Chimeric antigen receptor aiming at iPSC-NK cells and application of chimeric antigen receptor

Abstract

The invention relates to a chimeric antigen receptor aiming at iPSC-NK cells and application of the chimeric antigen receptor. Specifically, the invention provides a CAR structure containing a transmembrane region and an intracellular region from 2B4 protein, and the CAR structure can be efficiently expressed on iPSC cells and then differentiated into iNK cells containing the CAR structure. The CAR structure does not affect the differentiation and amplification process of iPSC to NK cells, and the engineered NK cells containing the CAR structure have enhanced cell killing ability, can be matched with various different scFvs and target different targets, and have application prospects in the field of disease treatment.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, and in particular to a chimeric antigen receptor targeting iPSC-NK cells and applications thereof. Background Art

[0002] Chimeric antigen receptor (CAR) is a common method to generate tumor target-specific immune cells by genetically modifying cells. CAR has been introduced into different types of immune cells, including T cells, NK cells, and macrophages. These CAR-transduced immune cells are named CAR-T, CAR-NK, and CAR-M, respectively.

[0003] In order to improve the specific killing of tumors, expressing CAR molecules on NK is an effective modification scheme. However, due to the characteristics of NK cells, they have natural resistance to the traditional method of transduction through retroviral vectors, resulting in very low transduction efficiency. In addition to direct transduction into NK cells, the CAR gene can also be edited into the iPSC genome by means of CRISPR / Cas9, and then the correctly edited CAR-iPSC is differentiated into CAR-iNK cells.

[0004] However, the CAR molecular structure that can be applied to the above technical path needs to meet many conditions, including: (1) the CAR molecule can be stably expressed in the iPSC stage to complete the screening of correctly edited iPSC clones; (2) the expression of the CAR molecule will not affect the differentiation and expansion process from iPSC to iNK cells, as well as the inherent natural immunity of iNK cells; (3) the CAR structure can maintain sufficient expression levels in the iNK stage and have good antigen recognition and killing functions; (4) the CAR molecular structure can be adapted to a variety of different scFv. At present, there is a lack of CAR structures that can meet the above conditions at the same time in this field.

[0005] Therefore, there is an urgent need in the art to develop CAR structures suitable for iPSCs and iPSC-derived iNK cells. Summary of the invention

[0006] The purpose of the present invention is to provide a CAR structure suitable for iPSCs and iNK cells derived from iPSCs.

[0007] In a first aspect of the present invention, an engineered iPSC cell is provided, wherein an exogenous expression vector is introduced into the engineered iPSC cell, wherein the exogenous expression vector comprises a gene encoding a chimeric antigen receptor (CAR);

[0008] The CAR comprises a structure as shown in Formula I:

[0009] L-EB-H-TM-ICD1-ICD2(I)

[0010] In the formula,

[0011] Each "-" is independently a connecting peptide or a peptide bond;

[0012] L is none or a signal peptide sequence;

[0013] EB is the extracellular binding domain;

[0014] H is the hinge region;

[0015] TM is the transmembrane region from the 2B4 protein;

[0016] ICD1 is derived from the intracellular region of the 2B4 protein;

[0017] ICD2 is a cytoplasmic signaling sequence derived from CD3ζ.

[0018] In another preferred embodiment, L is a signal peptide selected from the following group of proteins: CSF2RB, CD8, GM-CSF, CD4, CD28, CD137, NKG2D, or mutants / modified forms thereof, or a combination thereof.

[0019] In another preferred embodiment, the sequence of L is shown in SEQ ID NO:1.

[0020] In another preferred embodiment, the EB is a scFv targeting one or more target proteins.

[0021] In another preferred embodiment, the target protein includes: CD19, BCMA, CD22, CD20, EGFR, HER2, L1CAM, Mesothelin, GD2, CD171, EpCAM, CEA, PSMA, GPC3, ROR1, FAP, CD33, CD38, CD123, CD138, CD30, CD70, Nectin-4, MSLN, NCAM1, CEACAM5, NKG2D, MICA / B, KIRs, or a combination thereof.

[0022] In another preferred embodiment, the target protein includes: CD19, BCMA, or a combination thereof.

[0023] In another preferred embodiment, the sequence of the scFv targeting CD19 is shown in SEQ ID NO: 2.

[0024] In another preferred example, the sequence of the scFv targeting BCMA is shown in SEQ ID NO: 20.

[0025] In another preferred embodiment, the EB targets CD19 and BCMA simultaneously.

[0026] In another preferred embodiment, the H is a hinge region selected from the following proteins: CD8, CD28, CD137, IgG, or a combination thereof.

[0027] In another preferred embodiment, the H is derived from the hinge region of CD8 or CD28 protein.

[0028] In another preferred embodiment, the H is derived from the hinge region of CD8 and has an amino acid sequence as shown in SEQ ID NO:4.

[0029] In another preferred embodiment, the H is derived from the hinge region of CD28 and has an amino acid sequence as shown in SEQ ID NO:3.

[0030] In another preferred example, the TM is the transmembrane region of the 2B4 protein, having an amino acid sequence as shown in SEQ ID NO:6.

[0031] In another preferred embodiment, the ICD1 is the intracellular full-length sequence of the 2B4 protein, having an amino acid sequence as shown in SEQ ID NO:10.

[0032] In another preferred example, the ICD2 has an amino acid sequence as shown in SEQ ID NO:13.

[0033] In another preferred example, the CAR has an amino acid sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 22.

[0034] In another preferred embodiment, the exogenous expression vector is selected from the following group: a plasmid, a viral vector, a transposon, or a combination thereof.

[0035] In another preferred embodiment, the vector is a plasmid.

[0036] In another preferred embodiment, the introduction includes: gene editing introduction, electroporation, viral transduction, or a combination thereof.

[0037] In another preferred embodiment, the gene editing introduction is gene editing using the CRISPR / Cas9 system.

[0038] In another preferred embodiment, the introduction includes the step of: using the CRISPR / Cas9 system to knock the CAR encoding gene into the AAVS1 site.

