Chimeric antigen receptor targeting CD33 and mesothelin double targets and application thereof

Abstract

The invention provides a chimeric antigen receptor targeting CD33 and mesothelin double target spots and an application of the chimeric antigen receptor. On the basis of a double-target CAR design principle, the double-target CAR is constructed by utilizing two scFvs of CD33 and MSLN for the first time, and the double-target CAR can target tumor cells which only express CD33 or MSLN and also can kill tumor cells which express MSLN and CD33 more effectively. Meanwhile, CAR-T, CAR-NK, CAR-Macrophage and the like which are prepared by expressing the MSLN-CD33-CAR in immune cells can be used for killing cells expressing the CD33 or / and the MSLN in a targeted manner. Specifically, the CD33-MSLN-CAR is designed by using cord blood NK cells and ESC expression, and it is verified for the first time that the prepared CD33-MSLN-CAR UCB-NK cells have the advantage of targeted killing of tumor cells expressing CD33 and / or MSLN; it is verified that the prepared CD33-MSLN-CAR-ESC can be directionally differentiated into CD33-MSLN-CAR iNK cells, and the CD33-MSLN-CAR-ESC also has the advantage of targeted killing of tumor cells expressing CD33 and / or MSLN.

Description

Technical Field

[0001] The present invention belongs to the field of immune cell therapy and relates to a dual-targeted chimeric antigen receptor, in particular to a chimeric antigen receptor targeting CD33 and mesothelin dual targets and applications thereof. Background Art

[0002] The application of chimeric antigen receptor (CAR) technology has greatly improved the accuracy and effectiveness of immune cell therapy for tumors. A representative case is the expression of CAR elements in T cells. T cells expressing CAR are called Chimeric Antigen Receptor T (CAR-T), which can target and kill cells expressing target antigens. Similarly, CAR elements can also be expressed in NK cells and prepared into CAR-NK cells, which can also target cells expressing target antigens. This technology has played a positive role in the treatment of tumors, autoimmune diseases, etc.

[0003] The CAR element is mainly composed of the signal peptide (SP) of the antibody, a single chain fragment variable (scFv), a hinge, a transmembrane region, and an intracellular region. Among them, scFv is derived from the variable regions of the light chain (VL) and the variable regions of the heavy chain (VH) of the antibody, and the two chains are connected by a linker to form a single chain antibody. Therefore, the type of antigen epitope bound by the CAR molecule is determined by the scFv. Different scFvs bind to different antigen epitopes. CARs targeting different antigen epitopes can have the same components except for different scFvs. At present, hundreds of CARs have been designed for antigens of blood tumors and solid tumors, including CARs targeting single antigens (single target CARs) and CARs targeting dual antigens (dual target CARs). Among them, CD19-CAR targeting the CD19 antigen of B cells has become a tumor target for clinical treatment.

[0004] CD33 is a membrane protein expressed on myeloid cells and a key protein for identifying myeloid cells. At present, CAR designed for CD33 protein can effectively kill myeloid leukemia cells expressing CD33 after being prepared into CAR-T cells and CAR-NK cells, and has also shown certain positive effects in clinical treatment. However, since NK cells partially express CD33, there will be fratricide and reduced amplification efficiency in the preparation of CD33-CAR-NK. This is also a problem that needs to be solved in the preparation of CD33-CAR-NK using natural NK cells. Mesothelin (MSLN) is a protein expressed on the mesothelial cell membrane and is highly expressed in tumor cells of some solid tumors, such as mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, bile duct epithelial carcinoma, etc. At present, preclinical trials have shown that the use of MSLN-CAR-NK cells can effectively kill ovarian cancer cells and prolong the survival time of mouse tumor models constructed with human ovarian cancer. And the use of MSLN-CAR-NK cells to treat ovarian cancer has entered the clinical trial stage. The use of CAR technology to treat myeloid leukemia is a promising treatment. In addition to CD33, CLL1, and other target membrane proteins, MSLN protein is a newly discovered molecule expressed on the diseased cells of some acute myeloid leukemia (AML) patients, accounting for about 36% of the total, and the expression value of MSLN detected by transcripts is about 5-1077.6 per million TPM, so MSLN is also a unique target protein for targeting AML. Studies have shown that MSLN-CAR-T cells and MSLN-CAR-NK cells can effectively kill AML cell lines expressing MSLN. And they can effectively eliminate tumor cells in tumor model mice and inhibit tumor progression. However, since the MSLN protein will be cut off from the cell membrane to form free MSLN, when using immune cells expressing MSLN-CAR to target AML cells expressing MSLN, the binding efficiency will be reduced, weakening the killing effect. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a chimeric antigen receptor targeting CD33 and mesothelin and its application.

[0006] In a first aspect of the present invention, a chimeric antigen receptor targeting CD33 and mesothelin dual targets is provided, characterized in that the chimeric antigen receptor comprises a leader peptide (signal peptide, SP), an antigen binding region, a hinge region (Hinge), a transmembrane region (Transmembrane, TM), and an intracellular region (Activation) connected in sequence, and the antigen binding region comprises a first antigen binding region targeting CD33 and a second antigen binding region targeting mesothelin (mesothelin, MSLN).

[0007] Furthermore, the amino acid sequence of the signal peptide (SP) is shown in SEQ ID NO.10.

[0008] Furthermore, the encoding nucleotide sequence of the signal peptide (SP) is shown in SEQ ID NO.23.

[0009] Furthermore, the hinge region includes but is not limited to the hinge region of CD8, CD28, IgG1 or IgG4 protein, preferably the CD8 hinge region (CD8 hinge).

[0010] Furthermore, the amino acid sequence of the CD8 hinge region (CD8 hinge) is shown in SEQ ID NO.11.

[0011] Furthermore, the nucleotide sequence encoding the CD8 hinge region (CD8 hinge) is shown in SEQ ID NO.24.

[0012] Furthermore, the transmembrane region includes but is not limited to the transmembrane region of CD4, CD8, CD8a, CD28, NKG2D, CD3ζ (CD247) or ICOS protein, preferably the CD8 transmembrane region (CD8 TM).

[0013] Furthermore, the amino acid sequence of the CD8 transmembrane region (CD8 TM) is shown in SEQ ID NO.12.

[0014] Furthermore, the encoding nucleotide sequence of the CD8 transmembrane region (CD8 TM) is shown in SEQ ID NO.25.

