Enhanced SNTC cell targeting CD33 and CD38 antigens as well as preparation method and application of enhanced SNTC cell

Abstract

The invention discloses an enhanced SNTC cell targeting CD33 and CD38 antigens as well as a preparation method and application thereof, and particularly provides a pluripotent stem cell, and the pluripotent stem cell comprises the following steps: (1) down-regulating the expression of CD33 and CD38; (2) overexpressing a chimeric antigen receptor targeting CD33 and CD38, and / or overexpressing a chimeric antigen receptor targeting CD33 and a chimeric antigen receptor targeting CD38; and (3) overexpressing at least one of FB22 and IL-15RF. According to the pluripotent stem cell provided by the embodiment of the invention, the pluripotent stem cell can be directionally differentiated into an artificially modified innate immune cell capable of avoiding self-injury and killing, accurately identifying and removing tumor cells and enhancing survival and migration capabilities, and compared with an NK cell in the prior art, the pluripotent stem cell is low in transformation cost and has a good application prospect. The innate immune cells obtained after differentiation can treat tumors more effectively, and have high value for clinical treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an enhanced SNTC cell targeting CD33 and CD38 antigens, its preparation method, and its application. Background Technology

[0002] The CAR structure of CAR-NK cells typically consists of three parts: an extracellular antigen-binding domain (usually scFv), a transmembrane domain, and an intracellular activation domain, similar to the CAR structure used in CAR-T cells. Currently, most widely researched and used CAR-T therapies utilize autologous CAR-T cells. Studies have found that CAR-T cells prepared from single-target CARs exhibit tumor cell antigen escape during tumor treatment; that is, the antigen targeted by the CAR shows weakened or even absent expression in tumor cells, preventing CAR-T cells from recognizing tumor cells and limiting the therapeutic effect. Therefore, dual-target CARs have been designed to address this phenomenon, targeting two antigens of the same tumor cell. CAR-T cells prepared using dual-target CARs can effectively avoid antigen escape, thus effectively eliminating tumor cells. Furthermore, CAR-NK cells used in adoptive cell therapy offer advantages such as no GvHD (graft-versus-host disease), high safety, uniform quality, high efficacy, and few side effects.

[0003] In the treatment of myeloid leukemia, CD33 CAR-NK cells are currently the most studied. CD33 is a myeloid cell differentiation antigen with a molecular weight of 67 kDa, mainly distributed in myeloid hematologic cells, especially in the early stages of differentiation. Its intracellular region contains an immunotyrosine inhibitory motif (ITIM), so it may have functions such as regulating cell growth and differentiation by recruiting signaling molecules. CD33 is expressed in most patients with acute myeloid leukemia, but not on the surface of hematopoietic stem cells, mature granulocytes, or other tissues. Therefore, CD33 has become a good target for the treatment of myeloid leukemia. However, because NK cells partially express CD33, cannibalism and reduced expansion efficiency occur in the preparation of CD33 CAR-NK cells (PMID: 35058934). This is also a problem that needs to be solved in the preparation of CD33 CAR-NK cells using natural NK cells.

[0004] CD38 is a transmembrane glycoprotein involved in signal transduction and cell adhesion, and it is highly expressed on the cell surface of hematologic malignancies such as multiple myeloma (MM), acute myeloid leukemia (AML), Burkitt lymphoma (BL), and T-cell acute lymphoblastic leukemia (T-ALL). CD38 CAR-T cells are mostly used in research on the treatment of multiple myeloma. However, since mature myeloid cells and their precursor cells also express CD38, anti-CD38 CARs have the potential to cause myelosuppression. NK cells also highly express CD38, so cannibalism can occur between CD38 CAR-NK cells. Furthermore, single-target therapy can lead to tumor antigen escape.

[0005] Therefore, in order to solve the above problems, it is still necessary to further develop CAR cells that can effectively treat tumors without exhibiting cannibalism or reduced amplification efficiency. Summary of the Invention

[0006] The present invention aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a pluripotent stem cell.

[0007] This invention is based on the following discoveries of the inventors: Currently, chimeric antigen receptors (CARs) designed to target CD33 can effectively kill CD33-expressing myeloid leukemia cells after being loaded into T cells or NK cells. However, during the construction of CD33 CAR-NK, because NK cells themselves also partially express CD33 antigen, a "self-killing" phenomenon occurs between CAR-NK cells, significantly weakening their in vitro expansion capacity. Furthermore, NK cells generally highly express CD38 molecules. This characteristic not only triggers self-killing among CD38 CAR-NK cells but also poses a challenge in clinical combination therapy: when patients receive anti-CD38 monoclonal antibody therapy, the drug may inadvertently damage the infused CD38 CAR-NK cells, thereby weakening their actual tumor-clearing effect. Directly knocking out CD33 and CD38 genes in native NK cells is inefficient and costly, and existing technologies mostly focus on single modifications (such as knocking out only CD33, CD38, or only expressing CAR), lacking synergistic enhancement designs. To overcome the above problems, the inventors performed gene manipulation at the stem cell stage, knocking out CD33, CD38, and overexpressing functional molecules FB22 and IL-15RF, improving the efficiency and uniformity of gene knockout, significantly reducing the cost of introducing functional molecules, and enhancing cell efficacy.

[0008] Therefore, in a first aspect, the present invention provides a pluripotent stem cell. According to embodiments of the present invention, the pluripotent stem cell comprises: (1) downregulated expression of CD33 and CD38; (2) overexpression of chimeric antigen receptors targeting CD33 and CD38, and / or overexpression of chimeric antigen receptors targeting CD33 and chimeric antigen receptors targeting CD38; and (3) overexpression of at least one of FB22 and IL-15RF. The pluripotent stem cell according to embodiments of the present invention is capable of directed differentiation into artificially modified innate immune cells that avoid self-harm and kill, accurately recognize and eliminate tumor cells, and have enhanced survival and migration capabilities.

[0009] According to embodiments of the present invention, the above-mentioned pluripotent stem cells may further include at least one of the following additional technical features: According to an embodiment of the present invention, the pluripotent stem cells are selected from at least one of human embryonic stem cells, human induced pluripotent stem cells, and chemically induced reprogrammed pluripotent stem cells.

[0010] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD33 and CD38 includes a single-chain antibody targeting CD33 and a single-chain antibody targeting CD38; According to an embodiment of the present invention, the chimeric antigen receptor targeting CD33 includes a single-chain antibody targeting CD33.

[0011] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD38 includes a single-chain antibody targeting CD38.

[0012] According to an embodiment of the present invention, the single-chain antibody targeting CD33 includes a first heavy chain variable region and a first light chain variable region.

[0013] According to an embodiment of the present invention, the first heavy chain variable region and the first light chain variable region include a heavy chain variable region and a light chain variable region selected from Gemtuzumab ozogamicin (GO, hP67.6), lintuzumab (SGN-33), or M195.34 antibody.

[0014] According to an embodiment of the present invention, the C end of the first heavy chain variable region is connected to the N end of the first light chain variable region, or the C end of the first light chain variable region is connected to the N end of the first heavy chain variable region.

[0015] According to an embodiment of the present invention, the first heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% identity with it, and the first light chain variable region has an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it.

[0016] According to an embodiment of the present invention, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the linker peptide 4, and the C-terminus of the linker peptide 4 is connected to the N-terminus of the first light chain variable region, or the C-terminus of the first light chain variable region is connected to the N-terminus of the linker peptide 4, and the C-terminus of the linker peptide 4 is connected to the N-terminus of the first heavy chain variable region.

[0017] According to an embodiment of the present invention, the single-chain antibody targeting CD33 has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% identity with it.

[0018] According to an embodiment of the present invention, the single-chain antibody targeting CD38 includes a second heavy chain variable region and a second light chain variable region.

[0019] According to an embodiment of the present invention, the second heavy chain variable region and the second light chain variable region include a heavy chain variable region and a light chain variable region selected from Daratumumab, Isatuximab or CM313 antibody.

[0020] According to an embodiment of the present invention, the C end of the second heavy chain variable region is connected to the N end of the second light chain variable region, or the C end of the second light chain variable region is connected to the N end of the second heavy chain variable region.

[0021] According to an embodiment of the present invention, the second heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% identity with it, and the second light chain variable region has an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% identity with it.

[0022] According to an embodiment of the present invention, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the linker peptide 5, and the C-terminus of the linker peptide 5 is connected to the N-terminus of the second light chain variable region, or the C-terminus of the second light chain variable region is connected to the N-terminus of the linker peptide 5, and the C-terminus of the linker peptide 5 is connected to the N-terminus of the second heavy chain variable region.

[0023] According to an embodiment of the present invention, the single-chain antibody targeting CD38 has an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with it.

