Preparation method and application of CAR-SNTC cell capable of simultaneously targeting three targets

Abstract

The invention discloses a pluripotent stem cell. Wherein the pluripotent stem cells comprise down-regulated expression of CD33, CD38 and CD7, and over-expression of a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and at least one of IL-15RF and LAP-3. 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.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing CAR-SNTC cells that simultaneously target three targets and its application, and more specifically to a method for preparing artificially modified innate immune cells, artificially modified innate immune cells, pharmaceutical compositions, methods for enhancing the killing, proliferation, activation and migration of artificially modified innate immune cells, and their uses. Background Technology

[0002] Malignant hematologic malignancies such as acute myeloid leukemia (AML) and multiple myeloma (MM) have high relapse rates and poor prognoses, especially among elderly patients with extremely low survival rates (e.g., the 5-year survival rate for AML patients is less than 10%). Traditional chemotherapy and radiotherapy cause significant damage to normal cells, have strong side effects, and are difficult to overcome tumor heterogeneity.

[0003] Gene-modified cell therapy is a novel treatment method that treats diseases by modifying the genome of immune cells in patients or healthy individuals. This technology utilizes genetic engineering to introduce exogenous genes into immune cells or knock out regulatory factors within them, endowing these immune cells with new functions or enhanced therapeutic potential. Gene-modified cell technology is widely used for immunotherapy with immune cells such as NK cells, T cells, NKT cells, and macrophages. However, the efficiency of knocking out regulatory factors in immune cells is low, and the cost of introducing exogenous genes is high. Therefore, there is an urgent need to improve the efficiency of knocking out regulatory factors in immune cells and reduce the cost of introducing exogenous genes into immune cells to improve the therapeutic efficacy of immune cell therapy. Summary of the Invention

[0004] 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.

[0005] This invention is based on the following discoveries of the inventors: Simultaneous knockout of CD33, CD38, and CD7 in natural NK cells is inefficient and costly. Furthermore, existing technologies often focus on single modifications (such as knocking out only CD38, CD7, or CD33, or expressing only CAR), lacking synergistic enhancement designs, and the efficiency of performing multiple gene editing on NK cells is even lower. To overcome this problem, the inventors performed gene manipulation at the stem cell stage, knocking out CD38, CD7, and CD33 and introducing functional molecules IL-15RF and LAP-3, before inducing stem cells to differentiate into NK cells. This improved the efficiency and uniformity of gene knockout and significantly reduced the cost of introducing functional molecules.

[0006] In a first aspect, the present invention proposes a pluripotent stem cell. According to embodiments of the invention, the pluripotent stem cell comprises: downregulated expression of CD33, CD38, and CD7, and overexpression of at least one of a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3. The pluripotent stem cells according to embodiments of the invention are capable of directed differentiation into artificially modified innate immune cells that avoid self-harm and cell death, precisely recognize and eliminate tumor cells, and have enhanced survival and migration capabilities. According to embodiments of the invention, "knockout of CD33, CD38, and CD7, and overexpression of a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, IL-15RF, and LAP-3" exhibits significantly superior overall anti-tumor effects compared to knocking out other molecules and overexpressing CARs targeting other irrelevant targets, as well as overexpressing other fusion proteins and overexpressing other chemokines.

[0007] 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, CD38, and CD7 in the pluripotent stem cells to be treated; and overexpressing in the pluripotent stem cells at least one of a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3, in order 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.

[0008] 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.

[0009] In a fourth aspect, the present invention provides an artificially modified innate immune cell. According to embodiments of the invention, the artificially modified innate immune cell comprises downregulated expression of CD33, CD38, and CD7, and overexpression of at least one of a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3. 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 exhibit enhanced survival and migration abilities.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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

[0015] 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 1 This is a diagram showing the deletion of CD33, CD38, and CD7 gene fragments in hPSCs in Example 2.

[0016] Figure 2 This is a graph showing the results of flow cytometry analysis of CD19 CAR, CD38 CAR, and CD7 CAR expression in 3KO / 3CAR-S-hPSCs in Example 3.

[0017] Figure 3 This is a graph showing the results of flow cytometry detection of the 3KO / 3CAR-SNTC phenotype in Example 5.

[0018] Figure 4 This is a graph showing the results of flow cytometry detection of the 3KO / 3CAR-SNTC phenotype in Example 5.

[0019] Figure 5 This is a graph showing the results of flow cytometry detection of the 3KO / 3CAR-SNTC phenotype in Example 5.

[0020] Figure 6 This is a flow cytometry result showing that 3KO / 3CAR-SNTC cells in Example 6 can avoid cannibalism.

[0021] Figure 7 This is a diagram showing the results of 3KO / 3CAR-SNTC cells specifically and efficiently killing tumor cells in Example 7.

