Manipulated T cells with extended in vivo half-life and method for preparing them

Abstract

This invention relates to engineered T cells with an extended in vivo half-life and a method for preparing them. These engineered T cells can reduce the killing of allogeneic T cells while avoiding the killing of NK cells.

Description

[[Technical Field]]

[0001] The present application relates to modified immune cells, and particularly to immune cells modified to reduce graft-versus-host disease (GvHD), activation-induced immune effector cell death (AICD), and host-versus-graft reaction (HVG) in a host organism. [[Background Art]]

[0002] One of the biggest challenges in the development of universal CAR-T cell therapy (U-CART, also referred to as allogeneic chimeric antigen receptor T cell therapy) is the persistence of U-CART cells. This is mainly caused by three factors. First, U-CART cells may attack patient somatic cells via TCR and cause graft-versus-host disease (GvHD). Second, CAR-T cells activated by target cells exhibit activation-induced immune effector cell death (AICD, Activation Induced Cell Death), which is triggered by the co-expression of FasL (CD95L) and Fas (CD95) in activated T cells. FasL is a cytotoxic effector molecule, and Fas induces apoptosis of activated T cells via autocrine or paracrine FasL. Third, U-CART cells are attacked by the patient's T cells, causing a host-versus-graft reaction (HVG). This prevents U-CART cells from surviving long-term in the patient's body, greatly limiting their therapeutic effect. Currently, to address the persistence problem of U-CART cells, scientists generally select a method of knocking out the TCR gene of healthy donor T cells to prevent GvHD, and simultaneously intervening in the expression of B2M (which inhibits the expression of HLA-I class molecules) on the surface of U-CART cells to block recognition and attack by the patient's T cells.

[0003] However, the above strategy may lead to another type of effector cell attacking U-CART cells that do not express HLA molecules. These effector cells are natural killer (NK) cells. NK cells are effector immune cells that recognize cells with low HLA expression. Many virus-infected cells and tumor cells downregulate the expression of HLA molecules to evade recognition by T cells. U-CART cells whose expression of HLA-related molecules has been reduced by intervention may also be eliminated from the body by NK cell immunity through this mechanism.

[0004] Currently, there is no U-CART cell therapy that can simultaneously address the problems of AICD, heterologous T-cell attack, and NK cell attack. [Overview of the project]

[0005] This application provides engineered T cells with improved persistence and a method for preparing them. Because they reduce GvHD, AICD, and HVG without inducing NK cell killing, they offer improved in vivo persistence and therapeutic efficacy.

[0006] Specifically, a first aspect of this application relates to engineered T cells modified to eliminate or reduce the expression and / or function of the endogenous T cell receptor (TCR) protein and / or the SPPL3 protein. In some embodiments, the engineered T cells are γδ T cells, which are modified to eliminate or reduce the expression and / or function of the γ subunit (TCRγ) and / or TCRδ subunit (TCRδ) of the endogenous T cell receptor (TCR). In some embodiments, the engineered T cells are αβ T cells, which are modified to eliminate or reduce the expression and / or function of the α subunit (TCRα) and / or TCR β subunit (TCRβ) of the endogenous T cell receptor (TCR). In some embodiments, the modification includes modification of the SPPL3 genomic region on chromosome 12 of the engineered T cell. In some embodiments, the modification includes modification of the mRNA or pre-mRNA of the SPPL3 gene of the engineered T cell. In some embodiments, the engineered T cells are human-derived T cells, and the modification involves a nonsense mutation in the SPPL3 genomic region of chromosome 12 of the engineered T cell or in the mRNA (or premRNA) of the SPPL3 gene. In some embodiments, the engineered T cells are human-derived T cells, and the modification occurs between one or more nucleotide sites, or between any two nucleotide sites, in any one or more exon regions of the SPPL3 gene.

[0007] Preferably, the modifications are located at positions 120,903,845 to 120,904,358, 120,810,809 to 120,810,886, 120,791,469 to 120,791,557, 120,784,474 to 120,784,593, 120,783,674 to 120,783,752, and 120,782,655 to 120, on chromosome 12 of the GRCh38.p14 reference genome. It occurs at one nucleotide site or between any two nucleotide sites at positions 782,767, 120,768,953-120,769,059, 120,768,325-120,768,488, 120,767,394-120,767,593, 120,766,263-120,766,372, or 120,764,999-120,765,070. More preferably, the modification occurs at one nucleotide site, or between any two nucleotide sites, at positions 120,791,469 to 120,791,557, 120,783,674 to 120,783,752, or 120,784,474 to 120,784,593 of human chromosome 12. In some embodiments, the T cell is a human-derived T cell, and the modification includes modification of the TRAC genomic region on chromosome 14 of the engineered T cell. In some embodiments, the modification includes modification of the mRNA or pre-mRNA of the TRAC gene in the engineered T cell. In some embodiments, the T cell is a human-derived T cell, and the modification includes a nonsense mutation in the TRAC genomic region or the TRAC gene on chromosome 14 of the engineered T cell. In some embodiments, the T cells are human-derived T cells, and the modification occurs at one nucleotide site between positions 23016448 and 23016490 of chromosome 14 in the GRCh38.p14 reference genome, or between any two nucleotide sites. In some embodiments, the modification results in a frameshift mutation. In some embodiments, the modification results in a nonsense mutation.

[0008] In some embodiments, the T cells have normally expressed HMC-I class proteins. In some embodiments, the T cells have normally expressed HLA-A, HLA-B, HLA-C, and B2M. In some embodiments, the T cells are human-derived T cells, and the expression and / or function of the B2M protein within them is not reduced.

[0009] In some embodiments, the engineered T cells further comprise or express an engineered receptor. In some embodiments, the engineered receptor is one or more selected from chimeric antigen receptors (CARs), engineered TCRs, and T cell antigen couplers (TACs).

[0010] In some embodiments, the engineered T cells express a CAR, which comprises i) an extracellular antigen-binding domain that specifically recognizes one or more target antigens or antigen epitopes, ii) a transmembrane domain, and iii) an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain is one or more selected from the extracellular domain of an antigen ligand, a single-domain antibody (sdAb), a single-chain Fv (scFv), and Fab. In some embodiments, the engineered T cells contain one or more CARs, and the one or more CARs have different extracellular antigen-binding domains. In some embodiments, the CAR has one or more different extracellular antigen-binding domains. In some embodiments, the different extracellular antigen-binding domains recognize different target antigens and / or different antigen epitopes. In some embodiments, the extracellular antigen-binding domain is identified by prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA), CAM5, CD123, thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; type C lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit α (IL-13Rα); interleukin-11 receptor α (IL-11Rα); prostate-specific membrane antigen (PSMA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its variants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neuronal cell adhesion molecule (NCAM); carbonate anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe);Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; High molecular weight melanoma-associated antigen (HMWMAA); O-acetyl GD2 ganglioside (OAcGD2); Folic acid receptor; Tumor vascular endothelial marker 1 (TEM1 / CD248); Tumor vascular endothelial marker 7-related (TEM7R); Claudin6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; κ light chain (kappa light chain); CSPG4; EGP2, EGP40; FAP; FAR; FBP; Embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; Fibronectin; Tenascin; Oncoemulsified variant of tumor necrosis zone; G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexose moiety of globoH glycoceramide (GloboH); Mammary differentiation antigen (NY-BR-1); Uloplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenergic receptor β3 (ADRB3); Panexin 3 (PANX3) ); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); Sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie2); Melanoma carcinoma testicular antigen-1 (MAD-CT-1); Melanoma carcinoma testicular antigen-2 (MAD-CT-2); Fos-related antigen 1; P53 variant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Apoptosis-induced melanoma inhibitor (ML-IAP); ERG (Transmembrane protease serine 2 (TMPRSS2) ETS fusion gene);N-acetylglucosaminyltransferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelopathy virus tumor gene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 3 recognized by T cells (SART3); pairing box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin Bulin-like receptor 1 (LAIR1); IgA receptor Fc fragment (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like receptor 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like receptor 5 (FCRL5);It binds to one or more proteins selected from immunoglobulin λ-like peptide 1 (IGLL1) and CD155. In some embodiments, the transmembrane domain includes any transmembrane domain selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD6, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD47, CD52, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, CISH, and PD-1. In some embodiments, the intracellular signaling domain includes a primary intracellular signaling domain comprising any molecule selected from the group consisting of CD3ζ, CD3γ, CD3ε, CD3δ, FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, CD66d, FcγRIIa, DAP10, and DAP12. In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain comprising one or more costimulatory molecules selected from the group consisting of ligands that specifically bind to CARD11, CD2, CD4, CD7, CD19, CD27, CD28, CD30, CD40, CD160, ICAM-1, OX40, 4-1BB, SELPLG, LIGHT, HVEM, B7-H3, ICOS, PD-1, SLAMF7, LFA-1, NKG2C, CDS, GITR, BAFFR, NKp80, IPO-3, SLAMF8, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, CD83, SLAMF1, CTLA-4, LAG-3, PD-L2, PD-L1, DAP10, TRIM, ZAP70, CD83, and any combination thereof. In some embodiments, the CAR further includes a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is the hinge region of IgG, IgD, CD8α, or CD28.

[0011] In some embodiments, the manipulated T cells express a T cell antigen coupler (TAC) comprising (i) an antigen-binding domain, (ii) a TCR-binding domain (e.g., scFv), and (iii) a co-receptor domain (e.g., a hinge, transmembrane, and / or cytoplasmic region). In some embodiments, the TAC comprises (a) an extracellular ligand-binding domain containing an antigen-binding fragment (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes one or more target antigens (e.g., tumor antigens) or antigen epitopes (e.g., tumor epitopes); (b) an optional first adapter; (c) an extracellular TCR-binding domain (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes the extracellular domain of a TCR subunit (e.g., CD3ε); (d) an optional second adapter; (e) the extracellular domain or a portion thereof of an optional first TCR coreceptor (e.g., CD4, CD8); (f) a transmembrane domain containing the transmembrane domain of a second TCR coreceptor (e.g., CD4, CD8); and (g) an optional intracellular signaling domain containing the intracellular signaling domain of a third TCR coreceptor (e.g., CD4, CD8). In some embodiments, the manipulated T cells include one or more TACs, each having a different antigen-binding domain or ligand-binding domain. In some embodiments, the TACs have one or more different antigen-binding domains or ligand-binding domains. In some embodiments, the different antigen-binding domains or ligand-binding domains recognize different target antigens and / or different antigenic epitopes. In some embodiments, the antigen-binding domain or the ligand-binding domain is PSCA, CEA, CAM5, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD22, CD30, CD70, CD123, CD138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276), B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, HIV-1 Gag, MART-1, gp100, tyrosinase, mesothelin, EpCAM, PRSS21, vascular endothelial growth factor receptor,Lewis (Y) antigen, CD24, PDGFR-β, SSEA-4, MUC1, MUC6, EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII, NCAM, CAIX, LMP2, EphA2, Fucosyl GM1;sLe, Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TGS5, HMWMAA, OAcGD2, Folate receptor, CD248, TEM7R, Claudin6, Cla din18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA, CA9, κ light chain, CSPG4, EGP2, EGP40, FAP, FAR, FBP, embryonic AchR, HLA-A1, HLA-A2, MAGEA1, MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, fibronectin, tenascin, tumor necrosis zone Oncoemulsion variants, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, ETV6-AML, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, P53 variant, hTERT, sarcoma translocation breakpoint, ML-IAP, It specifically recognizes one or more target antigens selected from ERG, NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OYTES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and CD155. In some embodiments, the TCR subunit is one or more selected from the group consisting of CD3ε, CD3δ, CD3γ, TCRα, TCRβ, TCRγ, and TCRδ, and the first, second, and third TCR coreceptors are each independently one selected from the group consisting of CD4, CD8, and CD28. In some embodiments, the first, second,The first, second, and third TCR coreceptors are identical. In some embodiments, the first, second, and third TCR coreceptors are different. In some embodiments, the TAC further includes a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is the hinge region of IgG, IgD, CD8α, or CD28.

[0012] In some embodiments, the engineered T cells express an engineered T cell receptor (TCR) comprising: (a) an extracellular ligand-binding domain containing an antigen-binding fragment (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes); (b) an optional first adapter; (c) the extracellular domain or a portion thereof of an optional first TCR subunit (e.g., Cα, Cβ, Cδ, Cγ, CD3ε); (d) a transmembrane domain of a second TCR subunit (e.g., TCRα, TCRβ); and (e) an intracellular signaling domain comprising an intracellular signaling domain of a third TCR subunit (e.g., TCRα, TCRβ). Here, the first, second, and third TCR subunits are independently selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ, and CD3ζ. In some embodiments, the first, second, and third TCR subunits are identical (e.g., all CD3ε, all TCRα, or all TCRβ). In some embodiments, the first, second, and third TCR subunits are different. In some embodiments, the engineered T cell comprises one or more TCRs having different extracellular ligand-binding domains. In some embodiments, the TCRs have one or more different extracellular ligand-binding domains. In some embodiments, the different extracellular ligand-binding domains recognize different target antigens and / or different antigenic epitopes. In some embodiments, the antigen-binding domain or the ligand-binding domain may be PSCA, CEA, CAM5, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD22, CD30, CD70, CD123, CD138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276), B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, or HIV-1.Gag, MART-1, gp100, tyrosinase, mesothelin, EpCAM, PRSS21, vascular endothelial growth factor receptor, Lewis (Y) antigen, CD24, PDGFR-β, SSEA-4, MUC1, MUC6, EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII, NCAM, CAIX, LMP2, EphA2, fucosyl GM1;sLe, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TGS5, HMWM AA, OAcGD2, folate receptor, CD248, TEM7R, Claudin6, Claudin18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA, CA9, κ light chain, CSPG4, EGP2, EGP40, FAP, FAR, FBP, embryonic AchR, HLA-A1, HLA-A2, MAGEA1, MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, fibronectin, tenascin, oncoemulsion variants in tumor necrosis zone, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, ETV6-AML, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, P53 variant, hTERT, sarcoma translocation variant The TCR specifically recognizes one or more target antigens selected from the following: marker, ML-IAP, ERG, NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OYTES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and CD155. In some embodiments, the engineered TCR further includes a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α.

