Chimeric antigen receptors for NK cells and engineered NK cells

Abstract

The present invention provides chimeric antigen receptors for NK cells and engineered NK cells. The chimeric antigen receptor provided by the present invention comprises an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a signaling domain in sequence, wherein the hinge domain comprises or is a 4 1BB hinge domain, the transmembrane domain is selected from NKG2D, KIR2DS1, CD3ζ, CD28, 2B4, NKP44 and NKP30 transmembrane domains, the costimulatory domain is selected from 2B4, DAP12, 4 1BB, NTB A and CD2 costimulatory domains, and the signaling domain comprises or is a CD3ζ signaling domain. The engineered NK cells provided by the present invention can stably express the chimeric antigen receptor and have a strong cell killing effect.

Description

Technical Field

[0001] The present invention relates to genetically modified natural killer (NK) cells and methods for their manufacture. Specifically, the present invention relates to CAR-NK cells, methods for manufacturing these CAR-NK cells, and the use of these CAR-NK cells in medicine, particularly for treating cancer. Background Art

[0002] Natural Killer (NK) cells are a key member of the human immune cell family and serve as the first line of defense in the human immune system. They play a crucial role in immune surveillance and clearance in the body's anti-tumor immunity. Over the past decade or so, numerous international clinical studies have investigated the efficacy and safety of NK cell transfusions for the treatment of hematologic malignancies and solid tumors, demonstrating their effectiveness and safety. NK cells play a crucial role in the anti-tumor field, and the development of CAR technology has led to the emergence of CAR-NK. NK cells modified with CAR structures are theoretically capable of efficiently recognizing tumor cells and killing them through various means, including the release of cytotoxic mediators and the induction of target cell apoptosis. However, due to the natural properties of NK cells, CAR-NK therapy is less likely to produce a severe cytokine storm during treatment, potentially offering a higher safety profile in clinical use. Furthermore, since NK cells have a shorter survival time in the body than T cells, CAR-NK therapy also has a shorter survival time in the body than CAR-T. This characteristic may cause CAR-NK cells to be less effective than CAR-T cells, but from a safety perspective, this characteristic avoids a series of unknown risks that may be caused by the long-term presence of genetically modified immune cells in the human body.

[0003] Studies have shown that overexpressing exogenous CAR molecules in NK cells can enhance their tumor-targeting and killing abilities. Internationally, numerous clinical trials have been conducted on the use of CAR-NK cells for the treatment of hematologic malignancies and solid tumors. The CAR structures used in these trials can be broadly categorized into two main types. One type of CAR structure was originally designed for T cell activation, but is not fully suitable for NK cell activation and therefore cannot maximize NK cell function. The other type of CAR structure is specifically designed for NK cell activation, but this type of CAR structure is relatively rare.

[0004] In view of this, the field urgently needs more CAR structural designs that can be used for CAR NK cell preparation. Summary of the Invention

[0005] One aspect of the present invention provides a chimeric antigen receptor, which comprises an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a signaling domain in sequence, wherein the hinge domain comprises or is a 4-1BB hinge domain, the transmembrane domain is selected from NKG2D, KIR2DS1, CD3ζ, CD28, 2B4, NKP44 and NKP30 transmembrane domains, the costimulatory domain is selected from 2B4, DAP12, 4-1BB, NTB-A and CD2 costimulatory domains, and the signaling domain comprises or is a CD3ζ signaling domain.

[0006] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the hinge domain comprises or is a 4-1BB hinge domain, the transmembrane domain is selected from NKG2D, CD3ζ, 2B4, and NKP44 transmembrane domains, the costimulatory domain is selected from 2B4, DAP12, 4-1BB, and NTB-A costimulatory domains, and the signaling domain is a CD3ζ signaling domain.

[0007] In some embodiments, the structure of the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor provided by the present invention is selected from any one of the following structural formulas, wherein [] represents a domain, and ]-[ represents a connection between domains, preferably a bond or a peptide linker, such as a peptide linker composed of glycine and serine:

[0008] (a)[4-1BB]-[NKG2D]-[2B4]-[CD3ζ]

[0009] (b)[4-1BB-KIR2DS1]-[KIR2DS1]-[2B4]-[CD3ζ]

[0010] (c)[4-1BB]-[CD3ζ]-[2B4]-[CD3ζ]

[0011] (d)[4-1BB]-[CD28]-[2B4]-[CD3ζ]

[0012] (e)[4-1BB]-[2B4]-[2B4]-[CD3ζ]

[0013] (f)[4-1BB]-[NKP44]-[2B4]-[CD3ζ]

[0014] (g)[4-1BB]-[NKP30]-[2B4]-[CD3ζ]

[0015] (h)[4-1BB]-[NKG2D]-[DAP12]-[CD3ζ]

[0016] (i)[4-1BB]-[NKG2D]-[4-1BB]-[CD3ζ]

[0017] (j) [4-1BB]-[NKG2D]-[NTB-A]-[CD3ζ], and

[0018] (k)[4-1BB-KIR2DS1]-[KIR2DS1]-[CD2]-[CD3ζ].

[0019] In some embodiments, in the chimeric antigen receptor provided by the present invention, the antigen binding domain is a domain that binds to a tumor-specific antigen or a tumor-associated antigen, preferably an antigen-binding fragment of an antibody corresponding to the antigen, preferably scFv or VHH, preferably the tumor-specific antigen or tumor-associated antigen is selected from HER2, PSMA, BCMA, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, Claudin18.2, EpCAM, GD2, MSLN, EGFR, MUC1, EGFRVIII, CD38, Trop-2, c-MET, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA125, MG7-Ag, NY-CO-1, RCAS1, SDCCAG16, TAAL6 and TAG72; preferably, the antigen binding domain binds to HER2 antigen; preferably, the antigen binding domain is an anti-HER2 scFv, preferably comprising or being the amino acid sequence shown in SEQ ID NO.19.

[0020] In some embodiments, in the chimeric antigen receptor provided by the present invention, the antigen binding domain further comprises a signal peptide at its N-terminus, preferably the signal peptide is the signal peptide of CD8α, preferably comprising or being the amino acid sequence shown in SEQ ID NO.20.

[0021] In some embodiments, in the chimeric antigen receptor provided by the present invention:

[0022] (a) the 4-1BB hinge domain comprises or is the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 1;

[0023] (b) the hinge domain of 4-1BB-KIR2DS1 comprises or is the amino acid sequence of SEQ ID NO. 2 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 2;

[0024] (c) the NKG2D transmembrane domain comprises or is the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 5;

[0025] (d) the KIR2DS1 transmembrane domain comprises or is the amino acid sequence of SEQ ID NO. 6 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 6;

[0026] (e) the CD3ζ transmembrane domain comprises or is the amino acid sequence shown in SEQ ID NO. 7 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 7;

[0027] (f) the CD28 transmembrane domain comprises or is the amino acid sequence shown in SEQ ID NO. 8 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 8;

[0028] (g) the 2B4 transmembrane domain comprises or is the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO. 9;

[0029] (i) the NKP44 transmembrane domain comprises or is the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO. 10;

[0030] (j) the NKP34 transmembrane domain comprises or is the amino acid sequence shown in SEQ ID NO. 11 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 11;

[0031] (k) the 2B4 costimulatory domain comprises or is the amino acid sequence of SEQ ID NO. 13 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 13;

[0032] (1) the DAP12 costimulatory domain comprises or is the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO. 14;

[0033] (m) the 4-1BB costimulatory domain comprises or is the amino acid sequence of SEQ ID NO. 15 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 15;

[0034] (n) the NTB-A costimulatory domain comprises or is the amino acid sequence of SEQ ID NO. 16 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 16;

[0035] (o) the CD2 costimulatory domain comprises or is the amino acid sequence of SEQ ID NO. 17 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 17; and / or

[0036] (p) The CD3ζ signaling domain comprises or is the amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 18.

[0037] In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor comprises or is an amino acid sequence as shown in any one of SEQ ID NOs. 49 to 59, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 49 to 59.

[0038] In some embodiments, the chimeric antigen receptor comprises or is an amino acid sequence as shown in any one of SEQ ID NOs. 22 to 32, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 22 to 32.

[0039] In another aspect, the present invention provides an engineered NK cell that is modified to express a chimeric antigen receptor on the cell surface, wherein the chimeric antigen receptor is any of the chimeric antigen receptors provided by the present invention. In some embodiments, the NK cell is, for example, an NK cell differentiated from induced pluripotent stem cells, an NK cell derived from peripheral blood or cord blood, or an NK92 cell line.

