Chimeric antigen receptor for iPSC-nk cells and uses thereof

Abstract

The present application relates to a kind of chimeric antigen receptors for iPSC-NK cell and its application. Specifically, the present application provides the CAR structure comprising the transmembrane region and intracellular region from 2B4 protein, which can be efficiently expressed on iPSC cell, and then differentiated into iNK cell containing CAR structure. The CAR structure of the present application does not affect the process of iPSC differentiation and expansion into NK cell, and the engineered NK cell containing the CAR structure of the present application has enhanced cell killing ability, and can be adapted with a variety of different scFv, target different targets, and has application prospect in the field of disease treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell biology, in particular to a chimeric antigen receptor for iPSC-NK cells and application thereof. BACKGROUND

[0002] Chimeric antigen receptor (CAR) is a common method to produce tumor target specific immune cells by genetic modification of cells. CAR has been introduced into different types of immune cells, including T cells, NK cells and macrophages, and 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 cells is an effective modification scheme, however, due to the characteristics of NK cells, they have natural resistance to the traditional method of transduction by retroviral vectors, resulting in very low transduction efficiency. In addition to direct transduction to NK cells, CAR genes can also be edited into iPSC genomes by CRISPR / Cas9 means, and then 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 the edited correct iPSC clone; (2) the expression of the CAR molecule will not affect the differentiation and expansion process from iPSC to iNK cell, and the inherent natural immune ability of iNK cell itself; (3) the CAR structure can maintain sufficient expression level and has good antigen recognition and killing function in iNK stage; (4) the CAR molecular structure can be adapted to a variety of different scFv. At present, there is a lack of CAR structure that can meet the above conditions at the same time in the art.

[0005] Therefore, there is an urgent need in the art to develop a CAR structure suitable for iPSC and iPSC derived iNK cells. SUMMARY

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

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

[0008] The CAR contains a structure as shown in formula I:

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

[0010] wherein,

[0011] each “-” is independently a linking peptide or a peptide bond;

[0012] L is nothing or a signal peptide sequence;

[0013] EB is an extracellular binding domain;

[0014] H is a hinge region;

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

[0016] ICD1 is an intracellular region from 2B4 protein;

[0017] ICD2 is a cytoplasmic signaling sequence derived from CD3 zeta.

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

[0019] In another preferred embodiment, the sequence of L is set forth 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 proteins include 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 proteins include CD19, BCMA, or a combination thereof.

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

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

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

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

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

[0028] In another preferred embodiment, the H is a hinge region derived from CD8, having an amino acid sequence as set forth in SEQ ID NO: 4.

[0029] In another preferred embodiment, the H is a hinge region derived from CD28, having an amino acid sequence as set forth in SEQ ID NO: 3.

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

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

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

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

[0034] In another preferred embodiment, the exogenous expression vector is selected from the group consisting of 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 introducing comprises: gene editing introduction, electroporation, viral transduction, or a combination thereof.

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

[0038] In another preferred embodiment, the introducing comprises the step of: knocking in the CAR-encoding gene into an AAVS1 site using a CRISPR / Cas9 system.

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

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

[0041] In a second aspect of the present application, a method for preparing a CAR-NK cell is provided, comprising the step of: culturing the engineered iPSC cell of the first aspect of the present application to obtain the CAR-NK cell.

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

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

[0044] B. adding a cytokine combination to the reaction vessel to induce the CAR-iPSC cell mass to obtain the CAR-NK cell.

[0045] In another preferred embodiment, the culturing for 7-14 days.

[0046] In another preferred embodiment, the culturing for 26-30 days, for example, 28 days.

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

[0048] In a third aspect of the present application, a CAR-NK cell is provided, which is obtained by differentiating the engineered iPSC cell of the first aspect of the present application or prepared by the method of the second aspect of the present application.

[0049] In a fourth aspect of the present application, a pharmaceutical composition is provided, which comprises:

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

[0051] (b) a pharmaceutically acceptable carrier.

[0052] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of an injection, a lyophilized agent.

[0053] In another preferred embodiment, the pharmaceutical composition comprises 0.01-99.99% of the engineered cell of the first aspect of the present application; and 0.01-99.99% of a pharmaceutically acceptable carrier; the percentage is the mass percentage of the pharmaceutical composition.

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

[0055] In a fifth aspect of the present application, there is provided a use of the CAR-NK cell of the third aspect of the present application or the pharmaceutical composition of the fourth aspect of the present application, for the manufacture of a medicament or a preparation for preventing and / or treating a tumor.