[0039] In another preferred example, the CAR expression level on the engineered iPSC cells is detected by flow cytometry, and the mean fluorescence intensity (MFI) value is recorded as R1, R1≥2000, preferably R1≥2500, and more preferably R1≥3000.

[0040] In another preferred example, the control engineered iPSC cells express the CAR shown in SEQ ID NO: 14, and the CAR expression level on the control engineered iPSC cells is detected by flow cytometry, and the mean fluorescence intensity (MFI) value is recorded as R0, R1 / R0≥5, preferably R1 / R0≥10, and more preferably R1 / R0≥15.

[0041] In a second aspect of the present invention, a method for preparing CAR-NK cells is provided, comprising the steps of: performing differentiation culture on the engineered iPSC cells described in the first aspect of the present invention to obtain CAR-NK cells.

[0042] In another preferred embodiment, the differentiation culture comprises the steps of:

[0043] A. amplifying and culturing the CAR-iPSC in a reaction vessel to obtain CAR-iPSC cell aggregates;

[0044] B. Add a cytokine combination into the reaction container, induce differentiation culture of the CAR-iPSC cell mass, and obtain the CAR-NK cells.

[0045] In another preferred embodiment, the expansion culture is for 7-14 days.

[0046] In another preferred embodiment, the induced differentiation culture is for 26-30 days, such as 28 days.

[0047] In another preferred embodiment, the reaction container is a bioreactor.

[0048] In the third aspect of the present invention, a CAR-NK cell is provided. The CAR-NK cell is obtained by differentiation of the engineered iPSC cell described in the first aspect of the present invention, or is prepared by the method described in the second aspect of the present invention.

[0049] In a fourth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0050] (a) the CAR-NK cell according to the third aspect of the present invention; and

[0051] (b) a pharmaceutically acceptable carrier.

[0052] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection and lyophilized agent.

[0053] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the engineered cells as described in the first aspect of the present invention; and 0.01 to 99.99% of a pharmaceutically acceptable carrier; the percentages are the mass percentages of the pharmaceutical composition.

[0054] In another preferred embodiment, the concentration of the CAR-NK cells in the pharmaceutical composition is 1×10 3 -1×10 8 cells / mL, preferably 1×10 4 -1×10 7 cells / mL.

[0055] In the fifth aspect of the present invention, there is provided a use of the CAR-NK cell as described in the third aspect of the present invention or the pharmaceutical composition as described in the fourth aspect of the present invention for preparing a drug or preparation for preventing and / or treating tumors.

[0056] In another preferred embodiment, the tumor is a CD19 and / or BCMA positive tumor.

[0057] In another preferred embodiment, the tumor includes: a solid tumor, a blood tumor, or a combination thereof.

[0058] In the sixth aspect of the present invention, a method for treating a tumor or cancer is provided, the method comprising: administering the CAR-NK cell as described in the third aspect of the present invention or the pharmaceutical composition as described in the fourth aspect of the present invention to a subject in need thereof.

[0059] In another preferred embodiment, the subject in need is a tumor patient.

[0060] In another preferred embodiment, the tumor includes: a solid tumor, a blood tumor, or a combination thereof.

[0061] In another preferred embodiment, the subject includes a non-human mammal or a human.

[0062] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The technical process of editing iPSCs and differentiating them to obtain iPSC-CAR-NK is shown.

[0064] Figure 2 The expression levels of each group of anti-CD19 CAR molecules on iPSCs after gene editing and screening are shown.

[0065] Figure 3 The expression levels of each group of anti-CD19 CAR after site-directed knock-in on iPSCs are shown.

[0066] Figure 4The results of the detection of the expression levels of each group of anti-CD19 CAR on iPSC monoclonals are shown.

[0067] Figure 5 The expression efficiency of each group of anti-CD19 CAR on iNK is shown.

[0068] Figure 6 The cell killing ability of iNK cells expressing CD19 CAR in each group against K562 or Nalm-6 is shown.

[0069] Figure 7 The expression levels of anti-BCMA-CAR on iPSCs and iPSC-CAR-NKs are shown.

[0070] Figure 8 The expression efficiency of two anti-BCMA+CD19 dual CARs on iNK is shown.

[0071] Fig. 9 The results of the experiment on the killing ability of iNK cells expressing two BCMA+CD19 dual CARs against RPMI8226 cell line are shown. DETAILED DESCRIPTION

[0072] After extensive and in-depth research and a large number of screenings, the inventors have provided a CAR molecular structure that is particularly suitable for expression on iPSC, which contains a unique transmembrane region and intracellular region from the 2B4 protein, which can be used with scFv targeting different antigens. Compared with the commonly used CAR structure, the CAR with a specific structure of the present invention has an unexpectedly high expression rate on iPSC cells, with an increase of several times to dozens of times. In addition, after the CAR of the present invention is expressed on iPSC, the ability to differentiate into iNK cells is not affected, so that CAR-iNK cells differentiated from CAR-iPSC can be obtained efficiently. The engineered NK cells containing the CAR structure of the present invention have enhanced cell killing ability, and the performance is significantly improved compared with the currently commonly used control CAR structure. The present invention was completed on this basis.

[0073] CAR of the present invention

[0074] In the present invention, a CAR molecule design suitable for expression on iPSC or iNK cells is provided, which can not only be directly and efficiently expressed on iNK cells (NK cells derived from iPSC), but also be efficiently expressed on iPSC cells, and then differentiated into iNK cells containing a CAR structure.

[0075] Chimeric antigen receptors (CARs) are usually composed of three major regions: the extracellular region, the transmembrane region, and the intracellular region.

[0076] The extracellular region is mainly an antigen binding domain, which is used to recognize and bind to antigens on the surface of target cells, usually a single-chain variable fragment (scFv) of an antibody. The extracellular region may also optionally include a hinge region, which serves as a connection and enhances the flexibility and stability of the structure. The transmembrane region connects the intracellular and extracellular regions of the CAR. Commonly used transmembrane regions include the transmembrane regions of CD8 and CD28 proteins, but are not limited thereto. The intracellular region includes a signal transduction domain and a co-stimulatory molecule, which is responsible for the transmission of signals to further improve the signal transmission capability of a specific CAR and to give immune cells a stronger killing ability and duration.

