Polypeptide capable of binding to immune checkpoint and use thereof

Abstract

Provided is a fusion protein, which comprises a CD226 extracellular region, a transmembrane domain, and a co-stimulatory domain. The fusion protein can significantly enhance the tumor killing ability of a cell expressing an engineered receptor and prolong the survival time of the cell in vivo. Also provided are a nucleic acid molecule comprising a coding sequence of the fusion protein, an engineered cell expressing the fusion protein, and uses of the fusion protein, the nucleic acid molecule, and the engineered cell.

Description

Immune checkpoint-binding polypeptides and their applications Technical Field

[0001] The present application mainly relates to the field of cell therapy, and in particular to immune cells expressing the extracellular segment of CD226 or CD226 fusion protein and a therapy using the immune cells. Background Art

[0002] CAR-T cells, which express chimeric antigen receptors (CARs), have achieved breakthroughs in the treatment of hematologic malignancies. However, solid tumors are characterized by complex structures, tumor heterogeneity, and a complex tumor microenvironment, which hinder CAR-T infiltration and function. Furthermore, low oxygen and low pH levels can affect CAR-T survival in the body. Studies have shown that enhancing CAR-T survival or increasing its expansion in the body or at the tumor site can significantly improve the efficacy of CAR-T therapy in solid tumor treatment.

[0003] CD226 is a transmembrane glycoprotein composed of three domains. The first domain outside the CD226 molecule envelope is the structural basis for its ligand recognition, adhesion, immune synapse formation, and cytotoxicity. The intracellular region of CD226 contains four tyrosine residues and one serine residue. Known ligands for CD226 include CD155 and CD112. When CD226 binds to its ligand, the CD226 molecule moves to the lipid rafts on the cell membrane and recruits intracellular signaling molecules such as PTK and PKC, which are phosphorylated on the four tyrosine residues to activate the cell.

[0004] Summary of the Invention

[0005] This application mainly relates to the application of CD226 in engineered immune effector cells, and in particular to the application of CD226 in engineered immune effects. On the one hand, the present application constructs a CD226 fusion protein, which comprises a CD226 extracellular segment, a transmembrane structure and an intracellular signaling portion. On the other hand, the present application also constructs engineered immune cells, such as T cells, that express CD226 fusion protein and a CAR structure. The engineered cells have stronger anti-tumor effects and in vivo persistence against solid tumors. The targets of the CAR structure include but are not limited to solid tumor targets such as CEA, PSCA, CD70, and B7H3. The CD226 fusion protein can enhance the killing ability of CAR-T cells, competitively bind to CD155 and CD112 with TIGIT, and thus alleviate immunosuppression in the tumor microenvironment.

[0006] Specifically, this application provides:

[0007] 1. A fusion protein comprising or consisting of:

[0008] 1) CD226 extracellular region, transmembrane domain, and costimulatory domain; or

[0009] 2) Signal peptide, CD226 extracellular region, transmembrane domain and co-stimulatory domain.

[0010] In some embodiments, the signal peptide is a signal peptide derived from a secreted protein.

[0011] In some embodiments, the extracellular region and the transmembrane domain are connected by a hinge region.In some embodiments, the hinge region is the hinge region of IgG, IgD, CD8α or CD28, or a combination thereof.

[0012] In some embodiments, the extracellular region and the transmembrane domain are connected by a peptide bond. In some embodiments, the extracellular region and the transmembrane domain, and the costimulatory domain and the transmembrane domain are connected directly or indirectly. In some embodiments, the direct connection is connected by a covalent bond or a non-covalent bond. In some embodiments, the indirect connection is connected by a linker. In some embodiments, the linker is a short peptide chain. In some embodiments, the CD226 extracellular region is the extracellular region of human CD226. In some embodiments, the extracellular region of CD226 comprises the amino acid sequence of SEQ ID NO: 1 or 3, or a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 1 or 3, or an amino acid sequence having greater than 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.5%, 99.7%, 99.8%, or 99.9% or greater) sequence identity to the amino acid sequence of SEQ ID NO: 1 or 3.

[0013] 2. The fusion protein according to item 1, wherein the transmembrane domain comprises or is composed of a transmembrane domain selected from any one or more of the following molecules: ICOS, 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, PD-1, and CD226. In some embodiments, the transmembrane domain comprises or is the transmembrane domain of CD28. In some embodiments, the transmembrane domain comprises or is the transmembrane domain of human CD28. In some embodiments, the transmembrane domain comprises or is an amino acid sequence as set forth in SEQ ID NO: 17, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 17, or an amino acid sequence having greater than 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.5%, 99.7%, 99.8%, or 99.9% or greater) sequence identity to the amino acid sequence as set forth in SEQ ID NO: 17.

[0014] 3. The fusion protein according to item 1 or 2, wherein the costimulatory domain comprises a signal transduction domain selected from any one or more of the following molecules or consists of a signal transduction domain of one or more signaling molecules selected from the following:

[0015] CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80(KLRF1), CD 160. CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a ligand that specifically binds to CD83. In some embodiments, the costimulatory domain is a signal transduction domain of CD28. In some embodiments, the costimulatory domain is a signal transduction domain of human CD28. In some embodiments, the costimulatory domain comprises or is an amino acid sequence as set forth in SEQ ID NO: 18, or a conservatively substituted variant of an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence having greater than 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.5%, 99.7%, 99.8%, or 99.9% or greater) sequence identity to an amino acid sequence as set forth in SEQ ID NO: 18.

[0016] 4. The fusion protein according to any one of items 1 to 3, wherein the signal peptide comprises or is a signal peptide of a CD226 or CD8α molecule. In some embodiments, the signal peptide of the CD8α molecule is a signal peptide of a human CD8α molecule, and the signal peptide of the CD226 molecule is a signal peptide of a human CD226 molecule. In some embodiments, the signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 4, or a conservatively substituted variant of the amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having 85% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.5%, 99.7%, 99.8%, or 99.9% or more) sequence identity with the amino acid sequence as shown in SEQ ID NO: 4.

[0017] 5. The fusion protein according to any one of items 1 to 4, which comprises or is, from N-terminus to C-terminus:

[0018] 1) CD226 extracellular region, CD28 transmembrane domain, and CD28 signaling domain; or

[0019] 2) CD226 signal peptide, CD226 extracellular region, CD28 transmembrane domain and CD28 signal transduction domain.

[0020] 6. A fusion protein according to any one of items 1 to 5, wherein the extracellular region of CD226 comprises or is the amino acid sequence shown in SEQ ID NO: 1, a conservatively substituted variant of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having more than 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.5%, 99.7%, 99.8%, or 99.9% or more) sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0021] 7. The fusion protein according to claim 6, which comprises or is the amino acid sequence as shown in SEQ ID NO: 2, a conservatively substituted variant of the amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having more than 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.5%, 99.7%, 99.8%, or 99.9% or more) sequence identity to the amino acid sequence as shown in SEQ ID NO: 2.

[0022] In some embodiments, the CD226 extracellular region is further directly or indirectly linked to one or more polypeptides or proteins that bind to tumor antigens or immune checkpoint proteins. In some embodiments, the transmembrane domain is further directly or indirectly linked to one or more polypeptides or proteins that bind to tumor antigens or immune checkpoint proteins.

[0023] 8. An engineered receptor comprising the fusion protein of any one of items 1 to 6, and a primary signal transduction domain located at the C-terminus of the fusion protein.

[0024] 9. An engineered receptor according to claim 8, wherein the primary signal transduction domain comprises or consists of the signal transduction domains of one or more of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, CD79α, CD79β, CD66d, DAP10 and DAP12.

[0025] In some embodiments, the primary signaling domain is a portion of a signaling domain in a human protein that includes a tyrosine-based activation motif of an immunoreceptor.

[0026] 10. An engineered nucleic acid molecule comprising a molecule encoding the fusion protein of any one of items 1 to 7 or the engineered receptor of item 8 or 9. In some embodiments, the engineered nucleic acid molecule is DNA, RNA (eg, mRNA), or a hybrid molecule of RNA and DNA.

