Chimeric antigen receptors, compositions and methods

Abstract

The present disclosure describes chimeric antigen receptors for expression in natural killer (NK) cells, including pharmaceutical compositions of NK cells (and / or iPSC) modified to express chimeric antigen receptors, and methods involving these chimeric antigen receptors. Typically, the chimeric antigen receptor includes an extracellular domain comprising an antigen recognition region, a transmembrane domain connected to the extracellular domain, and an intracellular domain connected to the transmembrane domain. The intracellular domain may include a signaling peptide that activates NK cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of Chinese patent application No. 201680008013.0. This application claims priority to U.S. Provisional Patent Application No. 62 / 109,281 filed on January 29, 2015, which is incorporated herein by reference. Summary of the invention

[0003] In one aspect, the present disclosure describes a chimeric antigen receptor for expression in natural killer (NK) cells. Typically, the chimeric antigen receptor includes an extracellular domain comprising an antigen recognition region, a transmembrane domain connected to the extracellular domain, and an intracellular domain connected to the transmembrane domain. The intracellular domain may include a signaling peptide that activates NK cells.

[0004] In some embodiments, the antigen recognition domain can specifically bind to an antigen associated with a disease.

[0005] In some embodiments, the antigen recognition domain can specifically bind to a tumor antigen.

[0006] In some embodiments, the extracellular domain may further include a signal peptide or a leader sequence and / or a spacer sequence.

[0007] In some embodiments, the intracellular domain may include a signaling domain of a NK cell membrane-bound signaling adaptor protein such as 2B4, DAP10, DAP12, IL21R, CD137 (41BB) or CD3ζ.

[0008] In some embodiments, the transmembrane domain may include a transmembrane region of a natural cytotoxicity receptor expressed in NK cells, such as CD16, NKp44, NKp46, or NKG2D.

[0009] In another aspect, the present disclosure describes pharmaceutical compositions comprising NK cells (and / or iPSCs) modified so as to express any of the embodiments of the chimeric antigen receptor summarized above.

[0010] In another aspect, the present disclosure describes a method of providing immunotherapy to a subject with a condition. Generally, the method comprises administering to the subject a therapeutic composition as summarized above, wherein the antigen recognition region of the chimeric antigen receptor specifically binds to an antigen associated with the condition.

[0011] The above summary is not intended to describe every disclosed embodiment or every practice of the present invention. The following description more specifically exemplifies illustrative embodiments. In several places of this application, guidance is provided by listing examples, which can be used in various combinations. In each case, the given list is used only as a representative group and should not be interpreted as an exclusive list. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 (A) Exemplary natural cytotoxicity receptors and the effects of binding of these receptors to their ligands on NK cell degranulation and polarization and target cell killing. (B) Representative examples of natural cytotoxicity receptors and their corresponding signaling adaptors. (C) Third generation chimeric T cell antigen receptor constructs used in iPSC-derived NK cells.

[0013] Figure 2 .(A) Generalized schematic diagram of NK-activating CAR. (B) Schematic diagram of novel chimeric antigen receptor constructs. The chimeric antigen receptor was cloned into the pkt2 vector, which contains IR / DR for use with SB100X transposase, mCAGs promoter, chimeric antigen receptor (CAR) sequence, internal ribosome entry site (IRES), and GFP:Zeo selection marker. The chimeric antigen receptor fragment was obtained from UniProt and assembled by gBlock synthesis and traditional restriction enzyme cloning (IDT).

[0014] Figure 3 .Surface expression of chimeric antigen receptors in NK92 and iPS cells. NK92 cells or iPS cells were transfected using the SleepingBeaut transposon system using SB100X. Cells were then screened using Zeocin and flow cytometry was performed to assess the cell surface expression of various chimeric antigen receptors. Expression was assessed using a biotin-coupled polyclonal goat anti-mouse antibody that recognizes mouse IgG F(ab')2 fragments (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, cat#115-065-072). Streptavidin coupled to a fluorescent dye was used to detect the bound antibodies.

[0015] Figure 4CD107A release and IFN-γ production in NK92 cells. NK92 cell degranulation and cytokine production were evaluated by flow cytometry. NK92 cells were mixed 1:1 with mesothelin-negative (MA148), mesothelin-positive (A1847) ovarian cancer target cells, or protein A microbeads coupled or uncoupled to mesothelin / Fc chimeric protein. Cells were stained for CD107a and intracellularly for IFN-γ production.

