Immune cell with improved function and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a pluripotent stem cell containing a SOCS3 knockout and / or chimeric antigen receptor, an immune cell, a composition and pharmaceutical application. The invention provides a pluripotent stem cell or a derivative cell thereof. The SOCS3 gene in the pluripotent stem cell is knocked out. In the process that the pluripotent stem cells are differentiated into the NK cells, higher cell yield and higher activation receptor and costimulatory factor expression are achieved. The invention also provides an immune cell, the SOCS3 gene in the immune cell is knocked out, and the surface of the immune cell contains the chimeric antigen receptor. The immune cells have enhanced amplification ability, anti-tumor activity, and persistence (long-term survival in vitro or in vivo).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to pluripotent stem cells, immune cells, compositions, and pharmaceutical uses containing SOCS3 knockout and / or chimeric antigen receptors. Background Technology

[0002] Adoptive cell therapy, using immune cells from patients and donors, has achieved great success. However, the preparation of universal, batch-uniform, and functionally enhanced immune cells remains challenging, especially in terms of improving their in vitro and in vivo persistence. Lymphocytes such as T cells and natural killer (NK) cells can effectively kill tumor cells and play an important role in innate and adaptive immunity, making them the main effectors in current adoptive cell therapy. However, the ability of these cells to expand in vitro and in vivo, their persistence, and their anti-tumor capabilities still require further improvement.

[0003] Natural killer (NK) cells are naturally occurring non-T lymphocytes capable of rapidly killing virus-infected cells and tumor cells. They can recognize tumor cells through a set of activating and inhibiting receptors, thus initiating the killing of tumor cells. As a key component of the innate immune system, NK cells are important effectors of the lymphocyte population in anti-tumor and anti-infection immunity. However, NK cell depletion is often accompanied by tumor progression and chronic infection in patients, leading to limited immune surveillance and anti-tumor functions, thus restricting the potential of NK cells in immune surveillance, anti-tumor, and anti-infection. The great success of adoptive cell therapy (such as autologous chimeric antigen receptor (CAR) T cells) in patients with hematologic malignancies and the market launch of related cell therapy products have sparked great interest in using gene-edited immune cells to treat cancer. The success of CAR-T cells has also inspired efforts to explore the clinical applications of other immune cell types (such as NK cells). Compared with T cells, NK cells do not have TCR, and allogeneic transplantation does not produce graft-versus-host disease (GvHD), cytokine storms, neurotoxicity, etc., making them more suitable for preparation as universal immune cell therapy drugs. NK cells have multiple sources, including peripheral blood, umbilical cord blood, the NK92 cell line, and pluripotent stem cells. NK cells differentiated from pluripotent stem cells are easily gene-edited, and multiple batches of uniform NK cells can be produced by establishing tertiary cell banks. In recent years, several NK cell drugs differentiated from pluripotent stem cells have entered the clinical trial stage.

[0004] Cytokines are crucial for the preactivation of NK cells. Interleukin-2 (IL-2) and interleukin-15 (IL-15) can enhance the signal transduction of NK cell activation receptors, maintaining NK cell activity and function. IL-15 is structurally very similar to IL-2. The heterotrimeric receptor of IL-15 shares the same IL-2R / IL-15Rβ (CD122) and γ chain (CD132) as the IL-2 receptor. These shared receptor components share the JAK1 / JAK3 / STAT5 signaling pathway. IL-15 and IL-2 have similar functions, including stimulating T cell proliferation, producing cytotoxic T lymphocytes, stimulating B cell immunoglobulin synthesis, and promoting NK cell production and survival. However, IL-2 is prone to causing systemic toxicity; in vitro culture with continuous high-dose (10 ng / ml) IL-15 stimulation also leads to low NK cell survival and cell cycle arrest, accompanied by weakened signal transduction, reduced in vivo and in vitro function, and decreased antitumor activity. This indicates that sustained high-dose IL-15 culture leads to NK cell depletion (doi.org / 10.1172 / jci.insight.96219). In vivo, high-dose IL-15 can easily cause several side effects, including hypotension, thrombocytopenia, elevated transaminase levels, pancreatitis, chest pain accompanied by hypotension, and elevated troponin levels (https: / / doi.org / 10.1038 / s41573-019-0052-1). Therefore, improving the sensitivity of NK cells to IL-15 to maintain the activity and persistence of iNK cells in vivo under physiological conditions or at low doses of IL-15 is crucial for NK cell-based immunotherapy. SOCS family proteins, including CISH and SOCS1-7, can be induced by cytokines and subsequently inhibit cytokine signaling, forming a classic negative feedback loop. CISH has been identified as an immune checkpoint for NK cells (https: / / doi:10.1038 / ni.3470). Knocking out CISH in iPSCs enhances the sensitivity of differentiated NK cells to IL-15 and strengthens their proliferative capacity and anti-tumor activity. However, CISH knockout iPSCs experience a delay in the differentiation of CD56+ iNK cells into iNK cells (https: / / doi.org / 10.1016 / j.stem.2020.05.008), the specific reason for which remains unclear, suggesting that CISH knockout may be a contributing factor. - / - The impact on human NK cell development requires further investigation. Furthermore, for pluripotent stem cell-derived immune cells, improving the yield of differentiated immune cells and NK cells is crucial for the industrial-scale production of clinically usable immune cells.

[0005] Therefore, there is an urgent need to develop strategies to enhance the persistence, anti-tumor activity, and expansion performance of immune cells and NK cells, as well as methods to improve the differentiation of pluripotent stem cells into immune cells and NK cells. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, one object of this invention is to provide an immune cell, through gene modification, where engineered immune cells (especially NK cells) possess enhanced proliferative capacity, anti-tumor activity, and persistence (long-term survival in vitro or in vivo). Simultaneously, related gene modifications can increase the differentiation rate of iPSCs into immune cells and NK cells, and activate the expression of receptors and co-stimulatory factors.

[0007] Therefore, in a first aspect, the present invention provides a cell or a group thereof. According to an embodiment of the present invention, the cell has the following characteristics:

[0008] (i) The cells are: (a) induced pluripotent stem cells or commercial embryonic stem cells; or (b) derived cells obtained by differentiation of the cells described in (a);

[0009] (ii) The SOCS3 gene in the cells is knocked out; and / or

[0010] (iii) The cells contain chimeric antigen receptors and / or polynucleotides encoding chimeric antigen receptors.

[0011] According to an embodiment of the present invention, the cell is an induced pluripotent stem cell and the SOCS3 gene in the cell has been knocked out by gene editing or homologous recombination.

[0012] According to an embodiment of the present invention, the cells are immune cells obtained by induced differentiation of induced pluripotent stem cells or commercial embryonic stem cells.

[0013] According to an embodiment of the present invention, the immune cells include NK cells, T cells, and macrophages.

[0014] According to an embodiment of the present invention, the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the transmembrane domain is connected to the extracellular domain, and the intracellular domain is connected to the transmembrane domain.

[0015] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof capable of specifically recognizing tumor antigens, inflammation-related factors, viral surface glycoproteins.

[0016] According to an embodiment of the present invention, the tumor antigen is GPRC5D.

[0017] According to an embodiment of the invention, the cells contain expression of high-affinity, non-cleavable CD16 (hnCD16) or a variant thereof.

[0018] According to an embodiment of the present invention, the cell contains one or more endogenous genes that are inactivated or whose expression is suppressed, the endogenous genes including at least one selected from TGFBR2, PVR, NKG2A, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, TIGIT, CD38, and TCR genes.

[0019] According to an embodiment of the present invention, the endogenous gene is the CD38 gene.

[0020] According to an embodiment of the present invention, the cell expresses membrane-bound cytokines.

[0021] According to an embodiment of the present invention, the membrane-bound cytokine includes at least one selected from IL15, IL21, IL7, and IL3.

[0022] According to an embodiment of the present invention, the membrane-bound cytokine is IL15.

[0023] According to an embodiment of the present invention, in the cells: (A) the B2M and CIITA genes are knocked out or their expression is reduced; and (B) at least one of HLA-E, HLA-G, and CD47 is introduced, wherein HLA-E or HLA-G includes its non-cleavable form.

[0024] A second aspect of the present invention provides an immune cell. According to an embodiment of the present invention, the SOCS3 gene in the immune cell is knocked out, and the surface of the immune cell contains a chimeric antigen receptor.

[0025] The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the transmembrane domain is connected to the extracellular domain, and the intracellular domain is connected to the transmembrane domain.

[0026] The extracellular domain includes antibodies or antigen-binding fragments thereof capable of specifically recognizing tumor antigens, inflammation-related factors, and viral surface glycoproteins.

[0027] The effector immune cells (such as NK cells, T cells, and macrophages) used in adoptive cell therapy still have limited expansion, persistence, and anti-tumor capabilities both in vitro and in vivo. Specific methods are needed to enhance cell function and persistence to benefit patients. This invention provides methods for preparing SOCS3-deficient immune cells with enhanced in vitro or in vivo expansion, viability, and anti-tumor capabilities, as well as methods for further expressing pluripotent stem cells and their derived immune cells (such as NK cells) with various gene modifications, including chimeric antigen receptors and CD16, to prepare compositions for the treatment of tumors, viral infections, and autoimmune diseases. The SOCS3 knockout immune cells provided by this invention can enhance cell persistence, anti-tumor activity, and proliferative advantage; they also express higher levels of activating receptors and co-stimulatory receptors. Furthermore, the SOCS3 knockout pluripotent stem cells provided by this invention can achieve higher yields (number of generated iNK cells divided by the number of initial iPSCs) and higher expression of activating receptors and co-stimulatory factors when differentiated into immune cells or NK cells.

[0028] According to an embodiment of the present invention, the SOCS3 gene in the immune cells is knocked out by gene editing or homologous recombination.

[0029] According to an embodiment of the present invention, the tumor antigen is GPRC5D.

[0030] According to an embodiment of the present invention, the extracellular domain comprises a bispecific binding molecule containing a first binding region, the first binding region comprising an antibody or an antigen-binding fragment thereof capable of specifically recognizing GPRC5D. According to an embodiment of the present invention, the antibody contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 90% identity with it:

[0031] Heavy chain variable region CDR sequence: SEQ ID NO:1-3;

[0032] CDR sequence of the light chain variable region: SEQ ID NO:4-6.

[0033] (SEQ ID NO:1)SDYAWN

[0034] (SEQ ID NO:2)YISYSGSATYSPSLKS

[0035] (SEQ ID NO:3)GGIAGRGRWGAMDY

[0036] (SEQ ID NO:4)KASQNVGTNVA

[0037] (SEQ ID NO:5)SASYRDS

[0038] (SEQ ID NO:6)QQYKSYPLT

[0039] According to an embodiment of the present invention, the antibody comprises:

[0040] The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown as in SEQ ID NO:1, 2, and 3, or as amino acid sequences having at least 90% identity with SEQ ID NO:1, 2, and 3, respectively.

[0041] According to an embodiment of the present invention, the antibody comprises:

[0042] The light chain variable regions CDR1, CDR2, and CDR3 sequences are shown as in SEQ ID NO:4, 5, and 6, or as amino acid sequences having at least 90% identity with SEQ ID NO:4, 5, and 6, respectively.

[0043] According to an embodiment of the present invention, the antibody comprises:

[0044] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown as in SEQ ID NO:1, 2, and 3 or amino acid sequences having at least 90% identity with SEQ ID NO:1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown as in SEQ ID NO:4, 5, and 6 or amino acid sequences having at least 90% identity with SEQ ID NO:4, 5, and 6, respectively.

[0045] The GPRC5D antibody provided according to one embodiment of the present invention has heavy chain variable regions CDR1, CDR2, CDR3 as shown in SEQ ID NO:1, 2 and 3 and light chain variable regions CDR1, CDR2, CDR3 as shown in SEQ ID NO:4, 5 and 6. The antibody can specifically bind to GPRC5D on the surface of multiple myeloma cells. Engineered immune cells designed based on the antibody can specifically target multiple myeloma cells, kill target cells, and are expected to treat multiple myeloma patients who have relapsed after BCMA-targeted therapy.

[0046] According to an embodiment of the present invention, the antibody has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 7.

[0047] According to an embodiment of the present invention, the antibody has a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 8.

[0048] According to an embodiment of the present invention, the antibody is a single-chain antibody having an amino acid sequence as shown in SEQ ID NO: 9.

[0049] (SEQ ID NO:7)

[0050] QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSS

[0051] (SEQ ID NO:8)

[0052] DIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPLTFGQGTRLEIK

[0053] (SEQ ID NO:9)

[0054] DIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPLTFGQGTRLEIKGGGGSGGGGSGGGGS QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSS

[0055] This invention provides a novel GPRC5D antibody that specifically binds to GPRC5D. Engineered immune cells designed based on this GPRC5D antibody can specifically target and kill multiple myeloma cells, and hold promise for treating multiple myeloma patients who have relapsed after BCMA-targeted therapy. Through gene modification, the obtained engineered immune cells (especially NK cells) exhibit enhanced proliferative capacity, anti-tumor activity, and persistence (long-term survival in vitro or in vivo). Engineered immune cells based on the GPRC5D antibody possess specific killing properties against multiple myeloma.

[0056] According to an embodiment of the present invention, the bispecific binding molecule further includes a second binding region having the activity of binding antigens specifically expressed on lymphocytes.

[0057] According to an embodiment of the present invention, the antigen includes at least one selected from BCMA, CD38, CD138, CD3E, and FCGR3A.

[0058] According to an embodiment of the present invention, the second binding region comprises a single-chain antibody or a single-domain antibody.

[0059] According to an embodiment of the present invention, the first binding region and the second binding region are connected by a linker peptide.

[0060] According to an embodiment of the present invention, the extracellular domain of the chimeric antigen receptor is connected to the transmembrane domain via a hinge domain.

[0061] According to an embodiment of the present invention, the transmembrane domain is derived from a type I transmembrane protein or a synthetically produced transmembrane protein with a hydrophobic helix.

[0062] According to an embodiment of the present invention, the transmembrane domain is derived from the transmembrane domain of at least one protein from the group consisting of:

[0063] CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, H VEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.

[0064] According to an embodiment of the present invention, the transmembrane domain is derived from the transmembrane region of NKG2D.

[0065] According to an embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO: 10.

[0066] (SEQ ID NO:10)SNLFVASWIAVMIIFRIGMAVAIFCCFFFPS

[0067] According to an embodiment of the invention, the hinge domain originates from the hinge region of at least one protein from the group consisting of:

[0068] CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.

[0069] According to an embodiment of the present invention, the hinge domain originates from the hinge region of CD8;

[0070] According to an embodiment of the present invention, the hinge domain has an amino acid sequence as shown in SEQ ID NO: 11.

[0071] (SEQ ID NO:11)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0072] According to an embodiment of the present invention, the intracellular domain includes a co-stimulatory signaling domain and an intracellular signal transduction domain, which are connected by a linker peptide.

[0073] According to an embodiment of the present invention, the co-stimulatory signal domain includes an intracellular co-stimulatory signal domain of at least one protein selected from the group consisting of:

[0074] ligands of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40, and MyD88.

[0075] According to an embodiment of the present invention, the co-stimulation signal domain is derived from the intracellular co-stimulation signal domain of 2B4.

[0076] According to an embodiment of the present invention, the co-stimulatory signal domain has an amino acid sequence as shown in SEQ ID NO: 12.

[0077] (SEQ ID NO:12)

[0078] WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS

[0079] According to an embodiment of the present invention, the intracellular signal transduction domain includes a structural domain containing at least one ITAM motif.

[0080] According to an embodiment of the present invention, the intracellular signal transduction domain includes an intracellular signal transduction domain derived from at least one protein from the group consisting of;

[0081] CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, DAP10, DAP-12.

[0082] According to an embodiment of the present invention, the intracellular signal transduction domain is an intracellular signal transduction domain derived from CD3ζ.

[0083] According to an embodiment of the present invention, the intracellular signal transduction domain has an amino acid sequence as shown in SEQ ID NO: 13.

[0084] (SEQ ID NO:13)

[0085] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0086] According to an embodiment of the present invention, the chimeric antigen receptor further includes a signal peptide linked to the N-terminus of the extracellular domain of the chimeric antigen receptor.

[0087] According to an embodiment of the present invention, the signal peptide is a signal peptide derived from the CD8 protein.

[0088] According to an embodiment of the present invention, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 14.

[0089] (SEQ ID NO:14)MALPVTALLLPLALLLHAARP

[0090] According to an embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0091] (SEQ ID NO:15)

[0092] MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPLTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSNLFVASWIAVMIIFRIGMAVAIFCCFFFPSWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0093] (SEQ ID NO:16)

[0094] MALPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSTDFTLTISSLQPEDFATYYCQQYKSYPLTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWN WIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVKGRF TISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSNLFVASWIAVMIIFRIGMAVAIFCCFFFPSWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQS QSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0095] According to an embodiment of the present invention, the immune cells are obtained by isolating from peripheral blood, umbilical cord blood, or tissues and organs, or the immune cells are obtained by differentiating from stem cells or immune cell progenitor cells.

[0096] According to an embodiment of the present invention, the stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells that have not undergone in vivo development and are within 14 days of fertilization.

[0097] According to an embodiment of the present invention, the immune cells include at least one selected from NK cells, T cells, and macrophages.