[0015] Furthermore, the intracellular region includes but is not limited to CD3ζ (CD247) or FCER1G, preferably CD3ζ.

[0016] Furthermore, the amino acid sequence of CD3ζ is shown in SEQ ID NO.13.

[0017] Furthermore, the coding nucleotide sequence of CD3ζ is shown in SEQ ID NO.26.

[0018] Furthermore, the chimeric antigen receptor also includes a co-stimulatory region (co-stimulatory signal transmission region, Co-stimulation).

[0019] Furthermore, the co-stimulatory region is selected from one or a combination of two of 4-1BB, CD28, OX40, 2B4 and CD27.

[0020] Furthermore, the chimeric antigen receptor also includes a cytokine or protein ligand that is beneficial to the survival and activation of T cells or NK cells.

[0021] Furthermore, the cytokines or protein ligands that are beneficial to the survival and activation of T cells or NK cells include but are not limited to CXCR1, CXCR, PDL1, IL-2 or IL-15.

[0022] Furthermore, the antigen binding region has the following specific structure:

[0023] T1-scFv: From N-terminus to C-terminus, the VL of CD33 scFv (CD33 scFv-VL) was first connected to the VH of CD33 scFV (CD33 scFv-VH) through linker 1 to form CD33 scFv, and the VL of MSLN scFv (MSLNscFv-VL) was connected to the VH of MSLN scFV (MSLN-scFv-VH) through linker 3 to form MSLN scFv. CD33 scFv is then connected to MSLN scFv through linker 2 to form a CD33 scFv-VL-Linker 1-CD33scFv-VH-Linker 2-MSLN scFv-VL-MSLN scFv-VL-Linker 3-MSLN-scFv-VH (CD33 scFv-linker 2-MSLN scFv) structure. This structure is called Tandem, i.e. CD33-linker 2-MSLN TanscFv, abbreviated as T1-scFv.

[0024] Furthermore, the antigen binding region has the following specific structure:

[0025] T2-scFv: From N-terminus to C-terminus, the VL of MSLN scFv (MSLN scFv-VL) was first connected to the VH of MSLN scFV (MSLN-scFv-VH) through linker 3 to form MSLN scFv, and the VL of CD33 scFv (CD33scFv-VL) was connected to the VH of CD33 scFV (CD33 scFv-VH) through linker 1 to form CD33 scFv. The MSLN scFv fragment is then connected to CD33 scFv through linker 2 to form an MSLN scFv-VL-MSLNscFv-VL-Linker 3-MSLN-scFv-VH-Linker 2-CD33 scFv-VL-Linker 1-CD33 scFv-VH (MSLN scFv-linker 2-CD33 scFv) structure. This structure is called Tandem, i.e. MSLN-linker2-CD33 Tan scFv, abbreviated as T2-scFv.

[0026] Furthermore, the antigen binding region has the following specific structure:

[0027] L1-scFv: From N-terminus to C-terminus, the VL of CD33 scFv (CD33 scFv-VL) is linked to the VH of MSLN scFV (MSLN scFv-VH) through linker 4 to form CD33 VL-linker 4-MSLN VH, the VL of MSLN scFv (MSLN scFv-VL) is linked to the VH of CD33 scFv (CD33 scFv-VH) through linker 4 to form MSLN VL-linker 4-CD33 VH, and then CD33 VL-linker 4-MSLN VH and MSLN VL-linker 4-CD33 VH are linked through Linker 5 to form CD33 scFv-VL-Linker 4-MSLN scFv-VH-Linker 5-MSLN scFv-VL-Linker 4-CD33 scFv-VH structure, the existence form of this structure is called loop, namely CD33-Linker 5-MSLN Loop scFv, abbreviated as: L1-scFv.

[0028] Furthermore, the antigen binding region has the following specific structure:

[0029] L2-scFv: From N-terminus to C-terminus, the VH of MSLN scFv (MSLN scFv-VH) was linked to the VL of CD33 scFv (CD33 scFv-VL) through linker 4 to form MSLN VH-linker 4-CD33 VL, the VH of CD33scFv (CD33 scFv-VH) was linked to the VL of MSLN (MSLN scFv-VL) through linker 4 to form CD33VH-linker 4-MSLN VL, and then the MSLN VH-linker 4-CD33 VL and CD33 VH-linker4-MSLN VL were linked through Linker 5 to form MSLN scFv-VH-linker 4-CD33 scFv-VL-Linker 5-scFv-CD33VH-linker 4-scFv-MSLN VL structure, the existence form of this structure is called loop, that is, MSLN-Linker 5-CD33Loop scFv. Abbreviation: L2-scFv.

[0030] Furthermore, the CD33 scFv is a single-chain antibody sequence from any source that can bind to CD33 protein, including but not limited to: GO (hP67.6), lintuzumab (SGN-33), and M195.34.

[0031] Furthermore, the MSLN scFv is a single-chain antibody sequence from any source that can bind to the MSLN protein, including but not limited to: YP218, hYP218, clone SS, Ms501, Ms503, C2G4, and MX75.

[0032] Furthermore, the amino acid sequence of the CD33 scFv-VL is shown in SEQ ID NO.1.

[0033] Furthermore, the encoding nucleotide sequence of the CD33 scFv-VL is shown in SEQ ID NO.14.

[0034] Furthermore, the amino acid sequence of the CD33 scFv-VH is shown in SEQ ID NO.2.

[0035] Furthermore, the encoding nucleotide sequence of the CD33 scFv-VH is shown in SEQ ID NO.15.

[0036] Furthermore, the amino acid sequence of the MSLN scFv-VL is shown in SEQ ID NO.3.

[0037] Furthermore, the encoding nucleotide sequence of the MSLN scFv-VL is shown in SEQ ID NO.16.

[0038] Furthermore, the amino acid sequence of the MSLN scFv-VH is shown in SEQ ID NO.4.

[0039] Furthermore, the encoding nucleotide sequence of the MSLN scFv-VH is shown in SEQ ID NO.17.

[0040] Furthermore, the types of the linkers 1-5 include but are not limited to flexible or rigid.

[0041] Furthermore, the amino acid sequence of the linker 1 is shown in SEQ ID NO.5.

[0042] Furthermore, the encoding nucleotide sequence of linker 1 is shown in SEQ ID NO.18.