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

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

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

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

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

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

[0062] It should be understood that, within the scope of the present application, all combinations between the above technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 A technical flow chart showing the editing of iPSC and the differentiation to obtain iPSC-CAR-NK is shown.

[0064] Figure 2 Expression levels of anti-CD19 CAR molecules in each group on iPSC after gene editing and screening are shown.

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

[0066] Figure 4The results of detection of expression levels of each group of anti-CD19 CAR on iPSC monoclonal 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 each group of iNK expressing CD19 CAR on K562 or Nalm-6 is shown.

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

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

[0071] Figure 9 The experimental results of the killing ability of iNK cells expressing two BCMA+CD19 dual CAR respectively on RPMI8226 cell lines are shown. DETAILED DESCRIPTION

[0072] Through extensive and in-depth research, and through a large number of screenings, the present inventors provide a CAR molecular structure particularly suitable for expression on iPSC, which contains a unique transmembrane region and intracellular region from 2B4 protein, and can be combined with scFv targeting different antigens. Compared with the commonly used CAR structure, the CAR with the specific structure of the present application has unexpectedly high expression rate on iPSC cells, with a several-fold to several tens of fold increase in the enhancement. In addition, the expression of the CAR of the present application on iPSC does not affect the ability to differentiate into iNK cells, so that CAR-iNK cells differentiated from CAR-iPSC can be obtained with high efficiency. The engineered NK cells containing the CAR structure of the present application have enhanced cell killing ability, and the performance is significantly improved compared with the commonly used control CAR structure. On this basis, the present application is completed.

[0073] CAR of the present application

[0074] In the present application, a CAR molecular 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 can be efficiently expressed on iPSC cells, and then differentiated into iNK cells containing CAR structure.

[0075] Chimeric antigen receptor CAR is generally composed of three main regions, i.e. extracellular region, transmembrane region and intracellular region.

[0076] The extracellular region is mainly an antigen binding domain, which is used for recognition and binding with antigens on the surface of target cells, and is usually a single-chain variable fragment (scFv) of an antibody. The extracellular region can also optionally include a hinge region, which functions as a connection and enhances the flexibility and stability of the structure. The transmembrane region connects the intracellular and extracellular regions of the CAR, and commonly used transmembrane regions include the transmembrane regions of CD8 and CD28 proteins, but are not limited thereto. The intracellular region includes a signaling domain and a costimulatory molecule, and this region is responsible for signal transmission to further improve the signal transmission capacity of a specific CAR and to make the immune cells have stronger killing ability and survival time.

[0077] The CAR of the present application has an extracellular binding domain, a hinge region, a transmembrane region, an intracellular region, and a signaling sequence, and preferably, the CAR of the present application 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 nothing or a signal peptide sequence;

[0082] EB is an extracellular binding domain;

[0083] H is a hinge region;

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

[0085] ICD1 is an intracellular region from a 2B4 protein;

[0086] ICD2 is a cytoplasmic signaling sequence derived from CD3 zeta.

[0087] In an immature CAR molecule, the function of the signal peptide is mainly to guide the CAR protein to correctly enter the endoplasmic reticulum of the cell, so as to carry out subsequent folding, modification and transport. After the CAR is successfully positioned on the cell membrane, the signal peptide is usually cut off, so that the mature CAR molecule usually does not contain a signal peptide. In the CAR molecule of the present application, the type of signal peptide is not particularly limited. In an embodiment of the present application, the sequence of the selected signal peptide is shown in SEQ ID NO: 1.

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

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

[0090] In an embodiment of the present application, the CAR is a CAR targeting CD19, which has an amino acid sequence shown as SEQ ID NO: 15. In an embodiment of the present application, the CAR is a CAR targeting BCMA, which has an amino acid sequence shown as SEQ ID NO: 20. In an embodiment of the present application, the CAR is a dual-CAR targeting CD19 and BCMA simultaneously, which has a sequence shown as SEQ ID NO: 23.

[0091] The CAR molecule of the present application can be expressed at a high level on iNK cells or iPSC cells, and does not affect the differentiation of iPSCs into iNK cells, nor does it affect the innate immune ability 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 pathogenic infections in the human body. Because NK cells play a key role in the body's non-specific anti-tumor response, a large number of basic and clinical studies have applied NK cells to the treatment of hematopoietic system malignancies (leukemia, lymphoma and multiple myeloma, etc.) and solid tumors (such as melanoma, ovarian cancer and lung cancer, etc.). The research and preparation ideas of CAR-NK cells are similar to those of CAR-T cells. CAR-NK cells are prepared by gene editing to express CAR, and then CAR-NK cells with specific anti-tumor ability are infused into tumor patients to play an anti-tumor role, 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 optimization space. For example, CAR structure design, CAR gene transduction method and transduction efficiency are all key factors. The traditional method of CAR gene delivery is to use viral vectors to insert into the genome in the form of random integration, which has potential safety risks.

