Engineered immune cells for allogeneic transplantation

Abstract

The present invention provides engineered immune cells in which the expression of at least one MHC-associated gene and at least one NK-activating receptor-binding molecule is inhibited or silenced, thereby inhibiting NK cell killing of the engineered immune cells. The present invention further provides pharmaceutical compositions comprising the engineered immune cells, and uses of the engineered immune cells in the manufacture of medicaments for the treatment of cancer, infectious diseases, or autoimmune diseases.

Description

[Technical field]

[0001] The present invention is in the field of immunotherapy. More specifically, the present invention relates to an engineered immune cell in which the expression of at least one NK activating receptor binding molecule and at least one MHC-associated gene is inhibited or silenced, thereby inhibiting killing by NK cells. [Background technology]

[0002] In recent years, cancer immunotherapy technology has developed rapidly, and chimeric antigen receptor T cell (CAR-T)-related immunotherapy in particular has achieved excellent clinical efficacy in the treatment of blood tumors. CAR-T cell immunotherapy involves genetically modifying T cells in vitro to enable them to recognize tumor antigens, amplifying them to a certain number, and then reinjecting them into patients to kill cancer cells and achieve the goal of tumor treatment.

[0003] In 2017, two types of autologous CAR-T therapy were approved by the FDA and marketed in the United States. One type is for B-cell acute leukemia, and the other is for diffuse B-cell non-Hodgkin's lymphoma. Although these two types of CAR-T cells have excellent clinical therapeutic effects, they are very expensive and require a long production period, making large-scale popularization very difficult. Therefore, in order to solve the above problems, it is necessary to develop a universal CAR-T product for allogeneic transplantation. Universal CAR-T can be manufactured using T cells isolated from the peripheral blood of healthy donors, which realizes allogeneic reinfusion and significantly shortens the waiting time for patients to receive treatment. In addition, the survival rate and function of T cells obtained from healthy donors are better than those derived from patients, which can increase the CAR infection rate and improve the treatment effect.

[0004] However, the development of universal CAR-T cells still faces two problems: (1) After the engineered CAR-T cells enter the patient and proliferate to a certain extent, they may attack the patient's normal cells or tissues, causing graft-versus-host disease (GvHD); (2) The normal immune system in the patient's body may reject the allogeneic CAR-T cells, causing host-versus-graft disease (HvGD). Currently, the transplant risk is mainly reduced by knocking out TCR, MHC class I molecules and / or class II molecules (e.g., HLA, B2M, etc.). However, since MHC class molecules are important molecules for NK cells to recognize "self-cells", completely inactivating MHC class I molecules such as B2M will cause NK cells to regard the reinfused engineered immune cells as "foreign" and kill them, ultimately affecting the survival and persistence of the reinfused cells, thereby affecting the therapeutic effect.

[0005] Therefore, there is a need to develop novel engineered immune cells that can reduce or inhibit the killing effect of NK cells in the recipient, thereby reducing or avoiding the risk of immune rejection caused by allogeneic transplantation. Summary of the Invention

[0006] In a first aspect, the present invention provides an engineered immune cell, in which expression of at least one MHC associated gene and at least one NK activating receptor binding molecule is inhibited or silenced, inhibiting killing of the engineered immune cell by NK cells.

[0007] In one embodiment, the MHC-related gene is selected from HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

[0008] In one embodiment, the NK activating receptor binding molecule binds to an NK activating receptor selected from NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, 2B4, DNAM-1, CD2, and LFA-1. Preferably, the NK activating receptor binding molecule binds to an NK activating receptor selected from NKG2D, NKp30, 2B4, DNAM-1, CD2, and LFA-1.

[0009] In one embodiment, the NK activating receptor binding molecule is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1, H60, MULT1, B7-H6, BAG6, PfEMP1, HSPGS, AICL, CD112, CD155, CD48, CD58, CD59, ICAM1, ICAM2, ICAM3, STAT1, JAK1, IFNGR2, JAK2, and IFNGR1. Preferably, the NK activating receptor binding molecule is selected from CD112, CD155, CD48, MICA, MICB, CD58, B7-H6, ICAM1, ICAM2, and ICAM3.

[0010] In one embodiment, the engineered immune cells further include those in which expression of at least one TCR / CD3 gene is inhibited or silenced, wherein the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof.

[0011] In one preferred embodiment, the expression of at least one TCR / CD3 gene, at least one MHC-related gene and at least one NK-activating receptor binding molecule of the engineered immune cells is inhibited or silenced, wherein the TCR / CD3 gene is selected from TRAC, TRBC and combinations thereof, the MHC-related gene is selected from B2M, RFX5, RFXAP, RFXANK, CIITA and combinations thereof, and the NK-activating receptor binding molecule is selected from CD112, CD155, CD48, MICA / B, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, CD58, B7-H6, ICAM1, ICAM2 and ICAM3. In one embodiment, the expression of TRAC or TRBC, B2M and at least one NK-activating receptor binding molecule of the engineered immune cells is inhibited or silenced. In one embodiment, the expression of TRAC or TRBC, CIITA and at least one NK-activating receptor binding molecule of the engineered immune cells is inhibited or silenced. In a preferred embodiment, the expression of TRAC or TRBC, B2M, CIITA and at least one NK activating receptor binding molecule of the engineered immune cells is inhibited or silenced.In a preferred embodiment, the expression of TRAC or TRBC, B2M, RFX5 and at least one NK activating receptor binding molecule of the engineered immune cells is inhibited or silenced.

[0012] In one embodiment, the engineered immune cells of the invention are characterized by the following: CD52, GR, dCK and PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI The expression of one or more genes selected from immune checkpoint genes such as , SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3 is inhibited or silenced. Preferably, the expression of CD52, dCK, PD1, LAG3, TIM3, CTLA4, TIGIT or a combination thereof of the engineered immune cells is inhibited or silenced.

[0013] In one embodiment, the engineered cell further expresses an immune recognition receptor comprising a ligand binding domain, a transmembrane domain and an intracellular signaling domain.Preferably, the immune recognition receptor is a chimeric antigen receptor or a T cell receptor.More preferably, the immune recognition receptor is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain.

[0014] In one embodiment, the immune recognition receptor is TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut The antibody binds to a target selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, and any combination thereof. Preferably, the target is selected from CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, Folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2, NKG2D, EGFRvIII, CS1, BCMA, mesothelin, and any combination thereof.

[0015] In one embodiment, the transmembrane domain is selected from the transmembrane domains of the TCR alpha, beta, gamma, and delta chains, CD3 zeta, epsilon, gamma, and delta subunits, CD3 delta subunits, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154 proteins. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8 alpha, CD4, CD28, and CD278.

[0016] In one embodiment, the intracellular signaling domain is selected from the signaling domains of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d proteins.Preferably, the intracellular signaling domain is a signaling domain comprising CD3ζ.

[0017] In one embodiment, the costimulatory domain is a costimulatory signaling domain of one or more proteins selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof. Preferably, the costimulatory domain is a costimulatory signaling domain of CD27, CD28, CD134, CD137 or CD278, or a combination thereof.

[0018] In one embodiment, the immune cells are selected from T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells. Preferably, the immune cells are T cells selected from CD4+ / CD8+ T cells, CD4+ helper T cells, CD8+ T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells or αβ-T cells. In a second aspect, the present invention further provides a pharmaceutical composition comprising an engineered immune cell according to the present invention and one or more pharma- ceutically acceptable excipients.

[0019] In a third aspect, the present invention provides a method of treating a subject suffering from cancer, an infectious disease or an autoimmune disease, comprising administering to said subject an effective amount of an engineered immune cell or a pharmaceutical composition according to the present invention.Thus, the present invention further provides the use of an engineered immune cell or a pharmaceutical composition according to the present invention in the manufacture of a medicament for the treatment of cancer, an infectious disease or an autoimmune disease.