[0043] Furthermore, the amino acid sequence of linker 2 is shown in SEQ ID NO.6.

[0044] Furthermore, the encoding nucleotide sequence of linker 2 is shown in SEQ ID NO.19.

[0045] Furthermore, the amino acid sequence of linker 3 is shown in SEQ ID NO.7.

[0046] Furthermore, the encoding nucleotide sequence of linker 3 is shown in SEQ ID NO.20.

[0047] Furthermore, the amino acid sequence of linker 4 is shown in SEQ ID NO.8.

[0048] Furthermore, the encoding nucleotide sequence of linker 4 is shown in SEQ ID NO.21.

[0049] Furthermore, the amino acid sequence of linker 5 is shown in SEQ ID NO.9.

[0050] Furthermore, the encoding nucleotide sequence of linker 5 is shown in SEQ ID NO.22.

[0051] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.27-30.

[0052] In the second aspect of the present invention, a nucleic acid is provided, characterized in that the nucleic acid encodes the chimeric antigen receptor described in the first aspect of the present invention.

[0053] Furthermore, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.31-34.

[0054] In the third aspect of the present invention, a recombinant vector is provided, characterized in that it comprises the nucleic acid described in the second aspect of the present invention.

[0055] Furthermore, the recombinant vector includes but is not limited to lentivirus, retrovirus expression plasmid, and transposon system expression plasmid.

[0056] In the fourth aspect of the present invention, a recombinant cell is provided, wherein the cell expresses the chimeric antigen receptor described in the first aspect of the present invention or contains the nucleic acid described in the second aspect of the present invention or contains the recombinant vector described in the third aspect of the present invention.

[0057] Furthermore, the expression method includes but is not limited to constitutive expression, endogenous gene-driven expression, and small molecule compound or drug-induced expression.

[0058] Furthermore, the method of delivering the recombinant vector into the recombinant cell includes but is not limited to lentiviral infection, retroviral infection, adenoviral infection, Sendai virus infection, electrofection method, and chemical method.

[0059] Furthermore, the recombinant cells include recombinant immune cells or recombinant pluripotent stem cells.

[0060] Furthermore, the types of the recombinant immune cells include but are not limited to T cells, NK cells, and macrophages; preferably NK cells.

[0061] Furthermore, the types of recombinant pluripotent stem cells include but are not limited to embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), hematopoietic progenitor cells (HPC), lymphocyte progenitor cells (CLP), and common myeloid progenitor cells (CMP); preferably, embryonic stem cells.

[0062] In the fifth aspect of the present invention, an induced immune cell expressing the chimeric antigen receptor described in the first aspect of the present invention is provided, characterized in that the induced immune cell is prepared by induction of the recombinant pluripotent stem cell described in the fourth aspect of the present invention.

[0063] Furthermore, the induced immune cells include but are not limited to induced T cells (iT), induced NK cells (iNK), and induced macrophages; preferably, induced NK cells (iNK).

[0064] Furthermore, the induction method is in vitro induction or in vivo induction.

[0065] Furthermore, the in vitro induction method includes a monolayer induction method, an organoid induction method, or a combination of the two.

[0066] Furthermore, the in vivo induction method includes but is not limited to the human body and a bioreactor.

[0067] In the sixth aspect of the present invention, a pharmaceutical composition is provided, characterized in that the pharmaceutical composition contains the recombinant cells described in the fourth aspect of the present invention or the induced immune cells described in the fifth aspect of the present invention and a pharmaceutically acceptable carrier or excipient.

[0068] Furthermore, the pharmaceutical composition is an injection, a microneedle, a mucosal patch, an enema, a suppository, a gel, an oral agent, an aerosol, a drop, an ointment, an implant, a capsule or an aerosol.

[0069] In the seventh aspect of the present invention, provided is the use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant cell described in the fourth aspect of the present invention, the induced immune cell described in the fifth aspect of the present invention, and the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of anti-tumor drugs.

[0070] Furthermore, the tumor is a blood tumor or a solid tumor.

[0071] Furthermore, the blood tumor includes myeloid leukemia, T cell leukemia, B cell leukemia, T cell lymphoma, B cell lymphoma and the like.

[0072] Furthermore, the solid tumors include mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, bile duct epithelial carcinoma, breast cancer, gastric cancer, kidney cancer, intestinal cancer, liver cancer, glioma, etc.

[0073] In the eighth aspect of the present invention, a method for treating tumors is provided, which comprises the step of administering or transplanting a therapeutically effective amount of the recombinant cells described in the fourth aspect of the present invention, the induced immune cells described in the fifth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention to a subject in need thereof.

[0074] Furthermore, the tumor is a blood tumor or a solid tumor.

[0075] Furthermore, the blood tumor includes myeloid leukemia, T cell leukemia, B cell leukemia, T cell lymphoma, B cell lymphoma and the like.

[0076] Furthermore, the solid tumors include mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, bile duct epithelial carcinoma, breast cancer, gastric cancer, kidney cancer, intestinal cancer, liver cancer, glioma, etc.

[0077] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0078] Compared with the prior art, the present invention has the following advantages and progress:

[0079] The present invention is based on the dual-target CAR design principle, and for the first time uses two scFvs, CD33 and MSLN, to construct a dual-target CAR. In addition to being able to target tumor cells that only express CD33 or MSLN, this dual-target CAR can also more effectively kill tumor cells such as AML that express MSLN and CD33. At the same time, the MSLN-CD33-CAR structure can target both MSLN and CD33, and the CAR-T, CAR-NK, and CAR-Macrophage prepared by expressing MSLN-CD33-CAR in immune cells (T, NK, Macrophage) can target and kill cells that express CD33 or / and MSLN. On the other hand, the present invention uses umbilical cord blood NK cells (UCB-NK) and ESC to express the designed CD33-MSLN-CAR, and verifies for the first time that the prepared CD33-MSLN-CAR UCB-NK cells have the advantage of targeted killing of tumor cells expressing CD33 and / or MSLN; it verifies that the prepared CD33-MSLN-CAR-ESC can be directed to differentiate into CD33-MSLN-CAR iNK cells, and also has the advantage of targeted killing of tumor cells expressing CD33 and / or MSLN. Specifically, the MSLN-CD33-CAR-NK cells prepared by the present invention have a stronger killing ability for tumor cells expressing CD33 and MSLN; compared with CD33-CAR-NK cells, they have better amplification ability; compared with CD33-CAR-NK cells, their CAR expression ratio will not decrease. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0081] Figure 1 Four different CD33-MSLN-CAR structures (T1-CAR, T2-CAR, L1-CAR, L2-CAR) constructed based on the first-generation CAR structure using four scFvs (T1-scFv, T2-scFv, L1-scFv, L2-scFv);