[0095] CAR-iNK cells of the present application

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

[0097] iPSC (induced pluripotent stem cell) is a cell type with embryonic stem cell-like properties, which was first reported by the Yamanka laboratory at 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 stem cells with pluripotency. These transcription factors include Oct3 / 4, Sox2, Klf4 and c-Myc, which work together to reset the cell's gene expression pattern, returning it to a state similar to that of embryonic stem cells.

[0098] Under the culture conditions with the addition of special cytokines, iPSCs can differentiate into NK cells, i.e. the iNK cells of the present application. Specifically, iPSCs can aggregate together to form embryoid bodies (EBs) under the culture conditions with the addition of special cytokines. The EBs gradually grow larger and differentiate hematopoietic stem cells. After about 28 days of culture, the EBs can differentiate CD56-positive NK cells, which can be further activated and expanded. Ultimately, 1 starting iPSC cell can differentiate 1000-10000 iNK cells.

[0099] The current clinical treatment of NK cells is mainly derived from human tissues such as umbilical cord blood and peripheral blood. These naturally derived NK cells have large heterogeneity, high preparation cost, low yield, and preparation cycle limited by the availability of human tissues. In the present application, the iPSC-derived iNK cells can solve the above problems.

[0100] In the present application, the CAR-containing iNK cells can be obtained by the following two ways: one is to introduce CAR into iPSC to obtain CAR-iPSC, and then differentiate and culture to obtain CAR-iNK cells; the other is to directly introduce CAR into the iNK cells differentiated from iPSC.

[0101] In an embodiment of the present application, CAR is introduced into iPSC by CRISPR / Cas gene editing system, and correct edited iPSC clones are obtained, and then differentiated to form CAR-expressing iNK cells. In this process, the expression of CAR molecules in the present application does not affect the differentiation and expansion process of iPSC to iNK cells, and the inherent natural immune ability of iNK cells is not affected.

[0102] The CAR-iPSC differentiated CAR-iNK cells or directly introduced CAR-iNK cells of the present application have significantly improved cell-specific killing ability, for example, specific killing ability to tumor cells expressing specific antigens.

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

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

[0105] A. providing the CAR-iPSC cells of the present application;

[0106] B. culturing the CAR-iPSC in a reaction container for expansion to obtain a CAR-iPSC cell mass;

[0107] C. adding a cytokine combination to the CAR-iPSC cell mass in a reaction container for differentiation culture to obtain CAR-NK cells.

[0108] In one preferred embodiment, iPSCs are first transfected or genetically engineered using a vector encoding a CAR molecule; then the ideal CAR-iPSC monoclonal is obtained by screening through genome sequencing, CAR molecule expression level flow detection and other comprehensive means; finally, the CAR-iPSC is fed into a bioreactor for 3D culture to form a spherical iPSC mass. By adding a specific cytokine composition, the iPSCs are induced to undergo cell fate conversion for directional differentiation as follows: from iPSC to HSC (hematopoietic stem cell), to lymphoid progenitor cell, and finally to obtain CD3-CD56+characteristic iPSC-CAR-NK cell.

[0109] Therapeutic applications

[0110] The present application also provides therapeutic applications of the CAR-NK cells prepared by the methods of the present application, including for treating tumors or cancers.

[0111] The present application provides methods of treating tumors, comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-iNK cells of the present application. In the present application, the types of tumors include: solid tumors, blood tumors, or combinations thereof.

[0112] The CAR-iNK cells of the present application can be administered alone or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present application can include the target cell populations as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelators such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present application are preferably formulated for intravenous administration.

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

[0114] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present application to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis and condition of the patient (subject). It can generally be stated that: the pharmaceutical composition comprising the CAR-iNK cells described herein can be administered in an amount of 10 4 to 10 10at a dose of 1 x 105 5 up to 10 6 cells / kg body weight (including all integer values within those ranges). The pharmaceutical compositions of the present application can also be administered multiple times at these doses. The cells can be administered by using infusion techniques well known in the art of immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by monitoring the patient's signs of disease and adjusting the treatment accordingly by a person skilled in the medical arts.