[0020] Detailed Description of the Invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0021] NK cells express various activating and inhibitory receptors on their surface, and the killing function of NK cells is regulated and controlled by the balance between the positive and negative signals provided by these two types of receptors. Under normal circumstances, inhibitory receptors that recognize MHC class I molecules on the cell surface dominate the signaling balance. However, when MHC class I molecules are abnormally expressed, such as by being artificially inhibited or knocked out, the corresponding activating receptors become dominant and NK cells are activated, thereby exerting their cytotoxic effects and releasing cytokines. In the case of allogeneic transplantation of engineered immune cells, this cytotoxic effect of NK cells may kill the injected therapeutic cells, thereby reducing the therapeutic effect. Therefore, it is necessary to modify the engineered immune cells so that the risk of being killed by NK cells when reinjected into the patient's body can be reduced or avoided, thereby extending the survival time of the therapeutic immune cells and improving the therapeutic effect.

[0022] Thus, in a first aspect, the present invention provides an engineered immune cell, in which expression of at least one MHC associated gene and at least one NK activating receptor binding molecule is inhibited or silenced, thereby inhibiting killing of the engineered immune cell by NK cells. NK activating receptor binding molecule

[0023] The term "NK activating receptor binding molecule" as used herein refers to a molecule that can bind to an NK activating receptor and activate NK cell function (e.g., killing function). Non-limiting examples of NK activating receptors include NK cell surface receptors that have or bind to an immunoreceptor tyrosine-based activation motif (ITAM). Such receptors can be divided into the Ig superfamily and the C-type lectin superfamily according to molecular structure, and include, but are not limited to, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, 2B4, DNAM-1, CD2, LFA-1, etc.

[0024] In one embodiment, the NK activating receptor binding molecule is a molecule that binds to NKG2C, NKG2E or NKG2D. NKG2C, NKG2E and NKG2D all belong to the NKG2 family and bind to regulatory molecules through amino acids in the transmembrane domain, thereby activating downstream signaling pathways. Expression of NKG2C and NKG2E requires the formation of a heterodimer with CD94, and by binding to the ITAM-containing DAP12 molecule, two tyrosine residues in the ITAM are rapidly phosphorylated, ZAP-70 and Syk are recruited, and NK cells are activated. NKG2C and NKG2E recognize the ligand HLA-E in humans and the ligand Qa1b in mice. NKG2D is widely expressed not only in all human NK cells but also in the majority of γδ T cells and activated CD8+αβ T cells, and in mice it is expressed in all NK cells, some γδ T cells and some macrophages. NKG2D is expressed in the form of a homodimer and binds to two different adaptor proteins, DAP10 and DAP12, and mediates different functions through two signaling pathways. On the one hand, NKG2D binds to DAP10 through positively charged arginine residues in the transmembrane domain and phosphorylates DAP10, thus NK cells are activated to exert cytotoxic effects through a PI3K-dependent Ras-independent signaling pathway. On the other hand, NKG2D also binds to ITAM-containing DAP12 and recruits tyrosine kinases such as Syk and ZAP-70 through phosphorylation of tyrosine residues, triggering downstream signaling and inducing the release of cytokines and chemokines. The ligands of NKG2D in humans are MIC genes (including MICA and MICB) and ULBP genes (including ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6), and in mice, the ligands are Rae-1, H60 and MULT1. MICA and MICB are located on the HLA-B locus side of the MHC gene complex and share a high homology of 91%. ULBP genes are structurally similar to MHC class I molecules, and both contain α1 and α2 domains, but lack the α3 functional domain and β2 microglobulin.Thus, in one embodiment, the NK activating receptor binding molecule is selected from HLA-E, Qa1b, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1, H60 and MULT1. More preferably, the NK activating receptor binding molecule is selected from MICA, MICB, ULBP1 and ULBP2.

[0025] In one embodiment, the NK activating receptor binding molecule is a molecule that binds to NKp30, NKp44, NKp46 or NKp80. NKp30, NKp44, NKp46 and NKp80 all belong to the natural cytotoxicity receptors (NCR) family, and their expression levels on the cell surface are closely related to the function of NK cells. NKp30 is one of the important factors in the killing activity of NK cells, and its expression level is unbalanced in various tumors and virus infections, and may be involved in immune escape of tumors and viruses. The extracellular segment of NKp30 is a V-type immunoglobulin-like domain, which is linked to an arginine-rich transmembrane domain by a hydrophobic amino acid sequence. NKp30 binds to CD3ζ and transmits an activation signal into the cell via the latter's ITAM. Activating ligands of NKp30 include B7-H6, BAG6 (also called BAT3), PfEMP1, HSPGS, etc. The extracellular region of NKp44 contains a V-type domain, and the transmembrane domain has a charged lysine that binds to KAPAP / DAP12. Although the intracellular domain of NKp44 contains ITIM, it lacks an inhibitory function and does not weaken the activation signal transmitted by DAP12. Studies have shown that NKp44 can bind to viral hemagglutinin to exert the antiviral effect of NK cells. The extracellular region of NKp46 contains two C2-type Ig-like domains, the transmembrane domain contains a positively charged arginine residue, and the intracellular region does not contain an ITAM motif, so like NKp30, it must form a complex with molecules such as CD3ζ and / or FcεRIγ that contain ITAM in the intracellular segment to transmit the activation signal into the cell. NKp46 is expressed on the surface of all mature NK cells and is the key receptor for initiating the killing function of NK cells. NKp46 also binds to viral hemagglutinin to exert the antiviral effect of NK cells. NKp46 can also bind to HSPGS.NKp80 is expressed in the form of a homodimer on the surface of almost all NK cells, and after binding to its ligand activation-induced C-type lectin (AICL), it rapidly activates NK cells and enhances their cytotoxicity and ability to secrete proinflammatory cytokines. Thus, in one embodiment, the NK-activating receptor binding molecule is selected from B7-H6, BAG6, PfEMP1, HSPGS, and AICL, and preferably selected from B7-H6, BAG6, and AICL.

[0026] In one embodiment, the NK activating receptor binding molecule is a molecule that binds to 2B4. 2B4, also known as CD244, is a membrane protein that is widely expressed on the surface of NK cells, CD8+ T cells, monocytes, and granulocytes. The extracellular region of 2B4 contains a V-type immunoglobulin domain and a C2-type immunoglobulin-like domain, the transmembrane domain does not contain any charged amino acids, and the intracellular region contains an immunoreceptor tyrosine-based inhibitory switch motif (ITSM), which is recognized by the cytoplasmic SH2 domains of adaptor proteins such as SAP, EAT-2, and DRT. 2B4 crosslinking phosphorylates the tyrosine of ITSM and recruits adaptor proteins, where the complex formed between 2B4 and SAP can activate NK cells. 2B4 is not an independent receptor, but rather a helper activating receptor for NK cells, whose initiation depends on cooperation with other NCR receptors. The ligand for 2B4 is CD48, which is highly expressed in hematopoietic cell lines and some B lymphocytes. Thus, in one embodiment, the NK activating receptor binding molecule is CD48.