[0016] Figure 5 . Cr-51 release assay using NK92 cells. NK92, NK92 / 28 / 41BB / CD3 or NK92 / CAR4 cells were incubated with K562, K562 mesothelin+, MA148 or A1847 cells at the indicated ratios for 4 hours. The release of Cr-51 was then detected to assess cell killing. This experiment was performed according to Woll et al., 2009, Blood 113 (24): 6094-6101, except that iPSC-derived NK cells were used instead of hESC-derived NK cells.

[0017] Figure 6 .Exemplary other NK activating chimeric antigen receptors.

[0018] Figure 7 .Comparison of third-generation T cell CAR and Figure 2 Schematic diagram of an exemplary NK CAR construct reflected in .

[0019] Figure 8 . Schematic diagram of exemplary NK CAR constructs.

[0020] Fig. 9 . Schematic diagram of exemplary NK CAR constructs.

[0021] Fig.10 .Data showing the cytotoxicity of NK CAR against K562 cells.

[0022] Fig.11 .Data showing cytotoxicity of NK CAR against two ovarian cancer cell lines.

[0023] Fig.12 . Data showing expression of an exemplary NK CAR by induced pluripotent stem cells.

[0024] Fig.13 . Data showing surface expression of exemplary CARs by iPSC-derived NK cells.

[0025] Fig.14 .Exemplary generalized NK CAR vector constructs.

[0026] Fig.15 .Exemplary generalized NK CAR vector constructs. Tandem cHS4 insulators can inhibit CAR vector silencing, thereby improving the expression of the CAR in NK cells and iPSCs. DETAILED DESCRIPTION

[0027] The present disclosure describes chimeric antigen receptors designed to specifically incorporate NK cell activation domains. The chimeric antigen receptors can be incorporated into intracellular and / or transmembrane regions, including, for example, intracellular and / or transmembrane regions from CD16, NKp44, NKp46, and / or NKG2D connected to costimulatory or signaling domains from, for example, 2B4 (CD244), CD137 (41BB), IL21, DAP10, DAP12, and / or CD3ζ.

[0028] Chimeric antigen receptors (CARs) are engineered artificial receptors that can provide engineered specificity for immune cells expressing the CAR. Typically, immune cell populations can be collected from subjects with a particular form of cancer. The collected immune cells can be modified to express a chimeric antigen receptor that specifically binds to an antigen expressed by a tumor cell and then infused back into the subject. Modified immune cells expressing chimeric antigen receptors are better able to recognize and kill tumor cells that express antigens specifically recognized by the chimeric antigen receptor.

[0029] Chimeric antigen receptors have been designed to activate T cells for the treatment of refractory ALL (targeting CD19), pancreatic cancer (targeting mesothelin), and other malignancies. There are several chimeric antigen receptor constructs, but most are designed to activate T cells.

[0030] In contrast, the chimeric antigen receptors described herein are designed to be expressed in induced pluripotent stem cells (iPSCs), which then differentiate into NK cells. They can also be directly expressed in peripheral blood (PB)-NK cells, NK-92 cells, or other suitable NK cell lines. NK-92 cells or other NK cell lines have been used in clinical studies for anticancer treatment. NK cells expressing chimeric antigen receptors can then be used as immunotherapy for treating a variety of cancers. The chimeric antigen receptors described herein may include a signaling domain that can be used with the antigen recognition portion of various targeting antibodies. Specifically, the present disclosure describes an exemplary embodiment of a chimeric antigen receptor that reflects targeted mesothelin for the treatment of ovarian cancer. However, since mesothelin is expressed on many adenocarcinomas, the described embodiments may have a wider range of uses. In addition, the mesothelin targeting domain described is merely exemplary; other single-chain variable fragments (scFV) can be engineered into the NK-specific chimeric antigen receptor (NK-CAR) signal construct to target substantially any malignant tumor.

[0031] One feature of the NK cell chimeric antigen receptor described herein is that it can bypass the adaptor molecules / accessory proteins required for signal transduction to initiate the natural cytotoxicity receptor. Alternatively or in addition thereto, a transmembrane domain that is usually bound to the adaptor molecule / accessory protein is included so that the accessory protein can also bind, making it more likely that signal transduction will initiate. The NK cell chimeric antigen receptor designed to include, for example, CD3ζ enables bypassing other natural cytotoxicity receptors. The incorporation of transmembrane domains and other intracellular domains enables these NK cell chimeric antigen receptors to bind to the adaptor protein and provide better signal transduction than CD3ζ alone by activating multiple pathways.