[0024] According to an embodiment of the present invention, in the chimeric antigen receptor targeting CD33 and CD38, the C-terminus of the single-chain antibody targeting CD33 is linked to the N-terminus of the single-chain antibody targeting CD38.

[0025] According to an embodiment of the present invention, the C-terminus of the CD33-targeting single-chain antibody is linked to the N-terminus of the linker peptide 3, and the C-terminus of the linker peptide 3 is linked to the N-terminus of the CD33-targeting single-chain antibody.

[0026] According to an embodiment of the present invention, the C-terminus of the single-chain antibody targeting CD38 is connected to the N-terminus of the single-chain antibody targeting CD33.

[0027] According to an embodiment of the present invention, the C-terminus of the CD38-targeting single-chain antibody is linked to the N-terminus of the linker peptide 3, and the C-terminus of the linker peptide 3 is linked to the N-terminus of the CD38-targeting single-chain antibody.

[0028] According to an embodiment of the present invention, the C-terminus of the first light chain variable region is connected to the N-terminus of the single-chain antibody targeting CD38, and the C-terminus of the single-chain antibody targeting CD38 is connected to the N-terminus of the first heavy chain variable region.

[0029] According to an embodiment of the present invention, the C-terminus of the first light chain variable region is connected to the N-terminus of linker peptide 1, the C-terminus of linker peptide 1 is connected to the N-terminus of the second heavy chain variable region, the C-terminus of the second heavy chain variable region is connected to the N-terminus of linker peptide 2, the C-terminus of linker peptide 2 is connected to the N-terminus of the second light chain variable region, the C-terminus of the second light chain variable region is connected to the N-terminus of linker peptide 1, and the C-terminus of linker peptide 1 is connected to the N-terminus of the first heavy chain variable region.

[0030] According to an embodiment of the present invention, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the single-chain antibody targeting CD33, and the C-terminus of the single-chain antibody targeting CD33 is connected to the N-terminus of the second light chain variable region.

[0031] According to an embodiment of the present invention, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the linker peptide 1, the C-terminus of the linker peptide 1 is connected to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is connected to the N-terminus of the linker peptide 2, the C-terminus of the linker peptide 2 is connected to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the second light chain variable region.

[0032] According to embodiments of the present invention, the linker peptide 1, linker peptide 2, linker peptide 3, linker peptide 4, and linker peptide 5 are flexible linker peptides or rigid linker peptides.

[0033] According to an embodiment of the present invention, the amino acid sequence of linker peptide 1, linker peptide 3, linker peptide 4, and linker peptide 5 is (GGGGS)n, where n is a positive integer not less than 1, and preferably, n is 1 to 6.

[0034] According to a preferred embodiment of the present invention, the amino acid sequence of the linker peptide 1 is shown in SEQ ID NO:15.

[0035] According to a preferred embodiment of the present invention, the amino acid sequence of the linker peptide 2 is shown in SEQ ID NO:16.

[0036] According to a preferred embodiment of the present invention, the amino acid sequence of the linker peptide 3 is shown in SEQ ID NO:17.

[0037] According to an embodiment of the present invention, the amino acid sequence of the linker peptide 4 is shown in SEQ ID NO:22.

[0038] According to an embodiment of the present invention, the amino acid sequence of the linker peptide 5 is shown in SEQ ID NO:35.

[0039] According to embodiments of the present invention, the chimeric antigen receptor targeting CD33 and CD38 and / or the chimeric antigen receptor targeting CD33 and / or the chimeric antigen receptor targeting CD38 further includes at least one of signal peptide 1, hinge region, transmembrane region and intracellular region.

[0040] According to an embodiment of the present invention, the C-terminus of the signal peptide 1 is connected to the N-terminus of the chimeric antigen receptor targeting CD33 and CD38 and / or the chimeric antigen receptor targeting CD33 and / or the chimeric antigen receptor targeting CD38.

[0041] According to an embodiment of the present invention, the N-terminus of the hinge region is connected to the C-terminus of the chimeric antigen receptor targeting CD33 and CD38 and / or the chimeric antigen receptor targeting CD33 and / or the chimeric antigen receptor targeting CD38.

[0042] According to an embodiment of the present invention, the C end of the hinge region is connected to the N end of the transmembrane region.

[0043] According to an embodiment of the present invention, the C-terminus of the transmembrane region is connected to the N-terminus of the intracellular region.

[0044] According to an embodiment of the present invention, the signal peptide 1 has an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 80% identity with it.

[0045] According to an embodiment of the present invention, the hinge region includes a CD8 hinge region.

[0046] According to an embodiment of the present invention, the hinge region has an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% identity with it.

[0047] According to an embodiment of the present invention, the transmembrane region includes the CD8 transmembrane region.

[0048] According to an embodiment of the present invention, the transmembrane region has an amino acid sequence as shown in SEQ ID NO: 19 or an amino acid sequence having at least 80% identity with it.

[0049] According to an embodiment of the present invention, the intracellular region includes CD3ζ.

[0050] According to an embodiment of the present invention, the intracellular region has the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 80% identity with it.

[0051] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD33 has an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% identity with it.

[0052] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD38 has an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 80% identity with it.

[0053] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD33 and CD38 has an amino acid sequence as shown in any one of SEQ ID NO:24-27 or an amino acid sequence having at least 80% identity with it.

[0054] According to an embodiment of the present invention, the FB22 has an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having at least 80% identity with it.

[0055] According to an embodiment of the present invention, the IL-15RF includes signal peptide 2, IL-15, linker peptide 6, and IL-15Rα.

[0056] According to an embodiment of the present invention, the signal peptide 2 has an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 80% identity with it.

[0057] According to an embodiment of the present invention, the IL-15 has an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 80% identity with it.

[0058] According to an embodiment of the present invention, the linker peptide 6 has an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence having at least 80% identity with it.

[0059] According to an embodiment of the present invention, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity with it.

[0060] According to an embodiment of the present invention, the IL-15RF has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% identity with it.

[0061] According to an embodiment of the present invention, the pluripotent stem cell carries: (1) the nucleotide sequence shown in SEQ ID NO:7 and 8, or the nucleotide sequence shown in any one of SEQ ID NO:31 to 34; (2) at least one of the nucleotide sequences shown in SEQ ID NO:9 and 10.

[0062] In a second aspect, the present invention provides a method for preparing the pluripotent stem cells described in the first aspect. According to an embodiment of the present invention, the method includes: downregulating the expression of CD33 and CD38 in the pluripotent stem cells to be treated; overexpressing chimeric antigen receptors targeting CD33 and CD38 in the pluripotent stem cells to be treated, and / or overexpressing a chimeric antigen receptor targeting CD33 and a chimeric antigen receptor targeting CD38; and overexpressing at least one of FB22 and IL-15RF to obtain the pluripotent stem cells. The method according to the embodiments of the present invention is highly efficient, low-cost, and can effectively prepare the pluripotent stem cells described in the first aspect of the present invention.

[0063] According to embodiments of the present invention, the method for preparing pluripotent stem cells as described in the first aspect of the present invention may further include at least one of the following additional technical features: According to an embodiment of the present invention, the downregulation of CD33 and CD38 expression in the pluripotent stem cells to be treated is achieved by at least one of gene silencing, gene editing, small molecule inhibitors, and antibody drugs.

[0064] According to an embodiment of the present invention, the overexpression of chimeric antigen receptors targeting CD33 and CD38, and / or the overexpression of chimeric antigen receptors targeting CD33 and CD38 is achieved by introducing nucleic acid molecules encoding chimeric antigen receptors targeting CD33 and CD38, and / or the overexpression of chimeric antigen receptors targeting CD33 and CD38 into the pluripotent stem cells to be treated.

[0065] According to an embodiment of the present invention, the overexpression of at least one of FB22 and IL-15RF is achieved by introducing a nucleic acid molecule encoding at least one of FB22 and IL-15RF into the pluripotent stem cells to be treated.

[0066] According to embodiments of the present invention, the gene editing is selected from at least one of CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas13, base editor, and Prime Editing.

[0067] According to an embodiment of the present invention, the introduction is performed by at least one of electroporation, transfection, and infection.

[0068] In a third aspect, the present invention provides a method for preparing artificially modified innate immune cells. According to an embodiment of the present invention, the method includes: performing directed differentiation culture on pluripotent stem cells as described in the first aspect of the present invention or pluripotent stem cells prepared by the method described in the second aspect of the present invention to obtain the artificially modified innate immune cells. The method according to the embodiments of the present invention is highly efficient, low-cost, and can effectively prepare artificially modified innate immune cells that avoid self-harm and cell death, accurately identify and eliminate tumor cells, and have enhanced survival and migration abilities.