[0027] In one embodiment, the NK activating receptor binding molecule is a molecule that binds to DNAM-1. DNAM-1, also called CD226, is the major coactivation receptor that initiates NK cell function. DNAM-1 contains an extracellular domain of two immunoglobulin V-like domains, a transmembrane domain, and a cytoplasmic domain that contains potential phosphorylation sites for tyrosine and serine residues. DNAM-1 is expressed on a variety of blood cells, including T cells, NK cells, NKT cells, monocytes, granulocytes, and platelets. DNAM-1 is the common receptor for CD155 and CD112. CD112 is a herpes simplex virus receptor that belongs to the human nectin family and is involved in the Ca 2+ CD155 is an IgSF-independent adhesion molecule, whose family members interact through homotypic or heterotypic morphology to mediate cell-cell adhesion. CD155 is structurally similar to nectin, also known as nectin-like molecule 5 (NECL5). Thus, in one embodiment, the NK-activating receptor binding molecule is selected from CD112 and CD155.

[0028] In one embodiment, the NK activating receptor binding molecule is a molecule that binds to CD2. CD2, also called LFA-2, is a single-chain glycoprotein consisting of 327 amino acids and expressed on the surface of mature T cells, most thymocytes, and some NK cells. Ligands of CD2 include CD58 (also called LFA-3) and CD59. CD58 is widely expressed on the surface of non-hematopoietic cells and hematopoietic cells, and through specific recognition with CD2 molecules, activates phospholipase Cγ1, which mediates the production of inositol triphosphate and diacylglycerol, increases intracellular calcium ion concentration, and synergistically activates the transcription of IL-2, promoting T cell proliferation and cytokine secretion. The interaction between CD58 and CD2 molecules also promotes the formation of a TCR-polypeptide-MHC triple complex, further enhancing the antigen transduction effect. Research has shown that in the absence of expression of CD58 molecules, NK cells and T cells are not effectively activated. CD59 is a glycoprotein with a molecular weight of about 20KD that is anchored to the cell membrane by GPI. CD59 binds to CD2 near Gly95, and the CD2 molecule binds to T cells, thereby participating in the adhesion between T cells and target cells. According to research, the binding sites of CD58 and CD59 on CD2 partially overlap, and they jointly participate in the activation of the CD2 molecule and synergistically promote lymphocyte proliferation. Therefore, in one embodiment, the NK activating receptor binding molecule is selected from CD58 and CD59.

[0029] In one embodiment, the NK-activating receptor binding molecule is a molecule that binds to LFA-1. LFA-1 is composed of two non-covalently linked polypeptide chains: an alpha subunit (CD11a) and a beta subunit (CD18). Ligands for LFA-1 include members of the immunoglobulin superfamily ICAM1, ICAM2, and ICAM3, which are intercellular adhesion molecules that play an important role in localizing and adhering leukocytes to epithelial cells at the site of injury. ICAM1 can be expressed on a variety of cells, such as lymphocytes, endothelial cells, monocytes, and tumor cells, and is induced and regulated by cytokines. ICAM2 is expressed on leukocytes, endothelial cells, and platelets, while ICAM3 is expressed only on leukocytes, and neither is regulated or regulated by cytokine induction. Each of the three ligands binds to a different region within the alpha subunit of LFA-1. Interbinding of ICAM molecules to LFA-1 provides the costimulatory signals necessary for immune killing via cytotoxic T cells and NK cells, thereby activating the immune response. Among them, ICAM-1 is expressed in the form of a dimer and has the highest affinity with LFA-1. Research has shown that ICAM-1 can enhance the killing effect of NK cells via NKG2D against colorectal cancer cells. Therefore, in one embodiment, the NK activating receptor binding molecule is selected from ICAM1, ICAM2 and ICAM3.

[0030] In one embodiment, the NK activating receptor binding molecule is a molecule associated with the IFNγ signaling pathway. IFNγ plays an important role in the initiation and effector stages of immune responses, including activation of macrophages, activation of NK cells, differentiation of B cells and T cells, and upregulation of the expression of MHC class I and MHC class II molecules in various cell types. The IFNγ receptor contains two subunits, α (IFNGR1) and β (IFNGR2), both of which belong to the class II cytokine receptor family and each contain two extracellular fibronectin type III domains. The intracellular domain of IFNGR1 contains a domain that binds to the kinase JAK1 and the signal transduction and transcription regulator STAT1, while the intracellular domain of IFNGR2 contains a domain that binds to the kinase JAK2 and participates in signal transduction. During the NK cell killing process, a large amount of IFNγ is released, which binds to the IFNγR on the surface of tumor cells and activates the JAK-STAT pathway, thereby activating NK cells and further inducing tumor cell killing. Thus, in one embodiment, the NK activating receptor binding molecule is selected from STAT1, JAK1, IFNGR2, JAK2 and IFNGR1.

[0031] Thus, in one embodiment, the NK activating receptor binding molecule binds to an NK activating receptor selected from NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, 2B4, DNAM-1, CD2, and LFA-1. Preferably, the NK activating receptor binding molecule binds to an NK activating receptor selected from NKG2D, NKp30, 2B4, DNAM-1, CD2, and LFA-1. In another embodiment, the NK activating receptor binding molecule is a molecule associated with the IFNγ signaling pathway. Thus, in one embodiment, the NK-activating receptor binding molecule is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1, H60, MULT1, B7-H6, BAG6, PfEMP1, HSPGS, AICL, CD112, CD155, CD48, CD58, CD59, ICAM1, ICAM2, ICAM3, STAT1, JAK1, IFNGR2, JAK2, and IFNGR1. Preferably, the NK-activating receptor binding molecule is selected from CD112, CD155, CD48, MICA, MICB, CD58, B7-H6, ICAM1, ICAM2, and ICAM3.

[0032] In one embodiment, the NK-activating receptor binding molecule may also be, for example, CD111, CD113, CEACAM1, CEACAM7, SMAP2, IL23R, PCGF5, DET1, PTGFRN, CD84, CADM1, CD72, CD74, BTN3A3, CD47, CTSS, NTRK2, RTP4, TLR3 / CD283, TMEM140, TMPRSS3, BST2 / CD317, BTN3A1, CD40, Other known NK activating ligands may be used, including, but not limited to, EPST11, ERAP1, ERAP2, GJD3, HCP5, IFI6, IFITM1, IFITM2, IFITM3, LGALS3BP, C1QBP, CD24, CD55, CD9, GJA1, GPRC5B, HMMR, IGSF5, SYNGR3, TFRC / CD71, CD90, TMEM68, TMEM97, ANKRD27, and the like. MHC-related genes

[0033] The major histocompatibility complex (MHC) was originally characterized as a protein that plays a major role in transplantation responses. It is expressed on the surface of all higher vertebrates and is called H-2 in mice and HLA in human cells. There are two main types of MHC: class I and class II. Class I MHC proteins are heterodimers of two proteins: a transmembrane protein α chain encoded by the MHCI gene and an extracellular protein β2 microsphere protein chain encoded by a gene not within the MHC gene cluster. The α chain contains three domains, and foreign peptides bind to the two N-terminal most variable domains α1 and α2. Class II MHC proteins are also heterodimers and contain two transmembrane proteins encoded by genes within the MHC complex. Class I MHC / antigen complexes interact with cytotoxic T cells, while class II MHC transfers antigens to helper T cells. Also, class I MHC proteins tend to be expressed on almost all nucleated cells and platelets (and red blood cells in mice), whereas class II MHC proteins are more selectively expressed: generally, class II MHC proteins are expressed on B cells, some macrophages and monocytes, Langerhans cells, and dendritic cells.

[0034] The human class I HLA gene cluster comprises three major loci B, C and A. Class I molecules are heterodimers composed of an MHC alpha heavy chain (encoded by HLA-A, HLA-B or HLA-C) and a light chain (encoded by beta-2 microglobulin protein, B2M). The heavy chain is membrane anchored, is approximately 45 kDa and contains eight exons. Exon 1 encodes the leader peptide, exons 2 and 3 encode the alpha 1 and alpha 2 domains, both of which bind peptides, exon 4 encodes the alpha 3 domain, exon 5 encodes the transmembrane region, and exons 6 and 7 encode the cytoplasmic tail. Polymorphisms within exon 2 and exon 3 confer peptide binding specificity to each class of molecule. Thus, in one embodiment, inhibiting or silencing expression of MHC-related genes refers to inhibiting or silencing expression of one or more genes selected from HLA-A, HLA-B, HLA-C and B2M.

