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

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 caused by 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 activated T cells expressing FasL (CD95L) and Fas (CD95) at the same time. FasL is a killer effector molecule, and Fas can cause apoptosis of activated T cells through autocrine or paracrine FasL; Third, U-CART cells will be attacked by patient T cells, resulting in 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 response to the persistence problem 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. SUMMARY OF THE INVENTION

[0006] The present application provides an engineered T cell with enhanced persistence and a preparation method thereof, which reduces GvHD, AICD and HVG without incurring NK cell killing, 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, which has been modified to eliminate or reduce the expression and / or function of 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 of 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 cell is a T cell derived from a human, and the modification comprises a nonsense mutation in the SPPL3 genomic region on chromosome 12 of the engineered T cell or the mRNA (or pre-mRNA) of the SPPL3 gene. In some embodiments, the engineered T cell is a T cell derived from a human, and the modification occurs at any one or more nucleotide sites in any one or more exon regions of the SPPL3 gene, or between any two nucleotide sites.

[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, and 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 site 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 sites. 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 of the TRAC genomic region or the TRAC gene of chromosome 14 of the engineered T cell. In some embodiments, the T cell is a T cell derived from a human, and the modification occurs at any nucleotide position from position 23016448 to position 23016490 relative to the GRCh38.p14 reference genome chromosome 14, 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 normally expressed HMC-I class proteins. In some embodiments, the T cells have normally expressed HLA-A, HLA-B, HLA-C and B2M. In some embodiments, the T cells are human-derived T cells, and the expression and / or function of the B2M protein 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; CD 22; CD 30; CD 70; CD 123; 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);Claudin6, 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); globoH GloboH, hexose moiety of glycoceramide; NY-BR-1, mammary gland differentiation antigen; uroplakin2 (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 protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytotoxicity 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 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); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5);Immunoglobulin lambda-like polypeptide 1 (IGLL1) and CD155. In some embodiments, the transmembrane domain comprises a transmembrane domain from any molecule selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD6, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD47, CD52, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, CISH, and PD-1. In some embodiments, the 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 co-stimulatory signaling domain comprising one or more co-stimulatory 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 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, 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 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 an extracellular domain or a portion thereof of a first TCR co-receptor (e.g., CD4, CD8); (f) a transmembrane domain comprising a transmembrane domain of a second TCR co-receptor (e.g., CD4, CD8); and (g) an optional intracellular signaling domain comprising an 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, and the one or more TACs have different antigen binding domains or ligand binding domains. 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, CD123, CD 138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276),B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, HIV-1Gag, 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)C er, TGS5, HMWMAA, OAcGD2, folate receptor, CD248, TEM7R, Claudin6, Claudin18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA), CA9, κ light chain, CSPG4, EGP2, EGP40, FAP, FAR, FBP, embryonic AchR, HLA-A1, HLA-A2, MAGEA1, MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, fibronectin, tenascin, tumor Oncofetal variants in necrotic areas, 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 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, LILRA2, 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 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 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 or portion thereof of a first TCR subunit (e.g., Cα, Cβ, Cδ, Cγ, CD3ε); (d) a transmembrane domain of a second TCR subunit (e.g., TCRα, TCRβ); and (e) an intracellular signaling domain comprising an intracellular signaling domain of a third TCR subunit (e.g., TCRα, TCRβ); 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, CD70, CD 123, CD 138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276),B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, HIV-1Gag, 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, carcinoembryonic 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 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, 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 aforementioned first aspect, wherein the modification includes one or more selected from the following: destruction or knockout of coding genes, 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 enzyme cutting of the target site. In some embodiments, the modification comprises one or more of insertion, deletion, and substitution. In some embodiments, the modification comprises one or more of translocation, point mutation, fragment deletion, and fragment addition. In some embodiments, the modification results in one or more of frameshift mutation, loss of function mutation, dominant negative mutation, missense mutation, and nonsense mutation. In some embodiments, the modification comprises gene editing or RNA editing mediated by the CRISPR / Cas system, and 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 splicing site therein) or the mRNA or pre-mRNA sequence corresponding to the genomic region.Preferably, 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, 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 of human chromosome 12, or the 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 of human chromosome 14, or the 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, in addition to the guide sequence, does not include a tracrRNA and a tracr-partner 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 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, 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, ID 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 genome 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 includes 2'-O-methylation modification on the ribose of the nucleotide or 3' phosphorothioate bond modification between nucleotides or both. In some embodiments, the modification is 2'-O-methylation modification on the first three ribose nucleotides at the 5' end, 2'-O-methylation modification on the last three ribose nucleotides at the 3' end, 3' phosphorothioate modification between the first three nucleotides at the 5' end, and 3' phosphorothioate modification between the last three nucleotides at the 3' end.

[0016] The third aspect of the present application also provides 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 a SPPL3 protein or a functional fragment thereof. In some embodiments, the modification comprises one or more of an insertion, a deletion, and 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 site. In some embodiments, the modification occurs in the 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 change any genomic site of SPPL3 but downregulates mRNA by interfering with the transcription of SPPL3. In some embodiments, the modification does not interfere with any genomic site and mRNA level of SPPL3, but interferes with the process of mRNA translation into SPPL3 protein, thereby resulting in downregulation of SPPL3 protein levels. In some embodiments, the modification causes a mutation in the SPPL3 genomic region of human chromosome 12 or the mRNA or pre-mRNA sequence corresponding to the genomic region. In some embodiments, the modification causes a nonsense mutation in the SPPL3 genomic region of human chromosome 12 or the mRNA or pre-mRNA sequence corresponding to the genomic region. In some embodiments, the engineered cell has or expresses a normal level of MHC-I protein. In some embodiments, the engineered cell has normally expressed HLA-A, HLA-B, HLA-C and B2M. In some embodiments, the normal level of MHC-I protein does not cause NK cells to kill the engineered cells. 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: T cells, B cells, natural killer (NK) cells, macrophages, and DC cells. In some embodiments, the immune cell is an αβT cell, and the expression and / or function of the endogenous TCRα and / or TCRβ or its functional fragment of the αβT cell is eliminated or reduced. In some embodiments, the engineered T cell is a γδT cell, and it is modified, and the expression and / or function of its endogenous TCRγ and / or TCRδ is eliminated or reduced. In some embodiments, the engineered cell further comprises or expresses 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, 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: 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 does not include tracrRNA and tracr-partner sequences in addition to the guide 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 of the nucleotide or a 3' phosphorothioate bond modification between nucleotides or both. In some embodiments, the modification is a 2'-O-methylation modification on the first three nucleotide riboses at the 5' end, a 2'-O-methylation modification on the last three nucleotide riboses at the 3' end, a 3' phosphorothioate modification between the first three nucleotides at the 5' end, and a 3' phosphorothioate modification between the last three nucleotides at the 3' end.