[0027] In some embodiments, the engineered nucleic acid molecules are chemically modified. For example, in some embodiments, one or more thymidines of the engineered nucleic acid molecules are replaced by uridines. In some embodiments, one or more uridines of the engineered nucleic acid molecules are replaced by thymidines. In some embodiments, one or more guanosines of the engineered nucleic acid molecules are replaced by creatinines. In some embodiments, the chemical modification is to replace one or more nucleotides of the nucleic acid molecules with their corresponding nucleotide derivatives. For example, in some embodiments, one or more uridines of the engineered nucleic acid molecules are replaced by one or more selected from the group consisting of 5-(carboxyhydroxymethyl)uridine (CHM5U), 5-carboxymethylaminomethyluridine (CMNM5U), 5-carboxymethylaminomethyl-2-thiouridine (CMNM5S2U), dihydrouridine (DHU), 2'-O-methylpseudouridine (FM), 1-methylpseudouridine (M1F), 3-(3-amino-3-carboxyl-propyl)uridine ((ACP3)U), uridine-5-oxyacetic acid (O5U), uridine-5-oxyacetic acid methyl ester (MV), 5-methoxycarbonyl The uridine residues are 5-methyluridine (MCM5U), 5-methoxycarbonylmethyl-2-thiouracil (MCM5S2U), 5-methoxyuridine (MO5U), 5-methyl-2-thiouridine (S2T), 2-thiouridine (S2U), 4-thiouridine (S4U), 5-methyluridine (M5U), 2'-O-methyl-5-methyluridine (TM), 2'-O-methyluridine (UM), 5-methylaminomethyluridine (MAM5U), 5-methylaminomethyl-2-thiouridine (MAM5S2U), pseudouridine (P), and 5-methoxycarbonylmethyl-2-thioguanosine (MCM5S2U). In some embodiments, one or more guanosines of the engineered nucleic acid molecule are replaced by one or more selected from the group consisting of wybutoxosine (osyw), whibutoxosine (yw), 1-methylinosine (m1i), 2'-O-methylguanosine (gm), 1-methylguanosine (m1g), 2,2-dimethylguanosine (m22g), 2-methylguanosine (m2g), 7-methylguanosine (m7g) and β, D-galactose Q nucleoside (gal q), Q nucleoside (q) and β, D-mannose Q nucleoside (man q).In some embodiments, one or more adenosines of the engineered nucleic acid molecule are replaced by one or more selected from the group consisting of N6-isopentenyl adenosine (i6a), 1-methyladenosine (m1a), 2-methyladenosine (m2a), N6-methyladenosine (m6a), 2-methylthio-N6-isopentenyl adenosine (ms2i6a), N-((9-β-D-ribofuranosyl-2-thiomethylpurin-6-yl)carbamoyl)threonine (ms2t6a), N-((9-β-D-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine (mt6a), N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine (t6a), β, Q nucleoside (q), and D-mannose Q nucleoside (man q). In some embodiments, one or more cytidines of the engineered nucleic acid molecule are replaced by one or more of the following: 4-acetylcytidine (ac4c), 2'-O-methylcytidine (cm), 3-methylcytosine (m3c), N4-methylcytidine (m4c), 5-methylcytidine (m5c), β, and 2-thiocytidine (s2c). In some embodiments, the chemical modification includes a 2'-O-methylation modification on the ribose of the nucleotide or a 3'thiophosphate 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'thiophosphate modification between the nucleotides of the first three nucleotides at the 5' end, and a 3'thiophosphate modification between the nucleotides of the last three nucleotides at the 3' end.

[0028] In some embodiments, the nucleic acid molecule further encodes one or more second engineered receptors that bind to one or more tumor antigens and / or immune checkpoint proteins. In some embodiments, the second engineered receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a T cell antigen coupling agent (TAC). In some embodiments, the one or more immune checkpoint proteins bound by the second engineered receptor are not CD155. In some embodiments, the one or more immune checkpoint proteins bound by the second engineered receptor include CD155.

[0029] 11. An engineered cell comprising the fusion protein of any one of items 1 to 7 or the engineered receptor of item 8 or 9 and / or the engineered nucleic acid molecule of item 10.

[0030] 12. The engineered cell according to claim 11, further comprising one or more engineered receptors that bind to one or more target molecules, wherein the one or more target molecules are selected from tumor antigens and / or immune checkpoint proteins, wherein,

[0031] The tumor antigen is selected from the group consisting of: prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA) CAM5, CD123, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit alpha (IL-13Rα); interleukin-11 receptor alpha (IL-11Rα); prostate-specific membrane antigen (PSMA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and their mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFR-1, EGFR-2, EGFR-3, EGFR-4, EGFR-5, EGFR-6, EGFR-7, EGFR-8, EGFR-9, EGFR-10, EGFR-11, EGFR-12, EGFR-13, EGFR-14, EGFR-15, EGFR-16, EGFR-17, EGFR-18, EGFR-19, EGFR-21, EGFR-22, EGFR-23, EGFR-27, EGFR-28, EGFR-29, EGFR-31, EGFR-32, EGFR-33, EGFR-34, EGFR-35 FRvIII); 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);The hexose portion of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant 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 mutant; 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 myelocytotoxic viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); CD79a; CD79b; C D72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1); and CD155.

[0032] The immune checkpoint protein is selected from: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD 48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, TIGIT, and VSIG8.

[0033] In some embodiments, the one or more target molecules are selected from one or more of the following: CD70, PSCA, and CEA.

[0034] In some embodiments, the engineered receptor is selected from one or more of the following: CAR, TCR, and TAC.

[0035] 13. The engineered cell according to item 12, wherein the engineered receptor that binds to other molecules is a CAR that binds to CEA. In some embodiments, the CAR that binds to CEA comprises or is, from N-terminus to C-terminus: a domain that binds to CEA-hinge region-transmembrane region-signaling domain, or a domain that binds to CEA-transmembrane region-signaling domain. In some embodiments, the CEA is human CEA. In some embodiments, the domain that binds to CEA is an antibody or an antigen-binding fragment of the antibody. In some embodiments, the antigen-binding fragment is an scFv. In some embodiments, the signal transduction domain comprises or is a primary signal transduction domain. In some embodiments, the signal transduction domain comprises a primary signal transduction domain and a costimulatory domain. In some embodiments, the signal transduction domain consists of a primary signal transduction domain and a costimulatory domain. In some embodiments, the signal transduction domain comprises one or more costimulatory domains. In some embodiments, the CEA-binding CAR comprises or is, from N-terminus to C-terminus, CEA ScFv-8h-8TM-BBZ. CEAscFv represents an scFv composed of a heavy chain variable region and a light chain variable region of an anti-CEA antibody, and "-" represents a connection via a peptide bond or peptide chain. In some embodiments, the CEAscFv comprises or is the amino acid sequence shown in SEQ ID NO: 5.

[0036] 14. An engineered cell according to claim 13, wherein the CEA-binding CAR comprises or is an amino acid sequence as shown in SEQ ID NO: 6 or a conservatively substituted variant of SEQ ID NO: 6, or an amino acid sequence having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.5%, 99.7%, 99.8%, or 99.9%) sequence identity with SEQ ID NO: 6.

[0037] 15. The engineered cell according to item 12, wherein the engineered receptor that binds another molecule is a CAR that binds to PSCA. In some embodiments, the CAR that binds to PSCA comprises or is, from the N-terminus to the C-terminus: a PSCA-binding domain-hinge region-transmembrane region-signaling domain, or a PSCA-binding domain-transmembrane region-signaling domain. In some embodiments, the PSCA is human PSCA. In some embodiments, the PSCA-binding domain is an antibody or an antigen-binding fragment of such an antibody. In some embodiments, the antigen-binding fragment is an scFv. In some embodiments, the signaling domain comprises or is a primary signaling domain. In some embodiments, the signaling domain comprises a primary signaling domain and a costimulatory domain. In some embodiments, the signaling domain consists of a primary signaling domain and a costimulatory domain. In some embodiments, the signaling domain comprises one or more costimulatory domains. In some embodiments, the PSCA-binding CAR comprises or is, from N-terminus to C-terminus, PSCA ScFv-8h-8TM-BBZ. Here, "PSCA scFv" represents an scFv composed of one heavy chain variable region and one light chain variable region of an anti-PSCA antibody, and "-" represents a peptide bond or peptide chain connection. In some embodiments, the PSCA scFv comprises or is the amino acid sequence set forth in SEQ ID NO: 8.

[0038] 16. The engineered cell of claim 15, wherein the PSCA-binding CAR comprises or is an amino acid sequence as set forth in SEQ ID NO: 9 or a conservatively substituted variant of SEQ ID NO: 9, or an amino acid sequence having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.5%, 99.7%, 99.8%, or 99.9%) sequence identity to SEQ ID NO: 9.

[0039] 17. The engineered cell according to item 12, wherein the engineered receptor that binds to other molecules is a CAR that binds to CD70. In some embodiments, the CAR that binds to CD70 comprises or is, from N-terminus to C-terminus: a CD70-binding domain-hinge region-transmembrane region-signaling domain, or a CD70-binding domain-transmembrane region-signaling domain. In some embodiments, the CD70 is human CD70. In some embodiments, the CD70-binding domain is an antibody or an antigen-binding fragment of the antibody. In some embodiments, the antigen-binding fragment is an scFv. In some embodiments, the signal transduction domain comprises or is a primary signal transduction domain. In some embodiments, the signal transduction domain comprises a primary signal transduction domain and a costimulatory domain. In some embodiments, the signal transduction domain consists of a primary signal transduction domain and a costimulatory domain. In some embodiments, the signal transduction domain comprises one or more costimulatory domains. In some embodiments, the CD70-binding CAR comprises or is, from N-terminus to C-terminus, CD70ScFv-8h-8TM-BBZ. Wherein, CD70 scFv represents an scFv composed of one heavy chain variable region and one light chain variable region of an anti-CD70 antibody, and "-" indicates connection via a peptide bond or peptide chain. In some embodiments, the CD70 scFv comprises or is the amino acid sequence set forth in SEQ ID NO: 35.

[0040] 18. The engineered cell according to claim 17, wherein the CD70-binding CAR comprises or is an amino acid sequence as shown in SEQ ID NO: 12 or a conservatively substituted variant of SEQ ID NO: 12, or an amino acid sequence having at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.5%, 99.7%, 99.8%, or 99.9%) sequence identity to SEQ ID NO: 12.

[0041] The engineered cell according to any of the foregoing, wherein the costimulatory domain comprises a signaling domain of one or more molecules selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD160, CD18, CD29a, CD30b, IL3Rβ, IL3Rγ, IL4Rγ, IL5Rα, IL6Rα, IL7Rα, IL8Rα, IL9Rα, IL10Rα, IL11Rα, IL12Rα, IL13Rα, IL14Rα, IL15Rα, IL16Rα, IL17Rα, IL18Rα, IL19Rα, IL21Rα, IL22Rβ, IL23Rγ, IL24Rγ, IL25Rα, IL26Rα, IL27Rα, IL28Rα, IL29Rα, IL30Rα, IL31Rα, IL32Rα, IL33Rα, IL34Rα, IL35Rα, IL36Rα, IL37Rα, IL38Rα, IL39Rα, IL40Rα, IL41Rα, IL42Rα Id, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and ligands that specifically bind to CD83.