[0032] Although some T cell chimeric antigen receptor constructs can activate NK cells to a certain extent due to shared signaling domains, the chimeric antigen receptors described herein are specifically designed to activate NK cells. The chimeric antigen receptors designed to specifically activate NK cells can improve NK function and receptor uses in NK cell immunotherapy (e.g., cell-mediated refractory tumor killing).

[0033] Chimeric antigen receptors typically include an extracellular domain, a transmembrane domain and an intracellular domain. The intracellular domain usually resides in the cytoplasm of the cell. Once the antigen is recognized by the extracellular domain, the intracellular domain transmits the activation signal to the NK cell, which induces the NK cell to destroy the targeted tumor cells. Exemplary signal transduction intracellular domains include, for example, a membrane-bound signal adapter protein (including, for example, 2B4 (CD244), CD137 (41BB), IL21, DAP10, DAP12 and / or CD3ζ) signal transduction domain, or a portion thereof, including, for example, an immunoreceptor tyrosine activation motif (ITAM), a YxxM motif, a TxYxxV / I motif, FcRγ, NKp80 (by atypical hemi-ITAM signal transduction) and / or DNAM, etc.

[0034] The transmembrane domain spans the plasma membrane, connecting the intracellular domain to the extracellular domain. Exemplary transmembrane domains include, for example, the intracellular and / or transmembrane domains of natural cytotoxicity receptors (NCRs), such as CD16, NKp44, NKp46, NKG2D, NKp30, NKp80, and / or DNAM-1, or portions thereof that include, for example, one or more charged amino acids. In some embodiments, the charged amino acids may be lysine and / or arginine residues. In some cases, the transmembrane region may be derived from a transmembrane protein, meaning that it naturally has an extracellular C-terminus rather than an extracellular N-terminus. In this case, the direction of the transmembrane region may be reversed, for example, in Figure 6 The chimeric antigen receptor is marked with "Rev TM" in the NK cell membrane so that the chimeric antigen receptor is correctly oriented.

[0035] The extracellular domain generally includes a signal peptide and an antigen recognition region. In many embodiments, the extracellular domain may also include a spacer. The signal peptide guides the nascent polypeptide into the endoplasmic reticulum so that it can be correctly glycosylated and anchored into the plasma membrane. Generally speaking, any eukaryotic cell signal peptide can be used as long as it guides the protein to the endoplasmic reticulum. An exemplary signal peptide includes a CD8α leader sequence, but other signal peptide sequences may also be suitable. When the spacer is present, it connects the antigen recognition domain to the transmembrane domain. The spacer generally provides flexibility so that the antigen recognition region can be freely oriented to different directions, thereby enabling the antigen recognition region to bind to the antigen target. An exemplary spacer includes a CD8α hinge sequence, but other Ig hinge regions may also be suitable. The antigen recognition region may include any peptide sequence that can specifically bind to a specified target. As used herein, "specifically bind" and its variants refer to having any degree of differentiated or non-general affinity for a specific target. Thus, the antigen recognition region may include an antibody fragment, such as scFv or Fab, that specifically binds to a particular antigen (e.g., a tumor antigen, a viral antigen, a modified self-antigen, etc.). In some embodiments, the scFv may be derived from a monoclonal antibody. A chimeric antigen receptor may be designed to include an antigen recognition region that specifically binds to any given target. Thus, although Figure 2 , Figure 6 , Figure 7 and Figure 8An embodiment designed to specifically bind to mesothelin is shown, but the NK-activating chimeric antigen receptor can be designed to specifically bind to and thereby target any antigen associated with cells (e.g., tumorigenic cells or virus-infected cells) that are intended to be targets for NK cell-mediated killing. For example, NK cells and / or CARs have shown activity against a variety of solid tumors and virus-infected cells, including but not limited to HIV (human immunodeficiency virus), hepatitis B, hepatitis C, CMV (cytomegalovirus), EBV (Epstein-Barr virus), HPV (human papillomavirus), and the like.