[0069] According to an embodiment of the present invention, the directed differentiation culture is carried out by at least one of the monolayer induction method, the embryoid induction method, and the organoid induction method.

[0070] In a fourth aspect, the present invention provides an artificially modified innate immune cell. According to embodiments of the present invention, the artificially modified innate immune cell comprises (1) downregulated expression of CD33 and CD38; (2) overexpression of chimeric antigen receptors targeting CD33 and CD38, and / or overexpression of both a chimeric antigen receptor targeting CD33 and a chimeric antigen receptor targeting CD38; and (3) overexpression of at least one of FB22 and IL-15RF. The artificially modified innate immune cell according to embodiments of the present invention can effectively avoid self-harm and cell death, accurately identify and eliminate tumor cells, and has enhanced survival and migration capabilities.

[0071] In a fifth aspect, the present invention provides an artificially modified innate immune cell. According to an embodiment of the invention, the artificially modified innate immune cell is prepared by the method described in the third aspect of the invention. The artificially modified innate immune cell according to an embodiment of the invention can effectively avoid self-harm and cell death, accurately identify and eliminate tumor cells, and has enhanced survival and migration abilities.

[0072] In a sixth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises pluripotent stem cells as described in the first aspect of the present invention, and artificially modified innate immune cells as described in the fourth or fifth aspect of the present invention. The pharmaceutical composition according to embodiments of the present invention can effectively prevent self-harm and cell death, accurately identify and eliminate tumor cells, and enhance their survival and migration abilities.

[0073] In a seventh aspect, the present invention provides a method for enhancing the killing, proliferation, activation, and migration of artificially modified innate immune cells. According to an embodiment of the present invention, the method includes: preparing pluripotent stem cells using the method described in the second aspect of the present invention; and performing directed differentiation culture on the pluripotent stem cells. The method according to an embodiment of the present invention can enhance the killing, proliferation, activation, and migration capabilities of artificially modified innate immune cells.

[0074] According to an embodiment of the present invention, the directed differentiation culture is carried out by at least one of the monolayer induction method, the embryoid induction method, and the organoid induction method.

[0075] In an eighth aspect of the invention, the invention provides for the use of the pluripotent stem cells described in the first aspect of the invention, the artificially modified innate immune cells described in the fourth or fifth aspect of the invention, or the pharmaceutical composition described in the sixth aspect of the invention in the preparation of a medicament for the treatment or prevention of tumors.

[0076] According to embodiments of the present invention, the use in the preparation of the above-described pharmaceutical product may further include at least one of the following additional technical features: According to an embodiment of the present invention, the tumor includes a hematoma.

[0077] According to embodiments of the present invention, the hematologic malignancy includes at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma.

[0078] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0079] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1This is a schematic diagram of enhanced SNTC cells that express dual-target CARs (CD33-CD38 CAR) that simultaneously target CD33 and CD38 antigens and express FB22 and IL-15RF genes, as described in Example 1 of the present invention. Figure 2 This is a sequencing result diagram of the CD33 and CD38 genes knocked out in hPSCs cells in Example 1 of the present invention; Figure 3 In Embodiment 2 of this invention, four different CD33-CD38 CAR structures (T1-CAR, T2-CAR, L1-CAR, L2-CAR) are constructed based on the first-generation CAR structure and using four scFv (T1-scFv, T2-scFv, L1-scFv, L2-scFv). Figure 4 The protein expression levels of FB22 and IL-15RF in CD33KO-CD38KO-FB22-IL15RF-hPSC cells in Example 3 of this invention are shown. Figure 5 The protein expression level of CD33-CD38 CAR in CD33-CD38 CAR-hPSC cells in Example 4 of this invention; Figure 6 This shows the immunophenotype of SNTC cells in Example 5 of the present invention, as well as the protein expression levels of CD33, CD38, CD33-CD38 CAR, FB22, and IL-15RF. Figure 7 This is a graph showing the results of flow cytometry detection of self-injury and killing in SNTC and NTC cells in Example 6 of the present invention; Figure 8 This is a graph showing the results of flow cytometry analysis of the killing ability of SNTC and NTC cells against tumor cells in Example 7 of this invention. Figure 9 This is a graph showing the statistical results of the expansion of SNTC, NTC, FB22-NTC and IL15RF-NTC cells in an animal model in Example 8 of the present invention. Detailed Implementation

[0080] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0081] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0082] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0083] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0084] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0085] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0086] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Numerous algorithms exist for aligning sequences and determining sequence identity, including: the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0087] In this paper, the term "at least 80% identity" means at least 80% identity with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0088] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

[0089] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The chimeric antigen receptor, nucleic acid molecule, expression vector, or transgenic immune cell or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous injection.

[0090] In this document, the term "treatment" refers to the use of drugs to achieve desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of drugs or transgenic immune cells to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administration of drugs containing cells with chimeric antigen receptors as described herein to an individual in need.

[0091] This invention knocks out endogenous CD33 (P67) and CD38 (ADPRC1), which can prevent CD33 / CD38 CAR-NK cells from attacking CD33 and CD38-expressing cells in a "self-destructive" manner and eliminate bone marrow suppression, thereby improving the expansion and persistence of CD33 / CD38 CAR-NK cells. Based on this, a dual-target CAR targeting CD33 and CD38 is designed. Utilizing this dual-target CAR-NK cell approach can effectively avoid antigen escape, thus better eliminating tumor cells and preventing tumor recurrence. Furthermore, FB22 is a chemokine receptor widely expressed in blood cells and closely related to the chemotaxis and migration of blood cells. Overexpression of the FB22 gene can achieve longer-term persistence of innate immune cells (NTCs) in vivo. Interleukin-15 (IL-15) is a key cytokine for NK cell survival and proliferation; IL-15 signaling can be used to enhance NK cell-mediated tumor immunotherapy. IL-15RF is a recombinant structure that can be presented via interleukin-15 (IL-15) / interleukin-15 receptor α (IL-15Rα). Therefore, overexpression of the IL-15RF gene further enhances the survival and proliferation of NK cells in vivo. This application defines NK-like cells obtained through induced differentiation of pluripotent stem cells as NTC cells (innate immune cells). NK-like cells obtained by engineering and introducing the above-mentioned combined elements into pluripotent stem cells and then inducing differentiation are defined as enhanced NTC cells, i.e., SNTC (synthetic innate cells). Based on this, the present invention proposes a pluripotent stem cell, a method for preparing pluripotent stem cells, a method for preparing artificially modified innate immune cells, artificially modified innate immune cells, a pharmaceutical composition, a method for enhancing the killing, proliferation, activation and migration of artificially modified innate immune cells, and their uses.

[0092] pluripotent stem cells In a first aspect, the present invention proposes a pluripotent stem cell comprising: (1) downregulated expression of CD33 and CD38; (2) overexpression of a chimeric antigen receptor targeting CD33 and CD38, and / or overexpression of a chimeric antigen receptor targeting CD33 and a chimeric antigen receptor targeting CD38; and (3) overexpression of at least one of FB22 and IL-15RF. The pluripotent stem cells according to embodiments of the present invention are capable of directed differentiation into artificially modified innate immune cells that avoid self-harm and kill, accurately recognize and eliminate tumor cells, and have enhanced survival and migration capabilities. It should be noted that, in this document, "chimeric antigen receptor targeting CD33 and CD38" is abbreviated as "CD33CD38CAR", "chimeric antigen receptor targeting CD33" is abbreviated as "CD33CAR", and "chimeric antigen receptor targeting CD38" is abbreviated as "CD38CAR".

[0093] In this article, "CD38" refers to nicotinamide adenine dinucleotide nucleoside enzyme (NADase), a transmembrane glycoprotein. CD38 can serve as a target for malignant hematologic malignancies (especially multiple myeloma). It consists of approximately 300 amino acids and has an extracellular enzyme activity domain, a transmembrane region, and an intracellular short tail.

[0094] In this article, "CD33" belongs to the sialic acid-binding immunoglobulin lectin (Siglec) family, designated Siglec-3. Its extracellular domain contains one type V immunoglobulin (Ig) domain and one type C2 Ig domain, mediating the binding of sialylated ligands. Its transmembrane region is a single-pass transmembrane structure, and its intracellular domain contains the immunoreceptor tyrosine inhibitory motif (ITIM), transmitting inhibitory signals. Composed of approximately 350 amino acids, it is one of the commonly used targets for the treatment of acute myeloid leukemia (AML). High expression of CD33 can be detected in tumor cells of 80-90% of AML patients, making it a frequently used target for AML treatment.