[0013] A second aspect of this application also provides a method for preparing the engineered T cells described in the first aspect, wherein the modification includes one or more selected from disruption or knockout of a coding gene, inhibition of transcription of a coding gene, disruption or clearance of mRNA, inhibition of expression of a coding gene, and inhibition of the protein. In some embodiments, the modification is achieved by removing or reducing the expression of TCR or its functional fragment and SPPL3 or its functional fragment by RNA interference (RNAi). In some embodiments, the RNAi is achieved by silencing or inhibiting the expression of TCR or its functional fragment and SPPL3 or its functional fragment by small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA). In some embodiments, the modification is achieved by physical mutagenesis, chemical mutagenesis, or translocation. In some embodiments, the modification is achieved by a PCR method. In some embodiments, the modification is mediated by one or more selected from non-homologous end joining (NHEJ), homologous recombination repair (HDR), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR / Cas, and adenosine deaminase (ADAR). In some embodiments, the modification is gene editing, including, for example, base editing, PRIME editing, or restriction enzyme digestion at a target site. In some embodiments, the modification includes one or more of insertions, deletions, and substitutions. In some embodiments, the modification includes one or more of translocations, point mutations, fragment deletions, and fragment additions. In some embodiments, the modification results in one or more of frameshift mutations, loss-of-function mutations, dominant-negative mutations, missense mutations, and nonsense mutations. In some embodiments, the modification includes gene editing or RNA editing mediated by the CRISPR / Cas system, wherein the gene editing or RNA editing uses a guide RNA (gRNA) that targets SPPL3 and / or TRAC.In some embodiments, the SPPL3-targeting gRNA includes a guide sequence complementary to the SPPL3 genomic region of human chromosome 12 (e.g., any exon region, any intron region, any splice site) or the mRNA or pre-mRNA sequence corresponding to the said genomic region. Preferably, the SPPL3-targeting gRNA includes positions 120,903,845 to 120,904,358, 120,810,809 to 120,810,886, 120,791,469 to 120,791,557, 120,784,474 to 120,784,593, 120,783,674 to 120,783,752, 120,782,655 to 120,782,767, and 12 The SPPL3 genomic region includes positions 0,768,953 to 120,769,059, 120,768,325 to 120,768,488, 120,767,394 to 120,767,593, 120,766,263 to 120,766,372, or 120,764,999 to 120,765,070, or a guide sequence complementary to the mRNA or pre-mRNA sequence corresponding to the said genomic region. More preferably, the gRNA targeting SPPL3 includes a guide sequence complementary to the SPPL3 genomic region at positions 120,791,469–120,791,557, 120,783,674–120,783,752, or 120,784,474–120,784,593 of human chromosome 12, or to the mRNA or pre-mRNA sequence corresponding to the said genomic region. In some embodiments, the gRNA targeting TRAC includes a guide sequence complementary to the TRAC genomic region at positions 23016448–23016490 of human chromosome 14, or to the mRNA or pre-mRNA sequence corresponding to the said genomic region. In some embodiments, the CRISPR / Cas system is a type II CRISPR / Cas system, and the gRNA does not include a tracrRNA or tracr chaperone sequence in addition to the guide sequence. In some embodiments, the CRISPR / Cas system is a type II CRISPR / Cas system, and the gRNA is a single guide RNA (sgRNA).In some embodiments, the CRISPR / Cas system is a CRISPR / Cas9 system or a CRISPR / Cas12 system. In some embodiments, the guide sequences complementary to the SPPL3 genomic region are SEQ ID NOs.68, SEQ ID NOs.145, SEQ ID NOs.150, SEQ ID NOs.154, SEQ ID NOs.155, SEQ ID NOs.159, SEQ ID NOs.161, SEQ ID NOs.165, SEQ ID NOs.172, SEQ ID NOs.176, SEQ ID NOs.196, SEQ ID NOs.203, SEQ ID NOs.204, SEQ ID NOs.209, SEQ ID NOs.212, SEQ ID NOs.241, SEQ ID NOs.250, SEQ ID NOs.261, SEQ ID NOs.269, SEQ ID NOs.270, SEQ ID NOs.271, SEQ ID NOs.272, SEQ ID NOs.273, SEQ ID NOs.27 4. One or more selected from SEQ ID NOs: 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 297, 300, 305, 312, and 315.

[0014] In some embodiments, the guide sequences complementary to the SPPL3 genomic region are SEQ ID NOs: 150, 154, 155, 159, 161, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, and 294. In some embodiments, the guide sequence complementary to the SPPL3 genomic region is SEQ ID NOs: 155 or 261. In some embodiments, the guide sequences complementary to the TRAC genomic region are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, and / or SEQ ID NO: 260. In some embodiments, the gRNA is chemically modified. In some embodiments, the chemical modification includes 2'-O-methylation modification of the ribose of nucleotides, or 3' thiophosphate ester bond modification between nucleotides, or both. In some embodiments, the modification includes 2'-O-methylation modification of the ribose of the first three nucleotides at the 5' end, 2'-O-methylation modification of the ribose of the last three nucleotides at the 3' end, 3' thiophosphorylation modification between the first three nucleotides at the 5' end, and 3' thiophosphorylation modification between the last three nucleotides at the 3' end.

[0015] A third aspect of this application further provides a method for extending the in vivo half-life of engineered cells, the method comprising modifying the engineered cells to remove or reduce the expression and / or function of the SPPL3 protein or a functional fragment thereof. In some embodiments, the modification includes one or more insertions, deletions, and substitutions. In some embodiments, the modification includes one or more translocations, point mutations, fragment deletions, and fragment additions. In some embodiments, the modification results in one or more frameshift mutations, loss-of-function mutations, dominant-negative mutations, missense mutations, and nonsense mutations. In some embodiments, the modification is located at the SPPL3 genomic locus. In some embodiments, the modification occurs in mRNA transcribed from the SPPL3 gene. In some embodiments, the modification occurs in an exon. In some embodiments, the modification occurs in an intron. In some embodiments, the modification does not alter the genomic locus of SPPL3 but downregulates mRNA by interfering with the transcription of SPPL3. In some embodiments, the modification does not interfere with the genomic locus or mRNA level of SPPL3, but interferes with the translation from mRNA to SPPL3 protein, thereby resulting in downregulation of SPPL3 protein levels. In some embodiments, the modification causes mutations in the SPPL3 genomic region of human chromosome 12, or in the mRNA or premRNA sequence corresponding to that genomic region. In some embodiments, the modification causes nonsense mutations in the SPPL3 genomic region of human chromosome 12, or in the mRNA or premRNA sequence corresponding to that genomic region. In some embodiments, the engineered cells have or express normal levels of MHC-I protein. In some embodiments, the engineered cells have normally expressed HLA-A, HLA-B, HLA-C, and B2M. In some embodiments, normal levels of MHC-I protein do not cause killing of the engineered cells by NK cells. In some embodiments, the engineered cells are immune cells or their precursor cells.In some embodiments, the immune cells are one or more selected from T cells, B cells, natural killer (NK) cells, macrophages, and DC cells. In some embodiments, the immune cells are αβT cells, in which the expression and / or function of endogenous TCRα and / or TCRβ, or functional fragments thereof, are removed or reduced. In some embodiments, the engineered T cells are γδT cells, modified to remove or reduce the expression and / or function of their endogenous TCRγ and / or TCRδ. In some embodiments, the engineered cells further comprise or express an engineered receptor. In some embodiments, the engineered receptor is selected from one or more chimeric antigen receptors (CARs), engineered TCRs, and T cell antigen couplers (TACs).

[0016] A fourth aspect of this application also provides a guide RNA (gRNA) that targets SPPL3, which includes sequence numbers 68, 145, 150, 154, 155, 159, 161, 165, 172, 176, 196, 203, 204, 209, 212, 241, 250, 261, 269, 270, 271, 272, 273, and sequence number It contains one guide sequence selected from sequence number 274, sequence number 275, sequence number 276, sequence number 277, sequence number 278, sequence number 279, sequence number 280, sequence number 281, sequence number 282, sequence number 283, sequence number 284, sequence number 285, sequence number 286, sequence number 287, sequence number 288, sequence number 289, sequence number 290, sequence number 291, sequence number 292, sequence number 293, sequence number 294, sequence number 295, sequence number 297, sequence number 300, sequence number 305, sequence number 312, and sequence number 315. Preferably, it includes one guide sequence selected from SEQ ID NOs: 150, 154, 155, 159, 161, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, and 294.

[0017] In some embodiments, the gRNA is involved in the formation of a type II CRISPR / Cas complex. In some embodiments, the gRNA is involved in the formation of a CRISPR / Cas complex, and the Cas enzyme is Cas9 or Cas12. In some embodiments, the CRISPR / Cas system is a type II CRISPR / Cas system, and the gRNA does not contain a tracrRNA or tracr chaperone sequence other than the guide sequence. In some embodiments, the CRISPR / Cas system is a type II CRISPR / Cas system, and the gRNA is sgRNA. In some embodiments, the gRNA is chemically modified. In some embodiments, the chemical modification includes 2'-O-methylation modification of the ribose of a nucleotide, or 3' thiophosphate ester bond modification between nucleotides, or both. In some embodiments, the modifications include 2'-O-methylation of the ribose of the first three nucleotides at the 5' end, 2'-O-methylation of the ribose of the last three nucleotides at the 3' end, 3' thiophosphorylation between the first three nucleotides at the 5' end, and 3' thiophosphorylation between the last three nucleotides at the 3' end.

[0018] A fifth aspect of this application provides a nucleic acid composition comprising the gRNA described in the fourth aspect and a gRNA that targets a T cell receptor gene. In some embodiments, the T cell receptor gene is the TRAC gene. In some embodiments, the gRNA that targets the T cell receptor gene includes a guide sequence complementary to the TRAC genomic region at positions 23016448 to 23016490 of chromosome 14. In some embodiments, the guide sequence includes a polynucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, or SEQ ID NO: 260.

[0019] In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which is sequence number 68, sequence number 145, sequence number 150, sequence number 154, sequence number 155, sequence number 159, sequence number 161, sequence number 165, sequence number 172, sequence number 176, sequence number 196, sequence number 203, sequence number 204, sequence number 209, sequence number 212, sequence number 241, sequence number 250, sequence number 261, sequence number 269, sequence number 270, sequence number 271, sequence number 272, sequence number 273, sequence number 274, sequence number 275, sequence number 276, sequence number 277, sequence number 278, sequence number 279, sequence number 280, sequence number 281, sequence number 282, sequence number 283, sequence number 284, sequence number 285, sequence number 286, sequence number 287, sequence number 288, sequence number 289, sequence number 290, sequence number 291, sequence number 292, sequence number 29 3. The system includes a guide sequence shown in any one of SEQ ID NOs: 294, 295, 297, 300, 305, 312, and 315, preferably including a guide sequence shown in any one of SEQ ID NOs: 150, 154, 155, 159, 161, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, and 294, with the other targeting the gRNA of the T cell receptor gene. In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which comprises a guide sequence shown in SEQ ID NO: 155 or SEQ ID NO: 261, and the other which targets a gRNA of a T cell receptor gene.In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which comprises a guide sequence shown in SEQ ID NO: 155 or SEQ ID NO: 261, and the other comprising a guide sequence complementary to the TRAC genomic region at positions 23016448 to 23016490 of chromosome 14. In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which comprises a guide sequence shown in SEQ ID NO: 155 or SEQ ID NO: 261, and the other comprising a guide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 257. [Brief explanation of the drawing]

[0020] [Figure 1A] Figures 1A and 1B show the proliferation and viability of U-CART constructed using the CRISPR / Cas9 system from day 0 to day 9 before purification, respectively. sgRNA1-sgRNA17-SP3-3 represent double knockout of TRAC and SPLL3 by combining SEQ ID NO: 1, which targets TRAC, with SEQ ID NOs: 68, 145, 150, 154, 155, 159, 161, 165, 172, 176, 196, 203, 204, 209, 212, 241, and 250, which target SPLL3, respectively. [Figure 1B] Figures 1A and 1B show the proliferation and viability of U-CART constructed using the CRISPR / Cas9 system from day 0 to day 9 before purification, respectively. sgRNA1-sgRNA17-SP3-3 represent double knockout of TRAC and SPLL3 by combining SEQ ID NO: 1, which targets TRAC, with SEQ ID NOs: 68, 145, 150, 154, 155, 159, 161, 165, 172, 176, 196, 203, 204, 209, 212, 241, and 250, which target SPLL3, respectively. [Figure 2A]Figure 2A is a diagram showing the TCR gene knockout efficiency of U-CART constructed using the CRISPR / Cas9 system on day 9 before purification. [Figure 2B] Figure 2B is a diagram showing the SPPL3 gene knockout efficiency of U-CART constructed using the CRISPR / Cas9 system on day 9 before purification. [Figure 2C] Figure 2C is a diagram showing the percentage of CAR+ cells (CAR-positive cells) in U-CART constructed using the CRISPR / Cas9 system on day 9 before purification. [Figure 2D] Figure 2D is a diagram showing the viability of U-CART cells constructed using the CRISPR / Cas9 system on day 9 before purification. [Figure 2E] Figure 2E is a diagram showing the cell number of U-CART cells constructed using the CRISPR / Cas9 system on day 9 before purification. [Figure 3A] Figures 3A to 3B respectively show the proliferation and viability of U-CART constructed using the CRISPR / Cas12 system from day 0 to day 9 before purification. UCART-TCR-sg1, UCART-TCR-sg2, UCART-TCR-sg3, and UCART-TCR-sg4 respectively represent SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, and SEQ ID NO: 260, which are sequences targeting TRAC. SPPL3-sg1, SPPL3-sg2, SPPL3-sg3, and SPPL3-sg4 respectively represent SEQ ID NO: 290, SEQ ID NO: 269, SEQ ID NO: 261, and SEQ ID NO: 270, which are sequences targeting SPPL3. These sequences are combined to achieve double knockout of TRAC and SPPL3. [Figure 3B]Figures 3A to 3B respectively show the proliferation and viability rate of U-CART constructed using the CRISPR / Cas12 system from day 0 to day 9 before purification. UCART-TCR-sg1, UCART-TCR-sg2, UCART-TCR-sg3, and UCART-TCR-sg4 respectively represent SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, and SEQ ID NO: 260, which are sequences targeting TRAC. SPPL3-sg1, SPPL3-sg2, SPPL3-sg3, and SPPL3-sg4 respectively represent SEQ ID NO: 290, SEQ ID NO: 269, SEQ ID NO: 261, and SEQ ID NO: 270, which are sequences targeting SPPL3. These sequences are combined to achieve double knockout of TRAC and SPPL3. [Figure 4A] Figure 4A is a diagram showing the TCR gene knockout efficiency of U-CART constructed using the CRISPR / Cas12 system on day 9 before purification. [Figure 4B] Figure 4B is a diagram showing the SPPL3 gene knockout efficiency of U-CART constructed using the CRISPR / Cas12 system on day 9 before purification. [Figure 4C] Figure 4C is a diagram showing the proportion of CAR+ cells in U-CART constructed using the CRISPR / Cas12 system on day 9 before purification. [Figure 4D] Figure 4D is a diagram showing the viability rate of U-CART cells constructed using the CRISPR / Cas12 system on day 9 before purification. [Figure 4E] Figure 4E is a diagram showing the number of U-CART cells constructed using the CRISPR / Cas12 system on day 9 before purification. [Figure 5A] Figure 5A is a diagram showing changes in TCR knockout efficiency of U-CART constructed using the CRISPR / Cas9 system before and after purification. [Figure 5B] Figure 5B is a diagram showing changes in the viability rate of U-CART constructed using the CRISPR / Cas9 system before and after purification. [Figure 6A]Figures 6A and 6B show the proliferation rate (2A) and viability (2B) of U-CART constructed using the CRISPR / Cas9 system compared with CAR-T cells and T cells. [Figure 6B] Figures 6A and 6B show the proliferation rate (2A) and viability (2B) of U-CART constructed using the CRISPR / Cas9 system compared with CAR-T cells and T cells. [Figure 6C] Figure 6C shows the change in long-term survival rate of U-CART constructed using the CRISPR / Cas9 system. [Figure 7A] Figure 7A shows the change in TCR knockout efficiency before and after purification of U-CART constructed using the CRISPR / Cas12 system. [Figure 7B] Figure 7B shows the change in viability of U-CART before and after purification using the CRISPR / Cas12 system. [Figure 8A] Figures 8A and 8B show the proliferation rate (2A) and viability (2B) of U-CART cells constructed using the CRISPR / Cas12 system compared with CAR-T cells and T cells. [Figure 8B] Figures 8A and 8B show the proliferation rate (2A) and viability (2B) of U-CART cells constructed using the CRISPR / Cas12 system compared with CAR-T cells and T cells. [Figure 8C] Figure 8C shows the change in long-term survival rate of U-CART constructed using the CRISPR / Cas12 system. [Figure 9] Figure 9 shows the resistance of two donor-derived T cells to allogeneic T cell killing after different modifications. [Figure 10] Figure 10 shows the killing effect of NK cells on T cells derived from the same donor after different modifications. [Figure 11A] Figures 11A and 11B show the killing effect of PBMC cells on T cells after they have been modified by different modifications. [Figure 11B]Figures 11A and 11B show the killing effect of PBMC cells on T cells after they have been modified by different modifications. [Figure 12] Figure 12 shows the FasL-mediated killing effect on T cells after different modifications. [Figure 13] Figure 13 shows the in vitro killing effect of T cells with different modifications on target cells. Here, U-CART cells were constructed using the CRISPR / Cas9 system. [Figure 14] Figure 14 shows the in vitro killing effect of T cells after different modifications on target cells. Here, U-CART cells were constructed using the CRISPR / Cas12 system. [Figure 15] Figure 15 shows the proliferation of CAR+ cells when differently modified T cells were co-incubated with target cells in vitro. [Figure 16] Figure 16 shows the in vivo imaging results in mice. [Figure 17] Figure 17 shows the killing effects of T cells after different modifications on target cells in immunodeficient mice. [Modes for carrying out the invention]

