Engineered T cell with prolonged half-life in vivo and preparation method thereof

Abstract

The invention relates to an engineered T cell with prolonged half-life in vivo and a preparation method thereof. The engineered T cell can reduce the killing of allogenic T cells and avoid the killing of NK cells at the same time.

Description

Engineered T cells with extended in vivo half-life and preparation method thereof Technical Field

[0001] The present application relates to modified immune cells, and in particular to immune cells modified to reduce graft-versus-host disease (GvHD), activation-induced immune effector cell death (AICD), and host-versus-graft effect (HVG) in the host. Technical Background

[0002] In the development of universal CAR-T cell therapy (also known as U-CART, allogeneic chimeric antigen receptor T cell therapy), one of the biggest challenges is the persistence of U-CART cells. This is mainly due to three reasons: First, through TCR mediation, U-CART cells will attack the patient's somatic cells, leading to graft-versus-host disease (GvHD); Second, CAR-T cells activated by target cells have activation-induced immune effector cell death (AICD, Activation Induced Cell Death), which is caused by the simultaneous expression of FasL (CD95L) and Fas (CD95) by activated T cells. FasL is a killer effector molecule that can cause apoptosis of activated T cells through autocrine or paracrine FasL; Third, U-CART cells will be attacked by the patient's T cells, leading to host-versus-graft effect (HVG), which makes U-CART cells unable to survive in the patient's body for a long time, thereby greatly limiting its therapeutic effect. At present, in order to solve the problem of the persistence of U-CART cells, scientists generally choose to knock out the TCR gene in healthy donor T cells to avoid the occurrence of GvHD, and at the same time intervene in B2M expressed on the surface of U-CART cells (inhibiting the expression of HLA-I class molecules) to prevent them from being recognized and attacked by patient T cells.

[0003] However, the above strategy will cause another effector cell to attack U-CART cells that do not express HLA molecules. This effector cell is a natural killer cell (NK). NK cells are effector immune cells that can recognize cells with low HLA expression. Many virus-infected cells and tumor cells downregulate the expression of HLA molecules to escape T cell recognition. U-CART cells that have reduced the expression of HLA-related molecules due to intervention will also be cleared by the body through NK cell immunity due to this mechanism.

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

[0005] SUMMARY OF THE INVENTION

[0006] The present application provides an engineered T cell with enhanced persistence and a preparation method thereof, which does not induce NK cell killing while reducing GvHD, AICD and HVG, and thus has improved in vivo persistence and therapeutic effect.

[0007] Specifically, the first aspect of the present application relates to an engineered T cell that has been modified to eliminate or reduce the expression and / or function of an endogenous T cell receptor (TCR) protein and / or SPPL3 protein. In some embodiments, the engineered T cell is a γδT cell, and it has been modified to eliminate or reduce the expression and / or function of the γ subunit (TCRγ) and / or TCRδ subunit (TCRδ) of its endogenous T cell receptor (TCR). In some embodiments, the engineered T cell is an αβT cell, and it has been modified to eliminate or reduce the expression and / or function of the α subunit (TCRα) and / or β subunit (TCRβ) of the endogenous T cell receptor (TCR). In some embodiments, the modification comprises modification of the SPPL3 genomic region on chromosome 12 of the engineered T cell. In some embodiments, the modification comprises modification of the mRNA or mRNA precursor (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 comprises a nonsense mutation in the SPPL3 genomic region on chromosome 12 of the engineered T cells or the mRNA (or pre-mRNA) of the SPPL3 gene. In some embodiments, the engineered T cells are human-derived T cells, and the modification occurs at any one or more nucleotide positions, or between any two nucleotide positions, in any one or more exon regions of the SPPL3 gene.

[0008] Preferably, the modification occurs 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, 120,791,469 to 120,791,557, any nucleotide position from 82,655 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, or between any two nucleotide positions. More preferably, the modification occurs at any nucleotide position of human chromosome 12, 120,791,469 to 120,791,557, 120,783,674 to 120,783,752, or 120,784,474 to 120,784,593, or between any two nucleotide positions. In some embodiments, the T cell is a T cell derived from humans, and the modification comprises modification of the TRAC genomic region of chromosome 14 of the engineered T cell. In some embodiments, the modification comprises modification of the mRNA or pre-mRNA of the TRAC gene of the engineered T cell. In some embodiments, the T cell is a T cell derived from humans, and the modification comprises a nonsense mutation in the TRAC genomic region or the TRAC gene of chromosome 14 of the engineered T cell. In some embodiments, the T cell is derived from a human T cell, and the modification occurs at any nucleotide position between positions 23016448 and 23016490 relative to chromosome 14 of the GRCh38.p14 reference genome, or between any two nucleotide positions. In some embodiments, the modification results in a frameshift mutation. In some embodiments, the modification results in a nonsense mutation.

[0009] In some embodiments, the T cells have normal expression of HMC-class I proteins. In some embodiments, the T cells have normal expression of 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 therein is not reduced.

[0010] In some embodiments, the engineered T cells further comprise or express an engineered receptor. In some embodiments, the engineered receptor is selected from one or more of the following: a chimeric antigen receptor (CAR), an engineered TCR, and a T cell antigen conjugate (TAC).

[0011] In some embodiments, the engineered T cells express CAR, and the CAR comprises i) an extracellular antigen binding domain that specifically recognizes one or more target antigens or antigenic epitopes; ii) a transmembrane domain; and iii) an intracellular signaling domain. In some embodiments, the extracellular antigen binding domain is selected from one or more of the following: an 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 comprise 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 antigenic epitopes. In some embodiments, the extracellular antigen binding domain binds to one or more proteins selected from the group consisting of: prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA), CAM5, CD123, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit alpha (IL-13Rα); interleukin-11 receptor alpha (IL-11Rα); prostate-specific membrane antigen (PSMA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), M UC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic 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); folate receptor; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R);Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa 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; oncofetal 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); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); Adrenergic receptor β3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternate 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 cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane Transcription factor serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytotoxic viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4);Synovial sarcoma, breakpoint X 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the IgA receptor (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 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5);Immunoglobulin lambda-like polypeptide 1 (IGLL1) and CD 155. In some embodiments, the transmembrane domain comprises a transmembrane domain from any one of the molecules 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 comprises a primary intracellular signaling domain comprising a molecule from any one 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 from the group consisting of 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, a ligand that specifically binds to CD83, and any combination thereof. In some embodiments, the CAR further comprises 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.

[0012] In some embodiments, the engineered T cells express a T cell antigen conjugate (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 cytosolic region). In some embodiments, a TAC comprises: (a) an extracellular ligand binding domain comprising an antigen binding fragment (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes one or more target antigens (e.g., tumor antigens) or antigenic epitopes (e.g., tumor epitopes); (b) an optional first linker; (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) optionally a second linker; (e) optionally the extracellular domain or portion thereof of a first TCR co-receptor (e.g., CD4, CD8); (f) a transmembrane domain comprising the transmembrane domain of a second TCR co-receptor (e.g., CD4, CD8); and (g) an optional intracellular signaling domain comprising the intracellular signaling domain of a third TCR co-receptor (e.g., CD4, CD8). In some embodiments, the engineered T cells comprise one or more TACs, each of which has a different antigen binding domain or ligand binding domain. In some embodiments, the TAC has 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 specifically recognizes one or more target antigens selected from the following: PSCA, CEACAM5, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD 22, CD 30, CD 70, CD 123, CD 138, 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, Claudin 6, Claudin18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA), CA9, kappa light chain, CSPG4, EGP2, EGP40, FAP, FAR, FBP, embryonic AchR, HLA-A1, HLA-A2, MAGEA1, MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, SURVI VIN, TAG72, TEM1, fibronectin, tenascin, oncofetal variant of tumor necrosis, 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, M AD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, 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, LI LRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1 and CD155. In some embodiments, the TCR subunit is selected from one or more of the following groups: CD3ε, CD3δ, CD3γ, TCRα, TCRβ, TCRγ and TCRδ; and wherein the first, second and third TCR co-receptors are each independently selected from any one of the following groups: 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 comprises 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.

[0013] In some embodiments, the engineered T cells express an engineered T cell receptor (TCR) comprising: (a) an extracellular ligand binding domain comprising 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 linker; (c) an optional extracellular domain of a first TCR subunit (e.g., Cα, Cβ, Cδ, Cγ, CD3ε) or a portion thereof; (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); wherein the first, second, and third TCR subunits are independently selected from any one of the following groups: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ, and CD3ζ. In some embodiments, the first, second and third TCR subunits are the same (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, and the one or more TCRs have different extracellular ligand binding domains. In some embodiments, the TCR has 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 specifically recognizes one or more target antigens selected from the group consisting of PSCA, CEACAM5, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD 22, CD 30, CD 70, CD 123, CD 138, 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, Claudin 6, Claudin18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA), CA9, 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, oncofetal variant of tumor necrosis area, 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 mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG, NA17, PAX3, and androgen receptor antagonist. Hormone 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 comprises 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 from CD8α.

[0014] The second aspect of the present application also provides a method for preparing the engineered T cells of the first aspect, wherein the modification includes one or more selected from the following: destruction or knockout of the coding gene, inhibition of coding gene transcription, destruction or removal of mRNA, inhibition of coding gene expression, and inhibition of the protein. In some embodiments, the modification eliminates or reduces the expression of TCR or its functional fragment and / or SPPL3 or its functional fragment by RNA interference (RNAi). In some embodiments, the RNAi silences or inhibits the expression of TCR or its functional fragment and / or SPPL3 or its functional fragment by small interfering RNA (siRNA), short hairpin RNA (shRNA) or small RNA (miRNA). In some embodiments, the modification is achieved by physical mutagenesis, chemical mutagenesis or transposition. In some embodiments, the modification is achieved by PCR method. In some embodiments, the modification is mediated by one or more selected from the following: non-homologous end joining (NHEJ), homology-directed repair (HDR), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR / Cas, adenosine deaminase (ADAR). In some embodiments, the modification is gene editing, including, for example, base editing, PRIME editing, or enzymatic cleavage of a target site. In some embodiments, the modification comprises one or more of an insertion, a deletion, or a substitution. In some embodiments, the modification comprises one or more of a translocation, a point mutation, a fragment deletion, and a fragment addition. In some embodiments, the modification results in one or more of a frameshift mutation, a loss-of-function mutation, a dominant negative mutation, a missense mutation, and a nonsense mutation. In some embodiments, the modification comprises gene editing or RNA editing mediated by a CRISPR / Cas system, wherein the gene editing or RNA editing uses a guide RNA (gRNA) targeting SPPL3 and / or TRAC. In some embodiments, the gRNA targeting SPPL3 comprises a guide sequence complementary to the SPPL3 genomic region of human chromosome 12 (e.g., any exon region, any intron region, any splice site therein) or the mRNA or pre-mRNA sequence corresponding to the genomic region.Preferably, the gRNA targeting SPPL3 comprises a gRNA that is aligned with human chromosome 12 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, 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 SPPL3 genomic region or a guide sequence complementary to the mRNA or pre-mRNA sequence corresponding to the genomic region. More preferably, the gRNA targeting SPPL3 comprises 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 on human chromosome 12, or an mRNA or pre-mRNA sequence corresponding to the genomic region. In some embodiments, the gRNA targeting TRAC comprises a guide sequence complementary to the TRAC genomic region at positions 23016448 to 23016490 on human chromosome 14, or an mRNA or pre-mRNA corresponding to the genomic region. In some embodiments, the CRISPR / Cas system is a type II CRISPR / Cas system, wherein the gRNA does not comprise 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, wherein 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 sequence complementary to the SPPL3 genomic region is selected from one or more of 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: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285 NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:312 and SEQ ID NO:315.