[0095] The CARs (including their functional moieties and functional variants) of embodiments of the present invention can be obtained by methods known in the art. CARs can be prepared by any suitable method for preparing peptides or proteins. Suitable methods for de novo synthesis of peptides and proteins are described in references such as Chan et al., *Fmoc Solid Phase Peptide Synthesis*, Oxford University Press, Oxford, United Kingdom, 2000; *Peptide and Protein Drug Analysis*, edited by Reid, R., Marcel Dekker Inc., 2000; *Epitope Mapping*, edited by Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent 5,449,752. Furthermore, some CARs (including their functional moieties and functional variants) of the present invention can be isolated from and / or purified from sources such as plants, bacteria, insects, mammals such as rats, humans, etc. Isolation and purification methods are well known in the art. Alternatively, the CARs described herein (including their functional portions and functional variants) can be commercially synthesized by companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this regard, the CARs of the present invention can be synthesized, recombined, isolated, and / or purified.

[0096] This invention also includes, within the scope of this invention, functional variants of the CARs described herein. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein having a large or significant sequence identity or similarity to the parent CAR, said functional variant retaining the biological activity of the CAR variant. Functional variants encompass, for example, those variants of the CARs (parent CARs) described herein that retain the ability to recognize target cells to a similar degree, the same degree, or a higher degree than the parent CAR. Regarding the parent CAR, the amino acid sequence of the functional variant may, for example, have at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99%, or higher identity with the amino acid sequence of the parent CAR.

[0097] Functional variants, for example, comprise the amino acid sequence of a parent CAR having at least one conserved amino acid substitution. Alternatively or additionally, functional variants may comprise the amino acid sequence of a parent CAR having at least one non-conserved amino acid substitution. In this case, non-conserved amino acid substitutions that do not interfere with or inhibit the biological activity of the functional variant are preferred. Non-conserved amino acid substitutions can enhance the biological activity of the functional variant, resulting in an increase in the biological activity of the functional variant compared to the parent CAR.

[0098] The amino acid substitutions in the CAR of this invention are preferably conservative amino acid substitutions. Conservative amino acid substitutions are those known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can include replacing an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), replacing an amino acid with a nonpolar side chain with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Tip, Cys, Val, etc.), replacing a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), replacing an uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), replacing an amino acid with a β-branched side chain with another amino acid with a β-branched side chain (e.g., Ile, Thr, and Val), and replacing an amino acid with an aromatic side chain with another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr, etc.).

[0099] According to embodiments of the present invention, the above-mentioned pluripotent stem cells may further include at least one of the following additional technical features: According to an embodiment of the present invention, the pluripotent stem cells are selected from at least one of human embryonic stem cells, human induced pluripotent stem cells, and chemically induced reprogrammed pluripotent stem cells.

[0100] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD33 and CD38 comprises a single-chain antibody targeting CD33 and a single-chain antibody targeting CD38. Thus, the single-chain antibody targeting CD33 and the single-chain antibody targeting CD38 in the chimeric antigen receptor targeting CD33 and CD38 are in a fusion state.

[0101] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD33 includes a single-chain antibody targeting CD33.

[0102] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD38 includes a single-chain antibody targeting CD38.

[0103] According to specific embodiments of the present invention, the CAR of the present invention can be a separate chimeric antigen receptor targeting CD33 and a separate chimeric antigen receptor targeting CD38, or it can be a chimeric antigen receptor targeting both CD33 and CD38 obtained by fusing CD33 single-chain antibodies and CD38 single-chain antibodies. Its specific form is not particularly limited, as long as it enables the co-expression of CD33 and CD38 single-chain antibodies in pluripotent stem cells. Whether it is in a separate or fused form is included within the scope of the present invention.

[0104] According to an embodiment of the present invention, the single-chain antibody targeting CD33 includes a first heavy chain variable region and a first light chain variable region.

[0105] According to an embodiment of the present invention, the first heavy chain variable region and the first light chain variable region include heavy chain variable regions and light chain variable regions selected from Gemtuzumab ozogamicin (GO, hP67.6), lintuzumab (SGN-33) or M195.34 antibody.

[0106] In the CD33-targeting single-chain antibody described in this invention, the connection order of the heavy chain variable region and the light chain variable region is not particularly limited. According to an embodiment of the invention, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first light chain variable region, or the C-terminus of the first light chain variable region is connected to the N-terminus of the first heavy chain variable region. According to a specific embodiment of the invention, the C-terminus of the first heavy chain variable region is connected to the N-terminus of a linker peptide 4, and the C-terminus of the linker peptide 4 is connected to the N-terminus of the first light chain variable region, or the C-terminus of the first light chain variable region is connected to the N-terminus of the linker peptide 4, and the C-terminus of the linker peptide 4 is connected to the N-terminus of the first heavy chain variable region.

[0107] According to an embodiment of the present invention, the first heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% identity with it, and the first light chain variable region has an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it.

[0108] According to an embodiment of the present invention, the single-chain antibody targeting CD33 has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% identity with it.

[0109] According to an embodiment of the present invention, the single-chain antibody targeting CD38 includes a second heavy chain variable region and a second light chain variable region. According to an embodiment of the present invention, the second heavy chain variable region and the second light chain variable region include a heavy chain variable region and a light chain variable region selected from Daratumumab, Isatuximab, or CM313 antibody.

[0110] In the CD38-targeting single-chain antibody described in this invention, the connection order of the heavy chain variable region and the light chain variable region is not particularly limited. According to an embodiment of the invention, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the second light chain variable region, or the C-terminus of the second light chain variable region is connected to the N-terminus of the second heavy chain variable region. According to a specific embodiment of the invention, the C-terminus of the second heavy chain variable region is connected to the N-terminus of linker peptide 5, and the C-terminus of linker peptide 5 is connected to the N-terminus of the second light chain variable region, or the C-terminus of the second light chain variable region is connected to the N-terminus of linker peptide 5, and the C-terminus of linker peptide 5 is connected to the N-terminus of the second heavy chain variable region.

[0111] According to an embodiment of the present invention, the second heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% identity with it, and the second light chain variable region has an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% identity with it. According to an embodiment of the present invention, the single-chain antibody targeting CD38 has an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with it.

[0112] According to an embodiment of the present invention, the C-terminus of the single-chain antibody targeting CD33 is connected to the N-terminus of the single-chain antibody targeting CD38.

[0113] According to an embodiment of the present invention, the C-terminus of the CD33-targeting single-chain antibody is linked to the N-terminus of the linker peptide 3, and the C-terminus of the linker peptide 3 is linked to the N-terminus of the CD33-targeting single-chain antibody.

[0114] According to an embodiment of the present invention, in the chimeric antigen receptor targeting CD33 and CD38, the C-terminus of the single-chain antibody targeting CD38 is linked to the N-terminus of the single-chain antibody targeting CD33.

[0115] According to an embodiment of the present invention, the C-terminus of the CD38-targeting single-chain antibody is linked to the N-terminus of the linker peptide 3, and the C-terminus of the linker peptide 3 is linked to the N-terminus of the CD38-targeting single-chain antibody.

[0116] According to an embodiment of the present invention, the C-terminus of the first light chain variable region is connected to the N-terminus of the single-chain antibody targeting CD38, and the C-terminus of the single-chain antibody targeting CD38 is connected to the N-terminus of the first heavy chain variable region.

[0117] According to an embodiment of the present invention, the C-terminus of the first light chain variable region is connected to the N-terminus of linker peptide 1, the C-terminus of linker peptide 1 is connected to the N-terminus of the second heavy chain variable region, the C-terminus of the second heavy chain variable region is connected to the N-terminus of linker peptide 2, the C-terminus of linker peptide 2 is connected to the N-terminus of the second light chain variable region, the C-terminus of the second light chain variable region is connected to the N-terminus of linker peptide 1, and the C-terminus of linker peptide 1 is connected to the N-terminus of the first heavy chain variable region.

[0118] According to an embodiment of the present invention, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the single-chain antibody targeting CD33, and the C-terminus of the single-chain antibody targeting CD33 is connected to the N-terminus of the second light chain variable region.

[0119] According to an embodiment of the present invention, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the linker peptide 1, the C-terminus of the linker peptide 1 is connected to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is connected to the N-terminus of the linker peptide 2, the C-terminus of the linker peptide 2 is connected to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the second light chain variable region.

[0120] According to embodiments of the present invention, linker peptide 1, linker peptide 2, linker peptide 3, linker peptide 4, and linker peptide 5 are flexible linker peptides or rigid linker peptides. Those skilled in the art will understand that the linker peptides described herein serve a spatial linking function, and both flexible and rigid linker peptides can be used.

