Chimeric antigen receptors targeting cd33 and mesothelin dual targets and uses thereof

Abstract

The application provides a chimeric antigen receptor targeting CD33 and mesothelin double targets and application thereof. The application is based on a double-target CAR design principle, and CD33 and MSLN two scFvs are first used to construct a double-target CAR, which can target tumor cells expressing only CD33 or MSLN, and can more effectively kill tumor cells expressing MSLN and CD33. Meanwhile, the CAR-T, CAR-NK, CAR-Macrophage and the like prepared by expressing MSLN-CD33-CAR in immune cells can target and kill cells expressing CD33 or / and MSLN. Specifically, the application uses the umbilical cord blood NK cell and ESC to express the designed CD33-MSLN-CAR, and first verifies that the prepared CD33-MSLN-CAR UCB-NK cell has the advantage of targeting and killing tumor cells expressing CD33 and / or MSLN; and verifies that the prepared CD33-MSLN-CAR-ESC can be directionally differentiated into CD33-MSLN-CAR iNK cells, and also has the advantage of targeting and killing tumor cells expressing CD33 and / or MSLN.

Description

Technical Field

[0001] This invention belongs to the field of immune cell therapy and relates to a dual-target chimeric antigen receptor, specifically a chimeric antigen receptor targeting both CD33 and mesothelin and its applications. Background Technology

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

[0003] CAR elements are mainly composed of an antibody's signal peptide (SP), a single-chain fragment variable (scFv), a hinge, a transmembrane region, and an intracellular region. The scFv originates from the variable regions of the light chain (VL) and the variable regions of the heavy chain (VH) of the antibody, with the two chains linked by a linker to form a single-chain antibody. Therefore, the type of antigenic epitope that a CAR molecule binds to is determined by the scFv; different scFvs bind to different antigenic epitopes. CARs targeting different antigenic epitopes may have the same components except for the scFv. Currently, hundreds of CARs have been designed targeting antigens in hematological malignancies and solid tumors, including CARs targeting single antigens (single-target CARs) and CARs targeting two antigens (dual-target CARs). Among them, CD19-CAR, targeting the CD19 antigen of B cells, has become a tumor target for clinical therapeutic applications.

[0004] CD33 is a membrane protein expressed on myeloid cells and a key protein for identifying myeloid cells. Currently, CARs designed targeting the CD33 protein, after being prepared into CAR-T cells and CAR-NK cells, can effectively kill CD33-expressing myeloid leukemia cells and have shown some positive effects in clinical treatment. However, because NK cells partially express CD33, cannibalism and reduced amplification efficiency occur in the preparation of CD33-CAR-NK cells. This is a problem that needs to be solved in the preparation of CD33-CAR-NK cells using natural NK cells. Mesothelin (MSLN) is a protein expressed on the membrane of mesothelial cells and is highly expressed in the tumor cells of some solid tumors, such as mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, and cholangiocarcinoma. Preclinical trials have demonstrated that MSLN-CAR-NK cells can effectively kill ovarian cancer cells and prolong the survival time of mouse tumor models constructed from human ovarian cancer. Furthermore, the use of MSLN-CAR-NK cells to treat ovarian cancer has entered the clinical trial stage. CAR technology is a promising treatment for myeloid leukemia. Currently, besides CD33 and CLL1, MSLN protein is a newly discovered molecule expressed on diseased cells of some acute myeloid leukemia (AML) patients, accounting for approximately 36% of the total. Furthermore, the MSLN expression level detected by transcriptome analysis is approximately 5-1077.6 per million TPM, making MSLN a unique target protein for AML. Current research indicates that MSLN-CAR-T cells and MSLN-CAR-NK cells can effectively kill AML cell lines expressing MSLN and effectively eliminate tumor cells in tumor model mice, inhibiting tumor progression. However, because MSLN protein is cleaved from the cell membrane, forming free MSLN, the binding efficiency of MSLN-expressing immune cells targeting MSLN-expressing AML cells is reduced, weakening the killing effect. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a chimeric antigen receptor that targets both CD33 and mesothelin and its application.

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

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

[0008] Furthermore, the nucleotide sequence encoding 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.

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

[0011] Furthermore, the nucleotide sequence encoding the CD8 hinge region 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 proteins, preferably the CD8 transmembrane region (CD8™).

[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™) is shown in SEQ ID NO.25.

[0015] Furthermore, the intracellular region includes, but is not limited to, CD3ζ (CD247) or FCERT1G, preferably CD3ζ.

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

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

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

[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 cytokines or protein ligands that facilitate the survival and activation of T cells or NK cells.