[0098] In a third aspect, the invention provides a composition. According to embodiments of the invention, the composition contains the cells or populations thereof described in the first aspect and / or the immune cells described in the second aspect.

[0099] In a fourth aspect of the invention, the invention proposes the use of the cells or populations thereof described in the first aspect, the immune cells described in the second aspect, and the compositions described in the third aspect in the preparation of medicaments for treating diseases.

[0100] According to an embodiment of the present invention, the disease is a hematologic malignancy, a solid tumor, a viral infection, or a disease related to autoimmune abnormalities.

[0101] According to an embodiment of the present invention, the hematologic malignancy or solid tumor is a GPRC5D-related disease.

[0102] According to an embodiment of the present invention, the GPRC5D-related diseases include multiple myeloma.

[0103] SOCS3 knockout in immune cells that further express CAR can further enhance the anti-tumor activity of immune cells. SOCS3 knockout at the iPSC stage can increase the yield of differentiated NK cells and the expression of activating receptors and co-stimulatory factors. Furthermore, the differentiated iNK cells can differentiate and proliferate under low-concentration (1 ng / mL) IL15 conditions, exhibiting enhanced cell persistence, anti-tumor activity, and proliferative advantage. SOCS3 knockout combined with other gene modifications can yield more functionally enhanced immune cells (ADCC, tumor-specific killing, low immunogenicity, and high resistance to the tumor microenvironment).

[0104] Additional aspects and advantages of the invention 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 the invention. Attached Figure Description

[0105] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0106] Figure 1 The graph shows gene expression of the SOCS family genes (CISH, SOCS1-7) in Example 1 under different concentrations of IL15. TPM (transcripts per million) represents the relative abundance of the corresponding transcripts in RNA sequencing. The vertical axis of the graph represents the doubling of the TPM value of the gene shown relative to its own value at 0.1 ng / mL under different concentrations of IL15 stimulation. Error bars represent SD values ​​from three experimental replicates.

[0107] Figure 2 The electrophoretic patterns of PCR products from the EcoRI digestion of CISH and SOCS1-7 target regions in Example 3 are shown. The markers from top to bottom are 700bp, 500bp, 400bp, 300bp, 200bp, 150bp, 100bp, 75bp, 50bp, and 25bp.

[0108] Figure 3 A shows the sequencing peak diagram of the SOCS3 target region of the sample shown; B shows the genotype ratio analysis after sequencing of PCR products, with upward arrows indicating an increase in the ratio and downward arrows indicating a decrease in the ratio; C shows the changes in the genotype ratios of multiple SOCS3 mutants before and after SOCS3 KO iPSCs differentiate into iNK.

[0109] Figure 4 The figure shows the iNK amplification fold of iNK derived from SOCS family gene knockout iPSCs in Example 4 up to day 6.

[0110] Figure 5 The WT iNK and genetically modified iNK (SOCS3 KO or CISH) shown in Example 6 are illustrated. - / - Differentiation of CD56-positive cells under 10 ng / mL IL-15 conditions, cell viability (A) and cell proliferation rate at Day 41 (B);

[0111] Figure 6 Showing WT iNK and genetically modified iNK (SOCS3 KO or CISH) - / - Cells were amplified at 10 ng / mL IL-15. The cell proliferation curve (A) and changes in cell viability during iNK amplification (B) are shown.

[0112] Figure 7 Showing WT iNK and genetically modified iNK (SOCS3 KO or CISH) - / - Cells were cultured at 1 ng / mL IL-15, and the changes in iNK cell viability during amplification were observed. Cells were spheroidized using AggreWell during differentiation, and the changes in cell viability (A) and amplification rate (C) during iNK amplification were observed. Cells were also spheroidized using an orbital shaker during differentiation, and the changes in cell viability (B) and amplification rate (D) during iNK amplification were observed.

[0113] Figure 8AThis figure shows the killing effect of cryopreserved and thawed NK cells on the hematologic malignancy cell line MOLM-13 (human acute myeloid leukemia cells). Cryo: cryopreserved and thawed cells; CBNK: NK cells derived from umbilical cord blood; E:T ratio, NK cell to target cell ratio. P-values ​​in the figure were obtained using Tukey's multiple comparisons test through conventional one-way ANOVA statistical analysis. Error bars represent SD, and values ​​were obtained from three replicates.

[0114] Figure 8B This figure shows the killing effect of cryopreserved and thawed NK cells on the hematologic malignancy cell line RPMI 8226 (human myeloma cells). Cryo: cryopreserved and thawed cells; CBNK: NK cells derived from umbilical cord blood; E:T, the ratio of NK cells to target cells. The p-values ​​in the figure were obtained using Tukey's multiple comparisons test through conventional two-way ANOVA statistical analysis. Error bars represent SD, and values ​​were obtained from three replicates.

[0115] Figure 9 This study demonstrates the killing effect of cryopreserved and thawed NK cells on the solid tumor cell line U-87MG-Luc (human brain astroblastoma cells). Figure A shows the results after 6 hours of co-incubation, and Figure B shows the results after 24 hours of co-incubation. Cryo: cryopreserved and thawed cells; CBNK: NK cells derived from umbilical cord blood. The p-values ​​in the figures were obtained using Tukey's multiple comparisons test in a standard two-way ANOVA statistical analysis. Error bars represent standard deviations (SD), and values ​​were derived from three replicates.

[0116] Figure 10 The RNAseq analysis shows the expression levels of SOCS3 KO iNK activating receptor and co-stimulatory factor; the error bars represent SD values, which are derived from three experimental replicates.

[0117] Figure 11This diagram illustrates exogenous DNA templates used for homologous recombination-mediated site-specific integration at or near the SOCS3 locus, where A. Examples of several types of exogenous DNA inserted at or near the SOCS3 locus; B. Examples of several DNA templates expressing one or more peptides via exogenous promoters; C. A diagram illustrating homologous recombination-mediated targeted integration into SOCS3. The diagram illustrates the site-specific insertion of the peptide expression cassette CAG-anti-GPRC5D CAR-BGHpA (T0321) or CAG-anti-GPRC5D CAR-BGHpA-2A-hnCD16-BGHpA (T0401) into the SOCS3 locus, thereby disrupting SOCS3 expression while simultaneously expressing an anti-GPRC5D CAR molecule.

[0118] Figure 12 The expression levels of SOCS3 in different iNK cells were detected by qPCR.

[0119] Figure 13 The images show the expression of anti-GPRC5D scFV in donor iPSCs and gene-edited iPSCs (T0321iPSC and T0401iPSC) as detected by flow cytometry (A); the expression of CD16 on the surface of T0401 iPSC (B); and the expression of CD16 on the surface of iNK cells differentiated from iPSCs in Figure A (C).

[0120] Figure 14 The sequence peak diagram after the insertion of the 7bp sequence in A.T0401 iPSC is shown; B. CD38 expression levels of WTiNK and T0401 iNK were detected by flow cytometry.

[0121] Figure 15 The expression levels of CD56, CD3, CD16 and CD38 in T0401 iNK were shown by flow cytometry.

[0122] Figure 16 This shows the anti-GPRC5D scFV levels in a mixed population of T0321 and WT iPSCs (T0321 mix) before, during, and after iNK differentiation. + Changes in the proportion of (anti-GPRC5D scFV) cells;

[0123] Figure 17 The study showed the killing effect of WT iNK and T0321 mix iNK on MM.1S (human multiple myeloma cells); the error bar represents the SD, and the values ​​are from 3 experimental replicates. E:T represents the ratio of NK cells to target cells.

[0124] Figure 18 The results show: A. The killing effect of different iNK cells on MM.1S-Luc cells when co-incubated with different iNK cells at a 4:1 effector-target ratio for 4 h in the presence of different concentrations of daratumumab; B. The killing effect of different iNK cells on MM.1S-Luc cells in the presence of 2 μg / mL daratumumab in multiple rounds when co-incubated with different iNK cells at a 4:1 effector-target ratio. Each round of testing involved incubation for 24 h. Error bars represent SD values, which are derived from three experimental replicates. Detailed Implementation

[0125] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0126] 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 indicated technical features. 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 invention, unless otherwise stated, "a plurality of" means two or more.

[0127] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0128] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, 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.

[0129] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0130] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition 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.

[0131] the term

[0132] "scFv" stands for "Single-Chain Fragment Variable," which is a genetically engineered fusion protein. It is a small molecule composed of the variable regions of the antibody heavy chain and the light chain linked by a peptide chain.

[0133] "Antibody" includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding antigens, and single-chain antibody fragments, including single-chain variable fragments (scFv) and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody). Examples include bispecific antibodies, biantibodies, triantibodies and tetraantibodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to include its functional antibody fragment.

[0134] "Antibody fragment" refers to a molecule other than a complete antibody, which contains a portion of a complete antibody capable of binding an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dimers; linear antibodies; variable heavy chain regions; single-chain antibody molecules (such as scFv); and single-domain antibodies; and multispecific antibodies formed from antibody fragments. In a particular embodiment, the antibody is a single-chain antibody fragment containing a variable heavy chain region and / or a variable light chain region, such as scFv.

[0135] As used herein, "heavy chain variable region" refers to a region of an antibody molecule that contains at least one complementarity-determining region (CDR) of the antibody heavy chain variable structural domain. The heavy chain variable region may contain one, two, or three CDRs of the antibody heavy chain.

[0136] In this article, the term "single-domain antibody (sdAb)" refers to a naturally occurring heavy chain antibody found in camel / alpaca blood that lacks the light chain and consists only of the heavier H chain. The amino-terminal (N-terminus) amino acid sequence of the peptide chain varies considerably and is called the variable region (V region), while the carboxyl-terminus (C-terminus) is relatively stable and varies little, called the constant region (C region). The V region of the H chain is called VH. Within the variable region, certain areas exhibit even greater variability in amino acid composition and sequence, called the hypervariable region (HVR). The hypervariable region is the site of antigen-antibody binding and is therefore also called the complementarity-determining region (CDR). The heavy chain variable region has three CDR regions. CDR1 and CDR3 are slightly longer than those in humans, and CDR3 protrudes outward in the tertiary structure. Therefore, it is speculated that single-domain antibodies have higher antigen-binding specificity and affinity than conventional antibodies. In some embodiments, the single-domain antibody is a human single-domain antibody or an alpaca single-domain antibody, with or without humanization. The variable domains of single-domain antibodies exhibit the same general structure as a relatively conserved framework region (FR) linked by three hypervariable regions or CDRs. From the N-terminus to the C-terminus, the heavy chain contains domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignment of each domain conforms to the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991; or Chothia and Lesk, Journal of Molecular Biology, 196:901-917, 1987; Chothia et al., Nature, 342:878-883, 1989).

[0137] The “hypervariable region of an antibody” refers to the CDR amino acid residues of the antibody responsible for antigen binding. The hypervariable region contains amino acid residues from the CDR, such as 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain (as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and / or residues from the hypervariable ring, such as 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain (as described in Chothia et al., Journal of Molecular Biology 196:901-917 (1987)).

[0138] Monoclonal antibodies are antibodies derived from a substantially homogeneous group of antibodies, where all antibodies in the mixture share a single amino acid sequence derived from a single clone. Monoclonal antibodies are typically highly specific and target a single antigenic site or epitope. In contrast, polyclonal antibody formulations usually consist of a mixture of antibodies with different amino acid sequences targeting the same or different determinants (epitaxes). In addition to their specificity, monoclonal antibodies have the advantage of being synthesized in homologous cultures, free from contamination by other immunoglobulins with different specificities and characteristics.

[0139] As used herein, the term "hinge" refers to the region between the CH1 and CH2 regions of the antibody heavy chain. This region includes interchain disulfide bonds, is rich in proline, does not form α-helices, and readily extends and twists to a certain extent, which facilitates complementary binding between the antibody's antigen-binding site and the antigenic epitope. In the heavy chain single-domain antibody of this invention, the variable region of the heavy chain is connected to the Fc region via the hinge region.

[0140] In this invention, "antigen" can be a therapeutic target related to diseases such as hematologic malignancies, solid tumors, autoimmune diseases, and viral infections. For example, in tumor diseases, antigen specifically refers to tumor antigens; in autoimmune diseases, antigen specifically refers to inflammation-related factors; and in viral infections, antigen specifically refers to viral surface glycoproteins.

[0141] Chimeric antigen receptors, or CARs for short, are artificial receptor molecules manufactured using genetic engineering techniques. They include an antigen-binding domain (usually derived from a single-chain antibody variable region gene fragment, scFV; or from a single-domain antibody), a hinge domain, a transmembrane domain, and an intracellular signal transduction domain.

[0142] As described in this article, "reduced expression" refers to the elimination or reduction of the expression of one or more gene products encoded by a gene in a cell compared to the normal expression level of the gene's mRNA or protein. In some cases, this effect on gene expression is temporary or reversible, such as by inhibiting gene expression through small molecule compounds, RNAi, siRNA, shRNA, or specific proteins. In other cases, this effect on gene expression is permanent, such as through gene editing, introducing base insertions or deletions into the coding region of the target gene, causing frameshift mutations, or through homologous recombination, inserting a DNA sequence into the coding or non-coding region of the target gene, causing frameshift mutations, premature termination of transcription and / or translation, or translation into a completely different polypeptide, thus permanently inactivating the gene. The percentage of reduced gene expression caused by different methods can be 100%, 90% or more, 80% or more, 70% or more, 60% or more, 50% or more, 40% or more, 30% or more, or 20% or more.

[0143] "Inactivation" refers to the loss of function, or the loss of protein function.

[0144] As used herein, the term "nucleic acid" is used interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form, encompassing nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, said nucleic acids being synthetic, naturally occurring or unnatural, having similar binding properties to a reference nucleic acid, and being metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in a host cell. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences.

[0145] As used herein, the term "vector" refers to a delivery system that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) into and express that genetic element, for example, to produce a protein, RNA, or DNA encoded by that genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into the cell, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vector (e.g., expression vector) provided by the present invention contains a nucleic acid sequence encoding a fusion protein as described in the present invention, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF1α), and at least one selection marker.

[0146] As used in this article, the term "DNA fragment" is distinguished from circular plasmids. A DNA fragment refers to a single-stranded or double-stranded DNA fragment that can be used as a DNA template for homologous recombination.

[0147] As used herein, the term "homologous sequence" refers to a sequence in the DNA template that has at least 60%, 70%, 80%, or 90% similarity to the target site in homologous recombination. This sequence is a homologous arm sequence in the DNA template that enables the DNA template to be site-specifically integrated into the target site.

[0148] As used in this article, the term "frameshift mutation" refers to the insertion or deletion of one, two, or several pairs of bases (not three or multiples of three, i.e., the added or subtracted bases do not correspond to one or more triplet codons) at a point in the DNA sequence, causing a shift in the amino acid triplet codon during transcription, resulting in a complete change in the base sequence starting from the damaged point, and translation into abnormal amino acids.

[0149] As used in this article, the term "expression cassette" is a separate component of vector DNA, consisting of a regulatory sequence and the coding sequence of a foreign polypeptide. Once vector DNA containing an expression cassette is transferred into cells, it guides the cells to produce RNA and foreign polypeptides.

[0150] As used herein, the term "cell" refers to a eukaryotic cell, particularly a mammalian cell, such as a human cell.

[0151] As used herein, the term "immune cell" refers to cells derived from humans. More specifically, the immune cells of this invention can be derived from pluripotent stem cells through directed differentiation. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells, and macrophages. The immune cells of this invention can also be derived from human hematopoietic stem cells. These hematopoietic stem cells can be isolated from peripheral blood, umbilical cord blood, placental blood, or differentiated from pluripotent stem cells. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells, and macrophages. The immune cells of this invention can also be isolated from human blood, bone marrow, lymph nodes or lymphoid organs, or specific tissues (including but not limited to tumor tissue, abdominal cavity, liver, lungs, and other organs, muscles, etc.). For example, cells of innate or adaptive immunity, such as bone marrow or lymphocytes, typically T cells, NK cells, macrophages, etc. Preferably, according to this invention, the immune cells include T cells, NK cells, and macrophages. The immune cells of this invention can also be derived from immune cell progenitor cells, such as lymphocyte progenitor cells.

[0152] "Pluripotent stem cells" generally refer to human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Human ESCs are obtained from blastocysts during the development of human fertilized eggs, or from embryos obtained through nuclear transfer (embryonic stem cells within 14 days of fertilization that have not undergone in vivo development). Human iPSCs can be obtained from human cells through reprogramming by overexpressing specific transcription factors (such as OCT4, SOX2, KLF4, and MYC). Both human ESCs and iPSCs express OCT4, NANOG, Tra-1-60, and SSEA-4, while those that do not express SSEA-1. A significant characteristic of pluripotent stem cells is their ability to differentiate into three germ layer tissues or cells in vitro or in vivo.

[0153] In this invention, "hematopoietic stem cells" refers to CD34. + These cells can be isolated from hematopoietic stem cells, which can be isolated from peripheral blood, umbilical cord blood, placental blood, bone marrow, spleen, lymph, or other tissues, or differentiated from pluripotent stem cells.

[0154] "Progenitor cells" are cells that can differentiate into various types of immune cells, such as myeloid stem cells, lymphoid stem cells, and further, also refer to erythroid stem cells, granulocytic and monocyte stem cells, megakaryocyte stem cells, precursor B cells, precursor T cells, NK progenitor cells, and macrophage progenitor cells.