[0115] Administration of the subject compositions can be carried out in any convenient way, including by spray, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the pharmaceutical compositions of the present application are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the pharmaceutical compositions of the present application are preferably administered by intravenous injection. The pharmaceutical compositions of the present application can be injected directly into a tumor, lymph node or site of infection.

[0116] In certain embodiments of the present application, the activated and expanded cells using the methods described herein or other methods known in the art to expand iNK cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with or subsequent to) any number of relevant therapeutic modalities. In further embodiments, the CAR-iNK cells of the present application can be used in conjunction with chemotherapy, radiation, immunosuppressive agents, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the present application are used in conjunction with bone marrow transplantation, with chemotherapeutic agents, etc., e.g., in some embodiments, following transplantation, the subject receives infusion of expanded immune cells of the present application. In an additional embodiment, the expanded cells are administered prior to or following a surgical procedure.

[0117] The dosage of the above therapies administered to a patient will vary with the precise attributes of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented in accordance with accepted practices in the art. Generally, 1 x 105 6 to 1 x 105 10 CAR-iNK cells of the present application can be administered to a patient by, e.g., intravenous infusion.

[0118] The primary advantages of the present application include:

[0119] (1) The CAR molecules of the present application can be stably expressed at high levels in iPSC cells, and high CAR-expressing iPSC clones can be obtained, which in turn can be expanded and differentiated to obtain high yields of CAR-iNK cells;

[0120] (2) The expression of the CAR molecule of the present application does not affect the differentiation and expansion process from iPSC to iNK cell and the natural immune ability inherent in iNK cell itself;

[0121] (3) The CAR molecule structure of the present application can maintain sufficient expression level and has good antigen recognition and killing function in iNK stage;

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

[0123] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are usually according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions suggested by the manufacturer. Unless otherwise specified, percentages and fractions are weight percentages and weight fractions.

[0124] Example 1: Preparation process of iPSC-CAR-NK

[0125] In the present application, the preparation process of CAR-NK is shown in the following figure Figure 1The iPSCs are first transfected or genetically edited with a vector encoding the CAR molecule; then the CAR-iPSCs stably expressing the CAR molecule are selected; finally the CAR-iPSCs are fed into a bioreactor for 3D culture to form spherical iPSC clusters. By adding specific cytokine compositions, the iPSCs are induced to undergo cell fate conversion for directed differentiation as follows: iPSCs to HSCs (hematopoietic stem cells), then to lymphoid progenitor cells, and finally to iPSC-CAR-NK cells characterized by CD3-CD56+. In short, by adding cytokines SCF, VEGF, and BMP4 to the basal medium, the iPSCs are induced to differentiate into hematopoietic stem cells and complete the differentiation of lymphoid progenitor cells. Then, the combination of cytokines IL3, IL7, IL15, SCF, and FLT3L is used for the differentiation of NK cells. The basal medium formula refers to the components in Dan S. Kaufman's differentiation process (Hematopoietic and Nature Killer Cell Development from Human Pluripotent Stem Cells, 2013). The differentiation process lasts for 28 days, and then the iNKs are expanded using an expansion medium, which is the OptiVitro NK Cell Expansion Basal Kit NE01 (NE000-N061, EkoCyte). According to experimental requirements, the iNKs at appropriate expansion days are taken for functional experiments.

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

[0127] The CAR3 of the present application and the control CARs (CAR1, CAR4, CAR6, CAR7, and CAR10) are constructed using the scFv of the antibody FMC63 targeting CD19, and the element composition of each group of CAR molecules is shown in Table 1, in which Hinge is the hinge region, TM is the transmembrane region, and ICD is the intracellular domain.

[0128] Table 1 Construction of CARs of the present application and control CARs

[0129]

[0130] CAR3 is the structure of the present application containing 2B4 protein transmembrane domain and intracellular domain; the others are control CAR structures, among which CAR1 is the structure from Cappell, K. M., Kochenderfer, J. N. Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol 20, 359-371 (2023); CAR4, CAR6, CAR7 are structures derived from Dan S. Kaufman. Cell Stem Cell. 2018 Aug 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 sequences of CAR elements

[0133]

[0134]

