1:1500) and Golgi Plug TM (1:1000), and continue to incubate in the incubator for 4 hours. After 4 hours, Fc blocking antibody (2ul / well) was added to each well and incubated at 4°C for 15 minutes. After 15 minutes, dead-live dye, IgG1 isotype control antibody, CD56 antibody, and CD3 antibody were added and incubated at 4°C for 30 minutes. After 30 minutes, the cells were washed twice with FACS buffer (FACS buffer-1% FBS in DPBS), centrifuged at 300g for 4 minutes each time, and the supernatant was discarded. The cell pellet was resuspended with 100μL fixation / permeabilization solution and incubated in a 4°C refrigerator for 20 minutes. After 20 minutes, the cells were washed with 1× BD Perm / Wash buffer. TM Wash the cells twice with the buffer, centrifuging at 300g for 4 minutes each time, discard the supernatant, and use 100ul 1×BD Perm / Wash TM Resuspend the cell pellet in the buffer, add IFN-γBV421 antibody (1ul / well) to each reaction well, incubate in a 4°C refrigerator for 30 minutes in the dark, and then use 1×BD Perm / Wash TM Wash twice with buffer, centrifuge at 300g for 4 minutes each time, discard the supernatant, and use 200ul 1×BD Perm / Wash TM Resuspend the cell pellet in buffer and perform detection on the instrument.

[0198] The results are shown in FIG8B . When co-incubated with tumor cells Nalm6, CAR-iNK cells can secrete higher levels of CD107a and IFNγ cytokines, thereby killing tumor cells.

[0199] Example 13: Multiple rounds of CAR-iNK stimulation of tumor cells in vitro

[0200] The cell killing function test was performed according to Example 10. Effector cells and tumor cells were added according to E / T ratios of 4, 2, 1, 0.5, and 0.25, and five 96-well plates were plated at the same time. The 96-well plates were incubated at 37°C and 5% CO2. After 24 hours, one of the 96-well plates was removed for testing, and a new round of tumor cells (1E4 / well) was added to the remaining four plates and incubated at 37°C and 5% CO2. After 48 hours, the second 96-well plate was removed for testing, and a new round of tumor cells (1E4 / well) was added to the remaining three plates. This process was repeated until the last plate was tested.

[0201] The results are shown in Figure 8C. Under multiple rounds of Nalm6 tumor cell stimulation, CAR-iNK still showed a sustained specific killing effect.

[0202] Example 14: In vivo efficacy test of CAR-iNK

[0203] The in vivo efficacy experiment process is shown in Figure 9. NOG mice were selected for in vivo efficacy evaluation. A total of three groups were set up, namely solvent control group, iNK group, and CAR-iNK group, with a total of 6 mice in each group. The drug was administered by intravenous injection, and the cell dosage was 1E7 cells / mouse each time. On the first day (D-1), the experimental mice were irradiated (1.4 Gy), and tumor cells Nalm6-luc were inoculated on the second day (D0). On the third day (D1), iNK cells and CAR-iNK cells were administered intravenously for the first time. A total of 3 doses were administered, and the interval between each dose was 7 days. During this period, blood was collected from the mice for accompanying PK detection by flow cytometry. At the same time, the IVIS imaging system was used in combination with bioluminescent tumor cell lines to observe the growth of the tumor at the living level.

[0204] The results, as shown in Figures 10 and 11, demonstrate robust preclinical anti-tumor toxicity and persistence. Flow cytometry analysis revealed that the CAR-iNK population in peripheral blood was 100-200 cells / μl. In vivo efficacy and survival studies demonstrated that the CAR-iNK group significantly inhibited tumor growth and prolonged survival in mice compared to the iNK control group.

[0205] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A CAR-iNK cell, characterized in that: The CAR comprises: (1) scFv, (2) transmembrane domain, (3) a co-stimulatory domain, and (4) Signaling domain, The scFv comprises the VH and VL of humanized FMC63, and the signaling domain is modified with IL15RF.

2. The cell according to claim 1, characterized in that The amino acid sequence of the VH comprises one or more of the following sequences: (1) the amino acid sequence shown in SEQ ID NO. 1; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 1, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 1; (3) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO. 1, and which retains the activity of the amino acid sequence shown in SEQ ID NO. 1; The amino acid sequence of the VL comprises one or more of the following sequences: (1) the amino acid sequence shown in SEQ ID NO. 2; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 2, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 2; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO.

2.

3. The cell according to claim 2, characterized in that The scFv further comprises a connecting peptide; The connecting peptide is located between the light chain variable region and the heavy chain variable region of the scFv, and its sequence is shown in SEQ ID NO.

3.

4. The cell according to claim 3, characterized in that The scFv comprises one or more of the following sequences: (1) the amino acid sequence shown in SEQ ID NO. 4; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 4, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 4; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 4, and which retains the activity of the amino acid sequence shown in SEQ ID NO.

4.

5. The cell according to claim 1, characterized in that The CAR has the structure of the following formula I: L-scFv-H-TM-C-CD3ζ-IL15RF (I), in, Each "-" is independently a connecting peptide or a peptide bond; L is an optional signal peptide sequence; The scFv is the scFv according to claim 4; H is an optional hinge region; TM is the transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

6. The cell according to claim 5, characterized in that The CAR comprises one or more of the following sequences: (1) the amino acid sequence shown in SEQ ID NO. 5; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 5, and that retains the activity of the amino acid sequence shown in SEQ ID NO. 5; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO. 5, and which retains the activity of the amino acid sequence shown in SEQ ID NO.

5.

7. A method for preparing the cell according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: (a) CAR lentivirus was transduced into iPSCs to obtain CAR-iPSCs; (b) Selecting iPSC positive monoclonal clones that simultaneously express CAR19 and IL15RF; (c) Construction of CAR-iPSC seed bank; (d) Inducing CAR-iPSCs to differentiate into CAR-iNK cells.

8. The method according to claim 7, characterized in that The step (a) comprises: (a1) The chimeric antigen receptor CAR19 and IL15RF were constructed into a lentiviral vector; (a2) The plasmid was extracted and purified, mixed with the helper plasmid, and co-transfected into 293T cells; (a3) collecting the cell culture supernatant containing the virus particles, filtering and concentrating; (a4) The concentrated viral particles are used to transduce iPSCs to obtain CAR-iPSCs.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cell according to any one of claims 1 to 6, or the cell prepared by the method according to any one of claims 7 to 8, and a pharmaceutically acceptable carrier, diluent and / or excipient.

10. Use of the cell according to any one of claims 1 to 6, or the method according to any one of claims 7 to 8, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing and / or treating cancer or tumor-related diseases.

11. The use according to claim 10, characterized in that The disease is selected from B-cell lymphoma, including non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL); acute lymphoblastic leukemia (ALL), including CD19-positive B-cell ALL; chronic lymphocytic leukemia (CLL), including CD19-positive B-cell CLL; myeloma, including multiple myeloma (MM) and non-Hodgkin lymphoma cell leukemia (LCBCL); and other CD19-positive B-cell related diseases, including CD19-positive leukemia in acute myeloid leukemia (AML).

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