[0022] Figure 8 This is a diagram showing the significant expansion of 3KO / 3CAR-SNTC cells in a mouse model in Example 8. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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%.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] This 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.

[0035] pluripotent stem cells In a first aspect, the present invention provides a pluripotent stem cell. According to embodiments of the invention, the pluripotent stem cell comprises: downregulated expression of CD33, CD38, and CD7, and overexpression of at least one of a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3. The pluripotent stem cells according to embodiments of the invention are capable of directed differentiation into artificially modified innate immune cells that avoid self-harm and cell death, precisely recognize and eliminate tumor cells, and have enhanced survival and migration capabilities. It should be noted that, in this document, "chimeric antigen receptor targeting CD38" is abbreviated as "CD38 CAR," "chimeric antigen receptor targeting CD19" is abbreviated as "CD19 CAR," and "chimeric antigen receptor targeting CD7" is abbreviated as "CD7 CAR." According to embodiments of the present invention, "knocking out CD33, CD38, and CD7, and overexpressing chimeric antigen receptors targeting CD38, CD19, and CD7, as well as IL-15RF and LAP-3" exhibits significantly superior overall anti-tumor effects compared to knocking out other molecules, overexpressing CARs targeting other irrelevant targets, overexpressing other fusion proteins, and overexpressing other chemokines. The specific combination according to embodiments of the present invention is not a simple superposition of multiple functions, but rather produces an unexpected synergistic enhancement effect. The present invention creatively combines "knocking out CD33, CD38, and CD7" with "overexpressing CARs targeting CD38, CD19, and CD7," a design that precisely solves a core and challenging problem in multi-target CAR therapy. When immune cells simultaneously express multiple CARs targeting different antigens, if they also express these targets themselves, it will lead to more complex intercellular attacks (i.e., "cannibalism"), severely affecting the yield, stability, and in vivo persistence of cell products. This invention, by simultaneously knocking out three key targets—CD33, CD38, and CD7—that are highly expressed in various hematologic malignancies, fundamentally eliminates the resulting "self-cannibalization" of immune cells. These cells can be used in combination with CD33 antibodies to kill tumor cells, ensuring the stable coexistence of the obtained 3KO / 3CAR-SNTC cells and simultaneously exerting their effects against CD19. + (such as B-cell lymphoma), CD38 + (such as multiple myeloma) and CD7 +This invention enhances the multi-target killing ability of tumors (such as T-cell leukemia). This targeted, paired design of multiple knockout and multiple CAR expression offers a key advantage that cannot be achieved by combining CARs targeting other antigens with the knockout of other irrelevant molecules, laying a solid foundation for safe and efficient "universal" multi-target anti-cancer cell products. The invention deeply integrates three functional modules: multiple CARs (providing multiple specific recognition and killing), IL-15RF (providing autocrine proliferation and survival signals), and LAP-3 (enhancing migration and homing ability to tumor microenvironments such as bone marrow), forming a functionally tightly synergistic unit. The multiple CAR module constitutes a "multi-precision navigation system," enabling cells to simultaneously recognize and eliminate heterogeneous tumor cell populations expressing CD19, CD38, or CD7, significantly broadening the therapeutic spectrum and potentially reducing the risk of tumor immune escape. The IL-15RF module, as a "built-in sustaining system," continuously provides IL-15 signals through autocrine / paracrine mechanisms, greatly enhancing the survival, persistence, and proliferation of derived immune cells in the suppressive tumor microenvironment, reducing dependence on exogenous cytokines. The LAP-3 module, acting as a "highly efficient homing system," significantly enhances the migration and infiltration efficiency of cells into hematopoietic tissues such as bone marrow (the lesion sites of various hematological malignancies), ensuring that a sufficient number of effector cells can reach and act on the tumor site. The synergistic effect of these three modules achieves a comprehensive functional integration of "multi-precision navigation (3CAR) + sustained combat capability (IL-15RF) + efficient force delivery (LAP-3)." As shown in Example 8, 3KO / 3CAR-SNTC cells, possessing all three modules, exhibited significantly better in vivo expansion and survival than the control group (3KO / 3CAR-LAP3-NTC or 3KO / 3CAR-IL15RF-NTC) possessing only some components, demonstrating the synergistic enhancement effect of this combination in terms of in vivo persistence. This technical solution represents a deep optimization targeting key challenges in the treatment of malignant hematological malignancies. CD19, CD38, and CD7 are among the most important and mature therapeutic targets in B-cell malignancies, multiple myeloma / acute myeloid leukemia, and T-cell malignancies, respectively. This invention not only solves the problem of immune cell cannibalism through simultaneous knockout, but also enhances the adaptability and combat effectiveness of cells in the hematologic malignancy microenvironment through the addition of IL-15RF and LAP-3. As shown in Example 7, the 3KO / 3CAR-SNTC cells obtained by this scheme are effective against Raji (CD19) + ), RPMI8226 (CD38) + ) and CCRF-CEM (CD7 +These three representative hematologic malignancy cell lines all exhibited significantly enhanced specific killing activity, demonstrating their powerful broad-spectrum anti-tumor ability. In other words, the specific technical combination of "CD33 / CD38 / CD7 triple knockout + CD38 / CD19 / CD7 triple CAR + IL-15RF + LAP-3" provided by this invention is not an obvious simple replacement for the conventional approach of "knocking out other molecules to overexpress CARs targeting other irrelevant targets, and overexpressing other fusion proteins and other chemokines." The combination according to embodiments of this invention addresses the bottleneck of immune cell "self-destruction" in multi-target CAR therapy and integrates multiple targeting, durable survival, and efficient homing function, generating a series of unexpected synergistic effects. Ultimately, in the treatment of malignant hematologic malignancies, it brings significant and comprehensive improvements in cell product stability, in vivo persistence, tumor invasiveness, killing spectrum breadth, and final anti-tumor efficacy.