[0015] In some embodiments, the guide sequence complementary to the SPPL3 genomic region is SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 161, 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: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295 NO: 292, SEQ ID NO: 293, and SEQ ID NO: 294. In some embodiments, the guide sequence complementary to the SPPL3 genomic region is SEQ ID NO: 155 or SEQ ID NO: 261. In some embodiments, the guide sequence complementary to the TRAC genomic region is 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 comprises a 2'-O-methylation modification on the ribose sugars of the nucleotides or a 3' phosphorothioate linkage modification between nucleotides, or both. In some embodiments, the modification comprises a 2'-O-methylation modification on the first three ribose sugars of the 5' end, a 2'-O-methylation modification on the last three ribose sugars of the 3' end, a 3' phosphorothioate modification between the first three nucleotides of the 5' end, and a 3' phosphorothioate modification between the last three nucleotides of the 3' end.

[0016] A third aspect of the present application also provides a method for extending the in vivo half-life of an engineered cell, comprising modifying the engineered cell to eliminate or reduce the expression and / or function of an SPPL3 protein or a functional fragment thereof. In some embodiments, the modification comprises one or more of an insertion, a deletion, or a substitution. In some embodiments, the modification comprises one or more of a translocation, a point mutation, a fragment deletion, and a fragment addition. In some embodiments, the modification results in one or more of a frameshift mutation, a loss-of-function mutation, a dominant-negative mutation, a missense mutation, and a nonsense mutation. 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 any genomic locus of SPPL3 but downregulates mRNA by interfering with SPPL3 transcription. In some embodiments, the modification does not interfere with any genomic locus of SPPL3 or mRNA levels, but interferes with the translation of mRNA into SPPL3 protein, thereby downregulating SPPL3 protein levels. In some embodiments, the modification results in a mutation in the SPPL3 genomic region on human chromosome 12, or in an mRNA or pre-mRNA sequence corresponding to the genomic region. In some embodiments, the modification results in a nonsense mutation in the SPPL3 genomic region on human chromosome 12, or in an mRNA or pre-mRNA sequence corresponding to the genomic region. In some embodiments, the engineered cells have or express normal levels of MHC-I proteins. In some embodiments, the engineered cells have normal expression of HLA-A, HLA-B, HLA-C, and B2M. In some embodiments, the normal levels of MHC-I proteins do not induce killing of the engineered cells by NK cells. In some embodiments, the engineered cells are immune cells or precursor cells thereof. In some embodiments, the immune cells are selected from one or more of the following: T cells, B cells, natural killer (NK) cells, macrophages, and DC cells. In some embodiments, the immune cells are αβ T cells, and the expression and / or function of endogenous TCRα and / or TCRβ, or functional fragments thereof, of the αβ T cells are eliminated or reduced. In some embodiments, the engineered T cells are γδT cells, and they are modified so that the expression and / or function of their endogenous TCRγ and / or TCRδ are eliminated or reduced. 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 of a chimeric antigen receptor (CAR), an engineered TCR, and a T cell antigen conjugate (TAC).

[0017] The fourth aspect of the present application also provides a guide RNA (gRNA) targeting SPPL3, which comprises a guide sequence selected from any one of the following: 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: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280 NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:312 and SEQ ID NO:315.Preferably, it comprises a guide sequence selected from any one of the following: SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 161, 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: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294 NO:293 and SEQ ID NO:294.

[0018] In some embodiments, the gRNA participates in the formation of a type II CRISPR / Cas complex. In some embodiments, the gRNA participates in the formation of a CRISPR / Cas complex, wherein the Cas enzyme is Cas9 or Cas12. In some embodiments, the CRISPR / Cas system is a type II CRISPR / Cas system, wherein the gRNA, in addition to the guide sequence, does not include a tracrRNA and a tracr-companion sequence. In some embodiments, the CRISPR / Cas system is a type II CRISPR / Cas system, wherein the gRNA is an sgRNA. In some embodiments, the gRNA is chemically modified. In some embodiments, the chemical modification includes a 2'-O-methylation modification on the ribose sugars of the nucleotides or a 3' phosphorothioate bond modification between nucleotides, or both. In some embodiments, the modification includes a 2'-O-methylation modification on the first three ribose sugars of the 5' end, a 2'-O-methylation modification on the last three ribose sugars of the 3' end, a 3' phosphorothioate modification between the first three nucleotides of the 5' end, and a 3' phosphorothioate modification between the last three nucleotides of the 3' end.

[0019] The fifth aspect of the present application provides a nucleic acid composition comprising the gRNA of the fourth aspect and a gRNA targeting a T cell receptor gene. In some embodiments, the T cell receptor gene is a TRAC gene. In some embodiments, the gRNA targeting the T cell receptor gene comprises a guide sequence complementary to the TRAC genomic region at positions 23016448 to 23016490 on chromosome 14. In some embodiments, the guide sequence comprises a polynucleotide sequence as 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.

[0020] In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which comprises 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: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285 : The guide sequence set forth in any one of SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 300, SEQ ID NO: 305, SEQ ID NO: 312, and SEQ ID NO: 315;Preferably, it includes SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:159, SEQ ID NO:161, 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:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293 and SEQ In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which comprises a guide sequence as set forth in SEQ ID NO: 155 or SEQ ID NO: 261, and the other comprises a guide sequence complementary to the TRAC genomic region from position 23016448 to position 23016490 on chromosome 14. In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which comprises a guide sequence as set forth in SEQ ID NO: 155 or SEQ ID NO: 261, and the other comprises a guide sequence complementary to the TRAC genomic region from position 23016448 to position 23016490 on chromosome 14. In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which comprises a guide sequence as set forth in SEQ ID NO: 155 or SEQ ID NO: 261, and the other comprises a guide sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 257. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figures 1A-1B show the proliferation and viability of U-CART constructed using the CRISPR / Cas9 system on days 0-9 before purification. sgRNA1-sgRNA17-SP3-3 represent the combination of SEQ ID NO: 1 targeting TRAC with 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, and SEQ ID NO: 250 targeting SPPL3, respectively, to double knockout TRAC and SPLL3.

[0022] Figure 2A shows the gene knockout efficiency of TCR on day 9 before purification of U-CART constructed using the CRISPR / Cas9 system.

[0023] Figure 2B shows the gene knockout efficiency of SPPL3 on day 9 before purification of U-CART constructed using the CRISPR / Cas9 system.

[0024] Figure 2C shows the percentage of CAR+ cells on day 9 before purification of U-CART constructed using the CRISPR / Cas9 system.

[0025] Figure 2D shows the viability of U-CART cells constructed using the CRISPR / Cas9 system on day 9 before purification.

[0026] Figure 2E shows the cell number of U-CART cells constructed using the CRISPR / Cas9 system on day 9 before purification.

[0027] Figures 3A-3B show the proliferation and viability of U-CART constructed using the CRISPR / Cas12 system on days 0-9 before purification. UCART-TCR-sg1, UCART-TCR-sg2, UCART-TCR-sg3, and UCART-TCR-sg4 represent sequences targeting TRAC, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, and SEQ ID NO: 260, respectively; SPPL3-sg1, SPPL3-sg2, SPPL3-sg3, and SPPL3-sg4 represent sequences targeting SPPL3, SEQ ID NO: 290, SEQ ID NO: 269, SEQ ID NO: 261, and SEQ ID NO: 270, respectively. The above sequences were combined to double-knockout TRAC and SPLL3.

[0028] Figure 4A shows the gene knockout efficiency of TCR on day 9 before purification of U-CART constructed using the CRISPR / Cas12 system.

[0029] Figure 4B shows the gene knockout efficiency of SPPL3 on day 9 before purification using the U-CART constructed using the CRISPR / Cas12 system.

[0030] Figure 4C shows the percentage of CAR+ cells on day 9 before purification of U-CART constructed using the CRISPR / Cas12 system.

[0031] Figure 4D shows the viability of U-CART cells constructed using the CRISPR / Cas12 system on day 9 before purification.

[0032] Figure 4E shows the cell number of U-CART cells constructed using the CRISPR / Cas12 system on day 9 before purification.

[0033] Figure 5A shows the changes in TCR knockout efficiency before and after purification of U-CART constructed using the CRISPR / Cas9 system.

[0034] Figure 5B shows the changes in activity of U-CART constructed using the CRISPR / Cas9 system before and after purification.

[0035] Figures 6A and 6B show the comparison of U-CART and CAR-T constructed using the CRISPR / Cas9 system and the T cell proliferation rate (2A) and viability (2B).

[0036] Figure 6C shows the changes in the long-term viability of U-CART constructed using the CRISPR / Cas9 system.

[0037] Figure 7A shows the changes in TCR knockout efficiency before and after purification of U-CART constructed using the CRISPR / Cas12 system.

[0038] Figure 7B shows the changes in activity of U-CART constructed using the CRISPR / Cas12 system before and after purification.

[0039] Figures 8A and 8B show the comparison of U-CART and CAR-T constructed using the CRISPR / Cas12 system and T cell proliferation (2A) and viability (2B).

[0040] Figure 8C shows the changes in the long-term viability of U-CART constructed using the CRISPR / Cas12 system.

[0041] FIG9 shows the resistance of T cells from two donors to the cytotoxic effects of allogeneic T cells after being modified in different ways.

[0042] FIG10 shows the killing effect of NK cells on T cells derived from the same donor that have undergone different modifications.

[0043] Figures 11A and 11B show the killing effect of PBMC cells on T cells modified with different modifications.

[0044] FIG12 shows the FasL-mediated killing effect on T cells modified with different modifications.

[0045] Figure 13 shows the killing effect of differently modified T cells on target cells in vitro, where U-CART cells are constructed based on the CRISPR / Cas9 system.

[0046] Figure 14 shows the killing effect of differently modified T cells on target cells in vitro, where U-CART cells are constructed based on the CRISPR / Cas12 system.

[0047] Figure 15 shows the proliferation of CAR+ cells in T cells modified with different modifications when they are co-incubated with target cells in vitro.

[0048] FIG16 shows the results of in vivo mouse imaging.

[0049] FIG17 shows the killing effect of T cells modified with different methods on target cells in immunodeficient mice.

[0050] Detailed Description of the Invention

[0051] The inventors of this application have creatively provided a method for reducing the HVG effect of allogeneic T cells on engineered cells, which is different from knocking out MHC-I or inhibiting MHC-I expression and / or function. This method reduces HVG without increasing NK cell killing of the engineered cells. Specifically, this application also provides engineered T cells transformed by this method, methods for preparing such engineered T cells, and gRNAs and combinations thereof used in this method.

[0052] definition

[0053] The present application will be described in conjunction with specific embodiments and with reference to certain drawings, but the application is not limited thereto. Any reference signs in the claims should not be construed as limiting their scope. In the accompanying drawings, for illustrative purposes, the sizes of some elements may be exaggerated and not drawn to scale. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of a conflict, this document (including definitions) shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present application. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and embodiments disclosed herein are illustrative only and are not intended to be limiting.

[0054] As used herein, "SPPL3" stands for signal peptide peptidase-like 3, also known as IMP2, PSH1, or PSL4, and is a multi-spanning membrane protein located on the membranes of Golgi-associated vesicles, the plasma membrane, and the rough endoplasmic reticulum. SPPL3 has aspartic endopeptidase activity, intramembrane cleavage, and protein homodimerization activity. Many of its substrates are located in the Golgi apparatus and are involved in N- and O-linked sugar modifications and the biosynthesis of glycosaminoglycans. SPPL3 is essential for the cleavage and extracellular release of the lumenal domains of glycosyltransferases and glycosidases. Shedding of sugar-modifying enzymes impairs their activity in the Golgi apparatus. Increased SPPL3 expression is associated with hypoglycosylation of many secretory and membrane proteins; decreased SPPL3 expression is associated with hyperglycosylated proteins. In the absence of SPPL3, researchers noted an upregulation of neolactose-series glycosphingolipids (GSLs) on the cell surface, which in turn hindered the interaction of antibodies and receptors with HLA class I (HLA-I) glycoproteins and reduced CD8+ T cell activation. SPPL3 also has non-proteolytic functions, including interacting with stromal interaction molecule 1 (STIM1) and Orai1, enhancing TCR signaling to significantly induce calcium influx and NFAT activation, which are critical for lymphocyte signaling. An exemplary SPPL3 is human SPPL3, such as the SPPL3 with gene ID 121665 in the NCBI database. In some embodiments, the gene sequence of human SPPL3 is as shown in NC_000012.12 at positions 120762510 to 120904358, i.e., positions 120762510 to 120904358 of chromosome 12 of the GRCh38.p14 reference genome.