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

[0136] The structures of CAR1, CAR3, CAR4, CAR6, CAR7 and CAR10 were knocked in in iPSC by electroporation method, and after screening, stable CAR-iPSC cells were obtained, 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] iPSC was digested with StemPro Accutase cell dissociation reagent (Gibco, A1110501), centrifuged to remove supernatant, washed once with PBS, counted, and 1x10 6Cells were electroporated. The RNP system, consisting of sgRNA and Cas9 protein, was used. The sgRNA (5'-GGGGCCACTAGGGACAGGAT-3', SEQ ID NO:24) and Cas9 (IDT, 1081061) were mixed at a molar ratio of 2:1. 100 μl of the electroporation system required 250 pmol of sgRNA and 125 pmol of Cas9 protein. The mixture was thoroughly combined and incubated at room temperature for 15 min. Electroporation was then performed using the P3 primary cell 4D nuclear transfer kit (Lonza, V4XP-3032) following the manufacturer's instructions using the Lonza 4D nuclear transfer electroporator. After electroporation, cells were transferred to culture dishes coated with Laminin iMatrix-511 (Amsbio, 892012). The culture medium was prepared by adding clone R2 (Stemcell, 100-0691) at a ratio of 1:10. The cells were then cultured in an incubator at 37°C.

[0139] (2) The plasmid structure of SA-P2A-puro-promoter-anti-CD19-CAR was designed, and the element was knocked into the AAVS1 site by electroporation. The expression of puro was driven by the endogenous promoter of AAVS1, and the expression of anti-CD19-CAR was driven by the exogenous promoter. Cells with element knock-in possessed the puro resistance gene. Cells after electroporation were selected using puro to obtain anti-CD19-CAR knock-in cells. Stable knock-in cell lines could be obtained without the need for single-clone selection.

[0140] (3) The positive rate of CD19-CAR expression in iPSCs of each group was detected by flow cytometry. The expression levels of CD19-CAR in iPSCs of each group are shown below. Figure 2 As shown in the figure, the expression level of CAR molecules is characterized by MFI, with the horizontal axis representing the expression level of CAR molecules. The results show that the MFI value of CAR3 in iPSCs after electroporation is 4191. The MFI values ​​of the control CAR1, CAR4, CAR6, CAR7, and CAR10 are 197, 414, 292, 252, and 178, respectively. The results after normalization with 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 in iPSC after electroporation, at least 10 times higher than the control CAR, up to 23 times. It shows that the expression level of CAR3 structure in iPSC can obtain high level of stable expression. In addition, compared with CAR4, CAR6 increases the DAP10 intracellular region, but the expression level of CAR in iPSC decreases.

[0142] The iPSCs expressing CAR molecules with different structures after editing of 6 genes were differentiated, and the corresponding CAR-iNK cells were obtained, and then the function experiments were carried out. The iPSC differentiation and expansion are shown in Reference Example 1 and Figure 2 The iPSCs expressing CAR molecules with different structures after editing of 6 genes were differentiated, and the corresponding CAR-iNK cells were obtained, and then the function experiments were carried out. The iPSC differentiation and expansion are shown in Reference Example 1 and Figure 1 The iPSCs expressing CAR molecules with different structures after editing of 6 genes were differentiated, and the corresponding CAR-iNK cells were obtained, and then the function experiments were carried out. The iPSC differentiation and expansion are shown in Reference Example 1 and

[0143] The target cells and effector cells were counted and resuspended in R10 medium, and the specified ratio and number (effector to target ratio of 1:1, target cell number of 2x10 4 ), and placed in a 96U bottom plate. After centrifugation at 300g for 5min, the cells were cultured in a 37℃ incubator for 5hr. After removing the supernatant, the remaining cells were washed once with PBS, resuspended in 100μl of PBS, and then 50μl of One-glo (Promega, E6120) was added. After mixing, all the supernatant was transferred to a white plate and read by chemiluminescence method. According to the read value of the target cells without co-culturing with effector cells, the killing ability of each NK cell was calculated.

[0144] The results show that under the condition of effector to target ratio E:T=1:1, the killing efficiency of iNK expressing CAR3 is 80%, while the killing efficiency of iNK expressing CAR10 is less than 20%. Since the number of iPSC cells expressing CAR4, CAR6 and CAR7 is very small, the data of killing efficiency cannot be obtained.

[0145] The above results show that the CAR3 structure can not only be expressed at a high level in iPSC cells, but also does not affect the differentiation of CAR-iPSC to CAR-iNK cells, and the CAR-iNK cells obtained by differentiation have high killing ability, which shows that the CAR3 structure does not affect the differentiation and expansion process from iPSC to iNK cells. Therefore, the CAR3 of the present application is suitable for expression in iPSC, and then CAR-iNK cells are obtained.