[0077] The CAR of the present invention has an extracellular binding domain, a hinge region, a transmembrane region, an intracellular region and a signal transduction sequence. Preferably, the CAR of the present invention contains a structure as shown in Formula I:

[0078] L-EB-H-TM-ICD1-ICD2(I)

[0079] In the formula,

[0080] Each "-" is independently a connecting peptide or a peptide bond;

[0081] L is none or a signal peptide sequence;

[0082] EB is the extracellular binding domain;

[0083] H is the hinge region;

[0084] TM is the transmembrane region from the 2B4 protein;

[0085] ICD1 is derived from the intracellular region of the 2B4 protein;

[0086] ICD2 is a cytoplasmic signaling sequence derived from CD3ζ.

[0087] In immature CAR molecules, the function of the signal peptide is mainly to guide the CAR protein to correctly enter the endoplasmic reticulum of the cell for subsequent folding, modification and transport. After CAR is successfully localized to the cell membrane, the signal peptide is usually removed, so mature CAR molecules usually do not contain signal peptides. In the CAR molecules of the present invention, the type of signal peptide is not particularly limited. In one embodiment of the present invention, the sequence of the selected signal peptide is shown in SEQ ID NO: 1.

[0088] In the CAR molecule of the present invention, the extracellular binding domain can be a scFv targeting a single antigen, or a structure formed by scFvs targeting multiple antigens in series. In one embodiment of the present invention, the extracellular binding domain is a scFv targeting a single antigen, such as a scFv targeting CD19 or a scFv targeting BCMA. In one embodiment of the present invention, the extracellular binding domain simultaneously targets multiple antigens, such as simultaneously targeting CD19 and targeting BCMA; preferably, the scFvs targeting multiple antigens are connected in series to form the extracellular binding domain.

[0089] In the CAR molecule of the present invention, the transmembrane region and the intracellular region are from the 2B4 protein. As used herein, the 2B4 protein is also referred to as CD244, SLAMF4, and is an immune cell surface molecule belonging to one of the members of the immunoglobulin superfamily. The transmembrane region sequence of the 2B4 protein is shown in SEQ ID NO:6, and the intracellular full-length sequence of the 2B4 protein is shown in SEQ ID NO:10. The CAR molecule of the present invention comprising the transmembrane region of the 2B4 protein and the full-length intracellular sequence of 2B4 can be efficiently expressed on iPSC cells or iNK cells.

[0090] In one embodiment of the present invention, the CAR is a CAR targeting CD19, which has an amino acid sequence as shown in SEQ ID NO: 15. In one embodiment of the present invention, the CAR is a CAR targeting BCMA, which has an amino acid sequence as shown in SEQ ID NO: 20. In one embodiment of the present invention, the CAR is a dual CRA that simultaneously targets CD19 and BCMA, and its sequence is shown in SEQ ID NO: 23.

[0091] The CAR molecule of the present invention can be highly expressed on iNK cells or iPSC cells without affecting the differentiation of iPSCs into iNK cells or the inherent natural immunity of iNK cells.

[0092] Chimeric Antigen Receptor (CAR)-NK Cells

[0093] Natural killer (NK) cells are important immune cells in the body and are the first line of defense against tumors and pathogens. Since NK cells play a key role in the body's nonspecific anti-tumor response, a large number of basic and clinical studies have applied NK cells to the treatment of hematopoietic malignancies (leukemia, lymphoma, multiple myeloma, etc.) and solid tumors (such as melanoma, ovarian cancer, and lung cancer, etc.). The research and development and preparation ideas of CAR-NK cells are similar to those of CAR-T cells. CAR-NK cells are prepared by gene editing to make NK cells express CAR, and then CAR-NK cells with specific anti-tumor ability are infused back into tumor patients to exert anti-tumor effects, thereby achieving the purpose of treatment.

[0094] Although CAR-NK cells have made remarkable progress in the treatment of hematological malignancies and solid tumors in recent years, CAR-NK cells still face great challenges and room for optimization. For example, CAR structure design, CAR gene transduction method and transduction efficiency are all key influencing factors. The traditional method of CAR gene delivery is to use viral vectors to deliver and insert into the genome in the form of random integration, which poses potential safety risks.

[0095] CAR-iNK cells of the present invention

[0096] As used herein, the terms “iNK cells”, “iPSC-derived NK cells”, “NK cells differentiated from iPSCs”, and “iPSC-NK cells” are used interchangeably and refer to NK cells derived from iPSCs.

[0097] iPSC (induced pluripotent stem cell) is a cell type with characteristics similar to those of embryonic stem cells. It was first reported by the Yamanaka Laboratory of Kyoto University in 2006. By introducing a set of specific transcription factors into terminally differentiated somatic cells such as fibroblasts, these cells can be reprogrammed into pluripotent stem cells. These transcription factors include Oct3 / 4, Sox2, Klf4 and c-Myc, which work together to reset the gene expression pattern of cells and return them to a state similar to that of embryonic stem cells.

[0098] Under the culture conditions of adding special cytokines, iPSC can differentiate into NK cells, i.e., iNK cells of the present invention. Specifically, iPSC can aggregate with each other to form embryoid bodies (EBs), and under the culture conditions of adding special cytokines, EBs gradually grow larger and gradually differentiate into hematopoietic stem cells. After about 28 days of culture, EBs can differentiate into CD56-positive NK cells, which can be further activated and amplified, and finally one starting iPSC cell can differentiate into 1000-10000 iNK cells.

[0099] Currently, NK cells used in clinical treatment are mainly derived from human tissues such as umbilical cord blood and peripheral blood. These natural NK cells have great heterogeneity, high preparation costs, low yields, and preparation cycles are limited by the accessibility of human tissues. In the present invention, iNK cells derived from iPSC differentiation can solve the above problems.

[0100] In the present invention, iNK cells containing a CAR structure can be obtained by the following two approaches: one is to introduce CAR into iPSC to obtain CAR-iPSC, and then obtain CAR-iNK cells through differentiation culture; the other is to directly introduce CAR into iNK cells differentiated from iPSC.