[0055] "CRISPR system" or "CRISPR / Cas system" refers collectively to transcripts and other elements that participate in the expression of CRISPR-associated ("Cas") genes and / or direct their activity. For example, a CRISPR / Cas system may include a sequence encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-partner sequence (e.g., a partial direct repeat comprising a "direct repeat" and tracrRNA processing in an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in an endogenous CRISPR system), and other sequences and transcripts derived from the CRISPR locus.

[0056] In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Perfect complementarity is not necessarily required if there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. A CRISPR complex can comprise a guide sequence that hybridizes to the target sequence and complexes with one or more Cas proteins.

[0057] The term "guide sequence" refers to a contiguous nucleotide sequence within a guide RNA that is partially or fully complementary to a target sequence within a 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.

[0058] The term "guide RNA" is used interchangeably with gRNA herein and refers to a molecule based on nucleic acid, including but not limited to a sequence (e.g., a guide sequence or a spacer) that can form a protein-RNA complex with the Cas protein and that comprises a sequence that is fully complementary to the target sequence, to hybridize with the target sequence and guide the Cas protein-RNA complex to specifically bind to the target sequence. In some embodiments, the gRNA includes or is crRNA. In some embodiments, the gRNA includes two RNA chains, wherein the spacer sequence and the direct repeat (DR) sequence are in different RNA chains, e.g., crRNA chain and tracrRNA chain. In some embodiments, the gRNA is an RNA chain, e.g., sgRNA.

[0059] The terms "single guide RNA," "synthetic guide RNA," and "sgRNA" are used interchangeably to refer to a polynucleotide sequence comprising a guide sequence and any other sequences that are necessary for sgRNA function 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 a tracrRNA and a tracr partner sequence derived from a crRNA. The tracr sequence may comprise all or part of the sequence of a tracrRNA from a naturally occurring CRISPR / Cas system. The term "guide sequence" is the nucleotide sequence that specifies a target site in a guide RNA and is used interchangeably with the terms "guide" or "spacer." The term "tracr partner sequence" may also be used interchangeably with the term "direct repeat." As used herein, "sgRNA" iBAR ” refers to a single guide RNA with an iBAR sequence.

[0060] As used in this application, "universal CAR-T" or "U-CART" cells refer to CAR-T (or CART) cells whose GvHD and / or HVG effects are reduced relative to allogeneic individuals. The reduction can be achieved by, for example, eliminating or reducing TCR protein expression and / or function. In some embodiments, the U-CART is a type of engineered T cell provided herein. The term "CAR-T" or "CART" cell refers to a T cell that expresses and / or contains a chimeric antigen receptor (CAR) on the cell membrane.

[0061] As used herein, the term "wild type" is a term understood by those skilled in the art and refers to the typical form of an organism, strain, gene or trait as it occurs in nature, as distinguished from mutant or variant forms.

[0062] As used herein, the term "variant" is understood to mean a display that exhibits properties that deviate from the naturally occurring pattern.

[0063] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence through traditional Watson-Crick base pairing or other non-traditional types. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to 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 refers to two nucleic acids that hybridize under stringent conditions.

[0064] As used herein, "stringent conditions" for hybridization refer to conditions under which nucleic acids having complementarity with a target sequence primarily hybridize to the target sequence and substantially do not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary according to numerous factors. Generally, the longer the sequence, the higher the temperature at which the sequence will specifically hybridize to 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 1, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0065] "Hybridization" refers to the reaction in which one or more polynucleotides form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can comprise a double strand forming a double helix structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can constitute a step in a broader process, such as the initiation of PCR, or the cutting of a polynucleotide by an enzyme. A sequence that can hybridize to a given sequence is referred to as the "complementary sequence" of a given sequence.

[0066] As used herein, "construct" refers to a nucleic acid molecule (for example, DNA or RNA), or a vector capable of delivering such nucleic acid molecules. For example, when used in the context of gRNA or sgRNA, construct refers to a gRNA or sgRNA molecule, a nucleic acid molecule encoding gRNA or sgRNA (for example, isolated DNA or viral vector), or a vector capable of delivering a nucleic acid molecule encoding gRNA or sgRNA, such as a slow virus carrying a nucleic acid molecule encoding gRNA or sgRNA. When used in the context of protein, construct refers to a nucleic acid molecule comprising a nucleotide sequence that can be transcribed into RNA or expressed as a protein. Construct may include the necessary regulatory elements operably connected to a nucleotide sequence, and when construct is present in a host cell, the regulatory element allows transcription or expression of the nucleotide sequence. As used herein, "operably connected" refers to that the expression of a gene is under the control of a regulatory element (for example, a promoter) connected to its space. Regulatory elements may be located at 5' (upstream) or 3' (downstream) of the gene under its control. The distance between a regulatory element (e.g., a promoter) and a gene can 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 is derived from the gene. As is known in the art, changes in this distance can be accommodated without losing the function of the regulatory element (e.g., a promoter). The term "vector" is used to describe a nucleic acid molecule that can be engineered to contain one or more polynucleotides of a clone 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 containing one or more free ends, no free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other polynucleotide species known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, such as by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." A recombinant expression vector may comprise a nucleic acid of the present application in a form suitable for expressing the nucleic acid in a host cell, meaning that the recombinant expression vector includes one or more regulatory elements that can be selected for expression based on the host cell, i.e., are operably linked to the nucleic acid sequence to be expressed.

[0067] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a eukaryotic cell that can be cultured in vitro and modified using the methods described herein. The term "cell" includes the primary subject cell and its progeny.

[0068] As used herein, the term "autologous" means any material from the same individual that is later reintroduced into that individual.

[0069] "Allogeneic" refers to a transplant from different individuals of the same species. "Allogeneic T cells" refers to T cells from a donor that have a tissue human leukocyte antigen (HLA) type that matches the recipient. Typically, matching is based on the variability of three or more loci of the HLA gene, and preferably perfectly matches at these loci. In some cases, allogeneic transplant donors may be related (usually siblings with close HLA matching), syngeneic (monozygotic "identical" twins of the patient), or unrelated (donors with no blood relationship and a very close HLA matching degree). HLA genes are divided into two categories (type I and type II). In general, mismatching of type I genes (i.e., HLA-A, HLA-B, or HLA-C) can increase the risk of transplant rejection. Mismatching of type II HLA genes (i.e., HLA-DR or HLA-DQB1) can increase the risk of graft-versus-host disease (GvHD).

[0070] The term "donor subject" or "donor" refers herein to a subject whose cells are obtained for further in vitro engineering. A donor subject can be a patient to be treated with a cell population produced by the methods described herein (i.e., an autologous donor), or can be an individual who donates a blood sample (e.g., a lymphocyte sample) that will be used to treat a different individual or patient (i.e., an allogeneic donor) after a cell population is produced by the methods described herein. Those subjects who receive modified cells (modified immune cells as described herein or prepared by the present methods) can be referred to as "recipients" or "recipient subjects."

[0071] As used herein, the "phenotype" of a cell refers to an observable characteristic or property of a cell, such as its morphology, development (e.g., growth, proliferation, differentiation, or death), homeostasis, biochemical or physiological properties, phenology, or behavior. The phenotype may result from the expression of genes in the cell, the influence of environmental factors, or the interaction between the 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 the body. In some embodiments, the phenotype is death. In some embodiments, the phenotype is the effector function of an immune cell (e.g., cytokine release and / or cytotoxic killing), or a reduction or absence of effector function.

[0072] As used herein, the term "stimulation" refers to a primary response induced by connecting a cell surface moiety. For example, in the context of a receptor, this stimulation requires the connection of the receptor, and subsequent signal transduction events. With regard to the stimulation of T cells, this stimulation refers to the connection of a T cell surface moiety, which in one embodiment subsequently induces a signal transduction event, such as binding to a TCR / CD3 complex. In addition, the stimulation event can activate the cell and upregulate or downregulate the expression or secretion of a molecule, such as downregulating TGF-β. Therefore, even in the absence of a direct signal transduction event, the connection of the cell surface moiety may also lead to the reorganization of the cytoskeletal structure, or the aggregation of the cell surface moieties, each of which can be used to enhance, modify or alter subsequent cellular responses.

[0073] As used herein, the term "activated" refers to a state in which cells have sufficiently ligated cell surface moieties to induce significant biochemical or morphological changes. In the context of T cells, such activation refers to a state in which T cells have been sufficiently stimulated to induce cell proliferation. Activation of T cells may also induce cytokine production and regulatory or cytolytic effector functions. In the context of other cells, the term infers the upregulation or downregulation of specific physicochemical processes. The term "activated T cell" refers to a T cell that is currently undergoing cell division, cytokine production, regulatory or cytolytic effector functions, and / or has recently undergone an "activation" process.

[0074] As used herein, an "isolated" nucleic acid molecule is one that has been identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it was produced. Preferably, the isolated nucleic acid is not bound to all components associated with the production environment. An isolated nucleic acid molecule encoding a polypeptide herein is in a form different from the form or environment in which it is found in nature. Thus, an isolated nucleic acid molecule is different from a nucleic acid encoding a polypeptide herein that is naturally present in a cell.

[0075] Unless otherwise indicated, a nucleotide sequence "encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that a nucleotide sequence encoding a protein may contain one or more introns in some versions.

[0076] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell (e.g., an immune cell). A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include primary subject cells and their progeny.

[0077] As used herein, the term "CAR", i.e., a 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 selected from one or more of the following groups: an 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 from any one 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 from CD8α. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain from a molecule selected from any one of the group consisting of CD3ζ, CD3γ, CD3ε, CD3δ, FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, CD66d, FcγRIIa, DAP10, and DAP 12. In some embodiments, the primary intracellular signaling domain is from CD3ζ. In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain from one or more costimulatory molecules selected from the group consisting of 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, a ligand that specifically binds to CD83, and any combination thereof. In some embodiments, the costimulatory signaling domain is derived from 4-1BB. In some embodiments, CAR further comprises a hinge domain 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.

[0078] As used herein, an engineered "TCR" is an engineered T cell receptor comprising: (a) an extracellular ligand binding domain comprising 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) optionally a first linker; (c) optionally an extracellular domain of a first TCR subunit (e.g., Cα, Cβ, Cδ, Cγ, CD3ε) or a portion thereof; (d) a transmembrane domain of a second TCR subunit (e.g., TCRα, TCRβ); and (e) an intracellular signaling domain comprising the intracellular signaling domain of a third TCR subunit (e.g., TCRα, TCRβ); wherein the first, second, and third TCR subunits are independently selected from any one of the following groups: 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 TCR further comprises a hinge domain 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 from CD8α.

[0079] As used herein, "TAC" refers to a T cell antigen conjugate 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 cytosolic 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 comprising 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) optionally a first linker; (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) optionally a second linker; (e) optionally a first TCR co-receptor (e.g., CD3ε). 4, CD8) extracellular domain or a portion thereof; (f) a transmembrane domain comprising a transmembrane domain of a second TCR co-receptor (e.g., CD4, CD8); and (g) optionally an intracellular signaling domain comprising an intracellular signaling domain of a third TCR co-receptor (e.g., CD4, CD8); wherein the TCR subunit is selected from any one or more of the following groups: CD3ε, CD3δ, CD3γ, TCRα, TCRβ, TCRγ and TCRδ; and wherein the first, second and third TCR co-receptors are each independently selected from any one of the following groups: CD4, CD8 and CD28. In some embodiments, the first, second and third TCR co-receptors are the same. In some embodiments, the first, second and third TCR co-receptors are different. In some embodiments, the TAC further comprises a hinge domain (e.g., from CD8α) located between the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain.