[0040] In another aspect, the present invention provides an engineered NK cell population comprising the engineered NK cells provided by the present invention; preferably, the cell population comprises 1×10 5 Up to 1x10 7 cells.

[0041] The present invention also provides a polynucleotide encoding any one of the chimeric antigen receptors of the present invention. In some embodiments, the sequence of the polynucleotide is selected from any one of SEQ ID NOs. 30 to 42.

[0042] In another aspect, the present invention provides a vector comprising the polynucleotide provided by the present invention; preferably, the vector is a viral vector, preferably a retroviral or adeno-associated viral vector; preferably, the vector comprises an expression regulatory sequence, including a promoter.

[0043] The present invention also provides a method for preparing engineered NK cells, comprising using the viral vector provided by the present invention to transfect NK cells, so that the polynucleotide is expressed in the NK cells, thereby expressing the chimeric antigen receptor of the present invention on the cell surface; the NK cells are, for example, NK cells produced by induced pluripotent stem cell differentiation, NK cells derived from peripheral blood or umbilical cord blood, or NK92 cell lines.

[0044] The present invention also provides a method for preparing engineered NK cells, comprising using the viral vector provided by the present invention to transfect induced pluripotent stem cells (iPSCs), and inducing differentiation of the iPSCs into NK cells, wherein the chimeric antigen receptors of the present invention are expressed on the surface of the NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram showing the experimental process of testing various CAR structures of the present invention.

[0046] Figure 2 Flow cytometry was used to detect the expression of various CAR molecules on 293T cells.

[0047] Figure 3 The positive expression percentage of each CAR molecule in 293T cells is shown.

[0048] Figure 4 The expression abundance (MFI, mean fluorescence intensity) of each CAR molecule in 293T cells is shown.

[0049] Figure 5 The CAR expression positivity rates of 8 CAR molecules on PG13 cells are shown.

[0050] Figure 6 The expression of 8 CAR molecules on NK92 cells was detected by cytometry.

[0051] Figure 7 The positive rates and CAR protein abundance (MFI) of 8 CAR molecules are shown.

[0052] Figure 8 Shows the effects of 8 CAR molecules on NK92 cell viability.

[0053] Figure 9 Showing the killing effect of 6 NK92 cells expressing CAR molecules on target cells.

[0054] Figure 10 Shows the killing effect of two NK92 cells expressing CAR molecules on target cells.

[0055] Figure 11 The CAR expression positive rates of the three CAR molecules on PG13 cells are shown.

[0056] Figure 12 The expression of three CAR molecules on NK92 cells was detected by cytometry.

[0057] Figure 13 The positive rates of the three CAR molecules and the CAR protein abundance (MFI) are shown.

[0058] Figure 14 Shows the effects of three CAR molecules on NK92 cell viability.

[0059] Figure 15 Showing the killing effect of NK92 cells expressing three CAR molecules on target cells.

[0060] Figure 16 The CAR expression positivity rates of three other CAR molecules on PG13 cells are shown.

[0061] Figure 17 The expression of three other CAR molecules on NK92 cells was detected by cytometry.

[0062] Figure 18 The positive rates and CAR protein abundance (MFI) of three additional CAR molecules are shown.

[0063] Figure 19 The effects of three other CAR molecules on NK92 cell viability are shown.

[0064] Figure 20 The killing effect of another three NK92 cells expressing CAR molecules on target cells is shown. DETAILED DESCRIPTION

[0065] definition

[0066] The term "chimeric antigen receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CAR is a core component of chimeric antigen receptor NK cells (CAR-NK), which may include an antigen (e.g., tumor-specific antigen and / or tumor-associated antigen) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. In the present invention, the CAR can be combined with an intracellular domain based on the antigen specificity of an antibody. NK cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing target antigens.

[0067] The term "antigen binding domain" refers to a domain in the CAR structure that binds to an antigen expressed on the cell surface, which may be an antigen-binding fragment of an antibody against the antigen. The term "antibody" generally refers to a polypeptide molecule that can specifically recognize and / or neutralize a specific antigen. For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising its antigen-binding portion. The term "antibody" includes monoclonal antibodies and multispecific antibodies (eg, bispecific or trispecific antibodies), including but not limited to human antibodies, humanized antibodies, and chimeric antibodies. The antibody may be in the form of IgG or a heavy chain antibody. The antigen-binding fragment of an antibody includes but is not limited to scFv, Fab, Fab', (Fab)2, or VHH. In the CAR structure, the preferred antigen-binding domain is the scFv or VHH of an antibody.

[0068] The term "VHH" or "nanobody" refers to a single antigen-binding fragment of a heavy-chain antibody (HcAb) produced by camelids (such as alpacas and llamas) or sharks. A VHH or nanobody is a single heavy chain variable region of a heavy chain antibody, consisting of three hypervariable regions (CDR1, CDR2, CDR3) and four framework regions (FR1, FR2, FR3, and FR4) separating the hypervariable regions.

[0069] The term "transmembrane domain" generally refers to the domain in CAR that passes through the cell membrane and is connected to the intracellular signal transduction domain to play a role in transmitting signals.

[0070] The term "costimulatory domain" generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for lymphocytes to effectively respond to antigens.

[0071] The term "hinge domain" generally refers to the connecting region between the antigen binding domain and the transmembrane region.

[0072] The term "signaling domain" generally refers to a domain located inside a cell that can transduce signals. In the present invention, the intracellular signaling domain can transduce signals into the cell.

[0073] The term "tumor" includes both hematologic tumors and solid tumors. The terms "hematologic tumor," "hematopoietic tumor," or "lymphoid tumor" are used interchangeably and refer to tumors of hematopoietic or lymphoid tissue. Hematologic tumors include, but are not limited to, leukemias, lymphomas, myelodysplastic syndromes, or myelomas, preferably selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute T-cell leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute monocytic leukemia (AMoL), mantle cell lymphoma (MCL), histiocytic lymphoma, or multiple myeloma, preferably AML. Solid tumors include, but are not limited to, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma or small cell carcinoma, hepatocellular carcinoma, hepatoblastoma, colon adenocarcinoma, renal cell carcinoma, renal cell adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, glioblastoma, glioma, head and neck cancer, lung cancer, breast cancer, Merkel cell carcinoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, lung cancer, kidney cancer, renal adenocarcinoma, breast cancer, breast adenocarcinoma, mammary ductal carcinoma, non-small cell lung cancer, ovarian cancer, oral cancer, anal cancer, skin cancer, Ewing's sarcoma, gastric cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, and ovarian cancer. malignant neoplasms or metastatic-inducing secondary tumors of the following: ovarian adenocarcinoma, ovarian teratoma, ovarian teratoma, bladder papilloma, neuroblastoma, glioblastoma multiforme, glioblastoma, astrocytoma, epithelioid carcinoma, melanoma, or retinoblastoma.

[0074] The term "linker" is a chemical entity used to connect the antigen binding domain, hinge domain, transmembrane domain, costimulatory domain and / or intracellular signaling domain in the CAR structure. CAR may comprise one, two, three, four or five or more linkers. The linker can be a peptide linker having a length of about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intermediate length of amino acids. Exemplary peptide linkers include glycine polymers (G)n; glycine-serine polymers (G 1-5 S 1-5 )n, wherein n is an integer from 1 to 5; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art.

[0075] The term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0076] Chimeric antigen receptor

[0077] Some aspects of the present invention provide a chimeric antigen receptor (CAR), which is a fusion polypeptide that is particularly suitable for stable expression in NK cells to obtain CAR-modified NK cells (CAR-NK cells). Experiments have confirmed that the obtained CAR-NK can effectively kill target cells, such as ovarian cancer cells.

[0078] In one aspect, the present invention provides a chimeric antigen receptor, which comprises, in sequence, an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a signaling domain, wherein the hinge domain comprises or is a 4-1BB hinge domain, the transmembrane domain is selected from NKG2D, KIR2DS1, CD3ζ, CD28, 2B4, NKP44 and NKP30 transmembrane domains, the costimulatory domain is selected from 2B4, DAP12, 4-1BB, NTB-A and CD2 costimulatory domains, and the signaling domain comprises or is a CD3ζ signaling domain.