[0021] Furthermore, the cytokines or protein ligands that promote 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: Starting from the N end to the C end, first connect the VL (CD33 scFv-VL) of CD33 scFv to the VH (CD33 scFv-VH) of CD33 scFV through linker 1 to form CD33 scFv. Then connect the VL (MSLNscFv-VL) of MSLN scFv to the VH (MSLN-scFv-VH) of MSLN scFV through linker 3 to form MSLN scFv. Then, CD33 scFv is connected to MSLN scFv through linker 2 to form a structure 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). This structure exists in the form of a Tandem, namely CD33-linker 2-MSLN TanscFv, or simply T1-scFv.

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

[0025] T2-scFv: From N end to C end, first connect the VL (MSLN scFv-VL) of MSLN scFv to the VH (MSLN-scFv-VH) of MSLN scFV through linker 3 to form MSLN scFv. Then connect the VL (CD33scFv-VL) of CD33 scFv to the VH (CD33 scFv-VH) of CD33 scFV through linker 1 to form CD33 scFv. The MSLN scFv fragment is then linked to the CD33 scFv via linker 2, forming the 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). This structure is called a Tandem, i.e., MSLN-linker2-CD33 Tan scFv, or simply T2-scFv.

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

[0027] L1-scFv: From N to C, connect the VL (CD33 scFv-VL) of CD33 scFv to the VH (MSLN scFv-VH) of MSLN scFV via linker 4, forming CD33 VL-linker 4-MSLN VH. Then connect the VL (MSLN scFv-VL) of MSLN scFV to the VH (CD33 scFv-VH) of CD33 scFv via linker 4, forming MSLN VL-linker 4-CD33 VH. Finally, connect CD33 VL-linker 4-MSLN VH and MSLN VL-linker 4-CD33 VH via linker 5, forming CD33 scFv-VL-Linker 4-MSLN scFv-VH-Linker 5-MSLN scFv-VL-Linker. The 4-CD33 scFv-VH structure exists in the form of a loop, namely CD33-Linker 5-MSLN Loop scFv, or simply L1-scFv.

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

[0029] L2-scFv: From N to C, connect the VH (MSLN scFv-VH) of MSLN scFv to the VL (CD33 scFv-VL) of CD33 scFV via linker 4, forming MSLN VH-linker 4-CD33 VL. Connect the VH (CD33 scFv-VH) of CD33scFV to the VL (MSLN scFv-VL) of MSLN via linker 4, forming CD33VH-linker 4-MSLN VL. Then, connect MSLN VH-linker 4-CD33 VL and CD33 VH-linker 4-MSLN VL via linker 5, forming MSLN scFv-VH-linker 4-CD33 scFv-VL-Linker 5-scFv-CD33VH-linker. The 4-scFv-MSLN VL structure exists in the form of a loop, namely MSLN-Linker 5-CD33Loop scFv. Abbreviation: L2-scFv.

[0030] Furthermore, the CD33 scFv is a single-chain antibody sequence from any source capable of binding to the 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 capable of binding the MSLN protein, including but not limited to: YP218, hYP218, clone SS, Ms501, Ms503, C2G4, and MX75.

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

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

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

[0035] Furthermore, the encoding nucleotide sequence of 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 linkers 1-5 include, but are not limited to, flexible or rigid.

[0041] Furthermore, the amino acid sequence of 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 a second aspect of the invention, a nucleic acid is provided, characterized in that the nucleic acid encodes the chimeric antigen receptor described in the first aspect of the invention.

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

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

[0055] Furthermore, the recombinant vector includes, but is not limited to, lentiviruses, retroviral expression plasmids, and transposon system expression plasmids.

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

[0057] Furthermore, the expression methods include, but are not limited to, constitutive expression, endogenous gene-initiated expression, and small molecule compound or drug-induced expression.

[0058] Furthermore, the recombinant vector may be delivered into the recombinant cells by means including but not limited to lentiviral infection, retroviral infection, adenovirus infection, Sendai virus infection, electroporation, and chemical methods.

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

[0060] Furthermore, the types of 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 (ESCs), induced pluripotent stem cells (iPSCs), hematopoietic progenitor cells (HPCs), lymphoid progenitor cells (CLPs), and common myeloid progenitor cells (CMPs); preferably embryonic stem cells.

[0062] In a fifth aspect of the invention, an induced immune cell expressing the chimeric antigen receptor described in the first aspect of the invention is provided, characterized in that the induced immune cell is induced and prepared from recombinant pluripotent stem cells described in the fourth aspect of the 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 either in vitro induction or in vivo induction.