[0080] As used herein, "treatment" is a method for obtaining a beneficial or desired result including a clinical outcome. For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the degree of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing down the progress of the disease, improving the disease state, providing relief (partial or complete), reducing the dosage of one or more other drugs required for treating the disease, delaying the progress of the disease, improving the quality of life and / or prolonging survival. "Treatment" also includes reducing the pathological consequences of cancer or immune diseases.

[0081] The term "effective amount" as used herein refers to an amount sufficient to treat a particular disorder, condition or disease, such as an amount of a medicament (e.g., a modified immune cell as described herein or a pharmaceutical composition thereof) that improves, alleviates, reduces and / or delays one or more symptoms thereof (e.g., cancer, infectious disease, GvHD, transplant rejection, autoimmune disease or radiation sickness). When referring to cancer, an effective amount includes an amount sufficient to shrink the tumor and / or reduce the tumor growth rate (e.g., inhibit tumor growth) or prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An effective amount of an agent (e.g., a modified immune cell) or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down, and preferably prevent cancer cell infiltration into peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and preferably stop to a certain extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent. In the case of infectious diseases, such as viral infections, a therapeutically effective amount of the modified immune cells described herein or their compositions can reduce the number of cells infected by pathogens; reduce the production or release of pathogen-derived antigens; inhibit (i.e., slow down and preferably stop to a certain extent) the spread of pathogens to uninfected cells; and / or alleviate one or more symptoms associated with infection to a certain extent. In some embodiments, a therapeutically effective amount is an amount that prolongs patient survival.

[0082] As used herein, "individual" or "subject" refers to a mammal, including but not limited to humans, cows, horses, felines, canines, rodents, or primates. In some embodiments, the individual is a human.

[0083] As used herein, "patient" includes any person suffering from a disease (eg, cancer). The terms "subject," "individual," and "patient" are used interchangeably herein.

[0084] "Multiplicity of infection" or "MOI" are used interchangeably herein and refer to the ratio of a pathogen (e.g., a phage, virus, or bacterium) to its target (e.g., a cell or organism) for infection. For example, when referring to a group of cells inoculated with viral particles, the multiplicity of infection or MOI refers to the ratio between the number of viral particles (e.g., viral particles containing a sgRNA library) and the number of target cells present in the mixture during viral transduction.

[0085] Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps.

[0086] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0087] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, description referring to "about X" includes description of "X."

[0088] As used herein, reference to "other than" a value or parameter generally means and describes "other than" that value or parameter. For example, the method is not intended to treat type X cancer, which means that the method is intended to treat other types of cancer besides type X.

[0089] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0090] For the description of the numerical range of nucleotides herein, each intermediate number therebetween is explicitly considered. For example, for the range of 19-21nt, the number 20nt is also considered in addition to 19nt and 21nt, and for the range of MOI, each intermediate number therebetween is explicitly considered, whether it is an integer or a decimal.

[0091] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0092] One of ordinary skill in the art will understand that both uracil and thymine can be represented by "t", rather than using "u" to represent uracil and "t" to represent thymine; in the context of RNA, unless otherwise specified, it will be understood that "t" is used to represent uracil.

[0093] In this application, unless otherwise specified, when referring to genomic loci of human genes, the reference genome used is GRCh38.p14.

[0094] Engineered T cells

[0095] In one aspect, the present application provides an engineered T cell that has been modified to eliminate or reduce (e.g., reduce at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of any one) the expression and / or function of an endogenous T cell receptor (TCR) protein and / or SPPL3 protein. In some embodiments, the T cell is an αβT cell that has been modified to eliminate or reduce the expression and / or function of the α subunit (TCRα) and / or the β subunit (TCRβ) of the endogenous T cell receptor (TCR). In some embodiments, the T cell is an αβT cell that has been modified to eliminate or reduce the expression and / or function of the TCRα conserved region (TRAC) gene or the TRAC gene or its expression and / or function. In some embodiments, the T cells are γδ T cells that have been modified to eliminate or reduce the expression and / or function of endogenous γ subunit (TCRγ) and / or T cell receptor (TCR) δ subunit (TCRδ).

[0096] In some embodiments, the engineered T cells have normal expression of HMC class I proteins. In some embodiments, the engineered T cells have normal expression of B2M, HLA-A, HLA-B, and HLA-C. In some embodiments, the engineered T cells are human T cells and have normal expression of HLA class I proteins.

[0097] In some embodiments, the activation-induced cell death (AICD) of the engineered T cells is reduced by at least about 10% (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), for example, by at least about 20%, compared to T cells that do not have reduced or eliminated endogenous TCR and / or SPPL3 protein. In some embodiments, the AICD of the engineered T cells is reduced by at least about 10% (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), for example, by at least about 20%, compared to engineered T cells that do not have reduced or eliminated expression (RNA and / or protein expression) and / or function of SPPL3 protein.

[0098] In some embodiments, the engineered T cells, after transplantation into an allogeneic recipient, have at least about 10% (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) reduced HVG upon challenge with allogeneic T cells, e.g., by at least about 20%, compared to T cells that do not have reduced or eliminated endogenous TCR and / or SPPL3 protein. In some embodiments, the engineered T cells have at least about 10% (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) reduced HVG upon challenge with allogeneic T cells, e.g., by at least about 20%, compared to engineered T cells that do not have reduced or eliminated expression (RNA and / or protein expression) and / or function of SPPL3 protein.

[0099] In some embodiments, the engineered T cells persist in vivo at least about 10% longer (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 1.5 fold, 2 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more) compared to engineered T cells that do not have reduced or eliminated expression (RNA and / or protein expression) and / or function of endogenous TCR proteins and / or SPPL3 proteins.

[0100] In some embodiments, the engineered T cells of the present application have no significant difference in the degree of HVG caused by attack by allogeneic T cells compared to T cells in which the expression and / or function of MHC class I molecules and TCR proteins is reduced or eliminated, and the expression and / or function of SPPL3 protein is not reduced or eliminated; and the engineered T cells of the present application have a reduced or eliminated expression and / or function of MHC class I molecules and TCR proteins, and the expression and / or function of SPPL3 protein is not reduced or eliminated, and the natural killing caused by attack by NK cells 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%. Therefore, in some embodiments, in an in vivo environment, in a peripheral blood environment, in the presence of PBMCs, or in the presence of NK cells and T cells, the extent of killing by the engineered T cells of the present application by NK cells and T cells is essentially the same as that by T cells in which the expression and / or function of MHC class I molecules and TCR proteins is reduced or eliminated, and the expression and / or function of SPPL3 proteins is not reduced or eliminated, or is reduced by at least about 10% (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%), for example, by at least about 20%. In some embodiments, compared to T cells in which the expression and / or function of MHC class I molecules and TCR proteins is reduced or eliminated, and the expression and / or function of SPPL3 proteins is not reduced or eliminated, the engineered T cells of the present application are maintained for substantially the same time in allogeneic recipients or in allogeneic blood or PBMCs, or have a longer persistence, that is, they may exist for no significant difference or for a longer time, for example, they maintain no significant difference or a longer half-life in vivo.

[0101] In some embodiments, the engineered T cells have or express engineered receptors. In some embodiments, the engineered T cells are further modified to express engineered receptors. In some embodiments, the engineered receptors are selected from one or more of the following: 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 multivalent. In some embodiments, the engineered receptors are monospecific, for example, monovalent and monospecific, or multivalent and monospecific. In some embodiments, the engineered receptors are multispecific (for example, bispecific).

[0102] 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 proteins and / or SPPL3 proteins and / or other proteins, and / or the immune cells modified to express engineered receptors can be autologous or allogeneic.

[0103] Methods for preparing engineered T cells

[0104] In a second aspect, the present application also provides a method for preparing the above-mentioned engineered T cells.

[0105] In some embodiments, expression of endogenous TCR proteins and / or SPPL3 proteins in the engineered T cells is reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or inhibited by antisense RNA, miRNA, siRNA, or shRNA that specifically recognizes RNA encoding endogenous TCR and / or SPPL3 proteins.

[0106] In some embodiments, the function of endogenous TCR proteins and / or SPPL3 proteins is reduced or inhibited by chemically modified mRNA, such as chemically modified mRNA of a dominant negative inhibitor (e.g., a dominant negative variant or fragment thereof, or a dominant negative binding partner) of endogenous TCR proteins and / or SPPL3 proteins. In some embodiments, immune cells are modified to express a dominant negative SPPL3 protein variant or a dominant negative fragment thereof. In some embodiments, immune cells are modified to express a dominant negative binding partner of SPPL3 protein.

[0107] In some embodiments, the expression (RNA and / or protein expression) and / or function of endogenous TCR proteins and / or SPPL3 proteins is reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or inhibited by a small molecule compound, nucleic acid (or a vector comprising the same), lipid and / or protein molecule.

[0108] In some embodiments, the engineered T cells are genetically modified at the DNA locus encoding the endogenous TCR protein and / or SPPL3 protein. In some embodiments, the immune cells are genetically modified at the SPPL3 locus. In some embodiments, the locus encoding the endogenous TCR protein and / or SPPL3 protein is modified with a mutagen. Mutagens can be divided into three categories: physical (e.g., gamma rays, ultraviolet (UV) radiation), chemical (e.g., ethyl methanesulfonate (EMS)), and transposable elements (e.g., transposons, retrotransposons, T-DNA, retroviruses). In some embodiments, the mutagen or condition is ionizing radiation (IR), ultraviolet radiation, an alkylating agent (such as nitrogen mustard, methyl methanesulfonate (MMS), EMS, N-ethyl-N-nitrosourea (ENU)), an aromatic amine (e.g., 2-aminofluorene), a polycyclic aromatic hydrocarbon (PAH; e.g., dibenzo[a,l]pyrene, naphthalene, anthracene, pyrene), cross-linking, insertional mutagenesis (e.g., mediated by a transposon or virus), or other toxins (e.g., aflatoxin, N-nitrosamine). In some embodiments, the endogenous TCR protein and / or SPPL3 protein locus is 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 extensive mutagenesis (REM). In some embodiments, gene editing is performed by PCR methods. In some embodiments, gene editing is performed by non-PCR methods. Any known gene editing method can be used herein, including but not limited to non-homologous end joining (NHEJ)-mediated, homology-directed repair (HDR)-mediated, zinc finger nuclease (ZFN)-mediated, transcription activator-like effector nuclease (TALEN)-mediated or CRISPR / Cas-mediated gene editing. HDR can occur non-conservatively or conservatively. In some embodiments, HDR is mediated by a single-strand annealing (SSA) pathway. In some embodiments, HDR is mediated by a classical double-strand break repair (DSBR) pathway, a synthesis-dependent chain annealing (SDSA) pathway, or a break-induced repair (BIR) pathway. In some embodiments, the cell modification method described herein further comprises introducing a nucleic acid template (e.g., containing a desired mutation), such as inserting at a double-strand break (DSB) to modify the target genomic sequence (e.g., by HDR). Gene editing can introduce one or more mutations into a DNA locus encoding one or more target proteins (e.g., SPPL3 protein), including but not limited to insertions, deletions, substitutions (e.g., non-synonymous substitutions), truncations, translocations, point mutations, and the like. 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 comprises gene knockout (KO).In some embodiments, gene editing includes base editing (e.g., introducing non-synonymous substitutions). In some embodiments, base editing introduces stop codons, which can reduce the expression of functional RNA and / or protein. In some embodiments, base editing introduces mutations that affect RNA and / or protein function. 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 comprising i) dead Cas protein (dCas) and ii) adenine base editor (ABE) or adenine deaminase (ADA), or cytidine base editor (CBE) or cytidine deaminase (CDA), or a functional fragment thereof. The cytidine base editor can convert the target C:G base pair to a T:A base pair, while the adenine base editor can convert the A:T base pair to a G:C base pair. In short, these two types of base editors are able to target and install all possible conversion mutations (C to T, G to A, A to G, T to C, C to U, and A to U).