[0079] In some embodiments, the present invention provides a chimeric antigen receptor, which comprises an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a signaling domain in sequence, wherein the hinge domain is a 4-1BB hinge domain or a 4-1BB-KIR2DS1 chimeric hinge domain, the transmembrane domain is selected from NKG2D, KIR2DS1, CD3ζ, CD28, 2B4, NKP44 and NKP30 transmembrane domains, the costimulatory domain is selected from 2B4, DAP12, 4-1BB, NTB-A and CD2 costimulatory domains, and the signaling domain comprises or is a CD3ζ signaling domain.

[0080] In some embodiments, the present invention provides a chimeric antigen receptor, which comprises an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a signaling domain in sequence, wherein the hinge domain is a 4-1BB hinge domain, the transmembrane domain is selected from NKG2D, KIR2DS1, CD3ζ, CD28, 2B4, NKP44 and NKP30 transmembrane domains, the costimulatory domain is selected from 2B4, DAP12, 4-1BB, NTB-A and CD2 costimulatory domains, and the signaling domain comprises or is a CD3ζ signaling domain.

[0081] In a preferred embodiment, the present invention provides a chimeric antigen receptor, wherein the hinge domain comprises or is a 4-1BB hinge domain, the transmembrane domain is selected from NKG2D, CD3ζ, 2B4, and NKP44 transmembrane domains, the costimulatory domain is selected from 2B4, DAP12, 4-1BB, and NTB-A costimulatory domains, and the signaling domain is a CD3ζ signaling domain.

[0082] In a preferred embodiment, the present invention provides a chimeric antigen receptor, wherein the hinge domain is a 4-1BB hinge domain or a 4-1BB-KIR2DS1 chimeric hinge domain, the transmembrane domain is selected from NKG2D, CD3ζ, 2B4, and NKP44 transmembrane domains, the costimulatory domain is selected from 2B4, DAP12, 4-1BB, and NTB-A costimulatory domains, and the signaling domain is a CD3ζ signaling domain.

[0083] In a preferred embodiment, the present invention provides a chimeric antigen receptor, wherein the hinge domain is a 4-1BB hinge domain, the transmembrane domain is selected from NKG2D, CD3ζ, 2B4, and NKP44 transmembrane domains, the costimulatory domain is selected from 2B4, DAP12, 4-1BB, and NTB-A costimulatory domains, and the signaling domain is a CD3ζ signaling domain.

[0084] In some specific embodiments, the present invention provides a chimeric antigen receptor, wherein the structure of [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor is selected from any one of the following structural formulas, wherein [] represents a domain, and ]-[ represents a connection between domains:

[0085] (a)[4-1BB]-[NKG2D]-[2B4]-[CD3ζ]

[0086] (b)[4-1BB-KIR2DS1]-[KIR2DS1]-[2B4]-[CD3ζ]

[0087] (c)[4-1BB]-[CD3ζ]-[2B4]-[CD3ζ]

[0088] (d)[4-1BB]-[CD28]-[2B4]-[CD3ζ]

[0089] (e)[4-1BB]-[2B4]-[2B4]-[CD3ζ]

[0090] (f)[4-1BB]-[NKP44]-[2B4]-[CD3ζ]

[0091] (g)[4-1BB]-[NKP30]-[2B4]-[CD3ζ]

[0092] (h)[4-1BB]-[NKG2D]-[DAP12]-[CD3ζ]

[0093] (i)[4-1BB]-[NKG2D]-[4-1BB]-[CD3ζ]

[0094] (j) [4-1BB]-[NKG2D]-[NTB-A]-[CD3ζ], and

[0095] (k)[4-1BB-KIR2DS1]-[KIR2DS1]-[CD2]-[CD3ζ].

[0096] In a preferred embodiment, the present invention provides a chimeric antigen receptor, wherein the structure of [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor is selected from any one of the following structural formulas, wherein [] represents a domain, and ]-[ represents a connection between domains:

[0097] (a)[4-1BB]-[NKG2D]-[2B4]-[CD3ζ]

[0098] (c)[4-1BB]-[CD3ζ]-[2B4]-[CD3ζ]

[0099] (e)[4-1BB]-[2B4]-[2B4]-[CD3ζ]

[0100] (f)[4-1BB]-[NKP44]-[2B4]-[CD3ζ]

[0101] (h)[4-1BB]-[NKG2D]-[DAP12]-[CD3ζ]

[0102] (i) [4-1BB]-[NKG2D]-[4-1BB]-[CD3ζ], and

[0103] (j)[4-1BB]-[NKG2D]-[NTB-A]-[CD3ζ].

[0104] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the antigen binding domain is a domain that binds to a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). In some embodiments, the antigen binding domain is an antigen-binding fragment of an antibody to TSA or TAA, such as an scFv or VHH. Exemplary TSAs or TAAs include, but are not limited to, HER2, PSMA, BCMA, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, Claudin18.2, EpCAM, GD2, MSLN, EGFR, MUC1, EGFRVIII, CD38, Trop-2, c-MET, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA125, MG7-Ag, NY-CO-1, RCAS1, SDCCAG16, TAAL6, and TAG72.

[0105] In one exemplary embodiment, the antigen-binding domain of the chimeric antigen receptor provided herein binds to the HER2 antigen. In such an embodiment, the antigen-binding domain can be an anti-HER2 scFv. The amino acid sequence of an exemplary anti-HER2 scFv is shown in SEQ ID NO. 19.

[0106] In some embodiments, the chimeric antigen receptor provided herein further comprises a signal peptide (SP) at its N-terminus in the antigen-binding domain. The signal peptide facilitates the chimeric antigen receptor to penetrate the cell membrane and expose the antigen-binding domain to the cell surface. In some embodiments, the signal peptide is the signal peptide of CD8α. The amino acid sequence of an exemplary CD8α signal peptide is shown in SEQ ID NO. 20.

[0107] Therefore, in some embodiments, the present invention provides a chimeric antigen receptor, wherein the structure of [antigen binding domain]-[hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor is selected from any one of the following structural formulas, wherein [] represents a domain, and ]-[ represents a connection between domains:

[0108] (a)[SP-scFv]-[4-1BB]-[NKG2D]-[2B4]-[CD3ζ]

[0109] (b)[SP-scFv]-[4-1BB-KIR2DS1]-[KIR2DS1]-[2B4]-[CD3ζ]

[0110] (c)[SP-scFv]-[4-1BB]-[CD3ζ]-[2B4]-[CD3ζ]

[0111] (d)[SP-scFv]-[4-1BB]-[CD28]-[2B4]-[CD3ζ]

[0112] (e)[SP-scFv]-[4-1BB]-[2B4]-[2B4]-[CD3ζ]

[0113] (f)[SP-scFv]-[4-1BB]-[NKP44]-[2B4]-[CD3ζ]

[0114] (g)[SP-scFv]-[4-1BB]-[NKP30]-[2B4]-[CD3ζ]

[0115] (h)[SP-scFv]-[4-1BB]-[NKG2D]-[DAP12]-[CD3ζ]

[0116] (i)[SP-scFv]-[4-1BB]-[NKG2D]-[4-1BB]-[CD3ζ]

[0117] (j) [SP-scFv]-[4-1BB]-[NKG2D]-[NTB-A]-[CD3ζ], and

[0118] (k) [SP-scFv]-[4-1BB-KIR2DS1]-[KIR2DS1]-[CD2]-[CD3ζ].

[0119] In a preferred embodiment, the present invention provides a chimeric antigen receptor, wherein the structure of [antigen binding domain]-[hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor is selected from any one of the following structural formulas, wherein [] represents a domain, and ]-[ represents a connection between domains:

[0120] (a)[SP-scFv]-[4-1BB]-[NKG2D]-[2B4]-[CD3ζ]

[0121] (c)[SP-scFv]-[4-1BB]-[CD3ζ]-[2B4]-[CD3ζ]

[0122] (e)[SP-scFv]-[4-1BB]-[2B4]-[2B4]-[CD3ζ]

[0123] (f)[SP-scFv]-[4-1BB]-[NKP44]-[2B4]-[CD3ζ]

[0124] (h)[SP-scFv]-[4-1BB]-[NKG2D]-[DAP12]-[CD3ζ]

[0125] (i) [SP-scFv]-[4-1BB]-[NKG2D]-[4-1BB]-[CD3ζ], and

[0126] (j) [SP-scFv]-[4-1BB]-[NKG2D]-[NTB-A]-[CD3ζ].