[0065] Furthermore, the in vitro induction methods include monolayer induction, organoid induction, or a combination of both.

[0066] Furthermore, the in vivo induction methods include, but are not limited to, the human body and bioreactors.

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

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

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

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

[0071] Furthermore, the hematologic malignancies include myeloid leukemia, T-cell leukemia, B-cell leukemia, T-cell lymphoma, B-cell lymphoma, etc.

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

[0073] In an eighth aspect of the invention, a method for treating a tumor is provided, comprising the steps of administering or transplanting a therapeutically effective amount of the recombinant cells of the fourth aspect of the invention, the induced immune cells of the fifth aspect of the invention, or the pharmaceutical composition of the sixth aspect of the invention to a subject in need.

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

[0075] Furthermore, the hematologic malignancies include myeloid leukemia, T-cell leukemia, B-cell leukemia, T-cell lymphoma, B-cell lymphoma, etc.

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

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

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

[0079] This invention is based on the dual-target CAR design principle, and for the first time utilizes two scFvs, CD33 and MSLN, to construct a dual-target CAR. This dual-target CAR can not only target tumor cells expressing only CD33 or MSLN, but also more effectively kill AML and other tumor cells expressing both MSLN and CD33. Simultaneously, the MSLN-CD33-CAR structure can target both MSLN and CD33. CAR-T, CAR-NK, and CAR-Macrophage prepared by expressing MSLN-CD33-CAR in immune cells (T, NK, Macrophage) can target and kill cells expressing CD33 and / or MSLN. On the other hand, this invention utilizes CD33-MSLN-CAR designed using umbilical cord blood NK cells (UCB-NK) and ESC expression, and for the first time verifies that the prepared CD33-MSLN-CAR UCB-NK cells have the advantage of targeting and killing tumor cells expressing CD33 and / or MSLN; it also verifies that the prepared CD33-MSLN-CAR-ESC can be directed to differentiate into CD33-MSLN-CAR iNK cells, and also have the advantage of targeting and killing tumor cells expressing CD33 and / or MSLN. Specifically, the MSLN-CD33-CAR-NK cells prepared by this invention have a stronger killing ability against tumor cells expressing CD33 and MSLN; they have better expansion capacity compared to CD33-CAR-NK cells; and their CAR expression ratio is not reduced compared to CD33-CAR-NK cells. Attached Figure Description

[0080] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

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

[0082] Figure 2 The images show NK cells expressing T1-CAR, T2-CAR, L1-CAR, and L2-CAR, respectively (T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, L2-CAR-NK).

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

[0084] Figure 4 The results showed that, compared with CD33 CAR-NK cells, MSLN CAR-NK cells (Effector, E) were more effective at killing Nomo-1 acute myeloid tumor cell line (Target, T) expressing MSLN and CD33 proteins;

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

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

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

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

[0089] Figure 9 The results showed that L1-CAR-NK cells had a stronger expansion capacity than CD33-CAR-NK cells and a similar expansion capacity to MSLN CAR.

[0090] Figure 10 ESC cells expressing L1-CAR (L1-CAR-ESC) are shown;

[0091] Figure 11 The results showed that L1-CAR-ESC cells can differentiate into L1-CAR-expressing iNK cells (L1-CAR-iNK);

[0092] Figure 12 The study showed that L1-CAR-iNK cells (Effector, E) were more effective at killing Nomo-1 tumor cell line (Target, T) expressing MSLN and CD33 proteins than CD33 CAR-iNK cells.

[0093] Figure 13 The study showed that L1-CAR-iNK cells (Effector, E) were more effective than CD33 CAR-iNK cells in killing #1 cells (Target, T) of acute myeloid leukemia patients expressing MSLN and CD33 proteins.

[0094] Figure 14 The study showed that L1-CAR-iNK cells (Effector, E) were more effective than CD33 CAR-iNK cells in killing #2 cells (Target, T) of acute myeloid leukemia patients expressing MSLN and CD33 proteins.

[0095] Figure 15 The study showed that L1-CAR-iNK cells (Effector, E) were more effective than CD33 CAR-iNK cells in killing #3 cells (Target, T) of acute myeloid leukemia patients expressing MSLN and CD33 proteins.