[0121] According to embodiments of the present invention, linker peptide 1, linker peptide 2, linker peptide 3, linker peptide 4, and linker peptide 5 are preferably flexible linkers. According to embodiments of the present invention, the amino acid sequence of linker peptide 1, linker peptide 3, linker peptide 4, and linker peptide 5 is (GGGGS)n, where n is a positive integer not less than 1, preferably n is 1 to 6. According to a preferred embodiment of the present invention, the amino acid sequence of linker peptide 1 is as shown in SEQ ID NO:15. According to a preferred embodiment of the present invention, the amino acid sequence of linker peptide 2 is as shown in SEQ ID NO:16. According to a preferred embodiment of the present invention, the amino acid sequence of linker peptide 3 is as shown in SEQ ID NO:17. According to an embodiment of the present invention, the amino acid sequence of linker peptide 4 is as shown in SEQ ID NO:22. The amino acid sequence of linker peptide 5 is as shown in SEQ ID NO:35.

[0122] According to embodiments of the present invention, the chimeric antigen receptor targeting CD33 and CD38 and / or the chimeric antigen receptor targeting CD33 and / or the chimeric antigen receptor targeting CD38 further includes at least one of a signal peptide 1, a hinge region, a transmembrane region, and an intracellular region. It should be noted that the chimeric antigen receptor targeting CD33 and CD38, the chimeric antigen receptor targeting CD33, and / or the chimeric antigen receptor targeting CD38 in the present invention can be a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR, depending on the structure of their intracellular regions. The intracellular structure of the CAR is not particularly limited. For example, the intracellular region of the first-generation CAR contains only three ITAMs of CD3ζ or FcεRIγ, resulting in weak activation signals and easy depletion in vivo; the intracellular region of the second-generation CAR adds a co-stimulatory domain (CD28 or 4-1BB) before CD3ζ, balancing killing and survival; the intracellular region of the third-generation CAR adds two co-stimulatory domains (such as CD28+4-1BB or CD28+OX40) before CD3ζ; the fourth generation (TRUCK / Armored CAR) additionally embeds inducible cytokines (IL-12, IL-18) or immunomodulatory fragments, which can recruit innate immunity or disrupt the inhibitory microenvironment at the tumor site; according to a specific embodiment of the present invention, the C-terminus of the signal peptide 1 is linked to the N-terminus of the chimeric antigen receptor targeting CD33 and CD38 and / or the chimeric antigen receptor targeting CD33 and / or the chimeric antigen receptor targeting CD38. Thus, signal peptide 1 is located at the anterior end of the chimeric antigen receptors targeting CD33 and CD38 and / or the chimeric antigen receptors targeting CD33 and / or the chimeric antigen receptors targeting CD38, and is responsible for introducing the nascent CAR peptide into the secretory pathway. According to an embodiment of the invention, the N-terminus of the hinge region is connected to the C-terminus of the chimeric antigen receptors targeting CD33 and CD38 and / or the chimeric antigen receptors targeting CD33 and / or the chimeric antigen receptors targeting CD38. According to an embodiment of the invention, the C-terminus of the hinge region is connected to the N-terminus of the transmembrane region. According to an embodiment of the invention, the C-terminus of the transmembrane region is connected to the N-terminus of the intracellular region.

[0123] According to an embodiment of the present invention, the signal peptide 1 has an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 80% identity with it.

[0124] According to an embodiment of the present invention, the hinge region includes a CD8 hinge region. According to a specific embodiment of the present invention, the hinge region has an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% identity with it.

[0125] According to an embodiment of the present invention, the transmembrane region includes a CD8 transmembrane region, a CD28 transmembrane region, or a CD4 transmembrane region.

[0126] According to a specific embodiment of the present invention, the transmembrane region has an amino acid sequence as shown in SEQ ID NO: 19 or an amino acid sequence having at least 80% identity with it.

[0127] According to an embodiment of the present invention, the intracellular region includes CD3ζ or FcεRIγ. According to a specific embodiment of the present invention, the intracellular region has the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 80% identity with it.

[0128] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD33 has an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% identity with it.

[0129] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD38 has an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 80% identity with it.

[0130] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD33 and CD38 has an amino acid sequence as shown in any one of SEQ ID NO:24-27 or an amino acid sequence having at least 80% identity with it.

[0131] According to an embodiment of the present invention, the FB22 has an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having at least 80% identity with it.

[0132] According to embodiments of the present invention, the IL-15RF comprises signal peptide 2, IL-15, linker peptide 6, and IL-15Rα. It should be noted that in this invention, "IL-15RF" and "IL15RF" are synonymous. According to embodiments of the present invention, IL-15RF is a fusion protein of IL-15 and IL-15Rα, which continuously activates the IL-15 pathway through autocrine signaling, promoting the survival, proliferation, and functional activation of CAR-SNTC cells, reducing dependence on exogenous cytokines, and enhancing cell proliferation and survival.

[0133] According to an embodiment of the present invention, the signal peptide 2 has an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 80% identity with it.

[0134] According to an embodiment of the present invention, the IL-15 has an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 80% identity with it.

[0135] According to an embodiment of the present invention, the linker peptide 6 has an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence having at least 80% identity with it.

[0136] According to an embodiment of the present invention, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity with it.

[0137] According to an embodiment of the present invention, the IL-15RF has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% identity with it.

[0138] According to an embodiment of the present invention, the pluripotent stem cell carries: (1) the nucleotide sequence shown in SEQ ID NO:7 and 8, or the nucleotide sequence shown in any one of SEQ ID NO:31 to 34; (2) at least one of the nucleotide sequences shown in SEQ ID NO:9 and 10.

[0139] According to embodiments of the present invention, the specific manner in which the expression of CD33 and CD38 in the pluripotent stem cells is downregulated is not particularly limited. Any manner in which the expression of CD33 and CD38 in the pluripotent stem cells can be downregulated is included within the scope of protection of this application. For example, the downregulation of the expression of CD33 and CD38 in the pluripotent stem cells can be achieved by one or more of the following methods: single-base gene editing, zinc finger nucleases, transcription activator-like effector nucleases, CRISPR / Cas9 and CRISPR / Cas9 combined with iPSC and AAV vector technology. According to a specific embodiment of the present invention, the downregulation of CD33 and CD38 expression in the pluripotent stem cells is achieved by transducing sgRNAs targeting the CD33 and CD38 genes into the pluripotent stem cells. More specifically, a vector containing sgRNAs targeting the CD33 and CD38 genes and Cas12 is transduced into the pluripotent stem cells. The sgRNAs targeting the CD33 gene have the nucleotide sequences shown in SEQ ID NO:36-37, and the sgRNAs targeting the CD38 gene have the nucleotide sequences shown in SEQ ID NO:38-39.

[0140] Single-base gene editing refers to gene editing technology that can cause a single base change in the genome. The basic principle is to fuse cytosine deaminase (APOBEC) or adenosine deaminase with an existing Cas9n (D10A) to form a gene editing technology that relies on the CRISPR principle to modify a single base at position 4 to 7 away from the PAM end.

[0141] Zinc finger nucleases (ZFNs) consist of a DNA recognition domain and a non-specific endonuclease. The DNA recognition domain is composed of a series of Cys2-his2 zinc finger proteins tandemly. Each zinc finger protein recognizes and binds to a specific triplet base. For example, the most classic zinc finger nuclease is the fusion of a non-specific endonuclease FokI with a zinc finger-containing domain, the purpose of which is naturally to cut a specific sequence. The cut DNA can be repaired by the excision repair mechanism, which deletes the single-stranded portion at the cut site and then rejoins it. This method can be used to delete specific segments on chromosomes, thereby creating mutants or performing therapeutic procedures.

[0142] Transcription activator-like (TAL) effector nucleases (TALENs) are enzymes that can target and modify specific DNA sequences. They use TAL effectors—natural proteins secreted by plant bacteria—to recognize specific DNA base pairs. TAL effectors can be designed to recognize and bind to all target DNA sequences. Adding a nuclease to a TAL effector generates TALENs. TAL effector nucleases can bind to DNA and cut the DNA strand at specific sites, thereby introducing new genetic material.

[0143] The CRISPR / Cas9 system is widely present in prokaryotic genes and is an acquired immune defense mechanism evolved by bacteria and archaea to cope with constant attacks from viruses and plasmids. In these organisms, exogenous genetic material from bacteriophages is acquired and integrated into CRISPR sites; these sequence-specific fragments are transcribed into short CRISPR RNA (CRISPR-derived RNA). The crRNA binds to tracrRNA (trans-activating RNA) through base pairing to form double-stranded RNA. Then, the tracrRNA / crRNA complex directs the Cas9 protein to cleave the double-stranded DNA, thus performing gene editing.