[0109] In some embodiments, the engineered T cells are genetically modified at the RNA encoding endogenous TCR proteins and / or SPPL3 proteins. In some embodiments, the RNA encoding endogenous TCR proteins and / or SPPL3 proteins is modified by RNA editing. RNA editing can introduce one or more mutations into the RNA encoding endogenous TCR proteins and / or SPPL3 proteins, including but not limited to insertions, deletions, substitutions (such as non-synonymous substitutions), truncations, point mutations, etc. In some embodiments, the mutation is a frameshift mutation, a LOF mutation, a dominant negative mutation, a missense mutation, or a nonsense mutation. In some embodiments, RNA editing includes base editing (e.g., introducing non-synonymous substitutions, such as C to U, A to I). Any known RNA editing method can be used herein (see, e.g., 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), including but not limited to programmed RNA editing using endogenous ADARs (“LEAPER”; see, e.g., WO2020074001 and Qu et al. (Nat Biotechnol. 2019; 37(9): 1059-1069), or programmed A to I substitution RNA editing (“REPAIR”; see, 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”; see, for example, Merkle et al., “Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides,” Methods Mol Biol. 2021; 2181: 331-349), CRISPR-Cas-stimulated RNA targeting system (“CIRTS”; see, for example, Rauch et al., “Programmable RNA-Guided RNA Effector Proteins Built from Human Parts,” Cell. 2019; 178(1): 122-134.e12), RNA editing for specific CU exchange ("RESCUE"; see, e.g., Abudayyeh et al., "A cytosine deaminase for programmable single-base RNA editing," Science. 2019; 365(6451): 382-386), or CLUSTER (see, e.g., 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 of which are incorporated herein by reference in their entirety. In some embodiments, RNA editing is mediated by LEAPER. In some embodiments, RNA editing is mediated by CRISPR / Cas, such as by fusing an adenine base editor (ABE) or adenine deaminase (ADA), or a cytidine base editor (CBE) or cytidine deaminase (CDA), or a functional fragment thereof to a dead Cas (dCas, e.g., dCas13) protein.

[0110] Methods for making CRISPRs that recognize predetermined 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. So far, based on the outstanding functions and evolutionary modularity of the CRISPR-Cas system, two types (type 1 and type 2) and six types (I-VI) of CRISPR-Cas systems have been described. 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 which are incorporated herein by reference in their entirety. Among the Class 2 CRISPR-Cas systems, the Type II Cas9 system and the Type VA / B / E / J Cas12a / Cas12b / Cas12e / Cas12j system have been used for genome editing, providing broad prospects for biomedical research. Cas13a (C2c2) is a type VI-A RNA-guided RNA-targeting CRISPR effector that can be used for RNA editing as described herein. The CRISPR / Cas system used herein can produce double-strand breaks (DSBs) or single-strand breaks at predetermined nucleic acid sites. In some embodiments, the CRISPR / Cas system used herein is a CRISPR / Cas9 system. In some embodiments, the CRISPR / Cas system used herein is a CRISPR / Cas12 system.

[0111] In some embodiments, gene editing or RNA editing includes contacting the precursor T cells with the following substances under conditions that allow the introduction of gRNA constructs and optionally Cas components into precursor T cells: i) a guide RNA (gRNA) construct, wherein the gRNA construct comprises or encodes a gRNA comprising a guide sequence that is complementary to a target in a DNA locus or RNA encoding an endogenous TCR protein and / or SPPL3 protein; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein. In some embodiments, the precursor immune cells have expressed Cas proteins prior to the introduction of the gRNA construct. In some embodiments, the precursor immune cells do not express Cas proteins prior to the introduction of the gRNA construct and Cas components. In some embodiments, the precursor immune cells express an engineered receptor (e.g., CAR, engineered TCR, or TAC) prior to the introduction of the gRNA construct and / or Cas components. In some embodiments, the Cas protein has endonuclease activity. In some embodiments, the Cas protein is a fusion protein, such as a fusion protein comprising i) dCas (e.g., dCas13a, dCas9) and ii) ADA (e.g., TadA, such as 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 an sgRNA. In some embodiments, the gRNA comprises (or consists essentially of, or consists of) crRNA. In some embodiments, the gRNA comprises (or consists essentially of, or consists of) crRNA and tracrRNA.

[0112] In some embodiments, immune cells modified to eliminate or reduce (e.g., reduce at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) endogenous TCR proteins and SPPL3 have been modified or further modified to eliminate or reduce (e.g., reduce at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) one or more selected Expression (RNA and / or protein expression) and / or function of other proteins from the following group: PD-1, TIM-3, LAG-3, CTLA-4, CISH, SPPL3, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and ligands of NKp46. In some embodiments, the T cells have normally expressed HLA-I, and the normally expressed HLA-I does not result in killing of NK cells.

[0113] In some embodiments, the engineered T cells are further modified to express an engineered receptor. In some embodiments, the immune cells have already expressed an engineered receptor before being modified to reduce or eliminate the expression and / or function of endogenous TCR proteins and / or SPPL3 proteins. In some embodiments, the immune cells are modified to reduce or eliminate the expression and / or function of endogenous TCR proteins and / or SPPL3 proteins and are further modified to express an engineered receptor. In some embodiments, the modification to reduce or eliminate the expression and / or function of endogenous TCR proteins and / or SPPL3 proteins 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 endogenous TCR proteins and / or SPPL3 proteins and the modification to express the engineered receptor occur sequentially. Any engineered receptor that can transduce signals to immune cells (e.g., induce cell proliferation, cytokine production, and / or perform regulatory or cytolytic effector functions) and / or recognize 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 conjugate (TAC). In some embodiments, the engineered receptor is monovalent. In some embodiments, the engineered receptor is multivalent. In some embodiments, the engineered receptor is monospecific, for example, monovalent and monospecific, or multivalent and monospecific. In some embodiments, the engineered receptor is multispecific (for example, bispecific).

[0114] In some embodiments, the modification that reduces or eliminates the expression (RNA and / or protein expression) and / or function of endogenous TCR proteins and / or SPPL3 proteins does not downregulate or eliminate the expression (RNA and / or protein expression) and / or function of the engineered receptor (e.g., CAR). In some embodiments, the modification that reduces or eliminates the expression and / or function of endogenous TCR proteins and / or SPPL3 proteins downregulates 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 any one of less).

[0115] In some embodiments, the engineered receptor (e.g., CAR), gRNA construct or arRNA construct and / or Cas components are introduced into precursor T cells by transduction / transfection of nucleic acid (DNA or RNA) or a vector encoding the same (e.g., a non-viral vector, or a viral vector such as a lentiviral vector), or a virus comprising nucleic acid encoding the same (e.g., a lentivirus). In some embodiments, the engineered receptor (e.g., CAR), gRNA construct or arRNA construct and / or Cas components are introduced into precursor T cells by inserting proteins into the cell membrane while passing the cells through a microfluidic system such as a CELL The Cas component (e.g., Cas9 protein) is introduced into precursor T cells (e.g., see, U.S. Patent Application No. 20140287509).

[0116] The method of introducing a vector (e.g., a viral vector) or an isolated nucleic acid into a mammalian cell is known in the art. Nucleic acid or vectors as described herein can be transferred into T cells by physical, chemical or biological methods. Physical methods for introducing a vector (e.g., a viral vector) into a cell include calcium phosphate precipitation, lipofection, gene gun method, microinjection, electroporation, etc. The method of producing a cell comprising a vector and / or exogenous nucleic acid is well known in the art. In some embodiments, a vector (e.g., a viral vector) is introduced into a cell by electroporation. The biological method of introducing a vector into a cell includes the use of DNA and RNA vectors. The chemical means of introducing a vector (e.g., a viral vector) into a cell include colloidal dispersion systems, e.g., macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0117] In some embodiments, RNA molecules (for example, gRNA, arRNA or encoding Cas mRNA) can be prepared by conventional methods (for example, in vitro transcription) and then introduced into T cells by known methods such as electroporation. In some embodiments, the viral vector (lentiviral vector) or virus (for example, lentivirus) comprising the nucleic acid encoding any engineered receptor (for example, CAR), gRNA or arRNA and / or Cas protein as described herein is at least about 1, such as 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. Any one MOI (such as with an MOI of about 3) is contacted with precursor T cells.

[0118] In some embodiments, the transduced / transfected T cells are proliferated in vitro after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced / transfected T cells are cultured to proliferate for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days, for example, 7 days. In some embodiments, the transduced / transfected T cells are further evaluated or screened to purify the T cells described herein.

[0119] Reporter genes can be used to identify cells of potential transfection / transduction and to evaluate the function of regulatory sequences. Generally speaking, reporter genes are genes that are not present in or are not expressed by receptor organisms or tissues and encode polypeptides, the expression of which is demonstrated by some easily detectable properties, for example, enzymatic activity. After DNA / RNA is introduced into the receptor cells, the expression of the reporter gene is measured at the appropriate time. Suitable reporter genes can include: genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory 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 or commercially available using known techniques. Antibiotic selection markers can also be used to identify cells of potential transfection / transduction.

[0120] Other methods for confirming the presence of any nucleic acid described herein (e.g., gRNA construct or sgRNA construct) or mutation (e.g., inactivating mutation) in the target gene of the engineered T cells of the present application include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, PCR, DNA-seq or RNA-seq; biochemical analysis, such as immunological methods (e.g., ELISA and Western blotting), fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) to detect the presence or absence of specific peptides.

[0121] Methods for extending the in vivo half-life of engineered cells

[0122] Typically, after entering a patient's body, allogeneic engineered cells are often cleared from the donor due to conditions such as GvHD, AICD, and HVG. Consequently, they have poor in vivo persistence and insufficient time to function in the patient, limiting their clinical effectiveness. A common approach involves knocking out MHC class I molecules in allogeneic engineered cells to prevent HVG. However, knocking out MHC class I molecules can lead to NK cells recognizing and attacking cells that downregulate MHC class I molecules.

[0123] The present application provides a new method for extending the half-life of engineered T cells in vivo, which can avoid or reduce NK cell attack caused by the reduction of MHC class I molecular weight while eliminating HVG, thereby extending the half-life of the engineered cells in vivo by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 time, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times or more) relative to unengineered T cells. In addition, in some embodiments, the method of the present application can achieve similar or better effects compared to methods for extending the half-life of engineered cells in vivo by eliminating or reducing MHC class I molecules. In some embodiments, relative to a method for extending the in vivo half-life of engineered cells by eliminating or reducing MHC class I molecules, the method of the present application may be that the degree of HVG received by the engineered T cells remains unchanged, but the natural killing caused by NK cell attack received 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%.

[0124] In some embodiments, the engineered cell is an immune cell or a precursor cell thereof. In some embodiments, the immune cell is selected from one or more of the following groups: T cells, B cells, NK cells, macrophages, and DC cells. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell, and it is modified to eliminate or reduce the expression and / or function of endogenous TCR proteins. In some embodiments, the engineered cell expresses or has one or more engineered receptors selected from the following: CAR, TCR, and TAC. In some embodiments, the engineered T cell is a CAR-T cell, a TCR-T cell, or a TAC-T cell. In some embodiments, the engineered receptor is monovalent. In some embodiments, the engineered receptor is multivalent. In some embodiments, the engineered receptor is monospecific, for example, monovalent and monospecific, or multivalent and monospecific. In some embodiments, the engineered receptor is multispecific (for example, bispecific).

[0125] Specifically, the method comprises modifying the engineered cells to eliminate or reduce the expression and / or function of the SPPL3 protein or its functional fragment. In some embodiments, the method comprises any of the above methods for preparing engineered T cells.