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

[0147] The method of monoclonal screening after site-directed knock-in is used to detect the expression level of the CAR and the control CAR on the iPSC monoclonal. The structure of CAR1 and CAR3 is electroporated in iPSC, site-directed knock-in is carried out in the genome, after culture screening, monoclonal selection and genotype detection are carried out, and the expression level of the CAR molecule on the iPSC monoclonal is detected. The specific method is as follows:

[0148] (1) RNP system electroporation

[0149] The iPSC is digested by StemPro Accutase cell dissociation reagent (Gibco, A1110501), centrifuged to remove the supernatant, washed once with PBS, counted, and 1x10 6 cells were electroporated. RNP system was used for electroporation, which included sgRNA and Cas9 protein. sgRNA (5'-GGGGCCACTAGGGACAGGAT-3', SEQ ID NO: 24) and Cas9 (IDT, 1081061) were mixed in a molar ratio of 2:1. 250 pmol of sgRNA and 125 pmol of Cas9 protein were needed for 100 μl of electroporation system. The two were mixed uniformly, and room temperature was kept for 15 min. 2ug of plasmid expressing CAR1 and CAR3 structure was taken, and P3 primary cell 4D nucleic acid transfer kit (Lonza, V4XP-3032) was used for electroporation. Referring to the instruction, Lonza 4D nucleic acid transfer electroporator was used for electroporation. After electroporation, the cells were transferred to a culture dish, the culture dish was coated with Laminin iMatrix-511 (Amsbio, 892012), and the culture medium was used after adding clone R2 (Stemcell, 100-0691) at a ratio of 1:10. The cells were cultured in a 37℃ incubator.

[0150] (2) iPSC monoclonal selection

[0151] After 10 days of culture of the cells electroporated with CAR1 and CAR3 structure in step (1), monoclonal plating was carried out. The edited iPSC was digested, stained with FMC63 antibody (Bioswan, 200107), and the positive cells were plated into a 96-well plate using Namocell Single Cell Dispenser, with only one cell in each well. Continue to culture for 14 days, and select the single clone after the single clone grows. After washing with PBS, add 50 μl of Accutase digestion solution for digestion, and blow evenly with a gun head after digestion. 40 μl is transferred to a 12-well plate containing culture medium for culture, and 5 μl is transferred to a PCR tube for genotype identification.

[0152] (3) iPSC genotype identification

[0153] 5 μl sample taken during monoclonal selection was used for genomic rough extraction by PrepGEM DNA extraction kit, the kit item number is MicroGEM-PUN0500. 20 μl reaction system contains 5 μl sample, 2 μl 10X blue buffer, 0.4 μl PrepGEM enzyme, 12.6 μl water. After mixing the above reaction system uniformly, it is incubated in PCR instrument according to 75 degrees for 10 minutes, 95 degrees for 2 minutes, and after incubation, it can be used as a template for PCR experiment. Take 2 μl as a template, add DNA amplification enzyme and primer for PCR experiment, and identify the genotype of monoclonal.

[0154] The knock-in efficiency and expression amount of CAR molecules were detected by flow cytometry, and the results are shown in Figure 3 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%, and the editing efficiency of CAR3 is 7.24 times that of CAR1.

[0155] Consistent with the results of Example 2, the CAR3 structure can obtain the highest level of editing efficiency and stable expression in iPSC.

[0156] After knocking in CAR1 and CAR3 genes, the iPSC was selected and detected for monoclonal, and the expression level of CAR on the iPSC monoclonal was detected, and the results are shown in Figure 4 The expression level of CAR molecule is characterized by MFI, and the results show that among the three monoclonals (sc5, sc19 and sc20) containing the CAR3 structure of the application, the MFI values are 3416, 3941 and 3619, and the average value is 3658.66; while the average value of MFI of the control CAR1 monoclonal is only 384.33. Compared with CAR1, the expression amount of CAR3 of the application on iPSC has increased by an order of magnitude. It shows that the CAR3 structure can obtain higher level of stable expression in iPSC.

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

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

[0159] Vector construction and virus packaging: The synthetic sequences were directly replaced in the original target vector pMSCV-1928z (CAR1) to construct CAR3, CAR4, CAR6, CAR7 and CAR10, respectively, for retrovirus packaging, the process is as follows: take the well-grown 293T cells and inoculate in 10 cm dishes (6 x 10 6 / plates) and incubate overnight. The next day: check to ensure that the 293T in the culture dish reaches 70-80% confluence. Prepare the following transfection complex: A tube: 500 μl Opti-MEM + 10 μg gag-pol + 8 μg BaEVTR + 20 μg target vector (pMSCV, etc. 10 vectors), mix by pipette. B tube: 500 μl Opti-MEM + 40 μl PEI Pro, mix by pipette. Transfer the A tube solution to the B tube, mix with a pipette, and stand at room temperature for 15-20 min. Transfer the DNA-PEI Pro mixture to a 10 cm culture dish and mix gently. Incubate in a 37°C incubator for 6 hours, then remove the culture supernatant and wash with PBS, then add 10 ml of complete medium.