[0036] 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.

[0037] 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.

[0038] In this article, "CD7" belongs to the immunoglobulin superfamily (IgSF). Its extracellular domain contains a single Ig-like V-shaped domain that mediates cell-cell adhesion. Its transmembrane region is a single transmembrane structure, and its intracellular domain is a short tail containing signal transduction motifs. It consists of about 240 amino acids and is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma. CD7 is one of the commonly used targets for the treatment of malignant hematological malignancies.

[0039] 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.

[0040] 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 those variants of the CAR (parent CAR) described herein that retain the ability to recognize target cells to a degree similar to, the same as, or greater than that of the parent CAR. Regarding the parent CAR, the amino acid sequence of the functional variant may 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.

[0041] The functional variant may comprise the amino acid sequence of the parental CAR having at least one conserved amino acid substitution. Alternatively or additionally, the functional variant may comprise the amino acid sequence of the parental 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 parental CAR.

[0042] 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.).

[0043] According to an embodiment of the present invention, the pluripotent stem cells include: downregulated expression of CD33, CD38 and CD7, and overexpression of chimeric antigen receptors targeting CD38, CD19, CD7 and IL-15RF.

[0044] According to an embodiment of the present invention, the pluripotent stem cells include: downregulated expression of CD33, CD38 and CD7, and overexpression of chimeric antigen receptors targeting CD38, CD19, CD7 and LAP-3.

[0045] According to an embodiment of the present invention, the pluripotent stem cells include: downregulated expression of CD33, CD38 and CD7, and overexpression of chimeric antigen receptors targeting CD38, CD19, and CD7, as well as IL-15RF and LAP-3.

[0046] 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.

[0047] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD38 includes a single-chain antibody targeting CD38.

[0048] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD38 further comprises: a first transmembrane region and a first intracellular region, wherein the C-terminus of the single-chain antibody targeting CD38 is connected to the N-terminus of the first transmembrane region, and the C-terminus of the first transmembrane region is connected to the N-terminus of the first intracellular region.

[0049] 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: 1 or having at least 80% sequence homology with it.

[0050] MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKGGGGSG GGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS (SEQ ID NO: 1) According to an embodiment of the present invention, the first transmembrane region includes the CD8 transmembrane region.

[0051] According to an embodiment of the present invention, the first intracellular region includes a signal transduction domain.

[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: 2 or having at least 80% sequence homology with it.

[0053] MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQR SNWPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVK FSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 2).

[0054] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD38 has a nucleotide sequence as shown in SEQ ID NO: 9 or having at least 80% sequence homology with it.

[0055]

[0056] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD19 includes a single-chain antibody targeting CD19.

[0057] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD19 further includes: a second transmembrane region and a second intracellular region, wherein the C-terminus of the single-chain antibody targeting CD19 is connected to the N-terminus of the second transmembrane region, and the C-terminus of the second transmembrane region is connected to the N-terminus of the second intracellular region.

[0058] According to an embodiment of the present invention, the single-chain antibody targeting CD19 has an amino acid sequence as shown in SEQ ID NO: 3 or having at least 80% sequence homology with it.

[0059] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGS GGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 3) According to an embodiment of the present invention, the chimeric antigen receptor targeting CD19 has an amino acid sequence as shown in SEQ ID NO: 4 or having at least 80% sequence homology with it.

[0060] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNKSSQVFLKM NSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFS RSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 4).

[0061] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD19 has a nucleotide sequence as shown in SEQ ID NO: 10 or having at least 80% sequence homology with it.

[0062]

[0063] According to an embodiment of the present invention, the second transmembrane region includes the CD8 transmembrane region.

[0064] According to an embodiment of the present invention, the second intracellular region includes a signal transduction domain.

[0065] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD7 includes a single-chain antibody targeting CD7.