[0144] method In a second aspect, the present invention provides a method for preparing the pluripotent stem cells described in the first aspect. According to an embodiment of the present invention, the method includes: downregulating the expression of CD33 and CD38 in the pluripotent stem cells to be treated; overexpressing chimeric antigen receptors targeting CD33 and CD38 in the pluripotent stem cells to be treated, and / or overexpressing a chimeric antigen receptor targeting CD33 and a chimeric antigen receptor targeting CD38; and overexpressing at least one of FB22 and IL-15RF to obtain the pluripotent stem cells. The method according to the embodiments of the present invention is highly efficient, low-cost, and can effectively prepare the pluripotent stem cells described in the first aspect of the present invention.

[0145] According to an embodiment of the present invention, the downregulation of CD33 and CD38 expression in the pluripotent stem cells to be treated is achieved by at least one of gene silencing, gene editing, small molecule inhibitors, and antibody drugs.

[0146] According to embodiments of the present invention, the gene editing is selected from at least one of CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas13, base editor, and Prime Editing.

[0147] According to an embodiment of the present invention, the overexpression of chimeric antigen receptors targeting CD33 and CD38, and / or the overexpression of chimeric antigen receptors targeting CD33 and CD38 is achieved by introducing nucleic acid molecules encoding chimeric antigen receptors targeting CD33 and CD38, and / or the overexpression of chimeric antigen receptors targeting CD33 and CD38 into the pluripotent stem cells to be treated.

[0148] According to an embodiment of the present invention, the overexpression of at least one of FB22 and IL-15RF is achieved by introducing a nucleic acid molecule encoding at least one of FB22 and IL-15RF into the pluripotent stem cells to be treated.

[0149] According to an embodiment of the present invention, the introduction is performed by at least one of electroporation, transfection, and infection.

[0150] method In a third aspect, the present invention provides a method for preparing artificially modified innate immune cells. According to an embodiment of the present invention, the method includes: performing directed differentiation culture on pluripotent stem cells as described in the first aspect of the present invention or pluripotent stem cells prepared by the method described in the second aspect of the present invention to obtain the artificially modified innate immune cells. The method according to the embodiments of the present invention is highly efficient, low-cost, and can effectively prepare artificially modified innate immune cells that avoid self-harm and cell death, accurately identify and eliminate tumor cells, and have enhanced survival and migration abilities.

[0151] It should be clarified that the "innate immune cells (NTCs)" in this article refer to natural immune cells, which are a type of immune cell in the immune system that can rapidly recognize and defend against invading foreign cells (such as bacteria, viruses, and fungi) or abnormal cells (such as tumor cells) without prior contact with specific pathogens. They respond rapidly through non-specific defense mechanisms, forming the body's first line of immune defense. The "synthetic innate immune cells (SNTCs)" in this article refer to immune cells that have been selectively modified using genetic engineering or other biotechnological means to enhance their function, endow them with new characteristics, or improve their targeting.

[0152] According to an embodiment of the present invention, the directed differentiation culture is carried out by at least one of the monolayer induction method, the embryoid induction method, and the organoid induction method.

[0153] Artificially modified innate immune cells In a fourth aspect, the present invention provides an artificially modified innate immune cell comprising: (1) downregulation of CD33 and CD38 expression; (2) overexpression of chimeric antigen receptors targeting CD33 and CD38, and / or overexpression of chimeric antigen receptors targeting CD33 and CD38; and (3) overexpression of at least one of FB22 and IL-15RF. The artificially modified innate immune cell according to embodiments of the present invention can effectively avoid self-harm and cell death, accurately identify and eliminate tumor cells, and enhance survival and migration capabilities.

[0154] In a fifth aspect, the present invention provides an artificially modified innate immune cell. According to an embodiment of the invention, the artificially modified innate immune cell is prepared by the method described in the third aspect of the invention. The artificially modified innate immune cell according to an embodiment of the invention can effectively avoid self-harm and cell death, accurately identify and eliminate tumor cells, and has enhanced survival and migration abilities.

[0155] Pharmaceutical Composition In a sixth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises pluripotent stem cells as described in the first aspect of the present invention, and artificially modified innate immune cells as described in the fourth or fifth aspect of the present invention. The pharmaceutical composition according to embodiments of the present invention can effectively prevent self-harm and cell death, accurately identify and eliminate tumor cells, and enhance their survival and migration abilities.

[0156] According to embodiments of the present invention, pharmaceutically acceptable excipients are further included.

[0157] The pharmaceutical compositions of the present invention can be administered by any acceptable method of administration. The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as injections or lyophilized powders, and current methods for preparing these dosage forms are known or obvious to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the bioactive components contained therein to be bioavailable after administration to a patient.

[0158] method In a seventh aspect, the present invention provides a method for enhancing the killing, proliferation, activation, and migration of artificially modified innate immune cells. According to an embodiment of the present invention, the method includes: preparing pluripotent stem cells using the method described in the second aspect of the present invention; and performing directed differentiation culture on the pluripotent stem cells. The method according to an embodiment of the present invention can enhance the killing, proliferation, activation, and migration capabilities of artificially modified innate immune cells.

[0159] According to an embodiment of the present invention, the directed differentiation culture is carried out by at least one of the monolayer induction method, the embryoid induction method, and the organoid induction method.

[0160] use In an eighth aspect of the invention, the invention provides for the use of the pluripotent stem cells described in the first aspect of the invention, the artificially modified innate immune cells described in the fourth or fifth aspect of the invention, or the pharmaceutical composition described in the sixth aspect of the invention in the preparation of a medicament for the treatment or prevention of tumors.

[0161] According to an embodiment of the present invention, the tumor includes a hematoma.

[0162] According to embodiments of the present invention, the hematologic malignancy includes at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma.

[0163] The amino acid and nucleic acid sequences involved in this application are shown in Table 1.

[0164] Table 1

[0165] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0166] Example 1: Design and preparation of hPSCs with CD33 / CD38 double gene knockout like Figure 1 The diagram shows the overall process for preparing SNTC cells. In this example, the CD33 and CD38 genes are first knocked out. The specific process is as follows: 1.1 Design of hPSCs with CD33 / CD38 dual gene knockout (CD33 / CD38 KO-hPSCs) sgRNA spacer sequences were designed by selecting appropriate PAM sites on the CD33 and CD38 gene sequences. The spacer sequences targeted the exons of the CD33 and CD38 genes, and the target sequences on the CD33 and CD38 genes were unique. After artificially synthesizing the sgRNA products, CRISPR / Cas12 genome editing technology was used to knock out the above target genes.

[0167] Six sgRNA candidates were designed for the CD33 and CD38 genes, and their cleavage efficiency in 293T cells was determined. Vectors containing sgRNA and Cas12 were transduced into 293T cells via electroporation, and genomic DNA was extracted 3 days after transduction. The CD33 and CD38 gene sequences were then amplified by PCR, and the sgRNA cleavage efficiency was assessed by recognizing and cleaving incompletely matched DNA using the T7 endonuclease. Two gRNAs with the highest cleavage efficiency were selected for further knockout of hPSCs cells for each gene; their sequences are shown in Table 2.

[0168] Table 2: Selected sgRNA sequence information

[0169] 1.2 Preparation of hPSCs with CD33 / CD38 double gene knockout The target hPSCs were prepared based on the content in section 1.1. The specific operations are as follows: 1-2 hours prior to resuscitation, hPSCs were resuscitated by coating culture dishes with VTN and resuspended in Gibco™ Essential 8™ medium (E8). The cells were then seeded into the VTN-coated dishes. After culture at 37°C and 5% CO2 until confluence density reached 90%, hPSCs were digested into single cells using Triple digestion, centrifuged, and resuspended in electroporation buffer. Using a Celestrix (CTX-1500A LE+) electroporator, vectors containing the sgRNA and Cas12 sequences listed in Table 1 were transduced into hPSCs. Specific electroporation parameters were as follows: in Cell line mode, at 480V, each vector was added at a rate of 0.5-1 μg to a concentration of 1×10⁻⁶ cells / mL. 6 After suspending the cells, the hPSCs were electroporated. The electroporated hPSCs were then seeded in VTN-coated culture dishes and cultured at 37°C and 5% CO2. Because the vector contained a puromycin resistance gene sequence, 0.5-5 μg / mL puromycin was added to E8 medium on days 3-4 after electroporation for resistance selection. On days 7-8 after electroporation, single-clone hPSCs were picked and passaged. After 7-14 days of further culture and amplification, the genome of each single-clone hPSC was extracted for PCR identification. The results showed that fragments of the CD33 and CD38 genes in hPSCs were deleted. (See schematic diagram below.) Figure 2 This indicates that the two genes were successfully knocked out.