[0042] An engineered cell according to any of the foregoing, wherein the primary signaling domain comprises or consists of a signaling domain of one or more molecules selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, CD79α, CD79β, CD66d, DAP10, and DAP12.

[0043] The engineered cell according to any of the preceding items, wherein the hinge region is the hinge region of IgG, IgD, CD8α or CD28, or a combination thereof.

[0044] The engineered cell according to any of the foregoing, wherein the transmembrane domain comprises a transmembrane domain of one or more molecules selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD6, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD47, CD52, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, CISH, and PD-1.

[0045] 19. The engineered cell according to any one of items 11 to 18, which is a T cell, NK cell, macrophage, DC cell, B cell, or a precursor cell thereof.

[0046] 20. Use of the fusion protein of any one of items 1-7, the engineered receptor of item 8 or 9, the engineered nucleic acid molecule of item 10, or the engineered cell of any one of items 11-19 for the preparation of a medicament for treating cancer. In some embodiments, the cancer is a human cancer.

[0047] 21. The method according to claim 20, wherein the cancer is selected from one or more of the following:

[0048] Bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, and uterine cancer. In some embodiments, the cancer is human colorectal cancer. In some embodiments, the cancer is human bladder cancer. In some embodiments, the cancer is human clear cell renal adenocarcinoma.

[0049] In addition, the present application also provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the fusion protein according to any one of items 1 to 7, the engineered receptor according to item 8 or 9, the engineered nucleic acid molecule according to item 10, or the engineered cell according to any one of items 11 to 19. In some embodiments of the method for treating cancer, the cancer is selected from one or more of the following:

[0050] In some embodiments, the subject or patient is a human patient.

[0051] In addition, the present application also provides a drug for treating cancer, comprising the fusion protein of any one of items 1-7, the engineered receptor of item 8 or 9, the engineered nucleic acid molecule of item 10, or the engineered cell of any one of items 11-19. In some embodiments, the cancer is selected from one or more of the following:

[0052] In some embodiments, the subject or patient is a human patient.

[0053] 22. A method for prolonging the in vivo persistence of CAR-T cells, comprising expressing the fusion protein described in any one of items 1 to 7 or the engineered receptor described in item 8 or 9 on the CAR-T cell membrane.

[0054] 23. A method for improving the in vivo expansion ability of CAR-T cells, comprising expressing the fusion protein described in any one of items 1 to 7 or the engineered receptor described in item 8 or 9 on the CAR-T cell membrane.

[0055] 24. A method for enhancing the in vivo killing ability of CAR-T against target cells, comprising expressing the fusion protein described in any one of items 1 to 7 or the engineered receptor described in item 8 or 9 on the CAR-T cell membrane.

[0056] In some embodiments, the aforementioned in vivo refers to a cancer patient or subject. In some embodiments, the in vivo refers to a patient or subject in which CD155 is abnormally expressed in certain tissues and organs. In some embodiments, the cancer is selected from one or more of the following:

[0057] Bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, and uterine cancer.

[0058] In some embodiments, the CAR-T described in any one of items 22 to 24 comprises a CAR having the following structure from N-terminus to C-terminus: antigen binding domain-hinge region-transmembrane region-signal transduction domain, or antigen binding domain-transmembrane region-signal transduction domain. In some embodiments, the antigen binding domain comprises or is an antibody or an antigen binding fragment thereof, such as scFv. In some embodiments, the antigen binding domain comprises or is a ligand binding receptor domain, or a receptor binding ligand domain. In some embodiments, the signal transduction domain comprises or is a primary signal transduction domain. In some embodiments, the signal transduction domain comprises a primary signal transduction domain and a costimulatory domain. In some embodiments, the signal transduction domain consists of a primary signal transduction domain and a costimulatory domain. In some embodiments, the signal transduction domain comprises one or more costimulatory domains. In some embodiments, the CAR-T in any one of items 22 to 24 comprises or has the following structure from N-terminus to C-terminus: ScFv-8h-8TM-BBZ, wherein scFv represents an scFv composed of a heavy chain variable region and a light chain variable region of an anti-antibody, and "-" represents connection through a peptide bond or peptide chain.

[0059] 2A self-cleavage polypeptide is a kind of commonly used multi-gene expression scheme that translation level realizes multiple gene co-expression.The polypeptide was first found in foot-and-mouth disease virus (FMDV) in 1991, with an average length of 18-22 amino acids, and multiple different 2A peptides have been found, respectively derived from foot-and-mouth disease virus 2A (F2A), type 1 porcine teschovirus 2A (P2A), bright vein flat moth virus 2A (T2A), and horse-type rhinitis virus 2A (E2A). Its main working principle is that ribosomes are in the translation process, and when identifying 2A peptide ends, they can skip glycyl-prolyl peptide bond synthesis, and slide, thereby directly generating 2 independent proteins. The purpose of the application using 2A peptides is to obtain the engineered immune cells expressing CD226 fusion proteins, and finally the function of the engineered immune cells described in the test verification of embodiment is verified, and further verification of CD226 fusion proteins for the unpredictable effect of engineered immune cells. In addition to the above-mentioned 2A self-cleavage peptide, IRES (Internal ribosome entry site, IRES) can also be used. IRES can recruit ribosomes to translate mRNA, and can choose to express multiple proteins independently. The internal ribosome entry site (IRES) is used to separate the coding genes (also called ORFs) of target molecules such as CD226 fusion protein, at least one chimeric antigen receptor (CAR), and other fusion proteins. A single mRNA transcript will produce multiple proteins. The above-mentioned 2A peptide and IRES, as well as other small molecule sequences with similar functions, can be collectively referred to as linkers. In addition to using the above-mentioned linkers to achieve multi-gene expression, the above-mentioned engineered cells can also be achieved by separately transducing different genes into target cells, or by simultaneously transducing target genes with constructed vectors expressing different genes; whether using linkers or separately constructing expression vectors of multiple genes to transduce target cells, the purpose of obtaining engineered cells expressing CD226 can ultimately be achieved, and the functions and unpredictable effects of engineered cells are mainly based on the protein molecules expressed and the final engineered cells themselves.

[0060] The application verifies the function and effectiveness of engineered immune cells expressing CD226 fusion protein and engineered immune cells expressing CD226 fusion protein and chimeric antigen receptor. Although the examples herein are based on the verification of engineered T cells, the unpredictable effects of the engineered cells are mainly based on CD226 fusion protein and the combination of CD226 fusion protein and CAR. Therefore, the unpredictable effects described in the application can also be achieved in immune cells such as NK, DC, macrophages, NKT, γδT, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG1 shows a schematic diagram of engineered cells expressing CD226 fusion protein.

[0062] FIG2A and FIG2B show the proliferation fold and viability of CAR-T cells expressing CD226 fusion protein, wherein FIG2A shows the proliferation fold and FIG2B shows the viability.

[0063] FIG3 shows in vitro killing by CEA CAR-T cells expressing CD226 fusion protein.

[0064] FIG4 shows the in vitro cytokine secretion of CEA CAR-T expressing CD226 fusion protein.

[0065] FIG5 shows the in vivo efficacy of CEA CAR-T expressing CD226 fusion protein against colorectal cancer tumor-bearing models.

[0066] FIG6 shows the detection of CAR copy number when CEA CAR-T expressing CD226 fusion protein is used to treat a colorectal cancer mouse tumor-bearing model.

[0067] FIG. 7 shows the proliferation fold of PSCA CAR-T cells expressing CD226 fusion protein.

[0068] FIG8 shows in vitro killing by PSCA CAR-T cells expressing CD226 fusion protein.

[0069] FIG9 shows the in vitro cytokine secretion of PSCA CAR-T cells expressing CD226 fusion protein.

[0070] FIG10 shows the in vivo efficacy of PSCA CAR-T expressing CD226 fusion protein against bladder cancer tumor-bearing models.

[0071] FIG11 shows the detection of CAR copy number when PSCA CAR-T expressing CD226 fusion protein is used to treat a bladder cancer mouse tumor-bearing model.

[0072] FIG12 shows the in vitro cytokine secretion of CD70 CAR-T expressing CD226 fusion protein. Specific implementation plan

[0073] The present application relates to a transmembrane protein with an extracellular segment of CD226 as an antigen binding domain, the transmembrane protein is referred to as a fusion protein in this application, which comprises a CD226 extracellular region, a transmembrane domain and a co-stimulatory domain. The fusion protein can be expressed on the surface of CAR-T cells to enhance the tumor killing ability of the CAR-T cells and at the same time increase the duration of the CAR-T cells in the body. The present application also provides engineered cells expressing the fusion protein, such as CAR-T cells, nucleic acid molecules encoding the fusion protein, and uses of the fusion protein, nucleic acid molecules, and engineered cells.

[0074] definition

[0075] It should be understood that the present disclosure is not limited to the aspects described herein, which can of course vary. It should also be understood that the terminology used herein is used to describe particular aspects only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the technology belongs. All technical and patent disclosures cited herein are incorporated herein by reference in their entirety. Unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA within the skill of the art will be employed by those skilled in the art.