[0019] The fifth aspect of the present application provides a nucleic acid composition, which comprises the gRNA of the aforementioned 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 from position 23016448 to position 23016490 of 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 shown in SEQ ID NO: 155 or SEQ ID NO: 261, and the other of which targets a T cell receptor gene. In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which comprises a guide sequence as shown in SEQ ID NO: 155 or SEQ ID NO: 261, and the other of which comprises a guide sequence complementary to the TRAC genomic region at positions 23016448 to 23016490 of chromosome 14. In some embodiments, the nucleic acid composition comprises at least two gRNAs, one of which comprises a guide sequence as shown in SEQ ID NO: 155 or SEQ ID NO: 261, and the other of which comprises a guide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 257. ; BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A-1BThe proliferation and viability of U-CART constructed using the CRISPR / Cas9 system before purification on days 0-9 are shown respectively. Among them, sgRNA1-sgRNA17-SP3-3 respectively 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, SEQ ID NO: 250 targeting SPPL3 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 Shown is the gene knockout efficiency of SPPL3 on day 9 before purification of U-CART constructed using the CRISPR / Cas9 system.

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

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

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

[0027] Figure 3A-3BThe proliferation and viability of U-CART constructed using the CRISPR / Cas12 system on days 0-9 before purification are shown respectively. 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, SEQ ID NO: 270, respectively, and the above sequences are combined to double knockout TRAC and SPLL3.

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

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

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

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

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

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

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

[0035] Fig. 6A and 6B Comparison of U-CART and CAR-T constructed using the CRISPR / Cas9 system and T cell proliferation multiples (2A) and viability (2B) is shown.

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

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

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

[0039] Fig. 8A and 8B Comparison of U-CART and CAR-T constructed using the CRISPR / Cas12 system and T cell proliferation multiples (2A) and viability (2B) is shown.

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

[0041] Fig. 9 The results show that T cells from two donors, after being modified in different ways, have the ability to resist the cytotoxic effects of allogeneic T cells.

[0042] Fig.10 The killing effect of NK cells on T cells from the same donor that have been modified in different ways is shown.

[0043] Fig.11A and 11B Shows the killing effect of PBMC cells on T cells that have undergone different modifications.

[0044] Fig.12 Shows FasL-mediated killing of T cells modified differently.

[0045] Fig.13 The results show that T cells modified with different methods can kill target cells in vitro, among which U-CART cells are constructed based on the CRISPR / Cas9 system.

[0046] Fig.14 The results show that T cells modified with different methods can kill target cells in vitro, among which U-CART cells are constructed based on the CRISPR / Cas12 system.

[0047] Fig.15 The proliferation of CAR+ cells in T cells with different modifications is shown when they are co-incubated with target cells in vitro.

[0048] Fig.16 Shown are the results of in vivo imaging of mice.

[0049] Fig.17 The results show that T cells modified with different methods can kill target cells in immunodeficient mice. DETAILED DESCRIPTION OF THE INVENTION

[0051] The inventors of the present application creatively provide 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 the killing of the engineered cells by NK cells. Specifically, the present application also provides engineered T cells transformed by this method, a method for preparing the engineered T cells, and gRNA and combinations thereof used in the method.

[0052] definition

[0053] The present application will be described in conjunction with specific embodiments and with reference to certain drawings, but the present application is not limited thereto. Any reference signs in the claims should not be interpreted as limiting their scope. In the accompanying drawings, for the purpose of illustration, 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 as a whole. The materials, methods and embodiments disclosed herein are illustrative only and are not intended to be limiting.

[0054] As used herein, "SPPL3" is signal peptide peptidase-like 3, also known as IMP2, PSH1 or PSL4, and is a multi-spanning membrane protein located in 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 luminal domains of glycosyltransferases and glycosidases. The 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 noticed 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 interaction with matrix interaction molecule 1 (STIM1) and Orai1, enhancing TCR signaling to greatly induce calcium influx and NFAT activation that are critical for lymphocyte signaling. An exemplary SPPL3 is human SPPL3, such as SPPL3 with a gene ID of 121665 in the NCBI database. In some embodiments, the gene sequence of human SPPL3 is shown at positions 120762510 to 120904358 of NC_000012.12, 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 sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-partner sequences (e.g., partial direct repeats that contain "direct repeats" and tracrRNA processing in endogenous CRISPR systems), guide sequences (also referred to as "spacers" in endogenous CRISPR systems), 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. Complete complementarity is not necessarily required if there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. A CRISPR complex may comprise a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins.

[0057] The term "guide sequence" is a continuous nucleotide sequence in the guide RNA that has partial or complete complementarity with the target sequence in the target polynucleotide and can hybridize with the target sequence through base pairing promoted 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 nucleic acid-based molecule, including but not limited to being able to form a protein-RNA complex with the Cas protein and comprising a sequence (e.g., a guide sequence or a spacer) 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, for example, crRNA chains and tracrRNA chains. In some embodiments, the gRNA is an RNA chain, for example, sgRNA.

[0059] The terms "single guide RNA", "synthetic guide RNA" and "sgRNA" are used interchangeably and refer to a polynucleotide sequence comprising a guide sequence and any other sequences that are necessary for the function of the sgRNA and / or necessary for the interaction of the sgRNA 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, the second sequence comprising a tracr sequence derived from the tracrRNA and a tracr partner sequence derived from the 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 a nucleotide sequence that specifies a target site in a guide RNA and can be used interchangeably with the terms "guide" or "spacer". The term "tracr partner sequence" can 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 with reduced GvHD and / or HVG effects 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 kind of engineered T cell provided in this application. 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 represents 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). "Complete complementarity" 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 to two nucleic acids that hybridize under stringent conditions.