[0101] In one embodiment of the present invention, CAR is introduced into iPSC through the CRISPR / Cas gene editing system to obtain correctly edited iPSC clones, which are then differentiated to form iNK cells expressing CAR. In this process, the expression of the CAR molecule of the present invention does not affect the process of differentiation and expansion of iPSC into iNK cells, and the inherent natural immunity of iNK cells is not affected.

[0102] The CAR-iNK cells differentiated from the CAR-iPSCs of the present invention, or the directly introduced CAR-iNK cells, have significantly enhanced cell-specific killing capabilities, for example, specific killing capabilities against tumor cells expressing specific antigens.

[0103] Preparation method of CAR-NK cells of the present invention

[0104] In the present invention, a method for preparing CAR-iNK cells is provided, comprising the steps of:

[0105] A. Provide CAR-iPSC cells of the present invention;

[0106] B. amplifying and culturing the CAR-iPSC in a reaction container to obtain CAR-iPSC cell aggregates;

[0107] C. Add a cytokine combination into a reaction container, and perform differentiation culture on the CAR-iPSC cell mass to obtain CAR-NK cells.

[0108] In a preferred embodiment, iPSC is first transfected or gene-edited using a vector encoding a CAR molecule; then, the ideal CAR-iPSC monoclone is screened by comprehensive means such as genome sequencing and flow cytometry of CAR molecule expression levels; finally, the CAR-iPSC is fed into a bioreactor for 3D culture to form a spherical iPSC mass. By adding a specific cytokine composition, iPSC is induced to undergo directed differentiation through the following cell fate conversion: from iPSC to HSC (hematopoietic stem cell), then to lymphoid progenitor cells, and finally iPSC-CAR-NK cells characterized by CD3-CD56+ are obtained.

[0109] Therapeutic applications

[0110] The present invention also provides therapeutic applications of CAR-NK cells prepared by the method of the present invention, including use in treating tumors or cancers.

[0111] The present invention provides a method for treating a tumor, comprising administering a therapeutically effective amount of the CAR-iNK cells of the present invention to a subject in need thereof. In the present invention, the types of tumors include: solid tumors, hematological tumors, or a combination thereof.

[0112] The CAR-iNK cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0113] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.

[0114] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibitory effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that a pharmaceutical composition comprising the CAR-iNK cells described herein can be administered in an amount of 10 4 Up to 10 10The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6 The pharmaceutical compositions of the present invention may also be administered multiple times at these doses. Cells may be administered using well-known infusion techniques (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988) in immunotherapy. The optimal dose and treatment regimen for a specific patient may be easily determined by a medical technician by monitoring the patient's disease signs and thus adjusting the treatment.

[0115] The administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the pharmaceutical composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the pharmaceutical composition of the present invention is preferably administered by intravenous injection. The pharmaceutical composition of the present invention can be directly injected into a tumor, lymph node or infection site.

[0116] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding iNK cells to therapeutic levels are administered to patients in combination with any number of related treatment modalities (e.g., before, simultaneously, or after). In a further embodiment, the CAR-iNK cells of the present invention may be used in combination with: chemotherapy, radiation, immunosuppressants, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the present invention is used in combination with bone marrow transplantation, the use of chemotherapeutic agents, etc., for example, in some embodiments, after transplantation, the subject receives an injection of expanded immune cells of the present invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0117] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10 The CAR-iNK cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0118] The main advantages of the present invention include:

[0119] (1) The CAR molecule of the present invention can be stably expressed at a high level in iPSC cells, and iPSC clones with high CAR expression rate can be obtained, which can then be expanded and differentiated to obtain high-yield CAR-iNK cells;

[0120] (2) The expression of the CAR molecule of the present invention will not affect the differentiation and expansion process from iPSC to iNK cells and the inherent natural immune ability of iNK cells themselves;

[0121] (3) The CAR molecular structure of the present invention can maintain sufficient expression levels in the iNK stage and has good antigen recognition and killing functions;

[0122] (4) The CAR molecular structure of the present invention can be adapted to a variety of different scFvs and target different targets, and has application prospects in the field of tumor treatment.

[0123] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0124] Example 1: Process for preparing iPSC-CAR-NK

[0125] In the present invention, the schematic diagram of the process for preparing CAR-NK is as follows Figure 1As shown. First, iPSC is transfected or gene-edited using a vector encoding a CAR molecule; then, CAR-iPSCs that stably express CAR molecules are obtained through screening, and finally, CAR-iPSCs are fed into a bioreactor for 3D culture to form spherical iPSC clumps. By adding a specific cytokine composition, iPSCs are induced to undergo directed differentiation through the following cell fate conversion: from iPSC to HSC (hematopoietic stem cells), then to lymphoid progenitor cells, and finally iPSC-CAR-NK cells characterized by CD3-CD56+ are obtained. In short, cytokines SCF, VEGF, and BMP4 are added to the basal culture medium to induce iPSC cells to differentiate into hematopoietic stem cells and complete the differentiation of lymphoid progenitor cells. Then, a combination of cytokines IL3, IL7, IL15, SCF, and FLT3L are used to differentiate NK cells. The basal culture medium formula refers to the ingredients in Dan S. Kaufman's differentiation process (Hematopoietic and Nature Killer Cell Development from Human Pluripotent Stem Cells, 2013). The differentiation process lasted for 28 days, and then the iNK cells were expanded using expansion medium, and the expansion medium was OptiVitro NK cell expansion basic kit NE01 (NE000-N061, Yikesai). According to the experimental requirements, the iNK cells that have been expanded for an appropriate number of days were taken for functional experiments.

[0126] Example 2: Construction of anti-CD19 CAR structure

[0127] The scFv of the CD19-targeting antibody FMC63 was used to construct the CAR3 of the present invention and the control CAR (CAR1, CAR4, CAR6, CAR7 and CAR10). The element composition of each group of CAR molecules is shown in Table 1, wherein Hinge is the hinge region, TM is the transmembrane region, and ICD is the intracellular domain.