[0035] The human class II HLA cluster also contains three major loci: DP, DQ, and DR, and both class I and class II clusters are polymorphic. Class II molecules play a major role in the immune system by delivering exogenous peptides and are mainly expressed in antigen-presenting cells (e.g., B lymphocytes, dendritic cells, and macrophages). Class II molecules are heterodimers consisting of an α chain and a beta chain, both of which are membrane-anchored, and the α chain is about 33-35 kDa and contains five exons. Exon 1 codes for a leader peptide, exons 2 and 3 code for two extracellular regions, exon 4 codes for a transmembrane domain, and exon 5 codes for a cytoplasmic tail. Thus, in one embodiment, the expression of MHC-related genes is inhibited or silenced, referring to the expression of one or more genes selected from HLA-DPA, HLA-DQ, and HLA-DRA being inhibited or silenced.

[0036] The expression of class I and II MHC also depends on a variety of accessory proteins. For example, Tap1 and Tap2 subunits are part of the TAP transporter complex, which is required for loading peptide antigens into class I HLA complexes. LMP2 and LMP7 proteasome subunits are responsible for hydrolyzing antigenic proteins into peptides for display on HLA. It has been shown that reducing LMP7 reduces the expression of class I MHC on the cell surface. The expression of class II MHC is induced and expressed by several positive modulators, such as the RFX complex, CIITA. The RFX complex is composed of three subunits: RFXANK (also called RFXB), RFX5, and RFX accessory protein (also called RFXAP). The RFX complex promotes the expression of class II MHC molecules by facilitating the binding of other transcription factors to the promoter of MHC class II molecules and increasing the specificity of promoter binding. CIITA is the master controller of class II MHC expression. CIITA comprises an N-terminus rich in acidic amino acids, a PST region rich in Pro, Ser and Thr, a central GTP-binding region, and a C-terminus rich in Leu repeat sequences (LRRs), and the N-terminal acidic region and the PST region are transcription activation regions. Thus, in one embodiment, the expression of MHC-related genes is inhibited or silenced, which refers to the expression of one or more genes selected from TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK and CIITA being inhibited or silenced.

[0037] Thus, in one embodiment, the MHC-related genes are selected from MHC class I molecules such as HLA-A, HLA-B, HLA-C, B2M; MHC class II molecules such as HLA-DPA, HLA-DQ, HLA-DRA; and MHC accessory proteins such as TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA and combinations thereof. In another preferred embodiment, the MHC-related genes are selected from MHC class II molecules and MHC accessory proteins such as HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA. Engineered immune cells

[0038] The term "immune cell" as used herein refers to any cell that has one or more effector functions of the immune system (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). For example, the immune cell can be a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell or a NKT cell or an immune cell derived from a stem cell (e.g., an adult stem cell, an embryonic stem cell, an umbilical cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell or a hematopoietic stem cell). Preferably, the immune cell is a T cell. The T cell can be any T cell, for example, a T cell cultured in vitro, such as a T cell Jurkat, SupT1, etc., derived from a primary T cell or a T cell line cultured in vitro, or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, such as peripheral blood monocytes, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. T cells can also be enriched or purified. T cells can be at all stages of development, including, but not limited to, CD4+ / CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, and the like. In one preferred embodiment, the immune cells are human T cells. T cells can be obtained from the blood of a subject using a variety of techniques known to those skilled in the art, such as, for example, Ficoll separation.

[0039] When the engineered immune cells of the present invention express an immune recognition receptor (e.g., a chimeric antigen receptor or a T cell receptor), the immune recognition receptor can be introduced into the immune cells by conventional methods known in the art (e.g., transduction, transfection, transformation, etc.). "Transfection" is the process of introducing a nucleic acid molecule or polynucleotide (including a vector) into a target cell. For example, in the case of RNA transfection, it is the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into a host cell. The term is primarily used for non-viral methods in eukaryotic cells. The term "transduction" is usually used to describe the transfer of a nucleic acid molecule or polynucleotide via a virus. Transfection of animal cells usually involves opening transient pores or "holes" in the cell membrane to allow the uptake of a substance. Transfection can be performed using calcium phosphate, by electroporation, by squeezing the cells, or by mixing cationic lipids with the material to produce liposomes that fuse with the cell membrane and deposit their payload inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of a nucleic acid molecule or polynucleotide (including a vector) into bacteria or into non-animal eukaryotic cells (including plant cells). Thus, transformation is a genetic change that results from the uptake of exogenous genetic material (nucleic acid molecules) directly through the cell membrane from its surroundings into a bacterium or non-animal eukaryotic cell. Transformation is achieved by artificial means. For transformation to occur, the cell or bacterium must be in a state of competence. Techniques for prokaryotic transformation can include heat shock-mediated uptake, bacterial protoplasts and no fusion with wounded cells, microinjection, and electroporation.

[0040] In one embodiment, in addition to inhibiting or silencing expression of at least one MHC-associated gene and at least one NK activating receptor binding molecule, the engineered immune cells of the present invention can further include those in which expression of at least one TCR / CD3 gene is inhibited or silenced.

[0041] The T cell receptor (TCR) is a characteristic marker on the surface of all T cells. It binds to CD3 by non-covalent binding to form the TCR / CD3 complex, which then binds to a specific MHC-antigen peptide complex on the surface of an antigen-presenting cell, generating a specific antigen stimulating signal, activating the T cell, and exerting a killing effect. TCRs are heterodimers composed of two different peptide chains, and are usually divided into two categories, α / β type and γ / δ type, with more than 95% of peripheral T lymphocytes expressing TCRα / β. The TCRα chain is encoded by the TRAC gene, and the β chain is encoded by the TRBC gene. Each peptide chain of TCR contains a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region, with the cytoplasmic region being very short and not capable of transmitting an antigen stimulating signal. TCR molecules belong to the immunoglobulin superfamily, and their antigen specificity resides in the V region. The V region has three hypervariable regions, CDR1, CDR2 and CDR3, of which CDR3 has the greatest variation so that it can directly determine the antigen binding specificity of the TCR. When the TCR recognizes an MHC-antigen peptide complex, CDR1 and CDR2 recognize and bind to the MHC molecule, and CDR3 directly binds to the antigen peptide. CD3 contains four subunits, gamma, delta, epsilon and zeta, and usually exists in the form of dimers epsilon gamma, epsilon delta and zeta zeta. All four subunits contain a conservative immunoreceptor tyrosine-based activation motif (ITAM), two of which tyrosine residues are phosphorylated by tyrosine protein kinases to transmit activation signals to T cells. Thus, in one embodiment, the TCR / CD3 gene is selected from TRAC, TRBC, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta and combinations thereof.

[0042] In one preferred embodiment, the expression of at least one MHC-associated gene, at least one NK-activating receptor binding molecule and at least one TCR / CD3 gene of the engineered immune cells of the invention is inhibited or silenced, wherein the MHC-associated gene, the NK-activating receptor binding molecule and the TCR / CD3 gene are defined as above. In one preferred embodiment, the expression of at least one MHC associated gene, at least one NK activating receptor binding molecule and at least one TCR / CD3 gene of the engineered immune cells of the invention is inhibited or silenced, wherein the MHC associated gene is selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA and combinations thereof, the NK activating receptor binding molecule is selected from CD112, CD155, CD48, MICA, MICB, CD58, B7-H6, ICAM1, ICAM2, ICAM3 and combinations thereof, and the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ and combinations thereof.