[0096] Figure 16 This study demonstrated that L1-CAR-iNK cells can effectively inhibit tumor progression in an HL-60 tumor model expressing MSLN and CD33 proteins. Detailed Implementation

[0097] This invention provides a chimeric antigen receptor targeting both CD33 and mesothelin, and its applications. The invention is described in detail below with reference to embodiments to facilitate further understanding by those skilled in the art. However, the embodiments described below are only a part of the embodiments of this invention and should not be considered as any limitation on this invention. It should be noted that adjustments and improvements made by those skilled in the art based on the concept of this invention should be considered within the scope of protection of this invention. Specific technical operation steps and operators not specified in the embodiments are all performed in accordance with the general technical conditions described in the literature or relevant product instructions.

[0098] Example 1

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

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

[0101]

[0102] Table 2: Encoding nucleotide sequences of each component of the chimeric antigen receptor

[0103]

[0104]

[0105] Example 2

[0106] This embodiment employs four targeted CD33 and MSLN linkages designed in this 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), and 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) together with a lead peptide (SP) and CD8 hinge, CD8 TM, and CD3ζ in a 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 the CAR on the cell membrane to form as. Figure 1The diagram shows four types of CARs targeting CD33 and MSLN. The nucleotide sequences corresponding to the above structures were assembled and sent to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis. Specifically, the signal peptide (SP) in the CAR structure was linked to the 5' end of the four types of T1-scFv, T2-scFv, L1-scFv, and L2-scFv designed in the first step, forming SP-scFv (SP-T1-scFv, SP-T2-scFv, SP-L1-scFv, SP-L2-scFv). Then, the 3' end of the SP-scFv sequence was sequentially linked to a CD8 hinge, a CD8 transmembrane domain (CD8TM), and CD3ζ to form four CAR structures, specifically SP-T1-scFv-CD8 hinge-CD8TM-CD3ζ (T1-CAR, nucleotide sequence as shown in SEQ ID NO. 31:).

[0107]

[0108] Example 3

[0109] Retroviruses were packaged using retroviral expression vectors containing T1-CAR, T2-CAR, L1-CAR, and L2-CAR. These viruses were then used to infect NK cells derived from umbilical cord blood (UCBNK) to prepare MSLN-CD33-CAR-NK cells (T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, L2-CAR-NK). Specifically, retroviral expression vectors expressing CARs were constructed. The T1-CAR, T2-CAR, L1-CAR, and L2-CAR sequences synthesized in Example 1 were then homologously recombinated into the SFG (addgene, Catalog #22493) vector to form SFG-T1-CAR, SFG-T2-CAR, SFG-L1-CAR, and SFG-L2-CAR expression plasmids. The homologous recombination method was derived from a commercially available universal one-step seamless cloning kit (Yisheng Biotechnology, 10923ES), and the procedure was performed according to the kit's 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, please refer to Wang, Y., et al., Comparison of seven CD19 CAR designs in engineering NK cells for enhancing antitumour activity. Cell Prolif, 2024, 57(11): p.e13683. Finally, NK cells were infected with the virus. First, NK cells from umbilical cord blood were isolated. The isolated UCBNK cells were activated using K562-mIL21. After 6 days of activation, the CAR-carrying retrovirus was introduced into the NK cells by centrifugation. For specific methods, please refer to Wang, Y., et al., Comparison of seven CD19 CAR designs in engineering NK cells for enhancing antitumour activity. Cell Prolif, 2024, 57(11): p.e13683.The expression ratios of T1-CAR, T2-CAR, L1-CAR, and L2-CAR in CD56+ (NK) cells were detected by flow cytometry 48 hours after infection. Infected NK cells were collected and incubated 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 cells were centrifuged at 500g. The supernatant was discarded, and the cells were resuspended in PBS and analyzed by flow cytometry (BD LSRFortessa X-20 cytometer (BD Biosciences)). The results are shown below. Figure 2 As shown in the figure. The positive rates of T1-CAR were 38.0%, T2-CAR 46.8%, L1-CAR 43.4%, and T2-CAR 44.0%. Based on 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, and L2-CAR-NK cells can specifically kill Nomo-1 hematologic malignancies expressing CD33 and MSLN antigens in vitro. The T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells obtained in Example 3 were co-incubated with Nomo-1 tumor cells at different ratios to evaluate the specific tumor cell killing ability (cytotoxicity) of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells. Specific results are as follows: Figure 3As shown, the X-axis represents different E:T ratios (CAR-NK cells (Effector cells, E): Tumor cells (Target, T)); the Y-axis shows the percentage of dead tumor cells out of the total number of tumor cells; after co-incubation for 4 hours, the percentage of dead tumor cells out of the total number of tumor cells in each sample was detected by flow cytometry. This percentage is the cytotoxicity% of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells. When E:T = 0.2:1, the mean killing efficiencies 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 mean killing efficiencies 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 mean killing efficiencies of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells were 11.5%, 23.9%, 31.8%, and 29.2%, respectively; when E:T = 1.6:1, the mean killing efficiencies of T1-CAR-NK, T2-CAR-NK, L1-CAR-NK, and L2-CAR-NK cells were 14.1%, 29.0%, 40.5%, and 35.5%, respectively. Based on the above experimental results, all four types of NK cells expressing the CAR of this invention exhibited high killing efficiencies, with L1-CAR-NK showing the highest killing efficiency, followed by L2-CAR-NK, T2-CAR-NK, and T1-CAR-NK.