[0128] Table 1 Construction of CAR of the present invention and control CAR

[0129]

[0130] CAR3 is a structure containing the transmembrane domain and intracellular domain of 2B4 protein constructed by the present invention; the others are control CAR structures, among which CAR1 is a structure from Cappell, KM, Kochenderfer, JN Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol 20, 359-371 (2023); CAR4, CAR6, and CAR7 are structures derived from Dan S. Kaufman. Cell Stem Cell. 2018 A μg 2; 23 (2): 181-192.e5.doi: 10.1016 / j.stem.2018.06.002; CAR10 is one of the commonly used structures summarized in J Hematol Oncol. 2021 May 1; 14 (1): 73.doi: 10.1186 / s13045-021-01083-5.

[0131] The sequences of the elements of the CAR molecule are shown in Table 2.

[0132] Table 2 Amino acid sequence of CAR element

[0133]

[0134]

[0135] Example 3: Expression and differentiation of each group of anti-CD19-CAR on iPSCs

[0136] The structures of CAR1, CAR3, CAR4, CAR6, CAR7 and CAR10 were knocked into iPSC by electroporation, and stable CAR-iPSC cells were obtained after screening, and the expression level of each group of CAR on iPSC was detected. The experimental method is as follows:

[0137] (1) RNP system electroporation

[0138] iPSCs were digested with StemProAccutase cell dissociation reagent (Gibco, A1110501), centrifuged to remove the supernatant, washed once with PBS, counted, and 1×10 6The cells were electroporated. The RNP system was used for electroporation. The RNP included sgRNA and Cas9 protein. sgRNA (5'-GGGGCCACTAGGGACAGGAT-3', SEQ ID NO: 24) and Cas9 (IDT, 1081061) were mixed at a molar ratio of 2:1. 100μl electroporation system requires 250pmol of sgRNA and 125pmol of Cas9 protein. The two were mixed evenly and allowed to stand at room temperature for 15min. The P3 primary cell 4D nuclear transfer kit (Lonza, V4XP-3032) was used for electroporation. The Lonza 4D nuclear transfer electroporator was used for electroporation according to the instructions. After electroporation, the cells were transferred to a culture dish, which was coated with Laminin iMatrix-511 (Amsbio, 892012). The culture medium was added with clone R2 (Stemcell, 100-0691) at a ratio of 1:10, and the cells were cultured in a 37°C incubator.

[0139] (2) Design the plasmid structure of SA-P2A-puro-promoter-anti-CD19-CAR, knock the element into the AAVS1 site by electroporation, use the AAVS1 endogenous promoter to drive the expression of puro, and use the exogenous promoter to drive the expression of anti-CD19-CAR. The cells with the element knocked in have the puro resistance gene. After electroporation, the cells are screened with puro to obtain anti-CD19-CAR knocked-in cells, and a stable knocked-in cell line can be obtained without the need for monoclonal selection.

[0140] (3) Flow cytometry was used to detect the positive expression rate of CD19-CAR in each group on iPSC. The expression level of CD19-CAR in each group on iPSC was as follows: Figure 2 As shown, the expression level of CAR molecules is characterized by MFI, and the horizontal axis represents the expression amount of CAR molecules. The results show that the MFI value of CAR3 in iPSC after electroporation is 4191. The corresponding MFI values ​​of the control CAR1, CAR4, CAR6, CAR7, and CAR10 are 197, 414, 292, 252, and 178, respectively. The results after normalization with the results of CAR1 are shown in Table 1. Compared with the classic CAR1 structure, the expression level of CAR3 is 21.3 times that of CAR1, CAR4 is 2.1 times, CAR6 is 1.5 times, CAR7 is 1.3 times, and CAR10 is 0.9 times.

[0141] The above data show that CAR3 has the highest expression level in iPSC after electroporation, which is at least 10 times higher than the control CAR and up to 23 times higher. This shows that the expression level of the CAR3 structure in iPSC can achieve high-level stable expression. In addition, compared with CAR4, CAR6 has an increased DAP10 intracellular region, but the expression level of CAR in iPSC is reduced.

[0142] Will Figure 2 The 6 gene-edited iPSCs expressing different structural CAR molecules were differentiated to obtain the corresponding CAR-iNK cells, and then functional experiments were performed. iPSC differentiation and expansion refer to Example 1 and Figure 1 The iNK cells that have been expanded for an appropriate number of days were used to perform a killing experiment on Nalm-6 cells. The method is as follows:

[0143] The target cells and effector cells were counted and resuspended in R10 medium respectively, and the target cells were cultured at the specified ratio and number (the effector-target ratio was 1:1, and the number of target cells was 2×10 4 ), uniformly placed in a 96U bottom plate, centrifuged at 300g for 5min, and cultured in a 37°C incubator for 5hr. Take out the cultured cells, remove the supernatant, wash the remaining cells once with PBS, resuspend in 100μl PBS, add 50μl One-glo (Promega, E6120), mix well by pipetting, culture at room temperature for 10 minutes, transfer all supernatants to a white plate, and use the chemiluminescence method for reading, and calculate the killing ability of each NK cell based on the reading of target cells not co-cultured with effector cells.

[0144] The results showed that under the condition of effector-target ratio E:T=1:1, the killing efficiency of iNK expressing CAR3 reached 80%, while the killing efficiency of iNK expressing CAR10 was less than 20%. Since the number of iNK cells differentiated from iPSC cells expressing CAR4, CAR6, and CAR7 was very small, the killing efficiency data could not be obtained.

[0145] The above results show that the CAR3 structure can not only be expressed at a high level on iPSC cells, but also will not affect the differentiation of CAR-iPSC to CAR-iNK cells, and the differentiated CAR-iNK cells have a high level of killing ability, indicating that the CAR3 structure will not affect the differentiation and expansion process from iPSC to iNK cells. Therefore, the CAR3 of the present invention is suitable for expression on iPSC to obtain CAR-iNK cells.