[0170] Example 2: Design of P67-ADPRC1 CAR, FB22 and IL-15RF structures like Figure 3As shown, this embodiment uses four scFvs (T1-scFv, T2-scFv, L1-scFv, L2-scFv) designed in this application targeting P67 and ADPRC1, along with a lead peptide (SP) and CD8hinge, CD8TM, and CD3ζ from the first-generation CAR structure to form four first-generation CAR structures (T1-CAR, T2-CAR, L1-CAR, L2-CAR), mimicking the structure of CARs on the cell membrane. The protein sequences of P67 scFv-VL, P67 scFv-VH, ADPRC1 scFv-VL, and ADPRC1 scFv-VH, as well as the protein sequences of FB22 and IL-15RF, are shown in Table 1.

[0171] Example 3 Preparation of hPSCs overexpressing B22 and IL-15RF This embodiment, based on the hPSCs with P67 and ADPRC1 genes knocked out as described in Example 1, firstly constructs the FB22 gene and IL-15RF gene into homologous recombination vector plasmids, respectively. Then, constructs the sgRNA sequence targeting the AAVS1 site into the pX330 vector. Subsequently, using electroporation, the FB22 homologous recombination plasmid / IL-15RF homologous recombination plasmid and the pX330 plasmid targeting the AAVS1 gene are co-transformed into hPSCs. Flow cytometry is used to sort and obtain hPSCs stably expressing the FB22 gene and IL-15RF gene (P67KO-ADPRC1KO-FB22-IL15RF-hPSC). Figure 4 As shown, flow cytometry results indicated that the expression rate of FB22 was 98.6% and the expression rate of IL-15RF was 98.9%.

[0172] Example 4: Preparation of hPSC cells expressing P67-ADPRC1 CAR Based on the hPSCs obtained in Example 3 with P67 and ADPRC1 genes knocked out and FB22 and IL-15RF genes overexpressed, the nucleic acid molecule encoding the L2-CAR element (SEQ ID NO:34) was first constructed into the PB530A-2 vector (a transposon expression plasmid). The PB530 vector containing the L2-CAR element and the transposase plasmid (PBase) were electroporated into the hPSCs. Stable L2-CAR-expressing hPSCs (P67KO-ADPRC1KO-FB22-IL15RF-P67-ADPRC1 CAR-hPSC, abbreviated as P67-ADPRC1 CAR-hPSC) were obtained using flow cytometry. Figure 5As shown, flow cytometry results indicated that L2-CAR expression in hPSCs was 99.6%.

[0173] Example 5: Induction of P67-ADPRC1 CAR-hPSC differentiation to obtain SNTC cells This embodiment follows the technical steps of the patent "A RIC cell and its preparation method and application" (Chinese patent application number: 202210816564.7, international application number: PCT / CN2022 / 104873) to induce P67-ADPRC1 CAR-hPSCs to differentiate into SNTC cells. The results are as follows... Figure 6 As shown, on day 27, the harvested cells were analyzed by flow cytometry. The results showed that the immunophenotype of SNTC cells was CD45+CD3-CD56+CD16+ / - ( Figure 6 A), does not express the P67 and ADPRC1 genes ( Figure 6 B), while the expression rate of P67-ADPRC1 CAR was 99.1% ( Figure 6 C), the expression rate of the FB22 gene was 96.3% ( Figure 6 D), the expression rate of the IL-15RF gene was 70.8% ( Figure 6 E).

[0174] Example 6: SNTC cells can effectively avoid cannibalism. CFSE-labeled SNTC cells or NTC cells (target cells) were added to 96-well plates, with 100 μL of each containing 1 × 10^4 SNTC cells or NTC cells. Unlabeled SNTC cells were collected and counted. The cell concentration was adjusted according to the following effector-to-target ratios (E:T = 0.2:1, 0.4:1, 0.8:1, and 1.6:1) to ensure that each 100 μL contained the appropriate number of SNTC cells (effector cells). Following the above effector-to-target ratio, 100 μL of SNTC cells was added to each well of the 96-well plate containing SNTC cells or NTC cells. After mixing, the cells were incubated at 37°C for 4 hours. Cells were collected, centrifuged, and the supernatant was discarded. The cells were resuspended in 400 μL of 2% FBS / PBS containing 1 μL of DAPI (1 mg / mL, 400×). Flow cytometry was used to detect cytotoxicity (cytotoxicity was determined by the proportion of DAPI+ cells to CFSE+ cells). The results showed that, compared to NTC cells, SNTC cells could effectively avoid self-harm (…). Figure 7 ).

[0175] Example 7: SNTC cells can specifically and efficiently kill P67 and ADPRC1 positive AML cells. This embodiment verifies that SNTC cells can specifically kill HL-60 tumor cells expressing P67 and ADPRC1 antigens in vitro. SNTCs or NTCs (effector cells) were co-incubated with HL-60 tumor cells (target cells) at the following effector-target ratios (E:T = 0.2:1, 0.4:1, 0.8:1, and 1.6:1) for 12 hours, and the ability of SNTC cells to specifically kill HL-60 tumor cells (cytotoxicity) was evaluated. The results are as follows: Figure 8 As shown, after 12 hours of co-incubation, SNTC cells significantly enhanced the ability to kill P67 and ADPRC1-positive HL-60 tumor cell lines compared to NTC cells.

[0176] Example 8: SNTC cells significantly increased in vivo expansion in a mouse model. To verify whether SNTC cells could enhance the persistence of CAR-NTC cells in vivo, this study infused 5 × 10^5 luciferase-expressing SNTC, NTC, FB22-NTC, and IL15RF-NTC cells (all four cell types were induced from the differentiation of their respective luciferase-expressing hPSCs) into the tail vein of B-NDG severely immunodeficient mice (NOD.CB17-PrkdcscidIl2rgtm1 / Bcgen). Imaging analysis was performed using a small animal in vivo imaging system (IVIS Spectrum PerkinElmer) on days 1, 7, and 14 post-infusion. The results showed that compared to NTC, FB22-NTC, and IL15RF-NTC cells, the in vivo proliferation of SNTC cells was significantly increased. Figure 9 ).

[0177] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0178] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A pluripotent stem cell, characterized in that, include: (1) Downregulation of CD33 and CD38 expression; (2) Overexpression of chimeric antigen receptors targeting CD33 and CD38, and / or overexpression of chimeric antigen receptors targeting CD33 and chimeric antigen receptors targeting CD38; and (3) Overexpression of at least one of FB22 and IL-15RF.

2. The pluripotent stem cell according to claim 1, characterized in that, The pluripotent stem cells are selected from at least one of human embryonic stem cells, human induced pluripotent stem cells, and chemically induced reprogrammed pluripotent stem cells; Optionally, the chimeric antigen receptor targeting CD33 and CD38 includes a single-chain antibody targeting CD33 and a single-chain antibody targeting CD38. Optionally, the chimeric antigen receptor targeting CD33 includes a single-chain antibody targeting CD33; Optionally, the chimeric antigen receptor targeting CD38 includes a single-chain antibody targeting CD38; Optionally, the single-chain antibody targeting CD33 includes a first heavy chain variable region and a first light chain variable region; Optionally, the first heavy chain variable region and the first light chain variable region include a heavy chain variable region and a light chain variable region selected from Gemtuzumabozogamicin, lintuzumab (SGN-33) or M195.34 antibody; Optionally, the C-end of the first heavy chain variable region is connected to the N-end of the first light chain variable region, or the C-end of the first light chain variable region is connected to the N-end of the first heavy chain variable region. Optionally, the first heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% identity with it, and the first light chain variable region has an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity with it. Optionally, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the linker peptide 4, and the C-terminus of the linker peptide 4 is connected to the N-terminus of the first light chain variable region; or the C-terminus of the first light chain variable region is connected to the N-terminus of the linker peptide 4, and the C-terminus of the linker peptide 4 is connected to the N-terminus of the first heavy chain variable region. Optionally, the single-chain antibody targeting CD33 has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% identity with it; Optionally, the single-chain antibody targeting CD38 includes a second heavy chain variable region and a second light chain variable region; Optionally, the second heavy chain variable region and the second light chain variable region include a heavy chain variable region and a light chain variable region selected from Daratumumab, Isatuximab or CM313 antibody; Optionally, the C end of the second heavy chain variable region is connected to the N end of the second light chain variable region, or the C end of the second light chain variable region is connected to the N end of the second heavy chain variable region. Optionally, the second heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% identity with it, and the second light chain variable region has an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% identity with it. Optionally, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the linker peptide 5, and the C-terminus of the linker peptide 5 is connected to the N-terminus of the second light chain variable region, or the C-terminus of the second light chain variable region is connected to the N-terminus of the linker peptide 5, and the C-terminus of the linker peptide 5 is connected to the N-terminus of the second heavy chain variable region. Optionally, the single-chain antibody targeting CD38 has an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 80% identity with it.