[0021] The inventors of this application provide an original method for reducing the HVG effect on cells engineered with allogeneic T cells, distinct from methods for knocking out MHC-I or inhibiting MHC-I expression and / or function. This method reduces HVG without enhancing the NK cell-killing effect on the engineered cells. Specifically, this application also provides engineered T cells modified by this method, a method for preparing such engineered T cells, and gRNAs used in this method and combinations thereof.

[0022] definition This application is described using specific embodiments with reference to specific drawings, but is not limited thereto. No reference symbol in the claims should be construed as limiting the scope. In the drawings, the sizes of some elements may be exaggerated for illustrative purposes and may not be drawn to scale. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of any inconsistency, the provisions of this specification (including definitions) shall prevail. Methods and materials similar to or equivalent to those described herein may be used in carrying out or measuring the present invention, but preferred methods and materials are described below. All publications, patent applications, patents and other references referred to herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to limit the scope.

[0023] In this specification, "SPPL3" refers to signal peptide peptidase-like protein 3 (also known as IMP2, PSH1, or PSL4), a multi-pass transmembrane protein present in the Golgi-associated vesicle membrane, cell membrane, and rough endoplasmic reticulum. SPPL3 possesses aspartate endopeptidase activity, intramembrane cleavage, and protein homodimer activity. Many of its substrates are located in the Golgi apparatus and are involved in the modification of N- and O-linked glycans and glycosaminoglycan biosynthesis. SPPL3 is essential for the cleavage and extracellular release of the luminal domains of glycosyltransferases and glycosidases. Depletion of glycan-modifying enzymes can impair their activity in the Golgi apparatus. Increased SPPL3 expression is associated with low glycosylation of many secretory and membrane proteins, while decreased SPPL3 expression is associated with high glycosylation of proteins. Researchers have observed that SPPL3 deficiency leads to increased expression of neolactose sphingoglycolipid (GSL) on the cell surface. This inhibits the interaction between antibodies and receptors and HLA class I (HLA-I) glycoproteins, reducing CD8+ T cell activation. SPPL3 also has non-protein hydrolytic functions, such as interacting with matrix interaction molecule 1 (STIM1) and Orai1 to enhance TCR signaling and significantly inducing calcium influx and NFAT activation, which are important for lymphocyte signaling. An example of SPPL3 is human SPPL3, such as SPPL3 registered as gene ID 121665 in the NCBI database. In some embodiments, the human SPPL3 gene sequence is shown at positions 120762510 to 120904358 of NC_000012.12, i.e., positions 120762510 to 120904358 of chromosome 12 in the GRCh38.p14 reference genome.

[0024] The “CRISPR system” or “CRISPR / Cas system” is a collective term for transcripts and other elements involved in guiding the expression and / or activity of CRISPR-related ("Cas") genes. For example, the CRISPR / Cas system may include sequences encoding Cas genes, tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr chaperone sequences (e.g., in the endogenous CRISPR system, including “direct repeats” and partially direct repeat sequences processed by tracrRNA), guide sequences (also called “spacers” in the endogenous CRISPR system), and other sequences and transcripts derived from the CRISPR locus.

[0025] In CRISPR complex formation, the "target sequence" refers to a sequence designed to be complementary to the guide sequence, and hybridization between the target sequence and the guide sequence promotes CRISPR complex formation. Complete complementarity is not necessarily required, as long as sufficient complementarity exists to induce hybridization and promote CRISPR complex formation. The target sequence can include any polynucleotide, such as DNA polynucleotides or RNA polynucleotides. The CRISPR complex may include guide sequences that hybridize with the target sequence and form a complex with one or more Cas proteins.

[0026] The term "guide sequence" refers to a sequence of nucleotides in a guide RNA that has partial or complete complementarity to the target sequence of the target polynucleotide and can hybridize to the target sequence through base pairing facilitated by the Cas protein. In the CRISPR / Cas9 system, the target sequence is adjacent to the PAM site. The PAM sequence and its complementary sequence on the other strand together constitute the PAM site.

[0027] The term “guide RNA” is used herein interchangeably with gRNA and refers to, but is not limited to, a nucleic acid-based molecule that contains a sequence (e.g., a guide sequence or spacer) that is sufficiently complementary to the target sequence to hybridize to the target sequence and induce specific binding of the Cas protein-RNA complex to the target sequence, and that can form a protein-RNA complex with the Cas protein and is capable of hybridizing to the target sequence. In some embodiments, gRNA contains or is crRNA. In some embodiments, gRNA contains two RNA strands, and the spacer sequence and direct repeat (DR) sequence are located on different RNA strands, e.g., a crRNA strand and a tracrRNA strand. In some embodiments, gRNA is a single RNA strand, e.g., sgRNA.

[0028] The terms “single guide RNA,” “synthetic guide RNA,” and “sgRNA” are used interchangeably and refer to a polynucleotide sequence comprising a guide sequence and any other sequences necessary for the function of the sgRNA and / or necessary for the sgRNA to interact with one or more Cas proteins to form a CRISPR complex. In some embodiments, the sgRNA comprises a guide sequence fused to a second sequence comprising a tracr sequence derived from tracrRNA and a tracr chaperone sequence derived from crRNA. The tracr sequence may comprise all or part of the sequence of tracrRNA derived from the naturally occurring CRISPR / Cas system. The term “guide sequence” is a nucleotide sequence that identifies a target site in the guide RNA and is used interchangeably with the terms “guide” or “spacer.” The term “tracr chaperone sequence” is also used interchangeably with the term “direct repeat sequence.” iBAR "iBAR" refers to a single guide RNA that has an iBAR sequence.

[0029] In this specification, “Universal CAR-T” or “U-CART” cells refer to CAR-T (or CART) cells in which the GvHD and / or HVG effect is reduced compared to allogeneic individuals. This reduction can be achieved, for example, by removing or reducing the expression and / or function of TCR proteins. In some embodiments, the U-CART is a type of engineered T cell provided in this application. “CAR-T” or “CART” cells refer to T cells that express and / or contain a chimeric antigen receptor (CAR) on their cell membrane.

[0030] As used herein, the term “wild type” is a term understood by those skilled in the art and means a typical form of an organism, strain, gene, or characteristic found in nature, distinct from a variant or offshoot.

[0031] In this specification, the term "variant" should be understood as referring to a characteristic that deviates from patterns found in nature.

[0032] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either through conventional Watson-Crick base pairing or other unconventional methods. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid sequence (for example, about 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all consecutive residues in a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. As used herein, “substantially complementary” means a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or that two nucleic acids hybridize under stringent conditions.

[0033] As used herein, “stringent conditions” for hybridization refer to conditions under which nucleic acids complementary to the target sequence primarily hybridize with the target sequence and substantially do not hybridize with non-target sequences. Stringent conditions are generally sequence-dependent and vary by many factors. Generally, the longer the sequence, the higher the temperature at which it specifically hybridizes with its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology - Hybridization With Nucleic Acid Probes Part I, Second Chapter “Principles of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N, Y.

[0034] Hybridization refers to a reaction in which one or more polynucleotides form a stable complex through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogstein bonding, or other sequence-specific mechanisms. The complex can include double helical structures (double strands), multi-stranded complexes (three or more strands), self-hybridizing single strands, or any combination thereof. Hybridization reactions may constitute a step in a broader process, such as the initiation of PCR or enzymatic cleavage of polynucleotides. A sequence that can hybridize with a given sequence is called its "complementary sequence."

[0035] As used herein, “construction” refers to a nucleic acid molecule (e.g., DNA or RNA) or a vector capable of delivering such nucleic acid molecules. For example, when used in the context of gRNA or sgRNA, construction refers to a gRNA or sgRNA molecule, a nucleic acid molecule encoding gRNA or sgRNA (e.g., isolated DNA or a viral vector), or a vector capable of delivering a nucleic acid molecule encoding gRNA or sgRNA (e.g., a lentivirus carrying a nucleic acid molecule encoding gRNA or sgRNA). When used in the context of proteins, “construction” refers to a nucleic acid molecule containing a nucleotide sequence that can be transcribed into RNA or expressed as a protein. Construction may include essential regulatory elements operably ligated to the nucleotide sequence, which enable transcription or expression of the nucleotide sequence when the construction is present in a host cell. As used herein, “operably ligated” means that gene expression is under the control of a spatially ligated regulatory element (e.g., a promoter). The regulatory element may be located at the 5' end (upstream) or 3' end (downstream) of the gene under its control. The distance between a regulatory element (e.g., a promoter) and a gene may be approximately the same as the distance between the regulatory element (e.g., a promoter) and the gene it naturally controls, and the regulatory element originates from that gene. As is known in the art, changes in this distance can be adapted without impairing the function of the regulatory element (e.g., a promoter). The term “vector” is used to refer to a nucleic acid molecule that can be manipulated to contain one or more cloned polynucleotides that can be amplified in a host cell. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules with or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other polynucleotide species known in the art. A type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted by standard molecular cloning techniques, etc.Some vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication or free mammalian vectors). Other vectors (e.g., non-free mammalian vectors), after being introduced into a host cell, are integrated into the host cell's genome and replicate together with the host genome. Furthermore, some vectors can guide the expression of genes to which they are operably ligated. Such vectors are referred to herein as “expression vectors.” Recombinant expression vectors may contain the nucleic acids of this application in a form suitable for expression in a host cell. This means that a recombinant expression vector contains one or more regulatory elements, which can be selected based on the host cell, operably ligated for expression, i.e., to the nucleic acid sequence to be expressed.

[0036] "Host cell" refers to a cell that can, or is already, be a vector or receptor for a vector or isolated polynucleotide. The host cell may be a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a eukaryotic cell that is cultured in vitro and can be modified using the methods described herein. The term "cell" includes primary test cells and their progeny.

[0037] As used herein, the term “self” means any material that originates from the same individual and is subsequently reintroduced into that individual.

[0038] "Allogeneic" refers to a graft from a different individual of the same species. "Allogeneic T cells" refer to donor-derived T cells that match the recipient's tissue human leukocyte antigen (HLA) type. Matching is usually based on variability at three or more loci of the HLA gene, with perfect matching at these loci being preferred. In some cases, allogeneic transplant donors may be related (usually siblings with closely matching HLA types), genetically identical (identical twins of the patient), or unrelated (donors who are not related but are found to have a very high degree of HLA matching). HLA genes are classified into two categories: type I and type II. Generally, mismatch in type I genes (i.e., HLA-A, HLA-B, or HLA-C) increases the risk of transplant rejection. Mismatch in HLA type II genes (i.e., HLA-DR or HLA-DQB1) increases the risk of graft-versus-host disease (GvHD).

[0039] In this specification, the terms “donor subject” or “donor” refer to the subject from whom cells are collected for further in vitro manipulation. A donor subject may be a patient to be treated with the cell population produced by the method herein (i.e., an autologous donor), or an individual to provide a blood sample (e.g., a lymphocyte sample) to be used to treat another individual or patient with the cell population produced by the method herein (i.e., an allogeneic donor). A subject to be administered modified cells (e.g., modified immune cells described herein or prepared by the method herein) may be referred to as a “recipient” or “recipient subject.”

[0040] As used herein, the term "phenotype" of a cell refers to an observable characteristic or trait, such as cellular morphology, development (e.g., growth, proliferation, differentiation, or death), homeostasis, biochemical or physiological properties, phenomenology, or behavior. Phenotype may result from gene expression in a cell, the influence of environmental factors, or interactions between these two. In some embodiments, the phenotype is growth, differentiation, and / or maturation. In some embodiments, the phenotype is inhibition of growth or proliferation. In some embodiments, the phenotype is persistence in vivo. In some embodiments, the phenotype is death. In some embodiments, the phenotype is effector function of immune cells (e.g., cytokine release and / or cytotoxic killing) or a decrease or absence of effector function.

[0041] As used herein, the term “stimulation” refers to a primary response induced by the binding of cell surface regions. For example, in the context of receptors, such stimulation requires receptor binding and a subsequent signaling event. In the case of T cell stimulation, this stimulation, in one embodiment, refers to the binding of T cell surface regions, which then induces a signaling event, such as binding to the TCR / CD3 complex. Furthermore, the stimulation event can activate the cell and upregulate or downregulate the expression or secretion of molecules, such as the downregulation of TGF-β. Thus, even in the absence of a direct signaling event, the binding of cell surface regions may lead to the rearrangement of the cytoskeleton or the aggregation of cell surface regions, which can be utilized to enhance, modify, or alter subsequent cellular responses, respectively.