[0082] Figure 2 NK cells expressing T1-CAR, T2-CAR, L1-CAR, L2-CAR (T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, L2-CAR-NK), respectively, are shown;

[0083] Figure 3It was shown that T1-CAR-NK cells, T2-CAR-NK cells, L1-CAR-NK cells, and L2-CAR-NK cells (Effector, E) can effectively kill the Nomo-1 acute myeloid tumor cell line (Target, T) expressing MSLN and CD33 proteins;

[0084] Figure 4 It was shown that L1-CAR-NK cells were more effective than CD33 CAR-NK cells in killing the Nomo-1 acute myeloid tumor cell line (Target, T) expressing MSLN and CD33 proteins.

[0085] Figure 5 It was shown that L1-CAR-NK cells (Effector, E) were more effective in killing acute myeloid leukemia patient #1 cells (Target, T) expressing MSLN and CD33 proteins than CD33 CAR-iNK cells.

[0086] Figure 6 It was shown that L1-CAR-NK cells (Effector, E) were more effective in killing acute myeloid leukemia patient #2 cells (Target, T) expressing MSLN and CD33 proteins than CD33 CAR-iNK cells.

[0087] Figure 7 It was shown that L1-CAR-NK cells (Effector, E) were more effective in killing acute myeloid leukemia patient #3 cells (Target, T) expressing MSLN and CD33 proteins than CD33 CAR-iNK cells.

[0088] Figure 8 It was shown that the CAR expression of L1-CAR-NK cells during continuous culture was more stable than that of CD33 CAR-NK cells, and the expression stability was similar to that of MSLN CAR;

[0089] Fig. 9 It showed that the expansion capacity of L1-CAR-NK cells was stronger than that of CD33-CAR-NK cells and similar to that of MSLN CAR;

[0090] Fig.10 Shown are ESC cells expressing L1-CAR (L1-CAR-ESC);

[0091] Fig.11 It was shown that L1-CAR-ESC cells can be differentiated into iNK cells expressing L1-CAR (L1-CAR-iNK);

[0092] Fig.12 It was shown that L1-CAR-iNK cells (Effector, E) were more effective in killing Nomo-1 tumor cell lines (Target, T) expressing MSLN and CD33 proteins than CD33 CAR-iNK cells.

[0093] Fig.13 It was shown that L1-CAR-iNK cells (Effector, E) could more effectively kill acute myeloid leukemia patient #1 cells (Target, T) expressing MSLN and CD33 proteins compared with CD33 CAR-iNK cells.

[0094] Fig.14 It was shown that L1-CAR-iNK cells (Effector, E) were more effective in killing acute myeloid leukemia patient #2 cells (Target, T) expressing MSLN and CD33 proteins than CD33 CAR-iNK cells.

[0095] Fig.15 It was shown that L1-CAR-iNK cells (Effector, E) could more effectively kill acute myeloid leukemia patient #3 cells (Target, T) expressing MSLN and CD33 proteins compared with CD33 CAR-iNK cells.

[0096] Fig.16 It was shown that L1-CAR-iNK cells could effectively inhibit tumor progression in the HL-60 tumor model expressing MSLN and CD33 proteins. DETAILED DESCRIPTION

[0097] The present invention provides a chimeric antigen receptor targeting CD33 and mesothelin dual targets and its application. The present invention is specifically described below in conjunction with the embodiments to facilitate further understanding of the present invention by those skilled in the art. However, the embodiments described below are only part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by ordinary technicians in this field based on the concept of the present invention should be regarded as the scope of protection of the present invention. The specific technical operation steps and operators are not indicated in the embodiments, and they are all carried out according to the general technical conditions described in the literature of the field or the relevant product instructions.

[0098] Example 1

[0099] This example lists the amino acid sequences and encoding nucleotide sequences of proteins of each component structure of T1-scFv, T2-scFv, L1-scFv, L2-scFv and chimeric antigen receptor, as shown in Table 1 (amino acid sequence) and Table 2 (nucleotide sequence), respectively.

[0100] Table 1: Amino acid sequences of the components of the chimeric antigen receptor

[0101]

[0102] Table 2: Nucleotide sequences encoding components of chimeric antigen receptors

[0103]

[0104]

[0105] Example 2

[0106] This example uses four CD33 and MSLN-targeting scFvs designed in the present invention: T1-scFv (CD33 scFv-VL-Linker 1-CD33 scFv-VH-Linker 2-MSLN scFv-VL-MSLN scFv-VL-Linker 3-MSLN-scFv-VH), T2-scFv (MSLN scFv-VL-MSLN scFv-VL-Linker 3-MSLN-scFv-VH-Linker 2-CD33scFv-VL-Linker 1-CD33 scFv-VH), L1-scFv (CD33 scFv-VL-Linker 4-MSLN scFv-VH-Linker 5-MSLN scFv-VL-Linker 4-CD33 scFv-VH), and L2-scFv (MSLN scFv-VH-linker 4-CD33 scFv-VL-Linker 5-scFv-CD33 VH-linker 4-scFv-MSLN VL) respectively with the lead peptide (signal peptide, SP) and CD8 hinge, CD8 TM, CD3ζ in the first-generation CAR structure to form four types of first-generation CAR structures, specifically T1-CAR (amino acid sequence as shown in SEQ ID NO.27: MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVGGGGSGGGGSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIKGGGGSGGGGSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGVGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), T2-CAR (amino acid sequence as SEQ ID NO.28: MALPVTALLLPLALLLHAARPDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIKGGGGSGGGGSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGVGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), L1-CAR (amino acid sequence as SEQ ID NO.29: MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSGGGGSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGVGGSGSTSGSGKPGSGEGSTKGDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIKGGGGSDIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), and L2-CAR (amino acid sequence as SEQ ID NO.30: MALPVTALLLPLALLLHAARPQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGVGGSGGGGSEVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). Simulate the structure of CAR on the cell membrane to form as. Figure 1The four types of CAR targeting CD33 and MSLN shown. The nucleotide sequences corresponding to the above structures were assembled together and handed over to the company (Shanghai Sangon Biotechnology Co., Ltd.) for gene synthesis. The specific composition method is to connect the leader peptide (signal peptide, SP) in the CAR structure to the 5' end of the four types of T1-scFv, T2-scFv, L1-scFv and L2-scFv designed in the first step, respectively, to form SP-scFv (SP-T1-scFv, SP-T2-scFv, SP-L1-scFv, SP-L2-scFv), and then the 3' end of the SP-scFv sequence is sequentially connected to CD8 hinge (CD8 hinge), CD8 transmembrane domain (CD8TM), CD3ζ to form four CAR structures, specifically SP-T1-scFv-CD8 hinge-CD8 TM-CD3ζ (T1-CAR, nucleotide sequence as SEQ ID NO.31:

[0107]

[0108] Example 3

[0109] Retrovirus was packaged using a retroviral expression vector with T1-CAR, T2-CAR, L1-CAR, and L2-CAR, and NK cells (UCBNK) derived from umbilical cord blood (UCB) were infected with the virus to prepare MSLN-CD33-CAR-NK cells (T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, L2-CAR-NK). The specific implementation is as follows: first, a retroviral expression vector expressing CAR was constructed, and first, the T1-CAR, T2-CAR, L1-CAR and L2-CAR sequences synthesized in Example 1 were constructed into SFG (addgene, Catalog # 22493) vectors by homologous recombination to form SFG-T1-CAR, SFG-T2-CAR, SFG-L1-CAR and SFG-L2-CAR expression plasmids, and the homologous recombination method was derived from a commercial universal one-step seamless cloning kit (Yisheng Biology, 10923ES), and the operation method was performed according to the kit instructions. Then, the constructed SFG-T1-CAR, SFG-T2-CAR, SFG-L1-CAR and SFG-L2-CAR expression plasmids were used to package retroviruses. For specific packaging methods, see Wang, Y., et al., Comparison of seven CD19 CAR designs in engineering NK cells for enhancing anti-tumour activity. Cell Prolif, 2024. 57(11): p.e13683. Finally, the virus was used to infect NK cells. First, NK cells in umbilical cord blood were isolated, and the isolated UCBNK cells were activated with K562-mIL21. After 6 days of activation, the retrovirus carrying CAR was introduced into NK cells by centrifugal infection. For specific methods, see Wang, Y., et al., Comparison of seven CD19 CAR designs in engineering NK cells for enhancing anti-tumour activity. Cell Prolif, 2024. 57(11): p.e13683.48 hours after infection, the expression ratio of T1-CAR, T2-CAR, L1-CAR, and L2-CAR in CD56+ (NK) cells was detected by flow cytometry. The infected NK cells were collected and added with flow cytometry antibodies CD56-APC (brand: biolegend, catalog number: 985906), CD33 CAR-FITC (brand: Acro, catalog number: CD3-HF224), and MSLN CAR-PE (brand: Acro, catalog number: MSN-HP2H5). After incubation on ice for 30 minutes, 1 ml of PBS was added, and the supernatant was discarded after centrifugation at 500g. The cells were resuspended in PBS and detected by flow cytometer (BD LSRFortessa X-20cytometer (BD Biosciences)). The results are as follows. Figure 2 As shown. T1-CAR positive rate: 38.0%, T2-CAR positive rate: 46.8%, L1-CAR positive rate: 43.4%, T2-CAR positive rate: 44.0%. According to the above experimental results, the above CARs can be stably expressed in NK cells.

[0110] Example 4

[0111] This example verifies that T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, L2-CAR-NK cells can specifically kill blood tumor cells (Nomo-1) expressing CD33 and MSLN antigens in vitro. The T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, L2-CAR-NK cells obtained in Example 3 were co-cultured with Nomo-1 tumor cells at different ratios to evaluate the ability of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, L2-CAR-NK cells to specifically kill tumor cells (killing efficiency, Cytotoxicity). The specific results are as follows: Figure 3As shown, the X-axis represents different E:T ratios (CAR-NK cells (effector cells, Effector, E): tumor cells (Target, T)); the Y-axis shows the percentage of tumor cell death to tumor cell number; after 4 hours of co-incubation (Hours), the ratio of dead tumor cells to tumor cells in each sample was detected by flow cytometry, and this ratio is the killing ability (Cytotoxicity%) of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells. When E:T=0.2:1, the average killing efficiency of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells were 5.0%, 10.0%, 13.1%, and 13.2%, respectively; when E:T=0.4:1, the average killing efficiency of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells were 8.6%, 14.0%, 22.3%, and 22.1%, respectively; When E:T=0.8:1, the killing efficiency of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells is 11.5%, 23.9%, 31.8%, and 29.2% respectively; when E:T=1.6:1, the killing efficiency of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells is 14.1%, 29.0%, 40.5%, and 35.5% respectively. According to the above experimental results, the four NK cells expressing the CAR of the present invention have high killing efficiency, among which L1-CAR-NK has the highest killing efficiency, followed by L2-CAR-NK, T2-CAR-NK, and T1-CAR-NK.

[0112] Example 5

[0113] This example compares the ability of L1-CAR-NK cells, CD33 CAR-NK cells and MSLN CAR-NK cells to kill blood tumor cells (Nomo-1) expressing CD33 and MSLN antigens in vitro. L1-CAR-NK cells, CD33 CAR-NK cells and MSLN CAR-NK cells were co-cultured with Nomo-1 tumor cells at different ratios to evaluate the killing ability (killing efficiency, cytotoxicity) of the three CAR-NK cells. The specific results are as follows Figure 4As shown, the X-axis represents different E:T ratios (CAR-NK cells (effector cells, Effector, E): tumor cells (Target, T)); the Y-axis shows the percentage of tumor cell death to tumor cell number; after 4 hours of co-incubation (Hours), flow cytometry was used to detect the proportion of dead tumor cells to tumor cells in each sample, and this ratio is the killing ability (Cytotoxicity%) of L1-CAR-NK cells, CD33 CAR-NK cells and MSLN CAR-NK cells. When E:T=0.2:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 17.7%, 10.1%, and 10.0%, respectively; when E:T=0.4:1, the mean killing efficiencies of L1-CAR-NK, CD33CAR-NK, and MSLN CAR-NK cells were 27.8%, 21.6%, and 14.8%, respectively; when E:T=0.8:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 40.9%, 24.7%, and 20.6%, respectively; when E:T=1.6:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 50.5%, 29.0%, and 26.2%, respectively. According to the above experimental results, L1-CAR-NK has the highest killing efficiency, that is, the killing efficiency of dual-target CAR-NK is better than that of single-target CAR-NK cells.