[0155] As used in this article, "NK cells" refers to natural killer cells, generally CD56. + CD45 +Double positive, simultaneously expressing multiple activating receptors, such as NKG2D, NKp44, and NKp46. NK cells can differentiate from pluripotent stem cells (such as iPSCs), be isolated from peripheral blood, or be derived from CD34+. + It is derived from cell differentiation (such as umbilical cord blood or placental blood) or obtained from tumor cell lines (such as NK92).

[0156] In this article, "T cell" refers to CD4 + or CD8 + T cells, or double-negative T cells (CD3+) + CD4 - CD8 - The cells can be naive T cells (TN cells), T memory stem cells (TSCM cells), memory T cells (TCM cells), regulatory T cells (Treg cells), TCRT cells, NKT cells, tumor-infiltrating lymphocytes (TILs), or effector memory T cells (TEM cells) and combinations thereof.

[0157] In this article, "knockout" refers to a technique that inactivates or deletes a gene through specific means, denoted by KO. It can represent the inactivation or deletion of a single allele or a pair of alleles. Unless otherwise specified, "- / -" indicates the knockout of a pair of alleles.

[0158] In this article, "reduction" refers to a certain percentage decrease in gene expression or function relative to the wild type (e.g., any percentage between 10% and 100%).

[0159] In this article, "SOCS family genes" include CISH, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, etc.; unless otherwise specified, "SOCS" may also refer to any one or a combination of SOCS family genes.

[0160] In this article, "SOCS1-7" specifically refers to SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7.

[0161] In this article, "single or multiple CD3 peptide chains" refers to the ε, γ, δ and ζ peptide chains of CD3, a single peptide chain or a combination thereof.

[0162] “RNP” is a complex formed by a specific protein and a specific RNA. In this invention, it specifically refers to the complex of sgRNA and Cas9 protein.

[0163] "ADCC" refers to antibody-dependent cell-mediated cytotoxicity. In this invention, it refers to NK cell-mediated cytotoxicity in the presence of antibodies.

[0164] "Gene editing" refers to the editing (targeted modification) of target genes and their transcription products to achieve the addition, deletion, or substitution of specific DNA bases, thereby altering the sequence, expression level, or function of the target gene or regulatory element. Its basic principle is based on sequence-specific endonucleases, composed of sequence-specific DNA-binding domains and non-specific DNA-modifying domains, which recognize DNA target sites on chromosomes, cleave them, and induce DNA damage repair, thus achieving targeted editing of the specified genome.

[0165] As used in this article, the term "tumor antigen" is a general term for newly emerging and highly expressed proteins and polypeptides during malignant transformation of cells. Based on their relationship to tumors, tumor antigens are divided into two main categories: "tumor-specific antigens" (TSA) and "tumor-associated antigens" (TAA). TSA refers to antigens specific to tumor cells and not present on normal tissue cells. TAA refers to antigens not specific to tumor cells and also present on normal tissue cells, especially embryonic tissues. Therefore, TAA only shows quantitative changes between tumor cells and normal tissues. In terms of their source or characteristics, TAAs are generally proteins expressed in tumors induced by chemical and physical carcinogenic factors, proteins expressed in tumors induced by viruses, abnormal proteins encoded by oncogenes and mutant tumor suppressor genes, or proteins expressed after the activation of dormant genes. TAAs are generally embryonic proteins, differentiation proteins, proteins encoded by highly expressed oncogenes, or glycolipids and glycoproteins that are overexpressed or abnormally expressed (Principles of Immunology (4th Edition), edited by Zhou Guangyan, Beijing: Science Press, 2018.1; ISBN 978-7-03-053131-5; p339).

[0166] As used herein, the term "inflammatory-associated factor" refers to pro-inflammatory cytokines or inflammation-associated chemokines. The one or more inflammatory-associated factors are selected from interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-12 (IL-12), interleukin-18 (IL-18), granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17A (IL-17), CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL16, and CCL2-2.

[0167] As used in this article, the "effect-to-target ratio" can be expressed as E:T or Effetor:Target, and refers to the ratio of the number of effector cells (generally immune cells, such as NK cells in this article) to the number of target cells (generally tumor cells).

[0168] As used herein, the term "composition" refers to a form in which the biological activity of the active ingredient is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition will be applied.

[0169] As used herein, the term “treatment” means the temporary or permanent, partial or complete elimination, reduction, suppression or improvement of the clinical symptoms, manifestations or progression of an event, disease or condition.

[0170] In many embodiments, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has received or is currently receiving any form of treatment. As used herein, the terms "subject" or "patient" refer to a mammalian subject or patient. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, poultry, goats, and sheep. In some embodiments, the subject is a human. In some embodiments, the subject is a person suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0171] pluripotent stem cells

[0172] This invention provides a cell or a group thereof. According to an embodiment of the invention, the cell has the following characteristics:

[0173] (i) The cells are: (a) induced pluripotent stem cells or commercial embryonic stem cells; or (b) derived cells obtained by differentiation of the cells described in (a);

[0174] (ii) The SOCS3 gene in the cells is knocked out; and / or

[0175] (iii) The cells contain chimeric antigen receptors and / or polynucleotides encoding chimeric antigen receptors.

[0176] According to an embodiment of the present invention, the cell is an induced pluripotent stem cell, and the SOCS3 gene in the cell has been knocked out through gene editing or homologous recombination. It should be noted that the method of knocking out the SOCS3 gene in the cell is not limited to gene editing or homologous recombination, but can also be other methods known in the art.

[0177] According to an embodiment of the present invention, the cells are immune cells obtained by induced differentiation of induced pluripotent stem cells or commercial embryonic stem cells.

[0178] According to an embodiment of the present invention, the immune cells include NK cells, T cells, and macrophages. The immune cells are not limited to these cell types and may also be other types of immune cells.

[0179] According to an embodiment of the present invention, the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the transmembrane domain is connected to the extracellular domain, and the intracellular domain is connected to the transmembrane domain.

[0180] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof capable of specifically recognizing tumor antigens, inflammation-related factors, viral surface glycoproteins.

[0181] According to an embodiment of the present invention, the tumor antigen is GPRC5D.

[0182] According to an embodiment of the invention, the cells contain expression of high-affinity, non-cleavable CD16 (hnCD16) or a variant thereof.

[0183] According to an embodiment of the present invention, the cell contains one or more endogenous genes that are inactivated or whose expression is suppressed, the endogenous genes including at least one selected from TGFBR2, PVR, NKG2A, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, TIGIT, CD38, and TCR genes.

[0184] According to an embodiment of the present invention, the endogenous gene is the CD38 gene.

[0185] According to an embodiment of the present invention, the cell expresses membrane-bound cytokines.

[0186] According to an embodiment of the present invention, the membrane-bound cytokine includes at least one selected from IL15, IL21, IL7, and IL3.

[0187] According to an embodiment of the present invention, the membrane-bound cytokine is IL15.

[0188] According to an embodiment of the present invention, in the cells: (A) the B2M and CIITA genes are knocked out or their expression is reduced; and (B) the expression of at least one of HLA-E, HLA-G, and CD47 is introduced, wherein HLA-E or HLA-G includes its non-cleavable form.

[0189] immune cells

[0190] This invention provides an immune cell in which the SOCS3 gene is knocked out, and the surface of the immune cell contains a chimeric antigen receptor.

[0191] The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the transmembrane domain is connected to the extracellular domain, and the intracellular domain is connected to the transmembrane domain.

[0192] The extracellular domain includes antibodies or antigen-binding fragments thereof capable of specifically recognizing tumor antigens, inflammation-related factors, and viral surface glycoproteins. As a specific example, the tumor antigen may be GPRC5D.

[0193] According to a preferred embodiment of the present invention, the extracellular domain includes a bispecific binding molecule containing a first binding region, the first binding region comprising the antibody or its antigen-binding fragment capable of specifically recognizing GPRC5D.

[0194] According to a preferred embodiment of the present invention, the antibody specifically recognizing GPRC5D provided by the present invention comprises:

[0195] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown as in SEQ ID NO:1, 2, and 3 or amino acid sequences having at least 90% identity with SEQ ID NO:1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown as in SEQ ID NO:4, 5, and 6 or amino acid sequences having at least 90% identity with SEQ ID NO:4, 5, and 6, respectively.

[0196] It should be noted that any antibody or antibody fragment having the aforementioned heavy chain variable region CDR1, CDR2, CDR3 sequences and light chain variable region CDR1, CDR2, CDR3 sequences is included within the scope of protection of this invention. Examples include IgG, scFv, Fab, Fab', etc.

[0197] According to a preferred embodiment of the present invention, the antibody that specifically recognizes GPRC5D has a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 8.

[0198] According to a preferred embodiment of the present invention, the antibody is a single-chain antibody having the amino acid sequence shown in SEQ ID NO: 9. The GPRC5D single-chain antibody in this invention is also abbreviated as GPRC5D scFV.

[0199] According to an embodiment of the present invention, the bispecific binding molecule further includes a second binding region having the activity of binding antigens specifically expressed on lymphocytes.

[0200] According to embodiments of the present invention, antibodies that bind to antigens specifically expressed on lymphocytes are all included within the range of second binding regions of the bispecific binding molecules of the present invention.

[0201] According to a specific embodiment of the present invention, the antigen includes at least one selected from BCMA, CD38, CD138, CD3E, and FCGR3A. Preferably, the second binding region can be an intact antibody, a single-chain antibody, or a single-domain antibody. The first binding region and the second binding region can be linked by a linker peptide.

[0202] According to an embodiment of the present invention, the extracellular domain of the chimeric antigen receptor is connected to the transmembrane domain via a hinge domain.

[0203] In one embodiment of the invention, the CAR may include an extracellular targeting portion that recognizes the GPRC5D protein, and may also include a hinge domain. The hinge domain may be located between the extracellular antigen recognition domain and the transmembrane domain. For example, the hinge domain may include a hinge domain of one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region may be a hinge region derived from CD8. For example, the hinge domain may have an amino acid sequence as shown in SEQ ID NO: 11.

[0204] In this invention, the CAR may include a transmembrane domain, which may be derived from any type I transmembrane protein or a synthetic sequence predicted to form a hydrophobic helix. For example, the transmembrane region may include a transmembrane domain derived from one or more proteins from the following group: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane region may be a transmembrane region derived from NKG2D. For example, the transmembrane domain may have an amino acid sequence as shown in SEQ ID NO: 10.

[0205] In one embodiment of the invention, the intracellular domain includes a co-stimulatory signaling domain and an intracellular signal transduction domain, which are connected by a linker peptide. The CAR may include an extracellular targeting portion that recognizes the GPRC5D protein, and may also include a co-stimulatory signaling domain. The co-stimulatory signaling domain can provide a stimulatory signal. For example, the co-stimulatory signaling domain may include an intracellular co-stimulatory signaling domain of one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88. For example, the co-stimulatory signal region may be an intracellular co-stimulatory signal region derived from 2B4. For example, the co-stimulatory signal domain has an amino acid sequence as shown in SEQ ID NO: 12.

[0206] In this invention, the intracellular signal transduction domain comprises a domain containing at least one ITAM motif to transmit activation signals into the cell. For example, the intracellular signal transduction domain may comprise an intracellular signal transduction domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, DAP10, DAP-12, and other domains containing at least one ITAM. For example, the intracellular signal transduction domain may be a signal transduction domain derived from CD3ζ. For example, the intracellular signal transduction domain has an amino acid sequence as shown in SEQ ID NO: 13.

[0207] In this invention, the CAR may further include a signal peptide at the N-terminus of the targeting portion that binds to the GPRC5D protein. For example, the signal peptide may be a CD8-derived signal peptide. For example, the signal peptide may have an amino acid sequence such as SEQ ID NO: 14.

[0208] According to a specific embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0209] According to a specific embodiment of the present invention, the polypeptide encoding an anti-GPRC5D CAR comprises: a) an extracellular antigen domain containing a GPRC5D binding moiety, wherein the GPRC5D binding domain may contain an anti-GPRC5D single-chain variable region fragment (scFV) or sdAb; b) a hinge region; c) a transmembrane domain; d) a co-stimulatory signaling domain; and (d) an intracellular signal transduction domain. The amino acid sequence of the anti-GPRC5D scFV is shown in SEQ ID NO: 9.

[0210] According to a specific embodiment of the present invention, from the N-terminus to the C-terminus, the CAR may sequentially include a targeting portion that binds to the GPRC5D protein (e.g., the GPRC5D scFV of the present invention), the hinge region, the transmembrane domain, the co-stimulatory signaling domain, and the intracellular signal transduction domain.

[0211] In this invention, from the N-terminus to the C-terminus, the CAR may sequentially comprise a targeting portion that binds to the GPRC5D protein (e.g., the GPRC5D scFV of this invention) and a targeting portion that binds to a second antigen (antigen 2) (e.g., anti-antigen 2 scFV or sdAb), the hinge region, the transmembrane domain, the co-stimulatory signaling domain, and the intracellular signal transduction domain. The anti-antigen 2 scFV or sdAb may be a scFV or sdAb that targets and binds to an antigen specifically expressed on lymphocytes.

[0212] For example, from the N-terminus to the C-terminus, the CAR may sequentially include the GPRC5D scFV and a BCMA-binding scFV or sdAb, a hinge region derived from CD8, a transmembrane region derived from reverse NKG2D, a co-stimulatory signaling domain derived from 4-1BB, and an intracellular signal transduction domain derived from CD3ζ (as shown in SEQ ID NO: 16).

[0213] According to embodiments of the present invention, enhanced immune cells can be obtained using any of the following methods or combinations thereof:

[0214] 1) Randomly integrate one or more circular or linear exogenous DNA fragments into target cells;

[0215] 2) Integrate one or more circular or linear exogenous DNA fragments into target cells at specific sites. Site-specific integration can improve the efficiency of homologous recombination of exogenous DNA by introducing endonucleases at the target site; it can also improve the efficiency of homologous recombination by other physical means, such as electroporation.

[0216] 3) Introduce one or more double-strand breaks into target cells at specific sites using one or more endonucleases that can recognize specific sites. The target cells then generate base insertions or deletions at the specific sites by joining non-homologous ends.

[0217] 4) Introduce one or more double-strand breaks into target cells at specific sites using one or more endonucleases that can recognize specific sites, while providing exogenous circular or linear DNA fragments. The cells then generate base insertions or deletions at selected sites through homologous recombination.

[0218] The target cells at this point can be the immune cells, stem cells, or progenitor cells mentioned above, or donor cells used for reprogramming into iPSCs, or any derived cells from stem cells or progenitor cells during their differentiation into immune cells. The exogenous DNA fragment may contain a gene expression frame, i.e., at least components such as a promoter and an open reading frame; or it may not contain a gene expression frame.

[0219] According to embodiments of the present invention, the promoter in the gene expression frame can be CAG, CMV, EF1α, PGK, UBC, or other constitutive, inducible, time-, tissue-, or cell type-specific promoters; the open reading frame in the gene expression frame includes one or more polypeptides or proteins, including transcription factors, functional genes, safety switch proteins, cytokines, and those that can promote or enhance the function of immune cells or their source cells (such as in vivo and in vitro persistence, differentiation efficiency, expansion rate, tumor killing activity, tumor targeting, ADCC effect, etc.). The insertion site of the exogenous DNA fragment can be the AAVS1 site or other sites whose cell function is not affected by the insertion of exogenous genes; it can also be a functional gene locus or its adjacent location, where the functional gene is inactivated or its expression is defective after insertion; or it can be the stop codon position of a functional gene.

[0220] In some embodiments, this document provides genetically modified immune cells or cells of origin thereof, wherein the immune cells are T cells, including CD4+ cells. + or CD8 + T cells, or CD3 + CD4 - CD8 - T cells. Preferred cells may be selected from naive T cells (TN cells), T memory stem cells (TSCM cells), memory T cells (TCM cells), regulatory T cells (Treg cells), tumor-infiltrating lymphocytes (TILs), NKT cells, or effector memory T cells (TEM cells), and combinations thereof. The derived cells are pluripotent stem cells or hematopoietic stem cells derived from them; specifically, the pluripotent stem cells are iPSCs, clonal iPSCs, or iPSC cell lines, and the derived cells include CD34 cells. +Cells, including hematopoietic endothelial cells, hematopoietic stem cells and progenitor cells (HSPCs), hematopoietic pluripotent progenitor cells, T cell progenitor cells, T cells, and NKT cells. The derived cells can also be peripheral blood, umbilical cord blood, or T cells, T cell progenitor cells, hematopoietic pluripotent progenitor cells, and CD34 cells isolated from other tissues (including tumor tissue) or organs. + Cells, hematopoietic endothelial cells, hematopoietic stem cells, and progenitor cells.