[0127] In some specific embodiments, the present invention provides a chimeric antigen receptor, wherein the structure of [antigen binding domain]-[hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor is selected from any one of the following structural formulas, wherein [] represents a domain, and ]-[ represents a connection between domains:

[0128] (a)[CD8αSP-HER2 scFv]-[4-1BB]-[NKG2D]-[2B4]-[CD3ζ]

[0129] (b)[CD8αSP-HER2 scFv]-[4-1BB-KIR2DS1]-[KIR2DS1]-[2B4]-[CD3ζ]

[0130] (c)[CD8αSP-HER2 scFv]-[4-1BB]-[CD3ζ]-[2B4]-[CD3ζ]

[0131] (d)[CD8αSP-HER2 scFv]-[4-1BB]-[CD28]-[2B4]-[CD3ζ]

[0132] (e)[CD8αSP-HER2 scFv]-[4-1BB]-[2B4]-[2B4]-[CD3ζ]

[0133] (f)[CD8αSP-HER2 scFv]-[4-1BB]-[NKP44]-[2B4]-[CD3ζ]

[0134] (g)[CD8αSP-HER2 scFv]-[4-1BB]-[NKP30]-[2B4]-[CD3ζ]

[0135] (h)[CD8αSP-HER2 scFv]-[4-1BB]-[NKG2D]-[DAP12]-[CD3ζ]

[0136] (i)[CD8αSP-HER2 scFv]-[4-1BB]-[NKG2D]-[4-1BB]-[CD3ζ]

[0137] (j) [CD8αSP-HER2 scFv]-[4-1BB]-[NKG2D]-[NTB-A]-[CD3ζ], and

[0138] (k) [CD8αSP-HER2 scFv]-[4-1BB-KIR2DS1]-[KIR2DS1]-[CD2]-[CD3ζ].

[0139] In any of the above structural formulas, the connection between the domains can be a direct bond or a peptide linker as defined herein, for example a peptide linker composed of glycine (G) and serine (S), such as a G4S linker. In addition, in some embodiments, in a single chimeric antigen receptor, the connection between one or more domains is a direct bond, while the connection between another or other domains is a peptide linker. Those skilled in the art can determine the connection method between the domains based on routine experimentation.

[0140] In some embodiments, the 4-1BB hinge domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 1. In one embodiment, the 4-1BB hinge domain in the chimeric antigen receptor of the present invention is the amino acid sequence shown in SEQ ID NO. 1.

[0141] In some embodiments, the 4-1BB-KIR2DS1 hinge domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 2 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2. In one embodiment, the 4-1BB-KIR2DS1 hinge domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 2.

[0142] In some embodiments, the NKG2D transmembrane domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 5, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 5. In one embodiment, the NKG2D transmembrane domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 5.

[0143] In some embodiments, the KIR2DS1 transmembrane domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 6, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 6. In one embodiment, the KIR2DS1 transmembrane domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 6.

[0144] In some embodiments, the CD3ζ transmembrane domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 7. In one embodiment, the CD3ζ transmembrane domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 7.

[0145] In some embodiments, the CD28 transmembrane domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 8. In one embodiment, the CD28 transmembrane domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 8.

[0146] In some embodiments, the 2B4 transmembrane domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 9, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 9. In one embodiment, the 2B4 transmembrane domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 9.

[0147] In some embodiments, the NKP44 transmembrane domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 10, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 10. In one embodiment, the NKP44 transmembrane domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 10.

[0148] In some embodiments, the NKP34 transmembrane domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 11, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 11. In one embodiment, the NKP34 transmembrane domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 11.

[0149] In some embodiments, the 2B4 costimulatory domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 13, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 13. In one embodiment, the 2B4 costimulatory domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 13.

[0150] In some embodiments, the DAP12 costimulatory domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 14, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 14. In one embodiment, the DAP12 costimulatory domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 14.

[0151] In some embodiments, the 4-1BB costimulatory domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 15. In one embodiment, the 4-1BB costimulatory domain in the chimeric antigen receptor of the present invention is the amino acid sequence shown in SEQ ID NO. 15.

[0152] In some embodiments, the NTB-A costimulatory domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 16. In one embodiment, the NTB-A costimulatory domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 16.

[0153] In some embodiments, the CD2 costimulatory domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 17, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 17. In one embodiment, the CD2 costimulatory domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 17.

[0154] In some embodiments, the CD3 zeta signaling domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 18, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 18. In one embodiment, the CD3 zeta signaling domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 18.

[0155] In some embodiments, at least two (e.g., two, three, or four) of the corresponding hinge domains, transmembrane domains, costimulatory domains, and signaling domains in the chimeric antigen receptor of the present invention have the amino acid sequences described above for the corresponding domains. In some embodiments, the amino acid sequences of the corresponding hinge domains, transmembrane domains, costimulatory domains, and signaling domains in the chimeric antigen receptor of the present invention are the amino acid sequences described above for the corresponding domains. As described above, the connection between the domains can be a direct bond or a peptide linker as defined herein, such as a peptide linker consisting of glycine (G) and serine (S), such as a G4S linker. For example, in the chimeric antigen receptor of the present invention, there is a G4S linker between each domain.

[0156] In a preferred embodiment, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention comprises or is an amino acid sequence as shown in any one of SEQ ID NOs. 49 to 59, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence as shown in any one of SEQ ID NOs. 49 to 59. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is an amino acid sequence as shown in any one of SEQ ID NOs. 49 to 59.

[0157] In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is the amino acid sequence shown in SEQ ID NO. 49. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is the amino acid sequence shown in SEQ ID NO. 50. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is the amino acid sequence shown in SEQ ID NO. 51. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is the amino acid sequence shown in SEQ ID NO. 52. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is the amino acid sequence shown in SEQ ID NO. 53. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is an amino acid sequence as shown in SEQ ID NO. 54. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is an amino acid sequence as shown in SEQ ID NO. 55. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is an amino acid sequence as shown in SEQ ID NO. 56. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is an amino acid sequence as shown in SEQ ID NO. 57. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is an amino acid sequence as shown in SEQ ID NO. 58. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is the amino acid sequence shown in SEQ ID NO.59.

[0158] In some embodiments, the chimeric antigen receptor of the present invention is a HER2-targeting chimeric antigen receptor. In some embodiments, the HER2-targeting chimeric antigen receptor provided by the present invention comprises or is an amino acid sequence as set forth in any one of SEQ ID NOs. 22 to 32, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs. 22 to 32. In some embodiments, the amino acid sequence of the HER2-targeting chimeric antigen receptor of the present invention is set forth in any one of SEQ ID NOs. 22 to 32. In some embodiments, the HER2-targeting chimeric antigen receptor of the present invention comprises or is an amino acid sequence as set forth in SEQ ID NO. 22. In some embodiments, the HER2-targeting chimeric antigen receptor of the present invention comprises or is an amino acid sequence as set forth in SEQ ID NO. 23. In some embodiments, the HER2-targeting chimeric antigen receptor of the present invention comprises or is an amino acid sequence as set forth in SEQ ID NO. 24. In some embodiments, the HER2-targeting chimeric antigen receptor of the present invention comprises or is an amino acid sequence as set forth in SEQ ID NO. 25. In some embodiments, the HER2-targeting chimeric antigen receptor of the present invention comprises or is an amino acid sequence as set forth in SEQ ID NO. 26. In some embodiments, the chimeric antigen receptor targeting HER2 of the present invention comprises or is the amino acid sequence shown in SEQ ID NO.27. In some embodiments, the chimeric antigen receptor targeting HER2 of the present invention comprises or is the amino acid sequence shown in SEQ ID NO.28. In some embodiments, the chimeric antigen receptor targeting HER2 of the present invention comprises or is the amino acid sequence shown in SEQ ID NO.29. In some embodiments, the chimeric antigen receptor targeting HER2 of the present invention comprises or is the amino acid sequence shown in SEQ ID NO.30. In some embodiments, the chimeric antigen receptor targeting HER2 of the present invention comprises or is the amino acid sequence shown in SEQ ID NO.31. In some embodiments, the chimeric antigen receptor targeting HER2 of the present invention comprises or is the amino acid sequence shown in SEQ ID NO.32.