[0146] Example 4: Monoclonal screening and detection of each group of anti-CD19-CAR after site-directed knock-in on iPSCs

[0147] The expression levels of the CAR of the present invention and the control CAR on iPSC monoclonal clones were detected by using a method of monoclonal screening after site-specific knock-in. The structures of CAR1 and CAR3 were electroporated in iPSC, site-specific knock-in was performed in the genome, and after culture and screening, monoclonal selection and genotype detection were performed, and the expression level of the CAR molecule on iPSC monoclonal clones was detected. The specific method is as follows:

[0148] (1) RNP system electroporation

[0149] iPSCs were digested with StemProAccutase cell dissociation reagent (Gibco, A1110501), centrifuged to remove the supernatant, washed once with PBS, counted, and 1×10 6 The cells were electroporated. The RNP system was used for electroporation. The RNP included sgRNA and Cas9 protein. sgRNA (5'-GGGGCCACTAGGGACAGGAT-3', SEQ ID NO: 24) and Cas9 (IDT, 1081061) were mixed at a molar ratio of 2:1. 100μl electroporation system requires 250pmol of sgRNA and 125pmol of Cas9 protein. The two were mixed evenly and allowed to stand at room temperature for 15min. 2ug of plasmids expressing CAR1 and CAR3 structures were taken respectively, and electroporated using the P3 primary cell 4D nuclear transfer kit (Lonza, V4XP-3032). The Lonza 4D nuclear transfer electroporator was used for electroporation according to the instructions. After electroporation, the cells were transferred to a culture dish, which was coated with Laminin iMatrix-511 (Amsbio, 892012). The culture medium was added with clone R2 (Stemcell, 100-0691) at a ratio of 1:10, and the cells were cultured in a 37°C incubator.

[0150] (2) iPSC single clone selection

[0151] After 10 days of culture, the cells electroporated with CAR1 and CAR3 structures in step (1) were plated for monoclonal plating. After digestion and editing, iPSCs were stained with FMC63 antibodies (Bioswan, 200107), and positive cells were plated into 96-well plates using Namocell Single Cell Dispenser, with only one cell in each well. Culture was continued for 14 days, and monoclonal clones were selected after growth. Monoclones with good growth were selected, washed with PBS, and digested with 50 μl Accutase digestion solution. After digestion, the mixture was evenly blown with a pipette tip, 40 μl of which was transferred to a 12-well plate containing culture medium for culture, and 5 μl was transferred to a PCR tube for genotyping.

[0152] (3) iPSC genotype identification

[0153] The 5μl sample taken during the single clone selection was used for crude genome extraction using the PrepGEM DNA extraction kit, the kit number is MicroGEM-PUN0500. The 20μl reaction system contains 5μl sample, 2μl 10X blue buffer, 0.4μl PrepGEM enzyme, and 12.6μl water. After the above reaction system is mixed evenly, it is placed in a PCR instrument and incubated at 75 degrees for 10 minutes and 95 degrees for 2 minutes. After the incubation, it can be used as a template for PCR experiments. Take 2μl as a template, add DNA amplification enzymes and primers, etc. for PCR experiments to identify the genotype of the single clone.

[0154] The knock-in efficiency and expression level of CAR molecules were detected by flow cytometry. The results are as follows: Figure 3 As shown in the figure, it can be seen that CAR3 has the highest editing efficiency in iPSC, reaching 20.72%, while the editing efficiency of CAR1 is only 2.86%. The editing efficiency of CAR3 is 7.24 times that of CAR1.

[0155] Consistent with the results of Example 2, the CAR3 structure was able to achieve the highest level of editing efficiency and stable expression in iPSCs.

[0156] After knocking in the CAR1 and CAR3 genes, iPSCs were selected and tested for single clones. The expression level of CAR in iPSC single clones is shown in Figure 2. Figure 4 As shown. The expression level of CAR molecules characterized by MFI showed that in the three monoclones (sc5, sc19 and sc20) containing the CAR3 structure of the present invention, the MFI values ​​were 3416, 3941 and 3619, respectively, with an average value of 3658.66; while in the monoclone of the control CAR1, the average value of the MFI value was only 384.33. Compared with CAR1, the expression amount of CAR3 of the present invention on iPSC has achieved an order of magnitude increase. It shows that the CAR3 structure can obtain a higher level of stable expression in iPSC.

[0157] Example 5: Efficiency of each group of anti-CD19 CAR directly expressed on iNK

[0158] Six different CAR molecules were cloned into retroviral vectors, and the viruses were used to infect iNK cells to obtain groups of CAR-iNK cells, and the CAR positive rate of the cells was detected. The experimental method is as follows:

[0159] Vector construction and virus packaging: The synthesized sequence was directly replaced on the original target vector pMSCV-1928z (CAR1) to construct CAR3, CAR4, CAR6, CAR7 and CAR10, and retroviral packaging was performed separately. The process was as follows: 293T cells with good growth status were digested and inoculated in a 10 cm culture dish (6×10 6 / dish), culture overnight. The next day: Check to ensure that the 293T in the culture dish has reached 70-80% confluence. Prepare the following transfection complex: Tube A: 500μl Opti-MEM + 10μg gag-pol + 8μg BaEVTR + 20μg target vector (10 vectors including pMSCV), mix with a pipette. Tube B: 500μl Opti-MEM + 40μl PEIPro, mix with a pipette. Transfer the solution in tube A to tube B, mix with a pipette, and let stand at room temperature for 15-20 minutes. Transfer the DNA-PEIPro mixture to a 10cm culture dish and shake gently. Culture in a 37°C incubator. After 6 hours, remove the culture supernatant, wash with PBS, and then add 10ml of complete culture medium again.

[0160] Virus concentration: Collect 48hr or 72hr virus liquid from 293T culture dish into centrifuge tube; centrifuge at 4℃, 500g for 5 minutes, transfer the supernatant to a new centrifuge tube; filter the virus liquid into a new centrifuge tube using a 0.45um filter and syringe; add concentrate with 1 / 3 volume of virus liquid and mix with a pipette; after standing at 4℃ for 1hr, centrifuge at 4℃, 1500g for 45 minutes, remove the supernatant and retain the precipitate at the bottom of the centrifuge tube; add virus freezing solution with 1 / 100 volume of the original virus liquid to resuspend the precipitate at the bottom.