[0077] The term "CD226" refers to an immune activator receptor, also known as DNAM-1. It competes with TIGIT (T cell immunoreceptor with Ig and ITIM domains, also known as WUCAM, Vstm3, or VSIG9) for the same set of ligands: CD155 (PVR or poliovirus receptor) and CD112 (fibronectin-2 or PVRL2). However, compared to its binding to PVR, CD226 has a much weaker binding affinity to PVRL2 and PVRL3. Naturally, CD226 is a glycoprotein expressed on the surface of NK cells, platelets, monocytes, and some T cells. It belongs to the Ig superfamily. CD226 is composed of three domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains two immunoglobulin V-like domains and eight N-linked glycosylation sites. The intracellular domain contains four tyrosine residues and one serine residue. When CD226 binds to the ligand, the CD226 molecule moves to the lipid rafts on the cell membrane and recruits intracellular signaling molecules (such as PTK and PKC) to phosphorylate the four tyrosine residues, thereby activating the cells. CD226 can mediate the adhesion of platelets and megakaryocytes to vascular endothelial cells and play a role in the maturation of megakaryocytes. Exemplary CD226 includes human CD226. In some embodiments, CD226 is encoded by a gene with a gene ID of 10666 in the NCBI database.

[0078] As used herein, "CD226 extracellular region" can be used to refer to any peptide fragment in the CD226 extracellular region or any truncated form of the CD226 extracellular region, or the full-length CD226 extracellular region, as long as it retains the ability to bind to CD155. In some embodiments, the CD226 extracellular region comprises the amino acid sequence shown in SEQ ID NO: 1 or 3. In some embodiments, the CD226 extracellular region comprises an amino acid sequence corresponding to SEQ ID NO: 1 or 3 in any CD226 molecule. The "corresponding" refers to the position of the corresponding amino acid by sequence alignment. After the amino acid sequence of any CD226 molecule is made identical at as many positions as possible with the amino acid sequence of SEQ ID NO: 1 or 3 by introducing gaps or deleting amino acids into the amino acid sequence of any CD226 molecule (for exemplary methods, see, for example, BLAST, FASTA, and MEGLIGN methods), the amino acids in SEQ ID NO: 1 or 3 are numbered sequentially starting from 1 at the first amino acid at the nitrogen terminus. In the amino acid sequence of any CD226 molecule, the first and last amino acids at the same positions as those in the amino acid sequence of SEQ ID NO: 1 or 3, and all amino acids between the first and last amino acids, constitute the "amino acid sequence corresponding to SEQ ID NO: 1 or 3."

[0079] CD155, also known as "PVR," stands for poliovirus receptor, also known as Necl5 and Tage4. CD155 is a cell surface adhesion molecule that is dramatically overexpressed in several human malignancies, while expression is low or absent in most healthy tissues. Its overexpression promotes tumor cell invasion, migration, and proliferation and is associated with poor prognosis and increased tumor progression.

[0080] "CD112" is also known as "PVRL2", which is adhesion protein-2. It is a single-channel type I membrane protein with two Ig-like C2-type domains and one Ig-like V-type domain. It is one of the plasma membrane components of adhesion junctions.

[0081] As used herein, percentages of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identity, refer to a degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, the percentage of positions with identical bases or amino acid residues is determined as a ratio of the total number of positions after two sequences have been aligned to have identical residues at as many positions as possible, such as by introducing gaps. Percentages of "identity" can be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found on the Internet at the following address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0082] As used herein, a "variant" has at least one amino acid difference relative to a reference amino acid sequence, e.g., at least one amino acid addition, insertion, deletion, or substitution. For example, the amino acid substitution may be a conservative amino acid substitution, i.e., replacing the original corresponding amino acid with an amino acid having similar properties. "Conservative substitutions" may be polar to polar amino acids, such as glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine ​​(C, Cys), asparagine (N, Asn), and glutamine (Q, Gln); non-polar to non-polar amino acids, such as alanine (A, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), phenylalanine (F, Phe); acidic to acidic amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Gln); u); basic to basic amino acids, such as arginine (R, Arg), histidine (H, His), lysine (K, Lys); charged amino acids to charged amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys) and arginine (R, Arg); hydrophobic to hydrophobic amino acids, such as alanine (A, Ala), leucine (L, Leu), isoleucine (I, Ile), valine (V, Val), proline (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp) and methionine (M, Met). In some other embodiments, the variant may also comprise non-conservative substitutions. In some embodiments, the "variant" of the amino acid sequence may have at least about 90%, 95%, 96%, 97%, 98%, 99% sequence identity relative to the amino acid sequence. Compared to the amino acid sequence, the "variant" of the amino acid sequence may have an activity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or a range consisting of any two of the aforementioned values. As used herein, a "conservative substitution variant" of a protein, polypeptide or amino acid sequence refers to one or more amino acid residues in which the overall conformation and function of the protein or enzyme are changed by amino acid substitution, including but not limited to replacing the amino acids in the amino acid sequence of the parent protein in the manner described by the aforementioned "conservative substitution". Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different. For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm."Conservative substitution variants" also include polypeptides or enzymes with more than 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably more than 75%, preferably more than 85%, and even more than 90%, and having the same or substantially similar properties or functions as the native or parent protein or enzyme.

[0083] As used herein, "amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term "amino acid" includes the following 20 natural or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V). In some embodiments, the application, when referring to any of the above natural amino acids, also includes non-natural amino acids or amino acid analogs derived or modified therefrom. As used herein, "amino acid" also includes non-natural amino acids, modified amino acids (e.g., with modified side chains and / or backbones) and amino acid analogs. To further illustrate, amino acids are generally organic acids comprising substituted or unsubstituted amino, substituted or unsubstituted carboxyl and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, sulfhydryl, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halogen, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphino, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising photosensitive crosslinkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids comprising metals, amino acids containing novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photolabile (photocaged) and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids comprising carbon-linked sugars, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties.The amino acids described in the present application include, but are not limited to, 20 natural amino acids and 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), beta-alanine or beta-aminoalanine (bAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid or pipecolic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptaneic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisomethacrylic acid (bAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), methamphetamine (Des), 2,2'-diaminopimelic acid (Dpm), 2,3-diaminopropanesulfonic acid (Dpr), ethylglycine (EtGly), N- Ethyl aspartic acid (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), isoleucine (aIle), N-methylglycine or sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle) and ornithine (Orm). Therefore, in some embodiments, after the mutation, the amino acid mutation at the site comprises a substitution mutation converted to any of the above 20 natural amino acids and the above non-natural amino acids. In some embodiments, the amino acid mutation comprises a substitution mutation to any one of the following amino acids: G, A, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, H, Aad, bAad, bAla, Abu, 4Abu, Acp, Ahe, Aib, bAib, Apm, Dbu, Des, Dpm, Dpr, EtGly, EtAsn, Hyl, aHyl, 3Hyp, 4Hyp, Ide, aIle, MeGly, MeIle, MeLys, MeVal, Nva, Nle, and Orm.

[0084] In the context of this application, the terms "DNA" and "RNA" refer to single-stranded or double-stranded DNA or RNA molecules. Unless otherwise indicated, the terms "DNA" and "DNA molecule" refer to double-stranded DNA molecules composed of A, C, G and / or T nucleotides, while the terms "RNA" and "RNA molecule" refer to single-stranded RNA molecules composed of A, C, G and / or U nucleotides. As used herein, the A, C, G, T and U nucleotides refer to nucleotides containing adenine, guanine, cytosine, thymine and uracil as their respective nitrogenous bases.

[0085] RNA molecules include coding RNA or non-coding RNA (ncRNA), such as pre-mRNA, mature mRNA or long noncoding RNA (lncRNA).

[0086] As used herein, the "DNA and RNA hybrid molecule" is a molecule comprising a polynucleotide sequence consisting of deoxyribonucleotides and ribonucleotides. The DNA and RNA hybrid molecule can be obtained by:

[0087] Replace one or more deoxyribonucleotides in DNA with ribonucleotides;

[0088] Substituting one or more ribonucleotides in the RNA with deoxyribonucleotides; or

[0089] De novo synthesis using deoxyribonucleotides and ribonucleotides as raw materials through biological or chemical synthesis. It should be noted that the methods for obtaining DNA / RNA hybrid molecules are not limited to the above methods; DNA / RNA hybrid molecules obtained by any method fall within the scope of "DNA / RNA hybrid molecules" as defined in this application.

[0090] As used herein, when two nucleic acid molecules are described as having "the same genetic information", it means that the two nucleic acid molecules are complementary, or contain exactly the same base sequence, or that after one or more thymines in the base sequence of one nucleic acid molecule are converted to uracil, a nucleic acid molecule with exactly the same base sequence as the other nucleic acid molecule can be obtained. Therefore, any two of DNA, RNA, and hybrid molecules of DNA and RNA can have the same genetic information. Among them, the term "base sequence" refers to the order in which bases are arranged in a polynucleotide molecule. It should be understood by those skilled in the art that, unless otherwise specified, the base sequence or polynucleotide sequence described in this application can be used to represent thymine when used to describe a DNA sequence, but when the base sequence or polynucleotide sequence is used to describe RNA (e.g., mRNA), "T" will be replaced by "U" (uracil). Therefore, any DNA disclosed by a specific sequence number (SEQ ID NO) herein also discloses an RNA (e.g., mRNA or Poly (A) tail) sequence that is complementary or corresponding to the DNA, wherein each "T" in the DNA sequence is replaced by a "U".