[0043] In one embodiment, the engineered immune cells of the present invention are characterized by expression of CD52, GR, dCK, and immune checkpoint genes (e.g., PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, The present invention further includes those in which the expression of at least one gene selected from the group consisting of TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3 is inhibited or silenced.

[0044] Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art.For example, antisense RNA, RNA decoys, RNA aptamers, siRNA, shRNA, miRNA, transdominant negative proteins (TNPs), chimeric / fusion proteins, chemokine ligands, anti-infected cell proteins, intracellular antibodies (sFvs), nucleoside analogues (NRTIs), non-nucleoside analogues (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides and chemical agents) and protease inhibitors can be used to inhibit gene expression.In addition, gene silencing can also be achieved by, for example, meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzyme-mediated DNA fragmentation in the CRISPR system.

[0045] In one embodiment, the engineered cell further expresses an immune recognition receptor comprising a ligand binding domain, a transmembrane domain and an intracellular signaling domain.Preferably, the immune recognition receptor is a chimeric antigen receptor or a T cell receptor.More preferably, the immune recognition receptor is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain.

[0046] T cell receptor or TCR is defined as above. In the present invention, the TCR is a recombinant TCR that may comprise a heterologous ligand binding domain (e.g. an antibody).

[0047] The term "chimeric antigen receptor" or "CAR" as used herein refers to an artificially constructed hybrid polypeptide, which generally comprises one or more ligand-binding domains (e.g., the antigen-binding portion of an antibody), a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, each domain being linked by a linker. CARs can exploit the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition provides the ability of cells expressing CARs to recognize antigens independent of antigen therapy, circumventing a major mechanism of tumor escape. Additionally, when expressed in T cells, CARs advantageously do not dimerize with the α and β chains of the endogenous T cell receptor (TCR).

[0048] As used herein, "ligand binding domain" refers to any structure or functional variant thereof that can bind to a ligand (e.g., an antigen). The ligand binding domain may be an antibody structure, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric antibodies, and functional fragments thereof. For example, the ligand binding domain may include but is not limited to whole antibodies, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, linear antibodies, sdAbs (VH or VL), nanobodies (Nb), recombinant fibronectin domains, anticalins, and DARPINs, etc. Preferably, it is selected from Fab, scFv, sdAbs, and nanobodies. In the present invention, the ligand binding domain may be monovalent or bivalent, and may be a monospecific, bispecific, or multispecific antibody.

[0049] "Fab" refers to either of two identical fragments produced upon cleavage of an immunoglobulin molecule with papain, consisting of an intact light chain and the N-terminal portion of a heavy chain linked by disulfide bonds, where the N-terminal portion of the heavy chain contains the heavy chain variable region and CH1. Compared to intact IgG, Fab lacks the Fc fragment, has increased mobility and tissue penetration, and can bind antigen monovalently without mediating antibody effects.

[0050] A "single chain antibody" or "scFv" is an antibody composed of an antibody heavy chain variable region (VH) and a light chain variable region (VL) linked by a linker. The optimal length and / or amino acid composition of the linker can be selected. The length of the linker greatly influences the variable region folding and interaction of the scFv. In fact, the use of a relatively short linker (e.g., 5-10 amino acids) can prevent intrachain folding. For the selection of the size and composition of the linker, see, for example, Hollinger et al, 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Application Publications WO2006 / 020258 and WO2007 / 024715, the entire contents of which are incorporated herein by reference. An scFv can comprise a VH and a VL linked in any order, for example, VH-linker-VL or VL-linker-VH.

[0051] A "single domain antibody" or "sdAb" refers to an antibody that is naturally devoid of light chains, comprising only one heavy chain variable domain (VHH) and two conventional CH2 and CH3 domains; also referred to as a "heavy chain antibody".

[0052] "Nanobody" or "Nb" refers to a single cloned and expressed VHH structure that is the smallest known unit capable of binding a target antigen with the same structural stability and antigen-binding activity as the original heavy chain antibody.

[0053] The term "functional variant" or "functional fragment" refers to a variant that essentially contains a parent amino acid sequence, but contains at least one amino acid modification (i.e., substitution, deletion or insertion) compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. For example, in the case of an antibody, the functional fragment is its antigen-binding portion. In one embodiment, preferably, the amino acid modification is a conservative modification.

[0054] The term "conservative modifications" as used herein refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the chimeric antigen receptor of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0055] Thus, a "functional variant" or "functional fragment" has at least 75% sequence identity with the parent amino acid sequence, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and retains the biological activity, e.g., binding activity, of the parent amino acid.

[0056] The term "sequence identity" as used herein refers to the degree to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of the exact same sequence have 100% identity. Those skilled in the art have recognized that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197) and ClustalW.

[0057] The choice of ligand binding domain will depend on the cell surface marker on the target cell that is relevant to a particular disease state to be recognized, e.g., a tumor-specific or tumor-associated antigen. Thus, in one embodiment, the ligand binding domain of the present invention is selected from the group consisting of TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, Folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe antibody binds to one or more targets selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, Claudin18.2, NKG2D, and any combination thereof. Preferably, the target is selected from CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2 / neu), MUC1, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin18.2, NKG2D, and any combination thereof. Depending on the antigen to be targeted, the CAR of the present invention can be designed to include a ligand binding domain specific for the antigen. For example, if CD19 is the antigen to be targeted, a CD19 antibody can be used as the ligand binding domain of the present invention.

[0058] The term "transmembrane domain" as used herein refers to a polypeptide structure that allows expression of a chimeric antigen receptor on the surface of an immune cell (e.g., lymphocyte, NK cell or NKT cell) and directs the cellular response of the immune cell to a target cell. The transmembrane domain can be natural or synthetic and can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can transduce a signal when the chimeric antigen receptor binds to a target antigen. Particularly useful transmembrane domains in the present invention can be derived from, for example, TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 zeta subunit, CD3 epsilon subunit, CD3 gamma subunit, CD3 delta subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments. Alternatively, the transmembrane domain may be synthetic and comprise predominantly hydrophobic residues such as leucine and valine. Preferably, said transmembrane domain is derived from the CD8 α-chain.

[0059] In one embodiment, the chimeric antigen receptor of the present invention may further comprise a hinge region between the ligand-binding domain and the transmembrane domain. The term "hinge region" as used herein generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the ligand-binding domain. Specifically, the hinge region is intended to provide greater flexibility and accessibility to the ligand-binding domain. The hinge region may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. The hinge region may be derived in whole or in part from a natural molecule, for example, in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be a synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a portion of the hinge region of the CD8α chain, the FcγRIIIα receptor, IgG4 or IgG1, more preferably the CD8α hinge.

[0060] The term "intracellular signaling domain" as used herein refers to a protein portion that transmits an effector functional signal and instructs a cell to perform a specific function. The intracellular signaling domain is involved in primary intracellular signaling after the ligand binding domain binds to an antigen, resulting in activation of the immune cell and immune response. In other words, the intracellular signaling domain is involved in activating at least one of the normal effector functions of the immune cell expressing the CAR. For example, the effector function of a T cell can be a cytolytic activity or a helper activity, including the secretion of cytokines.

[0061] In one embodiment, the intracellular signaling domains comprised in the chimeric antigen receptors of the present invention can be the cytoplasmic sequences of T cell receptors and co-receptors, which act together to initiate primary signaling after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar functions. The intracellular signaling domains can comprise a number of immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of intracellular signaling domains of the present invention include those derived from FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD3zeta, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, the signaling domain of the CAR of the present invention comprises a CD3zeta signaling domain.