[0042] As used herein, the term “activation” refers to the state of a cell after sufficient ligation of its cell surface portion to induce significant biochemical or morphological changes. In the context of T cells, such activation refers to the state of a T cell that has been stimulated sufficiently to induce cell proliferation. T cell activation may also induce cytokine production, regulatory function, or cytolytic effector function. In the context of other cells, the term means the upregulation or downregulation of a particular physicochemical process. The term “activated T cell” refers to a T cell that is currently undergoing cell division, cytokine production, regulatory function, or cytolytic effector function, and / or has recently undergone the “activation” process.

[0043] The “isolated” nucleic acid molecules described herein are typically nucleic acid molecules identified and isolated from at least one contaminating nucleic acid molecule related to the environment in which they were produced. Preferably, the isolated nucleic acid does not bind to any components related to the production environment. The isolated nucleic acid molecules encoding the polypeptides described herein may be in a form different from that found in nature or in the environment. Therefore, the isolated nucleic acid molecules are different from the nucleic acids encoding the polypeptides described herein that are naturally present in cells.

[0044] Unless otherwise specified, the term "nucleotide sequence that codes for an amino acid sequence" includes all nucleotide sequences that exist in a degenerate form and code for the same amino acid sequence. The phrase "nucleotide sequence that codes for a protein or RNA" may include introns to the extent that a nucleotide sequence that codes for a protein may contain one or more introns in a particular version.

[0045] In this specification, the terms “transfection,” “transformation,” or “transduction” refer to the process of introducing or transferring a foreign nucleic acid into a host cell (e.g., an immune cell). A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with a foreign nucleic acid. These cells include primary test cells and their progeny.

[0046] In this specification, the term "CAR," i.e., chimeric antigen receptor, comprises i) an extracellular antigen-binding domain that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes), ii) a transmembrane domain, and iii) an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain is one or more selected from the group consisting of the extracellular domain of a ligand, a single-domain antibody (sdAb), a single-chain Fv (scFv), and a Fab. In some embodiments, the transmembrane domain is derived from any molecule selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD6, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD47, CD52, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, CISH, and PD-1. In some embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the intracellular signaling domain includes a primary intracellular signaling domain derived from any one molecule selected from the group consisting of CD3ζ, CD3γ, CD3ε, CD3δ, FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, CD66d, FcγRIIa, DAP10, and DAP12. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ.In some embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain derived from one or more co-stimulatory molecules selected from the group consisting of ligands that specifically bind to CARD11, CD2, CD4, CD7, CD19, CD27, CD28, CD30, CD40, CD160, ICAM-1, OX40, 4-1BB, SELPLG, LIGHT, HVEM, B7-H3, ICOS, PD-1, SLAMF7, LFA-1, NKG2C, CDS, GITR, BAFFR, NKp80, IPO-3, SLAMF8, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, CD83, SLAMF1, CTLA-4, LAG-3, PD-L2, PD-L1, DAP10, TRIM, ZAP70, CD83, and any combination thereof. In some embodiments, the co-stimulatory signaling domain is derived from 4-1BB. In some embodiments, the CAR further includes a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α or CD28.

[0047] In this specification, an engineered "TCR" is an engineered T cell receptor that expresses an engineered T cell receptor (TCR) comprising: (a) an extracellular ligand-binding domain containing an antigen-binding fragment (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes); (b) an optional first adapter; (c) the extracellular domain or a portion thereof of an optional first TCR subunit (e.g., Cα, Cβ, Cδ, Cγ, CD3ε); (d) a transmembrane domain of a second TCR subunit (e.g., TCRα, TCRβ); and (e) an intracellular signaling domain including an intracellular signaling domain of a third TCR subunit (e.g., TCRα, TCRβ). Here, the first, second, and third TCR subunits are independently selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ, and CD3ζ. In some embodiments, the first, second, and third TCR subunits are identical (e.g., all CD3ε, all TCRα, or all TCRβ). In some embodiments, the first, second, and third TCR subunits are different. In some embodiments, the manipulated TCR further includes a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α.

[0048] In this specification, “TAC” refers to a T cell antigen coupler comprising (i) an antigen-binding domain, (ii) a TCR-binding domain (e.g., scFv), and (iii) a co-receptor domain (e.g., hinge, transmembrane, and / or cytoplasmic region). See, for example, Helsen et al. Nat Commun. 2018;9(1):3049. In some embodiments, the TAC comprises: (a) an extracellular ligand-binding domain containing an antigen-binding fragment (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes one or more target antigens (e.g., tumor antigens) or antigen epitopes (e.g., tumor epitopes); (b) an optional first adapter; (c) an extracellular TCR-binding domain (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes the extracellular domain of a TCR subunit (e.g., CD3ε); (d) an optional second adapter; and (e) an optional first TCR coreceptor (e.g., CD4, CD8). (f) an extracellular domain or a portion thereof; a transmembrane domain including the transmembrane domain of a second TCR co-receptor (e.g., CD4, CD8); and (g) an optional intracellular signaling domain including the intracellular signaling domain of a third TCR co-receptor (e.g., CD4, CD8), wherein the TCR subunit is one or more selected from the group consisting of CD3ε, CD3δ, CD3γ, TCRα, TCRβ, TCRγ, and TCRδ, and the first, second, and third TCR co-receptors are each independently one selected from the group consisting of CD4, CD8, and CD28. In some embodiments, the first, second, and third TCR co-receptors are identical. In some embodiments, the first, second, and third TCR co-receptors are different. In some embodiments, the TAC further includes a hinge domain (e.g., derived from CD8α) located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain.

[0049] In this specification, “treatment” means a method for obtaining beneficial or desirable outcomes, including clinical outcomes. For the purposes of this application, beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by the disease; reduction of the severity of the disease; stabilization of the disease (e.g., prevention or delay of disease exacerbation); prevention or delay of disease progression (e.g., metastasis); prevention or delay of disease recurrence; delay or slowing of disease progression; improvement of the disease state; achievement of remission (partial or complete); reduction of the dosage of one or more other drugs required to treat the disease; delay of disease progression; improvement of quality of life; and / or extension of survival. “Treatment” also includes mitigation of pathological outcomes of cancer or immunological diseases.

[0050] As used herein, the term “effective dose” refers to an amount of a drug (such as modified immune cells or their pharmaceutical compositions described herein) sufficient to treat a particular disorder, condition, or disease, for example, to improve, alleviate, reduce, and / or delay one or more of its symptoms (e.g., cancer, infection, GvHD, graft rejection, autoimmune disease, or radiation sickness). When referring to cancer, an effective dose includes an amount sufficient to shrink a tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth), or to prevent or delay the growth of other undesirable cells. In some embodiments, an effective dose is sufficient to delay the onset. In some embodiments, an effective dose is sufficient to prevent or delay recurrence. An effective dose may be administered in one or more doses. An effective amount of a drug (e.g., modified immune cells) or composition may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow, and preferably block the invasion of cancer cells into peripheral organs to some extent; (iv) inhibit tumor metastasis (i.e., delay and preferably stop it to some extent); (v) inhibit tumor growth; (vi) prevent or delay tumor development and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. In the case of infections such as viral infections, an effective amount of the modified immune cells or composition described herein may reduce the number of cells infected with the pathogen, reduce the production or release of pathogen-derived antigens, inhibit (i.e., delay and preferably stop) the spread of the pathogen to uninfected cells, and / or alleviate to some extent one or more symptoms associated with the infection. In some embodiments, the effective amount is an amount that extends the patient's survival time.

[0051] As used herein, “individual” or “subject” refers to mammals, including but not limited to humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0052] As used herein, “patient” includes any human being suffering from a disease (e.g., cancer). The terms “subject,” “individual,” and “patient” are used interchangeably herein.

[0053] "Multiplicity of infection" or "MOI" is used interchangeably herein and refers to the ratio of a pathogen (e.g., a bacteriophage, virus, or bacterium) to its target of infection (e.g., cells or organisms). For example, when referring to a population of cells inoculated with viral particles, the multiplicity of infection or MOI refers to the ratio of the number of viral particles being introduced (e.g., viral particles containing an sgRNA library) to the number of target cells present in the mixture.

[0054] Where the term “includes” is used in this specification and in the claims, no other elements or steps are excluded.

[0055] It should be understood that the embodiments of this application described herein include embodiments consisting of and / or substantially consisting of.

[0056] In this specification, when a value or parameter is described as "approximately," it includes (and describes) variations in the value or parameter itself. For example, a description of "approximately X" includes a description of "X."

[0057] Where used herein, a reference to a value or parameter "not" generally means "other than" that value or parameter, and this is stated accordingly. For example, "This method is not used to treat type X cancer" means that this method is used to treat cancers other than type X cancer.

[0058] As used herein, the term "approximately X to Y" has the same meaning as "from approximately X to approximately Y".

[0059] In the description of numerical ranges for nucleotides in this specification, each number intervening between them is explicitly considered. For example, in the range of 19 to 21nt, the values ​​of 19nt and 21nt as well as 20nt are considered, and for the MOI range, each number between them, whether integer or decimal, is explicitly considered.

[0060] As used herein and in the appended claims, the singular forms “one,” “one,” and “this” include plural referents unless the context clearly indicates otherwise.

[0061] Those skilled in the art will understand that uracil can be represented by "u" and thymine by "t," while both uracil and thymine can be represented by "t." In the context of ribonucleic acid, unless otherwise specified, "t" will be understood to represent uracil.

[0062] In this application, unless otherwise specified, when referring to a genomic locus of a human gene, the reference genome used is GRCh38.p14.

[0063] Manipulated T cells In one embodiment, the application provides engineered T cells modified to eliminate or reduce the expression and / or function of the endogenous T cell receptor (TCR) protein and / or SPPL3 protein (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, the T cells are αβT cells modified to eliminate or reduce the expression and / or function of the α subunit (TCRα) and / or T cell receptor (TCR) β subunit (TCRβ) of the endogenous T cell receptor (TCR). In some embodiments, the T cells are αβT cells modified to eliminate or reduce the expression and / or function of the TCRα conserved region (TRAC) gene or the TRAC gene. In some embodiments, the T cells are γδT cells modified to eliminate or reduce the expression and / or function of the endogenous γ subunit (TCRγ) and / or δ subunit (TCRδ) of the T cell receptor (TCR).

[0064] In some embodiments, the engineered T cells have normally expressed HMC class I protein. In some embodiments, the engineered T cells have normally expressed B2M, HLA-A, HLA-B, and HLA-C. In some embodiments, the engineered T cells are human T cells having normally expressed HLA-I protein.

[0065] In some embodiments, compared to T cells in which endogenous TCR and / or SPPL3 protein are not reduced or removed, engineered T cells exhibit at least about 10% (e.g., any one of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) reduced AICD, for example, at least about 20%. In some embodiments, compared to engineered T cells in which SPPL3 protein expression (RNA and / or protein expression) and / or function are not reduced or removed, engineered T cells exhibit at least about 10% (e.g., any one of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) reduced AICD, for example, at least about 20%.

[0066] In some embodiments, compared to T cells in which endogenous TCR and / or SPPL3 protein are not reduced or removed, the engineered T cells, after transplantation into an allogeneic recipient, exhibit at least about 10% (e.g., any one of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) reduced HVG caused by attack by allogeneic T cells, for example, at least about 20%. In some embodiments, compared to engineered T cells in which SPPL3 protein expression (RNA and / or protein expression) and / or function are not reduced or removed, the engineered T cells exhibit at least about 10% (e.g., any one of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) reduced HVG caused by attack by allogeneic T cells, for example, at least about 20%.

[0067] In some embodiments, engineered T cells have at least about 10% longer in vivo persistence compared to engineered T cells in which the expression (RNA and / or protein expression) and / or function of endogenous TCR and / or SPPL3 protein is not reduced or eliminated.

[0068] In some embodiments, the engineered T cells of the present invention, in which the expression and / or function of MHC class I molecules and TCR proteins are reduced or eliminated, and the expression and / or function of SPPL3 proteins are not reduced or eliminated, show no significant difference in the degree of HVG caused by allogeneic T cell attack, and the engineered T cells of the present invention, in which the spontaneous killing effect caused by NK cell attack is reduced by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), for example, by at least about 20%. Accordingly, in some embodiments, the expression and / or function of MHC class I molecules and TCR proteins are reduced or eliminated in vivo, in peripheral blood, in the presence of PBMCs, or in the presence of both NK cells and T cells, resulting in substantially no difference, or at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) in the degree of NK cell and T cell killing received by the manipulated T cells of the present invention compared to T cells in which the expression and / or function of SPPL3 proteins are not reduced or eliminated, and for example, at least about 20%. In some embodiments, the expression and / or function of MHC class I molecules and TCR proteins are reduced or eliminated, and compared to T cells in which the expression and / or function of SPPL3 proteins are not reduced or eliminated, the manipulated T cells of the present invention maintain a substantially consistent duration or exhibit longer duration in allogeneic recipients or in allogeneic blood or PBMCs. In other words, the effect can be sustained for no significant difference or for a longer period of time, for example, maintaining a no significant difference or a longer half-life in vivo.

[0069] In some embodiments, the engineered T cells have or express an engineered receptor. In some embodiments, the engineered T cells are further modified to express an engineered receptor. In some embodiments, the engineered receptor is one or more selected from CAR, TCR, and TAC. In some embodiments, the engineered T cells are CAR-T cells, TCR-T cells, or TAC-T cells. In some embodiments, the engineered receptor is monovalent. In some embodiments, the engineered receptor is polyvalent. In some embodiments, the engineered receptor is monospecific, for example, monovalent and monospecific, or polyvalent and monospecific. In some embodiments, the engineered receptor is multispecific (e.g., bispecific).

[0070] In some embodiments, the engineered T cells are modified to reduce or eliminate the expression (RNA and / or protein expression) and / or function of endogenous TCR protein and / or SPPL3 protein and / or other proteins, and / or are modified to express engineered receptors. The immune cells may be autologous or allogeneic.

[0071] Method for preparing manipulated T cells In a second aspect, the present application also provides a method for preparing the aforementioned manipulated T cells.

[0072] In some embodiments, the expression of endogenous TCR protein and / or SPPL3 protein in the manipulated T cells is reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or inhibited by antisense RNA, miRNA, siRNA, or shRNA that specifically recognize the RNA encoding the endogenous TCR protein and / or SPPL3 protein.

[0073] In some embodiments, the function of endogenous TCR protein and / or SPPL3 protein is reduced or inhibited by chemically modified mRNA, for example, mRNA of a chemically modified dominant-negative inhibitor of endogenous TCR protein and / or SPPL3 protein (e.g., a dominant-negative mutant or fragment thereof, or a dominant-negative conjugation chaperone). In some embodiments, immune cells are modified to express a dominant-negative SPPL3 protein mutant or its dominant-negative fragment. In some embodiments, immune cells are modified to express a dominant-negative conjugation chaperone for SPPL3 protein.

[0074] In some embodiments, the expression (RNA and / or protein expression) and / or function of endogenous TCR protein and / or SPPL3 protein are reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or inhibited by small molecule compounds, nucleic acids (or vectors containing them), lipids, and / or protein molecules.