[0091] In this article, "coding" refers to i) the genetic information contained in the DNA sequence that can be transcribed into an RNA molecule, and / or ii) the genetic information contained in the RNA molecule that can be translated into an amino acid sequence. Therefore, as used herein, "coding sequence" can be used to refer to a ribonucleotide (RNA) sequence or a fragment thereof in an mRNA precursor or mature mRNA that can be translated into a protein, and can also refer to the complementary sequence of a deoxyribonucleotide (DNA) sequence or a fragment thereof that is used as a template for transcribing the mRNA precursor or mature mRNA. In addition, the "coding sequence" of the present application can further include polynucleotide sequences that encode proteins, functional nucleic acids, or fragments thereof, such as miRNA, shRNA, dsRNA, guide RNA, Poly (A) tail, 5'UTR, 3'UTR, etc. Among them, a DNA molecule containing genetic information that can be transcribed into an RNA molecule is called the "coding nucleic acid" of the RNA molecule; an RNA molecule containing genetic information that can be translated into an amino acid sequence is called the "coding nucleic acid" of the amino acid sequence.

[0092] Unless otherwise specified, "peptide", "polypeptide" and "protein" are used interchangeably in this application to refer to any natural active substance formed by two or more amino acids covalently linked by peptide bonds, which may or may not contain the secondary or tertiary structure of the protein molecule.

[0093] As used herein, the term "contacting" is used in accordance with its simple common meaning and refers to a process in which at least two different substances are allowed to become sufficiently close to react, interact or physically contact. It should be understood that the resulting reaction product can be produced directly by the reaction between the added reagents, or by an intermediate of one or more added reagents, which can be produced in a reaction mixture. The term "contacting" can include allowing two substances to react, interact or physically contact, wherein the two substances can be, for example, an engineered receptor (or engineered nucleic acid molecule) and a cell as provided herein. In an embodiment, contacting includes, for example, allowing an engineered nucleic acid molecule or an engineered peptide as described herein to enter a cell.

[0094] As used herein, "CAR-T cell persistence" refers to the duration of CAR-T cell persistence in a patient or subject, i.e., the period from in vivo administration of CAR-T cells to their eventual disappearance from the body. This can be calculated or measured by comparing CAR copy numbers. For example, an increase in CAR-T copy number after a specific period of in vivo use can be used to indicate an increase in CAR-T cell persistence.

[0095] In addition, in the present application, 8h represents the CD8 hinge region, 8TM represents the CD8 transmembrane domain, BB represents the 4-1BB costimulatory domain (or called the 4-1BB signal transduction domain), and Z or z represents the CD3ζ signal transduction domain.

[0096] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0097] Fusion protein

[0098] The present application also provides a fusion protein comprising the extracellular region of CD226, wherein the fusion protein is a transmembrane protein comprising an extracellular region, a transmembrane domain, and an intracellular region, and wherein the fusion protein itself does not comprise a primary signal transduction domain. In some embodiments, the fusion protein comprises or is, from the N-terminus to the C-terminus:

[0099] 1) CD226 extracellular region, transmembrane domain, and costimulatory domain; or

[0100] 2) Signal peptide, CD226 extracellular region, transmembrane domain and co-stimulatory domain.

[0101] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus:

[0102] 1) CD226 extracellular region, transmembrane domain, and costimulatory domain; or

[0103] 2) Secretory protein signal peptide, CD226 extracellular region, transmembrane domain, and co-stimulatory domain;

[0104] Wherein the CD226 extracellular region and the transmembrane domain, the transmembrane domain and the costimulatory domain are connected by direct and / or indirect means. The term "direct" connection or "direct connection" refers to a connection achieved only by a chemical bond, that is, the two proteins or polypeptides are not connected by other molecules, and the chemical bond can be a non-covalent bond (such as an ionic bond, a hydrogen bond, a hydrophobic bond or a van der Waals bond), or a covalent bond (such as a peptide bond). The term "indirect" connection or "indirect connection" refers to a connection through a linker, and the two proteins or polypeptides connected to each other using a linker are respectively connected to one end of the linker by a covalent or non-covalent bond. The "linker" can be a peptide linker (i.e., a peptide chain, such as a peptide chain consisting of 1 to 50 amino acids or derivatives thereof) or a non-peptide linker, and the linker can be cleavable (i.e., hydrolyzed by an enzyme in an organism, such as a mammalian organism) or non-cleavable. Exemplary non-peptide linkers include, but are not limited to, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylene polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethers, biodegradable polymers, polymeric lipids, chitin, and hyaluronic acid, or derivatives thereof, or combinations thereof.

[0105] In some embodiments, the CD226 extracellular region and the transmembrane domain comprise or are a hinge region, which can be the hinge region of any molecule, or the portion between the recognition domain and the transmembrane domain of any membrane receptor molecule. As used herein, a "hinge region" is generally a flexible portion in a protein that can participate in the propagation of conformational changes, such as from the active site to other parts of the protein structure (including other subunits), such as the segment between the CH1 and CH2 of the immunoglobulin heavy chain. In some embodiments, the hinge region is selected from the hinge region of one or more of the following molecules, or is composed of the hinge region of one or more of the following molecules: IgG, IgD, CD7, CD8α or CD28.

[0106] As used herein, "costimulatory domain" is generally derived from the co-stimulatory receptor of an immune cell, providing a second signal or secondary intracellular signal for activating immune cells (e.g., T cells). In some embodiments, the co-stimulatory domain includes one or more of the following, or is composed of one or more of the following: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITG AE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a signaling domain of a ligand that specifically binds to CD83.

[0107] As used herein, " transmembrane domain " is the part of transmembrane protein that connects the inside and outside of the cell, and is generally composed of an alpha helical fragment of amino acids, which are hydrophobic in nature and can therefore be embedded in the hydrophobic interior of the cell membrane. Typically, " transmembrane domain " can participate in regulating and / or conducting transmembrane signals. Exemplary transmembrane domains can be selected from the transmembrane domains of any one or more of the following molecules, or are composed of the transmembrane domains of any one or more of the following molecules: ICOS, CD226, TCR α, TCR β, TCR γ, TCR δ, CD3 ζ, CD3 ε, CD3 γ, CD3 δ, CD4, CD5, CD6, CD7, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD47, CD52, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, CISH, PD-1.

[0108] In some embodiments, the extracellular region of the fusion protein further comprises another one or more proteins, polypeptides or protein functional domains in addition to the CD226 extracellular region, and the CD226 extracellular region can be combined with another one or more proteins, polypeptides or protein functional domains to form a protein complex by any means. In some embodiments, the combination is that the CD226 extracellular region is connected to the another one or more proteins, polypeptides or protein functional domains by direct and / or indirect means. In some embodiments, the carbon (C) end of the CD226 extracellular region in the fusion protein is connected to the nitrogen (N) end of the another protein, polypeptide or protein functional domain. In some embodiments, the nitrogen (N) end of the CD226 extracellular region in the fusion protein is connected to the carbon (C) end of the another protein, polypeptide or protein functional domain. In some embodiments, the nitrogen (N) end of the CD226 extracellular region in the fusion protein is connected to the nitrogen (N) end of the another protein, polypeptide or protein functional domain. In some embodiments, the carbon (C) end of the CD226 extracellular region in the fusion protein is connected to the carbon (C) end of the other protein, polypeptide or protein functional domain. In some embodiments, the CD226 extracellular region in the fusion protein and the multiple proteins, polypeptides or protein functional domains are connected in series with each other. In some embodiments, the C-terminus and / or N-terminus of the CD226 extracellular region in the fusion protein are connected to at least two or more other proteins, polypeptides or protein functional domains, and the two or more other proteins, polypeptides or protein functional domains are not connected in series. In some embodiments, the one or more proteins are homologous proteins of the CD226 extracellular region or parts of the homologous proteins. In some embodiments, the one or more proteins are heterologous proteins of the CD226 extracellular region, or parts of the heterologous proteins.

[0109] In some embodiments, the one or more proteins, polypeptides or protein domains comprise a recognition polypeptide that specifically binds to the one or more proteins, and the one or more proteins can be tumor antigens, for example, one or more selected from the following: prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA), CD123, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit α (IL-13Rα); interleukin-11 receptor α (IL-11Rα); prostate-specific membrane antigen (PSMA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and their mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrins Type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain; 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); the hexose moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma 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 testis antigen-1 ( MAD-CT-1; melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutant; 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 myelocytic leukemia viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 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, X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA receptor The Fc fragment of the human leukocyte antigen (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1) and CD155.

[0110] In some embodiments, the one or more other proteins, polypeptides, or protein domains comprise a recognition polypeptide that specifically binds to one or more other proteins, and the one or more other proteins may be immune checkpoint proteins, such as one or more selected from the following:

[0111] 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, TIGIT, and VSIG8.

[0112] In some embodiments, the one or more other proteins, polypeptides or protein functional domains comprise a recognition polypeptide that specifically binds to one or more other proteins, and the one or more other proteins include any one or more immune checkpoint proteins selected from the above-mentioned ones, and any one or more tumor antigens mentioned above.

[0113] In some embodiments, the recognition polypeptide that specifically binds to another one or more proteins is an antibody or an antigen-binding fragment of the antibody, or a ligand or receptor or a fragment thereof that binds to the tumor antigen and / or immune checkpoint protein.

[0114] Engineered receptors

[0115] The present application also provides an engineered receptor comprising the aforementioned fusion protein. In some embodiments, the engineered receptor is further directly or indirectly connected to a primary signal transduction domain at the C-terminus of the aforementioned fusion protein. In some embodiments, the engineered receptor further comprises a TCR binding domain. In some embodiments, the TCR binding domain is located between the extracellular region and the costimulatory domain of the aforementioned fusion protein.

[0116] In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a T cell antigen coupling (TAC).