[0064] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity with a target sequence hybridizes primarily with the target sequence and does not substantially hybridize with 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 specifically hybridizes with its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part 1, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0065] "Hybridization" refers to a 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 contain 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 of these. Hybridization reactions can constitute steps in a broader process, such as the initiation of PCR, or the cutting of polynucleotides by an enzyme. A sequence that can hybridize with 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 (e.g., DNA or RNA), or a vector capable of delivering such nucleic acid molecules. For example, when used in the context of gRNA or sgRNA, construct refers to a gRNA or sgRNA molecule, a nucleic acid molecule encoding gRNA or sgRNA (e.g., 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. Constructs may include essential regulatory elements operably connected to nucleotide sequences, and when constructs are present in host cells, the regulatory elements allow transcription or expression of the nucleotide sequence. As used herein, "operably connected" refers to the expression of a gene being under the control of a regulatory element (e.g., 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 with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of directing the expression of those 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, which means that the recombinant expression vector includes one or more regulatory elements that may be selected for expression based on the host cell, i.e., 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. A 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 a 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" refer to T cells from a donor, which 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 very close HLA matching). 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 HLA type II 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 to a subject whose cells are obtained for further in vitro engineering in this article. The 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), and after the cell population is produced by the methods described herein, it will be used to treat a different individual or patient (i.e., an allogeneic donor). Those subjects who receive modified cells (modified immune cells as described herein or prepared by the present method) can be referred to as "recipients" or "recipient subjects".

[0071] As used herein, the "phenotype" of a cell refers to an observable characteristic or trait 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 come 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 vivo. In some embodiments, the phenotype is death. In some embodiments, the phenotype is an 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 portion. For example, in the context of a receptor, this stimulation requires the connection of a receptor, and a subsequent signal transduction event. Regarding the stimulation of T cells, this stimulation refers to the connection of a T cell surface portion, which in one embodiment subsequently induces a signal transduction event, such as binding to a TCR / CD3 complex. In addition, a stimulation event can activate cells and up-regulate or down-regulate the expression or secretion of a molecule, such as down-regulating TGF-β. Therefore, even in the absence of a direct signal transduction event, the connection of a cell surface portion may also result in the reorganization of the cytoskeletal structure, or the aggregation of a cell surface portion, each of which can be used to enhance, modify or change subsequent cell responses.

[0073] As used herein, the term "activated" refers to the state of a cell after sufficient ligation of cell surface moieties to induce significant biochemical or morphological changes. In the context of T cells, this activation refers to a state in which the T cell has been sufficiently stimulated to induce cell proliferation. The activation of T cells may also induce the production of cytokines and regulatory properties 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 properties or cytolytic effector functions, and / or has recently undergone an "activation" process.

[0074] "Isolated" nucleic acid molecules as described herein are nucleic acid molecules that are identified and separated from at least one contaminating nucleic acid molecule, which is usually associated with the environment in which it is produced. Preferably, the isolated nucleic acid is not combined with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides herein are in a form different from the form or environment in which they are found in nature. Therefore, the isolated nucleic acid molecules are different from the nucleic acids encoding the polypeptides herein that are naturally present in cells.

[0075] Unless otherwise specified, a nucleotide sequence "encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and 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 is transfected, transformed, or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0077] As used herein, the term "CAR", i.e., chimeric antigen receptor, comprises: i) an extracellular antigen binding domain that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes); ii) a transmembrane domain; and iii) an intracellular signaling domain. In some embodiments, the extracellular antigen binding domain is 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 following groups: CD3ζ, CD3γ, CD3ε, CD3δ, FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, CD66d, FcγRIIa, DAP10, and DAP12. In some embodiments, the primary intracellular signaling domain is from CD3ζ. In some embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain from one or more co-stimulatory 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 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 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 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 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" means 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., CD 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 result. For the purposes of this application, a beneficial or desired clinical result includes, but is 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 deterioration 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 the progression 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 progression of the disease, improving the quality of life and / or prolonging the survival period. "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 to improve, alleviate, reduce and / or delay one or more symptoms thereof (e.g., cancer, infectious disease, GvHD, transplant rejection, autoimmune disease or radiation sickness) of an agent (e.g., a modified immune cell or a pharmaceutical composition thereof as described herein). When referring to cancer, an effective amount includes an amount sufficient to shrink a 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 may 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 cells from infiltrating peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and preferably stop) tumor metastasis to a certain extent; (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) 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 the patient's survival.

[0082] As used herein, "individual" or "subject" refers to a mammal, including but not limited to: a human, a cow, a horse, a feline, a canine, a rodent, or a primate. 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 bacteria) to its infected target (e.g., a cell or organism). 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 involving "about X" includes description of "X".

[0088] As used herein, reference to "other than" a value or parameter generally means and describes "except for" that value or parameter. For example, the method is not used to treat type X cancer, which means that the method is used to treat other types of cancer other than 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 integer or 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] On the one hand, the present application provides an engineered T cell, which 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 endogenous T cell receptor (TCR) protein and / or SPPL3 protein. In some embodiments, the T cell is an αβT cell, which 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, and its TCRα conserved region (TRAC) gene or TRAC gene or its expression and / or function is eliminated or reduced. In some embodiments, the T cell is a γδ T cell that is modified so that the expression and / or function of the endogenous γ subunit (TCRγ) and / or T cell receptor (TCR) δ subunit (TCRδ) is eliminated or reduced.

[0096] In some embodiments, the engineered T cells have normally expressed HMC class I proteins. In some embodiments, the engineered T cells have normally expressed B2M, HLA-A, HLA-B, and HLA-C. In some embodiments, the engineered T cells are human T cells and have normally expressed HLA-I class 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, compared to T cells without reduced or eliminated endogenous TCR and / or SPPL3 protein, the engineered T cells, after transplantation into an allogeneic recipient, have their HVG reduced by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), for example, reduced by at least about 20%. In some embodiments, compared to engineered T cells without reduced or eliminated expression (RNA and / or protein expression) and / or function of SPPL3 protein, the engineered T cells have their HVG reduced by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), for example, reduced by at least about 20%.