[0036] Thus, for example, to better mediate NK cell cytotoxicity against tumors that include cells expressing mesothelin (e.g., ovarian cancer, pancreatic cancer, lung cancer, colon adenocarcinoma, mesothelioma, and other adenocarcinomas that express mesothelin), one can design and express in NK cells, for example Figure 2 , Figure 6 , Figure 7 and Figure 8 The chimeric antigen receptor shown in . The chimeric antigen receptor construct displayed contains a NK cell-specific transmembrane domain and an activation domain (activating domain), and can be expressed in the NK cell tumor line NK92. The transmembrane region and the intracellular region are taken from CD16, NKp44, NKp46 and / or NKG2D, and the activation domains of 2B4, DAP10, DAP12 and / or CD3ζ are combined in an attempt to maximize the activation of NK cells. Figure 3 Showing NK92 and iPS cell expression Figure 2 Chimeric antigen receptor shown in .

[0037] To evaluate the functionality of the chimeric antigen receptor, NK cells expressing the chimeric antigen receptor were tested against antigen-coated microbeads and mesothelin-expressing cell lines. Figure 4 showed that when NK cells expressing chimeric antigen receptors were mixed with mesothelin-positive targets, Figure 2 The chimeric antigen receptor enhances NK cell degranulation and cytokine production.

[0038] Figure 5 It was shown that NK92 cells expressing the NK-specific chimeric antigen receptor as described herein improved in vitro killing of mesothelin-positive target cells compared to the third-generation T cell-specific chimeric antigen receptor (NK92 / 28 / 41BB / CD3ζ) or non-transfected NK92 cells.

[0039] Fig.10The cytotoxicity of an exemplary NK CAR to K562 cells (upper left inset) and K562 cells expressing mesothelin as the target of the CAR (upper right inset) is shown. These results show a significant improvement in killing in a mesothelin-specific manner, with CAR7 and CAR9 being the most significant. The inset below is a summary of the results expressed in lytic units (Bryant et al., 1992, J Immunol Methods 146 (1): 91-103). CAR7 and CAR9 showed a significant improvement in killing compared to the third-generation T cell CAR (NK92 meso 3 rd ) showed significantly higher cytotoxicity. Cytotoxicity was measured by the Cr-51 release assay as described previously (Knorr et al., 2013, Stem Cells Transl Med 2(4):274-283; Woll et al., 2009, Blood 113(24):3094-6101; Woll et al., 2005, J Immunol 175(8):5095-5103). Fig.11 Similar results are shown using two ovarian cancer cell lines, meso-high (A1497) and meso-low (MA148). NK92 with different NK cell-based anti-meso CARs kills in a meso-specific manner. The bottom inset also reflects the summary expressed in lytic units. In addition, when expressed as Fig.10 and Fig.11 Upon stimulation of targets in the NK cell line, NK CAR mediated enhanced expression of CD107a and / or IFN-γ (data not shown).

[0040] In some embodiments, NK-specific chimeric antigen receptors as described herein can be expressed in iPSCs, which can then be differentiated into NK cells. iPSCs can be differentiated as described in Knorr et al., 2013, Stem Cells Transl Med. 2(4):274-283 or Ni et al., 2014, Stem Cells 32(4):1021-1031. Fig.12 Shown is the expression of an exemplary NK CAR by induced pluripotent stem cells (iPSCs), as demonstrated by the generation of CD45+CD56+ cells (top row, 5th inset). Fig.13 Shown is CAR surface expression by iPSC-derived NK cells. Fig.13 The bottom three rows show CAR expression only on the surface of iPSC-CAR4v2 (column 4, bottom three rows) compared to PB-NK cells and iPSC-NK cells that do not express CAR (unmodified control cells, column 4, rows 4 and 5). Fig.12 and 13Other illustrations in the figure show that other typical NK cell surface antigens / receptors on iPSC-CAR4v2 are similar to those present on unmodified control cells. These results indicate that iPSC-derived NK cells can display Fig.10 and Fig.11 The mesothelin-targeted-CAR-expressing NK cells in the same target-specific cytotoxicity.