[0117] As used herein, the term "CAR", i.e., chimeric antigen receptor, comprises: i) an 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 signal transduction domain. Wherein, the "intracellular signal transduction domain" includes a primary signal transduction domain and / or a costimulatory domain. In some embodiments, the intracellular signal transduction domain includes a primary signal transduction 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 signal transduction domain is from CD3ζ. As used herein, the "primary signaling domain" typically comprises an immune-receptor tyrosine-based activation motif (ITAM), which has the basic structure: YXXL / V, where Y represents tyrosine, L / V represents leucine or valine, and X represents any amino acid. Upon ligand binding, the tyrosine residue in the ITAM becomes phosphorylated by membrane-bound protein tyrosine kinases (PTKs), thereby recruiting other intracellular protein kinases or adaptor proteins to transmit activation signals into the cell.

[0118] As used herein, an engineered "TCR" is an engineered T cell receptor comprising: (a) an antigen binding domain (as used herein, an antigen binding domain comprises a domain that binds to an antigen, a ligand domain that binds to a receptor, or a receptor domain that binds to a ligand), which comprises 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 The extracellular antigen binding domain or part thereof of the first TCR subunit (e.g., Cα, Cβ, Cδ, Cγ, CD3ε); (d) the transmembrane domain of the second TCR subunit (e.g., TCRα, TCRβ); and (e) the intracellular signal transduction domain comprising the 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 antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is from CD8α. In some embodiments, the antigen binding domain comprises the aforementioned engineered CD226 extracellular region. In some embodiments, the antigen binding domain is the aforementioned engineered CD226 extracellular region.

[0119] As used herein, "TAC" refers to a T cell antigen conjugate comprising (i) an antigen binding domain, (ii) a TCR binding domain (e.g., scFv), and (iii) a co-receptor domain (e.g., hinge, transmembrane and / or cytosolic region). See, for example, Helsen et al. Nat Commun. 2018; 9(1): 3049. In some embodiments, the antigen binding domain comprises the aforementioned engineered CD226 extracellular region. In some embodiments, the antigen binding domain is the aforementioned engineered CD226 extracellular region. In some embodiments, a TAC comprises: (a) an antigen-binding domain (as used herein, an antigen-binding domain comprises a domain that binds to an antigen, a ligand domain that binds to a receptor, or a receptor domain that binds to a ligand), which comprises 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), Its specific recognition TCR subunit (e.g., CD3ε) extracellular antigen binding domain;(d) optionally a second linker;(e) optionally a first TCR co-receptor (e.g., CD4, CD8) extracellular antigen binding domain or a portion thereof;(f) a transmembrane domain comprising a second TCR co-receptor (e.g., CD4, CD8) transmembrane domain;and (g) optionally an intracellular signal transduction domain comprising a third TCR co-receptor (e.g., CD4, CD8) intracellular signal transduction domain;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 antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the antigen binding domain comprises the aforementioned engineered CD226 extracellular region. In some embodiments, the antigen binding domain is the aforementioned engineered CD226 extracellular region.

[0120] As used herein, the term "antigen binding domain" encompasses the concepts of "ligand binding domain" and "receptor binding domain", which are generally located in the extracellular segment of a cell (especially an immune cell) receptor and can specifically bind to a certain protein, and the scope of the certain protein is not limited in any way. Therefore, in some embodiments, the certain protein is a certain receptor, and the "antigen binding domain" is the part of the ligand of the certain receptor that specifically recognizes the certain receptor; in some embodiments, the protein is a certain ligand, and the "antigen binding domain" is the part of the receptor of the certain ligand that specifically recognizes the ligand; in some embodiments, the protein is an antibody or an antigen binding domain of the antibody, such as a single-chain antibody (scFv), Fab, F(ab')2, Fab', Fv, Fd, dAb or diabody.

[0121] Engineered nucleic acid molecules

[0122] This application also provides engineered nucleic acid molecules encoding the aforementioned fusion proteins or engineered receptors, wherein the nucleic acid molecules comprise a target protein coding sequence. In some embodiments, after the engineered nucleic acid molecules are introduced into a suitable host cell, the engineered nucleic acid molecules can be transcribed and translated into the engineered receptor or fusion protein. The term "engineered nucleic acid molecule" is used to distinguish it from "natural nucleic acid molecules." "Natural nucleic acid molecules" refer to nucleic acid molecules that exist in their natural form in nature. "Engineered nucleic acid molecule" is a restriction on the source or preparation method of the nucleic acid molecule, and does not constitute any restriction on its function or structure. Therefore, engineered nucleic acid molecules can be used to refer to any nucleic acid molecule that can be obtained through any or multiple bioengineering methods. It can have a polynucleotide sequence identical to a natural nucleic acid molecule, have modifications identical to a natural nucleic acid molecule, or even form a structure identical to a natural nucleic acid molecule. However, the difference between an engineered nucleic acid molecule and its corresponding natural nucleic acid molecule, or a natural nucleic acid molecule with the same polynucleotide sequence, is at least that the engineered nucleic acid molecule is not directly purified or extracted in its natural form from a naturally occurring animal or plant in nature.

[0123] In some embodiments, the engineered nucleic acid molecule is an engineered DNA molecule. In some embodiments, the DNA molecule can be replicated and / or expressed in a cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a eukaryotic cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a prokaryotic cell. In some embodiments, the DNA molecule can be expressed in a eukaryotic cell and can be replicated in a prokaryotic cell. Therefore, in addition to comprising the target protein coding sequence, the DNA molecule also comprises genetic manipulations or regulatory elements for replication and / or expression in prokaryotic and / or eukaryotic cells.

[0124] The engineered DNA molecule is made to replicate or efficiently replicate the necessary structural elements in the cell, which are known in the art and include, for example, an origin of replication (ORI). In some embodiments, the engineered DNA molecule further includes a marker gene or its fragment and / or a reporter gene or its fragment and a unique restriction endonuclease site that allows insertion of the DNA element, preferably a restriction endonuclease site in the form of a multiple cloning site (MCS). The marker gene is conducive to identifying cells containing the plasmid that comprises the marker gene and can be selected from, for example, antibiotic resistance genes. Each restriction endonuclease site in the MCS can be specifically recognized by different restriction endonucleases.

[0125] In some embodiments, the DNA molecule is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid consisting of a double-stranded DNA molecule. In some embodiments, the "plasmid" is a circular DNA molecule. In some embodiments, the "plasmid" can also encompass linear DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, cutting a circular plasmid with a restriction endonuclease, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid, as well as linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify the genomic genetic information stored in the nucleoid or nucleoid of a prokaryotic cell in a cell, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. Preferably, the DNA plasmid according to the application is a medium copy or high copy plasmid, more preferably a high copy plasmid. Examples of such high copy plasmids are vectors based on pUC, pTZ plasmids, or any other plasmids (e.g., pMB1, pCoIE1) containing an ORI that supports high copy plasmids.

[0126] In some embodiments, the engineered DNA molecule is a DNA molecule or a fragment thereof that constitutes a nucleoid or nucleoid of a prokaryotic organism, or a DNA molecule or a fragment thereof that constitutes a eukaryotic genome, that is, the target protein coding sequence or its complementary sequence can be replicated along with the prokaryotic genome.

[0127] In some embodiments, the engineered DNA molecule can be transcribed into mRNA. In some embodiments, the engineered DNA molecule further comprises a coding sequence of an element that can be used to initiate or regulate the expression of the protein, polypeptide or fragment thereof after transcription, the element including but not limited to 5'UTR, 3'UTR, poly (A) tail (or tailing signal) and the like. In some embodiments, the engineered DNA molecule comprises a coding sequence of at least one untranslated region (UTR). In some embodiments, the engineered DNA molecule comprises at least the coding sequence of the 5'UTR and the target protein coding sequence. In some embodiments, the engineered DNA molecule comprises at least the coding sequence of the 5'UTR, the target protein coding sequence, the coding sequence of the 3'UTR, the tailing signal (or the DNA sequence corresponding to the Ploy (A) tail sequence) from 5' to 3', and the start codon (5' end) and the stop codon (3' end) may also be respectively included at both ends of the target protein coding sequence, which are respectively the first three nucleotides and the last three nucleotides that can be translated of the mRNA molecule. 5'UTR generally comprises at least one ribosome binding site (RBS), such as the Shine-Dalgarno sequence in prokaryotes, or at least one translation initiation site, such as the Kozak sequence in eukaryotes. RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes at the time of translation initiation. Its activity can be optimized by changing the length and sequence of a given RBS or translation revelation site and the distance from the start codon. Alternatively or optionally, 5'UTR includes an internal ribosome entry site or IRES. 3'UTR may comprise one or more regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of the mRNA molecule, binding sites for regulatory RNA molecules (such as miRNA molecules), and / or signal sequences that participate in the intracellular transport of mRNA molecules.

[0128] On the basis of the aforementioned embodiment, in some embodiments, the target gene fragment further comprises one or more additional regulatory sequences, such as binding sites for amino acid sequences that enhance mRNA molecule stability, binding sites for amino acid sequences that enhance mRNA molecule translation, regulatory elements (such as riboswitches), and / or nucleotide sequences that have a positive impact on translation initiation. In addition, within the 5'UTR, preferably there is no functional upstream open reading frame, out-of-frame upstream translation initiation site, out-of-frame upstream start codon, and / or nucleotide sequences that produce secondary structures that reduce or prevent translation. The presence of such nucleotide sequences in the 5'UTR can have a negative impact on translation.