[0075] In some embodiments, the manipulated T cells are genetically modified at the DNA locus encoding the endogenous TCR protein and / or SPPL3 protein. In some embodiments, immune cells are genetically modified at the SPPL3 locus. In some embodiments, the locus of the endogenous TCR protein and / or SPPL3 protein is modified with a mutagenic substance. Mutagenic substances can be classified into three categories: physical (e.g., gamma rays, ultraviolet (UV) radiation), chemical (e.g., ethyl methanesulfonic acid (EMS)), and transposon elements (e.g., transposons, retrotransposons, T-DNA, retroviruses). In some embodiments, the mutagenic substance or condition is any of the following: ionizing radiation (IR), ultraviolet light, alkylating agents (e.g., nitrogen mustard gas, methylmethanesulfonic acid (MMS), EMS, N-ethyl-N-nitrosourea (ENU)), aromatic amines (e.g., 2-aminofluorene), polycyclic aromatic hydrocarbons (PAHs, e.g., dibenzo[a,l]pyrene, naphthalene, anthracene, pyrene), crosslinking, insertion mutations (e.g., mediated by transposons or viruses), or other toxins (e.g., aflatoxin, N-nitrosamine). In some embodiments, the endogenous TCR protein and / or SPPL3 protein loci are modified by gene editing. In some embodiments, gene editing is mediated by site-directed mutagenesis (SDM). In some embodiments, gene editing is mediated by random and broad mutagenesis (REM). In some embodiments, gene editing is achieved by PCR. In some embodiments, gene editing is achieved by non-PCR methods. This specification may use any known gene editing method, including but not limited to non-homologous end joining (NHEJ) mediated, homology-induced repair (HDR) mediated, zinc finger nuclease (ZFN) mediated, transcription activator-like effector nuclease (TALEN) mediated, or CRISPR / Cas mediated gene editing. HDR may occur in either a non-conservative or conservative manner. In some embodiments, HDR is mediated by the single-strand annealing (SSA) pathway.In some embodiments, HDR is mediated via the classical double-strand break repair (DSBR) pathway, the synthesis-dependent strand annealing (SDSA) pathway, or the break-inducible repair (BIR) pathway. In some embodiments, the cell modification methods described herein further include modifying a target genomic sequence (e.g., via HDR) by introducing a nucleic acid template (e.g., including a desired mutation), such as an insertion into a double-strand break (DSB) site. Gene editing can introduce one or more mutations, including but not limited to insertions, deletions, substitutions (e.g., nonsynonymous substitutions), truncations, translocations, and point mutations, into a DNA locus encoding one or more target proteins (e.g., the SPPL3 protein). In some embodiments, the mutation is a frameshift mutation, a loss-of-function (LOF) mutation, a dominant-negative mutation, a missense mutation, or a nonsense mutation. In some embodiments, gene editing includes gene knockout (KO). In some embodiments, gene editing includes base editing (e.g., introduction of nonsynonymous substitutions). In some embodiments, base editing introduces stop codons that can reduce the expression of functional RNA and / or proteins. In some embodiments, base editing introduces mutations that affect the function of RNA and / or proteins. In some embodiments, gene editing is mediated by CRISPR / Cas. In some embodiments, the Cas protein has endonuclease activity. In some embodiments, the Cas protein is a fusion protein containing i) a dead Cas protein (dCas) and ii) an adenine base editor (ABE) or adenosine deaminase (ADA), or a cytidine base editor (CBE) or cytidine deaminase (CDA), or functional fragments thereof. Cytidine base editors can convert target C:G base pairs to T:A base pairs, and adenine base editors can convert A:T base pairs to G:C base pairs. In summary, both classes of base editors can target and install any possible conversion mutation (C to T, G to A, A to G, T to C, C to U, and A to U).

[0076] In some embodiments, the manipulated T cells are genetically modified in the RNA encoding the endogenous TCR and / or SPPL3 protein. In some embodiments, the RNA encoding the endogenous TCR and / or SPPL3 protein is modified by RNA editing. RNA editing can introduce one or more mutations into the RNA encoding the endogenous TCR and / or SPPL3 protein, including but not limited to insertions, deletions, substitutions (such as nonsynonymous substitutions), cleavage, and point mutations. In some embodiments, the mutations are frameshift mutations, LOF mutations, dominant-negative mutations, missense mutations, or nonsense mutations. In some embodiments, RNA editing includes base editing (e.g., introduction of nonsynonymous substitutions such as C to U or A to I). Any known RNA editing method may be used in this specification (see, for example, Guillermo Aquino-Jarquin, “Novel Engineered Programmable Systems for ADAR-Mediated RNA Editing,” Mol Ther Nucleic Acids. 2020;19:1065-1072, the contents of which are incorporated herein by reference in their entirety).This includes leveraging endogenous ADARs for programmable RNA editing ("LEAPER," e.g., WO2020074001 and Qu et al. (see Nat Biotechnol. 2019;37(9):1059-1069), RNA editing for programmable A-to-I substitution ("REPAIR," e.g., Cox et al., "RNA editing with CRISPR-Cas13," Science. 2017;358(6366):1019-1027), recruiting endogenous ADARs to specific transcripts for oligonucleotide-mediated RNA editing ("RESTORE," e.g., Merkle et al., "Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides," Methods Mol Biol. 2021;2181:331-349), and CRISPR-Cas-induced RNA targeting systems ("CIRTS," e.g., Rauch Examples include, but are not limited to, RNA editing for specific C-to-U exchanges ("RESCUE," see Abudayyeh et al., "A cytosine deaminase for programmable single-base RNA editing," Science. 2019; 365(6451): 382-386), or CLUSTER (see P. Reautschnig et al., "CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo," Nat Biotechnol. 2022 May; 40(5): 759-768), the contents of each are incorporated herein by reference in their entirety. In some embodiments, RNA editing is mediated by LEAPERS.In some embodiments, RNA editing is mediated by CRISPR / Cas, for example, by fusing adenine base editor (ABE) or adenosine deaminase (ADA), or cytidine base editor (CBE) or cytidine deaminase (CDA), or functional fragments thereof, to a dead Cas (dCas, e.g., dCas13) protein.

[0077] Methods for constructing CRISPR systems that recognize specific DNA or RNA sites are known in the art. Any known CRISPR / Cas system suitable for gene editing or RNA editing can be used herein. To date, two classes (Class I and Class II) and six types (I-VI) of CRISPR-Cas systems have been described, based on the remarkable functional and evolutionary modularity of CRISPR-Cas systems. See, for example, Nidhi et el., “Novel CRISPR-Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives,” Int J Mol Sci. 2021;22(7):3327. The contents of this document are incorporated herein by reference in their entirety. Among the Class II CRISPR-Cas systems, the Type II Cas9 system and the Type VA / B / E / J Cas12a / Cas12b / Cas12e / Cas12j systems have been used for genome editing and offer a wide range of possibilities for biomedical research. Cas13a(C2c2) is a type VI-A RNA-inducible RNA-targeted CRISPR effector that can be used for RNA editing as described herein. The CRISPR / Cas systems used herein can induce double-strand breaks (DSBs) or single-strand breaks at a given nucleic acid site. In some embodiments, the CRISPR / Cas system used herein is the CRISPR / Cas9 system. In some embodiments, the CRISPR / Cas system used herein is the CRISPR / Cas12 system.

[0078] In some embodiments, gene editing or RNA editing involves contacting precursor T cells with i) a guide RNA (gRNA) construct (wherein the gRNA construct comprises or encodes a gRNA containing a guide sequence, the guide sequence being complementary to a target site in a DNA locus or RNA encoding an endogenous TCR protein and / or SPPL3 protein), and optionally ii) a Cas protein or a Cas component comprising a nucleic acid encoding a Cas protein, under conditions that allow for the introduction of a gRNA construct and / or Cas component into the precursor T cells. In some embodiments, the precursor immune cells already express the Cas protein before the introduction of the gRNA construct. In some embodiments, the precursor immune cells do not express the Cas protein before the introduction of the gRNA construct and / or Cas component. In some embodiments, the precursor immune cells express an engineered receptor (e.g., CAR, engineered TCR, or TAC) before the introduction of the gRNA construct and / or Cas component. In some embodiments, the Cas protein has endonuclease activity. In some embodiments, the Cas protein is a fusion protein, for example, i) dCas (e.g., dCas13a, dCas9) and ii) ADA (e.g., TadA, e.g., TadA8e) or CDA or a functional fragment thereof. In some embodiments, the Cas protein is Cas9, such as dCas9. In some embodiments, the gRNA is sgRNA. In some embodiments, the gRNA contains crRNA (or consists essentially of crRNA, or consists solely of crRNA). In some embodiments, the gRNA contains crRNA and tracrRNA (or consists essentially of crRNA and tracrRNA, or consists solely of crRNA and tracrRNA).

[0079] In some embodiments, immune cells modified to remove or reduce endogenous TCR proteins and SPPL3 (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) are modified to remove PD-1, TIM-3, LAG-3, CTLA-4, CISH, SPPL3, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6 The T cells are modified to remove or reduce the expression (RNA and / or protein expression) and / or function (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) of one or more other proteins selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and NKp46 ligands, or are further modified. In some embodiments, the T cells have normally expressed HLA-I, and the normally expressed HLA-I does not lead to the killing of NK cells.

[0080] In some embodiments, the engineered T cells are further modified to express the engineered receptor. In some embodiments, immune cells express the engineered receptor before being modified to reduce or eliminate the expression and / or function of the endogenous TCR protein and / or SPPL3 protein. In some embodiments, immune cells are modified to reduce or eliminate the expression and / or function of the endogenous TCR protein and / or SPPL3 protein, and then further modified to express the engineered receptor. In some embodiments, the modification to reduce or eliminate the expression and / or function of the endogenous TCR protein and / or SPPL3 protein and the modification to express the engineered receptor occur simultaneously. In some embodiments, the modification to reduce or eliminate the expression and / or function of the endogenous TCR protein and / or SPPL3 protein and the modification to express the engineered receptor occur sequentially. Any engineered receptor capable of signaling to immune cells (e.g., cell proliferation, induction of cytokine production, and / or performance of regulatory or cytolytic effector functions) and / or recognition of target antigens can be used herein. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen coupler (TAC). In some embodiments, the engineered receptor is monovalent. In some embodiments, the engineered receptor is polyvalent. In some embodiments, the engineered receptor is monospecific, for example, monovalent and monospecific, or polyvalent and monospecific. In some embodiments, the engineered receptor is multiplespecific (e.g., bispecific).

[0081] In some embodiments, modifications that reduce or eliminate the expression (RNA and / or protein expression) and / or function of the endogenous TCR protein and / or SPPL3 protein do not downregulate or eliminate the expression (RNA and / or protein expression) and / or function of the engineered receptor (e.g., CAR). In some embodiments, modifications that reduce or eliminate the expression and / or function of the endogenous TCR protein and / or SPPL3 protein downregulate the expression (RNA and / or protein expression) and / or function of the engineered receptor by up to about 30% (e.g., up to about 25%, 20%, 15%, 10%, 5%, 1%, or less).

[0082] In some embodiments, the engineered receptor (e.g., CAR), gRNA construct or arRNA construct, and / or Cas component are introduced into precursor T cells via transduction / transfection of nucleic acid (DNA or RNA) or encoding vector (e.g., non-viral vector or viral vector such as a lentiviral vector), or a virus containing encoding nucleic acid (e.g., a lentivirus). In some embodiments, the Cas component (e.g., Cas9 protein) is introduced into precursor T cells by inserting the protein into the cell membrane while passing the cell through a microfluidic system such as CELL SQUEEZE® (see, for example, U.S. Patent Application No. 20140287509).

[0083] Methods for introducing vectors (e.g., viral vectors) or isolated nucleic acids into mammalian cells are known in the art. The nucleic acids or vectors described herein can be introduced into T cells by physical, chemical, or biological methods. Physical methods for introducing vectors (e.g., viral vectors) into cells include calcium phosphate precipitation, lipofection, gene guns, microinjection, and electroporation. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. In some embodiments, vectors (e.g., viral vectors) are introduced into cells by electroporation. Biological methods for introducing vectors into cells include the use of DNA vectors and RNA vectors. Chemical methods for introducing vectors (e.g., viral vectors) into cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, and beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vector is liposomes (e.g., artificial membrane vesicles).

[0084] In some embodiments, RNA molecules (e.g., gRNA, arRNA, or mRNA encoding Cas) are prepared by conventional methods (e.g., in vitro transcription) and then introduced into T cells by known methods such as electroporation. In some embodiments, a viral vector (lentiviral vector) or virus (e.g., lentivirus) containing any of the engineered receptors (e.g., CAR), gRNA, or arRNA, and / or nucleic acids encoding the Cas protein described herein is brought into contact with precursor T cells at an MOI of at least about 1, for example, at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, or 10 (e.g., an MOI of about 3).

[0085] In some embodiments, transfected T cells are grown in vitro after introduction of a vector or isolated nucleic acid. In some embodiments, transfected T cells are cultured and grown for at least 1, 2, 3, 4, 5, 6, 7, 10, 12, or 14 days, for example, 7 days. In some embodiments, transfected T cells are further evaluated or screened to purify the T cells described herein.

[0086] Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene encoding a polypeptide that is not present in or expressed by the receptor organism or tissue, and whose expression is demonstrated by readily detectable characteristics such as enzymatic activity. Reporter gene expression is measured at an appropriate time after DNA / RNA has been introduced into the receptor cell. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (GFP) (e.g., Ui-Tei et al. FEBS Letters 479:79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Antibiotic selection markers can also be used to identify potentially transfected cells.

[0087] Other methods for confirming the presence of the nucleic acids described herein (e.g., gRNA constructs or sgRNA constructs) or mutations (e.g., inactivating mutations) in the target genes of the manipulated T cells of this application include molecular biological assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR, PCR, DNA-seq, or RNA-seq; and biochemical analyses such as immunological methods (ELISA, Western blotting, etc.), fluorescence-activated cell sorting (FACS), and magnetically activated cell sorting (MACS) for detecting the presence or absence of specific peptides.

[0088] Methods for extending the in vivo half-life of manipulated cells Typically, allogeneic engineered cells are often eliminated from the donor via GvHD, AICD, HVG, etc., after being introduced into the patient's body. As a result, their persistence in vivo is reduced, the duration of action in the patient is insufficient, and the clinical effect is limited. A common method to avoid HVG is to knock out HMC class I molecules in allogeneic engineered cells. However, knocking out MHC class I molecules may lead to cells with downregulated HMC class I molecules being recognized and attacked by NK cells.