[0066] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD7 further includes a third transmembrane region and a third intracellular region, wherein the C-terminus of the single-chain antibody targeting CD7 is connected to the N-terminus of the third transmembrane region, and the C-terminus of the third transmembrane region is connected to the N-terminus of the third intracellular region.

[0067] According to an embodiment of the present invention, the single-chain antibody targeting CD7 has an amino acid sequence as shown in SEQ ID NO: 5 or having at least 80% sequence homology with it.

[0068] ALPVTALLLPLALLLHAARPGAQPAMAAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRGGGG SGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSASGAD (SEQ ID NO: 5) According to an embodiment of the present invention, the third transmembrane region includes the CD8 transmembrane region.

[0069] According to an embodiment of the present invention, the third intracellular region includes a signal transduction domain.

[0070] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD7 has an amino acid sequence as shown in SEQ ID NO: 6 or having at least 80% sequence homology with it.

[0071] ALPVTALLLPLALLLHAARPGAQPAMAAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATY YCQQYSKLPYTFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNA RNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSASGADTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY CRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 6).

[0072] According to an embodiment of the present invention, the chimeric antigen receptor targeting CD7 has a nucleotide sequence as shown in SEQ ID NO: 11 or having at least 80% sequence homology with it.

[0073]

[0074] According to an embodiment of the present invention, the IL-15RF has an amino acid sequence as shown in SEQ ID NO: 7 or having at least 80% sequence homology with it.

[0075] MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSS LTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 7).

[0076] According to an embodiment of the present invention, the IL-15RF has a nucleotide sequence as shown in SEQ ID NO: 12 or having at least 80% sequence homology with it.

[0077]

[0078] According to embodiments of the present invention, the IL-15RF includes a signal peptide, IL-15, a linker peptide, and IL-15Rα. It should be noted that in this invention, "IL-15RF" and "IL15RF" are synonymous.

[0079] According to an embodiment of the present invention, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 22 or having at least 80% sequence homology with it.

[0080] MDWTWILFLVAAATRVHS (SEQ ID NO: 22) According to an embodiment of the present invention, the IL-15 has an amino acid sequence as shown in SEQ ID NO: 23 or having at least 80% sequence homology with it.

[0081] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 23) According to an embodiment of the present invention, the linker peptide has an amino acid sequence as shown in SEQ ID NO: 24 or having at least 80% sequence homology with it.

[0082] SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 24) According to an embodiment of the present invention, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 25 or having at least 80% sequence homology with it.

[0083] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGS QLMPSKSSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 25) According to an embodiment of the present invention, IL-15RF is a fusion protein of IL-15 and IL-15Rα. It continuously activates the IL-15 pathway through autocrine signaling, promotes the survival, proliferation and functional activation of CAR-SNTC cells, reduces dependence on exogenous cytokines, and enhances the cell's proliferation and survival capabilities.

[0084] According to an embodiment of the present invention, the LAP-3 has an amino acid sequence as shown in SEQ ID NO: 8 or having at least 80% sequence homology with it.

[0085] MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFAN VSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS (SEQ ID NO: 8).

[0086] According to an embodiment of the present invention, the LAP-3 has a nucleotide sequence as shown in SEQ ID NO: 13 or having at least 80% sequence homology with it.

[0087]

[0088] According to embodiments of the present invention, the LAP-3 includes an extracellular domain, a transmembrane domain, and an intracellular domain. It should be noted that LAP-3 can promote the migration and homing of immune cells, and overexpression of LAP-3 molecules on pluripotent stem cells can further derive cells with enhanced ability to migrate to the bone marrow.

[0089] 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, CD38, and CD7 in the pluripotent stem cells to be treated; and overexpressing in the pluripotent stem cells at least one of a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3, in order 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.

[0090] According to an embodiment of the present invention, the downregulation of CD33, CD38 and CD7 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.

[0091] 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.

[0092] According to embodiments of the present invention, the overexpression of at least one of the chimeric antigen receptor targeting CD38, the chimeric antigen receptor targeting CD19, the chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3 is achieved by introducing nucleic acid molecules encoding the chimeric antigen receptor encoding CD38, the chimeric antigen receptor encoding CD19, the chimeric antigen receptor encoding CD7, IL-15RF, and LAP-3 into pluripotent stem cells to be treated.

[0093] According to an embodiment of the present invention, the introduction is performed by at least one of electroporation, transfection, and infection.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] Artificially modified innate immune cells In a fourth aspect, the present invention provides an artificially modified innate immune cell. According to embodiments of the invention, the artificially modified innate immune cell comprises downregulated expression of CD33, CD38, and CD7, and overexpression of at least one of a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3. 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 exhibit enhanced survival and migration abilities.

[0098] Artificially modified innate immune cells 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.

[0099] 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.