[0099] In some embodiments, the engineered T cells have a persistence in vivo that is 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) than 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, compared with T cells in which the expression and / or function of MHC class I molecules and TCR proteins are reduced or eliminated, and the expression and / or function of SPPL3 proteins are not reduced or eliminated, the engineered T cells of the present application are not significantly different in the degree of HVG caused by attack by allogeneic T cells; and compared with T cells in which the expression and / or function of MHC class I molecules and TCR proteins are reduced or eliminated, and the expression and / or function of SPPL3 proteins are not reduced or eliminated, the natural killing caused by attack by NK cells of the engineered T cells of the present application is reduced by at least about 10% (for example, 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 NK cells and T cells of the engineered T cells of the present application is substantially the same as that of T cells in which the expression and / or function of MHC class I molecules and TCR proteins are reduced or eliminated, and the expression and / or function of SPPL3 proteins are not reduced or eliminated, or 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%. In some embodiments, compared with T cells in which the expression and / or function of MHC class I molecules and TCR proteins are reduced or eliminated, and the expression and / or function of SPPL3 proteins are not reduced or eliminated, 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, the expression of endogenous TCR protein and / or SPPL3 protein of the engineered T cells is reduced (e.g., reduced by at least about any one 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 protein.

[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 a 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 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or inhibited by small molecule compounds, nucleic acids (or vectors comprising the same), lipids and / or protein molecules.

[0108] In some embodiments, the engineered T cells are genetically modified at a DNA locus encoding an endogenous TCR protein and / or SPPL3 protein. In some embodiments, the immune cells are genetically modified at the SPPL3 locus. In some embodiments, the loci of the endogenous TCR protein and / or SPPL3 protein are modified by a mutagen. Mutagens can be divided into three categories: physical (e.g., gamma rays, ultraviolet (UV) radiation), chemical (e.g., ethyl methane sulfonate (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 (e.g., 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-nitrosamines). 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 accomplished by PCR methods. In some embodiments, gene editing is accomplished 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 includes introducing a nucleic acid template (e.g., containing a desired mutation), such as inserting at a double-strand break (DSB) to modify a target genomic sequence (e.g., by HDR). Gene editing can introduce one or more mutations in 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 can 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 RNA encoding endogenous TCR proteins and / or SPPL3 proteins. In some embodiments, RNA encoding endogenous TCR proteins and / or SPPL3 proteins is modified by RNA editing. RNA editing can introduce one or more mutations in RNA encoding endogenous TCR proteins and / or SPPL3 proteins, including but not limited to insertion, deletion, substitution (such as non-synonymous substitution), truncation, point mutation, 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 programmatic editing of RNA using endogenous ADARs ("LEAPER"; see, e.g., WO2020074001 and Qu et al. (Nat Biotechnol. 2019; 37(9): 1059-1069), or RNA editing with programmed A to I substitutions ("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, e.g., Merkle et al., "Precise RNAediting by recruiting endogenous ADARs with antisense oligonucleotides," Methods Mol Biol. 2021; 2181: 331-349), CRISPR-Cas stimulated RNA targeting system ("CIRTS"; see, e.g., 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. Reautschniget 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 with 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 (class 1 and class 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. In the Class 2 CRISPR-Cas system, 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-targeted 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, wherein the guide sequence 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 before the introduction of the gRNA construct. In some embodiments, the precursor immune cells do not express Cas proteins before the introduction of the gRNA construct and Cas components. In some embodiments, the precursor immune cells express engineered receptors (e.g., CAR, engineered TCR, or TAC) before 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 is substantially composed of, or is composed of) crRNA. In some embodiments, the gRNA comprises (or is substantially composed of, or is composed 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 The 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 the ligand of NKp46. In some embodiments, the T cell has a normally expressed HLA-I, and the normally expressed HLA-I does not cause killing of NK cells.

[0113] In some embodiments, the engineered T cells are further modified to express engineered receptors. In some embodiments, the immune cells have expressed engineered receptors before being modified to reduce or eliminate the expression and / or function of endogenous TCR proteins and / or SPPL3 proteins. In some embodiments, 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 engineered receptors. In some embodiments, the modification of reducing or eliminating the expression and / or function of endogenous TCR proteins and / or SPPL3 proteins and the modification of expressing engineered receptors occur simultaneously. In some embodiments, reducing or eliminating the expression and / or function of endogenous TCR proteins and / or SPPL3 proteins and the modification of expressing engineered receptors 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 (e.g., 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 engineered receptors (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 engineered receptors 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, engineered receptors (e.g., CAR), gRNA constructs or arRNA constructs, and / or Cas components are introduced into precursor T cells by transduction / transfection of nucleic acids (DNA or RNA) or vectors encoding them (e.g., non-viral vectors, or viral vectors such as lentiviral vectors), or viruses (e.g., lentiviruses) comprising nucleic acids encoding them. In some embodiments, the cells are simultaneously passed through a microfluidic system, such as a microfluidic system, by inserting proteins into the cell membrane. A Cas component (e.g., Cas9 protein) is introduced into precursor T cells (e.g., see U.S. Patent Application No. 20140287509).

[0116] Methods for introducing vectors (e.g., viral vectors) or isolated nucleic acids into mammalian cells are known in the art. Nucleic acids or vectors described herein can be transferred into T cells by physical, chemical or biological methods. Physical methods for introducing vectors (e.g., viral vectors) into cells include calcium phosphate precipitation, lipofection, gene gun method, microinjection, electroporation, etc. The method for producing cells containing vectors and / or exogenous nucleic acids is well known in the art. In some embodiments, vectors (e.g., viral vectors) are introduced into cells by electroporation. Biological methods for introducing vectors into cells include the use of DNA and RNA vectors. Chemical means for introducing vectors (e.g., viral vectors) into cells include colloidal dispersion systems, e.g., macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, and the lipid-based systems include 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 (e.g., gRNA, arRNA or mRNA encoding Cas) can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into T cells by known methods such as electroporation. In some embodiments, a viral vector (lentiviral vector) or virus (e.g., lentivirus) comprising a nucleic acid encoding any engineered receptor (e.g., 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 (e.g., 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 the 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 any one of 14 days, such as 7 days. In some embodiments, the transduced / transfected T cells are further evaluated or screened to purify the T cells described herein.