[0114] Example 6

[0115] This example verifies that L1-CAR-NK cells can specifically kill blood tumor cells of patients expressing CD33 and MSLN antigens in vitro. L1-CAR-NK cells were co-cultured with patient tumor cells to evaluate the ability of L1-CAR-NK cells to specifically kill tumor cells (killing efficiency, Cytotoxicity) of three patients with acute myeloid leukemia (patient #1, patient #2, patient #3). The specific results are as follows: Figure 5 (Patient #1), Figure 6 (Patient #2), Figure 7(Patient #3), wherein the X-axis represents different E:T ratios (L1-CAR-NK cells (effector cells, Effector, E): tumor cells (Target, T)); the Y-axis shows the percentage of tumor cell death to tumor cell number; after 12 hours of co-incubation (Hours), flow cytometry was used to detect the proportion of dead tumor cells to tumor cells in each sample, and this ratio is the killing ability (Cytotoxicity%) of L1-CAR-NK cells.Patient #1: When E:T = 0.5:1, the average killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 36.2%, 26.0%, and 15.6%, respectively; when E:T = 1:1, the average killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 42.9%, 33.2%, and 20.1%, respectively; when E:T = 2:1, the average killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 49.0%, 37.3%, and 25.1%, respectively; when E:T = 5:1, the average killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN The mean killing efficiency of CAR-NK cells was 52.4%, 45.3%, and 31.0% respectively; Patient #2: When E:T = 0.5:1, the mean killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells was 28.4%, 20.5%, and 7.9% respectively; When E:T = 1:1, the mean killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells was 35.7%, 20.6%, and 9.3% respectively; When E:T = 2:1, the mean killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells was 47.7%, 39.0%, and 14.3% respectively; When E:T = 5:1, the mean killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN The average killing efficiency of CAR-NK cells was 57.1%, 49.0%, and 18.2% respectively; Patient #3: When E:T = 0.5:1, the average killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells was 38.7%, 31.9%, and 25.2% respectively; When E:T = 1:1, the average killing efficiency of L1-CAR-NK, CD33CAR-NK, and MSLN CAR-NK cells was 52.0%, 45.9%, and 40.3% respectively; When E:T = 2:1, the average killing efficiency of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells was 56.5%, 51.7%, and 42.4% respectively; When E:T = 5:1, the average killing efficiency of L1-CAR-NK, CD33CAR-NK, and MSLN The average killing efficiency of CAR-NK cells is 67.1%, 61.9% and 52.2% respectively. According to the above experimental results, CD33-CAR-NK, MSLN-CAR-NK and L1-CAR-NK can kill tumor cells taken from tumor patients, and L1-CAR-NK has the highest killing efficiency.

[0116] Example 7

[0117] This example verifies that the CAR expression ratio of L1-CAR-NK cells during continuous in vitro culture is more stable than that of CD33-NK cells and is similar to that of MSLN-CAR-NK cells. Figure 8 As shown, NK cells expressing CAR (L1-CAR, CD33 CAR, MSLN CAR) were first sorted out and cultured continuously, and the expression ratios of CAR in L1-CAR-NK, CD33 CAR-NK, and MSLNCAR-NK cells were detected on the 3rd day (Day 3), 6th day (Day 6), 9th day (Day 9), and 12th day (Day 12) of culture, respectively. It can be seen that the average expression ratio of L1-CAR in L1-CAR-NK cells on Day 3, Day 6, Day 9, and Day 12 is 95.7%, 94.5%, 91.4%, and 89.9%, respectively; the average expression ratio of CD33-CAR in CD33-CAR-NK cells on Day 3, Day 6, Day 9, and Day 12 is 93.3%, 82.2%, 63.7%, and 55.5%, respectively; the average expression ratio of MSLN-CAR in MSLN-CAR-NK cells on Day 3, Day 6, Day 9, and Day 12 is 95.9%, 95.1%, 95.4%, and 94.3%, respectively. The results show that L1-CAR-NK cells can stably express CAR.

[0118] Example 8

[0119] This example verifies that the expansion ability of L1-CAR-NK cells during continuous culture in vitro is stronger than that of CD33-NK cells, and is similar to that of MSLN-CAR-NK cells. Fig. 9As shown, NK cells expressing CAR (L1-CAR, CD33CAR, MSLN CAR) were first sorted out and cultured continuously. On the 3rd day (Day 3), 6th day (Day 6), 9th day (Day 9), and 12th day (Day 12) of culture, the number of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were counted and the expansion multiple was calculated. It can be seen that the mean expansion times of L1-CAR-NK cells on Day 3, Day 6, Day 9, and Day 12 are 1.98, 5.98, 9.65, and 19.95, respectively; the mean expansion times of CD33-CAR-NK cells on Day 3, Day 6, Day 9, and Day 12 are 1.59, 3.43, 6.33, and 11.67, respectively; the mean expansion times of MSLN-CAR-NK cells on Day 3, Day 6, Day 9, and Day 12 are 2.15, 6.69, 13.06, and 20.49, respectively. The results show that L1-CAR-NK cells can be effectively expanded.