[0100] According to embodiments of the present invention, pharmaceutically acceptable excipients are further included.

[0101] 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 readily apparent 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] According to an embodiment of the present invention, the tumor includes a hematoma.

[0106] According to embodiments of the present invention, the hematologic malignancy includes, but is not limited to, at least one of acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma.

[0107] 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.

[0108] Example 1: Design scheme for CD33 / CD38 / CD7 triple gene knockout in hPSCs gRNAs were designed by selecting appropriate PAM sites on the CD33, CD38, and CD7 gene sequences, and then CRISPR / Cas12 genome editing technology was used to knock out the target genes.

[0109] Six gRNA candidates were designed for the CD33, CD38, and CD7 genes, and their cleavage efficiency in 293T cells was determined. Vectors containing gRNA and Cas12 were transduced into 293T cells via electroporation, and genomic DNA was extracted three days after transduction. The CD33, CD38, and CD7 gene sequences were then amplified by PCR, and the gRNA cleavage efficiency was assessed by recognizing and cleaving incompletely matched DNA using the T7 endonuclease. Two gRNAs with the highest cleavage efficiency for each of the three genes (Table 1) were selected for further knockout of hPSCs cells.

[0110] Table 1: Selected gRNA sequence information

[0111] Example 2: Construction of hPSCs with CD33, CD38 and CD7 knockout molecules (CD33 / CD38 / CD7 KO-hPSCs, denoted as 3KO-hPSCs) hPSCs were resuscitated by coating culture dishes with VTN and resuspended in Gibco™ Essential 8™ medium (E8) before being seeded into VTN-coated culture dishes. After culturing at 37°C and 5% CO2 for 6–7 days, hPSCs were digested into single cells using Tryple, centrifuged, and resuspended in electroporation buffer. Using a Celestrix (CTX-1500A LE+) electroporator, vectors containing the gRNA and Cas12 sequences listed in Table 1 were transduced into hPSCs via electroporation. 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 Electroporation was performed on the suspended hPSCs cells. 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, CD38, and CD7 genes in hPSCs were deleted. (See the schematic diagram for details.) Figure 1 This indicates that the three genes were successfully knocked out.

[0112] Example 3: Importing CD19 CAR, CD38 CAR, CD7 CAR, LAP-3 and IL-15RF into CD33 / CD38 / CD7 KO-hPSCs (denoted as 3KO / 3CAR-S-hPSCs) 1. Vectors containing CAR elements targeting CD19, CD38, and CD7 were introduced into CD33 / CD38 / CD7-KO hPSCs via electroporation. First, the nucleotide sequences encoding the CAR proteins for CD19, CD38, and CD7 were ligated into the piggyBac vector using homologous recombination molecular biology methods, forming PB-CD19 CAR, PB-CD38 CAR, and PB-CD7 CAR recombinant vectors.

[0113] The amino acid sequence of the chimeric antigen receptor targeting CD38 (CD38 CAR) is shown in SEQ ID NO: 2.

[0114] The nucleotide sequence encoding the CD38 CAR protein is shown in SEQ ID NO:9.

[0115] The amino acid sequence of the chimeric antigen receptor targeting CD19 (CD19 CAR) is shown in SEQ ID NO: 4.

[0116] The nucleotide sequence encoding the CD19 CAR protein is shown in SEQ ID NO: 10.

[0117] The amino acid sequence of the chimeric antigen receptor targeting CD7 (CD7 CAR) is shown in SEQ ID NO:6.

[0118] The nucleotide sequence encoding the CD7 CAR protein is shown in SEQ ID NO: 11.

[0119] The piggyBac plasmid and transposase plasmid (PB210PA-1, SBI brand) containing the nucleotide sequences of CD19 CAR, CD38 CAR and CD7 CAR were electroporated into CD33 / CD38 / CD7KO-hPSCs using an electroporator (11-0106, Celetrix brand). On day 7 after electroporation, hPSCs expressing CD19 CAR, CD38 CAR and CD7 CAR were sorted by flow cytometry. The specific sorting process is as follows: First, the hPSCs to be sorted are incubated and stained with FITC-Labeled Human CD38 Protein, His Tag (CD8-HF2H5, Acro brand), APC-Labeled Human CD7 Protein, His Tag (CD7-HA2H6, Acro brand), Rabbit anti-mouse FMC63, scFv monoclonal antibody, and PE (200106, Bioswan brand) antibody. After incubation for 15 minutes, the antibodies are washed off with PBS, and the hPSCs are resuspended in DAPI solution (422801, Biolegend brand). The cells are then sorted using a flow cytometer (MA900, Sony) to separate DAPI-negative CD19 CAR, CD38 CAR, and CD7 CAR triple-positive cells. The sorted cells were cultured in culture medium (Essential 8™ Medium, A1517001, Gibco brand) to obtain CD33 / CD38 / CD7 KO-hPSC clones (3KO / 3CAR-hPSCs) expressing CD19 CAR, CD38 CAR and CD7 CAR.