[0129] The target protein coding sequence comprises codons that can be translated into an amino acid sequence. The target protein coding sequence may comprise all codons that encode naturally occurring amino acids, or may comprise some or all of artificially synthesized codons. In some embodiments, some or all of the codons are codon-optimized. In some embodiments, some or all of the codons encode unnatural amino acids.

[0130] In some embodiments, the engineered DNA molecule further comprises structural elements on the 5' end of the target gene segment that are necessary to initiate or regulate transcription of the RNA, and these structural elements are known in the art. In some embodiments, the structural elements comprise at least a promoter. Promoters and their sequences are known in the art and include weak promoters, medium-strength promoters, strong promoters, mini promoters, or core promoters. In some specific embodiments, the promoter is a strong promoter. In some embodiments, the promoter can initiate transcription of the target protein coding sequence in prokaryotes. In some embodiments, the promoter can initiate transcription of the target protein coding sequence in eukaryotic cells. The "promoter" comprises at least one transcription recognition site followed by a transcription factor binding site. The recognition and binding sites can interact with amino acid sequences that mediate or regulate transcription. Compared to the recognition site, the binding site is closer to the target gene segment. The binding site can be, for example, a Pribnow box in prokaryotes or a TATA box in eukaryotes. For example, in some embodiments, when using the Pribnow box, the transcription recognition site can be located at about 35bp upstream of the transcription start site, and the transcription factor binding site can be located at about 10bp upstream of the transcription start site. In some embodiments, the promoter comprises at least one other regulatory element, such as an upstream element rich in AT at about 40 and / or 60 nucleotides before the transcription start site, and / or an additional regulatory element that enhances promoter activity between the recognition site and the binding site. In some embodiments, the promoter is a strong promoter, that is, the promoter comprises a sequence that promotes the transcription of the aforementioned target protein coding sequence. Strong promoters are well known to those skilled in the art, such as OXB18, OXB19, and OXB20 promoters derived from the RecA promoter of Escherichia coli, or can be identified or synthesized by conventional laboratory procedures. In some embodiments, the promoter is a T7 promoter. In some embodiments, the promoter also comprises additional regulatory elements before it, such as an enhancer that can promote the transcription of the aforementioned target protein coding sequence in a DNA plasmid.

[0131] In some embodiments, the eukaryotic cell is a yeast cell.In some embodiments, the DNA molecule is a yeast display vector.

[0132] In addition, the present application also provides engineered RNA molecules encoding the aforementioned engineered receptors or fusion proteins. In some embodiments, the engineered RNA molecules are transcribed from the aforementioned engineered DNA molecules. In some embodiments, the engineered RNA molecules have the same sequence as the RNA molecules transcribed from the aforementioned engineered DNA molecules. In some embodiments, the engineered RNA is mRNA. As used herein, "mRNA" (messenger RNA) is any RNA, naturally occurring, non-naturally occurring, or modified, that encodes at least one protein, polypeptide, or fragment thereof, and that has the ability to be translated to produce the encoded protein, polypeptide, or fragment thereof in vitro, in vivo, in situ, or ex vivo. Therefore, the mRNA can be mature mRNA or pre-mature mRNA, and the elements or structures that it must contain or optionally contain are known in the art. In some embodiments, the mRNA contains coding sequences for multiple necessary functional components to express, regulate, or enhance the expression level of the protein, polypeptide, or fragment thereof. Such functional components include, but are not limited to, a 5' cap, a 5' untranslatable region (UTR), a 3' untranslatable region (UTR), and the like. Both the 5'UTR and the 3'UTR are usually transcribed from genomic DNA and are elements present in the pre-mature mRNA.

[0133] The term "5' cap" is located at the 5' end of the mRNA and contains a methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate, forming a 5',5'-triphosphate linkage with its adjacent nucleotide. There are generally three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), referred to as type O, type I, and type II, respectively. Type O refers to unmethylated ribose of the terminal nucleotide, type I refers to methylation of the ribose of one terminal nucleotide, and type II refers to methylation of the ribose of both terminal nucleotides. In some embodiments, the 5' cap can be accomplished simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to produce a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA), or m7G(5')ppp(5')(2'-OMeA)pG (CleanCapAG) according to the manufacturer's protocol. For example, in some embodiments, 5' capping of the modified RNA can be accomplished post-transcriptionally using a vaccinia virus capping enzyme to produce an O-type cap structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Type I cap structures can be generated using both vaccinia virus capping enzyme and 2'-O methyl-transferase to produce m7G(5')ppp(5')(2'-OMeA)pG. Type II cap structures can be generated from type I cap structures by 2'-O-methylation of the 5'-third to last nucleotide using 2'-O methyl-transferase. Type III cap structures can be generated from type II cap structures by 2'-O-methylation of the 5'-fourth to last nucleotide using 2'-O methyl-transferase.

[0134] In some embodiments, the mRNA also includes a stabilizing element. Stabilizing elements may include, for example, a histone stem-loop. In some embodiments, the mRNA includes a coding region, at least one histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the mRNA includes a combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop. Although the two have alternative mechanisms in nature, their synergistic effect can increase protein expression to levels exceeding those observed with either element alone. The synergistic effect of the combination of poly(A) and at least one histone stem-loop is independent of the order of the elements or the length of the poly(A) sequence. In some embodiments, the histone stem-loop is typically derived from a histone gene and includes two adjacent partially or completely reverse-complementary sequences separated by a spacer (composed of a short sequence) that form a loop through intramolecular base pairing. Unpaired loop regions are generally unable to base pair with either of the stem-loop elements. The stability of the stem-loop structure generally depends on the length, the number of mismatches or bulges, and the base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) can occur. In some embodiments, the at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length.

[0135] In some embodiments, one or more AU-rich sequences of the mRNA can be removed. These sequences are sometimes referred to as AURES, which are destabilizing sequences found in the 3' UTR. AURES can be removed from the mRNA. Alternatively, AURES can be retained in the mRNA.

[0136] In some embodiments, the mRNA is configured in a lipid nanoparticle (LNP). In some embodiments, lipids are mixed with the mRNA to form lipid nanoparticles. In some embodiments, RNA is formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles are first formed into empty lipid nanoparticles and are combined or wrapped with the mRNA of the vaccine just before administration (e.g., within a few minutes to an hour).

[0137] The lipid nanoparticles generally comprise ionizable lipids, non-cationic lipids, sterols and PEG lipid components and target nucleic acids, such as the above-mentioned mRNA. Lipid nanoparticles of the present disclosure can be produced using components, compositions and methods as generally known in the art, see, for example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / US2017 / 037551, PCT / US2017 / 037552, PCT / US2017 / 037551, PCT / US2017 / 0 S2016 / 014280, PCT / US2017 / 038426, PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 52117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575, and PCT / US2016 / 069491, all of which are incorporated herein by reference in their entirety.

[0138] In some embodiments, the engineered nucleic acid molecule may also be a hybrid molecule of DNA and RNA, wherein the hybrid molecule of DNA and RNA carries the same genetic information as the engineered DNA molecule or the engineered RNA molecule.

[0139] In some embodiments, the engineered nucleic acid molecule encodes an amino acid sequence as set forth in any one or more of SEQ ID NOs: 1 to 12, or an amino acid sequence having greater than 85% sequence identity to an amino acid sequence as set forth in any one or more of SEQ ID NOs: 1 to 12. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence as set forth in any one or more of SEQ ID NOs: 25 to 34, or a polynucleotide sequence having greater than 85% sequence identity to a polynucleotide sequence as set forth in any one or more of SEQ ID NOs: 25 to 34.

[0140] Engineered cells

[0141] The present application also provides an engineered cell, wherein the engineered cell expresses or comprises the aforementioned fusion protein or the aforementioned engineered receptor on its cell membrane, or the engineered cell comprises the aforementioned engineered nucleic acid molecule.

[0142] In some embodiments, the engineered cells are engineered immune cells. In some embodiments, the engineered cells are T cells, NK cells, macrophages, DC cells, B cells, or their precursor cells. In some embodiments, the engineered cells are CAR-T or CAR-NK cells targeting one or more epitopes of CD155. In some embodiments, the engineered cells are TCR-T cells targeting one or more epitopes of CD155. In some embodiments, the engineered cells are TAC-T cells targeting one or more epitopes of CD155.

[0143] In some embodiments, the engineered cells are CAR-T or CAR-NK cells targeting one or more different antigens, which contain a fusion protein or engineered receptor as described above, and one or more CARs targeting other tumor antigens. In some embodiments, the engineered cells are CAR-T or CAR-NK cells targeting one or more different antigens, which contain a fusion protein or engineered receptor as described above, and one or more CARs targeting immune checkpoint proteins. In some embodiments, the engineered cells are CAR-T or CAR-NK cells targeting one or more different antigens, which contain a fusion protein or engineered receptor as described above, one or more CARs targeting other tumor antigens, and one or more CARs targeting immune checkpoint proteins.

[0144] In some embodiments, the engineered cell is a TCR-T cell targeting one or more different antigens, which comprises a fusion protein or engineered receptor as described above, and the TCR-T further comprises one or more TCRs targeting other tumor antigens. In some embodiments, the engineered cell is a TCR-T cell targeting one or more different antigens, which comprises a fusion protein or engineered receptor as described above, and the TCR-T further comprises a TCR targeting one or more immune checkpoint proteins. In some embodiments, the engineered cell is a TCR-T cell targeting one or more different antigens, which comprises a fusion protein or engineered receptor as described above, and the TCR-T further comprises one or more TCRs targeting other tumor antigens, and one or more TCRs targeting immune checkpoint proteins.