[0089] This application provides a novel method for extending the in vivo half-life of engineered T cells, which can remove HVG while avoiding or reducing NK cell attack caused by a decrease in MHC class I molecular weight, thereby extending the in vivo half-life of engineered cells by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1x, 1.5x, 2x, 5x, 10x, 20x, 50x, or more) compared to unengineered T cells. Furthermore, in some embodiments, the method of this application can achieve an effect equivalent to or better than methods for extending the in vivo half-life of engineered cells by removing or reducing MHC class I molecules. In some embodiments, compared to methods for extending the in vivo half-life of manipulated cells by eliminating or reducing MHC class I molecules, the methods of the present application may include reducing the spontaneous killing effect caused by NK cell attack by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), for example, at least about 20%, while maintaining the same level of HVG accepted by the manipulated T cells.

[0090] In some embodiments, the engineered cells are immune cells or their precursor cells. In some embodiments, the immune cells are one or more selected from the group consisting of T cells, B cells, NK cells, macrophages, and DC cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are T cells that have been modified to eliminate or reduce the expression and / or function of endogenous TCR proteins. In some embodiments, the engineered cells express or have one or more engineered receptors selected from CAR, TCR, and TAC. In some embodiments, the engineered T cells are CAR-T cells, TCR-T cells, or TAC-T cells. In some embodiments, the engineered receptors are monovalent. In some embodiments, the engineered receptors are polyvalent. In some embodiments, the engineered receptors are monospecific, e.g., monovalent and monospecific, or polyvalent and monospecific. In some embodiments, the engineered receptors are multispecific (e.g., bispecific).

[0091] Specifically, this method involves modifying the engineered cells to remove or reduce the expression and / or function of the SPPL3 protein or its functional fragment. In some embodiments, this method includes any of the above methods for preparing engineered T cells.

[0092] Guide RNA This application also provides guide RNAs (e.g., sgRNAs) for the CRISPR / Cas system, and constructs encoding CRISPR / Cas guide RNAs to target the SPPL3 gene by cleavage or base editing to induce mutations in SPPL3, particularly frameshift mutations (referred to as SPPL3-targeting gRNAs). When SPPL3 is knocked out in genetically modified cells using the sgRNAs provided in this application, and genetically modified cells that do not express SPPL3 are screened using SPPL3 as a marker, the SPPL3 knockout efficiency still reaches 99.37% even after culturing the screened genetically modified cells for several days (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 days). Furthermore, the guide RNAs provided in this application do not cause any detectable off-target effects. In some embodiments, the guide RNAs provided in this application include guide sequences shown in either SEQ ID NOs. 6-256 or SEQ ID NOs. 261-316. In some embodiments, the guide RNA provided in this application is SEQ ID NO: 68, SEQ ID NO: 145, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 196, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 209, SEQ ID NO: 212, SEQ ID NO: 241, SEQ ID NO: 250, SEQ ID NO: 261, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: Includes a guide sequence represented by any one of the following: 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 297, 300, 305, 312, and 315.In some embodiments, the guide RNA provided in this application includes a guide sequence shown in any one of SEQ ID NOs: 150, 154, 155, 159, 161, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, and 294. In some embodiments, this application also provides a combination of guide RNAs including a gRNA that targets SPPL3 and a gRNA that targets a TCR gene, where the TCR gene is one or more selected from, for example, TCRα, TCRβ, TCRγ, and TCRδ. In some embodiments, the gRNA targeting the TCR gene targets the TRAC gene.

[0093] In this application, "targeting" a gene using gRNA means that the guide sequence in the gRNA can bind complementarily to the coding region and / or non-coding region of that gene, or to the mRNA or premRNA of that gene.

[0094] Depending on the CRISPR / Cas system, the gRNA may contain other components in addition to the guide sequence. These other components may include, for example, additional sequence elements that facilitate the formation of a CRISPR complex with the Cas protein. In some embodiments, the gRNA (e.g., sgRNA) includes a second sequence containing a repeat-anti-repeat stem-loop. The repeat-anti-repeat stem-loop includes a tracr-mate sequence fused to a tracr sequence, the tracr sequence being complementary to the tracr-mate sequence via a loop region. In some embodiments, the gRNA (e.g., sgRNA) includes a crRNA containing a direct repeat (DR) sequence that can interact with the Cas protein. In some embodiments, the gRNA (e.g., sgRNA) includes both crRNA and tracrRNA, which may reside on the same RNA strand (e.g., forming the sgRNA) or on two RNA strands. The DR sequence may be derived from a DR sequence natively associated with the corresponding Cas protein. In some embodiments, the DR sequence is located at the 5' end of the spacer sequence. In some embodiments, the DR sequence is located at the 3' end of the spacer sequence. In some embodiments, the DR sequence contains one or more mutations relative to a reference (e.g., wild-type) DR sequence, including, for example, one or more mutations such as 5' and / or 3' extension, 5' and / or 3' cleavage, nucleotide insertion, nucleotide deletion, nucleotide substitution, or a combination thereof, compared to the reference DR sequence. In some embodiments, the DR sequence contains a repeat sequence and an anti-repeat sequence. In some embodiments, the repeat sequence of the DR sequence is substantially partially or completely complementary to the anti-repeat sequence of the DR sequence and hybridizes with the anti-repeat sequence of the DR sequence to form a repeat:anti-repeat duplex (also called a "stem"). In some embodiments, the length of the repeat:anti-repeat duplex is approximately 6 to 20 nucleotides, for example, approximately 6, 8, 10, 12, 14, 16, 18, 20 nucleotides, or more. In some embodiments, the repeat sequence and anti-repeat sequence of the DR sequence are linked directly or indirectly via a linker such as a loop sequence.When repeat and anti-repeat sequences are indirectly linked via a linker, the length of the repeat:anti-repeat double strand does not include the length of the linker. In some embodiments, the repeat sequence of the DR sequence and the anti-repeat sequence of the DR sequence are indirectly linked via a loop sequence. In some embodiments, the length of the loop sequence is approximately 4 to approximately 10 nucleotides, for example, approximately 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the loop sequence contains the GAA sequence. In some embodiments, the DR sequence of the gRNA contains a stem loop. In some embodiments, the DR sequence contains one or more stem loops, such as approximately 1 to approximately 5 stem loops.

[0095] Typically, in the context of the endogenous CRISPR / Cas9 system, the formation of a CRISPR complex (which includes a guide sequence that hybridizes to a target sequence and forms a complex with one or more Cas proteins) results in the cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs). The tracr sequence may contain or consist of all or part of a wild-type tracr sequence (e.g., any of approximately 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence) and may also form part of the CRISPR complex by hybridizing, for example, all or part of a tracr mate sequence operably linked to the guide sequence along at least part of the tracr sequence. In some embodiments, the tracr sequence has sufficient complementarity to hybridize with the tracr mate sequence and participate in the formation of the CRISPR complex. Similar to target sequences, complete complementarity is not considered necessary as long as there is sufficient complementarity for it to function. In some embodiments, a tracr sequence, when optimally aligned, has at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence. Determining the optimal alignment is within the scope of knowledge of those skilled in the art. There are, but are not limited to, publicly available and commercially available alignment algorithms and programs, such as ClustalW, Smith-Waterman, Bowtie, Geneious, Biopython, and SeqMan from Matlab. In some embodiments, the length of the tracr sequence is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 nucleotides, or more. Any known tracr-mate and tracr-sequences derived from the S.pyogenes CRISPR / Cas9 system, as described in US8697359, or any of the naturally occurring CRISPR system-derived tracr-mate and tracr-sequences described herein may be used.

[0096] In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript, resulting in hybridization between them that produces a transcript having secondary structures such as stem-loops (also called hairpins) referred to as "repeat-anti-repeat stem-loops."

[0097] A "stem-loop" refers to a nucleic acid having a secondary structure containing nucleotide regions, which are known or predicted to pair to form a double helix (stem region) linked by unpaired single-stranded nucleotide regions (loop regions). In this specification, the terms "hairpin," "hairpin-loop," and "foldback" structure are also used to refer to stem-loops. Such structures are well known in the art, and these terms are used in accordance with their well-known meanings in the art. As is known in the art, stem-loops do not require precise base pairing. Therefore, the stem may contain one or more base mismatches. Furthermore, the base pairing may be precise, i.e., there may be no mismatches at all.

[0098] In some embodiments, the sgRNA comprises at least one, two, or more stem-loops, e.g., two, three, four, or five stem-loops. In some embodiments, the sgRNA has up to five hairpins. In some embodiments, the sgRNA construct further comprises a transcription termination sequence, such as a poly-T sequence, e.g., six T nucleotides.

[0099] In some embodiments, the Cas protein is Cas9 or Cas12, and each sgRNA includes a guide sequence fused to a second sequence, the second sequence including a repeat-anti-repeat stem-loop that interacts with Cas9 or Cas12. Invariant guide RNA hairpin sequences can be provided based on common sense in the art, for example, as disclosed by Nishimasu et al. (Nishimasu H, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156:935-949). Any invariant hairpin sequence can be used as long as it can bind to the Cas nuclease after transcription.

[0100] In some embodiments, the sgRNA comprises a guide sequence, a repeat-anti-repeat-stem loop, and stem loop 1, stem loop 2, and / or stem loop 3, from 5' to 3'.

[0101] Furthermore, this application also provides a CRISPR system comprising the above-mentioned gRNA or a construct thereof, and a Cas enzyme or its coding nucleic acid or a construct thereof. In some embodiments, the CRISPR system is a CRISPR / Cas9 system. In some embodiments, the CRISPR system is a CRISPR / Cas12 system. [Examples]

[0102] The following examples and exemplary embodiments are intended purely to illustrate the present application and should not be construed as limiting the application in any way. The following examples and detailed description are provided illustratively, not as an limitation.

[0103] Furthermore, the abbreviations for cell processing used in the following examples and their corresponding meanings are as follows: TSKO: Double knockout with TCR and SPPL3; TKO: TCR knockout; TBKO: Double knockout of the TCR and B2M genes; TFKO: Double knockout of the TCR and Fas genes; TBSKO: Triple knockout of the TCR, B2M, and SPPL3 genes.

[0104] Example 1: Preparation of U-CART cells 1. Isolation and activation of T cells from healthy donors Collection of umbilical cord blood from healthy donors: After obtaining umbilical cord blood from a blood bank, it was transported to a GMP-compliant laboratory in a cold chain logistics vehicle equipped with temperature control for T cell separation.

[0105] 1.1 Preparation of peripheral blood mononuclear cells Saline solution was added to the transported umbilical cord blood using a pipette to dilute the umbilical cord blood and saline solution to a 1:1 (V / V) ratio. The hemoglobin was slowly added to a lymphocyte separation tube and centrifuged at 800 × g for 20 minutes. The buffy coat cells from the top of the lymphocyte separation were then aspirated and transferred to a new 50 mL centrifuge tube. T cell culture medium (Miltenyi, 170-076-306) was added, and the mixture was centrifuged at 400 × g for 5 minutes. The supernatant was discarded, and the cell pellet settled at the bottom of the centrifuge tube was collected to obtain peripheral blood mononuclear cells.

[0106] 1.1.2 Isolation and Activation of T Cells The obtained peripheral blood mononuclear cells were counted using a cell counter (Nexcelom, model: Cellometer K2), and then sorted into T cells. The specific procedure is as follows: The cell pellet was prepared using Easy buffer (manufacturer: StemCell, catalog number: 16F72331) in a 5 × 10⁶ size. 7The cells were adjusted to a density of 1 / ml and transferred to a 5ml flow cytometry tube using a 5ml pipette. T cell separation reagent was added at a concentration of 50μl / ml and incubated at room temperature for 5 minutes. Selection magnetic beads were added at a concentration of 40μl / ml and the beads were uniformly mixed within 30 seconds. The cell suspension was added to 2.5ml with Easy buffer and placed directly on the magnetic column for 3 minutes. Next, the cells were transferred to a 15ml centrifuge tube to obtain T cells. After selection, the cells were mixed with a 1000μl pipette, counted, centrifuged (400×g, 5 minutes), and the supernatant was discarded to obtain a T cell pellet. The T cell pellet was resuspended in T cell culture medium. Next, T cell activator (magnetic beads coated with anti-CD3 / CD28 antibody) was added in a 1:1 ratio to activate the T cells. Subsequently, the T cells were further cultured in a 37°C, 5% CO2 incubator. CAR molecules were introduced into the activated T cells using a lentivirus. The CAR molecule used in the examples of this application is a CAR that specifically binds to CD19. Its extracellular antigen-binding domain is the scFV of the anti-CD19 monoclonal antibody FMC63, and its hinge region and transmembrane region are the hinge region and transmembrane region of the CD8α molecule, respectively. The intracellular signal transduction domain consists of a 4-1BB costimulatory domain and a CD3Z tandem domain.

[0107] 1.2 Gene knockout in T cells The TRAC, B2M, and SPPL3 genes in the T cells obtained in Step 1.1.2 were knocked out using CRISPR / Cas9 gene editing technology. The specific procedure is as follows:

[0108] 1.2.1 Design of sgRNAs and plasmid construction targeting the TCR α chain constant coding region (i.e., TRAC) gene, the HLA constant coding region B2M gene, and the SPPL3 constant coding region gene. All sgRNAs designed to target the coding sequences of each exon in the TRAC, B2M, and SPPL3 coding regions were designed using the CRISPR RGEN tool. For the CRISPR / Cas9 system, refer to sequence numbers 1-256 in the sequence listings herein for sequences that target sgRNA regions. Among these, the sgRNAs represented by sequence numbers 68, 145, 150, 154, 155, 159, 161, 165, 172, 176, 196, 203, 204, 209, 210, 212, 241, and 250 are used to target complementary sequences within the SPPL3 genomic region. Additionally, the sgRNAs represented by sequence numbers 1 and 22 are used to target complementary sequences within the TRAC genomic region. For the CRISPR / Cas12 system, please refer to sequence numbers 257-316 in the sequence listing herein for sequences that target sgRNAs. Among these, the sgRNAs represented by sequence numbers 261, 262, 263, and 264 are used to target complementary sequences within the SPPL3 genomic region, while the sgRNAs represented by sequence numbers 257, 258, 259, and 260 are used to target complementary sequences within the TRAC genomic region.

[0109] 1.2.2 sgRNAs with high knockout efficiency and stability were prepared by chemical modification with 2'-O-methylation and / or internucleotide phosphorothioates.

[0110] 1.2.3 Restriction enzyme digestion was performed to linearize the Cas9 plasmid, GFP plasmid, Xba1 (manufacturer: NEB, catalog number: cat#R0145S), and Cutsmart buffer (manufacturer: NEB, catalog number: cat#B7204s). A 50 μl reaction system is shown in Table 1.

[0111] [Table 1]

[0112] The enzyme digestion product was bathed in water at 37°C for 4 hours, and 2 μl of the digested product was subjected to agarose gel electrophoresis at a voltage of 110 U for 30 minutes. A single band was observed in the gel imaging system, indicating that all plasmids were completely enzymatically digested and linearized.

[0113] The above reaction products were washed and purified.

[0114] The purified product was used for in vitro transcription (IVT) using the HISCRIBE® T7 ARCA mRNA kit (manufacturer: NEB, catalog number: cat#E2060S). Table 2 shows the 20 μl system.