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

[0161] After the packaged retrovirus was frozen for 48 hr, it was diluted and infected with K562 cells for titer detection, the infection conditions were as follows: take 3 x 10 4 K562 cells, resuspend in 100 μl of fresh medium, add 100 μl of concentrated retrovirus and polybrene (final concentration 5 μg / ml), centrifuge at 800g for 30 min at 32°C, and continue to culture in a 37°C, 5% CO2 incubator. The positive rate of K562 CAR cells was detected by flow cytometry (Anti-Mouse FMC63 scFv Monoclonal Antibody) 3 days after infection, and the virus titer was calculated according to the positive cell percentage and dilution factor for subsequent infection of iNK cells.

[0162] iNK virus infection: Retronectin (Takara, Cat#T100B) was diluted with PBS to a final concentration of 15 μg / ml, 250 μl / well of the working solution of Retronectin at a final concentration of 15 μg / ml was added to a 24-well cell culture plate, and the 24-well plate was placed at 4°C overnight. The Retronectin coating solution in the 24-well plate was discarded, and 500 μl of PBS containing 2% BSA was added as a blocking solution at room temperature for 30 minutes. The blocking solution was discarded, and the plate was washed twice with PBS containing 2.5% HEPES. The Retrovirus was thawed at room temperature, and the required virus volume was added to the well at an MOI of 10, and the volume was made up to 1 ml with PBS containing 2.5% HEPES, and then centrifuged at 2000g at 32°C for 2 hours. When the centrifugation was about to be completed, the activated NK cells for 3 days were counted, and the required cell amount was taken at an MOI of 10, centrifuged at 500g at room temperature for 5 minutes, and then resuspended in NK complete medium to a cell density of 5x10 5 / ml after the supernatant was discarded. 6 -2x10 6 / ml.

[0163] The CAR positive rate detection results are shown in Figure 5 and Table 3.

[0164] Table 3 CAR expression rate of each group

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

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

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

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

[0169] The killing ability of the 6 CAR-containing iNK cells obtained in Example 5 against K562 and Nalm-6 was detected by fluorescent enzyme. The experimental method was the same as that in Example 3.

[0170] The results are shown in Figure 6 The killing ability against K562 cells represents the non-specific killing ability of iNK cells, Figure 6 The results of the left graph show that iNK expressing CAR molecules of different structures have similar non-specific killing ability against K562, with no significant difference, indicating that the CAR molecules of different structures do not affect the natural immune killing function of iNK. Nalm-6 cells are CD19 positive cells, and in the killing experiment against Nalm-6, Figure 6 The results of the right graph show that under the same effector-target ratio killing conditions, CAR3 structure shows the strongest specific killing ability.

[0171] The above results show that CAR-iNK cells containing CAR3 structure of the application have an unaffected natural immune killing function, and a specific killing ability significantly higher than that of 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 is replaced by the scFv sequence of anti-BCMA antibody to construct the structure of anti-BCMA-CAR3.

[0174] The sequence of anti-BCMA CAR3 is shown in SEQ ID NO: 20:

[0175] (SEQ ID NO:20).

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

[0177] iPSCs expressing anti-BCMA-CAR3 were constructed using the method mentioned in Example 3, as follows. Figure 7 As shown in the left figure, the expression rate of BCMA-CAR3 in iPSCs was 93.78%. Using the method described in Example 1, the above-mentioned BCMA-CAR3-iPSCs were differentiated and expanded to obtain iNK cells expressing anti-BCMA-CAR3, as shown in the figure. Figure 7 As shown in the right figure, the expression rate of BCMA-CAR3 molecule on iNK is 91.53%. This indicates that the CAR3 structure can support the high expression of anti-BCMA antibody scFv in iPSC and iNK stages.

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

[0179] The present application also constructs a dual CAR structure targeting BCMA and CD19, uses the scFv of antibody FMC63 targeting CD19 and the scFv of anti-BCMA antibody to construct the dual CAR (anti-BCMA+CD19 dual CAR3) and the control dual CAR (anti-BCMA+CD19 dual CAR1) of the present application of the present application. The antigen binding domain of the dual CAR is composed of the scFv of FMC63 and the scFv of anti-BCMA antibody, and other elements are 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 scFv against BCMA is shown as SEQ ID NO: 21:

[0183] DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS (SEQ ID NO: 21).