[0112] Example 5

[0113] This example compares the in vitro killing ability of L1-CAR-NK cells, CD33 CAR-NK cells, and MSLN CAR-NK cells against hematologic malignancies (Nomo-1) expressing CD33 and MSLN antigens. L1-CAR-NK cells, CD33 CAR-NK cells, and MSLN CAR-NK cells were co-incubated with Nomo-1 tumor cells at different ratios, and the killing ability (cytotoxicity) of the three CAR-NK cell types was evaluated. Specific results are shown below. Figure 4As shown, the X-axis represents different E:T ratios (CAR-NK cells (Effector cells, E): Tumor cells (Target, T)); the Y-axis shows the percentage of dead tumor cells out of the total number of tumor cells; after co-incubation for 4 hours, the percentage of dead tumor cells out of the total number of tumor cells in each sample was detected by flow cytometry. This percentage represents the 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 were 27.8%, 21.6%, and 14.8%, respectively; when E:T = 0.8:1, the mean killing efficiencies were 40.9%, 24.7%, and 20.6%, respectively; and when E:T = 1.6:1, the mean killing efficiencies were 50.5%, 29.0%, and 26.2%, respectively. Based on the above experimental results, L1-CAR-NK has the highest killing efficiency, meaning that dual-target CAR-NK cells have a higher killing efficiency than single-target CAR-NK cells.

[0114] Example 6

[0115] This embodiment verifies that L1-CAR-NK cells can specifically kill hematological malignancies expressed by patients with CD33 and MSLN antigens in vitro. L1-CAR-NK cells were co-incubated with tumor cells from patients, and the ability of L1-CAR-NK cells to specifically kill tumor cells (cytotoxicity) from three patients with acute myeloid leukemia (patient #1, patient #2, and patient #3) was evaluated. Specific results are as follows: Figure 5 (Patient #1) Figure 6 (Patient #2) Figure 7As shown in (Patient #3), the X-axis represents different E:T ratios (L1-CAR-NK cells (Effector cells, E): Tumor cells (Target, T)); the Y-axis shows the percentage of dead tumor cells out of the total number of tumor cells; after co-incubation for 12 hours, the percentage of dead tumor cells out of the total number of tumor cells in each sample was detected by flow cytometry, and this percentage is the killing capacity (Cytotoxicity%) of L1-CAR-NK cells.In patient #1, when E:T = 0.5:1, the mean killing efficiencies 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 mean killing efficiencies 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 mean killing efficiencies 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 mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 49.0%, 37.3%, and 25.1%, respectively; and when E:T = 5:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 49.0%, 37.3%, and 25.1%, respectively. The mean killing efficiencies of CAR-NK cells were 52.4%, 45.3%, and 31.0%, respectively; when the E:T ratio was 0.5:1 in patient #2, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 28.4%, 20.5%, and 7.9%, respectively; when the E:T ratio was 1:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 35.7%, 20.6%, and 9.3%, respectively; when the E:T ratio was 2:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 47.7%, 39.0%, and 14.3%, respectively; when the E:T ratio was 5:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 35.7%, 20.6%, and 9.3%, respectively; when the E:T ratio was 2:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 47.7%, 39.0%, and 14.3%, respectively; when the E:T ratio was 5:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 31.0%, 42.4%, 45.3%, and 31.0%, respectively. The mean killing efficiencies of CAR-NK cells were 57.1%, 49.0%, and 18.2%, respectively. In patient #3, when E:T = 0.5:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 38.7%, 31.9%, and 25.2%, respectively. When E:T = 1:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 52.0%, 45.9%, and 40.3%, respectively. When E:T = 2:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were 56.5%, 51.7%, and 42.4%, respectively. When E:T = 5:1, the mean killing efficiencies of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells were... The mean killing efficiencies of CAR-NK cells were 67.1%, 61.9%, and 52.2%, respectively. Based on these experimental results, CD33-CAR-NK, MSLN-CAR-NK, and L1-CAR-NK can all kill tumor cells obtained from cancer patients, with L1-CAR-NK exhibiting the highest killing efficiency.