[0119] Reporter gene can be used to identify the cells of potential transfection / transduction and to evaluate the function of regulatory sequences. Generally speaking, reporter gene is a gene that is not present in or is not expressed by a receptor organism or tissue and encodes a polypeptide, and the expression of the polypeptide is demonstrated by some characteristics that are easy to detect, for example, enzyme activity. After DNA / RNA is introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include: genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (GFP) (for example, Ui-Tei et al.FEBS Letters 479:79-82 (2000)). Suitable expression systems are well-known and can be prepared or commercially obtained 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 blots, RT-PCR, PCR, DNA-seq or RNA-seq; biochemical analysis, such as detecting the presence or absence of specific peptides by immunological methods (e.g., ELISA and Western blot), fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

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

[0122] Generally, after entering the patient's body, allogeneic engineered cells are usually cleared from the donor due to GvHD, AICD, HVG and other reasons, resulting in poor in vivo persistence, insufficient time to act in the patient's body, and thus limited clinical effects. Common practices include knocking out the HMC class I molecules of allogeneic engineered cells to avoid HVG. However, when MHC class I molecules are knocked out, it can lead to NK cells recognizing and attacking cells that downregulate HMC 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 attacks caused by the reduction of MHC class I molecular weight while eliminating HVG, thereby extending the half-life of engineered cells in vivo by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times or more) relative to unengineered T cells. And, in some embodiments, relative to the method of extending the half-life of engineered cells in vivo by eliminating or reducing MHC class I molecules, the method of the present application can achieve similar or better effects. In some embodiments, relative to the method of 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 one of the above methods for preparing engineered T cells.

[0126] Guide RNA

[0127] The present application also provides a guide RNA (e.g., sgRNA) for a CRISPR / Cas system and a construct encoding a CRISPR / Cas guide RNA to hit the gene SPPL3 by cutting or base editing, and to produce a mutation in SPPL3, especially a frameshift mutation, referred to as a gRNA targeting SPPL3. The sgRNA provided by the present application is used to knock out SPPL3 in engineered cells, and the engineered cells that do not express SPPL3 are screened with SPPL3 as a marker. After the screened engineered cells are cultured for several days (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 days), the knockout efficiency of SPPL3 still reaches 99.37%. In addition, the guide RNA provided by the present application does not cause detectable off-target. In some embodiments, the guide RNA provided by the present application comprises a guide sequence 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, 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: NO:293 and the guide sequence shown in any one of SEQ ID NO:294. In some embodiments, the present application also provides a combination of guide RNAs, which comprises the above-mentioned gRNA targeting SPPL3 and a gRNA targeting a TCR gene. Wherein, 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 the TCR gene targets the TRAC gene.

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

[0129] According to the difference of CRISPR / Cas system, gRNA includes other components in addition to the guide sequence. The other components include, for example, additional sequence elements that promote the formation of CRISPR complexes with Cas proteins. In some embodiments, the gRNA (e.g., sgRNA) includes a second sequence containing a repeat-reverse-repeat stem loop. The repeat-reverse-repeat stem loop includes a tracr pairing sequence fused to a tracr sequence, and the tracr sequence is complementary to the tracr pairing sequence through a loop region. In some embodiments, gRNA (e.g., sgRNA) includes a crRNA comprising a direct repeat (DR) sequence that can interact with Cas proteins. In some embodiments, gRNA (e.g., sgRNA) includes crRNA and tracrRNA, which can be on the same RNA chain (e.g., forming sgRNA), or on two RNA chains. The DR sequence can be 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, compared to the reference DR sequence. In some embodiments, the DR sequence contains a repeat sequence and an anti-repeat sequence. In some embodiments, the repeat sequence of the DR sequence is partially or completely substantially complementary to and hybridizes with the anti-repeat sequence of the DR sequence to form a repeat: anti-repeat double strand (also referred to as a "stem"). In some embodiments, the length of the repeat: anti-repeat double strand 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 through a linker, such as a loop sequence. In the case where the repeat sequence and the anti-repeat sequence are indirectly connected through a linker, the length of the repeat: anti-repeat double strand does not include the length of the linker. In some embodiments, the repeat sequence of the DR sequence and the anti-repeat sequence of the DR sequence are indirectly connected through a loop sequence. In some embodiments, the length of the loop sequence is about 4 to about 10 nucleotides, 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 part 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 part of a tracr mate sequence operably linked to a 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 the optimal arrangement 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 may be used, such as a tracr mate sequence and tracr sequence from a Streptococcus pyogenes (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), referred to as a "repeat-anti-repeat stem-loop."

[0132] "Stem-loop" refers to a nucleic acid with a secondary structure that includes 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 exact base pairing. Therefore, the stem may include one or more base mismatches. In addition, the base pairing may be exact, i.e., not include any mismatches.