[0120] 2. The LAP-3 sequence (amino acid sequence as shown in SEQ ID NO: 8) was integrated into the AAVS1 safe site of the 3KO / 3CAR-hPSCs genome via electroporation using CRISPR / Cas9 and homologous recombination technology. Specifically, the homologous recombination vector encoding the LAP-3 protein (nucleotide sequence as shown in SEQ ID NO: 13) and the CRISPR / Cas9 vector targeting the hAAVS1 site (Cas 9 sgRNA1: CACCGTCACCAATCCTGTCCCTAG (SEQ ID NO: 20); Cas 9 sgRNA2: AAACCTAGGGACAGGATTGGTGAC (SEQ ID NO: 21)) were electroporated together into 3KO / 3CAR-hPSCs cells, and the hPSCs expressing LAP-3 were sorted using flow cytometry. The specific sorting process is as follows: First, the hPSCs to be sorted are incubated and stained with a flow cytometry antibody against LAP-3 (clone number: 12G5, Biolegend brand). After incubation for 15 minutes, the antibody is washed off with PBS, and the hPSCs are resuspended in DAPI solution (422801, Biolegend brand). The cells are then sorted using a flow cytometer (MA900, Sony) to separate DAPI-negative and LAP-3-positive cells. The sorted cells are then cultured in culture medium (Essential 8™ Medium, A1517001, Gibco brand) to obtain 3KO / 3CAR-hPSC clones expressing LAP-3 (3KO / 3CAR-LAP3-hPSCs).

[0121] 3. The piggyBac vector (PB530A-2, SBI brand) containing IL-15RF (the amino acid sequence of IL-15RF is shown in SEQ ID NO: 7) was introduced into 3KO / 3CAR-LAP3-hPSCs via electroporation. Specifically, the piggyBac plasmid encoding the nucleic acid molecule of IL-15RF protein (nucleotide sequence shown in SEQ ID NO: 12) and the transposase plasmid (PB210PA-1, SBI brand) were first electroporated into 3KO / 3CAR-LAP3-hPSCs using an electroporator (11-0106, Celetrix brand). On day 7 after electroporation, hPSCs expressing IL-15 were sorted using flow cytometry. The specific sorting process is as follows: First, the hPSCs to be sorted are incubated and stained with IL-15 Monoclonal Antibody (34559) and PE (MA5-23561, Invitrogen) antibodies. After incubation for 15 minutes, the antibodies are washed off with PBS, and the hPSCs are resuspended in DAPI solution (422801, Biolegend). The cells are then sorted using a flow cytometer (MA900, Sony) to separate DAPI-negative and IL-15-positive cells. The sorted cells are then cultured in culture medium (Essential 8™ Medium, A1517001, Gibco) to obtain IL-15-expressing 3KO / 3CAR-LAP3-hPSC clones (3KO / 3CAR-S-hPSCs). Flow cytometry analysis revealed that the expression rates of CD19 CAR, CD38 CAR, and CD7 CAR in 3KO / 3CAR-S-hPSCs were 99.6%, 99.4%, and 86.4%, respectively; the expression rate of LAP-3 was 95.5%; and the expression rate of IL15 was 77.3%. For detailed results, please refer to [link to relevant documentation]. Figure 2 .

[0122] Example 4: Construction of 3KO / 3CAR-IL15RF-hPSCs The process in this embodiment is the same as in Example 3, except that the IL15RF sequence (amino acid sequence as shown in SEQ ID NO:7) is not integrated into the AAVS1 safe site of the 3KO / 3CAR-hPSCs genome using CRISPR / Cas9 and homologous recombination technology without electroporation. The other processes are the same as in Example 3 to construct 3KO / 3CAR-IL15RF-hPSCs.