[0116] Example 7

[0117] This embodiment 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 similar to that of MSLN-CAR-NK cells. Specific results are as follows: Figure 8 As shown, NK cells expressing CAR (L1-CAR, CD33 CAR, MSLN CAR) were first sorted out and cultured continuously. The expression ratio of CAR in L1-CAR-NK, CD33 CAR-NK, and MSLNCAR-NK cells was detected on Day 3, Day 6, Day 9, and Day 12, respectively. The mean expression rates of L1-CAR in L1-CAR-NK cells at Day 3, Day 6, Day 9, and Day 12 were 95.7%, 94.5%, 91.4%, and 89.9%, respectively; the mean expression rates of CD33-CAR in CD33-CAR-NK cells at Day 3, Day 6, Day 9, and Day 12 were 93.3%, 82.2%, 63.7%, and 55.5%, respectively; and the mean expression rates of MSLN-CAR in MSLN-CAR-NK cells at Day 3, Day 6, Day 9, and Day 12 were 95.9%, 95.1%, 95.4%, and 94.3%, respectively. These results indicate that L1-CAR-NK cells can stably express CAR.

[0118] Example 8

[0119] This embodiment verifies that the expansion capacity of L1-CAR-NK cells during continuous in vitro culture is stronger than that of CD33-NK cells, and similar to that of MSLN-CAR-NK cells. Specific results are as follows: Figure 9As shown, NK cells expressing CAR (L1-CAR, CD33CAR, MSLN CAR) were first sorted out and cultured continuously. On day 3, day 6, day 9, and day 12, the number of L1-CAR-NK, CD33 CAR-NK, and MSLN CAR-NK cells was counted and the expansion fold was calculated. The mean fold increases of L1-CAR-NK cells on Day 3, Day 6, Day 9, and Day 12 were 1.98, 5.98, 9.65, and 19.95, respectively; the mean fold increases of CD33-CAR-NK cells on Day 3, Day 6, Day 9, and Day 12 were 1.59, 3.43, 6.33, and 11.67, respectively; and the mean fold increases of MSLN-CAR-NK cells on Day 3, Day 6, Day 9, and Day 12 were 2.15, 6.69, 13.06, and 20.49, respectively. These results indicate 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 (ESCs) (sourced from the National Stem Cell Bank and suitable for clinical application) via electroporation. Specifically, the L1-CAR encoding nucleotide sequence (nucleotide sequence as shown in SEQ ID NO. 33) was first ligated into the piggyBac vector using homologous recombination molecular biology methods, forming the PB-L1-CAR recombinant vector. The PB-L1-CAR plasmid and transposase plasmid (PB210PA-1, SBI brand) were then electroporated into ESCs using an electroporator (11-0106, Celestix brand). On day 7 post-electroporation, ESCs expressing L1-CAR were sorted using flow cytometry. The specific sorting process is as follows: First, the ESCs to be sorted are incubated and stained with fluorescent (Allophycocyanin, APC)-conjugated CD33 protein (catalog number CD33-HA2H3-25tests, BioPlasty Biotechnology Co., Ltd.) and fluorescent (Phycoerythrin, PE)-conjugated Mesothelin protein (catalog number MSN-HP2H5-25tests, BioPlasty Biotechnology Co., Ltd.). After incubation for 15 minutes, the antibodies are washed off with PBS, and the ESCs are resuspended in DAPI solution (422801, Biolegend brand). The cells are then sorted using a flow cytometer (MA900, Sony), separating DAPI-negative cells that are double-positive for CD33 and MSLN. The sorted cells are then cultured in ESC medium (Essential 8). TM Medium (A1517001, Gibco brand) was cultured to obtain ESCs expressing L1-CAR (L1-CAR-ESC). See also Figure 10 The proportion of L1-CAR expression in L1-CAR-ESC was found to be 99.9% using flow cytometry. Based on the experimental results, this embodiment successfully constructed L1-CAR-ESC.

[0122] Example 10

[0123] This example demonstrates that L1-CAR-ESC cells can be induced to differentiate into L1-CAR-expressing iNK cells (L1-CAR-iNK cells). Specific results are as follows: Figure 11As shown, L1-CAR-ESCs were 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 embodiment, 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.