[0120] Example 9

[0121] The piggyBac vector (PB530A-2, SBI brand) carrying L1-CAR was transferred into human embryonic stem cells (ESC) (sourced from the National Stem Cell Bank, which can be used for clinical applications) by electroporation. The specific operation was to first connect the L1-CAR encoding nucleotide sequence (nucleotide sequence such as SEQ ID NO.33) to the piggyBac vector by a molecular biological method of homologous recombination to form a PB-L1-CAR recombinant vector. The PB-L1-CAR plasmid and the transposase plasmid (PB210PA-1, SBI brand) were electroporated into ESC using an electroporator (11-0106, Celetrix brand). On the 7th day after electroporation, the ESC expressing L1-CAR was sorted out using a flow cytometer. The specific sorting process is to first use fluorescent (Allophycocyanin, APC) coupled CD33 protein (Cat. No. CD33-HA2H3-25tests, Biopsies Biotech Co., Ltd.) and fluorescent (Phycoerythrin, PE) coupled Mesothelin protein (Cat. No. MSN-HP2H5-25tests, Biopsies Biotech Co., Ltd.) to incubate and stain the ESC to be sorted. After incubation for 15 minutes, wash off the antibody with PBS, resuspend the ESC with DAPI solution (422801, Biolegend brand), and sort the cells with flow cytometer (MA900, Sony), and sort out DAPI-negative CD33 and MSLN double-positive cells. The sorted cells are cultured in ESC culture medium (Essential 8 TM Medium, A1517001, Gibco brand) to obtain ESCs expressing L1-CAR (L1-CAR-ESCs). Fig.10 , the proportion of L1-CAR expressed in L1-CAR-ESC detected by flow cytometry was 99.9%. According to the experimental results, L1-CAR-ESC was successfully constructed in this example.

[0122] Example 10

[0123] This example shows that L1-CAR-ESC cells can be induced to differentiate into iNK cells expressing L1-CAR (L1-CAR-iNK). Fig.11As shown, L1-CAR-ESC was induced into iNK cells using organoid chimera technology, and the proportion of induced iNK cells expressing L1-CAR was 81.0%. For the specific iNK induction method used in this example, see Huang, D., et al., Lateral plate mesoderm cell-based organoid system for NK cell regeneration from human pluripotent stem cells. Cell Discov, 2022.8(1): p.121.

[0124] Embodiment 11

[0125] This example verifies that L1-CAR-iNK cells can specifically kill Nomo-1 tumor cells expressing CD33 and MSLN antigens in vitro, and their killing ability is stronger than that of CD33 CAR-iNK cells. L1-CAR-iNK and CD33 CAR-iNK were co-incubated with Nomo-1 tumor cells to evaluate the specific Nomo-1 tumor cell ability (killing efficiency, Cytotoxicity) of L1-CAR-iNK cells. The specific results are as follows: Fig.12 As shown, the X-axis represents different E:T ratios (L1-CAR-iNK cells (effector cells, Effector, E): tumor cells (Target, T)); the Y-axis shows the percentage of tumor cell death to tumor cell number; after 12 hours of co-incubation (Hours), the ratio of dead tumor cells to tumor cells in each sample was detected by flow cytometry, and this ratio was the killing ability (Cytotoxicity%) of L1-CAR-iNK cells. When E:T=0.2:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells were 8.6% and 5.8%, respectively; when E:T=0.4:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells were 15.2% and 10.9%, respectively; when E:T=0.8:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells were 28.6% and 18.6%, respectively; when E:T=1.6:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells were 43.9% and 30.1%, respectively; the results showed that L1-CAR-iNK had the highest killing activity.

[0126] Example 12

[0127] This example verifies that L1-CAR-iNK cells can specifically kill tumor cells of acute myeloid leukemia patients (three patients, patient #1, patient #2, and patient #3) expressing CD33 and MSLN proteins in vitro, and the killing ability is stronger than that of CD33 CAR-iNK cells. L1-CAR-iNK and CD33 CAR-iNK cells were co-cultured with Nomo-1 tumor cells to evaluate the ability of L1-CAR-iNK cells to specifically kill tumor cells of patients (killing efficiency, Cytotoxicity). The specific results are as follows: Fig.13(Patient #1), 14 (Patient #2), 15 (Patient #3), wherein the X-axis represents different E:T ratios (L1-CAR-iNK cells (effector cells, Effector, E): tumor cells (Target, T)); the Y-axis shows the percentage of tumor cell death to tumor cell number; after 12 hours of co-incubation (Hours), the ratio of dead tumor cells to tumor cells in each sample was detected by flow cytometry, and this ratio was the killing ability (Cytotoxicity%) of L1-CAR-iNK cells. Patient #1: When E:T = 0.5:1, the average killing efficiency of L1-CAR-iNK and CD33CAR-iNK cells were 63.9% and 53.6%, respectively; when E:T = 1:1, the average killing efficiency of L1-CAR-iNK and CD33CAR-iNK cells were 67.4% and 56.9%, respectively; when E:T = 2:1, the average killing efficiency of L1-CAR-iNK and CD33CAR-iNK cells were 70.2% and 61.7%, respectively; when E:T = 5:1, the average killing efficiency of L1-CAR-iNK and CD33CAR-iNK cells were 75.3% and 68.0%, respectively; Patient #2: When E:T = 0.5:1, the average killing efficiency of L1-CAR-iNK and CD33CAR-iNK cells were 75.3% and 68.0%, respectively. The average killing efficiency of CAR-iNK cells was 63.8% and 55.6% respectively; when E:T = 1:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells was 71.0% and 62.4% respectively; when E:T = 2:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells was 73.1% and 65.7% respectively; when E:T = 5:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells was 74.5% and 67.7% respectively; Patient #3: when E:T = 0.5:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells was 71.9% and 60.0% respectively; when E:T = 1:1, the average killing efficiency of L1-CAR-iNK and CD33 The average killing efficiency of CAR-iNK cells was 73.7% and 63.7% respectively; when E:T=2:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells was 74.6% and 65.3% respectively; when E:T=5:1, the average killing efficiency of L1-CAR-iNK and CD33 CAR-iNK cells was 75.5% and 65.9% respectively. The results showed that CD33 CAR-iNK and L1-CAR-iNK could effectively kill tumor cells taken from tumor patients, and L1-CAR-iNK showed the strongest killing activity.

[0128] Example 13

[0129] This example verifies that L1-CAR-iNK cells can specifically kill HL-60 tumor cells expressing CD33 and MSLN antigens in vivo. Fig.16 As shown, HL-60 cells expressing CD33 and MSLN were first used to construct a tumor model in mice (tail vein injection, 500,000 cells per mouse). After the construction was successful, L1-CAR-iNK cells were injected into tumor mice through the tail vein (10 million cells per mouse), and the tumor progression of the tumor mice was detected using small animal in vivo imaging technology. For specific operations of in vivo imaging technology, please refer to Huang, D., et al., Lateral plate mesoderm cell-based organoid system for NK cell regeneration from human pluripotent stem cells. Cell Discov, 2022.8(1): p.121. According to the experimental results, it can be seen that compared with untreated mice, L1-CAR-iNK cells can effectively kill tumor cells in the body, thereby significantly delaying the tumor progression of the treated mice.