[0041] Conventional transfection methods can be used to introduce polynucleotides encoding NK CAR constructs into NK cells or iPSCs. Thus, although this article is described in the context of an exemplary embodiment in which a polynucleotide encoding CAR is transfected into cells using the Sleeping Beauty transposon system, any suitable transfection method can be used to modify NK cells (and / or iPSCs). Fig.14 Exemplary vector constructs that can be used to modify NK cells (and / or iPSCs) to express chimeric antigen receptors are shown. Fig.15 The construction of an alternative exemplary vector is shown, which also includes a tandem cHS4 insulator (Aker et al., 2007, Hum Gene Ther 18(4):333-343), which can inhibit CAR vector silencing, thereby improving the expression of the CAR in NK cells and iPSCs.

[0042] The NK cells and / or iPSs modified to express chimeric antigen receptors described herein can be formulated into pharmaceutical compositions together with a "carrier" for delivery to a subject having a condition characterized at least in part by a cell being a target of NK cell toxicity. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. It is well known in the art to use these media and / or agents for pharmaceutically active substances. In addition to any conventional vehicle or agent being incompatible with the active ingredient, it is considered that the active ingredient is used in a therapeutic composition. Supplementary active ingredients may also be incorporated into the composition.

[0043] "Pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to an individual along with NK cells (and / or iPSCs) modified to express a chimeric antigen receptor without causing any undesirable biological effect or interacting in a deleterious manner with any other component of the pharmaceutical composition in which it is contained.

[0044] The pharmaceutical composition can be formulated into various forms suitable for preferred routes of administration. Thus, the composition can be administered by known routes, such as parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.) or topical (e.g., intratracheal, intrapulmonary, etc.). The composition can also be administered by sustained or delayed release.

[0045] The preparation can be conveniently presented in a unit dosage form and can be prepared by methods well known in the pharmaceutical field. The method for preparing a composition with a pharmaceutically acceptable carrier includes the step of combining NK cells (and / or iPSC) modified to express a chimeric antigen receptor with a carrier constituting one or more auxiliary components. Generally speaking, the preparation can be prepared by combining NK cells (and / or iPSC) uniformly and / or closely with, for example, a liquid carrier.

[0046] The pharmaceutical composition comprising NK cells (and / or iPSC) modified to express chimeric antigen receptors can be provided in a suitable form, including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or a mixture in any form. The composition can be delivered in a formulation with any pharmaceutically acceptable excipient, carrier or vehicle.

[0047] The amount of NK cells (and / or iPSC) modified to express chimeric antigen receptors applied to a subject may vary with various factors, including but not limited to, the subject's weight, physical condition, and / or age, whether one or more chimeric antigen receptors are administered, and / or the route of administration. Therefore, the absolute amount of NK cells (and / or iPSC) included in a given unit dosage form may vary widely and depends on factors such as the subject's species, age, weight and physical condition, and method of administration. Accordingly, it is impractical to generally describe the amount of NK cells (and / or iPSC) modified to express chimeric antigen receptors that are effective for each and / or all possible applications. However, a person of ordinary skill in the art can easily determine a suitable amount by appropriately considering these factors.

[0048] In some embodiments, the method may include administering a sufficient number of NK cells (and / or iPSCs) modified to express a chimeric antigen receptor to provide, for example, about 10 5 cells / kg to about 10 10 In some embodiments, the method comprises administering a sufficient number of NK cells (and / or iPSCs) modified to express a chimeric antigen receptor to provide, for example, about 10 cells / kg to the subject. 7 cells / kg to about 10 8The dose of cells / kg is, for example, about 1×10 7 cells / kg to about 8×10 7 The dose was cells / kg.

[0049] Alternatively, the dose can be calculated using actual body weight obtained before the start of the treatment course. For doses calculated in this manner, the body surface area (m2) is calculated using the Dubois method before the start of the treatment course. 2 ): m 2 =(Weight kg 0.425 ×Heightcm 0.725 )×0.007184.

[0050] In some embodiments, the pharmaceutical composition comprising NK cells (and / or iPSC) modified to express a chimeric antigen receptor can be administered, for example, in a single dose to multiple doses per week, although in some embodiments the pharmaceutical composition can be administered at a frequency beyond this range to implement the method. In certain embodiments, the pharmaceutical composition can be administered from about 1 time per month to about 5 times per week.

[0051] Typically, the pharmaceutical composition is administered to a subject in an amount and dosage regimen effective to reduce, limit progression, alleviate, or resolve any degree of symptoms or clinical signs of a condition. As used herein, "alleviation" refers to any reduction in degree, severity, frequency, and / or likelihood of symptoms or clinical signs characteristic of a particular condition. "Symptoms" refers to any subjective evidence of a disease or patient's condition. "Signs" or "clinical signs" refer to objective physical findings related to a specific condition that are not discovered by the patient himself.