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

[0185] The anti-BCMA+CD19 dual CAR molecules of two different structures of CAR1 and CAR3 are cloned into a retrovirus vector, iNK cells are infected with the virus, and the expression positive rate is detected, and the experimental method is the same as in Example 5.

[0186] The expression rate detection results of the dual CAR are shown in Table 5. Figure 8 The results show that the expression level of anti-BCMA+CD19 dual CAR3 is higher than that of anti-BCMA+CD19 dual CAR1 structure, which indicates that the CAR3 structure can adapt to various scFvs, and can be expressed at a high level on iNK.

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

[0188] The iNK cells containing the anti-BCMA+CD19 dual CAR1 and anti-BCMA+CD19 dual CAR3 structure constructed in Example 9 were used to perform a function experiment with RPMI8226 cells as target cells, and the experimental method was the same as that in Example 3.

[0189] The results are shown in Figure 9 The results show that under the condition of an effector to target ratio of 3:1, iNK with CAR3 structure has stronger cytotoxicity, indicating that CAR3 structure has stronger killing ability to target cells.

[0190] Discussion

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

[0192] Firstly, CAR3 molecule is suitable for transduction into iNK cells by retrovirus, and the expression rate on iNK cells is several to dozens of times higher than that of the control CAR. Secondly, CAR3 molecule is suitable for transduction into iPSC cells by gene editing, and the expression rate on iPSC is also significantly higher than that of the control CAR, and the transduction of CAR3 molecule does not affect the process of iPSC cell differentiation into iNK cells, so the CAR can be edited into the iPSC genome by CRISPR / Cas9, and then the stable CAR-iPSC is differentiated into CAR-iNK cells.

[0193] In addition, the CAR3 structure of the present application can not only be adapted to the scFv of FMC63 monoclonal antibody, but also can be adapted to various other forms of scFv, such as multiple scFv 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-BCMA CAR3 (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] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSGGGGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFLVIIVILSALFLGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0213] All documents referred to in this disclosure are incorporated herein by reference as if each were individually incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that embodiments can be practiced without materials details, quantifications, and other specific items described herein.

Claims

1. An engineered iPSC cell, characterized in that, An exogenous expression vector containing a chimeric antigen receptor (CAR) encoding gene was introduced into the engineered iPSC cells. The CAR contains a structure as shown in Formula I: L-EB-H-TM-ICD1-ICD2(I) In the formula, Each "-" represents a peptide bond independently; L represents the absence of a signal peptide sequence; EB is an extracellular binding domain; the EB is a CD19-targeting scFv, and the sequence of the scFv is shown in SEQ ID NO:2; H is the hinge region derived from CD8, which has the amino acid sequence shown in SEQ ID NO:4; TM is a transmembrane region derived from the 2B4 protein, which has the amino acid sequence shown in SEQ ID NO:6; ICD1 originates from the intracellular region of the 2B4 protein and has the amino acid sequence shown in SEQ ID NO:10; ICD2 is a cytoplasmic signaling sequence derived from CD3ζ; The engineered iPSC cells were differentiated into CAR-NK cells.

2. The engineered iPSC cell as described in claim 1, characterized in that, The sequence of L is shown in SEQ ID NO:

1.

3. The engineered iPSC cell as described in claim 2, characterized in that, The ICD2 has an amino acid sequence as shown in SEQ ID NO:

13.

4. The engineered iPSC cell as described in claim 1, characterized in that, The CAR has an amino acid sequence as shown in SEQ ID NO:

15.

5. The engineered iPSC cell as described in claim 1, characterized in that, The importation includes the following steps: using a CRISPR / Cas9 system to knock the CAR-encoded gene into the AAVS1 site.

6. The engineered iPSC cell as described in 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 denoted as R1, where R1 ≥ 2000.

7. A method for preparing CAR-NK cells, characterized in that, The procedure includes: differentiating and culturing the engineered iPSC cells as described in claim 1 to obtain CAR-NK cells.

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

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (a) the CAR-NK cells as described in claim 8; and (b) Pharmaceutically acceptable carriers.

10. Use of the CAR-NK cells as described in claim 8 or the pharmaceutical composition as described in claim 9, characterized in that, Used to prepare drugs or preparations for the prevention and / or treatment of tumors, wherein the tumor is a CD19 positive tumor.

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