[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, wherein the second sequence comprises a repeat-reverse-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 sequences can be used as long as they can bind 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, which comprises 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 treatment 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 transport 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. Centrifuge at 800×g for 20 minutes, draw up the white film layer cells above the lymphocyte separation solution, transfer to a new 50 ml centrifuge tube, add T cell culture medium (Miltenyi Biotec, 170-076-306), 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 with Easy buffer (manufacturer: StemCell, catalog number: 16F72331). 7 / ml, use a 5ml pipette to transfer the cells to a 5ml flow tube; add T cell separation reagent at a concentration of 50ul / ml, and incubate at room temperature for 5 minutes after adding; add sorting magnetic beads, mix the magnetic beads within 30 seconds, and add a concentration of 40μl / ml; use Easy buffer to make up the cell solution to 2.5 ml, put it directly on the magnetic column for 3 minutes after adding, and then pour the cells into a 15ml centrifuge tube, and the obtained is T cells; after sorting, use a 1000μl pipette to mix and count, centrifuge (400×g, 5min) and remove the supernatant to obtain T cell precipitation. Resuspend the T cell precipitation in the culture medium of T cells. Then, add T cell activation factor (magnetic beads coated with anti-CD3 / CD28 antibodies) at a ratio of 1:1. At this time, the T cells are in an activated state, and the T cells are placed in a 37℃, 5% CO2 incubator for continued amplification and culture. Use lentivirus to transduce CAR molecules into activated T cells. The CAR molecule used in the embodiments of the present application is a CAR that specifically binds to CD19, and the extracellular antigen binding domain used is the scFV of the anti-CD19 monoclonal antibody FMC63 antibody, whose hinge region and transmembrane region are respectively the hinge region and transmembrane region of the CD8α molecule, and the intracellular signal transduction domain is composed of a 4-1BB co-stimulatory 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 for 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 TRAC, B2M and each exon of the SPPL3 coding region were designed by CRISPR RGENTools, wherein, 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 NO: 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 SEQ ID NO:257-316 at the end of the article, 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 Use chemical methods to perform 2'-O-methylation and / or internucleotide phosphorothioate modification on sgRNA to obtain sgRNA with high knockout efficiency and stability.

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

[0157] Table 1:

[0158] Element volume Cas9 plasmid 5μg 10*cutsmart buffer 5μl Xba1 2μl Add water to the total reaction volume 5μl

[0159] Incubate in a 37°C water bath for 4 hours, take 2 μl of the digested product for agarose gel electrophoresis, set the voltage to 110 U, and perform electrophoresis for 30 minutes. A single band is observed under a gel imager, indicating that the digestion is complete and all plasmids are linearized.

[0160] The above reaction product is taken and cleaned and purified.

[0161] The purified product was taken and in vitro transcription (ie, IVT) was performed using HISCRIBETM T7 ARCAmRNA Kit (manufacturer: NEB, product number: cat#E2060S). The 20 μl system is shown in Table 2.

[0162] Table 2:

[0163] Element volume 2*ARCAMix 10μl Template DNA 0.5 μg T7 RNA Polymerase Mix 2μl Nuclease-free water Up to 20 μl Total reaction volume 20μl

[0164] The above reaction system was placed on a PCR instrument and reacted for 4 hours at 37°C. After 4 hours, 2 μl of DNase 1 was added to the reaction system and reacted at 37°C for 20 minutes.

[0165] Take the above reaction product and perform the operations shown in Table 3.

[0166] Table 3:

[0167] Element volume Nuclease-free water 63μl IVT reaction 22μl 10*Poly(A) polymerase buffer 10μl Poly(A) polymerase 5μl Total reaction volume 100μl

[0168] The reaction system was placed on a PCR instrument for 2 hours. The reaction product was cleaned and purified and placed in a -80°C refrigerator for later use.

[0169] 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 cells and 8 μg RNA (2 μg each of Cas9 mRNA, TCR sgRNA, B2M sgRNA, and SPPL3 sgRNA), and then use the electroporator BTX Agile pulse MAX (manufacturer: BTX, model: 47-0200NINT) to introduce cas9 mRNA and sgRNA into the cells under 200-400V / 0.5-2ms electroporation conditions. Using the antigen screening principle, T cells that are negative for TCR and / or B2M and / or SPPL3 and positive for CD4 and CD8 are screened out to obtain U-CART cells.

[0170] The cells were taken and the cell genome was extracted using TIANamp Genomic DNA Kit (manufacturer: TIAN GEN, catalog number: cat# DP304-03). The cell genome extracted above was taken using synthetic primers, and 2*Esay Taq SuperMix (+dye) (manufacturer: TRANS, catalog number Code# AS111) was used to PCR amplify the genomic regions of TRAC, B2M and SPPL3 containing the corresponding sgRNA, respectively, with a 50 μl reaction system, as shown in Table 4.

[0171] Table 4:

[0172]

[0173] The reaction conditions are as follows:

[0174]

[0175]

[0176] 1.2.5 Take the PCR products after the reaction for NGS sequencing to verify the knockout efficiency of TCR, HLA, and SPPL3 at the molecular level. After 8 days of cell culture, the obtained T cells were monitored for quality control. The results showed that the efficiency of knocking out TCR alone was about 97.18%, and the efficiency of knocking out SPPL3 was about 96.98%. It can be seen that in this system, the CRISPR / Cas9 technology can successfully edit the TRAC gene of TCR, the B2M gene of HLA, and the constant coding sequence of SPPL3, including insertion mutations and deletion mutations, and both caused frameshift mutations, thereby inhibiting the expression of TCR, HLA, and SPPL3 at the gene level.

[0177] 1.2.6 Detection of off-target rates of TCR and SPPL3 genes in U-CART cells

[0178] At the same time, potential off-target sites of TRAC (SEQ ID NO: 1 and 2) and SPPL3 (SEQ ID NO: 155 and 166) on the whole human genome 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 the knockout of non-specific genes that were off-target. The results showed that the TRAC and SPPL3 genes in the U-CART cells obtained by screening after gene editing of T cells 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 were completely knocked out, and no gene mutations at potential off-target sites were found. The specific results are shown in Tables 5 and 6.

[0179] Table 5: Off-target analysis results of TRAC knockout T cells

[0180]

[0181]

[0182] Table 6: Off-target analysis results of SPPL3 knockout T cells

[0183]

[0184] 1.2.7 Determination of relevant vitality indicators of U-CART cells constructed using CRISPR / Cas9 system and CRISPR / Cas12 system before purification

[0185] 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 the proliferation, viability, TCR KO efficiency at day 9, and SPPL3 at day 9. The KO efficiency, CAR+ cell percentage on day 9, U-CART cell viability on day 9, and U-CART cell number on day 9 were measured. The results are shown in Figure 1A-Figure 2E As shown; for the CRISPR / Cas12 system, four sgRNAs targeting the complementary sequence 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 the complementary sequence 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 knock out SPPL3 and TCR, and the proliferation, viability, 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 of the obtained U-CART cells were measured, and the results are shown in FIG. Figure 3A-Figure 4E shown.