[0062] In one embodiment, the chimeric antigen receptor of the present invention comprises one or more costimulatory domains. The costimulatory domain may be an intracellular functional signaling domain from a costimulatory molecule, including the entire intracellular portion of said costimulatory molecule or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand in a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. Non-limiting examples of costimulatory domains of the invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70. Preferably, the costimulatory domain of the CAR of the invention is derived from 4-1BB, CD28, CD27, OX40 or a combination thereof. In one embodiment, the CAR of the invention comprises a 4-1BB, CD28 or 4-1BB+CD28 costimulatory domain.

[0063] In one embodiment, the CAR of the present invention may further comprise a signal peptide, which, when expressed in a cell such as a T cell, guides the nascent protein to the endoplasmic reticulum and then to the cell surface. The core of the signal peptide may comprise a long hydrophobic amino acid segment, which tends to form a single α-helix. At the end of the signal peptide, there is generally an amino acid segment that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave during or after translocation to generate a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides useful in the present invention are well known to those skilled in the art, and include, for example, signal peptides derived from CD8α, IgG1, GM-CSFRα, etc.

[0064] In one embodiment, the CAR of the present invention can further include a switch structure for regulating and controlling the expression time of the CAR. For example, the switch structure can be in the form of a dimerization domain that induces a conformational change by binding to its corresponding ligand, exposing the extracellular binding domain to bind to the target antigen, thereby activating the signaling pathway. Alternatively, the switch domain can be used to connect the binding domain and the signaling domain, respectively, and only when the switch domains bind to each other (e.g., in the presence of an inducing compound), the binding domain and the signaling domain can be linked by a dimer to activate the signaling pathway. The switch structure can be in the form of a masking peptide. The masking peptide can mask the extracellular binding domain to prevent binding to the target antigen, and when the masking peptide is cleaved, for example, by a protease, the extracellular binding domain can be exposed, resulting in a "normal" CAR structure. Various switch structures known to those skilled in the art can also be used in the present invention.

[0065] In one embodiment, the CAR of the present invention may contain a suicide gene, i.e., express a cell death signal that can be induced by an exogenous substance to eliminate the CAR cells when necessary (e.g., when severe toxic side effects occur). For example, the suicide gene may take the form of an inserted epitope, such as the CD20 epitope, RQR8, etc., and, if necessary, the CAR cells can be eliminated by adding antibodies or reagents that target these epitopes. The suicide gene may be herpes simplex virus thymidine kinase (HSV-TK), which can be induced by ganciclovir treatment to cause cell death. The suicide gene may be iCaspase-9, which can be induced to dimerize by chemical inducers such as AP1903, AP20187, etc., to activate downstream Caspase 3 molecules and cause apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention. Pharmaceutical Compositions

[0066] The present invention further provides a pharmaceutical composition comprising an engineered immune cell according to the present invention as an active agent and one or more pharma- ceutically acceptable excipients.Accordingly, the present invention further encompasses the use of said engineered immune cell in the manufacture of a pharmaceutical composition or medicament.

[0067] The term "pharmaceutical acceptable excipient" as used herein refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing undesirable local or systemic effects) and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutical acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adherents, glidants, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonicity agents, absorption retardants, stabilizers and tonicity adjusters. It is well known to those skilled in the art to select the appropriate excipients to prepare the desired pharmaceutical composition of the present invention. Exemplary excipients for use in pharmaceutical compositions of the invention include saline, buffered saline, dextrose and water. In general, the selection of a suitable excipient will depend, inter alia, on the active agent being used, the disease being treated and the desired form of the pharmaceutical composition.

[0068] The pharmaceutical compositions according to the invention are suitable for administration by a variety of routes. Usually, administration is performed parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

[0069] The pharmaceutical compositions according to the invention can be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts or in a form particularly suitable for the required administration method. The known processes for preparing the pharmaceutical products of the invention include, for example, conventional mixing, dissolving, granulating, dragee-making, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. The pharmaceutical compositions comprising the immune cells described herein are usually provided in the form of a solution, preferably containing a pharma-ceutically acceptable buffer.

[0070] The pharmaceutical composition according to the present invention can also be administered in combination with one or more other drugs suitable for the treatment and / or prevention of diseases. Preferred examples of drugs suitable for combination include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E, and the like. E), vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase and RNase; radionuclides such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs such as antibody-directed enzyme prodrugs; immunostimulants such as platelet factor 4, melanoma growth stimulating protein; known anti-cancer agents such as antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides. The pharmaceutical composition of the present invention may also be used in combination with one or more other therapeutic methods, such as chemotherapy and radiotherapy. Therapeutic Applications

[0071] The invention further provides a method of treating a subject suffering from cancer, an infectious disease or an autoimmune disease comprising administering to said subject an effective amount of an engineered immune cell or pharmaceutical composition according to the invention.Thus, the invention further encompasses the use of said engineered immune cell or pharmaceutical composition in the manufacture of a medicament for treating cancer, an infectious disease or an autoimmune disease. In one embodiment, an effective amount of the immune cells and / or pharmaceutical compositions of the invention are administered directly to a subject.

[0072] In another embodiment, the treatment method of the present invention is an ex vivo treatment. Specifically, the method includes the steps of (a) providing a sample containing immune cells; (b) inhibiting or silencing the expression of at least one NK-activating receptor binding molecule and at least one MHC-related gene (and any TCR / CD3 gene and / or other gene) of the immune cells in vitro, introducing any immune recognition receptor (preferably a chimeric antigen receptor) into the immune cells, and obtaining modified immune cells; and (c) administering the modified immune cells to a subject. Preferably, the immune cells provided in step (a) are selected from macrophages, dendritic cells, monocytes, T cells, NK cells, or NKT cells. The immune cells can be obtained from a sample (particularly a blood sample) of a subject by a conventional method known in the art. In addition, the immune cells are generally selected to be compatible with the immune system of the subject, i.e., the immune cells do not induce an immunogenic response. For example, "universal recipient cells" can be used, i.e., lymphocytes that are universally compatible and capable of growing and expanding in vitro to perform the desired biological effector functions. The use of such cells does not require obtaining and / or providing the subject's own lymphocytes. The ex vivo introduction in step (c) can be carried out by introducing the nucleic acid or vector described herein into immune cells via electroporation, or by infecting immune cells with a viral vector. The viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector as described above. Other methods include the use of transfection reagents (e.g., liposomes) or transient RNA transfection.

[0073] In one embodiment, the immune cells are allogeneic cells, preferably T cells, macrophages, dendritic cells, monocytes, or NK cells, NKT cells, more preferably T cells, NK cells or NKT cells.

[0074] As used herein, the term "allogeneic" refers to any material that is derived from a different animal or different patient of the same species as the individual that introduces the material.Two or more individuals are considered to be allogeneic to each other if the genes at one or more loci are different.In some cases, the genetic difference of allogeneic material from each individual of the same species is sufficient to cause antigen interaction.

[0075] The term "subject" as used herein refers to a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. A non-human mammal may be advantageously used as a subject in an animal model of cancer. Preferably, the subject is a human.