[0221] In some embodiments, this document provides genetically modified immune cells or cells derived therefrom, wherein the immune cells are NK cells, and the derived cells are pluripotent stem cells or derived cells, specifically, wherein the pluripotent stem cells are iPSCs or ESCs, iPSC or ESC cell lines, and the derived cells include CD34+ cells, hematopoietic endothelial cells, hematopoietic stem and progenitor cells (HSPCs), hematopoietic pluripotent progenitor cells, NK cell progenitor cells, and NK cells. The derived cells may also be NK cells, NK progenitor cells, hematopoietic pluripotent progenitor cells, or CD34+ cells isolated from peripheral blood, umbilical cord blood, or other tissues (including tumor tissue) or organs. + Cells, including hematopoietic endothelial cells, hematopoietic stem cells, and progenitor cells. In some embodiments, this document provides genetically modified immune cells or cells derived from them, wherein the immune cells are macrophages, including M1 macrophages and M2 macrophages. Their sources include macrophages from various tissues, such as bone marrow, lymph nodes, liver, spleen, lung, and circulatory system macrophages, microglia, etc.; they also include pluripotent stem cells or macrophages derived from hematopoietic stem cells, particularly iPSCs, cloned iPSCs, or iPSC cell lines, and pluripotent stem cell-derived cells including CD34. + Cells, including hematopoietic endothelial cells, hematopoietic stem and progenitor cells (HSPCs), hematopoietic pluripotent progenitor cells, macrophage progenitor cells, and macrophages. The derived cells can also be macrophages, macrophage progenitor cells, and hematopoietic pluripotent progenitor cells isolated from peripheral blood, umbilical cord blood, or other tissues (including tumor tissue) or organs. CD34 cells are also present. + Cells, hematopoietic endothelial cells, hematopoietic stem cells, and progenitor cells.

[0222] In some embodiments, the immune cells of the present invention are embryonic stem cells, induced pluripotent cells (iPSCs), cloned iPSCs or iPSC cell lines, or derived cells obtained by differentiating any of the above-described embryonic stem cells or iPSCs.

[0223] Any of the cells described above encodes at least a chimeric antigen receptor polypeptide that can recognize tumor antigens, inflammation-related factors, and viral surface glycoproteins.

[0224] The tumor antigen may be GPRC5D, and the chimeric antigen receptor may be a polypeptide encoding an anti-GPRC5D CAR or an anti-GPRC5D CAR.

[0225] In some embodiments of derived cells obtained from iPSC differentiation, the derived cells have an enhanced killing effect on tumor cells expressing GPRC5D compared to the corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other tissue or organ.

[0226] In some embodiments of the iPSCs and their derived cells that include SOCS3 knockout, the cells further comprise one or more of the following gene edits: (i) B2M and CIITA knockout or low-content; (ii) introduction of expression of at least one of HLA-E, HLA-G, and CD47, wherein HLA-E or HLA-G includes its non-cleavable form; (iii) introduction of expression of high-affinity non-cleavable CD16 (hnCD16) or a variant thereof; (iv) introduction of expression of a gene antigen receptor (CAR, or TCR, or NCR); (v) introduction of expression of membrane-bound cytokines (such as IL15, IL21, IL7, IL3, etc.); (vi) knockout or reduced expression of any of the following genes: SOCS1 SOCS2, SOCS4, SOCS5, SOCS6, SOCS7, CISH, TGFBR2, PVR, NKG2A, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, TIGIT, CD38, TCR deletion or decreased expression; and (vi)4-1BBL, NKG2D, NKp44, NKp46, CD3 single or multiple peptide chains, CD47, CD113, CD131, CD137, CD80, CD86, CD68, CD226, PDL1, A2AR, CAR, TCR, Fc receptor, conjugate and surface trigger receptor for coupling with universal conjugate, at least one of the following: introduction or increase of expression.

[0227] In some embodiments of the cells or populations thereof, one or more cells containing the CAR anti-disease antigen are primary NK cells or NK cells or populations thereof derived from pluripotent stem cells, hematopoietic stem cells and progenitor cells, NK cell progenitor cells, and these cells, compared with their unmodified counterparts, have at least one of the following characteristics: a stronger killing ability against tumor cells expressing CAR-targeted tumor antigens.

[0228] In some embodiments, the genetically modified immune cells or their source cells provided herein contain one or more endogenous genes that are inactivated or whose expression is suppressed. These endogenous genes include at least one of the following genes: SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, CISH, TGFBR2, B2M, CIITA, PVR, NKG2A, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, TIGIT, CD38, and TCR.

[0229] In some implementations, the genetically modified immune cells or their source cells generated using the methods provided herein contain SOCS3 knockout, SOCS3 inactivation, or SOCS3 expression suppression.

[0230] In some embodiments, the immune cells or populations thereof of the present invention are derived cells obtained by differentiation from totipotent or pluripotent stem cells (such as iPSCs or ESCs), iPSC or ESC monoclonal cells, iPSC or ESC cell lines, or derivative cells of any of the above; and wherein any of the above-mentioned cells contains at least SOCS3 knockout. In some embodiments of derivative cells obtained from iPSC differentiation, the derivative cells have the ability to survive and expand at low IL15 levels (≤1 ng / mL) and have enhanced killing ability against target cells compared to corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other tissue or organ.

[0231] In some embodiments, the immune cells or populations thereof described in this invention are embryonic stem cells, induced pluripotent cells (iPSCs), cloned iPSCs or iPSC cell lines, or derived cells obtained by differentiating any of the above-described embryonic stem cells or iPSCs; and wherein the above-described cells at least contain SOCS3 knockout, the cells further contain a polynucleotide encoding a high-affinity, cleavable hnCD16 or a variant thereof, and optionally, in some embodiments of additional genome editing as described above and throughout this invention, the cells may contain any one or more exogenous polynucleotides integrated into the SOCS3 site. In some embodiments of derived cells obtained by differentiating from the above-described iPSCs, the derived cells have an enhanced ADCC effect compared to corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other tissue or organ.

[0232] In one embodiment of the cells or populations thereof, cells comprising SOCS3 knockout and / or encoding hnCD16 or a variant thereof further comprise chimeric antigen receptors (CARs), wherein the CAR can be any one or more of the following: (1) CARs driven by T cell, NK cell, macrophage, or Treg cell-specific promoters, or CARs driven by CAG, CMV, EF1α, PGK, UBC, or other constitutive, inducible, time-, or tissue-type-specific promoters; (2) CARs that bind to bispecific or trispecific antigens; (3) CARs that are site-directed to sites such as human genome TCR, SOCS3, B2M, CIITA, immune checkpoints, tumor microenvironment-related genes (such as TGFBR2), or human genome safe harbors (such as AAVS1); (4) different CARs expressed under the same promoter; (5) different CARs expressed under different promoters; (6) intracellular CARs such as immune checkpoints, co-stimulatory factors, or adhesion molecules. (6) CARs with multiple targets are expressed on the cell surface; (7) CARs that can be induced by small molecules; (8) CARs co-expressed with another CAR; (9) CARs co-expressed in the cell with cytokines, cytokine receptors, adhesion molecules, hnCD16 or their variants in the same or different vectors; (10) CARs that are specific to GPRC5D and / or BCMA; (11) CARs that are specific to GPC3; and / or (12) CARs that are specific to any one or at least two of the following antigens: CD19, BCMA, GPRC5D, AFP, ASGR1, CD4, CD5, CD7, CD10, CD34, CD70, CD20, CD22, CD30, CD33, CD38, CD133, CD123, CD117, CD147, CD171, CD267, CLL-1, AXL, B7-H3, CD44v6, CEA, CAIX, CLDN18.2. CLDN6, DLL3, DR5, EGFR, EpCAM, ErbB, FAP, FBP, FRα, GD2, gp100, HER2, IL-1-RAP, IL-13Rα2, ITGB7, k-IgG, LeY, LFA1, LMP 1. MAGE-A3, MAGE-A1, MAGE-A4, MART1, MET, MG7, MMP2, MSLN, MUC1, MUC16, MUC16ecto, NECTIN4, NKG2D, NKG2DL (NKG2D ligand), P MEL, PSCA, PSMA, ROR1, ROR2, TM4SF1, TnMUC1, TRBC1, VEGFR2, CLD18, antigens expressed on target cells after infection by various pathogens (such as pathogen gp350 expressed after EBV infection), and inflammation-related factors (IL-1, TNF-α, IFN-γ, IL-12, IL-18, GMCSF, IL-6, IL-8, IL-17, CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL16, and CCL2-2, etc.).

[0233] In some embodiments of the cells or populations thereof, one or more cells containing SOCS3 knockout are primary NK cells or NK cells or populations thereof derived from pluripotent stem cells, hematopoietic stem cells and progenitor cells, NK cell progenitor cells, and these cells, compared with their unmodified counterparts, have at least one of the following characteristics: 1) they can differentiate, survive and expand under low concentrations of IL15 (≤1 ng / mL); 2) they have stronger expansion capacity in vitro or in vivo; and 3) they have stronger tumor-killing properties.

[0234] In one embodiment of the cells or population thereof, the SOCS3 knockout cells further include at least two genes knocked out of B2M, CIITA, and PVR; and express at least one gene or peptide of exogenous CD47, HLA-E, and HLA-G. The cells are immune cells, preferably NK cells. In one embodiment, the NK cells may be NK cells differentiated from iPSCs or ESCs. In one embodiment, the NK cells may be NK cells differentiated from CD34+ cells.

[0235] In one embodiment of the cells or population thereof, the SOCS3 knockout cells further comprise at least one gene knocked out from the following: TGFBR2, CD39, A2AR, NKG2A, TIGIT, KIR, PD-1, LAIR1, CD161, CTLA-4, TIM3, and LAG-3. The cells are immune cells, preferably NK cells. In one embodiment, the NK cells may be NK cells differentiated from iPSCs or ESCs. In one embodiment, the NK cells may be NK cells differentiated from CD34+ cells.

[0236] In one embodiment of the cells or population thereof, the SOCS3 knockout cells further express at least one gene or peptide of the exogenous peptide iCasp9 (inducible caspase 9) or tEGFR. The cells are immune cells, preferably NK cells. In one embodiment, the NK cells may be NK cells differentiated from iPSCs or ESCs. In one embodiment, the NK cells may be NK cells differentiated from CD34+ cells.

[0237] In some embodiments, iPSCs or their derivative cells include SOCS3 knockout (SOCS3 KO or SOCS3 KO). - / - ).

[0238] In some embodiments, iPSCs or their derivatives contain SOCS3 knockout and hnCD16 or its variant expression.

[0239] In some embodiments, iPSCs or their derivative cells comprise SOCS3 knockout and CD38 knockout (CD38 knockout). - / - ), and hnCD16 or its variants are expressed.

[0240] In some embodiments, iPSCs or their derivatives contain SOCS3 knockout, hnCD16 or its variant expression, B2M knockout, CIITA knockout, and at least one of CD47, or HLA-E, or HLA-G (including non-cleavable forms) expressed.

[0241] In some embodiments, iPSCs or their derivatives contain SOCS3 knockout, hnCD16 or a variant thereof, and CAR expression.

[0242] In some embodiments, iPSCs or their derivatives contain SOCS3 knockout, TGFBR2 knockout, or reduced expression.

[0243] In some embodiments, iPSCs or their derivatives contain SOCS3 knockout and TGFBR2 knockout or reduced expression, hnCD16 or its variants expressed, and / or CAR expressed.

[0244] In some embodiments, iPSCs or their derivatives may contain expression of hnCD16 or its variants as described above and throughout the invention, CD38 knockout, SOCS3 knockout, or CAR.

[0245] In some embodiments, iPSCs or their derivatives contain expression of at least one of the following genes as described above and throughout the present invention: SOCS3 knockout, CD38 knockout, B2M knockout, CIITA knockout, hnCD16 or its variants, CAR expression, and CD47, or HLA-E, or HLA-G (including non-cleavable forms).

[0246] In some of the above embodiments of cells containing CAR, the CAR is specific for GPRC5D.

[0247] In some of the above embodiments of cells containing CAR, the CAR is specific for GPRC5D and / or BCMA.

[0248] In some of the above embodiments of cells containing CAR, the CAR is specific for GPC3;

[0249] In some of the above embodiments of cells containing CAR, the CAR is specific for GPC3 and / or ASGR1.

[0250] In some of the above embodiments of cells containing CAR, the CAR is specific for GPC3 and / or AFP.

[0251] In some of the above-described embodiments of cells containing CAR, the CAR is specific for at least one protein or fragment thereof among EGFRvIII, IL-13Rα2, B7H3, GD2, and HER2.

[0252] In some embodiments comprising cells with SOCS3 knockout, hnCD16 or a variant thereof expression, and CAR expression, the CAR is specific to at least one of the following antigens: CD19, BCMA, GPRC5D, CD123, CD138, CD20, CD22, CD30, CD33, CD38, CD4, CD7, CLL-1, CD10, CD34, CS1, CD16, CD5, IL-1-RAP, ITGB7, κ-IgG, TACI, TRBC1, MUC1, NKG2DL (NKG2D ligand), PD-L1, CD133, CD117, LeY, CD70, ROR1, CD147, CD171, CD267, AXL, B7-H3, CD44v6, CEA, CAIX, CLDN18.2, CLDN6, DLL3, DR5, EGFR, EpCAM, ErbB, F AP, FBP, FRα, GD2, gp100, HER2, IL-13Rα2, LFA1, LMP1, MAGE-A3, MAGE-A1, MAGE-A4, MART1, MET, MG7, MMP2, MSLN, MUC1, MUC16, MUC16ecto, NECTIN4, NKG2D, PMEL, PSCA, PSMA, ROR1, ROR2, TM4SF1, TnMUC1, VEGFR2, CLD18, antigens expressed on target cells after infection by various pathogens (such as pathogens expressed after EBV infection), and inflammation-related factors (IL-1, TNF-α, IFN-γ, IL-12, IL-18, GMCSF, IL-6, IL-8, IL-17, CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL16, and CCL2-2, etc.).

[0253] In some embodiments comprising cells with SOCS3 knockout, TGFBR2 knockout or reduced expression, hnCD16 or its variant expression, or CAR expression, the CAR is specific to at least one of the following antigens: TACI, TRBC1, MUC1, NKG2DL (NKG2D ligand), PD-L1, CD133, CD117, LeY, CD70, ROR1, CD147, CD171, CD267, AXL, B7-H3, CD44v6, CEA, CAIX, CLDN18.2, CLDN6, DLL3, DR5, EGFR, EpCAM, ErbB, FAP, FBP, FRα, GD2, gp100, HER2, IL-13Rα2, LFA1, LMP1, MAGE -A3, MAGE-A1, MAGE-A4, MART1, MET, MG7, MMP2, MSLN, MUC1, MUC16, MUC16ecto, NECTIN4, NKG2D, PMEL, PSCA, PSMA, ROR1, ROR2, TM4SF1, TnMUC1, VEGFR2, CLD18, antigens expressed on target cells after infection by various pathogens (such as pathogens expressed after EBV infection), and inflammation-related factors (IL-1, TNF-α, IFN-γ, IL-12, IL-18, GMCSF, IL-6, IL-8, IL-17, CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL16 and CCL2-2, etc.).

[0254] The present invention also provides a method for obtaining or manufacturing the cells described above. The method includes directed differentiation of one or more iPSCs containing SOCS3 knockout, optionally, these iPSCs also having the following genetic modifications or characteristics: (i) B2M and CIITA knockout or low-content types; (ii) introduction of expression of at least one of HLA-E, HLA-G, and CD47, wherein HLA-E or HLA-G includes its non-cleavable form; (iii) introduction of expression of high-affinity non-cleavable CD16 (hnCD16) or a variant thereof; (iv) introduction of gene antigen receptor (CAR, TCR, NCR) expression; (v) introduction of expression of some or all peptides of exogenous cytokines expressed on the cell surface; (vi) introduction of SOCS family genes (SOCS1, SOCS2, etc.) other than SOCS3. (i) The expression of at least one of the following: SOCS4, SOCS5, SOCS6, SOCS7, CIS), TGFBR2, PVR, NKG2A, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, TIGIT, CD38, TCR is absent or reduced; and (vii) 4-1BBL, NKG2D, NKp44, NKp46, CD3 single or multiple peptide chains, CD47, CD113, CD131, CD137, CD80, CD86, CD68, CD226, PDL1, A2AR, CAR, TCR, Fc receptor, conjugate and surface trigger receptor for coupling with universal conjugate is introduced or increased.

[0255] This invention further provides a method for obtaining one or more iPSCs containing SOCS3 knockout. This includes using any of the following gene editing methods or combinations thereof: (1) gene knockout; (2) gene knock-in. Gene knockout can be accomplished by at least one of the following methods: i) complete or partial deletion of the target gene coding sequence, promoter sequence, or enhancer sequence; ii) insertion of a foreign DNA fragment into the target gene coding sequence, promoter sequence, or enhancer sequence; iii) other possible gene modification methods that result in a decrease of at least 50%, 40%, 30%, 20%, or 10% in the expression or function of the gene. Gene knock-in can be accomplished by at least one of the following methods: i) inserting a foreign DNA fragment into a target site using homologous recombination, where targeting the target site can be achieved by placing homologous sequences flanking the foreign DNA fragment; ii) inserting a foreign DNA fragment into a site of double-strand break in the genome using non-homologous recombination; iii) randomly integrating a foreign DNA fragment into the host genome using tools such as viruses, transposons, or plasmids.