[0161] After the packaged retrovirus was frozen for 48 hours, it was serially diluted and infected with K562 cells for titer detection. The infection conditions were as follows: 3×10 4 K562 cells were resuspended in 100 μl of fresh culture medium, and 100 μl of concentrated retrovirus and polybrene (final concentration 5 μg / ml) were added. The cells were centrifuged at 800 g at 32°C for 30 minutes, and the culture dish was placed in a 37°C, 5% CO2 constant temperature incubator for continued culture. On the third day after infection, the K562 CAR cell positive ratio (Anti-Mouse FMC63 scFv MonoclonalAntibody) was detected by flow cytometry, and the virus titer was calculated based on the percentage of positive cells and the dilution multiple for subsequent infection of iNK cells.

[0162] iNK virus infection: First dilute Retronectin (Takara, Cat#T100B) with PBS to a final concentration of 15μg / ml, add 250μl / well of Retronectin working solution with a final concentration of 15μg / ml to the suspended 24-well cell culture plate, and place the 24-well plate at 4°C overnight. Discard the Retronectin coating solution in the 24-well plate, add 500μl of PBS blocking solution containing 2% BSA and block at room temperature for 30 minutes. Discard the blocking solution and wash the plate twice with PBS containing 2.5% HEPES. Add the required virus volume of Retrovirus melted at room temperature to the wells at an MOI of 10, and make up the volume to 1ml with PBS containing 2.5% HEPES, then centrifuge at 32°C, 2000g for 2 hours. When the centrifugation is about to be completed, mix the NK cells activated for 3 days and count them. Take the required amount of cells at MOI = 10, centrifuge at 500g for 5 minutes at room temperature, discard the supernatant and resuspend in NK complete medium to a cell density of 5×10 5 / ml. Take out the centrifuged 24-well plate, discard the virus solution, add the NK cells after adjusting the density to the 24-well plate, 1ml / well, and centrifuge at 32°C, 1000g for 10 minutes. The centrifuged 24-well plate was cultured in a 37°C, 5% CO2 incubator. After 3 days, the NK cells were counted and the CAR positive rate was tested. The remaining cells were transferred to a 6-well plate for expansion culture, and the cell density was maintained at 1×10 6 -2×10 6 / ml.

[0163] CAR positive rate test results are as follows Figure 5 And as shown in Table 3.

[0164] Table 3 CAR expression rate in each group

[0165] serial number Expression efficiency CAR1 12.17％ CAR3 48.68％ CAR4 4.14％ CAR6 2.88％ CAR7 2.08％ CAR10 1.91％

[0166] The results showed that the CAR-iNK cells containing the CAR3 structure had the highest CAR positive rate, reaching 48.68%, while the highest in the control CAR was only 12.17% and the lowest was 1.91%. The expression rate of the CAR3 structure was at least 4 times higher than that of the control CAR, and even more than 20 times higher, indicating that the CAR3 structure was unexpectedly suitable for expression on iNK cells.

[0167] In addition, the number of iNK cells successfully transfected with CAR6, CAR7, and CAR10 was small, indicating that these CAR molecules are not suitable for expression on iNK cells, while the CAR3 molecule has no significant effect on the number of iNK cells. The above results show that compared with other structures, the CAR3 structure of the present invention is a CAR molecular structure that is more suitable for expression on iNK.

[0168] Example 6: Cell killing ability of each group of anti-CD19 CAR-iNK

[0169] Luciferase was used to detect the killing ability of the six iNK cells containing CAR molecules obtained in Example 5 against K562 and Nalm-6. The experimental method was the same as that in Example 3.

[0170] The results are as follows Figure 6 The killing ability of iNK cells against K562 cells indicates the non-specific killing ability of iNK cells. Figure 6 The results on the left show that iNK cells expressing CAR molecules with different structures have similar nonspecific killing abilities against K562 cells, with no significant difference, indicating that the expression of CAR molecules with different structures does not affect the natural immune killing function of iNK cells. Nalm-6 cells are CD19 positive cells. In the killing experiment against Nalm-6, Figure 6 The results in the right figure show that under the killing conditions of the same effector-target ratio, the CAR3 structure exhibits the strongest specific killing ability.

[0171] The above results show that the CAR-iNK cells containing the CAR3 structure of the present invention have unaffected natural immune killing function and a specific killing ability significantly higher than that of the control CAR-iNK cells.

[0172] Example 7: Construction of anti-BCMA-CAR structure

[0173] Referring to the structure of anti-CD19 CAR3 in Example 2, the FMC63 scFv sequence was replaced with the scFv sequence of the anti-BCMA antibody to construct the structure of anti-BCMA-CAR3.

[0174] The anti-BCMACAR3 sequence is shown in SEQ ID NO: 20:

[0175] (SEQ ID NO:20).

[0176] Example 8: Expression of anti-BCMA-CAR in iPSCs and iNKs

[0177] The method mentioned in Example 3 was used to construct iPSC expressing anti-BCMA-CAR3. Figure 7 As shown in the left figure, the expression rate of BCMA-CAR3 molecules in iPSC is 93.78%. The above BCMA-CAR3-iPSC was differentiated and expanded using the method mentioned in Example 1 to obtain iNK cells expressing anti-BCMA-CAR3. Figure 7 As shown in the right figure, the expression rate of BCMA-CAR3 molecules on iNK is 91.53%, indicating that the CAR3 structure can support the high expression of anti-BCMA antibody scFv in the iPSC and iNK stages.

[0178] Example 9: Construction of anti-BCMA+CD19 dual CAR structure

[0179] The present invention also constructs a dual CAR structure targeting BCMA and CD19, and uses the scFv of the CD19-targeting antibody FMC63 and the scFv of the anti-BCMA antibody to construct the dual CAR (anti-BCMA+CD19dual CAR3) and the control dual CAR (anti-BCMA+CD19 dual CAR1) of the present invention. The antigen binding domain of the dual CAR is composed of the scFv of FMC63 and the scFv of the anti-BCMA antibody in series, and the composition of the other components is shown in Table 4, wherein Hinge is the hinge region, TM is the transmembrane region, and ICD is the intracellular domain.