[0123] Example 5: Induction of 3KO / 3CAR-S-hPSCs into 3KO / 3CAR-SNTC cells High-purity lateral plate mesodermal cells were obtained by monolayer induction of hPSCs, 3KO / 3CAR-S-hPSCs prepared in Example 3, 3KO / 3CAR-IL15RF-hPSCs prepared in Example 4, and 3KO / 3CAR-LAP3-hPSCs prepared in Example 3 for 2 days. 2×10 4 The mesoderm of each lateral plate and 5×10 5 OP9 feeder cells were prepared as organoid units and seeded onto a Transwell nested membrane to form a gas-liquid interface for hematopoietic induction differentiation. After 25 days of induction, mature NTC cells, 3KO / 3CAR-SNTC cells, 3KO / 3CAR-IL15RF-NTC cells, and 3KO / 3CAR-LAP3-NTC cells were obtained, respectively. On Day 27, flow cytometry was used to detect the phenotypes of 3KO / 3CAR-SNTC cells, 3KO / 3CAR-IL15RF-NTC cells, and 3KO / 3CAR-LAP3-NTC cells. This example exemplifies the results for 3KO / 3CAR-SNTC cells; for detailed results, please refer to [link to relevant documentation]. Figure 3 The expression of CD33, CD38, CD7 molecules, CD19 CAR, CD38 CAR, CD7 CAR, LAP-3, and IL-15RF was also observed. The results showed that, compared to innate immune cells (NTCs) induced from unmodified hPSCs, 3KO / 3CAR-SNTC cells did not express CD33, CD38, and CD7 molecules. For details, please refer to [link to relevant documentation]. Figure 4 3KO / 3CAR-SNTC cells expressed CD19CAR, CD38CAR, CD7CAR, LAP-3, and IL-15RF. See details for further information. Figure 5 .

[0124] Example 6: 3KO / 3CAR-SNTC cells can avoid cannibalism NTCs and 3KO / 3CAR-SNTCs in target cells were stained with CFSE and counted. RPMI-1640 medium containing 10% FBS was used at a concentration of 1×10⁻⁶ NTCs per 100 μL. 4The cell suspension volume was adjusted by increasing the concentration of individual cells. 100 μL of 3KO / 3CAR-SNTC cells (target cells) were added to each well of a U-bottom 96-well plate. The target cells for the control group were NTC cells induced from unmodified hPSCs. The 3KO / 3CAR-SNTC cells were counted, and the cell concentration was adjusted according to the following effector-to-target ratios (E:T = 0.2:1, 0.8:1, and 1.6:1) to ensure that each 100 μL contained the appropriate amount of 3KO / 3CAR-SNTC cells. Following the above effector-target ratio, 100 μL of 3KO / 3CAR-SNTC cell suspension (effector cells) was added to each of the 96-well culture dishes containing NTC or 3KO / 3CAR-SNTC target cells. After mixing, the cells were incubated at 37°C for 4 hours. Cells were harvested, centrifuged at 500×g, 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×). NK cell killing activity was detected by flow cytometry (cytotoxicity was determined by the proportion of DAPI+ cells to CFSE+ cells). Results showed that 3KO / 3CAR-SNTC cells significantly killed NTC cells, but there was no significant killing effect among 3KO / 3CAR-SNTC cells, indicating that 3KO / 3CAR-SNTC cells can effectively avoid self-killing. For detailed results, please refer to [link to relevant documentation]. Figure 6 .

[0125] Example 7: 3KO / 3CAR-SNTC cells specifically and efficiently kill tumor cells This embodiment verifies that 3KO / 3CAR-SNTC cells can specifically kill Raji, RPMI8226, and CCRF-CEM tumor cells expressing CD19, CD38, and CD7 antigens in vitro. 3KO / 3CAR-SNTC or NTC cells were co-incubated with Raji, RPMI8226, and CCRF-CEM tumor cells at the following effector-to-target ratios (E:T = 0.4:1, 0.8:1, and 1.6:1) for 12 hours. The ability of 3KO / 3CAR-SNTC cells to specifically kill Raji, RPMI8226, and CCRF-CEM tumor cells (cytotoxicity) was evaluated. After 12 hours of co-incubation, compared with NTC cells, 3KO / 3CAR-SNTC cells significantly enhanced the ability to kill Raji, RPMI8226, and CCRF-CEM tumor cell lines. For detailed results, please refer to [link to relevant documentation]. Figure 7 .

[0126] Example 8: 3KO / 3CAR-SNTC cells significantly prolonged in vivo duration in a mouse model. As described in Example 3, hPSCs expressing luciferase (LUCI), 3KO / 3CAR-LAP3-hPSCs, 3KO / 3CAR-IL15RF-hPSCs, and 3KO / 3CAR-S-hPSCs were constructed respectively. NTCs expressing luciferase, 3KO / 3CAR-LAP3-NTCs, 3KO / 3CAR-IL15RF-NTCs, and 3KO / 3CAR-SNTCs were induced to express luciferase according to the method described in Example 5, and 5 × 10⁻⁶ NTCs were then used. 5 The corresponding cells were infused into the tail vein of B-NDG severely immunodeficient mice (NOD.CB17-PrkdcscidIl2rgtm1 / Bcgen), and 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, 3KO / 3CAR-LAP3-NTC, and 3KO / 3CAR-IL15RF-NTC, 3KO / 3CAR-SNTC cells significantly expanded in vivo. For details, please refer to [link to relevant documentation]. Figure 8 .

[0127] 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.

[0128] 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: Downregulation of CD33, CD38 and CD7 expression, and overexpression of at least one of the following: a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3.