[0159] Other aspects of the present invention provide a chimeric antigen receptor, which comprises, in sequence, an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a signaling domain, wherein the hinge domain comprises or is an IgD hinge domain, the transmembrane domain is an NKG2D or DAP10 transmembrane domain, the costimulatory domain is a 2B4 costimulatory domain, and the signaling domain comprises or is a CD3ζ signaling domain.

[0160] In some embodiments, the present invention provides a chimeric antigen receptor, which comprises an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a signaling domain in sequence, wherein the hinge domain is an IgD hinge domain, the transmembrane domain is an NKG2D or DAP10 transmembrane domain, the costimulatory domain is a 2B4 costimulatory domain, and the signaling domain comprises or is a CD3ζ signaling domain.

[0161] In some embodiments, the present invention provides a chimeric antigen receptor, which comprises an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a signaling domain in sequence, wherein the hinge domain is an IgD-DAP10 chimeric hinge domain, the transmembrane domain is an NKG2D or DAP10 transmembrane domain, the costimulatory domain is a 2B4 costimulatory domain, and the signaling domain comprises or is a CD3ζ signaling domain.

[0162] In some specific embodiments, the present invention provides a chimeric antigen receptor, wherein the structure of [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor is selected from any one of the following structural formulas, wherein [] represents a domain, and ]-[ represents a connection between domains:

[0163] (a) [IgD]-[NKG2D]-[2B4]-[CD3ζ], and

[0164] (b) [IgD-DAP10]-[DAP10]-[2B4]-[CD3ζ].

[0165] In some embodiments, the present invention provides a chimeric antigen receptor, wherein the antigen binding domain is a domain that binds to a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). In some embodiments, the antigen binding domain is an antigen-binding fragment of an antibody to TSA or TAA, such as an scFv or VHH. Exemplary TSAs or TAAs include, but are not limited to, HER2, PSMA, BCMA, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, Claudin18.2, EpCAM, GD2, MSLN, EGFR, MUC1, EGFRVIII, CD38, Trop-2, c-MET, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA125, MG7-Ag, NY-CO-1, RCAS1, SDCCAG16, TAAL6, and TAG72.

[0166] In one exemplary embodiment, the antigen-binding domain of the chimeric antigen receptor provided herein binds to the HER2 antigen. In such an embodiment, the antigen-binding domain can be an anti-HER2 scFv. The amino acid sequence of an exemplary anti-HER2 scFv is shown in SEQ ID NO. 19.

[0167] In some embodiments, the chimeric antigen receptor provided herein further comprises a signal peptide (SP) at its N-terminus in the antigen-binding domain. The signal peptide facilitates the chimeric antigen receptor to penetrate the cell membrane and expose the antigen-binding domain to the cell surface. In some embodiments, the signal peptide is the signal peptide of CD8α. The amino acid sequence of an exemplary CD8α signal peptide is shown in SEQ ID NO. 20.

[0168] Therefore, in some embodiments, the present invention provides a chimeric antigen receptor, wherein the structure of [antigen binding domain]-[hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor is selected from any one of the following structural formulas, wherein [] represents a domain, and ]-[ represents a connection between domains:

[0169] (a) [SP-scFv]-[IgD]-[NKG2D]-[2B4]-[CD3ζ], and

[0170] (b) [SP-scFv]-[IgD-DAP10]-[DAP10]-[2B4]-[CD3ζ].

[0171] In some specific embodiments, the present invention provides a chimeric antigen receptor, wherein the structure of [antigen binding domain]-[hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor is selected from any one of the following structural formulas, wherein [] represents a domain, and ]-[ represents a connection between domains:

[0172] (a) [CD8αSP-HER2 scFv]-[IgD]-[NKG2D]-[2B4]-[CD3ζ], and

[0173] (b) [CD8αSP-HER2 scFv]-[IgD-DAP10]-[DAP10]-[2B4]-[CD3ζ].

[0174] In any of the above structural formulas, the connection between the domains can be a direct bond or a peptide linker as defined herein, for example a peptide linker composed of glycine (G) and serine (S), such as a G4S linker. In addition, in some embodiments, in a single chimeric antigen receptor, the connection between one or more domains is a direct bond, while the connection between another or other domains is a peptide linker. Those skilled in the art can determine the connection method between the domains based on routine experimentation.

[0175] In some embodiments, the IgD hinge domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 3, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 3. In one embodiment, the IgD hinge domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 3.

[0176] In some embodiments, the IgD-DAP10 chimeric hinge domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 4, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 4. In one embodiment, the IgD-DAP10 chimeric hinge domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 4.

[0177] In some embodiments, the NKG2D transmembrane domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 5, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 5. In one embodiment, the NKG2D transmembrane domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 5.

[0178] In some embodiments, the DAP10 transmembrane domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 12, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 12. In one embodiment, the DAP10 transmembrane domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 12.

[0179] In some embodiments, the 2B4 costimulatory domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 13, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 13. In one embodiment, the 2B4 costimulatory domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 13.

[0180] In some embodiments, the CD3 zeta signaling domain in the chimeric antigen receptor of the present invention comprises or is the amino acid sequence set forth in SEQ ID NO. 18, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 18. In one embodiment, the CD3 zeta signaling domain in the chimeric antigen receptor of the present invention is the amino acid sequence set forth in SEQ ID NO. 18.

[0181] In some embodiments, at least two (e.g., two, three, or four) of the corresponding hinge domains, transmembrane domains, costimulatory domains, and signaling domains in the chimeric antigen receptor of the present invention have the amino acid sequences described above for the corresponding domains. In some embodiments, the amino acid sequences of the corresponding hinge domains, transmembrane domains, costimulatory domains, and signaling domains in the chimeric antigen receptor of the present invention are the amino acid sequences described above for the corresponding domains. As described above, the connection between the domains can be a direct bond or a peptide linker as defined herein, such as a peptide linker consisting of glycine (G) and serine (S), such as a G4S linker. For example, in the chimeric antigen receptor of the present invention, there is a G4S linker between each domain.

[0182] In a preferred embodiment, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention comprises or is an amino acid sequence as shown in SEQ ID NO. 60 or 61, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO. 60 or 61. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is an amino acid sequence as shown in SEQ ID NO. 60. In some embodiments, the [hinge domain]-[transmembrane domain]-[costimulatory domain]-[signaling domain] of the chimeric antigen receptor of the present invention is an amino acid sequence as shown in SEQ ID NO. 61.

[0183] In some embodiments, the chimeric antigen receptor of the present invention is a chimeric antigen receptor targeting HER2. In some embodiments, the chimeric antigen receptor targeting HER2 provided by the present invention comprises or is an amino acid sequence as shown in SEQ ID NO. 33 or 34, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO. 33 or 34. In some embodiments, the amino acid sequence of the chimeric antigen receptor targeting HER2 of the present invention is shown in either SEQ ID NO. 33 or 34. In some embodiments, the chimeric antigen receptor targeting HER2 of the present invention comprises or is an amino acid sequence as shown in SEQ ID NO. 33. In some embodiments, the chimeric antigen receptor targeting HER2 of the present invention comprises or is an amino acid sequence as shown in SEQ ID NO. 34.

[0184] Polynucleotides and vectors

[0185] Another aspect of the present invention provides a nucleic acid molecule encoding a chimeric antigen receptor provided by the present invention. As used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably in the present invention to refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides.

[0186] In some embodiments, the present invention provides a polynucleotide encoding a chimeric antigen receptor, which can be any of the chimeric antigen receptors described in the above section "Chimeric Antigen Receptors".

[0187] In some embodiments, the present invention provides DNA encoding a chimeric antigen receptor, which can be any of the chimeric antigen receptors described in the "Chimeric Antigen Receptor" section above.

[0188] In some embodiments, the present invention provides RNA encoding a chimeric antigen receptor, which can be any of the chimeric antigen receptors described in the "Chimeric Antigen Receptors" section above.

[0189] In some embodiments, the present invention provides a polynucleotide encoding a chimeric antigen receptor, which encodes an amino acid sequence as shown in any one of SEQ ID NOs. 22 to 34 or an amino acid sequence having at least 80% sequence identity with any one of them.

[0190] In some embodiments, the polynucleotide provided by the present invention has a nucleotide sequence as shown in any one of SEQ ID NOs. 36 to 48.