[0186] 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 can be used for the next purification experiment.

[0187] 1.3 U-CART cells prepared by double knockout of TCR gene and SPPL3 gene were purified according to the following experimental steps:

[0188] 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 dosage of 100μL / mL, place on a shaker at a speed of 25RMP, incubate at room temperature for 30min, wash twice with 1% HSA buffer, resuspend the cells with 1% HSA buffer, add Anti-Biotin Reagent at a dosage of 200μL / mL, place on a shaker at a speed of 25RMP, incubate at room temperature for 30min; wash once with 1% HSA buffer, resuspend the cells with 1% HSA buffer; pass the cells through LS columns (Miltenyi Biopharmaceuticals, Catalog No.: 130-042-401), collect negative cells for live cell counting, and detect the purification efficiency by flow cytometry.

[0189] The results are as follows Figure 5A-Figure 5B and Figure 7A-7B As shown, the viability of U-CART cells does not decrease after purification.

[0190] Example 2 Functional verification of U-CART

[0191] 2.1 Proliferation and viability detection

[0192] The U-CART cells (U-CART) in which the TRAC and SPPL3 genes were completely knocked out prepared in Example 1, the unmodified T cells (T), and the ordinary CAR-T cells (CART) without any gene knockout were tested for proliferation ability and cell viability under the same other conditions. The results are as follows Figure 6A-6B and Figure 8A-B As shown, it can be seen that knocking out the TCR gene and SPPL3 gene will not affect the proliferation and cell viability of U-CART cells. Figure 6C and Figure 8C It shows that after knocking out the TCR gene and SPPL3 gene of UCAR-T cells, the cell viability of U-CART will not decrease with the expansion of U-CART cells.

[0193] 2.2 Detection of the ability to resist allogeneic T cell killing

[0194] In order to evaluate whether the SPPL3-knockout U-CART cells will be cleared by the patient's T cells after being infused into the patient's body, the 1G4-NY-ESO-1 system (Wei Wensheng Laboratory, Peking University) was used as an experimental model. The experimental system uses T cells expressing 1G4 TCR, which specifically kill 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, T cells cannot present NY-ESO-1 antigen peptides to the cell surface, thereby resisting the killing of 1G4 TCR-T cells.

[0195] The specific experimental plan is: resuspend the donor's 1G4 TCR-T cells in AIM5+5% FBS medium, and adjust the cell concentration to 5*10^6 / mL. Resuspend the NY-ESO-1 positive HLA-A02-01 typed T cells that have undergone different gene editing in AIM5+5% FBS medium, and adjust the cell concentration of each group to 1*10^6 / mL. 1G4 TCR-T cells and NY-ESO-1T cells were inoculated into 96-well U-shaped plates according to the effector-target ratio (1G4:NY-ESO-1=5:1). Among them, 100 μL of 1G4 TCR-T and NY-ESO-1T cells were added to each well, totaling 200 μL per well. After being added to the incubator and incubated for 24 hours, 20 μL of cells were taken out from each well for K2 counting to calculate the total number of living cells. The remaining 180 μL was used to detect the ratio of 1G4TCR-T and NY-ESO-1 positive T by flow cytometry (Note: 1G4 TCR-T is labeled with GFP, and the flow detection is FITC positive; NY-ESO-1 positive T is labeled with BFP, and the flow detection is PB450 positive). By comparing with the total number of 1*10^5 NY-ESO-1 positive T cells at the time of plating, the killing rate of NY-ESO-1 positive T cells in each experimental group was calculated.

[0196] The results are as follows Fig. 9 As shown, knocking out SPPL3 can significantly enhance the ability of T cells to resist allogeneic T cells. In terms of enhancing the ability of T cells to resist allogeneic T cells, knocking out SPPL3 can achieve an effect equivalent to or slightly weaker than knocking out B2M.

[0197] 3. Detection of the ability to resist NK cell killing

[0198] NK cells and U-CART cells were plated at a ratio of NK:T=10:1, 2E5 U-CART cells were plated in each group, AIM-V+5% FBS culture medium was used, and the cells were incubated at 37°C overnight. Samples were collected after 24 hours. After blowing evenly, 200uL was taken from each well to detect the killing ratio by flow cytometry. Cell killing was detected by counting FACS.

[0199] The results are as follows Fig.10 As shown, % killing percentage = (number of U-CART cells in control wells - number of U-CART cells in interaction wells) / number of U-CART cells in control wells * 100%.

[0200] 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.

[0201] 4. Detection of the ability to resist the killing effect of allogeneic PBMC

[0202] In order to evaluate the effect of allogeneic T cells and NK cells on the killing of U-CART cells after SPPL3 knockout U-CART cells were infused into patients, U-CART cells were co-cultured with allogeneic PBMCs, and the killing of U-CART cells by allogeneic PBMCs was counted. The specific experimental plan was as follows: (1) First, three types of U-CART cells, namely, U-CART cells with TCR and SPPL3 knockout (TSKO), U-CART cells with only TCR knockout (TKO), and U-CART cells with TCR and B2M knockout (TBKO), were stained with CellTrace ( Fig.11A , TKO-CellTrace-CFSE, TBKO-CellTrace-Far Red, TSKO-CellTrace-VIOLET); or (2) firstly, 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 ( Fig. 11B , TKO-CellTrace-CFSE, TBKO-CellTrace-Far Red, TBSKO-CellTrace-VIOLET); then the three U-CART cells were mixed in a ratio of 1:1:1, and the mixed U-CART cells and allogeneic PBMCs were co-incubated in a 37°C, 5% CO2 incubator at different ratios (the ratio of PBMC to U-CART cell number, represented by E:T in the figure). After 120 hours, the number of live cells of TKO, TBKO, and TSKO / TBSKO was counted by flow cytometry absolute counting method. Compared with the control group without allogeneic PBMCs, the killing percentage % = (the number of live cells in the control group - the number of live cells in the experimental group) / the number of live cells in the control group * 100% was used to count the killing of the four U-CART cells by allogeneic PBMCs.