[0130] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modification and change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A chimeric antigen receptor targeting CD33 and mesothelin, characterized in that: The chimeric antigen receptor comprises a leader peptide, an antigen binding region, a hinge region, a transmembrane region, and an intracellular region which are sequentially connected, wherein the antigen binding region comprises a first antigen binding region targeting CD33 and a second antigen binding region targeting mesothelin; Preferably, the hinge region comprises CD8, CD28, IgG1 or IgG4; Preferably, the transmembrane region comprises CD4, CD8, CD8a, CD28, NKG2D, CD247 or ICOS; Preferably, the intracellular region includes CD247 or FCER1G.

2. The chimeric antigen receptor according to claim 1, characterized in that The antigen binding region has the following specific structure: T1-scFv: From N-terminus to C-terminus, first, the VL of CD33 scFv (CD33 scFv-VL) is linked to the VH of CD33scFv (CD33 scFv-VH) through linker 1 to form CD33 scFv, and the VL of MSLN scFv (MSLN scFv-VL) is linked to the VH of MSLN scFv (MSLN-scFv-VH) through linker 3 to form MSLN scFv; CD33scFv is then linked to MSLN scFv through linker 2 to form CD33 scFv-VL-Linker 1-CD33 scFv-VH-Linker 2-MSLN scFv-VL-MSLN scFv-VL-Linker3-MSLN-scFv-VH (CD33 scFv-linker 2-MSLN scFv) structure, the existence form of this structure is called Tandem, i.e. CD33-linker 2-MSLN Tan scFv; or: T2-scFv: From N-terminus to C-terminus, first, the VL of MSLN scFv (MSLN scFv-VL) was linked to the VH of MSLN scFV (MSLN-scFv-VH) through linker 3 to form MSLN scFv, and the VL of CD33scFv (CD33 scFv-VL) was linked to the VH of CD33 scFV (CD33 scFv-VH) through linker 1 to form CD33 scFv; then, the MSLN scFv fragment was linked to CD33 scFv through linker 2 to form MSLN scFv-VL-MSLN scFv-VL-Linker 3-MSLN-scFv-VH-Linker 2-CD33 scFv-VL-Linker 1-CD33 scFv-VH (MSLN scFv-linker 2-CD33 scFv) structure, the existence form of this structure is called Tandem, i.e. MSLN-linker 2-CD33 TanscFv; or: L1-scFv: From N-terminus to C-terminus, the VL of CD33 scFv (CD33 scFv-VL) is linked to the VH of MSLN scFV (MSLN scFv-VH) through linker 4 to form CD33 VL-linker 4-MSLN VH, the VL of MSLN scFV (MSLN scFv-VL) is linked to the VH of CD33 scFv (CD33 scFv-VH) through linker 4 to form MSLNVL-linker 4-CD33 VH, and then CD33 VL-linker4-MSLN VH and MSLN VL-linker4-CD33 VH are linked through Linker 5 to form CD33 scFv-VL-Linker 4-MSLN scFv-VH-Linker 5-MSLN scFv-VL-Linker 4-CD33 scFv-VH structure, the existence form of this structure is called loop, i.e. CD33-Linker 5-MSLNLoop scFv; or: L2-scFv: From N-terminus to C-terminus, the VH of MSLN scFv (MSLN scFv-VH) was linked to the VL of CD33scFv (CD33 scFv-VL) through linker 4 to form MSLN VH-linker 4-CD33 VL, the VH of CD33 scFv (CD33 scFv-VH) was linked to the VL of MSLN (MSLN scFv-VL) through linker 4 to form CD33 VH-linker 4-MSLN VL, and then the MSLN VH-linker 4-CD33 VL and CD33 VH-linker 4-MSLN VL were linked through Linker 5 to form MSLN scFv-VH-linker 4-CD33scFv-VL-Linker 5-scFv-CD33 VH-linker 4-scFv-MSLN VL structure, the existence form of this structure is called loop, namely MSLN-Linker 5-CD33Loop scFv.

3. The chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor also includes a co-stimulatory region and / or a cytokine or protein ligand that is beneficial to the survival and activation of T cells or NK cells; Preferably, the co-stimulatory region is selected from one or a combination of two of 4-1BB, CD28, OX40, 2B4 and CD27; Preferably, the cytokines or protein ligands that are beneficial to the survival and activation of T cells or NK cells include CXCR1, CXCR, PDL1, IL-2 or IL-15.

4. The chimeric antigen receptor according to claim 1, characterized in that The amino acid sequences of the chimeric antigen receptors are shown in SEQ ID NOs. 27-30.

5. A nucleic acid, characterized in that The nucleic acid encodes the chimeric antigen receptor according to any one of claims 1 to 4.

6. A recombinant vector, characterized in that: Comprising the nucleic acid of claim 5.

7. A recombinant cell, characterized in that The cell expresses the chimeric antigen receptor according to any one of claims 1 to 4, or contains the nucleic acid according to claim 5, or contains the recombinant vector according to claim 6.

8. The recombinant cell according to claim 7, characterized in that The recombinant cells include recombinant immune cells or recombinant pluripotent stem cells.

9. An inducible immune cell expressing the chimeric antigen receptor according to any one of claims 1 to 4, characterized in that: The induced immune cells are prepared by inducing the recombinant pluripotent stem cells according to claim 8.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the recombinant cell according to claim 7 or 8 or the induced immune cell according to claim 9 and a pharmaceutically acceptable carrier or excipient.

11. Use of the chimeric antigen receptor according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant vector according to claim 6, the recombinant cell according to claim 7 or 8, the induced immune cell according to claim 9, and the pharmaceutical composition according to claim 10 in the preparation of an anti-tumor drug; Preferably, the tumor is a blood tumor or a solid tumor; Further preferably, the blood tumor includes myeloid leukemia, T cell leukemia, B cell leukemia, T cell lymphoma, B cell lymphoma; Further preferably, the solid tumors include mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, cholangiocarcinoma, breast cancer, gastric cancer, kidney cancer, intestinal cancer, liver cancer, and glioma.

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