[0115] [Table 2]

[0116] The above reaction system was placed in a PCR instrument and reacted at 37°C for 4 hours. After 4 hours, 2 μl of DNase 1 was added to the reaction system and reacted at 37°C for 20 minutes.

[0117] The reaction products described above were processed as shown in Table 3.

[0118] [Table 3]

[0119] The above reaction system was set up in a PCR instrument and reacted for 2 hours. After washing and purifying the reaction product, it was stored in a refrigerator at -80°C for later use.

[0120] 1.2.4 The T cells cultured in step 1.1.2 were collected in a 50 mL centrifuge tube and centrifuged at 300 × g for 7 minutes, after which the supernatant was discarded. The cells were washed twice with DPBS solution (manufacturer: Gibco, catalog number: 1924294), and then the cell density was reduced to 2.5 × 10⁶ using electroporation reagent. 7The concentration was adjusted to / mL. Cas9 mRNA and sgRNA were mixed with T cells, and the final concentration was 2.5 × 10⁶ per 100 μL. 6 Cells and 8 μg of RNA (2 μg each of Cas9 mRNA, TCR sgRNA, B2M sgRNA, and SPPL3 sgRNA) were prepared. Then, Cas9 mRNA and sgRNA were introduced into the cells using a BTX Agile Pulse MAX electroporator (manufacturer: BTX, model: 47-0200NINT) at 200-400 V and 0.5-2 ms. Based on the principle of antigen screening, T cells that were negative for TCR and / or B2M and / or SPPL3 and positive for CD4 and CD8 were selected to obtain U-CART cells.

[0121] The genome was extracted from the above cells using the TIANamp Genomic DNA Kit (Manufacturer: TIAN GEN, Catalog No.: cat#DP304-03). Using synthetic primers, the extracted cell genome was PCR-amplified using 2*Esay Taq Super Mix (+dye) (Manufacturer: TRANS, Catalog No.: Code#AS111) to amplify the genomic regions containing the corresponding sgRNAs for TRAC, B2M, and SPPL3. The 50 μl reaction system is shown in Table 4.

[0122] [Table 4]

[0123] The reaction conditions were as follows: 95℃, 3 minutes 95°C, 30 seconds, 35 cycles Variable, 30 seconds, 72°C

[0124] 1.2.5 The PCR products after the reaction were subjected to NGS sequencing to verify the molecular knockout efficiency of TCR, HLA, and SPPL3. After 8 days of cell culture, the obtained T cells were monitored for quality control. The results showed that the efficiency of TCR knockout alone was approximately 97.18%, and the efficiency of SPPL3 knockout was approximately 96.98%. This system demonstrated that the constant coding sequences of the TCR TRAC gene, the HLA B2M gene, and SPPL3 can be edited, including insertion and deletion mutations, using CRISPR / Cas9 technology, inducing frameshift mutations, thereby inhibiting the expression of TCR, HLA, and SPPL3 at the gene level.

[0125] 1.2.6 Detection of off-target rates of TCR and SPPL3 genes in U-CART cells Simultaneously, potential off-target sites for TRAC (SEQ ID NOs. 1 and 2) and SPPL3 (SEQ ID NOs. 155 and 166) across the entire human genome were predicted, and predicted off-target regions that could potentially affect the expression of other genes were amplified and analyzed. The objective was to confirm at the molecular level that knockout of TRAC and SPPL3 does not induce knockout of off-target, nonspecific genes. As a result, T cells were gene-edited using sgRNAs targeting TRAC (including any of SEQ ID NOs. 1-4) and SPPL3 (including any of SEQ ID NOs. 6-256) as sgRNAs, and screening was performed. The resulting U-CART cells showed complete knockout of the TRAC and SPPL3 genes. Furthermore, no gene mutations were observed in potential off-target sites. Specific results are shown in Tables 5 and 6.

[0126] [Table 5]

[0127] [Table 6]

[0128] 1.2.7 Determination of pre-purification related viability indicators for U-CART cells constructed using CRISPR / Cas9 and CRISPR / Cas12 systems In the CRISPR / Cas9 system, SPPL3 and TCR were double knocked out by combining 17 sgRNAs (SEQ ID NO: 68, 145, 150, 154, 155, 159, 161, 165, 172, 176, 196, 203, 204, 209, 212, 241, 250) targeting the complementary sequence of the SPPL3 genomic region with one sgRNA (SEQ ID NO: 1) targeting the complementary sequence of the TCR genomic region. The proliferation, viability, TCR KO efficiency on day 9, SPPL3 KO efficiency on day 9, CAR+ cell percentage on day 9, U-CART cell viability on day 9, and U-CART cell count on day 9 of the resulting U-CART cells were measured. The results are shown in Figures 1A to 2E. In the CRISPR / Cas12 system, SPPL3 and TCR were double knocked out by combining four sgRNAs (SEQ ID NOs. 261, 262, 263, and 264) targeting the complementary sequence of the SPPL3 genomic region and four sgRNAs (SEQ ID NOs. 257, 258, 259, and 260) targeting the complementary sequence of the TCR genomic region. The proliferation, viability, TCR KO efficiency, SPPL3 KO efficiency, CAR+ cell percentage, U-CART cell viability, and U-CART cell count of the resulting U-CART cells were measured. The results are shown in Figures 3A to 4E.

[0129] These results indicate that U-CART cells edited using both the CRISPR / Cas9 and CRISPR / Cas12 systems achieved the expected experimental results and can be used for subsequent purification experiments.

[0130] 1.3 U-CART cells prepared by double knockout of the TCR gene and SPPL3 gene were purified according to the following experimental procedure. U-CART cells were washed with 1% HSA buffer and resuspended at a density of 1E8 / mL. TCRα / β-biotin was added at a concentration of 100 μL / mL and incubated at 25 rpm for 30 minutes at room temperature using a shaker. The cells were washed twice with 1% HSA buffer and resuspended in a buffer containing 1% HSA. Antibiotin reagent was added at a concentration of 200 μL / mL and incubated at 25 rpm for 30 minutes at room temperature using a shaker. The cells were washed once with 1% HSA buffer and resuspended in a buffer containing 1% HSA. The cells were passed through an LS column (Miltenyi, catalog number: 130-042-401), negative cells were collected, the number of viable cells was counted, and the purification efficiency was evaluated by flow cytometry.

[0131] The results are shown in Figures 5A-5B and 7A-7B, demonstrating that the viability of U-CART cells did not decrease after purification.

[0132] Example 2: Functional Verification of U-CART 2.1 Detection of proliferation and survival rate Using U-CART cells (U-CART) in which the TRAC and SPPL3 genes were completely knocked out, unmanipulated T cells (T), and normal CAR-T cells (CART) without gene knockout, the proliferation capacity and cell viability were detected under the same conditions. The results are shown in Figures 6A-6B and 8A-8B. It can be seen that knockout of the TCR and SPPL3 genes does not affect the proliferation and viability of U-CART cells. Figures 6C and 8C show that cell viability does not decrease with proliferation of U-CART cells after knockout of the TCR and SPPL3 genes.

[0133] 2.2 Detection of resistance to allogeneic (or heterogeneous) T cell killing To evaluate whether U-CART cells, after SPPL3 knockout, were eliminated by patient T cells after injection, the 1G4-NY-ESO-1 system (Wei Wensheng Laboratory, Peking University) was used as an experimental model. This system uses T cells expressing the 1G4 TCR, which specifically kills NY-ESO-1 positive HLA-A 02-01 genotype T cells. Knocking out the B2M gene prevented NY-ESO-1 positive HLA-A 02-01 genotype T cells from presenting the NY-ESO-1 antigen peptide on their cell surface, making them resistant to the killing effect of 1G4 TCR-T cells.

[0134] The specific experimental protocol was as follows: Donor 1G4 TCR-T cells were resuspended in AIM 5+5% FBS medium and the cell concentration was adjusted to 5 × 10^6 / mL. Different gene-edited NY-ESO-1 positive HLA-A 02-01 genotype T cells were resuspended in AIM 5+5% FBS medium and the cell concentration of each group was adjusted to 1 × 10^6 / mL. 1G4 TCR-T cells and NY-ESO-1 T cells were seeded in a 96-well U-shaped plate at an effector-to-target ratio (1G4:NY-ESO-1 = 5:1). 100 μL each of 1G4 TCR-T cells and NY-ESO-1 T cells were added to each well, for a total of 200 μL per well. After co-culturing in an incubator for 24 hours, 20 μL of cells were taken from each well and the total number of viable cells was measured by K2 counting. The remaining 180 μL was used to measure the ratio of 1G4 TCR-T cells to NY-ESO-1 positive T cells using flow cytometry (Note: 1G4 TCR-T cells had a GFP tag and were FITC-positive by flow cytometry, while NY-ESO-1 positive T cells had a BFP tag and were PB450-positive by flow cytometry). The NY-ESO-1 positive T cell elimination rate in each experimental group was calculated by comparing the total number of NY-ESO-1 positive T cells (1 × 10^5) at the time of seeding.

[0135] The results shown in Figure 9 demonstrate that SPPL3 knockout significantly enhances the resistance of T cells to allogeneic T cell toxicity. In terms of increasing the resistance of T cells to allogeneic T cell toxicity, SPPL3 knockout may be equivalent to or slightly weaker than B2M knockout.

[0136] 3. Detection of resistance to NK cell killing NK cells and U-CART cells were seeded in an NK:T ratio of 10:1, with 2E5 U-CART cells seeded per group. The cells were cultured overnight in an incubator at 37°C in AIM-V + 5% FBS medium. Samples were collected after 24 hours. After uniform pipetting, 200 μL was taken from each well, and the vitiligo rate was determined by flow cytometry analysis. Cell vitiligo was detected by FACS counting.

[0137] The results are shown in Figure 10. Here, the % mortality rate is calculated as (number of U-CART cells in the control well - number of U-CART cells in the interaction well) / number of U-CART cells in the control well * 100%.

[0138] The results showed that B2M knockout enhanced the killing activity of allogeneic NK cells, knockout of both B2M and SPPL3 relatively decreased the killing activity of NK cells, and knockout of SPPL3 alone without completely knocking out B2M significantly reduced the killing activity of allogeneic NK cells and improved the persistence of U-CART cells in patients.

[0139] 4. Detection of resistance to the lethal effect of the same type of PBMC To evaluate the combined killing effect of allogeneic T cells and NK cells when U-CART cells are injected into patients after SPPL3 knockout, U-CART cells were co-cultured with allogeneic PBMCs, and the killing effect of allogeneic PBMCs on U-CART cells was statistically analyzed. The specific experimental protocol is as follows. (1) First, CellTrace staining was performed on three types of U-CART cells: U-CART cells with TCR and SPPL3 knocked out (TSKO), U-CART cells with only TCR knocked out (TKO), and U-CART cells with TCR and B2M knocked out (TBKO) (Figure 11A, TKO-CellTrace-CFSE, TBKO-CellTrace-Far Red, TSKO-CellTrace-VIOLET); or (2) First, CellTrace staining was performed on three types of U-CART cells: U-CART cells with TCR, B2M, and SPPL3 knocked out (TBSKO), U-CART cells with only TCR knocked out (TKO), and U-CART cells with TCR and B2M knocked out (TBKO) (Figure 11B, TKO-CellTrace-CFSE, TBKO-CellTrace-Far Red, TBSKO-VIOLET). Next, three types of U-CART cells were mixed in a 1:1:1 ratio. The mixed U-CART cells were co-cultured with allogeneic PBMCs in different ratios (shown as E:T in the figure) in a 37°C, 5% CO2 incubator. After 120 hours, the number of viable cells in TKO, TBKO, and TSKO / TBSKO was measured by flow cytometry. Compared to a control group without allogeneic PBMCs, the killing effect of allogeneic PBMCs on the four types of U-CART cells was statistically calculated according to the formula: Killing rate (%) = (Number of viable cells in the control group - Number of viable cells in the experimental group) / Number of viable cells in the control group * 100%.

[0140] The results are shown in Figures 11A and 11B, indicating that when PBMC cells are used for cell killing, knocking out both TCR and SPPL3 (TSKO) significantly reduces the killing effect of PBMCs on U-CART cells compared to knocking out only TCR (TKO). However, knocking out B2M in addition to TCR (TBKO) enhances the killing effect of PBMCs on U-CART cells, and knocking out SPPL3 in addition to TCR and B2M (TBSKO) partially weakens the killing effect of PBMCs on U-CART cells.

[0141] From this, it appears that SPPL3 knockout in a PBMC environment or in vivo environment can reduce the killing effect of endogenous immune cells against allogeneic T cells with normal HLA-I expression (TSKO cells in this example) and T cells with depleted or suppressed HLA-I expression (B2M knockout).

[0142] 5. Detection of resistance to the lethal effect mediated by FasL The experimental protocol for this application is as follows: 1. 1E7 CAR-T cells were collected from each of the TKO / TFKO / TSKO populations, resuspended in 10 mL, and then seeded in 1 mL portions into each well of a 24-well plate. 2. The concentration of Biolegend Recombinant Human FASL was adjusted to 1000 μg / mL using complete T cell culture medium and prepared as a stock solution. 3. FASL stock solution was diluted to 500, 200, 100, and 50 μg / mL in complete T cell culture medium. 4. FASL was added to 24-well plates containing cells at a volume ratio of 1:1000, so that the final FASL concentrations for each group were 1000, 500, 200, 100, 50, and 0 ng / mL. A 5.24-well plate was incubated at 37°C in 5% CO2 for 24 hours. 6. After 24 hours, the 24-well plate was removed, and the cells were uniformly pipetted using a 1 mL pipette. 20 μL of cell suspension was added to 20 μL of AOPI dye, and the cell viability of each group was measured using a K2 counter, and the results were recorded.

[0143] The results are shown in Figure 12, indicating that SPPL3 knockout can partially achieve the effect of FasL knockout, that is, it can counteract Fas signaling pathway-induced apoptosis in U-CART cells.

[0144] 6. Detection of U-CART's in vitro lethality In Example 1, U-CART cells in which the TRAC and SPPL3 genes were completely knocked out (U-CART), CAR-T cells without gene knockout (CART), and unmanipulated T cells were used as effector cells, and Nalm6 (human acute B lymphoblastic leukemia cell line) was used as the target cell for verification. The specific method is as follows.

[0145] Cell resuscitation: Frozen U-CART cells, T cells, and target cells Nalm6 (human acute B lymphoblastic leukemia cell line) were rapidly thawed in a 37°C water bath. Once the ice crystals in the cryovial had almost completely disappeared, the cells were transferred to a 15 mL centrifuge tube, centrifuged at 400 × g for 5 minutes to collect the cells, and then resuspended in fresh culture medium to count the number of cells.

[0146] Co-culture of target cells and effector cells: The effector cells and target cells described above were added to a cell culture plate in an E:T ratio of 1:2. The cell culture plate was transferred to a cell culture incubator at 37°C and 5% CO2 and incubated.