[0052] In the foregoing description, specific embodiments may be described in isolation for clarity. Unless otherwise expressly stated that features of a specific embodiment are incompatible with features of another embodiment, certain embodiments may include a combination of compatible features described herein in conjunction with one or more embodiments.

[0053] For any method described herein comprising separate steps, these methods can be performed in any feasible order. Optionally, any combination of two or more steps can be performed simultaneously.

[0054] The present invention is illustrated by the exemplary embodiments described above. It should be understood that the specific examples, materials, amounts, and operations should be interpreted broadly in accordance with the scope and spirit of the invention described herein.

[0055] As used herein, the term "and / or" refers to one or all of the listed elements or any combination of two or more of the listed elements; when appearing in the specification and claims, the term "comprising" and its variations do not have a limiting meaning; unless otherwise indicated, "a", "an", "the" and "at least one" are used interchangeably to refer to one or more than one; the recitation of numerical ranges by endpoints includes all numbers subsumed within the range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0056] The complete disclosures of all patents, patent applications, and publications referred to herein, as well as electronically provided materials (e.g., including, for example, nucleotide sequence submissions in GenBank and RefSeq, amino acid sequence submissions in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. If there is any inconsistency between the disclosure of the present application and the disclosure of any document incorporated herein by reference, the disclosure of the present application applies. The foregoing detailed description and examples are given only for the purpose of clear understanding. No unnecessary limitations should be understood therefrom. The present invention is not limited to the precise details shown and described, as variations obvious to those skilled in the art will be included in the invention defined by the claims.

[0057] Unless otherwise indicated, all numbers used in the specification and claims expressing quantities of components, molecular weights, etc., should be understood as being modified in all cases by the term "about". Therefore, unless otherwise indicated, the numerical parameters listed in the specification and claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and not attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0058] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain the range necessarily resulting from the standard deviation found in their respective measurements.

[0059] Unless otherwise indicated, all headings are for the convenience of the reader and should not be used to limit the meaning of the text following the heading.

Claims

1. A polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises, from the N-terminus to the C-terminus: (a) an extracellular domain comprising an antigen recognition region at the N-terminus; (b) a transmembrane domain comprising the transmembrane region of NKG2D in an inverted orientation compared to native NKG2D which naturally has an extracellular C-terminus; and (c) an intracellular domain comprising a signaling peptide that activates natural killer cells, wherein the intracellular domain comprises at least a portion of: (i) intracellular domain (ICD) of 2B4 (CD244); (ii) 41BB (CD137) ICD; (iii) DAP12 ICD; (iv) 2B4 ICD and 41BB ICD; (v) IL21R ICD; or (vi) 41BB ICD and 2B4 ICD.

2. The polynucleotide of claim 1, wherein the antigen recognition region specifically binds to an antigen associated with a disease.

3. The polynucleotide of claim 1, wherein the antigen recognition region specifically binds to a tumor antigen.

4. The polynucleotide of any one of claims 1 to 3, wherein the extracellular domain further comprises a signal peptide or a leader sequence.

5. The polynucleotide of any one of claims 1-3, wherein the extracellular domain further comprises a spacer sequence.

6. The polynucleotide of any one of claims 1-3, wherein the intracellular domain further comprises at least a portion of the signaling domain of CD3ζ.

7. An induced pluripotent stem cell (iPSC) or NK cell comprising the polynucleotide according to any one of claims 1 to 6.

8. A method for producing the iPSC or NK cell according to claim 7, comprising introducing the polynucleotide according to any one of claims 1 to 6 into the iPSC or NK cell.

9. The method of claim 8, further comprising differentiating iPSCs into NK cells comprising the CAR.

10. A pharmaceutical composition comprising the iPSC or NK cell according to claim 7.

11. The pharmaceutical composition of claim 10, wherein the NK cells are derived from iPSCs.

12. Use of the pharmaceutical composition of claim 10 in the preparation of a medicament for providing immunotherapy to a subject having a condition, wherein the chimeric antigen receptor comprises an antigen recognition region that specifically binds an antigen associated with the condition.

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