[0256] This invention further provides a method for obtaining one or more iPSCs containing SOCS3 knockout. This includes further gene modification of at least one of the following, or a combination thereof: i) knocking out or reducing the expression of TGFBR2; ii) introducing a high-affinity, non-cleavable CD16 or a variant thereof; iii) introducing a CAR; iv) introducing a portion or all of the peptide of a cell surface-expressed exogenous cytokine or its receptor; v) knocking out B2M and CIITA, and / or PVR; and introducing the expression of at least one of HLA-E, HLA-G, and CD47, wherein HLA-E or HLA-G includes its non-cleavable form. The selected gene modifications can be performed all at once or in steps. The introduced exogenous gene expression cassette or exogenous polypeptide can be expressed independently or co-expressed in a bicistronic construct; expression can be driven by an exogenous promoter or stably expressed or tissue-specifically expressed using an endogenous gene promoter. The exogenous promoter can be any one of the following promoters or a combination thereof: CAG, CMV, EF1α, PGK, UBC, or other constitutive, inducible, time-, tissue-, or cell-type specific promoters. In some embodiments, targeted gene modification can be performed using engineered endonucleases (such as regularly clustered short palindromic repeat (CRISPR) systems, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), homing endonucleases (Meganucleases), homologous recombination, or any other functional variations of these methods.

[0257] Furthermore, this invention provides an in vitro gene editing system for engineering cells to obtain gene-edited cells. The system includes: 1) DNA, or mRNA, or RNP (such as gRNA and Cas proteins) encoding an engineered nuclease, and / or 2) a DNA template for homologous recombination. The DNA template comprises left and right homologous arms and a foreign DNA sequence between them. Figure 11 A (in the following text) Figure 11 A refers to Figure 11 Figure A in the text, in the following text Figure 11 B. Figure 11 C and Figure 11 A. The expressions are consistent, respectively referring to... Figure 11 Figure B in the middle Figure 11 Figure C in the diagram illustrates several structures of the DNA template. When the engineered nuclease targeting sequence is located at the SOCS3 locus, the left homologous arm (SOCS3-LA) and right homologous arm (SOCS3-RA) are homologous sequences near the cleavage site of the engineered nuclease targeting the SOCS3 locus; between SOCS3-LA and SOCS3-RA is (i) a nucleic acid sequence containing several base insertions, deletions, or mutations ( Figure 11Aa), where the insertion or deletion of bases is generally not 3 bp or a multiple thereof; however, in some cases, the insertion or deletion of 3 bp or a multiple thereof can also achieve the purpose of disrupting gene expression; or (ii) one or more nucleic acid sequences encoding one or more exogenous polypeptides. Figure 11 (b, c, d in A). The genome-edited cells are human embryonic stem cells (ESCs) or induced pluripotent cells (iPSCs), cloned iPSC / ESCs or iPSC / ESC cell lines, or derived cells obtained by differentiating any of the iPSCs described above; the genome-edited cells contain the above-mentioned nucleic acid sequence (…) at a selected location in the SOCS3 locus. Figure 11 A) Targeted integration and SOCS3 knockout; wherein the genome-edited cells are capable of differentiating into CD34 + iPSCs of cells. In some embodiments, the one or more nucleic acid sequences encoding one or more exogenous proteins are in-frame linked to and driven by an endogenous promoter of SOCS3 after integration; or the selected position in the SOCS3 locus is in an exon of SOCS3. In some embodiments, the one or more nucleic acid sequences encoding one or more exogenous polypeptides are driven by an exogenous promoter after integration; wherein the integration position in the SOCS3 locus is in an exon of SOCS3. In some embodiments, the integration of a nucleic acid sequence containing several bases can cause a frameshift mutation in SOCS3, wherein the integration position in the SOCS3 locus is in an exon of SOCS3.

[0258] This invention further provides a CRISPR-mediated gene editing method to obtain the aforementioned iPSC containing SOCS3 knockout. In some CRISPR-mediated embodiments, the obtained SOCS3 knockout is a monoallelic knockout or a bialic knockout. In some CRISPR-mediated embodiments, SOCS3 knockout is achieved by CRISPR binding to the target sequence and generating a double-strand break, and further by introducing an insertion or deletion mutation at the target sequence through non-homologous end joining or homologous recombination, or by introducing a foreign DNA fragment with a homologous arm. The target sequence is located at the SOCS3 locus, its promoter region, its enhancer region, or a region adjacent to the SOCS3 locus where altering its sequence can reduce SOCS3 expression by at least 50%, 40%, or 30%. Optionally, the target sequence may be:

[0259] SEQ ID NO:17( ACAGCAAGTTTCCCGCCGCC ),

[0260] Or SEQ ID NO:18 ( CAGCAGGTTCGCCTCGCCGC ),

[0261] or SEQ ID NO:19( CTTTCTGATCCGCGACAGCT )。

[0262] When Target sequence is SEQ ID NO:17, or SEQ ID NO:18, or SEQ ID NO:19, a DNA sequence in which the homologous arm sequence has at least 30%, 40%, 50%, 60%, 70%, 80%, 90% identity with sequence SEQ ID NO:20, or at least 30%, 40%, 50%, 60%, 70%, 80%, 90% identity with SEQ ID NO:21.

[0263] (SEQ ID NO:20)

[0264] CCCTCCCCTTCCCGGGTCATTCCCGGCAGGGAGGTGACGAGGTAGGGGCAGAGCGGATGGAAGCCGGAGATCCCAGGTTCCCGGAATACTCCGGCTGGGGCCTTCGGGCTTCTCCTGTCCCCTCCCTACCCCCGTGCCTCGGGTTTCTCCCTCCGTCCACACCGCCCGGGGCTACTGGACTGAGCGGCGCCCAGGCAGTCCCGGGGGCCCTTCTCCTGCTCCCCACCCGGCCACACTCCTGGAGACCTAACTTCCGCGCGCGAGTTTCCCACGCTGCGCCCTTGCAGTGCGCGCCTGGGAAGGGGCTGCCCGGGGCCACCCTGCCGGCAGGGGCGGGAGCCGTGCGGGCTCCGTGAGGCGCCTGGATCGGAGCGCGGGCCCAGGAGAGGGCCCCCGGGGCAGTGGGTGCCCCAGTCGCTCGGCGAAGGCAGGGGAGCCGGGGCGGGCCGGGCGCGCTGGAGGGTTCCGGGCACTCAACGCGCTCGCGCCTTCCTCTCCGCAGCCCCCCGGGATGCGGTAGCGGCCGCTGTGCGGAGGCCGCGAAGCAGCTGCAGCCGCCGCCGCGCAGATCCACGCTGGCTCCGTGCGCCATGGTCACCC ACAGCAAG TTTCCCGCCGCCGGGATGAGCCGCCCCCTGGACACCAGCCTGCGCCTCAAGACCTTCAGCTCCAAGAGCGAGTACCAGCTGGTGGTGAACGCAGTGCGCAAGCTGCAGGAGAGCGGCTTCTACTGGAGCGCAGTGACCG GCG

[0265] (SEQ ID NO:21)

[0266] GCGAGGCGAACCTGCTG CTCAGTGCCGAGCCCGCCGGCAC CTTTCTGATCCGCGACAGCT CGGACCAGCGCCACTTCTTCACGCTCAGCGTCAAGACCCAGTCTGGGACCAAGAACCTGCGCATCCAGTGTGAGGGGGGCAGCTTCTCTCTGCAGAGCGATCCCCGGAGCACGCAGCCCGTGCCCCGCTTCGACTGCGTGCTCAAGCTGGTGCACCACTACATGCCGCCCCCTGGAGCCCCCTCCTTCCCCTCGCCACCTACTGAACCCTCCTCCGAGGTGCCCGAGCAGCCGTCTGCCCAGCCACTCCCTGGGAGTCCCCCCAGAAGAGCCTATTACATCTACTCCGGGGGCGAGAAGATCCCCCTGGTGTTGAGCCGGCCCCTCTCCTCCAACGTGGCCACTCTTCAGCATCTCTGTCGGAAGACCGTCAACGGCCACCTGGACTCCTATGAGAAAGTCACCCAGCTGCCGGGGCCCATTCGGGAGTTCCTGGACCAGTACGATGCCCCGCTTTAAGGGGTAAAGGGCGCAAAGGGCATGGGTCGGGAGAGGGGACGCAGGCCCCTCTCCTCCGTGGCACATGGCACAAGCACAAGAAGCCAACCAGGAGAGAGTCCTGTAGCTCTGGGGGGAAAGAGGGCGGACAGGCCCCTCCCTCTGCCCTCTCCCTGCAGAATGTGGCAGGCGGACCTGGAATGTGTTGGAGGGAAGGGGGAGTACCACCTGAGTCTCCAGCTTCTCCGGAGGA

[0267] In some CRISPR-mediated embodiments, the editing further includes knocking in any of the following genes, or knocking in an expression cassette containing that gene, or a combination thereof, at at least one of the following sites: B2M, CIITA, NKG2A, TGFBR2, PVR, CD38, or a genomic safe harbor (such as the AAVS1 site): hnCD16 or a variant thereof, CAR, CD47, HLA-E or non-cleavable HLA-E, HLA-G or non-cleavable HLA-G, tEGFR or iCas9 (inducible Caspase 9 gene), membrane-bound IL15 (mbIL15). The gene sequence may be: hnCD16 or a variant thereof, CAR, CD47, HLA-E, HLA-G or non-cleavable HLA-G, tEGFR or iCas9, mbIL15.

[0268] Furthermore, this invention provides a donor plasmid or donor DNA fragment for engineering cells to obtain gene-edited cells, comprising: (i) a left homologous arm (SOCS3-LA) and a right homologous arm (SOCS3-RA) targeting SOCS3 at a nuclease cleavage site in the SOCS3 locus of the cell genome, wherein between SOCS3-LA and SOCS3-RA is: i) a nucleic acid sequence containing several base insertions, deletions, or mutations (e.g., ...). Figure 11 Aa) or ii) one or more nucleic acid sequences encoding one or more exogenous polypeptides, or their expression cassettes (e.g., Figure 11 (a, b, c, d). As an example, the one or more nucleic acid sequences encoding one or more exogenous polypeptides may be: Figure 11 As shown in B, the exogenous polypeptide is expressed by an exogenous promoter; wherein the genome-edited cell is an induced pluripotent cell (iPSC), a cloned iPSC, or an iPSC cell line; wherein the genome-edited cell comprises targeted integration of one or more nucleic acid sequences encoding one or more exogenous polypeptides at a selected location in the SOCS3 locus, and SOCS3 knockout; wherein the genome-edited cell is capable of differentiating into CD34 + iPSCs of cells. In some embodiments, the one or more nucleic acid sequences encoding one or more exogenous proteins are in-frame linked to and driven by an endogenous promoter of SOCS3 after integration; or the selected position in the SOCS3 locus is in an exon of SOCS3. In some embodiments, the one or more nucleic acid sequences encoding one or more exogenous polypeptides are driven by an exogenous promoter after integration; wherein the integration position in the SOCS3 locus is in an exon of SOCS3.

[0269] The present invention also provides a method for obtaining or manufacturing the cells described above. This method includes directed differentiation of one or more iPSCs comprising SOCS3 knockout and / or chimeric antigen receptors capable of recognizing tumor antigens, inflammation-related factors, and viral surface glycoproteins.

[0270] This invention further provides a method for obtaining one or more iPSCs containing SOCS3 knockout and / or chimeric antigen receptors that can recognize tumor antigens, inflammation-related factors, and viral surface glycoproteins. This includes introducing a DNA sequence encoding a CAR polypeptide against the above antigens into the iPSC using any of the following gene editing methods or combinations thereof: 1) inserting a foreign DNA fragment into a target site using homologous recombination, where targeting the target site can be achieved by flanking the foreign DNA fragment with homologous sequences; 2) inserting a foreign DNA fragment into a site of double-strand break in the genome using non-homologous recombination; 3) randomly integrating a foreign DNA fragment into the host genome using tools such as viruses, transposons, or plasmids.

[0271] This invention further provides a method for obtaining one or more iPSCs containing SOCS3 knockout and / or peptides that can recognize tumor antigens, inflammation-related factors, and viral surface glycoprotein receptors. This includes further gene modification of at least one of the following, or a combination thereof: 1) introducing high-affinity, cleavable CD16 or a variant thereof; 2) knocking out CD38; 3) knocking out B2M and CIITA, and / or PVR; and introducing expression of at least one of HLA-E, HLA-G, and CD47, wherein HLA-E or HLA-G includes its cleavable form; 4) membrane-bound IL15 (mbIL15). The selected gene modifications can be performed all at once or in steps. The introduced exogenous gene expression cassette or exogenous peptide can be expressed independently or co-expressed in a bicistronic construct; expression can be driven by an exogenous promoter or stably expressed or tissue-specifically expressed using an endogenous gene promoter. The exogenous promoter can be any one of the following promoters or a combination thereof: CAG, CMV, EF1α, PGK, UBC, or other constitutive, inducible, time-, tissue-, or cell-type specific promoters. In some embodiments, targeted gene modification can be performed using engineered endonucleases (such as regularly clustered short palindromic repeat (CRISPR) systems, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), homing endonucleases (Meganucleases), homologous recombination, or any other functional variations of these methods.

[0272] According to a specific embodiment of the present invention, the present invention further provides a piggyBac-mediated method for expressing anti-GPRC5D CAR, thereby obtaining one or more iPSCs containing the aforementioned anti-GPRC5D CAR polypeptide. In some piggyBac-mediated embodiments, the DNA sequence encoding the anti-GPRC5D CAR is located in the transposable region of the piggyBac (the region between the inverted terminal repeat sequences at both ends). Subsequently, with the assistance of a transposase, the piggybac randomly integrates the DNA sequence encoding the anti-GPRC5D CAR into the TTAA site of the host genome.

[0273] According to a specific embodiment of the present invention, the GPRC5D CAR expression cassette is cloned into the piggyBac vector carrying resistance (such as Puromycin resistance) to obtain the plasmid anti-GPRC5D CAR. Subsequently, the plasmid anti-GPRC5DCAR and the plasmid expressing transposase (PBase) are co-transfected into a healthy human-derived iPSC using a nuclear transfection instrument and a nuclear transfection kit. The transfection steps can be referred to the kit instructions. After resistance selection, amplification and culture are performed to obtain iPSCs expressing the anti-GPRC5D CAR molecule (anti-GPRC5D CAR-iPSC). The proportion of the obtained iPSCs expressing the anti-GPRC5D CAR molecule is identified using FACS using mouse anti-anti-GPRC5D scFV antibody (primary antibody) and PE Goat anti-mouse IgG (secondary antibody).

[0274] DNA encoding the anti-GPRC5D CAR can be introduced using various tools, such as donor plasmids, piggyBac, dsDNA, ssDNA, and various integrating or non-integrating viruses. Depending on the chosen introduction tool, PBase can be selected to assist in piggyBac introduction, while nucleases can be selected to form single-strand or double-strand breaks to assist in other non-viral introduction methods.

[0275] Figure 11 A illustrates several forms of targeted integration of exogenous DNA sequences at the SOCS3 locus to disrupt SOCS3 expression. The exogenous DNA is flanked by homologous arms targeting specific sites at the SOCS3 locus to facilitate homologous recombination. The exogenous DNA can be: Figure 11 Aa. Nucleic acid sequences containing several base insertions, deletions, or mutations ( Figure 11 Aa), where the insertion or deletion of bases is generally not 3 bp or a multiple thereof; however, in some cases, the insertion or deletion of 3 bp or a multiple thereof can also achieve the purpose of disrupting gene expression. As an example, please refer to Table 1 of this article for the ssODN sequence targeting SOCS3; Figure 11Figures b, c, and d in A illustrate the coding sequences or expression frames of polypeptides or combinations thereof containing homologous arms. Figure 11 Figure B illustrates an expression cassette containing a single polypeptide or combination thereof with homologous arms. See the diagram for an example. Figure 11 Ba, Figure 11 Bb, Figure 11 Bc, Figure 11 Bd, Figure 11 Be, Figure 11 Bf.

[0276] The site-directed homologous recombination method can be found in Example 3 of this invention and can be fine-tuned depending on the gene editing system used. For example, if a CRISPR / Cas9 plasmid targeting SOCS3 is used in combination with... Figure 11 For plasmid B, use 4 μg each. If using the RNP system, use 10-20 μg Cas9 protein, 10-20 μg sgRNA, 4 μg plasmid, or 1-6 μg dsDNA (containing... Figure 11 (Element B). After co-transferring the above gene modification system into iPSCs, the cells were enriched using 0.5 μg / mL Puro or directly expanded. Cells were then seeded at 200, 500, and 1000 cells / well and cultured at 37°C with 10 μM Y27632 at 5% CO2 for approximately one week. Single clones were picked and transferred to 96-well plates, and DNA was collected for genotyping.