[0147] Continuous addition of target cells: Cells were collected every 48 hours, counted, and analyzed by flow cytometry to determine the proportion of target cells and CAR+ cells. Based on the CAR+% of each cell type and the total cell count, effector cells and target cells were added back to the cell culture plate in an E:T ratio of 1:2.

[0148] The above procedure was repeated until the remaining CAR+ cells were insufficient for co-culture experiments.

[0149] After each co-culture, the following information was collected and calculated. The percentage of remaining target cells was statistically calculated as the percentage of CD4-CD8 cells, i.e., the percentage of target cells among all cell types. Total number of CAR-positive cells: Total number of CAR-positive cells = Percentage of CAR-positive cells (%) × Total number of cells in the sample. The cumulative proliferation factor of CAR-positive cells = (Total number of CAR-positive cells currently / Total number of CAR-positive cells at the time of previous seeding) × Cumulative proliferation factor of CAR-positive cells at the time of previous seeding. (The cumulative proliferation factor of CAR-positive cells at time 0 is defined as 1.)

[0150] The results of various T cell killing capacity tests and the proliferation of CAR-positive (CAR+) T cells are shown in Figures 13-14 and 15. As can be seen from Figures 13-14, CART cells and U-CART cells showed superior killing effects compared to T cells. Although U-CART cells were gene-edited, their ability to kill target cells did not differ significantly from that of normal control CART cells. Analysis of target cell survival rates revealed that U-CART cells significantly killed Nalm-6 target cells, and long-term killing experiments showed that less than 10% of target cells remained after cell killing by U-CART cells. Figure 15 shows that T cells without transduction of the CAR molecule did not show proliferation of CAR+ cells, nor did they show any inhibitory effect on target cells. As a result, as shown in Figure 8, the proportion of target cells increased over time, and after 144 hours of co-culture, target cells accounted for more than 90% of the total cells. Figure 15 shows that U-CART cells maintained CAR+ cell (CAR-positive cell) proliferation up to 288 hours after co-culture, and the peak proliferation rate was consistent with that of CAR-T cells. In conclusion, U-CART cells exhibit good proliferation in response to target cell stimulation and possess long-term and significant inhibitory function against tumor cells.

[0151] 7. In vivo killing efficacy assay of U-CART In this example, Nalm-6 tumors were induced in human acute B lymphoblastic leukemia cells using highly immunodeficient NCG mice. Next, control T cells, CAR-T cells, and U-CART cells prepared from the same healthy donor were injected, and the tumor-suppressive effect of U-CART cells in tumor-bearing mice was evaluated.

[0152] Twenty female NCG mice aged 6-8 weeks were used, with 1 × 10⁶ mice per mouse. 6 Modeling was performed by injecting 6 × 10⁶ Nalm6-LAE cells (Nalm6-Luciferase-LAE) into the tail vein. Four days after tumor cell inoculation, in vivo imaging was performed to detect tumor volume, and the mice were randomly divided into four groups of 5 based on fluorescence values, with each group consisting of 6 × 10⁶ mice, and CAR-T cells were used to analyze each of them. 6 CAR+ cells (mouse) and U-CART 2.0 cells (6 × 10⁻⁶) 6 CAR+ cells (in mice) were administered. In addition, unedited T cells or U-CART cell cryopreservation solution from the same volunteers were administered to the animals, with 5 animals in each group, designated as the T cell control group and the tumor-bearing control group, respectively. All animals received one tail vein injection, and the day of administration was recorded as day 1. After administration, tumor volume was evaluated weekly by in vivo imaging, the survival status of the mice was observed and recorded daily, and the animals' body weight was measured and recorded twice a week. Animals that lost more than 20% of their body weight during this period were considered dead and euthanized.

[0153] The results are shown in Figure 17. As can be seen from the in vivo imaging results (Figure 16), after injection of CAR-T cells and U-CART cells, tumor growth rates were faster in both the tumor-bearing control group and the T-cell control group compared to the test group. Furthermore, the tumor growth rate in the T-cell control group was consistent with that of the tumor-bearing control group, suggesting that T cells themselves do not have a killing function against tumor cells. Compared to the CAR-T cell group, the animals in the U-CART cell group did not show tumor recurrence by day 39 after administration. In immunodeficient mice, TCR and SPPL3 double knockout U-CART cells showed significantly stronger tumor-killing ability, comparable to that of non-knockout CAR-T cells. From this, it is clear that gene editing did not weaken the in vivo killing ability of CAR-T cells, nor did it affect their long-term survival in vivo.

[0154] The sequences used in the above embodiments of this application are shown in the following sequence listing. It should be understood that the following sequences are merely illustrative sequences in the embodiments of this application and do not limit the scope of this application in any way. Nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When representing an RNA sequence, "T" represents uridine.

[0155] [Table 7]

[0156] [Table 8]

[0157] [Table 9]

[0158] [Table 10]

[0159] [Table 11]

[0160] Table 12

[0161] Table 13

[0162] Table 14

[0163] Table 15

[0164] Table 16

[0165] Table 17

[0166] Table 18

[0167] Table 19

Claims

1. Engineered T cells modified to remove or reduce the expression and / or function of endogenous T cell receptor (TCR) protein or its functional fragment, and / or SPPL3 protein or its functional fragment.

2. The engineered T cell according to claim 1, wherein the expression and / or function of the α subunit of the endogenous T cell receptor (TCR) (TCRα) and / or the β subunit of the endogenous T cell receptor (TCR) (TCRβ) are removed or reduced after modification.

3. Furthermore, the engineered T cells according to claim 1 or 2, wherein the expression and / or function of the β-2 microglobulin (B2M) is maintained, or removed or reduced.

4. The engineered T cell according to any one of claims 1 to 3, further comprising or expressing an engineered receptor.

5. The manipulated T cell according to claim 4, wherein the manipulated receptor is one or more selected from a chimeric antigen receptor (CAR), a manipulated TCR, and a T cell antigen coupler (TAC).

6. A method for preparing engineered T cells according to any one of claims 1 to 5, wherein the modification includes removing or reducing the expression and / or function of the TCR protein or a functional fragment thereof and / or the SPPL3 protein or a functional fragment thereof by one or more selected from disruption or knockout of a coding gene, frameshift mutation or knockout, inhibition of transcription of a coding gene, disruption or clearance of mRNA, inhibition of expression of a coding gene, and inhibition of the protein.

7. The method according to claim 6, wherein the modification removes or reduces the expression of TCR or its functional fragment and SPPL3 or its functional fragment by RNA interference (RNAi), and optionally the RNAi silences or inhibits the expression of TCR or its functional fragment and SPPL3 or its functional fragment by small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA).

8. The modification includes removing or reducing the expression of TCR or its functional fragment and SPPL3 or its functional fragment by a gene editing method or RNA editing method via the CRISPR / Cas system, wherein the gene editing method or RNA editing method uses a guide RNA (gRNA) that targets SPPL3 and / or TRAC, and the gRNA is located at positions 120,903,845 to 120,904,358, 120,810,809 to 120,810,886, 120,791,469 to 120,791,557, 120,784,474 to 120,784,593, and 120 of human chromosome 12. The method according to claim 6, using a guide sequence complementary to the SPPL3 genomic region at positions 783,674 to 120,783,752, 120,782,655 to 120,782,767, 120,768,953 to 120,769,059, 120,768,325 to 120,768,488, 120,767,394 to 120,767,593, 120,766,263 to 120,766,372, or 120,764,999 to 120,765,070, and / or using a guide sequence complementary to the TRAC genomic region at positions 23016448 to 23016490 of human chromosome 14.

9. The guide sequences complementary to the SPPL3 genomic region are sequence numbers 68, 145, 150, 154, 155, 159, 161, 165, 172, 176, 196, 203, 204, 209, 212, 241, 250, 261, 269, 270, 271, 272, 273, 274, 275, One or more selected from SEQ ID NOs: 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 297, 300, 305, 312, and 315, and / or The guide sequence complementary to the TRAC genome region is one or more selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, and SEQ ID NO:

260. The method according to claim 8.

10. The method according to claim 8, wherein the gRNA uses a guide sequence complementary to the SPPL3 genomic region at positions 120,791,469 to 120,791,557, 120,783,674 to 120,783,752, or 120,784,474 to 120,784,593 of human chromosome 12.

11. A guide sequence complementary to the aforementioned SPPL3 genomic region is One or more selected from SEQ ID NOs: 150, 154, 155, 159, 161, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, and 294. The method according to claim 10.

12. The method according to any one of claims 8 to 11, wherein the gRNA is chemically modified.

13. The method according to claim 12, wherein the chemical modification includes a 2'-O-methylation modification of a nucleotide ribose, or a 3'-thiophosphate ester bond modification between nucleotides, or both.

14. The method according to claim 13, wherein the modifications are a 2'-O-methylation modification of the ribose of the first three nucleotides at the 5' end, a 2'-O-methylation modification of the ribose of the last three nucleotides at the 3' end, a 3' thiophosphorylation modification between the first three nucleotides at the 5' end, and a 3' thiophosphorylation modification between the last three nucleotides at the 3' end.

15. The method according to any one of claims 8 to 14, wherein the CRISPR / Cas system is a CRISPR / Cas9 system or a CRISPR / Cas12 system.

16. A method for extending the in vivo half-life of manipulated cells, comprising modifying the manipulated cells to remove or reduce the expression and / or function of the SPPL3 protein or a functional fragment thereof.

17. The method according to claim 16, wherein the modification removes or reduces the expression and / or function of the SPPL3 protein or its functional fragment by one or more selected from disruption of the coding gene, frameshift mutation or knockout, inhibition of transcription of the coding gene, disruption or clearance of mRNA, inhibition of coding gene expression, and inhibition of the protein, and optionally, the modification removes or reduces the expression of SPPL3 or its functional fragment by RNA interference (RNAi), for example, the RNAi silencing or inhibiting the expression of SPPL3 or its functional fragment by small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA).

18. The modification includes removing or reducing the expression of SPPL3 or its functional fragment by a gene editing method or RNA editing method via the CRISPR / Cas system, wherein the gene editing method or RNA editing method uses a guide RNA (gRNA) that targets SPPL3, for example, the gRNA is located at positions 120,903,845 to 120,904,358, 120,810,809 to 120,810,886, 120,791,469 to 120,791,557, 120,784,474 to 120,784,593, and 120,783,674 of human chromosome 12. The method according to claim 16, wherein a guide sequence complementary to the SPPL3 genomic region at positions 1-120,783,752, 120,782,655-120,782,767, 120,768,953-120,769,059, 120,768,325-120,768,488, 120,767,394-120,767,593, 120,766,263-120,766,372, or 120,764,999-120,765,070 is used to knock out the SPPL3 coding gene or induce a frameshift mutation in the SPPL3 coding gene.

19. The method according to claim 16, wherein the gRNA uses a guide sequence complementary to the SPPL3 genomic region at positions 120,791,469 to 120,791,557, 120,783,674 to 120,783,752, or 120,784,474 to 120,784,593 of human chromosome 12.

20. The method according to any one of claims 16 to 19, wherein the manipulated cells have or express normal levels of MHC-I protein.

21. The method according to any one of claims 16 to 20, wherein the manipulated cells are immune cells or their progenitor cells.

22. The method according to claim 21, wherein the immune cells are one or more selected from T cells, B cells, natural killer (NK) cells, macrophages, and dendritic cells (DCs).

23. The method according to claim 22, wherein the immune cells are αβT cells, and the expression and / or function of endogenous TCRα and / or TCRβ or functional fragments thereof in the αβT cells are removed or reduced.

24. The method according to any one of claims 16 to 23, wherein the manipulated cells further comprise or express a manipulated receptor.

25. The manipulated receptor, The method according to claim 24, wherein the method is one or more selected from a chimeric antigen receptor (CAR), an engineered TCR, and a T cell antigen coupler (TAC).

26. Sequence ID 68, Sequence ID 145, Sequence ID 150, Sequence ID 154, Sequence ID 155, Sequence ID 159, Sequence ID 161, Sequence ID 165, Sequence ID 172, Sequence ID 176, Sequence ID 196, Sequence ID 203, Sequence ID 204, Sequence ID 209, Sequence ID 212, Sequence ID 241, Sequence ID 250, Sequence ID 261, Sequence ID 269, Sequence ID 270, Sequence ID 271, Sequence ID 272, Sequence ID 273, Sequence ID 274, Sequence ID 275, Sequence IDs 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 297, 300, 305, 312, and 315 A guide RNA (gRNA) for removing or reducing the expression of the SPPL3 protein or its functional fragment by a gene editing method or RNA editing method via the CRISPR / Cas system, comprising any nucleotide sequence selected from the following.

27. A composition comprising the gRNA described in claim 26.

28. A composition comprising a gRNA that targets a gene encoding a T cell receptor (TCR) or a functional fragment thereof, and / or a gRNA that targets a gene encoding the SPPL3 protein or a functional fragment thereof.

29. The gRNA targeting SPPL3 is located at positions 120,903,845 to 120,904,358, 120,810,809 to 120,810,886, 120,791,469 to 120,791,557, 120,784,474 to 120,784,593, 120,783,674 to 120,783,752, and 120,782,655 to 120,78 The composition according to claim 28, comprising a guide sequence complementary to the SPPL3 genomic region at position 2,767, 120,768,953 to 120,769,059, 120,768,325 to 120,768,488, 120,767,394 to 120,767,593, 120,766,263 to 120,766,372, or 120,764,999 to 120,765,070.

30. The gRNA that targets SPPL3 is Sequence ID 68, Sequence ID 145, Sequence ID 150, Sequence ID 154, Sequence ID 155, Sequence ID 159, Sequence ID 161, Sequence ID 165, Sequence ID 172, Sequence ID 176, Sequence ID 196, Sequence ID 203, Sequence ID 204, Sequence ID 209, Sequence ID 212, Sequence ID 241, Sequence ID 250, Sequence ID 261, Sequence ID 269, Sequence ID 270, Sequence ID 271, Sequence ID 272, Sequence ID 273, Sequence ID 274, Sequence ID 275, Sequence IDs 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 297, 300, 305, 312, and 315 The composition according to claim 29, comprising any guide array selected from the following.

31. The composition according to claim 29, wherein the gRNA uses a guide sequence complementary to the SPPL3 genomic region at positions 120,791,469 to 120,791,557, 120,783,674 to 120,783,752, or 120,784,474 to 120,784,593 of human chromosome 12.

32. A guide sequence complementary to the aforementioned SPPL3 genomic region is Sequence IDs 150, 154, 155, 159, 161, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, and 294 The composition according to claim 31, which is one or more selected from the following.

33. The composition according to any one of claims 28 to 32, wherein the T cell receptor gene is the TRAC gene.

34. The composition according to claim 33, wherein the gRNA targeting the T cell receptor gene includes a guide sequence complementary to the TRAC genomic region at positions 23016448 to 23016490 of chromosome 14.

35. The composition according to claim 34, wherein the guide sequence includes the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, or SEQ ID NO: 260.

Images