[0126] guide RNA

[0127] The present application also provides guide RNAs (e.g., sgRNAs) for CRISPR / Cas systems and constructs encoding CRISPR / Cas guide RNAs to target the gene SPPL3 by cleavage or base editing and generate mutations, particularly frameshift mutations, in SPPL3, referred to as gRNAs targeting SPPL3. The sgRNAs provided herein are used to knock out SPPL3 in engineered cells, and engineered cells that do not express SPPL3 are screened using SPPL3 as a marker. After culturing the screened engineered cells for several days (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 days), the knockout efficiency of SPPL3 still reaches 99.37%. Furthermore, the guide RNAs provided herein do not cause detectable off-target effects. In some embodiments, the guide RNAs provided herein comprise guide sequences as shown in any one of SEQ ID NOs: 6 to 256 or SEQ ID NOs: 261 to 316.In some embodiments, the guide RNA provided herein comprises 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: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285 NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:312 and SEQ ID NO:315.In some embodiments, the guide RNA provided herein comprises SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 161, 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: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294 The guide sequence is set forth in any one of SEQ ID NO: 293 and SEQ ID NO: 294. In some embodiments, the present application also provides a combination of guide RNAs comprising the aforementioned gRNA targeting SPPL3 and a gRNA targeting a TCR gene. The TCR gene is selected from, for example, one or more of the following: TCRα, TCRβ, TCRγ, and TCRδ. In some embodiments, the gRNA targeting a TCR gene targets the TRAC gene.

[0128] In this application, gRNA "targets" a gene, which means that the guide sequence in the gRNA can complementarily bind to the coding and / or non-coding regions of the gene, or the mRNA or pre-mRNA of the gene.

[0129] Depending on the CRISPR / Cas system, gRNA may include other components in addition to the guide sequence. The other components include, for example, additional sequence elements that promote 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 pairing sequence fused to the tracr sequence, and the tracr sequence is complementary to the tracr pairing sequence through the loop region. In some embodiments, the gRNA (e.g., sgRNA) includes a crRNA comprising a direct repeat (DR) sequence capable of interacting with the Cas protein. In some embodiments, the gRNA (e.g., sgRNA) includes crRNA and tracrRNA, which may be on the same RNA chain (e.g., forming an sgRNA), or on two RNA chains. The DR sequence may be derived from a DR sequence naturally 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 comprises one or more mutations relative to a reference (e.g., wild-type) DR sequence, for example, one or more mutations comprising 5' and / or 3' extensions, 5' and / or 3' truncations, nucleotide insertions, nucleotide deletions, nucleotide substitutions, or a combination thereof, relative 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 partially or completely substantially complementary to and hybridizes with the anti-repeat sequence of the DR sequence to form a repeat: anti-repeat duplex (also referred to as a "stem"). In some embodiments, the length of the repeat: anti-repeat duplex is about 6 to 20 nucleotides, such as about any one of 6, 8, 10, 12, 14, 16, 18, 20 nucleotides, or longer. In some embodiments, the repeat sequence of the DR sequence and the anti-repeat sequence of the DR sequence are directly or indirectly connected via a linker, such as a loop sequence. In the case where the repeat sequence and the anti-repeat sequence are indirectly connected via a linker, the length of the repeat: anti-repeat duplex 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 connected via a loop sequence. In some embodiments, the loop sequence is about 4 to about 10 nucleotides in length, for example, any one of about 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the loop sequence comprises a sequence of GAA. In some embodiments, the DR sequence of the gRNA comprises a stem loop. In some embodiments, the DR sequence comprises one or more stem loops, such as about 1 to about 5 stem loops.

[0130] Typically, in the context of an endogenous CRISPR / Cas9 system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of) the target sequence. A tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., any of about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridizing along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity with the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex. As with the target sequence, it is believed that full complementarity is not required, as long as there is sufficient functionality. In some embodiments, when optimally aligned, the tracr sequence has at least about any of 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence. Determining optimal alignment is within the capabilities of those skilled in the art. For example, there are publicly and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in Matlab, Bowtie, Geneious, Biopython, and SeqMan. In some embodiments, the length of the tracr sequence is about or more than about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides. Any known tracr mate sequence and tracr sequence from a naturally occurring CRISPR system can be used, such as a tracr mate sequence and tracr sequence from the S. pyogenes CRISPR / Cas9 system, such as those described in US8697359 and herein.

[0131] In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript such that hybridization between the two produces a transcript with a secondary structure, such as a stem-loop (also known as a hairpin), termed a "repeat-anti-repeat stem-loop."

[0132] "Stem-loop" refers to a nucleic acid with a secondary structure comprising a region of nucleotides that are known or predicted to pair and form a duplex (stem portion) connected by an unpaired single-stranded nucleotide region (loop portion). The terms "hairpin," "hairpin loop," and "foldback" structures are also used herein to refer to stem-loops. Such structures are well known in the art, and these terms are used according to 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 include one or more base mispairings. In addition, the base pairing may be precise, i.e., not include any mispairings.

[0133] In some embodiments, the sgRNA comprises at least one, two, or more stem-loops, such as 2, 3, 4, or 5 stem-loops. In some embodiments, the sgRNA has up to 5 hairpins. In some embodiments, the sgRNA construct further comprises a transcription termination sequence, such as a polyT sequence, for example, 6 T nucleotides.

[0134] In some embodiments, wherein the Cas protein is Cas9 or Cas12, each sgRNA comprises a guide sequence fused to a second sequence comprising a repeat-anti-repeat stem loop that interacts with Cas9 or Cas12. An invariant guide RNA hairpin sequence can be provided according to common knowledge 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 they are capable of binding to the Cas nuclease after transcription.

[0135] In some embodiments, the sgRNA comprises, from 5' to 3', a guide sequence, a repeat-anti-repeat stem-loop. In some embodiments, the sgRNA comprises, from 5' to 3', a guide sequence, a repeat-anti-repeat stem-loop, and stem-loop 1, stem-loop 2, and / or stem-loop 3.

[0136] In addition, the present application also provides a CRISPR system comprising the above-mentioned gRNA or its construct, and a CAS enzyme or its encoding nucleic acid or construct. In some embodiments, the CRISPR system is a CRISPR / Cas9 system. In some embodiments, the CRISPR system is a CRISPR / Cas12 system. Example

[0137] The following examples and exemplary embodiments are intended to be purely exemplary of the present application and, therefore, should not be considered to limit the present application in any way.The following examples and detailed description are provided by way of illustration and not by way of limitation.

[0138] In addition, as used in the following examples, the abbreviations and corresponding meanings of cell treatments are as follows:

[0139] TSKO: TCR and SPPL3 double knockout;

[0140] TKO: TCR knockout;

[0141] TBKO: TCR and B2M gene double knockout;

[0142] TFKO: TCR and Fas gene double knockout;

[0143] TBSKO: triple knockout of TCR, B2M and SPPL3 genes.

[0144] Example 1 Preparation of U-CART cells

[0145] 1. Isolation and Activation of Healthy Donor T Cells

[0146] Collection of umbilical cord blood from healthy donors: After obtaining the umbilical cord blood from the blood bank, it is transported to the GMP laboratory via a cold chain logistics transfer vehicle equipped with constant temperature equipment for T cell separation.

[0147] 1.1 Preparation of peripheral blood mononuclear cells

[0148] Use a pipette to draw up physiological saline and add it to the transported umbilical cord blood. Dilute the umbilical cord blood and physiological saline at a ratio of 1:1 (V / V). Slowly add the blood cell dilution solution to the lymphocyte separation tube. After centrifugation at 800×g for 20 minutes, aspirate the buffy coat cells above the lymphocyte separation solution and transfer it to a new 50 ml centrifuge tube. Add T cell culture medium (Miltenyi Biotec, 170-076-306) and centrifuge at 400×g for 5 minutes. Discard the supernatant and retain the cell pellet at the bottom of the centrifuge tube to obtain peripheral blood mononuclear cells.

[0149] 1.1.2 Isolation and activation of T cells

[0150] The peripheral blood mononuclear cells were counted using a cell counter (Nexcelom, model: Cellometer K2) and then T cells were sorted. The specific steps were as follows: the cell pellet was adjusted to a density of 5*10 7 / ml, transfer the cells to a 5ml flow cytometry tube using a 5ml pipette. Add T cell isolation reagent at a concentration of 50ul / ml and incubate at room temperature for 5 minutes. Add sorting magnetic beads, mix thoroughly within 30 seconds, and add to a concentration of 40μl / ml. Make up the cell suspension to 2.5ml with Easy Buffer and place directly on the magnetic column for 3 minutes. Then, transfer the cells into a 15ml centrifuge tube to obtain T cells. After sorting, mix thoroughly with a 1000μl pipette, count the cells, centrifuge (400×g, 5min), and remove the supernatant to obtain the T cell pellet. Resuspend the T cell pellet in T cell culture medium. Then, add T cell activating factor (magnetic beads coated with anti-CD3 / CD28 antibodies) at a 1:1 ratio. The T cells are now activated and placed in a 37°C, 5% CO2 incubator for further expansion. The activated T cells are transduced with the CAR molecule using a lentiviral vector. The CAR molecule used in the examples of the present application is a CAR that specifically binds to CD19. The extracellular antigen binding domain used is the scFV of the anti-CD19 monoclonal antibody FMC63, whose hinge region and transmembrane region are respectively the hinge region and transmembrane region of the CD8α molecule, and the intracellular signaling domain is composed of a 4-1BB costimulatory domain and a CD3Z tandem structure.

[0151] 1.2 Gene knockout of T cells

[0152] Use CRISPR / Cas9 gene editing technology to knock out the TRAC, B2M, and SPPL3 genes in the T cells obtained in step 1.1.2. The specific steps are as follows:

[0153] 1.2.1 Design of sgRNA and plasmid construction targeting the TCR α chain constant coding region (i.e., TRAC) gene, HLA constant coding region B2M gene, and SPPL3 constant coding region gene.

[0154] The sgRNAs designed for the coding sequences of each exon of the TRAC, B2M and SPPL3 coding regions were all designed using CRISPR RGEN Tools. For the CRISPR / Cas9 system, the sequences of the sgRNA targeting regions are shown in the sequence listing at the end of the article as SEQ ID NOs: 1-256, wherein the sgRNAs represented by 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: 210, SEQ ID NO: 212, SEQ ID NO: 241 and SEQ ID NO: 250 are used to target the complementary sequence of the SPPL3 genomic region. The sgRNA represented by NO: 2 is used to target the complementary sequence of the TRAC genomic region; for the CRISPR / Cas12 system, the sequences of the sgRNA targeting regions are shown in the sequence listing at the end of the article as SEQ ID NO: 257-316, among which the sgRNA represented by SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, and SEQ ID NO: 264 are used to target the complementary sequence of the SPPL3 genomic region, and the sgRNA represented by SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, and SEQ ID NO: 260 are used to target the complementary sequence of the TRAC genomic region.

[0155] 1.2.2 Chemically modify sgRNAs with 2'-O-methylation and / or internucleotide phosphorothioate to produce sgRNAs with high knockout efficiency and stability.

[0156] 1.2.3 Take the Cas9 plasmid and GFP plasmid, Xba1 (manufacturer: NEB, cat#R0145S), and cutsmart buffer (manufacturer: NEB, cat#B7204s) for enzyme digestion and linearization. The 50 μl reaction system is shown in Table 1.

[0157] Table 1:

[0158] Incubate in a 37°C water bath for 4 hours, take 2 μl of the digested product and perform agarose gel electrophoresis with the voltage set to 110 U for 30 minutes. A single band was observed under a gel imager, indicating that the digestion was complete and all plasmids were linearized.

[0159] The above reaction product is taken and cleaned and purified.

[0160] The purified product was taken and in vitro transcription (ie, IVT) was performed using HISCRIBE™ T7 ARCA mRNA Kit (manufacturer: NEB, cat# E2060S). The 20 μl system is shown in Table 2.

[0161] Table 2:

[0162] The 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.

[0163] Take the above reaction product and perform the operations shown in Table 3.

[0164] Table 3:

[0165] The reaction system was placed in a PCR instrument for 2 hours. The reaction product was cleaned and purified and stored in a -80°C refrigerator for later use.