[0277] In some embodiments, the genome-edited cells obtained using donor plasmids or donor DNA fragments as provided, and the hematopoietic and immune cells derived therefrom, further comprise chimeric antigen receptors (CARs), wherein the CAR has at least one of the following characteristics and wherein the CAR can be any one or more of the following: (1) CARs driven by T cell, NK cell, macrophage, or Treg cell-specific promoters, or CARs driven by CAG, CMV, EF1α, PGK, UBC, or other constitutive, inducible, time-, or tissue-type-specific promoters; (2) CARs that bind to bispecific or trispecific antigens; (3) CARs that are site-directedly inserted into human genome TCR, B2M, CIITA, immune checkpoints, tumor microenvironment-related genes (such as TGFBR2), human genome safe harbors (such as AAVS1), etc.; (4) different CARs expressed under the same promoter; (5) CARs that are associated with immune suppression. (6) Multi-target CARs are expressed on the cell surface by binding to intracellular components such as inhibitors, co-stimulatory factors, or adhesion molecules; (7) CARs that can be induced to be expressed by small molecules; (8) CARs co-expressed with another CAR; (9) CARs co-expressed in the cell with cytokines, cytokine receptors, or adhesion molecules in the same or different vectors; (10) CARs that are specific to GPC3 or EGFRvIII; and / or (11) CARs that are specific to any of the following: CD19, BCMA, GPRC5D, AFP, ASGR1, CD4, CD5, CD7, CD10, CD34, CD70, CD20, CD22, CD30, CD33, CD38, CD133, CD123, CD117, CD147, CD171, CD267, CLL-1, AXL, B7-H3, CD44v6, CEA, CAIX, CLDN18.2. CLDN6, DLL3, DR5, EGFR, EpCAM, ErbB, FAP, FBP, FRα, GD2, gp100, HER2, IL-1-RAP, IL-13Rα2, ITGB7, k-IgG, LeY, LFA1, LMP 1. MAGE-A3, MAGE-A1, MAGE-A4, MART1, MET, MG7, MMP2, MSLN, MUC1, MUC16, MUC16ecto, NECTIN4, NKG2D, NKG2DL (NKG2D ligand), P MEL, PSCA, PSMA, ROR1, ROR2, TM4SF1, TnMUC1, TRBC1, VEGFR2, CLD18, antigens expressed on target cells after infection by various pathogens (such as gp350 expressed after EBV infection), and inflammation-related factors (IL-1, TNF-α, IFN-γ, IL-12, IL-18, GMCSF, IL-6, IL-8, IL-17, CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL16, and CCL2-2, etc.).

[0278] In some embodiments of the donor plasmid or donor DNA fragment, the donor plasmid or donor DNA fragment further comprises one or more of the following: (1) an exogenous promoter that drives the expression of one or more nucleic acid sequences encoding one or more exogenous polypeptides, wherein the exogenous promoter may be CAG, CMV, EF1α, PGK, UBC or other constitutive, inducible, time-, tissue-, or cell type-specific promoters; (2) a linker sequence inserted between two nucleic acid sequences encoding the exogenous polypeptide; the linker sequence may be a 2A sequence encoding a self-cleaving 2A peptide, or the linker sequence may be an internal ribosome entry sequence (IRES); (3) an insulator at the 3' end of the left homologous arm and an insulator at the 5' end of the right homologous arm; and (iv) a polyadenylate (polyA) signal.

[0279] In some embodiments using donor plasmids or donor DNA fragments, cells resulting from gene editing of iPSCs or iPSC-derived blood cells exhibit one or more of the following phenotypes: (i) viable and amplifiable at low IL15 levels (e.g., 1 ng / mL); (ii) SOCS3 knockout and expression of hnCD16 or a variant thereof; (iii) SOCS3 knockout and expression of hnCD16 or a variant thereof, with TGFBR2 expression reduced by at least 50%, 40%, 30%, and 20%. In some embodiments using donor plasmids or donor DNA fragments, cells resulting from gene editing of iPSCs or iPSC-derived blood cells further comprise one or more of the following: (i) B2M, CIITA knockout or low-expression, and expression of exogenous CD47; (ii) expression of HLA-E, or HLA-G, or non-cleavable HLA-G; (iii) SOCS family genes other than SOCS3 (SOCS1, SOCS2, SOCS4, SOCS5, SOCS6, SOCS7, CIS), TGFBR1, TGFBR2, PVR, N... The deletion or reduced expression of at least one of KG2A, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, TIGIT, CD38, and TCR; and the introduction or increase of expression of at least one of (v)4-1BBL, NKG2D, NKp44, NKp46, CD3 single or multiple peptide chains, CD47, CD113, CD131, CD137, CD80, CD86, CD68, CD226, PDL1, A2AR, CAR, TCR, Fc receptor, conjugate, and surface trigger receptor for coupling with a universal conjugate.

[0280] In some embodiments using donor plasmids or donor DNA fragments, the gene-edited iPSC-derived blood cells contain derived CD34. + Cells, derived hematopoietic stem cells and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, or derived B cells. In one embodiment, the hematopoietic cells of the genome-edited iPSC derived from self-differentiation contain derived NK cells or derived T cells and have at least one of the following characteristics compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue: (1) can be cultured and proliferated in IL15 containing only 1 ng / mL; (2) have a higher expansion rate in vitro or in vivo; (3) have longer survival time in vitro or in vivo; (4) increased cytotoxicity; (5) improved tumor penetration; (6) acquire or enhance ADCC effects; and (7) are more resistant to tumor immunosuppression.

[0281] Another aspect of the present invention provides a method for generating genome-edited iPSCs or hematopoietic cells derived therefrom using the donor plasmid or donor DNA fragment as described, the method comprising: introducing a donor plasmid or donor DNA fragment into an iPSC, culturing the iPSC for a sufficient time to achieve targeted integration of one or more nucleic acid sequences encoding one or more exogenous polypeptides at the SOCS3 locus, and simultaneously knocking out SOCS3 expression in the iPSC, thereby obtaining genome-edited iPSCs. In some embodiments, the method may further comprise guiding the resulting genome-edited iPSCs to differentiate into hematopoietic cells comprising the same targeted integration of one or more nucleic acid sequences encoding one or more exogenous polypeptides at the SOCS3 locus and SOCS3 knockout as the genome-edited iPSCs. In some embodiments of the method, the method further comprises introducing a CRISPR system, ZFN, TALEN, homing nuclease, or any other functional variant thereof.

[0282] Composition

[0283] This invention provides SOCS3-deficient immune cells with enhanced in vitro or in vivo expansion capacity, viability, and antitumor activity, comprising an effective amount of a pharmaceutical composition administered to a human subject in need, said pharmaceutical composition comprising human SOCS3 knockout immune cells and a pharmaceutically acceptable carrier. Preferably, the immune cells are natural killer cells (NK cells).

[0284] In some embodiments, the present invention provides a composition for therapeutic use comprising immune cells, such as NK cells, and other pharmaceutically acceptable excipients. In some embodiments, the subject suitable for receiving cell therapy suffers from at least one of the following categories of diseases: hematologic malignancies, solid tumors, infectious diseases, and autoimmune diseases. The composition may also comprise clinically acceptable peptides, cytokines, growth factors, feeder cells or alternatives thereof, immune checkpoint inhibitors, antibody drugs (anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibodies), etc. In some embodiments of the composition for therapeutic use, the antibody used with the provided cells may include at least one of the following drugs: rituximab, vetuzumab, oflavumab, utuzumab, oxcartuzumab, obbituzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, denutoximab, avelumumab, daratumumab, esatuzumab, etocilizumab, and raltoximab.

[0285] In some embodiments, the present invention relates to compositions for treating patients, wherein the NK cells or NK cells comprising SOCS3 knockout are derived from human induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), peripheral blood cells, umbilical cord blood cells, or placental blood cells.

[0286] In some embodiments, the present invention relates to compositions for treating patients who have cancer that is a hematopoietic tumor or a solid tumor, or who are infected with a pathogenic virus, or who have an autoimmune disease.

[0287] In some embodiments, the present invention relates to compositions for treating patients, wherein SOCS3 knockout NK cells are more sensitive to cytokine stimulation and have stronger amplification and antitumor functions compared to unmodified NK cells.

[0288] In some embodiments, the present invention relates to a pharmaceutical composition comprising human SOCS3 knockout NK cells and at least one pharmaceutically acceptable excipient.

[0289] This invention further provides a CRISPR-mediated gene editing method for constructing one or more iPSCs containing chimeric antigen receptor peptides that can recognize tumor antigens, inflammation-related factors, and viral surface glycoproteins. In some CRISPR-mediated embodiments, CRISPR binds to the target sequence and generates a double-strand break, and further inserts a DNA sequence encoding an anti-GPRC5D CAR at the target sequence via non-homologous end joining or homologous recombination. The target sequence can be any of the following sites: AAVS1 site, or any of the following sites: CISH, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, NKG2A, B2M, CIITA, CD38, TGFBR2, PVR, TIM3, TIGIT, TCR, etc.

[0290] In some CRISPR-mediated embodiments, the editing further includes expressing any of the following genes at the same site as described above, and the anti-GPRC5D CAR peptide, in tandem or parallel expression: hnCD16 or a variant thereof, mbIL15, CD47, HLA-E, HLA-G or non-cleavable HLA-G, tEGFR or iCas9.

[0291] In some CRISPR-mediated embodiments, the editing further includes expressing at least one of the following genes or peptides at at least one of the above-mentioned different sites, such as AAVS1, CISH, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7, NKG2A, B2M, CIITA, CD38, TGFBR2, PVR, TIM3, TIGIT, and TCR: hnCD16 or a variant thereof, mbIL15, CD47, HLA-E, HLA-G or non-cleavable HLA-G, tEGFR, or iCas9.

[0292] Furthermore, this invention provides a donor plasmid or donor DNA fragment for engineering cells to obtain genome-edited cells, comprising: 1) one or more nucleic acid sequences encoding one or more exogenous polypeptides and 2) left and right homologous arm sequences targeting a target site. The genome-edited cells are induced pluripotent cells (iPSCs), cloned iPSCs, or iPSC cell lines; the genome-edited cells include targeted integration of the one or more nucleic acid sequences encoding one or more exogenous polypeptides at the target site; and the genome-edited cells are capable of differentiating into CD34 cells. + iPSCs of a cell. In some embodiments, the one or more nucleic acid sequences encoding one or more exogenous proteins are in-frame linked to and driven by an endogenous promoter at the target locus after integration; or the selected position at the target locus is located in an exon of the target gene. In some embodiments, the one or more nucleic acid sequences encoding one or more exogenous polypeptides are driven by an exogenous promoter after integration; wherein the integration position at the target locus is located in an exon of the target gene.

[0293] In some embodiments, the present invention provides a piggybac plasmid for engineering cells to obtain genome-edited cells, comprising one or more nucleic acid sequences encoding one or more exogenous polypeptides. The exogenous polypeptides include a CAR against GPRC5D, and / or at least one of hnCD16 or a variant thereof, CD47, HLA-E, HLA-G or non-cleavable HLA-G, tEGFR, iCas9, and mbIL15. The genome-edited cells are induced pluripotent cells (iPSCs), cloned iPSCs, or iPSC cell lines; the genome-edited cells comprise the random integration of the one or more nucleic acid sequences encoding one or more exogenous polypeptides into the host cell genome; the genome-edited cells are capable of differentiating into CD34... + iPSCs in cells.

[0294] In some embodiments using donor plasmids, donor DNA fragments, or piggyBac, the gene-edited iPSC-derived blood cells contain derived CD34. + Cells, derived hematopoietic stem cells and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, or derived B cells. In one embodiment, the hematopoietic cells of the genome-edited iPSC derived from self-differentiation contain derived NK cells or derived T cells and, compared with their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue, have at least one of the following characteristics, said characteristics including at least: (1) specific killing of tumor cells expressing GPRC5D; (2) obtaining or enhancing ADCC effects.

[0295] The present invention further provides a composition for manufacturing gene-edited iPSCs or hematopoietic cells differentiated from them, and said composition comprises a donor plasmid, or donor DNA, or piggyBac plasmid as described herein. Furthermore, a kit is provided herein comprising a donor plasmid, or donor DNA fragment, or piggyBac plasmid as described herein, and one or more culture media for iPSC culture, maintenance, or differentiation. Further provided are donor plasmids, or donor DNA, or piggyBac plasmids for use in pharmaceuticals, and host cells.

[0296] This invention provides a method for generating genome-edited iPSCs or hematopoietic cells derived therefrom using the donor plasmid, donor DNA fragment, or piggyBac plasmid as described, the method comprising: 1) introducing the donor plasmid, donor DNA fragment, or piggyBac plasmid into iPSCs, and 2) culturing the iPSCs for a sufficient time to obtain genome-edited iPSCs. In some embodiments, the method may further comprise guiding the resulting genome-edited iPSCs to differentiate into hematopoietic cells having the same genotype and phenotype as the genome-edited iPSCs. In some embodiments of the method, the method further comprises introducing a CRISPR system, ZFN, TALEN, homing nuclease, or any other functional variant thereof.

[0297] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with 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.

[0298] Example 1: Expression of SOCS family genes under different concentrations of IL-15 stimulation

[0299] One WT iPSC (iPSN0050, from Shenzhen Sanqi Biotechnology Co., Ltd.) was taken and differentiated into NK cells using the following method:

[0300] 1) When the iPSC confluence reaches 75%-85%, wash once with DMEM / F12 medium, add Accutase or TrypLE digestive enzyme, and incubate at 37°C in a 5% CO2 incubator for digestion. Then centrifuge and discard the supernatant, and use mTeSR supplemented with 5 μM Y27632, 50 ng / mL BMP4, and 10 μM CHIR99021. TM 1. Resuspend cells in culture medium to obtain single-cell iPSCs;

[0301] 2) Transfer single-cell iPSCs to AggreWell TM In microplates, according to AggreWell TM Follow the instructions for 400 or 800 to ensure the single cells transferred to each well are evenly distributed throughout the well. For reference, 250,000 iPSCs can be used per well of a 24-well plate. Centrifuge the cell-loaded microplate at 100g for 3 minutes and incubate statically at 37°C, 5% CO2, and 95% humidity for 1–2 days. This step can also be performed using a shaker to form spheres: adjust the concentration of the single-cell suspension, transfer 1.5 million single-cell iPSCs to a 6-well plate, and supplement each well with 5 μM Y27632, 50 ng / mL BMP4, and 10 μM CHIR99021 mTeSR. TM Prepare 1 culture medium to a density of 3 mL / well. Place the culture plate on a shaking incubator at speeds of 60 rpm, 70 rpm, 80 rpm, and 90 rpm for incubation. Select wells with uniformly sized spheres for further incubation.

[0302] Then, the medium was changed to the second differentiation medium, and thereafter the medium was changed every 2 days for 4-5 days. The components of the second differentiation medium were: StemPro-34 complete medium supplemented with 1% ITS-X, 1 μM β-mercaptoethanol, 50 ug / mL ascorbic acid, 1xGlutaMAX, 50 ng / mL VEGF, 50 ng / mL bFGF, 50 ng / mL BMP4, and 35 nM UM171.

[0303] 3) After 3 days, switch to the third differentiation medium. Third differentiation medium: StemPro-34 TMSFM complete culture medium supplemented with 1% ITS-X, 1μM β-mercaptoethanol, 50ug / mL ascorbic acid, 1x GlutaMAX, 50ng / mL VEGF, 50ng / mL bFGF, 50ng / mL SCF, 10ng / mL FLT3L, and 30ng / mL TPO.

[0304] 4) After the above three stages of culture, generally on days 10-14 after differentiation, depending on CD34... + Depending on the FACS results, CD34 + When the proportion exceeds 80%, the fourth differentiation medium can be used to further differentiate into NK cells. This process generally lasts 3-5 weeks. The fourth differentiation medium is: DMEM / F12 + 15% human AB serum + 5 ng / mL IL-3 (used in the first week) + 20 ng / mL IL-7 + 10 ng / mL FLT3L + 50 ng / mL SCF + IL15. The IL15 concentrations used are: 0.1 ng / mL, 0.5 ng / mL, 0.9 ng / mL, 2 ng / mL, 5 ng / mL, and 10 ng / mL. The specific concentration can be adjusted according to different experimental needs.

[0305] Cell RNA was collected and sequenced on days 36-38 after differentiation (see Example 2 for specific methods). The expression changes of SOCS family genes under different concentrations of IL-15 stimulation were analyzed. Figure 1 This study demonstrates the use of different concentrations of IL15 during WT iNK culture to detect the expression of SOCS family genes (CISH, SOCS1-7) under varying IL15 stimulation. The expression value of each SOCS family gene in iNK cells differentiated with 0.1 ng / mL IL15 was set to 1. Figure 1 The results showed that the expression of CISH, SOCS1, SOCS2, and SOCS3 increased exponentially with increasing IL15 concentration. Compared to 0.1 ng / mL IL15, 5 ng / mL IL15 stimulated a 4.3-fold, 3.6-fold, 7.1-fold, and 10.9-fold increase in the expression of CISH, SOCS1, SOCS2, and SOCS3, respectively. This indicates that SOCS3 expression showed the highest fold increase during iNK differentiation using 5 ng / mL IL15, suggesting that SOCS3 expression levels may be most significantly affected by IL15 levels during iNK differentiation.

[0306] Example 2: RNA Sequencing

[0307] The RNA sequencing in Example 1 was performed by BGI Genomics. The standard procedure is as follows:

[0308] 1. Cell lysis: Collect NK cells, wash 2-3 times with PBS, centrifuge to remove supernatant, and add an appropriate amount of TRIzol lysis buffer (per 1×10⁻⁶ cells) to the cell sample tube. 6 Add 1 mL of TRIzol to each cell, vortex to mix, and incubate at room temperature for 5 minutes to allow the cells to fully lyse. Store at -80°C.

[0309] 2. Sample extraction and purification

[0310] 1) Transfer the cell lysate obtained in step 1 into an EP tube containing 300 μL of chloroform / isoamyl alcohol (24:1) and mix by vigorous inversion.

[0311] 2) Centrifuge at 4℃, 12000×g for 8 min. If the intermediate layer is thick and the aqueous phase is turbid, extract again with an equal volume of chloroform / isoamyl alcohol (24:1).