[0203] The results are as follows Fig.11A and Fig. 11B As shown, 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; but 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.

[0204] 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 inhibited HLA-I expression (B2M is knocked out) by the body's immune cells.

[0205] 5. Detection of the ability to resist FasL-mediated killing

[0206] The test plan for this application is as follows:

[0207] 1. Take 1E7 cells of TKO / TFKO / TSKO CAR-T cells, resuspend to 10 mL, and plate in a 24-well plate, 1 mL per well;

[0208] 2. Use T cell complete medium to adjust the concentration of Biolegend Recombinant Human FASL to 1000 μg / mL as the stock solution;

[0209] 3. Dilute the FASL stock solution with T cell complete culture medium to 500, 200, 100, and 50 μg / mL;

[0210] 4. Add FASL to the 24-well plate 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, 0 ng / mL;

[0211] 5. Place the 24-well plate in an incubator at 37°C and 5% CO2 and incubate for 24 hours;

[0212] 6. After 24 hours, take out the 24-well plate, blow the cells evenly with a 1 mL pipette, take 20 uL of the cell suspension and add 20 uL of AOPI dye, use a K2 counter to count the cell viability of each group of cells, and record the counting results.

[0213] The results are as follows Fig.12As shown, it is shown that knocking out SPPL3 can partially achieve the effect of knocking out FasL, that is, it can resist the apoptosis of U-CART cells induced by the Fas signaling pathway.

[0214] 6. U-CART in vitro killing ability test

[0215] 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:

[0216] Resuscitation of cells: Rapidly thaw the frozen U-CART cells, T cells and target cells Nalm6 (human acute B lymphoblastic leukemia cell line) in a 37°C water bath. When there are almost no ice crystals in the cryopreservation tubes, transfer the cells to 15 mL centrifuge tubes, centrifuge at 400 × g for 5 min to collect the cells, resuspend them in fresh culture medium, and count them.

[0217] Co-incubation of target cells and effector cells: Add the effector cells and target cells to the cell culture plate at an E:T ratio of 1:2. Transfer the cell plate to a 37°C, 5% CO2 cell culture incubator for incubation.

[0218] Continuous addition of target cells: Every 48 hours, collect cells for counting and flow cytometry, and calculate the target cell ratio and CAR+ cell ratio. According to the CAR+% of each cell and the total number of cells, add the effector cells and target cells to the cell culture plate again at an E:T ratio of 1:2.

[0219] Repeat the above steps until the number of remaining CAR+ cells is insufficient to continue the co-culture experiment.

[0220] After each co-incubation, the following information was collected and calculated:

[0221] Residual percentage of target cells: Count the ratio of CD4-CD8-, that is, the percentage of target cells in all cell species;

[0222] Total number of CAR+ cells: Total number of CAR+ cells = CAR+% * total number of sample cells;

[0223] Cumulative proliferation fold of CAR+ cells = (total number of current CAR+ cells / total number of CAR+ cells at the last plating) * cumulative proliferation fold of CAR+ cells at the last time. (The cumulative proliferation fold of CAR+ cells at 0 hours is defined as 1).

[0224] Results of different T cell killing ability tests and CAR-positive (CAR+) T cell proliferation Figure 13-Figure 14 and Fig.15 As shown. Figure 13-Figure 14 It can be seen that 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 proportion of target cells shows that U-CART cells have a significant effect on killing Nalm-6 target cells, and in the entire long-term killing experiment, the killing effect of U-CART cells on target cells is less than 10%; Fig.15 It shows that the T cells without CAR molecules have no CAR+ cell proliferation and the T cells have no killing inhibitory effect on target cells, which leads to the increase of the target cell ratio over time in Figure 8. After 144 hours of co-incubation, the target cell ratio of the total cells is higher than 90%. Fig.15 The CAR+ cell proliferation of U-CART cells can be maintained for up to 288 hours after co-incubation, and the peak proliferation multiple is basically consistent with CAR-T. In summary, U-CART cells have good proliferation under the stimulation of target cells and have long-term and significant killing and inhibitory functions on tumor cells.

[0225] 7. U-CART in vivo killing ability detection

[0226] In this example, highly immunodeficient NCG mice were used to bear human acute B lymphoblastic leukemia cells Nalm-6, and then control T cells, CAR-T cells and U-CART cells prepared from the same healthy donor were injected to evaluate the tumor inhibitory effect of U-CART cells in tumor-bearing mice.

[0227] Twenty 6-8 week old female NCG mice were used in the experiment. 1×10 Nalm6-LAE cells (Nalm6-Luciferase-LAE) were injected into the tail vein. 6 / mouse for modeling. 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 in each group; accompanied by unedited T cells or U-CART cell cryopreservation fluid from the same volunteer, 5 animals in each group, T cell control group and tumor-bearing control group respectively. 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 weight of animals was weighed and recorded twice a week. If the weight loss exceeded 20% during the period, the animal was recorded as dead and euthanized.

[0228] The results are as follows Fig.17 As shown, after the injection of CAR-T and U-CART cells, according to the results of in vivo imaging ( Fig.16 ) It can be seen that compared with the test substance, the tumor growth rate of the tumor control group and the T cell control group was faster, and the tumor growth rate of the T cell control group was consistent with that of the tumor control group, indicating that T cells themselves have no killing function on tumor cells; compared with the CART cell group, the animals in the U-CART cell group did not have tumor recurrence on the 39th day after administration, and the TCR and SPPL3 double knockout U-CART cells had a very strong ability to kill tumor cells in immunodeficient mice that was not weaker than that of CAR-T cells without gene knockout. It can be seen that gene editing did not weaken the killing ability of CAR-T cells in vivo and did not affect their long-term survival in vivo.

[0229] The sequences used in the above examples of the present application are shown in the following sequence table. It should be understood that the following sequences are only exemplary sequences of the present application's embodiments, rather than any limitation to the present application's embodiments. The nucleic acid sequences in the following sequence table may represent DNA sequences or RNA sequences, and when they represent RNA sequences, "T" therein represents uridine.

[0230] Sequence Listing:

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

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 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.

Images