[0124] Example 11

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

[0126] Example 12

[0127] This embodiment verifies that L1-CAR-iNK cells can specifically kill tumor cells from three patients (patient #1, patient #2, and patient #3) expressing CD33 and MSLN proteins in vitro, and that their killing ability is stronger than that of CD33 CAR-iNK cells. L1-CAR-iNK and CD33 CAR-iNK cells were co-incubated with Nomo-1 tumor cells, and the ability of L1-CAR-iNK cells to specifically kill patient tumor cells (killing efficiency, cytotoxicity) was evaluated. Specific results are as follows: Figure 13As shown in (Patient #1), 14 (Patient #2), and 15 (Patient #3), the X-axis represents different E:T ratios (L1-CAR-iNK cells (Effector cells, E): Tumor cells (Target, T)); the Y-axis shows the percentage of dead tumor cells out of the total number of tumor cells; after co-incubation for 12 hours, the percentage of dead tumor cells out of the total number of tumor cells in each sample was detected by flow cytometry, and this percentage is the killing capacity (Cytotoxicity%) of L1-CAR-iNK cells. In patient #1, when E:T = 0.5:1, the mean killing efficiencies of L1-CAR-iNK and CD33CAR-iNK cells were 63.9% and 53.6%, respectively; when E:T = 1:1, the mean killing efficiencies of L1-CAR-iNK and CD33CAR-iNK cells were 67.4% and 56.9%, respectively; when E:T = 2:1, the mean killing efficiencies of L1-CAR-iNK and CD33CAR-iNK cells were 70.2% and 61.7%, respectively; when E:T = 5:1, the mean killing efficiencies of L1-CAR-iNK and CD33CAR-iNK cells were 75.3% and 68.0%, respectively. In patient #2, when E:T = 0.5:1, the mean killing efficiencies of L1-CAR-iNK and CD33CAR-iNK cells were 63.9% and 53.6%, respectively. The mean killing efficiencies of CAR-iNK cells were 63.8% and 55.6%, respectively; when E:T = 1:1, the mean killing efficiencies of L1-CAR-iNK and CD33 CAR-iNK cells were 71.0% and 62.4%, respectively; when E:T = 2:1, the mean killing efficiencies of L1-CAR-iNK and CD33 CAR-iNK cells were 73.1% and 65.7%, respectively; when E:T = 5:1, the mean killing efficiencies of L1-CAR-iNK and CD33 CAR-iNK cells were 74.5% and 67.7%, respectively; in patient #3, when E:T = 0.5:1, the mean killing efficiencies of L1-CAR-iNK and CD33 CAR-iNK cells were 71.9% and 60.0%, respectively; when E:T = 1:1, the mean killing efficiencies of L1-CAR-iNK and CD33 CAR-iNK cells were 71.9% and 60.0%, respectively. The mean killing efficiencies of CAR-iNK cells were 73.7% and 63.7%, respectively. At an E:T ratio of 2:1, the mean killing efficiencies of L1-CAR-iNK and CD33 CAR-iNK cells were 74.6% and 65.3%, respectively. At an E:T ratio of 5:1, the mean killing efficiencies of L1-CAR-iNK and CD33 CAR-iNK cells were 75.5% and 65.9%, respectively. The results showed that CD33 CAR-iNK and L1-CAR-iNK could effectively kill tumor cells obtained from cancer patients, and L1-CAR-iNK exhibited the strongest killing activity.

[0128] Example 13

[0129] This embodiment verifies that L1-CAR-iNK cells can specifically kill HL-60 tumor cells expressing CD33 and MSLN antigens in vivo. Specific results are as follows: Figure 16 As shown, a tumor model was first constructed in mice using HL-60 cells expressing CD33 and MSLN (500,000 cells injected per mouse via tail vein). After successful construction, L1-CAR-iNK cells were injected into tumor-bearing mice via tail vein (10 million cells injected per mouse). Small animal in vivo imaging technology was used to detect tumor progression in the tumor-bearing mice. For specific operation 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, compared with untreated mice, L1-CAR-iNK cells can effectively kill tumor cells in vivo, thereby significantly delaying tumor progression in treated mice.