2. The pluripotent stem cell according to claim 1, characterized in that, include: Downregulation of CD33, CD38 and CD7 expression, and overexpression of chimeric antigen receptors targeting CD38, CD19, CD7 and IL-15RF. Optionally, this includes: downregulation of CD33, CD38 and CD7 expression, and overexpression of chimeric antigen receptors targeting CD38, CD19, CD7 and LAP-3. Optionally, this includes: downregulation of CD33, CD38 and CD7 expression, and overexpression of chimeric antigen receptors targeting CD38, CD19, CD7, IL-15RF and LAP-3. Optionally, 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.

3. The pluripotent stem cells according to claim 1 or 2, characterized in that, The chimeric antigen receptor targeting CD38 includes: a single-chain antibody targeting CD38; Optionally, the chimeric antigen receptor targeting CD38 has an amino acid sequence as shown in SEQ ID NO: 2 or having at least 80% sequence homology with it; Optionally, the chimeric antigen receptor targeting CD19 includes: a single-chain antibody targeting CD19; Optionally, the chimeric antigen receptor targeting CD19 has an amino acid sequence as shown in SEQ ID NO: 4 or having at least 80% sequence homology with it; Optionally, the chimeric antigen receptor targeting CD7 includes: a single-chain antibody targeting CD7; Optionally, the chimeric antigen receptor targeting CD7 has an amino acid sequence as shown in SEQ ID NO: 6 or having at least 80% sequence homology with it; Optionally, the IL-15RF includes a signal peptide, IL-15, a linker peptide, and IL-15Rα; Optionally, the IL-15RF has an amino acid sequence as shown in SEQ ID NO: 7 or having at least 80% sequence homology with it; Optionally, the LAP-3 has an amino acid sequence as shown in SEQ ID NO: 8 or having at least 80% sequence homology with it.

4. The pluripotent stem cell according to claim 3, characterized in that, The single-chain antibody targeting CD38 has an amino acid sequence as shown in SEQ ID NO: 1 or having at least 80% sequence homology with it; Optionally, the single-chain antibody targeting CD19 has an amino acid sequence as shown in SEQ ID NO: 3 or having at least 80% sequence homology with it; Optionally, the single-chain antibody targeting CD7 has an amino acid sequence as shown in SEQ ID NO: 5 or having at least 80% sequence homology with it; Optionally, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 22 or having at least 80% sequence homology with it; Optionally, the IL-15 has an amino acid sequence as shown in SEQ ID NO: 23 or having at least 80% sequence homology with it; Optionally, the linker peptide has an amino acid sequence as shown in SEQ ID NO: 24 or having at least 80% sequence homology with it; Optionally, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 25 or having at least 80% sequence homology with it.

5. A method for preparing pluripotent stem cells according to any one of claims 1 to 4, characterized in that, include: Downregulate the expression of CD33, CD38, and CD7 in untreated pluripotent stem cells; Overexpression of at least one of a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3 in the pluripotent stem cells to be treated is carried out in order to obtain the pluripotent stem cells.

6. The method according to claim 5, characterized in that, The downregulation of CD33, CD38, and CD7 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 at least one of the chimeric antigen receptors targeting CD38, CD19, CD7, IL-15RF, and LAP-3 is achieved by introducing nucleic acid molecules encoding the chimeric antigen receptors targeting CD38, CD19, CD7, IL-15RF, and LAP-3 into the pluripotent stem cells to be treated.

7. The method according to claim 6, characterized in that, The gene editing is selected from at least one of CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas13, base editor, and Prime Editing; Optionally, the importation is performed by at least one of electroporation, transfection, and infection.

8. 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 4 or the pluripotent stem cells prepared by the method described in any one of claims 5 to 7 are subjected to directed differentiation culture in order to obtain artificially modified innate immune cells.

9. The method according to claim 8, 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.

10. An artificially modified innate immune cell, characterized in that, include: Downregulation of CD33, CD38 and CD7 expression, and overexpression of at least one of the following: a chimeric antigen receptor targeting CD38, a chimeric antigen receptor targeting CD19, a chimeric antigen receptor targeting CD7, and IL-15RF and LAP-3.

11. An artificially modified innate immune cell, characterized in that, Prepared by the method according to claim 8 or 9.

12. A pharmaceutical composition, characterized in that, include: The pluripotent stem cells according to any one of claims 1 to 4, and the artificially modified innate immune cells according to claim 10 or 11.

13. 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 described in any one of claims 5 to 7; as well as The pluripotent stem cells were then subjected to directed differentiation culture.

14. The method according to claim 13, 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.

15. Use of the pluripotent stem cells of any one of claims 1 to 4, the artificially modified innate immune cells of claim 10 or 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for the treatment or prevention of tumors.

16. The use according to claim 15, characterized in that, The tumors include hemangiomas; Optionally, the hematologic malignancy includes at least one of acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma.

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