[0312] 3) Transfer the supernatant to a new 1.5 mL centrifuge tube, add 2 / 3 of the supernatant volume of isopropanol (for trace tissue or cell samples, 2 μL of 5 mg / mL glycogen can be added to assist precipitation), gently invert to mix, and place in a -20℃ refrigerator for at least 2 hours.

[0313] 4) Centrifuge at 17500×g for 25 min at 4℃, discard the supernatant, wash with 0.9 mL of 75% ethanol, and suspend the precipitate by inverting the container.

[0314] 5) Centrifuge at 17500×g for 3 min at 4℃ (if the precipitation is good, you can wash it again with 75% ethanol and centrifuge at 17500×g for 3 min at 4℃).

[0315] 6) Discard the supernatant, briefly centrifuge, remove any remaining liquid, and air dry for 3-5 minutes;

[0316] 7) Dissolve the precipitate in 20-200 μL of DEPC water or RNase-free water.

[0317] 3. Library construction and sequencing

[0318] 1) Select the appropriate testing method for quality inspection based on the sample and product requirements;

[0319] 2) Take a certain amount of RNA sample, denature it at an appropriate temperature to open its secondary structure, and enrich the mRNA using oligo(dT) magnetic beads;

[0320] 3) Add a fragmentation reagent to the mRNA obtained in the previous step, react at an appropriate temperature for a certain period of time to fragment the mRNA;

[0321] 4) Prepare a one-strand synthesis reaction system and set the reaction program to synthesize one-strand cDNA; prepare a two-strand synthesis reaction system and set the reaction program to synthesize two-strand cDNA.

[0322] 5) Prepare the reaction system and set the reaction program to repair the ends of the double-stranded cDNA and add an A base to the 3' end; prepare the adapter ligation reaction system and set the reaction program to ligate the adapter to the cDNA.

[0323] 6) Prepare the PCR reaction system and set the reaction program to amplify the product;

[0324] 7) Select appropriate testing methods to conduct quality inspection on the library according to product requirements;

[0325] 8) After denaturing the PCR product into single strands, prepare a cyclization reaction system and set the reaction program to obtain single-stranded circular products, and digest the uncyclized linear DNA molecules.

[0326] 9) Sequencing: Single-stranded circular DNA molecules replicate through rolling circles to form a DNA nanosphere (DNB) containing multiple copies. The resulting DNBs are then added into the mesh wells on a high-density DNA nanochip using high-density DNA nanochip technology, and sequenced using combined probe-anchored polymerization (cPAS) technology.

[0327] 4. Data Filtering

[0328] The raw data obtained from sequencing were filtered using SOAPnuke (v1.5.6) [Li R,Li Y,Kristiansen K,Wang J.(2008).SOAP: short oligonucleotide alignment program.Bioinformatics.24(5):713-4] to remove: 1) reads containing adapters (adapter contamination); 2) reads with an unknown base N content greater than 5%; 3) low-quality reads (reads with a quality value lower than 15 accounting for more than 20% of the total number of bases in the read are considered low-quality reads), thus obtaining clean data. Subsequently, Dr. Tom's multi-omics data mining system (https: / / biosys.bgi.com) was used for data analysis, plotting, and mining.

[0329] 5. TPM value acquisition

[0330] TPM values ​​of SOCS family genes were extracted using the Dr. Tom multi-omics data mining system. Clicking on a specific gene yielded the TPM values ​​for each sample. Graphpad Prism 9 was used for plotting (see...). Figure 1 ).

[0331] Example 3: SOCS family gene knockout

[0332] SOCS family gene knockout is achieved through homologous recombination mediated by CRISPR / Cas9 and ssODN (single-stranded oligonucleotides). The sgRNA sequences targeting each SOCS family gene are shown in Table 1. Based on the sgRNA sequence targeting a specific gene, two oligonucleotides were designed and synthesized, and then cloned into the CRISPR / Cas9 backbone plasmid (SEQ ID NO:22). For specific methods, please refer to the literature FAnn Ran et al. Genome engineering using the CRISPR-Cas9 system. NATURE PROTOCOLS. 8, 2281-2308 (2013) to obtain the CRISPR / Cas9 plasmid targeting the SOCS family.

[0333] Subsequently, 4 μg of CRISPR / Cas9 plasmids targeting each gene and 0.3 nmol of ssODN (synthesized by Sangon Biotech (Shanghai) Co., Ltd.) targeting the corresponding gene were co-transfected into a healthy human-derived iPSC (iPSN0050) via nuclear transfection (Lonza, Nucleofector II). The CRISPR / Cas9 plasmids simultaneously express sgRNA and Cas9 protein targeting specific sites and are purified with Puromycin resistance for enriching target cells. The nuclear transfection kit used was LONZAVPH5022, and the transfection procedure can be found in the kit's instructions. Each ssODN contains a homologous arm targeting the gene and incorporates a frameshift mutation and a GAATTC restriction enzyme site, which can be recognized by the restriction endonuclease EcoRI. Cells were cultured at 37°C and 5% CO2 for 2 days using mTeSR1 + 10 μg Y27632 + 0.5 μg / ml Puromycin. Puromycin was then removed, and once cells showed clustered growth, Y27632 was removed. mTeSR1 was used, with the medium changed daily. Cells were passaged to approximately 80% confluence, and approximately 1 million cells were collected, lysed, and genomic DNA (Pomega, A1120) was extracted. Genomic DNA Purification Kit). Using the aforementioned cell genome as a template, homologous recombination target regions were amplified by PCR, and homologous recombination efficiency was identified by EcoRI digestion. CRISPR / Cas9 recognition sites, ssODN sequences, PCR sequences, and expected fragment lengths after EcoRI digestion are shown in Table 1. EcoRI digestion results are shown in [Table 1]. Figure 2 The results show that homologous recombination-positive cells were present in all sample populations. Further, iPSCs (CISH iPSCs) with a CISH allele knockout pair were obtained through single-cell cloning. - / -The SOCS1-7 iPSC population includes both homologous recombination-based knockout iPSCs and non-homologous end joining-based knockout iPSCs. To confirm the genotypes of this cell population, the SOCS3 KO iPSC population was selected, and the PCR products were sequenced. The proportion of each genotype in the SOCS3 KO iPSC population was analyzed using the TIDE online analysis tool (http: / / shinyapps.datacurators.nl / tider / ). It was found that homologous recombination accounted for approximately 50.7%, wild-type for approximately 27.7%, and non-homologous end joining for approximately 21.6% (of which cells with insertions or deletions of 3 or multiples thereof accounted for approximately 7.3%). The overall proportion of cells undergoing gene editing leading to KO was 72.3%. Figure 3 (Left figure in B). Unless otherwise specified, the SOCS3 KO iPSC used subsequently refers to this population.

[0334] Table 1 below shows the CISH and SOCS1-7 gene knockout-related sgRNA sequences, ssODN sequences, PCR primer sequences for amplifying the gene editing target region, and the expected fragment size of the PCR product of the gene editing target region digested with EcoRI after ssODN integration. The underlined bases in the gray band of the ssODN sequence represent exogenous insertion sequences used to create frameshift mutations.

[0335] Table 1. Sequences and identification information related to CISH and SOCS1-7 gene knockout.

[0336]

[0337]

[0338] CRISPR / Cas9 plasmid (SEQ ID NO:22)

[0339]

[0340] aggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtacccaccacatctaccacctgagaaagaactgg

[0341] tggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccga

[0342] caacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccat

[0343] cctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagc

[0344] ctgggcctgacccccaacttcaagagcaacttgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctg

[0345] gcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcacca

[0346] aggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaa

[0347] agagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaaga

[0348] tggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctg

[0349] ggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactac

[0350] gtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaaggg

[0351] cgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccg

[0352] tgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaa

[0353] gaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaac

[0354] gcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctg

[0355] acactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccg

[0356] gctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaa

[0357] acttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaat

[0358] ctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgt

[0359] gatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggca

[0360] gccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccagg

[0361] aactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgac

[0362] aagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagag

[0363] aaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaa

[0364] agcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggt

[0365] gtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgat

[0366] caaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggc

[0367] taccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacgg

[0368] cgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgca

[0369] gacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcga

[0370] cagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatg

[0371] gaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttc

[0372] gagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctgg

[0373] ccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagat

[0374] cagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccga

[0375] gaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggt

[0376] gctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgacaaaaggccggcggccacgaaaa

[0377] aggccggccaggcaaaaaagaaaaaggaattcggcagtggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgacc

[0378] gagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacac

[0379] cgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacgg

[0380] cgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctg

[0381] gccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggca

[0382] agggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttct

[0383] acgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgagaattctaactag

[0384] agctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcct

[0385] aataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagc

[0386] aggcatgctggggagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcc

[0387] cgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtat

[0388] ttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccag

[0389] cgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgc

[0390] tttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttcttta

[0391] atagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagggattttgccgatttcggtctattggttaaaaaatgagctgatt

[0392] taacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacaccc

[0393] gccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcacc

[0394] gtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcgggg

[0395] aaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagag

[0396] tatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgc

[0397] cgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggct

[0398] ggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcat

[0399] ctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcg

[0400] catcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctc

[0401] aaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtg

[0402] gccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacg

[0403] gtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgactgtcagaccaagtttactcatatatactttagattgatttaaaacttcat

[0404] ttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaa

[0405] ggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttt

[0406] ccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacct

[0407] cgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggc

[0408] tgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaa

[0409] gggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgt

[0410] cgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggcct

[0411] tttgctggccttttgctcacatgt

[0412] Example 4: iNK with SOCS family gene knockout and amplification

[0413] SOCS family gene knockout iNK cells are differentiated from SOCS family gene knockout iPSC cells; the specific differentiation method is described in Example 1. During WT iNK differentiation, the concentration of IL-15 in the fourth culture medium was 10 ng / mL, while the concentration of IL-15 in the fourth culture medium during SOCS family gene knockout iNK differentiation was 1 ng / mL. After 36 days of differentiation, the amplification capacity of the iNK cells was tested: equal amounts of iNK cells were cultured in irradiated aAPCs (K562 cells expressing membrane-bound IL-21 and 4-1BBL) and amplification medium (GT-T551 H3 medium supplemented with 5% FBS and 1000 IU / mL IL-2, 1 ng / mL or 10 ng / mL IL-15). The amount of aAPC used was 0.7-1.5 times that of the iNK cells. On day 6 of culture, the number of iNK cells in each group was counted, and the fold increase was calculated. Figure 4 The results show that SOCS3KO iNK has a stronger iNK amplification capacity compared to other SOCS family KO genes. This suggests that SOCS3 KO can enhance the amplification capacity of iNK.

[0414] Example 5: SOCS3 KO iNK proliferation capacity analysis

[0415] To observe whether iPSCs with SOCS3 KO caused by frameshift mutations or short insertion sequences have a proliferative advantage during iNK differentiation, the SOCS3 KO mixed genotype population iPSCs obtained in Example 3 were used for testing. The sequencing peak diagram of the SOCS3-targeting region in this mixed population at the iPSC stage is shown below. Figure 3 Figure A shows the main genotypes and proportions in the SOCS3 KOiPSC and iNK populations analyzed using the TIDE online analysis tool. Figure 3 Figures B and C show the results. In the iPSC stage, the proportion of wild-type (WT) cells was approximately 27.7%, and the proportion of cells lacking 3bp (Del 3bp) was approximately 7.3%. Upon differentiation into iNK cells (differentiation method see Example 1), the proportion of WT genotype cells decreased to 8.4%, and the proportion of Del 3bp genotype cells decreased to less than 2%. Figure 3 (AC). This indicates that the SOCS3 KO genotype with frameshift mutations gained a proliferative advantage during differentiation into NK cells. The Del 3bp genotype, being a non-frameshift mutation, likely had less impact on SOCS3 function; therefore, the proportion of cells with this genotype decreased similarly to WT cells. This further demonstrates that SOCS3 KO NK cells possess stronger differentiation and proliferation capabilities compared to wild-type NK cells.

[0416] Example 6: Effect of SOCS3 gene knockout on iNK yield

[0417] Further comparisons were made of iNK differentiated at 10 ng / mL IL15 (using the differentiation method in Example 1), and at 41 days of differentiation, WT iNK, SOCS3 KO iNK, and CISH... - / - iNK has similar CD56 expression levels ( Figure 5 (Figure A) However, SOCS3 cells showed relatively high viability. Nevertheless, the cell proliferation rates varied significantly at this point. SOCS3 KO iNK cells proliferated 210.6 times compared to iPSCs, WT iNK cells proliferated 157.7 times, while CISH cells... - / - iNK only multiplied by 49.1 times ( Figure 5 (See Figure B in the middle). This shows that SOCS3 KO can improve iNK yield, and SOCS3 KO provides a greater benefit than CISH KO.

[0418] Example 7: Effect of SOCS3 KO on iNK durability

[0419] To further evaluate the impact of SOCS3 KO on iNK activity and amplification capacity, further studies were conducted on WT iNK, SOCS3KO iNK, and CISH. - / - iNK cells were tested for amplification capacity. iNK cells were obtained using the differentiation method described in Example 1, and by day 41 of differentiation, CD56... + The cell proportion, proliferation rate relative to the initial iPSCs, and iNK cell viability are shown in Table 2.

[0420] Table 2 iNK information used for amplification

[0421]

[0422]

[0423] Equal volumes of iNK cells (using 1 ng / mL or 10 ng / mL IL-15 in the fourth medium) were cultured in irradiated aAPC cells (K562 cells expressing membrane-bound IL-21 and 4-1BBL) and expansion medium (GT-T551 H3 medium supplemented with 5% FBS and 1000 IU / mL IL-2, and 1 ng / mL or 10 ng / mL IL-15). The amount of aAPC cells used was 0.7–1 times that of iNK cells, and the IL-15 concentration was consistent in both the expansion and fourth mediums. Cell counts were performed and cell viability was recorded on days 0, 6, 8, 10, 13, 16, 19, and 21, and cell proliferation was calculated.

[0424] according to Figure 6The results showed that under 10 ng / mL IL-15 conditions, SOCS3 KO iNK cells exhibited the highest fold increase (27.2-fold) on Day 16 and maintained high cell viability throughout culture up to 21 days, while WT iNK and CISH cells... - / - iNK amplification rate is very low and survival time is also greatly shortened. Figure 6 (See Figures A and B). Under the condition of 1 ng / mL IL15, the viability and cell expansion rate of SOCS3 KO iNK cells, whether differentiated from AggreWell spheres or oscillatory spheres, remained at a high level. Figure 7 (AD diagram).

[0425] Example 8: SOCS3 KO enhances iNK cell killing activity against tumor cells

[0426] To further observe the effect of SOCS3 KO on the antitumor activity of NK cells, bioimaging methods were used to detect the killing activity of CBNK, WT iNK, and SOCS3 KO iNK on target cells. Target cells were selected from hematologic malignancy cell lines MOLM-13-Luc (human acute myeloid leukemia cells) and RPMI 8226-Luc (human multiple myeloma cells), and solid tumor cell line U-87MG-Luc (human glioblastoma cell line).

[0427] The Luciferase assay for NK cell cytotoxicity is briefly described as follows: First, prepare a target cell suspension (target cell viability not less than 90%). After counting, seed the cells into the wells of a 96-well plate according to the specified effector-to-target ratio. Each effector-to-target ratio should correspond to at least three replicate wells per effector cell. Adjust the medium (RMPI 1640 + 10% FBS) in each well to 100 μL. Then, add the required number of effector cells (e.g., CBNK, WT iNK, SOCS3 KO iNK) according to the specified effector-to-target ratio. Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for the specified time (e.g., 6h or 24h). Take the luciferin substrate and dilute it 100-fold with cell culture medium. Add 50 μL of luciferin substrate dilution to each well and react at room temperature in the dark for 10 minutes. Set the microplate reader to lumens and the time to 1000ms, then read the values. Cytotoxicity % = (Control group - Cytotoxic group) / Control group * 100.

[0428] Figure 8A The results showed that SOCS3 KO iNK after cryopreservation and thawing had a dose-dependent killing effect on the MOLM-13-Luc cell line, and its killing activity was significantly better than CBNK (P<0.0001) and WT iNK (P<0.01) at an effector-to-target ratio of 5:1.

[0429] Figure 8B The study showed that SOCS3 KO iNK had a dose-dependent killing effect on the RPMI 8226-Luc cell line, and its killing activity was significantly better than CBNK (P<0.0001) and WT iNK (P<0.0001) at an effector-to-target ratio of 5:1; and its killing activity was significantly better than WT iNK (P<0.01) at effector-to-target ratios of 2:1 and 1:1.

[0430] Figure 9 The study showed that SOCS3 KO iNK, after cryopreservation and thawing, exhibited a dose-dependent killing effect on the U-87MG-Luc cell line. Under co-incubation conditions of 6 hours, at an effector-to-target ratio of 2:1, the killing activity was significantly superior to CBNK (P<0.01) and WT iNK (P<0.0001); at an effector-to-target ratio of 1:1, the killing activity was significantly superior to WT iNK (P<0.01). Figure 9 (Figure A). Under co-incubation conditions of 24 hours, at an effector-to-target ratio of 2:1, the killing activity was significantly superior to CBNK (P<0.05) and WT iNK (P<0.0001); at an effector-to-target ratio of 1:1, the killing activity was significantly superior to CBNK (P<0.001) and WT iNK (P<0.0001); at an effector-to-target ratio of 0.5:1, the killing activity was significantly superior to WT iNK (P<0.001). Figure 9 (Figure B in the middle)

[0431] The above results indicate that SOCS3 KO can enhance the anti-tumor performance of NK cells, showing enhanced killing effect on both hematologic malignancies and solid tumors.