[0191] The present invention also provides a vector comprising any one of the above-mentioned polynucleotides. In some embodiments, the vector is a vector for delivery. The present invention also provides a vector for delivering the polynucleotides of the present invention to a subject and / or a subject's cell. Examples of such vectors include, but are not limited to, plasmids, autonomously replicating sequences, transposable elements, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or PI-derived artificial chromosomes (PACs), phages such as lambda phage or M13 phage and viral vectors.

[0192] Examples of classes of animal viruses that can be used as viral vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAV), herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0193] Retrovirus is a common tool for gene delivery. In a specific embodiment, retrovirus is used to deliver the polynucleotides encoding the CAR of the present invention to cells. As used herein, the term "retrovirus" refers to an RNA virus whose genomic RNA is reverse transcribed into a linear double-stranded DNA copy and then its genomic DNA is covalently integrated into the host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus". Provirus serves as a template for RNA polymerase II and guides the expression of RNA molecules, which encode the structural proteins and enzymes required for the production of new viral particles. Illustrative retroviruses suitable for specific embodiments include but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus (spumavirus), Friend murine leukemia virus, mouse stem cell virus (MSCV) and Rous sarcoma virus (RSV) and slow virus.

[0194] The selected nucleic acid sequence can be inserted into a vector and packaged in a retroviral particle using techniques known in the art. The recombinant virus can then be separated and delivered to a cell, for example, in vitro or ex vivo. Standard assays (such as RT-PCR, FACS, Northern blotting, Western blotting, ELISA or immunohistochemistry) for detecting mRNA, DNA or gene products of nucleic acids encoding CAR can be used to assess the efficient expression of any CAR polypeptides described herein.

[0195] Exemplary lentiviral vectors for use in the methods of the present invention comprise at least the following portions of the lentiviral genome: a) a 5' long terminal repeat (LTR); b) a packaging sequence psi; c) a Rev response element (RRE); d) a promoter operably linked to a gene of interest; and e) a 3' long terminal repeat (LTR). In a preferred embodiment, the U3 region of the 5' LTR is replaced with a heterologous promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, or a simian virus 40 (SV40); thereby rendering the lentiviral transcription tat-independent. In another preferred embodiment, the 3' LTR sequence contains a deletion of the U3 region (i.e., the vector is a self-inactivating vector or SIN vector). The lentiviral vector may further comprise a lentiviral central polypurine tract (cPPT) and a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE).

[0196] A transient or stable packaging system can be used for the manufacture of lentiviral vectors. In a transient system, at least the following co-transfection packaging cells (e.g., HEK293 cells or HEK293 T HEK293-SF, TE671, HT1080, or HeLa) are used: a packaging plasmid encoding lentiviral Gag / Pol, a plasmid encoding the target envelope protein, and a transfer plasmid carrying the necessary lentiviral genomic elements and target genes as disclosed above. In another transient system, the lentiviral regulatory protein Rev can be expressed in trans on a fourth separate plasmid. Examples of suitable env genes include, but are not limited to, VSV-G env, MLV4070 env, RD114 env, RD114-TR, RD114pro, baculovirus 5GP64 env, GALV, or an envelope protein derived from measles virus. Co-transfection can be performed using methods well known in the art, such as using calcium phosphate or commercially available preparations. Suitable packaging cell lines for the production of lentiviral vectors are disclosed in, for example, WO 2012 / 028681 or WO 2004 / 022761.

[0197] CAR-NK cells and cell populations

[0198] Another aspect of the present invention provides engineered NK cells that are modified to express a chimeric antigen receptor on the cell surface, wherein the chimeric antigen receptor is any chimeric antigen receptor provided by the present invention. Such engineered NK cells are referred to as CAR-NK cells in the present invention.

[0199] In some embodiments, the present invention provides CAR-NK cells that express any one of the chimeric antigen receptors described in the "Chimeric Antigen Receptor" section on the cell surface.

[0200] In some embodiments, the CAR-NK cells provided by the present invention are derived from NK cells of peripheral blood or cord blood, or from NK cells differentiated from induced pluripotent stem cells, or from the NK92 cell line.

[0201] Another aspect of the present invention provides an NK cell population, which is essentially composed of NK cells and comprises a plurality of modified NK cells according to any of the above embodiments of the present invention.

[0202] The term "substantially consisting of NK cells" means that in a specified number of cell populations, NK cells (including wild-type NK cells and modified NK cells of the present invention) account for no less than about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the cell population.

[0203] In some embodiments, the cell population comprises about 1x10 5 Up to 1x10 7 cells, for example 1x10 5 2x10 5 3x10 5 4x10 5 5x10 5 6x10 5 7x10 5 8x10 5 9x10 5 1x10 6 2x10 6 3x10 6 4x10 6 5x10 6 6x10 6 7x10 6 8x10 6 9x10 6 or 1x10 7 In other embodiments, the cell population contains less than about 1 x 10 5 In other embodiments, the cell population contains more than 1x10 7 cells.

[0204] In some embodiments, in the NK cell population, the modified NK cells account for at least 30% of the cell population, for example, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99%.

[0205] In some embodiments, when the cell population is delivered in vivo, its duration in vivo is at least 7 days, at least 14 days, at least 21 days, at least 28 days, or at least 35 days. Preferably, in some embodiments, when the cell population is delivered in vivo, its duration in vivo is at least 35 days or longer. Preferably, in some embodiments, when the cell population is delivered in vivo, the cell population expands in vivo and continues to expand for at least 7 days, at least 14 days, at least 21 days, at least 28 days, or at least 35 days.

[0206] As described above, the vector of the present invention with the nucleic acid encoding CAR can be introduced into the cell by methods known in the art, non-limiting examples of which include viral transduction, electroporation transfection, liposome delivery, polymer carriers, chemical carriers, lipid complexes, polymer complexes, dendrimers, nanoparticles, emulsions, natural endocytosis or phagocytosis pathways, cell penetrating peptides, microinjection, microneedle delivery, particle bombardment, etc. For example, electroporation transfection can be used.

[0207] In some embodiments, the present invention provides a method for preparing the CAR-NK cells of the present invention, comprising using any of the viral vectors described in the above "Polynucleotides and Vectors" section (e.g., a retroviral vector or an adeno-associated viral vector) to transfect NK cells, so that the polynucleotide encoding the chimeric antigen receptor of the present invention carried by the viral vector is expressed in the NK cells, thereby expressing the chimeric antigen receptor of the present invention on the cell surface. As described above, the transfected NK cells can be NK cells derived from peripheral blood or cord blood, or NK cells differentiated from induced pluripotent stem cells, or derived from the NK92 cell line.

[0208] In the case of using induced pluripotent stem cells, the CAR-NK cells of the present invention can also be prepared by the following method. For example, in some embodiments, the present invention provides a method for preparing CAR-NK cells, comprising transfecting induced pluripotent stem cells using any one of the viral vectors (e.g., retroviral vectors or adeno-associated viral vectors) described in the above "polynucleotides and vectors" subsection, and inducing differentiation of iPSCs into NK cells, wherein the chimeric antigen receptor of the present invention is expressed on the surface of the NK cells.

[0209] Therapies and uses

[0210] Another aspect of the present invention provides a method for treating a tumor in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of any engineered NK cell or cell population described above.

[0211] Another aspect of the present invention provides a method for treating a tumor in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising any engineered NK cell or cell population described above, wherein the administration preferably continues for a time sufficient to treat the tumor.

[0212] The administration of the engineered NK cells or cell groups of the present invention can be carried out in any way, for example, by parenteral or non-parenteral administration, including by aerosol inhalation, injection, infusion, intake, infusion, implantation or transplantation. For example, it can be administered to the patient via artery, intradermal, subcutaneous, intratumor, intramedullary, intranodal, intramuscular, by intravenous (iv) injection or intraperitoneal administration. In one aspect, the cell of the present invention or cell group is administered by iv injection. In one aspect, the cell of the present invention or cell group is administered to the subject by intradermal injection or subcutaneous injection. In some embodiments, the cell of the present invention or cell group can be, for example, directly injected into a tumor, lymph node, tissue, organ or site of infection.

[0213] In some embodiments, the administration can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer. The course of treatment can also be repeated, as in chronic administration. Repeated administration can be of the same dose or of a different dose.

[0214] The cells or cell populations of the invention can be administered in combination with at least one additional therapeutic agent.