[0166] 1.2.4 Collect the T cells cultured in step 1.1.2 into a 50 ml centrifuge tube, centrifuge at 300 × g for 7 minutes, discard the supernatant, wash twice with DPBS solution (manufacturer: Gibco, catalog number: 1924294), and then adjust the cell density to 2.5 × 10 7 Cas9 mRNA and sgRNA were mixed with T cells to a final concentration of 2.5×10 cells / mL. 6 The cells were injected with 8 μg of RNA (2 μg each of Cas9 mRNA, TCR sgRNA, B2M sgRNA, and SPPL3 sgRNA). The cas9 mRNA and sgRNA were then introduced into the cells using the BTX Agile pulse MAX electroporator (manufacturer: BTX, model: 47-0200NINT) under 200-400V / 0.5-2ms electroporation conditions. The antigen screening principle was used to screen out T cells that were negative for TCR and / or B2M and / or SPPL3 and positive for CD4 and CD8 to obtain U-CART cells.

[0167] The cells were harvested and genomic DNA was extracted using the TIANamp Genomic DNA Kit (manufacturer: TIAN GEN, cat. #DP304-03). Using synthetic primers, the extracted cell genome was PCR-amplified using 2*Esay Taq Super Mix (+dye) (manufacturer: TRANS, cat. #AS111) for the genomic regions containing the corresponding sgRNAs for TRAC, B2M, and SPPL3, respectively. The reaction mixture was set up in 50 μl, as shown in Table 4.

[0168] Table 4:

[0169] The reaction conditions are as follows:

[0170] 1.2.5 PCR products were collected for NGS sequencing to verify the knockout efficiency of TCR, HLA, and SPPL3 at the molecular level. After 8 days of cell culture, the resulting T cells underwent quality control monitoring. 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 indicates that in this system, CRISPR / Cas9 technology can successfully edit the constant coding sequences of the TCR TRAC gene, the HLA B2M gene, and SPPL3, including insertion and deletion mutations, all of which resulted in frameshift mutations, thereby inhibiting the expression of TCR, HLA, and SPPL3 at the genetic level.

[0171] 1.2.6 Detection of off-target rates of TCR and SPPL3 genes in U-CART cells

[0172] At the same time, potential off-target sites on the human genome for TRAC (SEQ ID NO: 1 and 2) and SPPL3 (SEQ ID NO: 155 and 166) were predicted, and the predicted off-target site regions that may affect the expression of other genes were amplified and analyzed. The purpose was to confirm at the molecular level that the knockout of TRAC and SPPL3 did not introduce off-target non-specific gene knockout. The results showed that after the gene editing of T cells was completed using sgRNA targeting TRAC (including any one of SEQ ID NO: 1-4) and sgRNA targeting SPPL3 (including any one of SEQ ID NO: 6-256) as sgRNA, the TRAC and SPPL3 genes in the U-CART cells obtained by screening were completely knocked out. At the same time, no gene mutations at potential off-target sites were found. The specific results are shown in Tables 5 and 6.

[0173] Table 5: Off-target analysis results of TRAC knockout T cells

[0174] Table 6: Off-target analysis results of SPPL3 knockout T cells

[0175] 1.2.7 Determination of relevant viability indicators of U-CART cells constructed using CRISPR / Cas9 system and CRISPR / Cas12 system before purification

[0176] For the CRISPR / Cas9 system, 17 sgRNAs targeting the complementary sequence of the SPPL3 genomic region (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) and 1 sgRNA targeting the complementary sequence of the TCR genomic region (SEQ ID NO: 1) were combined to double knockout SPPL3 and TCR, and the obtained U-CART cells had a significant effect on proliferation, viability, TCR KO efficiency on day 9, and SPPL3 knockout on day 9. The KO efficiency, CAR+ cell% on day 9, U-CART cell viability on day 9, and U-CART cell number on day 9 were measured, and the results are shown in Figures 1A to 2E; for the CRISPR / Cas12 system, four sgRNAs targeting complementary sequences of the SPPL3 genomic region (SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264) and four sgRNAs targeting complementary sequences of the TCR genomic region (SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260) were combined to double knockout SPPL3 and TCR, and the proliferation and viability of the obtained U-CART cells, TCR KO efficiency on day 9, SPPL3 KO efficiency on day 9, CAR+ cell% on day 9, U-CART cell viability on day 9, and U-CART cell number on day 9 were measured, and the results are shown in Figures 3A to 4E.

[0177] The above results show that the U-CART edited by the CRISPR / Cas9 system and the CRISPR / Cas12 system have achieved the expected experimental results, and the next purification experiment can be carried out.

[0178] 1.3 U-CART cells prepared by double knockout of TCR gene and SPPL3 gene were purified according to the following experimental steps:

[0179] After washing the U-CART cells with 1% HSA buffer, resuspend the cells at a density of 1E8 / mL. Add TCRα / β-Biotin at a volume of 100 μL / mL, place on a shaker at a speed of 25 RMP, incubate at room temperature for 30 minutes, wash twice with 1% HSA buffer, resuspend the cells in a buffer containing 1% HSA, add Anti-Biotin Reagent at a volume of 200 μL / mL, place on a shaker at a speed of 25 RMP, and incubate at room temperature for 30 minutes; wash once with 1% HSA buffer, and resuspend the cells in a buffer containing 1% HSA; pass the cells through an LS column (Miltenyi Biotec, Cat. No. 130-042-401), collect negative cells for viable cell counting, and detect purification efficiency by flow cytometry.

[0180] The results are shown in Figures 5A-5B and 7A-7B, which show that the viability of U-CART cells does not decrease after purification.

[0181] Example 2 Functional Verification of U-CART

[0182] 2.1 Proliferation and viability detection

[0183] Take the U-CART cells (U-CART) in which the TRAC and SPPL3 genes prepared in Example 1 are completely knocked out, unmodified T cells (T), and common CAR-T cells (CART) without any gene knockout, and carry out proliferation ability and cell viability detection when other conditions are completely consistent. As shown in Figures 6A-6B and 8A-8B, it can be seen that knocking out TCR genes and SPPL3 genes does not affect the proliferation and cell viability of U-CART cells. Figure 6C and Figure 8C show that after UCAR-T cells knock out TCR genes and SPPL3 genes, the cell viability of U-CART does not decrease with the amplification of U-CART cells.

[0184] 2.2 Detection of the ability to resist allogeneic T cell killing

[0185] To evaluate whether SPPL3-knockout U-CART cells would be eliminated by the patient's T cells after infusion into the patient, the 1G4-NY-ESO-1 system (Wei Wensheng Laboratory, Peking University) was used as an experimental model. The experimental system uses T cells expressing the 1G4 TCR, which specifically kills NY-ESO-1-positive HLA-A 02-01-typed T cells. After the B2M gene is knocked out of NY-ESO-1-positive HLA-A 02-01-typed T cells, the T cells are unable to present NY-ESO-1 antigen peptides on the cell surface, thereby resisting the killing of 1G4 TCR-T cells.

[0186] The specific experimental plan was as follows: 1G4 TCR-T cells from the donor were resuspended in AIM5 medium supplemented with 5% FBS at a cell concentration of 5 x 10^6 / mL. NY-ESO-1-positive HLA-A02-01-typed T cells that had undergone different gene editing were resuspended in AIM5 medium supplemented with 5% FBS at a cell concentration of 1 x 10^6 / mL for each group. 1G4 TCR-T cells and NY-ESO-1 T cells were seeded into 96-well U-shaped plates at a target-to-effect ratio of 5:1 (1G4:NY-ESO-1). 100 μL of 1G4 TCR-T cells and NY-ESO-1 T cells were added to each well, totaling 200 μL per well. After incubation in the incubator for 24 hours, 20 μL of cells were removed from each well for K2 counting to calculate the total number of viable cells. The remaining 180 μL was used to analyze the ratio of 1G4 TCR-T cells to NY-ESO-1-positive T cells by flow cytometry (Note: 1G4 TCR-T cells are GFP-tagged and FITC-positive by flow cytometry; NY-ESO-1-positive T cells are BFP-tagged and PB450-positive by flow cytometry). The killing rate of NY-ESO-1-positive T cells in each experimental group was calculated by comparing the total number of 1*10^5 NY-ESO-1-positive T cells at the time of plating.

[0187] The results, shown in Figure 9, indicate that knocking out SPPL3 significantly enhances T cell resistance to allogeneic T cell cytotoxicity. In terms of enhancing T cell resistance to allogeneic T cell cytotoxicity, knocking out SPPL3 achieves an effect comparable to or slightly weaker than knocking out B2M.

[0188] 3. Detection of the ability to resist NK cell killing

[0189] NK cells and U-CART cells were plated in a 10:1 NK:T ratio, with 2E5 U-CART cells per group plated in AIM-V + 5% FBS medium. The cells were incubated overnight at 37°C. Samples were collected 24 hours later. After agitation, 200 μL of the aliquot was taken from each well to determine the killing ratio by flow cytometry. Cell killing was then determined by counting and FACS.

[0190] The results are shown in FIG10 , where % killing percentage = (number of U-CART cells in the control wells − number of U-CART cells in the interaction wells) / number of U-CART cells in the control wells * 100%.

[0191] The results showed that knocking out B2M would lead to an enhanced killing effect of allogeneic NK cells; knocking out B2M and SPPL3 at the same time would lead to a relative weakening of the killing effect of NK cells; not knocking out B2M at all and only knocking out SPPL3 could significantly reduce the killing effect of allogeneic NK cells, thereby enhancing the persistence of U-CART cells in the patient's body.

[0192] 4. Detection of the ability to resist the killing effect of allogeneic PBMC

[0193] To evaluate the effect of SPPL3 knockout U-CART cells on the combined killing effect of allogeneic T cells and NK cells after infusion into patients, U-CART cells were co-incubated with allogeneic PBMCs, and the killing effect of allogeneic PBMCs on U-CART cells was counted. The specific experimental scheme is as follows: (1) first, U-CART cells with TCR and SPPL3 knockout (TSKO) and three types of U-CART cells, namely, U-CART cells with only TCR knockout (TKO) and U-CART cells with TCR and B2M knockout (TBKO), were stained with CellTrace (Figure 11A, TKO-CellTrace-CFSE, TBKO-CellTrace-Far Red, TSKO-CellTrace-VIOLET); or (2) U-CART cells with TCR, B2M and SPPL3 knockout (TBSKO) and three types of U-CART cells, namely, U-CART cells with only TCR knockout (TKO) and U-CART cells with TCR and B2M knockout (TBKO), were stained with CellTrace (Figure 11B, TKO-CellTrace-CFSE, TBKO-CellTrace-Far Red, Red, TBSKO-CellTrace-VIOLET); the three U-CART cells were then mixed in a 1:1:1 ratio, and the mixed U-CART cells were co-incubated with allogeneic PBMCs at different ratios (the ratio of PBMC to U-CART cell number, represented by E:T in the figure) in a 37 ° C, 5% CO2 incubator. After 120 hours, the number of viable cells of TKO, TBKO, and TSKO / TBSKO was counted by absolute flow cytometry. Compared with the control group without allogeneic PBMCs, the killing percentage was calculated according to the formula % = (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%, and the killing of the four U-CART cells by allogeneic PBMCs was counted.

[0194] The results are shown in Figures 11A and 11B, indicating that when PBMC cells are used for killing, compared with only knocking out TCR (TKO), knocking out TCR and SPPL3 (TSKO) can significantly reduce the killing effect of PBMC on U-CART cells, but further knocking out B2M (TBKO) on the basis of knocking out TCR enhances the killing effect of PBMC on U-CART cells; however, knocking out SPPL3 (TBSKO) on the basis of knocking out TCR and B2M can also partially weaken the killing effect of PBMC on U-CART.

[0195] It can be seen that knocking out SPPL3 in the PBMC environment or in vivo environment can reduce the killing of allogeneic T cells with normal HLA-I expression (such as TSKO cells in this example) and T cells with eliminated or suppressed HLA-I expression (B2M knocked out) by the body's immune cells.