[0432] Example 9: Expression analysis of SOCS3 KO iNK activation receptor and co-stimulatory factors

[0433] Using the differentiation method described in Example 1, WT iPSCs and SOCS3 KO iPSCs were differentiated into iNK cells. During the WT iNK differentiation process, the concentration of IL-15 in the fourth culture medium was 1 ng / mL and 10 ng / mL, respectively; during the SOCS3 KO iNK differentiation process, the concentration of IL-15 in the fourth culture medium was 1 ng / mL. Using the RNA-seq sequencing and analysis method described in Example 2, the expression levels of iNK activation receptors and co-stimulatory factors were obtained. Figure 10The results showed that, under the same culture conditions (1 ng / mL IL15 in the fourth medium for iNK differentiation), SOCS3 KO iNK showed increased expression of NKG2C, NKG2D, NKp46, NKp44, NKp30, NKp80, NTB-A, CRACC, CRTAM, CD160, KIR2DS4, KIR3DS1, KIR2DS2, KIR2DL4, 2B4, 4-1BB, and CD16 compared to WT NK. Compared to WTiNK induced by 10 ng / mL IL15 (the concentration used in differentiation medium 4), SOCS3 KO iNK differentiated by 1 ng / mL IL15 showed increased expression of NKG2C, NKG2D, NKp46, NKp44, NKp30, NKp80, CRACC, CRTAM, CD160, KIR2DS4, KIR3DS1, KIR2DS2, KIR2DL4, and 4-1BB. Figure 10 The above data indicate that SOCS3 KO can enhance the expression levels of most iNK activating receptors and co-stimulatory factors.

[0434] Example 10: Homologous recombination at SOCS3 site

[0435] Using the nuclear transfection and cell culture methods described in Example 3, a CRISPR / Cas9 plasmid targeting SOCS3 (sgRNA recognition sequence as shown in SEQ ID NO:18) and a plasmid containing... Figure 11 Ba plasmids (homological arm sequences Seq ID NO 20 (SOCS3-LA), SEQ ID NO 21 (SOCS3-RA)) were transfected into WT iPSCs (iPSC0050), resulting in a single clone (T0321 iPSC) that integrated at the SOCS3 site. Similarly, using a CRISPR / Cas9 plasmid targeting SOCS3 (sgRNA recognition sequence as shown in SEQ ID NO:18) and Figure 11 Bb plasmid was transfected into CD38 KO iPSC (construction method can be found in CISH in Example 3). - / - The iPSC acquisition method uses the following sgRNA sequence: TGTACTTGACGCATCGCGCC (as shown in SEQ ID NO:55); and the ssODN sequence: CAGTATCCTGGTCCTGATCCTCGTCGTGGTGCTCGCGGTGGTCGTCTAAGCTTCCGAGGTGGCGCC AGCAGTGGAGCGGTCCGGGCACCACCAAGCGCT (as shown in SEQ ID NO:56) to obtain T0401 iPSCs.

[0436] The T0321 iPSC SOCS3 locus contains a site-directed integration of an anti-GPRC5D CAR cassette (CAG-anti-GPRC5DCAR-BGHpA); the T0401 iPSC SOCS3 locus contains a site-directed integration of an anti-GPRC5D CAR cassette and an hnCD16 expression cassette (CAG-anti-GPRC5D CAR-2A-CD16-BGHpA), with the CAR molecule and hnCD16 linked at 2A. Schematic diagrams of the donor plasmid structures and homologous recombination of T0321 and T0401 are shown below. Figure 11 C. For the iNK cells derived from these two clones, SOCS3 expression loss was detected using qPCR (primer sequences SOCS3-F: GTGGTGAACGCAGTGCGCAA (as shown in SEQ ID NO: 57), SOCS3-R: TGCCGGATCAGAAAGGTGCC (as shown in SEQ ID NO: 58)). Figure 12 FACS confirmed that T0321 iPSC and T0401 iPSC expressed an anti-GPRC5D CAR molecule (anti-GPRC5D scFV). + , Figure 13 A). However, although the T0401 iPSC contains elements that express hnCD16, the level of CD16 expression in the T0401 iPSC is still low ( Figure 13 B) This may be related to the need for stable expression of CD16A on the cell membrane surface. This may be related to CD247 (https: / / doi.org / 10.1073 / pnas.1706483114). After differentiation into iNK, the hnCD16 level in T0401 iNK increased to 95.84% ( Figure 13 (C), and further increased to nearly 100% after amplification. Figure 15 ), while the CD16 expression level in iNK cells without hnCD16 overexpression remains low ( Figure 13 (C)

[0437] Figure 14 It is evident that the T0401 iPSC inserted the "TAAGCTT" sequence at exon 1 of CD38, resulting in a frameshift mutation in CD38. Figure 14 (A), FACS confirmed the absence of T0401 iNK CD38 expression ( Figure 14 (B) Figure 15 FACS analysis showed that T0401iNK highly expressed CD56, CD16, and anti-GPRC5D CAR, and barely expressed CD38.

[0438] The expression of the anti-GPRC5D CAR molecule was detected using an anti-GPRC5D scFV antibody (prepared by Kangyuan Bochuang), and the secondary antibody was PE anti-mouse IgG Fc (Biolegend). The expression of CD16 was detected using PE-anti-human CD16 Antibody (Mouse IgG1,κ; Biolegend); the expression of CD56 was detected using APC anti-human CD56 (NCAM) Antibody (Biolegend); the expression of CD3 was detected using PE anti-human CD3 Antibody (Biolegend); and the expression of CD38 was detected using APC anti-human CD38 Antibody (Biolegend). All corresponding isotype controls were purchased from Biolegend.

[0439] Example 11: Long fragment insertion of SOCS3 KO iNK exhibits proliferative advantage

[0440] To further confirm that SOCS3 KO iNKs can gain a proliferative advantage during differentiation after the insertion of a long fragment at the SOCS3 locus, WT iPSCs mixed with T0321 iPSCs were differentiated into iNKs using the method provided in Example 1, wherein the IL15 concentration in the fourth culture medium was 1 ng / mL. Figure 16 As can be seen, at the onset of differentiation (Day 0), the proportion of T0321 iPSCs was approximately 4.91%. By day 16, 55.83% of the cells were positive for anti-GPRC5D scFV, and by day 41, 35.42% of the cells were positive for anti-GPRC5D scFV. This result indicates that during the differentiation process from iPSCs to iNK cells, the long-fragment inserted SOCS3 KO iNK (T0321) has a proliferative advantage over WT iNK and other non-long-fragment inserted iNKs. To test the anti-GPRC5D activity of T0321 iNK, the in vitro killing activity of the NK cell population (T0321 mix iNK) containing T0321 iNK cells against MM.1S-Luc was determined using the Luciferase method described in Example 8. Figure 17 It is evident that T0321 mixiNK exhibits enhanced killing activity compared to WT iNK, demonstrating the activity of the anti-GPRG5D CAR molecule in T0321 iNK.

[0441] Example 12: iNK cells overexpressing hnCD16 exhibit enhanced ADCC effect.

[0442] The steps for determining NK cell ADCC using the Luciferase method are briefly as follows: First, prepare the culture medium. Add 2 μg / mL of CD38 monoclonal antibody (Daratumumab) to complete medium (RMPI 1640 + 10% FBS) and mix thoroughly. Prepare a target cell suspension (target cell viability not less than 90%) using the above-prepared medium. After counting, seed the cells into the wells of a 96-well plate according to the specified effector-to-target ratio. Each effector-to-target ratio should correspond to at least 3 replicate wells for each effector cell. Adjust the medium in each well to 100 μL, and then add the required number of effector cells (e.g., various types of NK cells) according to the specified effector-to-target ratio. Resuspend the effector cells in the same complete medium. Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for the specified time (e.g., 6 h or 24 h). Take the luciferin substrate and dilute it 100-fold with cell culture medium. Add 50 μL of luciferin substrate dilution to each well and react at room temperature in the dark for 10 minutes. Set the microplate reader light source to 1 μm and the time to 1000 ms, then read the values. Cytotoxicity % = (Control group - Killed group) / Control group * 100. Figure 18 Figure A shows that without the addition of daratumumab, T0321 iNK and T0401 iNK have similar killing effects on MM.1S-luc. When 0.2-10 μg / mL daratumumab is added, the killing effect of T0401 iNK on MM.1S-luc is significantly improved compared to T0321 iNK. Furthermore, except for T0401 iNK, the killing ability of the other three iNK groups on MM.1S-luc decreases with increasing daratumumab concentration. Figure 18 From A, we can see that: 1) iNK cells can exert an ADCC effect in the presence of antibodies; 2) WT iNK cells are not sensitive to MM.1S-Luc cells, and SOCS3 KO cells do not significantly enhance the killing effect of NK cells on target cells; 3) Insertion of an anti-GPRC5D CAR expression cassette into the SOCS3 site can enhance the killing effect of NK cells on target cells; 4) Insertion of a CD16 or hnCD16 expression cassette into the SOCS3 site can enhance the ADCC effect of NK cells; 5) Daratumumab has certain toxicity to non-CD38 KO NK cells, which may be due to Daratumumab-induced CD38 KO NK cells. + This is caused by NK cells killing each other.

[0443] For multiple rounds of killing assays, centrifuge the culture plate from which the killing efficiency has been determined, and then carefully aspirate approximately 150 μL of the culture supernatant. Next, gently mix the remaining culture medium in the wells using a 100 μL pipette, transfer it to a new well, add another 100 μL of the target cell suspension, and return the plate to the incubator for the next round of ADCC assays. Each round is considered a 24-hour interval, until three rounds are completed. Figure 18 The results showed that T0401 iNK had a stronger multi-round killing (continuous killing) ability compared to WT iNK and T0321 iNK, further demonstrating that inserting the hnCD16 expression cassette at the SOCS3 site can enhance the ADCC effect of NK cells.

[0444] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," 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.

[0445] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A cell or a group of cells consisting of the same, characterized in that: The cells have the following characteristics: (i) The cell is: (a) an induced pluripotent stem cell or a commercially available embryonic stem cell; or (b) a derivative cell differentiated from the cell described in (a); (ii) the SOCS3 gene in the cell is knocked out; and / or (iii) the cell comprises a chimeric antigen receptor and / or a polynucleotide encoding a chimeric antigen receptor.

2. The cell or a population consisting of the cell according to claim 1, characterized in that: The cells are induced pluripotent stem cells and the SOCS3 gene in the cells is knocked out by gene editing or homologous recombination.

3. The cell or a population consisting of the cell according to claim 1, characterized in that: The cells are immune cells obtained by inducing differentiation of induced pluripotent stem cells or commercial embryonic stem cells. Optionally, the immune cells include NK cells, T cells, and macrophages; Optionally, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the transmembrane domain is connected to the extracellular domain, and the intracellular domain is connected to the transmembrane domain; Optionally, the extracellular domain includes an antibody or an antigen-binding fragment thereof that can specifically recognize a tumor antigen, an inflammation-related factor, or a viral surface glycoprotein; Optionally, the tumor antigen is GPRC5D.

4. The cell or a population consisting of the cell according to claim 1, characterized in that: The cells comprise expression of high affinity, uncleavable CD16 (hnCD16) or a variant thereof.

5. The cell or a population consisting of the cell according to claim 1, characterized in that: The cells contain one or more endogenous genes that are inactivated or whose expression is inhibited, and the endogenous genes include at least one selected from TGFBR2, PVR, NKG2A, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, TIGIT, CD38, and TCR genes; Optionally, the endogenous gene is the CD38 gene.

6. The cell or a population consisting of the cell according to claim 1, characterized in that: The cells comprise expression of a membrane-bound cytokine; Optionally, the membrane-bound cytokine comprises at least one selected from IL15, IL21, IL7, and IL3; Optionally, the membrane-bound cytokine is IL15.

7. The cell or a population consisting of the cell according to claim 1, characterized in that: In the cells: (A) B2M and CIITA genes are knocked out or expression is reduced; and (B) expression of at least one of HLA-E, HLA-G, and CD47 is introduced, wherein HLA-E or HLA-G includes its uncleavable form.

8. An immune cell, characterized in that The SOCS3 gene in the immune cell is knocked out, and the surface of the immune cell contains a chimeric antigen receptor, The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the transmembrane domain is connected to the extracellular domain, and the intracellular domain is connected to the transmembrane domain. The extracellular domain includes antibodies or antigen-binding fragments thereof that can specifically recognize tumor antigens, inflammation-related factors, and virus surface glycoproteins.

9. The immune cell according to claim 8, characterized in that The SOCS3 gene in the immune cells is knocked out by gene editing or homologous recombination; Optionally, the tumor antigen is GPRC5D; Optionally, the extracellular domain comprises a bispecific binding molecule comprising a first binding region comprising an antibody or an antigen-binding fragment thereof that is capable of specifically recognizing GPRC5D.

10. The immune cell according to claim 8, characterized in that The antibody comprises a CDR sequence selected from at least one of the following or an amino acid sequence having at least 90% identity thereto: Heavy chain variable region CDR sequence: SEQ ID NO: 1-3; CDR sequences of light chain variable region: SEQ ID NO: 4-6; Optionally, the antibody comprises: The heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences that are at least 90% identical to SEQ ID NOs: 1, 2 and 3; Optionally, the antibody comprises: The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences that are at least 90% identical to SEQ ID NOs: 4, 5 and 6.

11. The immune cell according to claim 8, characterized in that The antibodies include: The heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences that are at least 90% identical to SEQ ID NOs: 1, 2 and 3, and the light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences that are at least 90% identical to SEQ ID NOs: 4, 5 and 6; Optionally, the antibody has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 7; Optionally, the antibody has a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 8; Optionally, the antibody is a single-chain antibody having an amino acid sequence as shown in SEQ ID NO:

9.

12. The immune cell according to claim 9, characterized in that The bispecific binding molecule further comprises a second binding region, which has the activity of binding to an antigen specifically expressed on lymphocytes; Optionally, the antigen comprises at least one selected from BCMA, CD38, CD138, CD3E, and FCGR3A; Optionally, the second binding region comprises a single chain antibody or a single domain antibody; Optionally, the first binding region and the second binding region are connected via a connecting peptide.

13. The immune cell according to claim 8, characterized in that The extracellular domain of the chimeric antigen receptor is connected to the transmembrane domain via a hinge domain; Optionally, the transmembrane domain is derived from a type I transmembrane protein or an artificially synthesized transmembrane protein having a hydrophobic helix; Optionally, the transmembrane domain is derived from the transmembrane domain of at least one protein in the following group: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, H VEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM; Optionally, the transmembrane domain is derived from the transmembrane region of NKG2D; Optionally, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO: 10; Optionally, the hinge domain is derived from the hinge region of at least one protein in the following group: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT; Optionally, the hinge domain is derived from the hinge region of CD8; Optionally, the hinge domain has the amino acid sequence shown in SEQ ID NO:

11.

14. The immune cell according to any one of claims 8 to 13, characterized in that The intracellular domain includes a co-stimulatory signal domain and an intracellular signal transduction domain, and the co-stimulatory signal domain and the intracellular signal transduction domain are connected by a connecting peptide; Optionally, the costimulatory signal domain comprises an intracellular costimulatory signal domain of at least one protein selected from the following group: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand of CD83, CD40, and MyD88; Optionally, the costimulatory signaling domain is derived from the intracellular costimulatory signaling domain of 2B4; Optionally, the costimulatory signal domain has an amino acid sequence as shown in SEQ ID NO: 12; Optionally, the intracellular signaling domain comprises a domain comprising at least one ITAM motif; Optionally, the intracellular signaling domain comprises an intracellular signaling domain derived from at least one protein of the following group; CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, DAP10, DAP-12; Optionally, the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ; Optionally, the intracellular signaling domain has an amino acid sequence as shown in SEQ ID NO: 13; Optionally, the chimeric antigen receptor further comprises a signal peptide, which is linked to the N-terminus of the extracellular domain of the chimeric antigen receptor; Optionally, the signal peptide is a signal peptide derived from CD8 protein; Optionally, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 14; Optionally, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO:15 or SEQ ID NO:

16.

15. The immune cell according to claim 8, characterized in that The immune cells are obtained by separation from peripheral blood, umbilical cord blood, tissues, or organs, or the immune cells are obtained by differentiation of stem cells or immune cell progenitor cells; Optionally, the stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells within 14 days of fertilization that have not undergone in vivo development; Optionally, the immune cells include at least one selected from NK cells, T cells, and macrophages.

16. A composition, characterized in that A group comprising the cell according to any one of claims 1 to 7 or consisting of the cell and / or the immune cell according to any one of claims 8 to 15.

17. Use of the cell or a group of cells as claimed in any one of claims 1 to 7, the immune cell as claimed in any one of claims 8 to 15, or the composition as claimed in claim 16 in preparing a drug for treating a disease, characterized in that: The disease is a blood tumor or solid tumor, a viral infection, or a disease related to autoimmune abnormality.

18. The use according to claim 17, characterized in that The hematological tumor or solid tumor is a GPRC5D-related disease; Optionally, the GPRC5D-associated disease comprises multiple myeloma.

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