[0215] In some embodiments, the at least one additional therapeutic agent is surgery, chemotherapy, immunotherapy, androgen deprivation therapy, radiotherapy, or any combination thereof. In some embodiments, the immunotherapy is selected from anti-PD-1 monoclonal antibody (e.g., Nivolumab or Pembrolizumab), anti-CTLA-4 monoclonal antibody (e.g., Ipilimumab), anti-PD-L1 monoclonal antibody (e.g., Avelumab or Atezolizumab), anti-VEGF monoclonal antibody (Bevacizumab), and combinations thereof.

[0216] In some embodiments, the delivery of one treatment is still ongoing when the delivery of the second treatment begins, so that there is an overlap in the administration. This is sometimes referred to as "simultaneous" or "simultaneous delivery" in this article. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective due to combined administration. For example, compared to a similar situation observed when the second treatment is administered in the absence of the first treatment, or when the first treatment is used, the second treatment is more effective, for example, an equivalent effect is observed when less of the second treatment is used, or the second treatment alleviates the symptoms to a greater extent. In some embodiments, the degree to which the symptoms are alleviated or other parameters associated with the disease are reduced is greater than the degree of alleviation or reduction that would be observed when delivering one treatment without the presence of another treatment. The effects of the two treatments can be partially additive, completely additive, or greater than the result of the addition. The delivery can make the effect of the first treatment delivered still detectable when the second treatment is delivered.

[0217] Accordingly, the present invention further provides the use of any of the engineered NK cells or cell populations described above in the preparation of a drug for treating tumors. The present invention also provides the use of any of the chimeric antigen receptors described above or a polynucleotide encoding the same in the preparation of a drug for treating tumors.

[0218] The present invention also provides any engineered NK cell or cell population described above for use in treating tumors. The present invention also provides any chimeric antigen receptor or polynucleotide encoding the same described above for use in treating tumors.

[0219] In the above embodiments of the therapy and use, the tumor can be a blood tumor or a solid tumor. Exemplary blood tumors include, but are not limited to, leukemia, lymphoma, myelodysplastic syndrome or myeloma, preferably selected from the following leukemia, lymphoma or myeloma: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute T-cell leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute monocytic leukemia (AMoL), mantle cell lymphoma (MCL), histiocytic lymphoma or multiple myeloma, preferably AML.

[0220] Exemplary solid tumors include, but are not limited to, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma or small cell carcinoma, hepatocellular carcinoma, hepatoblastoma, colon adenocarcinoma, renal cell carcinoma, renal cell adenocarcinoma, colorectal cancer, colorectal adenocarcinoma, glioblastoma, glioma, head and neck cancer, lung cancer, breast cancer, Merkel cell carcinoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, lung cancer, kidney cancer, renal adenocarcinoma, breast cancer, breast adenocarcinoma, mammary adenocarcinoma, mammary ductal carcinoma, non-small cell lung cancer, ovarian cancer, oral cancer, anal cancer, skin cancer, Ewing's sarcoma, gastric cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, and ovarian cancer. malignant neoplasms or metastatic-inducing secondary tumors of the following: ovarian adenocarcinoma, ovarian teratoma, ovarian teratoma, bladder papilloma, neuroblastoma, glioblastoma multiforme, glioblastoma, astrocytoma, epithelioid carcinoma, melanoma, or retinoblastoma.

[0221] Sequence Listing

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

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[0244] Example

[0245] Example 1. Synthesis of Her2-CAR and Testing Process of CAR-NK Cells

[0246] The inventors obtained 13 CAR structures targeting HER2 through preliminary screening, named Her2-CAR+number (or CAR+number for short). These CAR structures are shown in Table 1. They have the same signal peptide (CD8α signal peptide, SEQ ID NO.20) and antigen binding domain (anti-HER2 scFv, SEQ ID NO.19, derived from trastuzumab). Her2-CAR1 is derived from the literature and serves as a positive control. Her2-CAR2, 3, 5, 7-11, 13, 19, 21, 23 and 26 are CAR structures designed according to the present invention. SP represents a signal peptide, scFv represents a single-chain variable fragment, Hinge represents a hinge domain, TM represents a transmembrane domain, cSD represents a costimulatory domain, and SD represents a signal transduction domain. The amino acid sequences of these CAR structures are shown in SEQ ID NOs. 21 to 34, respectively, and their nucleotide sequences are shown in SEQ ID NOs. 35 to 48, respectively.

[0247] Table 1. Domain composition of the synthetic CARs of the present invention

[0248]

[0249] The experimental process is as follows Figure 1 As shown, a brief description is given below.

[0250] (1) After 14 retroviral shuttle vector plasmids expressing the Her2-CAR structure were successfully cloned and synthesized, the plasmids were transfected into 293T cells together with the vsvg envelope protein plasmid and the gag-pol plasmid to package the retrovirus expressing the Her2-CAR with the envelope protein being vsvg.

[0251] (2) PG13 cells were infected with the vsvg retrovirus expressing Her2-CAR to obtain a retrovirus whose envelope protein was GaLV and expressed Her2-CAR.

[0252] (3) NK92 cells were infected with GaLV retrovirus expressing Her2-CAR to obtain CAR-expressing NK92 cells.

[0253] (4) Detect the positive rate of CAR molecule expression in NK92 cells.

[0254] (5) Co-culture NK92 cells expressing CAR molecules and tumor target cells (SKOV3 cells expressing luciferase SKOV3-Luci). After 24 hours of co-culture, the intensity of the fluorescent signal released by the tumor cells is detected by in vitro experiments to determine the strength of the CAR-NK92 cells' ability to kill tumor cells, thereby screening out CAR structural molecules that can enhance the killing function of NK cells.

[0255] Example 2. Expression of HER2-CAR and CAR-NK cell killing effect

[0256] Since CAR molecules are artificially synthesized fusion proteins, the protein stability of different CAR molecules may vary greatly. Therefore, we first transfected 14 plasmids expressing CAR molecules into 293T cells, and then detected the expression of CAR molecules in 293T cells by flow cytometry 48 hours later ( Figure 2-Figure 4 ), Her2-CAR 1, 8, 9, 10, 11, 19, 21, and 23 molecules showed good stability and were ideal candidate target CAR molecules. In subsequent experiments, we divided Her2-CAR 1, 8, 9, 10, 11, 19, 21, and 23 into one group for comparative screening, and divided the remaining CAR molecules into two groups for comparative screening.

[0257] First, we transfected 8 retroviral shuttle vector plasmids expressing Her2-CAR 1, 8, 9, 10, 11, 19, 21, and 23, together with the vsvg envelope protein plasmid and gag-pol plasmid, into 293T cells to package and obtain retroviruses expressing the above 8 different Her2-CAR molecules with the envelope protein of vsvg. Then, these 8 vsvg retroviruses were used to infect PG13 cells respectively. After 48 hours, we detected the CAR expression positive rate of the 8 Her2-CARs in PG13 cells ( Figure 5 ), the experimental results showed that all the above 8 different vsvg retroviruses successfully infected PG13 cells, although the positive rate of CAR molecule No. 19 was slightly lower.

[0258] We then expanded the culture of PG13 cells expressing CAR molecules and concentrated the cell culture supernatant to obtain GaLV retroviruses expressing the above 8 different Her2-CAR molecules. These 8 new GaLV retroviruses were then used to infect NK92 cells. One week after NK92 cells were infected with retroviruses, flow cytometry was used to detect the CAR positive rate and CAR protein abundance (MFI) ( Figure 6-Figure 7 ), the experimental results showed that except for the low positive rates of CAR structures 1 and 19, the positive rates of the other CAR structures were all above 30%. Therefore, in the subsequent experiments, we put the six structures of Her2-CAR 8, 9, 10, 11, 21, and 23 in one group for comparison, and put the two structures of Her2-CAR 1 and 19 in another group for comparison. Before the killing experiment, we tested the viability of these 8 NK92 cells expressing CAR molecules ( Figure 8 ), the results showed that the viability of NK92 cells expressing different CAR structures was around 90%, which was comparable to the viability of wild-type untreated NK92 cells, indicating that transfection of NK92 cells with retroviruses expressing CAR molecules would not significantly reduce cell viability.