[0196] 5. Detection of the ability to resist FasL-mediated killing

[0197] The test plan for this application is as follows:

[0198] 1. Take 1E7 cells of TKO / TFKO / TSKO CAR-T cells, resuspend them to 10 mL, and plate them in a 24-well plate, 1 mL per well;

[0199] 2. Adjust the concentration of Biolegend Recombinant Human FASL to 1000 μg / mL using complete T cell culture medium as a stock solution.

[0200] 3. Dilute the FASL stock solution with T cell complete culture medium to 500, 200, 100, and 50 μg / mL;

[0201] 4. Add FASL to 24-well plates containing cells at a volume ratio of 1:1000, so that the final concentration of FASL in each group is 1000, 500, 200, 100, 50, and 0 ng / mL;

[0202] 5. Place the 24-well plate in a 37°C, 5% CO2 incubator and incubate for 24 hours;

[0203] 6. After 24 hours, remove the 24-well plate and homogenize the cells using a 1 mL pipette. Take 20 μL of the cell suspension and add 20 μL of AOPI dye. Use a K2 counter to count the cell viability of each group of cells and record the count results.

[0204] The results are shown in Figure 12, indicating that knocking out SPPL3 can partially achieve the effect of knocking out FasL, that is, it can resist U-CART cell apoptosis induced by the Fas signaling pathway.

[0205] 6. U-CART in vitro killing ability test

[0206] The TRAC and SPPL3 genes prepared in Example 1 were completely knocked out of U-CART cells (U-CART), CAR-T cells (CART) without gene knockout, and unmodified T cells as effector cells, and Nalm6 (human acute B lymphoblastic leukemia cell line) as target cells for verification. The specific method is as follows:

[0207] Thaw cells: Rapidly thaw frozen U-CART cells, T cells, and target cells (Nalm6, a human acute B-lymphocytic leukemia cell line) in a 37°C water bath. When there are almost no ice crystals in the cryovials, transfer the cells to a 15 mL centrifuge tube and centrifuge at 400 × g for 5 min to collect the cells. Resuspend the cells in fresh culture medium and count them.

[0208] Co-incubation of target cells and effector cells: Add the effector cells and target cells to a cell culture plate at an E:T ratio of 1:2. Transfer the cell plate to a 37°C, 5% CO2 cell culture incubator and incubate.

[0209] Continuous addition of target cells: Every 48 hours, cells were collected for counting and flow cytometry analysis to calculate the target cell ratio and the CAR+ cell ratio. Based on the CAR+% of each cell and the total number of cells, effector cells and target cells were added to the cell culture plate again at an E:T ratio of 1:2.

[0210] Repeat the above steps until the number of remaining CAR+ cells is insufficient to continue the co-incubation experiment.

[0211] After each co-incubation, the following information was collected and calculated:

[0212] Percentage of target cells remaining: Calculate the ratio of CD4-CD8-, that is, the percentage of target cells in all cell types;

[0213] Total number of CAR+ cells: Total number of CAR+ cells = CAR+% * total number of sample cells;

[0214] Cumulative proliferation fold of CAR+ cells = (total number of CAR+ cells at the current time of plating / total number of CAR+ cells at the last time of plating) * cumulative proliferation fold of CAR+ cells at the last time of plating. (The cumulative proliferation fold of CAR+ cells at 0 hours is defined as 1).

[0215] The results of different T cell killing ability 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, the killing effect of CART and U-CART cells is better than that of T cells. Although U-CART has undergone gene editing, its ability to kill target cells is not significantly different from that of the ordinary control group CART. Analysis of the residual target cell ratio shows that U-CART cells have a significant effect on killing Nalm-6 target cells, and in the entire long-term killing experiment, the U-CART cell killing effect on target cells is less than 10%; Figure 15 shows that T cells that are not transduced with CAR molecules have no CAR+ cell proliferation and the T cells have no killing inhibitory effect on target cells. Therefore, the target cell ratio in Figure 8 increases over time. After 144 hours of co-incubation, the target cell ratio of the total cells is higher than 90%. The CAR+ cell proliferation of the U-CART cells in Figure 15 can be maintained for up to 288 hours after co-incubation, and the peak proliferation multiple is basically the same as that of CAR-T. In summary, U-CART cells have good expansion under target cell stimulation and have long-term and significant killing and inhibitory functions on tumor cells.

[0216] 7. U-CART in vivo killing ability detection

[0217] In this example, highly immunodeficient NCG mice were used to bear human acute B-lymphoblastic leukemia cells Nalm-6. Control T cells, CAR-T cells, and U-CART cells prepared from the same healthy donor were then injected to evaluate the tumor-suppressing effect of U-CART cells in tumor-bearing mice.

[0218] Twenty 6-8 week old female NCG mice were used in the experiment and 1×10 Nalm6-LAE cells (Nalm6-Luciferase-LAE) were injected into the tail vein. 6 / mouse was modeled. On the 4th day after tumor cell inoculation, in vivo imaging was performed to detect tumor burden, and the mice were randomly divided into 4 groups according to the imaging fluorescence value. CAR-T (6×10 6 CAR+ cells / mouse) and U-CART 2.0 (6×10 6 CAR+ cells / mouse), 5 animals per group; accompanied by administration of unedited T cells or U-CART cell cryopreservatives from the same volunteer, 5 animals per group, respectively, as a T cell control group and a tumor-bearing control group. All animals were administered a single tail vein injection, and the day of administration was recorded as day 1. After administration, tumor burden was detected by in vivo imaging once a week; the survival of mice was observed and recorded every day; the animal weight was weighed and recorded twice a week. If the weight loss exceeded 20% during this period, the animal was recorded as dead and euthanized.

[0219] The results are shown in Figure 17. After the injection of CAR-T and U-CART cells, according to the in vivo imaging results (Figure 16), the tumor growth rate of the tumor-bearing control group and the T cell control group was faster than that of the test substance, and the tumor growth rate of the T cell control group was consistent with that of the tumor-bearing control group, indicating that T cells themselves have no killing function against tumor cells; compared with the CART cell group, no tumor recurrence was observed in the animals in the U-CART cell group on day 39 after administration. TCR and SPPL3 double knockout U-CART cells have a strong ability to kill tumor cells in immunodeficient mice that is not weaker than that of CAR-T cells without gene knockout. It can be seen that gene editing does not weaken the killing ability of CAR-T cells in vivo and does not affect their long-term survival in vivo.

[0220] The sequences used in the above examples of the present application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences of the embodiments of the present application and are not intended to limit the present application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When representing RNA sequences, "T" represents uridine.

[0221] Sequence Listing:

Claims

1. An engineered T cell, which has been modified to eliminate 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, after modification, the expression and / or function of the α subunit (TCRα) of the endogenous T cell receptor (TCR) and / or the β subunit (TCRβ) of the endogenous T cell receptor (TCR) are eliminated or reduced.

3. The engineered T cell according to claim 1 or 2, further, the expression and / or function of its beta-2 microglobulin (B2M) is retained, or is eliminated or reduced.

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

5. The engineered T cell of claim 4, wherein the engineered receptor is selected from one or more of the following: a chimeric antigen receptor (CAR), an engineered TCR, and a T cell antigen conjugate (TAC).

6. A method for preparing the engineered T cell according to any one of claims 1 to 5, wherein the modification comprises eliminating or reducing the expression and / or function of the TCR protein or its functional fragment and / or the SPPL3 protein or its functional fragment by one or more selected from the following: disruption or knockout of the encoding gene, frameshift mutation or knockout, inhibition of transcription of the encoding gene, disruption or elimination of mRNA, inhibition of expression of the encoding gene and inhibition of the protein.

7. The method according to claim 6, wherein the modification eliminates 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 small RNA (miRNA).

8. The method according to claim 6, wherein the modification comprises eliminating or reducing the expression of TCR or its functional fragment and SPPL3 or its functional fragment by a gene editing method or an RNA editing method mediated by a CRISPR / Cas system, wherein the gene editing method or the RNA editing method uses a guide RNA (gRNA) targeting SPPL3 and / or TRAC, and the gRNA uses a target RNA that is related to human chromosome 12 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, 1 120,783,674 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 SPPL3 genomic region; and / or using a guide sequence complementary to the TRAC genomic region on human chromosome 14 at position 23016448 to 23016490.

9. The method according to claim 8, wherein: The guide sequence complementary to the SPPL3 genomic region is selected from one or more of the following: 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:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO: NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID and / or The guide sequence complementary to the TRAC genomic region is selected from one or more of the following: 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.

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

11. The method according to claim 10, wherein: The guide sequence complementary to the SPPL3 genomic region is selected from one or more of the following: SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:159, SEQ ID NO:161, 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:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293 and SEQ ID NO:

294.

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

13. The method according to claim 12, wherein the chemical modification comprises 2'-O-methylation modification on the ribose of the nucleotide or 3' phosphorothioate bond modification between nucleotides, or both.

14. The method of claim 13, wherein the modification is 2'-O-methylation modification on the first three nucleotide riboses at the 5' end, 2'-O-methylation modification on the last three nucleotide riboses at the 3' end, 3' phosphorothioate modification between the nucleotides of the first three nucleotides at the 5' end, and 3' phosphorothioate modification between the nucleotides of 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 half-life of an engineered cell in vivo, comprising modifying the engineered cell to eliminate or reduce the expression and / or function of SPPL3 protein or a functional fragment thereof.

17. The method according to claim 16, wherein the modification eliminates or reduces the expression and / or function of the SPPL3 protein or its functional fragment by one or more selected from the group consisting of: disruption of the encoding gene, frameshift mutation or knockout, inhibition of transcription of the encoding gene, disruption or elimination of mRNA, inhibition of expression of the encoding gene and inhibition of the protein, optionally, the modification eliminates or reduces the expression of SPPL3 or its functional fragment by RNA interference (RNAi), for example, the RNAi silences or inhibits the expression of the SPPL3 protein or its functional fragment by small interfering RNA (siRNA), short hairpin RNA (shRNA) or small RNA (miRNA).

18. The method according to claim 16, wherein the modification eliminates or reduces the expression of SPPL3 protein or its functional fragment by a gene editing method or an RNA editing method mediated by a CRISPR / Cas system, wherein the gene editing method or the RNA editing method uses a guide RNA (gRNA) targeting SPPL3, for example, the gRNA uses a target gene targeting human chromosome 12 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 The invention also provides a guide sequence complementary to the SPPL3 genomic region at position 120,783,674 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, to knock out the SPPL3 encoding gene or cause a frameshift mutation in the SPPL3 encoding gene.

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

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

21. The method according to any one of claims 16-20, wherein the engineered cell is an immune cell or a precursor cell thereof.

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

23. The method according to claim 22, wherein the immune cell is an αβT cell, and the expression and / or function of endogenous TCRα and / or TCRβ or functional fragments thereof of the αβT cell is eliminated or reduced.

24. The method of any one of claims 16-23, wherein the engineered cell further comprises or expresses an engineered receptor.

25. The method of claim 24, wherein the engineered receptor is selected from one or more of the following: Chimeric antigen receptors (CARs), engineered TCRs, and T cell antigen conjugates (TACs).

26. A guide RNA (gRNA) for eliminating or reducing the expression of SPPL3 protein or its functional fragment by a gene editing method mediated by a CRISPR / Cas system, comprising a nucleotide sequence selected from any one of the following: 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:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:312 and SEQ ID NO:

315.

27. A composition comprising the gRNA of claim 26.

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

29. The composition of claim 28, wherein the gRNA targeting SPPL3 comprises a gRNA that is compatible with human chromosome 12 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, A guide sequence complementary to the SPPL3 genomic region at position 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.

30. The composition according to claim 29, wherein the gRNA targeting SPPL3 comprises any one guide sequence selected from the following: 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:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:300, SEQ ID NO:305, SEQ ID NO:312 and SEQ ID NO:

315.

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

32. The composition of claim 31, wherein: The guide sequence complementary to the SPPL3 genomic region is selected from one or more of the following: SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:159, SEQ ID NO:161, 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:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293 and SEQ ID NO:

294.

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

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

35. The composition of claim 34, wherein the guide sequence comprises a nucleotide sequence as 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