[0076] In one embodiment, the cancer is a cancer associated with expression of a target that binds to the ligand binding domain.For example, the cancers include brain glioma, blastoma, sarcoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, renal cancer, laryngeal cancer, liver tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (e.g., (lips, tongue, mouth, pharynx), ovarian cancer, pancreatic cancer, prostate cancer, mesothelioma, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system malignancies, vulvar cancer, Waldenstrom's macroglobulinemia, lymphoma (Hodgkin lymphoma and B-cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade non-Hodgkin's lymphomas such as lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky NHL), mantle cell lymphoma, AIDS-related lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, MALT lymphoma, marginal zone lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, etc.), leukemias (acute nonlymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia (including undifferentiated and partially differentiated), acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute lymphoblastic ... The present invention relates to a method for treating a patient with a leukemia, including, but not limited to, acute leukemias such as acute myeloid leukemia, erythroleukemia, acute megakaryocytic leukemia, chronic leukemias such as chronic myeloid leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia, and other specific types of leukemias such as hairy cell leukemia, prolymphocytic cell leukemia, plasma cell leukemia, adult T-cell leukemia, eosinophilic leukemia, basophilic leukemia, blastic plasmacytoid dendritic cell neoplasm, malignant lymphoproliferative disorders, myelodysplasia, multiple myeloma, myelodysplasia, post-transplant lymphoproliferative disorders (PTLD), and other disorders associated with target expression.Preferably, the disease that can be treated with the engineered immune cells or pharmaceutical compositions of the present invention is selected from leukemia, lymphoma, multiple myeloma, myelodysplasia, brain glioma, pancreatic cancer, ovarian cancer, mesothelioma, breast cancer, lung cancer, prostate cancer, melanoma, myeloma, sarcoma, gastric cancer, etc.

[0077] In one embodiment, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

[0078] In one embodiment, the autoimmune disease includes, but is not limited to, type I diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vascular inflammation, pernicious anemia, and systemic lupus erythematosus.

[0079] In one embodiment, the method further comprises administering to the subject one or more additional chemotherapeutic agents, biologic agents, drugs or treatments, in this embodiment, the chemotherapeutic agents, biologic agents, drugs or treatments are selected from radiation therapy, surgery, antibodies and / or small molecules, and any combination thereof.

[0080] The present invention will be described in detail below by way of examples with reference to the drawings. It should be noted that those skilled in the art will recognize that the accompanying drawings and examples are merely illustrative and do not limit the present invention. The features of the embodiments and embodiments of the present application can be combined with each other unless there is a contradiction. [Brief description of the drawings]

[0081] [Figure 1] 1 shows the expression levels of NK-activating receptor-binding molecules on the surface of T cells. [Diagram 2] 1 shows the inhibitory effect of the DKO-T cells of the present invention on the killing effect of NK cells. [Diagram 3] 1 shows the inhibitory effect of the QKO-T cells of the present invention on the killing effect of NK cells. [Figure 4]1 shows scFv expression levels of CAR-dKO and CAR-sKO T cells of the present invention. [Diagram 5] 1 shows the killing effect of CAR-dKO and CAR-sKO T cells of the present invention on target cells. [Figure 6] 1 shows cytokine release levels following co-culture of CAR-dKO and CAR-sKO T cells of the invention with target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] The T cells used in all examples of the present invention were primary human CD4+CD8+ T cells isolated from healthy donors using leukapheresis with Ficoll-Paque™ PREMIUM (GE Healthcare, item number 17-5442-02). Example 1. Expression levels of NK activating receptor binding molecules

[0083] T cells were activated with DynaBeads CD3 / CD28 CTSTM (Gibco, item no. 40203D) and cultured at 37℃ and 5% CO2. T cells were collected at different culture time points, and the expression levels of various NK activating receptor binding molecules were detected by flow cytometry. The results are shown in Figure 1.

[0084] As can be seen from Figure 1, the NK activating receptor binding molecules MICA / B, CD48, CD112, CD155, ICAM1, ICAM2, ICAM3, B7-H6, and CD58 are constitutively or inducibly expressed on T cells. Example 2. Construction of DKO-T cells in which MHC-related genes and NK-activating receptor-binding molecules are knocked out

[0085] T cells were activated with DynaBeads CD3 / CD28 CTSTM (Gibco, item number 40203D) and cultured for 3 days at 37°C and 5% CO2. Then, 10 μg of Cas9 protein, 5 μg of B2M sgRNA, and 5 μg of sgRNA targeting NK activating receptor binding molecule were electrotransfected into the activated T cells at 400V, 0.7ms using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX) to obtain DKO-T cells in which B2M and NK activating receptor binding molecule were double knocked out. B2M only knockout T cells (KO-T) and wild-type T cells without knockout (NT) were used as controls. The sgRNA sequences used in the present invention are shown in Table 1.

[0086] After DKO-T, B2M-KO-T and NT cells were cultured at 37℃ and 5% CO2 for 11 days, the gene knockout efficiency of B2M and various NK activating receptor binding molecules was detected by flow cytometry using the corresponding antibodies (see Table 2). The results are shown in Table 3.

[0087] Table 1. sgRNA sequences used in the present invention JPEG0007623020000001.jpg106170

[0088] NOTE: Considering the high homology of the MICA and MICB genes, the sgRNA designed in this example can knock out both the MICA and MICB genes simultaneously.

[0089] Table 2. Information on antibodies used in flow cytometry JPEG0007623020000002.jpg100170

[0090] Table 3. Gene knockout efficiency in DKO-T cells JPEG0007623020000003.jpg65170 Example 3. Inhibitory effect of DKO-T cells of the present invention on NK cell killing effect

[0091] The inhibitory effect of the DKO-T cells produced according to the present invention on the NK cell killing effect was detected according to the following method: The DKO-T cells and KO-T cells produced according to the present invention were labeled with Far-Red (Invitrogen, item number C34564). The labeled DKO-T cells and KO-T cells were then added at 1 × 10 4 The cells were plated in a 96-well plate at a concentration of 100 cells / well, and co-cultured with NK92 cells at an effector-target ratio of 2:1. After 16-18 hours, the proportion of T cells in the culture medium was detected by a flow cytometry device, and the killing rate of NK cells was calculated. The results are shown in Figure 2.

[0092] It can be seen that when only HLA (e.g., B2M) is knocked out, the killing rate of NK cells against T cells reaches 80%. In contrast, further knockout of NK activating receptor binding molecules (i.e., DKO-T cells of the present invention) can significantly inhibit this killing effect. Example 4. Construction of QKO-T cells in which TCR, MHC-related genes, and NK-activating receptor binding molecules are knocked out

[0093] T cells were activated with DynaBeads CD3 / CD28 CTSTM (Gibco, item no. 40203D) and cultured continuously for 3 days at 37°C and 5% CO2. Then, 10μg of Cas9 protein, 2.5μg of B2M sgRNA, 2.5μg of TRAC sgRNA, 2.5μg of RFX5 sgRNA, and 2.5μg of CD155 sgRNA were electrotransfected into the activated T cells at 400V, 0.7ms using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX) to obtain QKO-T cells with B2M, TCR, RFX5, and CD155 knockout. In a similar manner, KO-T cells with B2M single knockout and DKO-T cells with B2M / CD155 double knockout were obtained. The gene knockout efficiency in these cells was detected by flow cytometry, and the results are shown in Table 4 below.

[0094] Table 4. Gene knockout efficiency in QKO-T cells JPEG0007623020000004.jpg35170

[0095] It can be seen that the corresponding genes in the KO-T cells and QKO-T cells prepared according to the present invention are effectively knocked out. Then, according to the method described in Example 3, the inhibitory effect of the KO-T cells and the QKO-T cells prepared above on the NK cell killing effect was detected. The results are shown in Figure 3.