3. The pluripotent stem cell according to claim 1, characterized in that, In the chimeric antigen receptor targeting CD33 and CD38, the C-terminus of the single-chain antibody targeting CD33 is linked to the N-terminus of the single-chain antibody targeting CD38. Optionally, the C-terminus of the CD33-targeting single-chain antibody is linked to the N-terminus of the linker peptide 3, and the C-terminus of the linker peptide 3 is linked to the N-terminus of the CD33-targeting single-chain antibody. Optionally, the C-terminus of the single-chain antibody targeting CD38 is linked to the N-terminus of the single-chain antibody targeting CD33; Optionally, the C-terminus of the CD38-targeting single-chain antibody is linked to the N-terminus of the linker peptide 3, and the C-terminus of the linker peptide 3 is linked to the N-terminus of the CD38-targeting single-chain antibody. Optionally, the C-terminus of the first light chain variable region is connected to the N-terminus of the single-chain antibody targeting CD38, and the C-terminus of the single-chain antibody targeting CD38 is connected to the N-terminus of the first heavy chain variable region. Optionally, the C-terminus of the first light chain variable region is connected to the N-terminus of linker peptide 1, the C-terminus of linker peptide 1 is connected to the N-terminus of the second heavy chain variable region, the C-terminus of the second heavy chain variable region is connected to the N-terminus of linker peptide 2, the C-terminus of linker peptide 2 is connected to the N-terminus of the second light chain variable region, the C-terminus of the second light chain variable region is connected to the N-terminus of linker peptide 1, and the C-terminus of linker peptide 1 is connected to the N-terminus of the first heavy chain variable region. Optionally, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the CD33-targeting single-chain antibody, and the C-terminus of the CD33-targeting single-chain antibody is connected to the N-terminus of the second light chain variable region. Optionally, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the linker peptide 1, the C-terminus of the linker peptide 1 is connected to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is connected to the N-terminus of the linker peptide 2, the C-terminus of the linker peptide 2 is connected to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the second light chain variable region. Optionally, the linker peptide 1, linker peptide 2, linker peptide 3, linker peptide 4, and linker peptide 5 are flexible linker peptides or rigid linker peptides. Optionally, the amino acid sequence of linker peptide 1, linker peptide 3, linker peptide 4, and linker peptide 5 is (GGGGS)n, where n is a positive integer not less than 1, and preferably, n is 1 to 6. Preferably, the amino acid sequence of the linker peptide 1 is shown in SEQ ID NO:15; Preferably, the amino acid sequence of the linker peptide 2 is shown in SEQ ID NO:16; Preferably, the amino acid sequence of the linker peptide 3 is shown in SEQ ID NO:17; Preferably, the amino acid sequence of the linker peptide 4 is shown in SEQ ID NO:22; Preferably, the amino acid sequence of the linker peptide 5 is shown in SEQ ID NO:

35.

4. The pluripotent stem cells according to any one of claims 1 to 3, characterized in that, The chimeric antigen receptor targeting CD33 and CD38 and / or the chimeric antigen receptor targeting CD33 and / or the chimeric antigen receptor targeting CD38 further includes at least one of signal peptide 1, hinge region, transmembrane region and intracellular region. Optionally, the C-terminus of the signal peptide 1 is linked to the N-terminus of the chimeric antigen receptor targeting CD33 and CD38 and / or the chimeric antigen receptor targeting CD33 and / or the chimeric antigen receptor targeting CD38. Optionally, the N-terminus of the hinge region is connected to the C-terminus of the chimeric antigen receptor targeting CD33 and CD38 and / or the chimeric antigen receptor targeting CD33 and / or the chimeric antigen receptor targeting CD38. Optionally, the C end of the hinge region is connected to the N end of the transmembrane region; Optionally, the C-terminus of the transmembrane region is connected to the N-terminus of the intracellular region; Optionally, the signal peptide 1 has an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 80% identity with it; Optionally, the hinge region includes the CD8 hinge region; Optionally, the hinge region has an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% identity with it; Optionally, the transmembrane region includes the CD8 transmembrane region; Optionally, the transmembrane region has an amino acid sequence as shown in SEQ ID NO: 19 or an amino acid sequence having at least 80% identity with it; Optionally, the intracellular region includes CD3ζ; Optionally, the intracellular region has the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 80% identity with it; Optionally, the chimeric antigen receptor targeting CD33 has an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% identity with it; Optionally, the chimeric antigen receptor targeting CD38 has an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 80% identity with it; Optionally, the chimeric antigen receptor targeting CD33 and CD38 has an amino acid sequence as shown in any one of SEQ ID NO:24-27 or an amino acid sequence having at least 80% identity with it; Optionally, the FB22 has an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having at least 80% identity with it; Optionally, the IL-15RF includes signal peptide 2, IL-15, linker peptide 6, and IL-15Rα; Optionally, the signal peptide 2 has an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 80% identity with it; Optionally, the IL-15 has an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 80% identity with it; Optionally, the linker peptide 6 has an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence having at least 80% identity with it; Optionally, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 30 or an amino acid sequence having at least 80% identity with it; Optionally, the IL-15RF has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% identity with it.

5. The pluripotent stem cell according to claim 1, characterized in that, The pluripotent stem cells carry: (1) The nucleotide sequences shown in SEQ ID NO:7 and 8, or the nucleotide sequences shown in any one of SEQ ID NO:31 to 34; (2) At least one of the nucleotide sequences shown in SEQ ID NO:9 and 10.

6. A method for preparing pluripotent stem cells according to any one of claims 1 to 5, characterized in that, include: Downregulate the expression of CD33 and CD38 in untreated pluripotent stem cells; Overexpression of chimeric antigen receptors targeting CD33 and CD38 in the pluripotent stem cells to be treated, and / or overexpression of chimeric antigen receptors targeting CD33 and chimeric antigen receptors targeting CD38; And overexpression of at least one of FB22 and IL-15RF in order to obtain the pluripotent stem cells.

7. The method according to claim 6, characterized in that, The downregulation of CD33 and CD38 expression in the pluripotent stem cells to be treated is achieved through at least one of gene silencing, gene editing, small molecule inhibitors, and antibody drugs; Optionally, the overexpression of chimeric antigen receptors targeting CD33 and CD38, and / or the overexpression of chimeric antigen receptors targeting CD33 and CD38 is achieved by introducing nucleic acid molecules encoding chimeric antigen receptors targeting CD33 and CD38, and / or the overexpression of chimeric antigen receptors targeting CD33 and CD38 into the pluripotent stem cells to be treated; Optionally, the overexpression of at least one of FB22 and IL-15RF is achieved by introducing a nucleic acid molecule encoding at least one of FB22 and IL-15RF into the pluripotent stem cells to be treated.

8. The gene editing according to claim 7 is selected from at least one of CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas13, base editor and Prime Editing; the import is performed by at least one of electroporation, transfection and infection.

9. A method for preparing artificially modified innate immune cells, characterized in that, include: The pluripotent stem cells described in any one of claims 1 to 5 or the pluripotent stem cells prepared by the method described in any one of claims 6 to 8 are subjected to directed differentiation culture in order to obtain artificially modified innate immune cells.

10. The method according to claim 9, characterized in that, The directed differentiation culture is carried out using at least one of the following methods: monolayer induction, embryoid induction, and organoid induction.

11. An artificially modified innate immune cell, characterized in that, include: (1) Downregulation of CD33 and CD38 expression; (2) Overexpression of chimeric antigen receptors targeting CD33 and CD38, and / or overexpression of chimeric antigen receptors targeting CD33 and chimeric antigen receptors targeting CD38; and (3) Overexpression of at least one of FB22 and IL-15RF.

12. An artificially modified innate immune cell, characterized in that, Prepared by the method according to claim 9 or 10.

13. A pharmaceutical composition, characterized in that, include: The pluripotent stem cells according to any one of claims 1 to 5, and the artificially modified innate immune cells according to claim 11 or 12.

14. A method for enhancing the killing, proliferation, activation, and migration of artificially modified innate immune cells, characterized in that, include: Pluripotent stem cells are prepared using the method according to any one of claims 6 to 8; as well as The pluripotent stem cells were then subjected to directed differentiation culture.

15. The method according to claim 14, characterized in that, The directed differentiation culture is carried out using at least one of the following methods: monolayer induction, embryoid induction, and organoid induction.

16. Use of the pluripotent stem cells of any one of claims 1 to 5, the artificially modified innate immune cells of claim 11 or 12, or the pharmaceutical composition of claim 13 in the preparation of a medicament for the treatment and / or prevention of tumors.

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