[0180] Table 4 Construction of anti-BCMA+CD19 dual CAR structure

[0181]

[0182] The sequence of the anti-BCMA scFv is shown in SEQ ID NO: 21:

[0183] DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEG STKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS(SEQ ID NO:21).

[0184] The sequence of anti-BCMA+CD19 dual CAR1 is shown in SEQ ID NO:22, and the sequence of anti-BCMA+CD19dual CAR3 is shown in SEQ ID NO:23.

[0185] Two anti-BCMA+CD19 dual CAR molecules with different structures, CAR1 and CAR3, were cloned into a retroviral vector, and the virus was used to infect iNK cells to detect the expression positive rate. The experimental method was the same as that in Example 5.

[0186] The results of dual CAR expression rate detection are as follows Figure 8 The results showed that the expression level of anti-BCMA+CD19 dual CAR3 was higher than that of anti-BCMA+CD19 dual CAR 1 structure, indicating that the CAR3 structure can adapt to a variety of scFvs and can be expressed at high levels on iNK.

[0187] Example 10: Cell killing ability of iNK cells expressing BCMA+CD19 dual CAR

[0188] Using the iNK cells containing anti-BCMA+CD19 dual CAR1 and anti-BCMA+CD19 dual CAR3 structures constructed in Example 9, functional experiments were performed using RPMI8226 cells as target cells. The experimental method was the same as in Example 3.

[0189] The results are as follows Fig. 9 The results showed that under the condition of effector-target ratio of 3:1, the iNK with CAR3 structure had stronger cytotoxicity, indicating that the CAR3 structure had stronger killing ability on target cells.

[0190] discuss

[0191] The low transduction efficiency of CAR molecules on NK cells is an urgent problem to be solved in the field of engineered NK cell therapy. The present invention constructs a CAR molecular structure CAR3 containing the full length of the transmembrane region and intracellular region of the 2B4 protein. Compared with the CAR molecular structure commonly used in autologous CAR-T or CAR-NK cells, the CAR3 structure of the present invention can be efficiently expressed on iNK or iPSC, and is a CAR molecular structure more suitable for iPSC-CAR-iNK.

[0192] First, the CAR3 molecule is suitable for transduction into iNK cells via retrovirus, and its expression rate on iNK cells is several to dozens of times higher than that of the control CAR. Secondly, the CAR3 molecule is suitable for transduction into iPSC cells via gene editing, and its expression rate on iPSC is also significantly higher than that of the control CAR. Moreover, the transduction of the CAR3 molecule does not affect the process of iPSC cells differentiating into iNK cells. Therefore, CRISPR / Cas9 can be used to edit CAR into the iPSC genome, and then the stable CAR-iPSC can be differentiated into CAR-iNK cells.

[0193] In addition, the CAR3 structure of the present invention can not only be adapted to the scFv of the FMC63 monoclonal antibody, but also to a variety of other forms of scFv, such as multiple scFvs in series, and the constructed CAR molecules have advantages in expression level and killing function.

[0194] CAR molecule sequence

[0195] Anti-CD19 CAR1 (SEQ ID NO: 14)

[0196] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0197] Anti-CD19 CAR3 (SEQ ID NO:15)

[0198] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0199] Anti-CD19 CAR4 (SEQ ID NO:16)

[0200] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPFFFCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0201] Anti-CD19 CAR6 (SEQ ID NO:17)

[0202] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPFFFCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0203] Anti-CD19 CAR7 (SEQ ID NO:18)

[0204] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPFFFCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0205] Anti-CD19 CAR10 (SEQ ID NO:19)

[0206] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0207] Anti - BCMACAR3 (SEQ ID NO:20)

[0208] MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0209] Anti-BCMA + CD19 dual CAR1 (SEQ ID NO: 22)

[0210] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0211] Anti-BCMA + CD19 dual CAR3 (SEQ ID NO:23)

[0212] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA TYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDF RGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKG EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY AMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIY EDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0213] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An engineered iPSC cell, characterized in that: An exogenous expression vector is introduced into the engineered iPSC cells, wherein the exogenous expression vector comprises a gene encoding a chimeric antigen receptor (CAR); The CAR comprises a structure as shown in Formula I: L-EB-H-TM-ICD1-ICD2(I) In the formula, Each "-" is independently a connecting peptide or a peptide bond; L is none or a signal peptide sequence; EB is the extracellular binding domain; H is the hinge region; TM is the transmembrane region from the 2B4 protein; ICD1 is derived from the intracellular region of the 2B4 protein; ICD2 is a cytoplasmic signaling sequence derived from CD3ζ.

2. The engineered iPSC cell according to claim 1, characterized in that The EB is a scFv targeting one or more target proteins, and the target proteins include: CD19, BCMA, or a combination thereof.

3. The engineered iPSC cell according to claim 1, characterized in that: The H is the hinge region derived from CD8.

4. The engineered iPSC cell according to claim 1, characterized in that The CAR has an amino acid sequence as shown in any one of SEQ ID NO: 15, 20 or 23.

5. The engineered iPSC cell according to claim 1, characterized in that The introduction includes the steps of: using the CRISPR / Cas9 system to knock the CAR encoding gene into the AAVS1 site.

6. The engineered iPSC cell according to claim 1, characterized in that The CAR expression level on the engineered iPSC cells was detected by flow cytometry, and the mean fluorescence intensity (MFI) value was recorded as R1, R1≥2000.

7. A method for preparing CAR-NK cells, characterized in that: The method comprises the steps of: performing differentiation culture on the engineered iPSC cells according to claim 1 to obtain CAR-NK cells.

8. A CAR-NK cell, characterized in that: The CAR-NK cells are obtained by differentiation of the engineered iPSC cells described in claim 1, or are prepared by the method described in claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (a) the CAR-NK cell according to claim 8; and (b) a pharmaceutically acceptable carrier.

10. A use of the CAR-NK cell according to claim 8 or the pharmaceutical composition according to claim 9, characterized in that: Used for preparing medicines or preparations for preventing and / or treating tumors.

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