[0130] The above description is merely a preferred embodiment of the present invention and does not limit the invention in any way. Modifications and alterations made to the above embodiments by those skilled in the art based on the technical essence of the present invention should all fall within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor targeting both CD33 and mesothelin MSLN, characterized in that, The chimeric antigen receptor comprises a lead peptide, an antigen-binding region, a hinge region, a transmembrane region, and an intracellular region connected in sequence. The antigen-binding region includes a first antigen-binding region targeting CD33 and a second antigen-binding region targeting mesothelin. The hinge region includes CD8, CD28, IgG1, or IgG4; The transmembrane region includes CD4, CD8, CD8a, CD28, NKG2D, CD247, or ICOS; The intracellular region includes CD247 or FCER1G; The specific structure of the antigen-binding region is as follows: T1-scFv: From the N end to the C end, firstly, the VL of CD33 scFv is connected to the VH of CD33scFV through linker 1 to form CD33 scFv. Then, the VL of MSLN scFv is connected to the VH of MSLN scFV through linker 3 to form MSLN scFv. Next, CD33 scFv is connected to MSLN scFv through linker 2 to form the structure CD33 scFv-VL-Linker 1-CD33 scFv-VH-Linker 2-MSLN scFv-VL-Linker 3-MSLN-scFv-VH. This structure is called a Tandem, namely CD33-linker 2-MSLN Tan scFv. or: T2-scFv: From the N end to the C end, firstly, the VL of MSLN scFv is connected to the VH of MSLN scFV through linker 3 to form MSLN scFv. Then, the VL of CD33 scFv is connected to the VH of CD33 scFV through linker 1 to form CD33 scFv. Next, the MSLN scFv fragment is connected to CD33 scFv through linker 2 to form the MSLNscFv-VL-Linker 3-MSLN-scFv-VH-Linker 2-CD33 scFv-VL-Linker 1-CD33 scFv-VH structure. This structure is called Tandem, i.e., MSLN-linker 2-CD33 Tan scFv. or: L1-scFv: From N to C, the VL of CD33 scFv is connected to the VH of MSLN scFV through linker 4, forming CD33 VL-linker 4-MSLN VH. The VL of MSLN scFV is connected to the VH of CD33 scFv through linker 4, forming MSLN VL-linker 4-CD33VH. Then, CD33 VL-linker 4-MSLN VH and MSLN VL-linker 4-CD33VH are connected together through linker 5 to form the structure CD33 scFv-VL-Linker 4-MSLN scFv-VH-Linker 5-MSLN scFv-VL-Linker 4-CD33 scFv-VH. This structure is called a loop, namely CD33-Linker 5-MSLN Loop scFv. or: L2-scFv: From N to C, the VH of MSLN scFv is connected to the VL of CD33scFV through linker 4, forming MSLN VH-linker 4-CD33 VL. The VH of CD33 scFV is connected to the VL of MSLN through linker 4, forming CD33 VH-linker 4-MSLN VL. Then, MSLN VH-linker 4-CD33 VL and CD33 VH-linker 4-MSLN VL are connected together through linker 5 to form the MSLN scFv-VH-linker 4-CD33 scFv-VL-Linker 5-scFv-CD33VH-linker 4-scFv-MSLN VL structure. This structure is called a loop, i.e., MSLN-Linker 5-CD33 Loop scFv. The amino acid sequence of CD33 scFv-VL is shown in SEQ ID NO.1; The amino acid sequence of CD33 scFv-VH is shown in SEQ ID NO.2; The amino acid sequence of the MSLN scFv-VL is shown in SEQ ID NO.3; The amino acid sequence of the MSLN scFv-VH is shown in SEQ ID NO.

4.

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

3. The chimeric antigen receptor according to claim 1, characterized in that, The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.27-30.

4. A nucleic acid, characterized in that, The nucleic acid encodes the chimeric antigen receptor as described in any one of claims 1-3.

5. A recombinant vector, characterized in that, It includes the nucleic acid described in claim 4.

6. A recombinant cell, characterized in that, The cells express the chimeric antigen receptor as described in any one of claims 1-3, or contain the nucleic acid as described in claim 4, or contain the recombinant vector as described in claim 5.

7. The recombinant cell according to claim 6, characterized in that, The recombinant cells include recombinant immune cells or recombinant pluripotent stem cells.

8. An inducible immune cell expressing the chimeric antigen receptor according to any one of claims 1-3, characterized in that, The induced immune cells are prepared by induction from the recombinant pluripotent stem cells as described in claim 7.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the recombinant cells of claim 6 or 7 or the induced immune cells of claim 8, and a pharmaceutically acceptable carrier or excipient.

10. Use of the chimeric antigen receptor according to any one of claims 1-3, the nucleic acid according to claim 4, the recombinant vector according to claim 5, the recombinant cell according to claim 6 or 7, the induced immune cell according to claim 8, and the pharmaceutical composition according to claim 9 in the preparation of an antitumor drug; The tumor is a hematologic malignancy; The hematologic malignancy is acute myeloid leukemia.

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