[0259] We then conducted a tumor cell killing experiment. The target tumor cells were SK-OV-3 cells (human ovarian cancer cells) that were positive for Her2 protein expression and also expressed luciferase. The experimental process was as follows: First, we co-cultured NK92 cells expressing CAR molecules with SK-OV-3 cells. In the experiment, we set up three different NK92 cell and tumor cell culture ratios, namely, effector-target ratios (2:1, 1:1, and 1:2). In theory, the higher the effector-target ratio, the stronger the tumor cell killing ability observed in the end. After 24 hours of co-culture of NK92 cells and SK-OV-3 cells, we performed Killing effect detection, the principle of killing effect detection is as follows: After NK92 cells and SK-OV-3 cells are co-cultured for 24 hours, we add luciferin substrate to the co-cultured cell well plate. The luciferase present in the SK-OV-3 living cells will catalyze the decomposition of the luciferase substrate and then produce fluorescence. Then the fluorescence signal intensity is detected using an instrument. If the fluorescence signal is stronger, it means that there are more living SK-OV-3 cells and the killing effect of NK92 cells is worse. The weaker the fluorescence signal, the fewer living SK-OV-3 cells and the better the killing effect of NK92 cells. In the experiment, WT NK92 cells that do not express CAR molecules are negative control cells. It should be noted that NK92 cells often have a certain background killing ability against tumor cells compared to T cells. The experimental results show that compared with WT NK92 cells, the killing ability of NK92 cells expressing the eight CAR molecules Her2-CAR1, 8, 9, 10, 11, 19, 21, and 23 is significantly improved, and the results obtained under three different effector-target ratio experimental conditions are relatively consistent ( Figure 9-10 ).

[0260] We then screened the remaining CAR structural sequences. We transfected the retroviral shuttle vector plasmids expressing the three structures of Her2-CAR 3, 5, and 7, along with the vsvg envelope protein plasmid and gag-pol plasmid, into 293T cells to package the vsvg retrovirus expressing the three different Her2-CAR molecules. These three retroviruses were then used to infect PG13 cells. After 48 hours, we detected the CAR expression positive rate of the three PG13 cells ( Figure 11 ), the experimental results showed that the above three different vsvg retroviruses all successfully infected PG13 cells, and the CAR positive rates of PG13 cells were all above 50%.

[0261] We then expanded the remaining PG13 cells and concentrated the culture supernatant to obtain GaLV retroviruses expressing the three different Her2-CAR molecules. These three new GaLV retroviruses were then used to infect NK92 cells. One week after NK92 cells were infected with retroviruses, flow cytometry was used to detect the CAR positive rate and CAR protein abundance (MFI) ( Figure 12-13 ), the results showed that the positive rate of Her2-CAR 3, 5, and 7 in NK92 was higher than 20%.

[0262] Before the killing experiment, we performed a viability test on three NK92 cells expressing Her2-CAR 3, 5, and 7 ( Figure 14 ), the results showed that the viability of NK92 cells expressing different CAR structures was around 90%, which was comparable to the viability of wild-type untreated NK92 cells. We then conducted a tumor cell killing experiment, using Her2-positive SK-OV-3 cells (human ovarian cancer cells) expressing luciferase. The experimental results showed that compared with WT NK92 cells, the killing ability of NK92 cells expressing the three CAR molecules Her2-CAR 3, 5, and 7 was significantly improved, and the results obtained under three different effector-target ratio experimental conditions were relatively consistent ( Figure 15 ), among which NK92 cells expressing Her2-CAR 3 had the strongest ability to kill tumor cells, followed by Her2-CAR7, and Her2-CAR 5 was relatively the worst.

[0263] Finally, we screened the remaining three structures Her2-CAR 2, 13, and 26. We transfected the retroviral shuttle vector plasmids expressing Her2-CAR 2, 13, and 26, along with the vsvg envelope protein plasmid and gag-pol plasmid, into 293T cells to package the vsvg retrovirus expressing the three different Her2-CAR molecules. These three retroviruses were then used to infect PG13 cells. After 48 hours, we detected the CAR expression positive rates of the three PG13 cells ( Figure 16 ), the experimental results showed that the above three different vsvg retroviruses successfully infected PG13 cells, and the CAR positive rates of PG13 cells were all above 50%.

[0264] We then expanded the remaining PG13 cells and concentrated the culture supernatant to obtain GaLV retroviruses expressing the above-mentioned Her2-CAR2, 13, and 26 molecules. These three new GaLV retroviruses were then used to infect NK92 cells. One week after NK92 cells were infected with retroviruses, flow cytometry was used to detect the CAR positive rate and CAR protein abundance (MFI) ( Figure 17-18 ), the results showed that the positive rates of the three CAR structures of Her2-CAR 2, 13, and 26 in NK92 were close to or higher than 20%.

[0265] Before the killing experiment, we tested the viability of NK92 cells expressing three structures of Her2-CAR 2, 13, and 26 ( Figure 19 ), the results showed that the viability of NK92 cells expressing different CAR structures was around 90%, which was comparable to the viability of wild-type untreated NK92 cells. We then conducted a tumor cell killing experiment, using Her2-positive SK-OV-3 cells (human ovarian cancer cells) expressing luciferase. The experimental results showed that compared with WT NK92 cells, the ability of NK92 cells expressing the three CAR molecules Her2-CAR 2, 13, and 26 to kill tumor cells was significantly improved, and the results obtained under three different effector-target ratio experimental conditions were relatively consistent ( Figure 20 ), among which NK92 cells expressing Her2-CAR 13 had the strongest ability to kill tumor cells, followed by Her2-CAR 2, and Her2-CAR 26 was relatively the worst.

[0266] Through experiments, we successfully screened 13 new CAR structures (Her2-CAR 2, 3, 5, 7, 8, 9, 10, 11, 13, 19, 21, 23, 26) that can significantly enhance the function of NK cells. Previous studies have shown that CAR molecules containing the intracellular co-stimulatory domain of the 2B4 protein can significantly enhance the killing function of NK cells. This study shows that even if the CAR molecule does not use the co-stimulatory domain of the 2B4 protein but uses other proteins such as NTB-A (Her2-CAR 23) or CD2 (Her2-CAR26), it can also well enhance the killing function of NK cells, indicating that the intracellular co-stimulatory domain of the 2B4 protein is not irreplaceable. In addition, the present invention also found that the CAR structure using the 4-1BB hinge domain or the IgD hinge domain can be stably expressed in NK cells, and the obtained CAR-NK has a strong killing effect on target cells.

Claims

1. A chimeric antigen receptor comprising, in sequence, a signal peptide, an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and a signaling domain, wherein the chimeric antigen receptor has a structural formula of [CD8α signal peptide]-[HER2scFv]-[4-1BB]-[CD3ζ]-[2B4]-[CD3ζ], wherein [] represents a structural domain, and ]-[ represents a connection between structural domains, wherein the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:

24.

2. An engineered NK cell, which is modified to express a chimeric antigen receptor on the cell surface, wherein the chimeric antigen receptor is the chimeric antigen receptor of claim 1; wherein the NK cell is an NK cell differentiated from induced pluripotent stem cells, an NK cell derived from peripheral blood or umbilical cord blood, or an NK92 cell line.

3. An engineered NK cell population comprising the engineered NK cells of claim 2.

4. The engineered NK cell population of claim 3, wherein the cell population comprises 1x10 5 to 1x10 7 cells.

5. A polynucleotide encoding the chimeric antigen receptor according to claim 1.

6. The polynucleotide according to claim 5, whose nucleotide sequence is shown in SEQ ID NO:

38. A vector comprising the polynucleotide according to claim 5 or 6.

8. The vector according to claim 7, wherein the vector is a viral vector.

9. The vector according to claim 8, wherein the viral vector is a retroviral or adeno-associated viral vector.

10. The vector according to claim 9, wherein the vector comprises an expression control sequence.

11. The vector of claim 10, wherein the regulatory sequence comprises a promoter.

12. A method for preparing engineered NK cells, comprising transfecting NK cells with the viral vector of claim 11, causing the polynucleotide to be expressed in the NK cells, thereby expressing the chimeric antigen receptor of claim 1 on the cell surface.

13. The method according to claim 12, wherein the NK cells are NK cells differentiated from induced pluripotent stem cells, NK cells derived from peripheral blood or cord blood, or NK92 cell lines.

14. A method for preparing engineered NK cells, comprising transfecting induced pluripotent stem cells (iPSCs) with the viral vector of claim 11, and inducing differentiation of the iPSCs into NK cells, wherein the chimeric antigen receptor of claim 1 is expressed on the surface of the NK cells.

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