[0096] It has been found that QKO-T cells, in which NK-activating receptor binding molecules, TCR genes, and multiple MHC-related genes are simultaneously knocked out, can also significantly inhibit the killing effect of NK cells. Example 5. Construction of CAR-dKO T cells and verification of their function 5.1 Construction of CAR-sKO T cells and CAR-dKO T cells

[0097] Sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), item number: PPL00157-4a): CD8α signal peptide (SEQ ID NO:12), anti-CD19 scFv (SEQ ID NO:13), CD8α hinge region (SEQ ID NO:14), CD8α transmembrane domain (SEQ ID NO:15), 4-1BB intracellular region (SEQ ID NO:16), CD3ζ intracellular signaling domain (SEQ ID NO:17), and the correct insertion of the target sequences was confirmed by sequencing.

[0098] 3mL of Opti-MEM (Gibco, item no. 31985-070) was added to a sterile tube to dilute the above plasmids, and packaging vector psPAX2 (Addgene, item no. 12260) and envelope vector pMD2.G (Addgene, item no. 12259) were added according to the ratio of plasmid:viral packaging vector:viral envelope vector=4:2:1. Then, 120μL X-treme GENE HP DNA Transfection Reagent (Roche, item no. 06366236001) was added, immediately mixed uniformly, and incubated at room temperature for 15 minutes, and then the plasmid / vector / transfection reagent mixture was added dropwise to the culture flask of 293T cells. The viruses were collected at 24 hours and 48 hours, combined, and ultracentrifuged (25000g, 4℃, 2.5 hours) to obtain concentrated lentivirus.

[0099] Concentrated lentivirus was transfected into sKO-T cells (B2M single knockout) and dKO-T cells (B2M / ICAM3 double knockout) to obtain CAR-sKO and CAR-dKO T cells, respectively. Unmodified wild-type T cells were used as a negative control (NT).

[0100] After culturing for 11 days under 37℃ and 5% CO2 conditions, the expression level of scFv in CAR-T cells was detected by flow cytometry using Biotin-SP(long spacer)AffiniPure Goat Anti-Mouse IgG,F(ab')2Fragment Specific(min x Hu,Bov,Hrs Sr Prot)(Jackson Immunoresearch, item number 115-065-072) as the primary antibody and APC Streptavidin (BD Pharmingen, item number 554067) or PE Streptavidin (BD Pharmingen, item number 554061) as the secondary antibody. The results are shown in Figure 4. It can be seen that the scFv is efficiently expressed in the CAR T cells produced according to the present invention. 5.2 Detection of the killing ability of CAR-T cells against target cells

[0101] First, 1 × 10 Nalm6 target cells carrying the fluorescein gene were cultured in 4 The NT cells, CAR-sKO T cells and CAR-dKO T cells were plated on a 96-well plate at a concentration of 1000 / well, and then co-cultured with the NT cells, CAR-sKO T cells and CAR-dKO T cells at an effector-target ratio of 2:1 (i.e., the ratio of effector T cells to target cells). After 16-18 hours, the fluorescence values ​​were measured using a microplate reader. The killing efficiency was calculated using the formula: (mean fluorescence value of target cells - mean fluorescence value of samples) / mean fluorescence value of target cells x 100%. The results are shown in Figure 5.

[0102] As can be seen, both CAR-sKO and CAR-dKO T cells are capable of specific killing of target cells, and the additional knockout of the NK activating receptor ICAM3 does not adversely affect the killing ability of CAR-T cells. 5.3 Detection of cytokine release levels from CAR-T cells

[0103] 1 x 10 Nalm6 target cells 5 The NT cells, CAR-sKO T cells, and CAR-dKO T cells of the present invention were plated in a 96-well plate at a concentration of 1:1 / well and co-cultured with the target cells at a ratio of 1:1, and the cell co-culture supernatants were collected after 18-24 hours.

[0104] A 96-well plate was coated with the capture antibody Purified anti-human IL2 Antibody (Biolegend, item number 500302) or Purified anti-human IFN-γ Antibody (Biolegend, item number 506502) and incubated overnight at 4°C. The antibody solution was then removed and 250 μL of PBST (1XPBS containing 0.1% Tween) solution containing 2% BSA (sigma, item number V900933-1kg) was added and incubated at 37°C for 2 hours. The plate was then washed three times with 250 μL of PBST (1XPBS containing 0.1% Tween). 50 μL of cell co-culture supernatant or standard was added to each well and incubated at 37°C for 1 hour, after which the plate was washed three times with 250 μL of PBST (1XPBS containing 0.1% Tween). Then, 50μL of detection anti-Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, item number ab25017) was added to each well and incubated at 37℃ for 1 hour, and the plate was washed three times with 250μL of PBST (1XPBS containing 0.1% Tween). HRP Streptavidin (Biolegend, item number 405210) was further added and incubated at 37℃ for 30 minutes, after which the supernatant was discarded and washed five times with 250μL of PBST (1XPBS containing 0.1% Tween). 50μL of TMB substrate solution was added to each well. After reacting at room temperature for 30 minutes in the dark, 50μL of 1mol / L H2SO4 was added to each well to stop the reaction. The absorbance at 450 nm was detected by a microplate reader within 30 minutes after the reaction was stopped, and the content of cytokines was calculated according to a standard curve (constructed according to the readings and concentrations of the standards). The results are shown in FIG. 6.

[0105] As can be seen, both CAR-sKO T cells and CAR-dKO T cells could significantly increase the cytokine release levels of CAR T cells against target cells, and further knockout of the NK activating receptor ICAM3 had no adverse effect on the cytokine release levels of CAR-T cells.

[0106] It should be noted that the above is merely a preferred embodiment of the present invention, and is not intended to limit the present invention, and those skilled in the art may have various modifications and changes to the present invention. Those skilled in the art will understand that any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should all be included in the protection scope of the present invention.

Claims

1. 1. An engineered immune cell, comprising: expression of at least one MHC-associated gene and at least one NK activating receptor binding molecule is inhibited or silenced, inhibiting NK cell killing of the engineered immune cells; the at least one NK activating receptor binding molecule is selected from CD48, ICAM2 and B7-H6; The MHC-associated gene is B2M or a combination of B2M and RFX5; An engineered immune cell characterized in that

2. The engineered immune cells further include those in which expression of at least one TCR / CD3 gene is inhibited or silenced; 2. The engineered immune cell of claim 1, wherein the TCR / CD3 genes are selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof.

3. The engineered immune cells are CD52, GR, dCK, PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3 3. The engineered immune cell of claim 1 or 2, further comprising one or more genes selected from the group consisting of IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36, IL37, IL38, IL39 ...

4. The engineered immune cell of any one of claims 1 to 3, further expressing an immune recognition receptor comprising a ligand binding domain, a transmembrane domain and an intracellular signaling domain.

5. The engineered immune cell of claim 4, wherein the immune recognition receptor is a chimeric antigen receptor or a T cell receptor.

6. The engineered immune cell of claim 5, wherein the immune recognition receptor is a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain.

7. The immune recognition receptors include TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB 2 (Her2 / neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut5. The engineered immune cell of claim 4, which binds to a target selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, and any combination thereof.

8. The engineered immune cell of any one of claims 1 to 7, characterized in that the immune cell is selected from a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell or a NKT cell.

9. 8. The engineered immune cell of any one of claims 1 to 7, wherein the immune cell is a T cell selected from a CD4+ / CD8+ T cell, a CD4+ helper T cell, a CD8+ T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a γδ-T cell or an αβ-T cell.

10. 10. The engineered immune cell of any one of claims 1 to 9, wherein the immune cell is derived from an adult stem cell, an embryonic stem cell, an umbilical cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell or a hematopoietic stem cell.

11. A pharmaceutical composition comprising the engineered immune cells of any one of claims 1 to 10 and one or more pharma- ceutically acceptable excipients.

12. 11. Use of the engineered immune cell of any one of claims 1 to 10 in the manufacture of a medicament for the treatment of cancer, an infectious disease or an autoimmune disease.

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