[0145] In some embodiments, the engineered cells are TAC-T cells targeting one or more different antigens, which contain a fusion protein or engineered receptor as described above, and the TAC-T further contains one or more TACs targeting other tumor antigens. In some embodiments, the engineered cells are TAC-T cells targeting one or more different antigens, which contain a fusion protein or engineered receptor as described above, and the TAC-T further contains a TAC targeting one or more immune checkpoint proteins. In some embodiments, the engineered cells are TAC-T cells targeting one or more different antigens, which contain a fusion protein or engineered receptor as described above, and the TAC-T further contains one or more TACs targeting other tumor antigens, and one or more TACs targeting immune checkpoint proteins.

[0146] In some embodiments, the engineered cell expresses or contains the aforementioned fusion protein or engineered receptor on its cell membrane, and the fusion protein or engineered receptor activates or inhibits the downstream signaling pathway of the fusion protein or engineered receptor after binding to CD155.

[0147] In some embodiments, the engineered cells are selected from: T cells, NK cells, macrophages, DC cells, B cells, or precursor cells thereof.

[0148] In some embodiments, the tumor antigens described herein are selected from one or more of the following:

[0149] PSCA, CEA, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD 22, CD 30, CD 70, CD 123, CD 138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276), B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, HIV-1 Gag, MART-1, gp100, tyrosinase, mesothelin, EpCAM, PRSS21, vascular endothelial growth factor receptor, Lewis (Y) antigen, CD24, PDGFR-β, SSE A-4, MUC1, MUC6, EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII, NCAM, CAIX, LMP2, EphA2, fucosyl GM1, sLe, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TGS5, HMWMAA, OAcGD2, folate receptor, CD248, TEM7R, Claudin 6, Claudin18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA), CA9, kappa light chain, CSPG4, EGP2, EGP40, FAP, FAR, FBP, embryonic AchR, HLA-A1, HLA-A2, MAGEA1, MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, fibronectin, tenascin, oncofetal variant of tumor necrosis, 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.

[0150] In some embodiments, the immune checkpoint proteins described herein are selected from one or more of the following: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD 48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, CD226, and VSIG8.

[0151] In some embodiments, the engineered cells comprise the aforementioned fusion proteins or engineered receptors and a CAR, TCR, or TAC that binds to CEA, PSCA, or CD70. In some embodiments, the engineered cells comprise the aforementioned fusion proteins or engineered receptors and a CAR that binds to CEA. In some embodiments, the structure of the CAR that binds to CEA is as follows: CEA ScFv-8h-8TM-BBZ. In some embodiments, the engineered cells comprise the aforementioned fusion proteins or engineered receptors and a CAR that binds to PSCA. In some embodiments, the structure of the CAR that binds to CEA is as follows: PSCA ScFv-8h-8TM-BBZ. In some embodiments, the engineered cells comprise the aforementioned fusion proteins or engineered receptors and a CAR that binds to CD70. In some embodiments, the structure of the CAR that binds to CD70 is as follows: CD70 ScFv-8h-8TM-BBZ. In the present application, CEA scFv, PSCA scFv and CD70 scFv refer to scFvs formed by connecting the heavy and light chains of antibodies targeting CEA, PSCA and CD70, respectively.

[0152] use

[0153] This application also provides the use of the aforementioned engineered fusion proteins, engineered receptors, engineered nucleic acid molecules, and engineered cells in the preparation of drugs for treating cancer. In some embodiments, the cancer is selected from one or more of the following:

[0154] Bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, and uterine cancer.

[0155] In some embodiments, the cancer involves tumor cells that highly express CD155.

[0156] In addition, the present application also provides the use of the aforementioned fusion protein, engineered receptor, or engineered cell as a TIGIT antagonist, which prevents TIGIT on the surface of immune cells from binding to CD155, thereby reversing tumor immunosuppression caused by the binding of TIGIT to CD155. Therefore, in some embodiments, the fusion protein, engineered receptor, or engineered cell can be administered in combination with other anticancer agents.

[0157] Example

[0158] Example 1: Functional verification of CAR-T cells targeting CEA using CD226 fusion protein

[0159] Cell expansion and viability assays

[0160] Three vectors containing the gene sequences of CEA ScFv-8h-8TM-BBZ-P2A-CD226-28TM-28 (CEA CD226-CAR-T), CEA ScFv-8h-8TM-BBZ (CEA CAR-T), and CD226-28TM-28z (CD226-z) were constructed, and viruses were prepared. Among them, CEAscFv is the scFv of the anti-CEA antibody, 8h represents the CD8 hinge region, 8TM represents the CD8 transmembrane domain, BB represents the 4-1BB costimulatory domain (or called the 4-1BB signal transduction domain), Z or z represents the CD3ζ signal transduction domain, CD226 represents the extracellular region of CD226, 28TM represents the CD28 transmembrane domain, 28 represents the CD28 costimulatory domain (or called the CD28 signal transduction domain), P2A represents the 2A peptide, and the specific sequences of the aforementioned structures used in the examples of the present application are shown in the sequence table at the end of the article. Specifically, the amino acid sequence of CEA ScFv-8h-8TM-BBZ-P2A-CD226-28TM-28 is shown in SEQ ID NO: 7, the amino acid sequence of CEA ScFv-8h-8TM-BBZ is shown in SEQ ID NO: 6, and the amino acid sequence of the CD226 fusion protein (CD226-28TM-28) is shown in SEQ ID NO: 2. Furthermore, the amino acid sequence of CEA scFv is shown in SEQ ID NO: 5, the amino acid sequence of 8h is shown in SEQ ID NO: 13, the amino acid sequence of 8TM is shown in SEQ ID NO: 14, the amino acid sequence of BB is shown in SEQ ID NO: 15, the amino acid sequence of Z is shown in SEQ ID NO: 16, the amino acid sequence of P2A is shown in SEQ ID NO: 24, the amino acid sequence of CD226 is shown in SEQ ID NO: 1, the amino acid sequence of 28TM is shown in SEQ ID NO: 17, and the amino acid sequence of 28 is shown in SEQ ID NO: 18.

[0161] Among them, the CEA CAR nucleotide sequence is shown in SEQ ID NO: 29, the CEA CD226 CAR nucleotide sequence is shown in SEQ ID NO: 30, the CD226 nucleotide sequence is shown in SEQ ID NO: 25, and the CD226 fusion protein nucleotide sequence is shown in SEQ ID NO: 26.

[0162] The specific virus preparation method is as follows:

[0163] In this example, lentivirus packaging was performed using the calcium phosphate method, as described in the Molecular Cloning Manual (3rd edition, by J. Sambrook et al.). Specifically, 293T cells were cultured in DMEM medium supplemented with 10% FBS (w / v) until healthy. The target plasmids were then mixed with the three lentiviral packaging plasmids, pMDLg / pRRE, pRSV-Rev, and pMD2.G, in appropriate proportions. CaCl2 and 2×HBS were then added. After mixing thoroughly, the mixture was allowed to stand at room temperature and then added to the culture medium of the treated 293T cells. After 4-6 hours, the medium was replaced with 10 mL of DMEM medium supplemented with 10% FBS. After 48 or 72 hours, the cell supernatant was collected. The viral supernatant was collected and purified. The purified virus was aliquoted into 1.5 mL EP tubes and stored at -80°C until ready for use.

[0164] After the lentivirus is concentrated, the titer is tested using 293T and / or CHO cells. 5 293T and / or CHO cells were inoculated into 24-well plates. The concentrated virus was infected with 293T and / or CHO cells at a volume of 1, 2.5, and 10 μl / well (DEAE promoter was required). Two days after infection, the infected 293T and / or CHO cells were collected and analyzed by flow cytometry. Total CAR expression was detected using Protein-L, and the viral titer was calculated. The titer was calculated as follows: Titer (TU / ml) = 1×10 5 × positive rate × dilution factor ÷ virus volume × 1000. The virus titers of the above CAR structures are shown in Table 1.

[0165] Lymphocytes were separated by gradient centrifugation. After centrifugation, the second layer of white lymphocytes was taken and washed with physiological saline to obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, and then infected with activated PBMCs at a certain multiplicity of infection (3-5 MOI). The total number of cells and viability were counted by cell counting on the 4th, 6th, and 8th days after viral infection / transduction. The results are shown in Figure 2: Figure 2A is the cell expansion multiple, and Figure 2B is the cell viability. The cells express CD226 fusion protein, wherein the schematic diagram of the CD226 fusion protein is shown in the structure on the right side of Figure 1, comprising an extracellular structural region, a transmembrane region, and an intracellular structural region. The extracellular region is the CD226 polypeptide described in the present invention; the structure on the left side of Figure 1 is a CAR structure targeting tumor targets such as CEA, which is mainly composed of an extracellular ScFv structural region, a hinge structural region, a transmembrane structural region, and an intracellular structural region. The intracellular structural region can contain two domains.

[0166] The results in Figure 2 show that there was no significant difference between the CEA CD226-CAR-T group and the control CAR-T group (CEA CAR-T and CD226-z group) and the CT group, and all had excellent expansion and viability.

[0167] In vitro efficacy verification

[0168] DLD1-Luc-GFP is a human colorectal adenocarcinoma epithelial cell line that expresses CD155 on its cell surface but is negative for CEA on its membrane. Based on DLD1 cells, we overexpressed CEA in them, making them double-positive cells expressing both CD155 and CEA. Both cells were modified with Luc-GFP and are referred to as DLD1 and DLD1-CEA. DLD1-CEA (human colorectal adenocarcinoma epithelial cells, positive for CEA and CD155) and DLD1 (human colorectal adenocarcinoma epithelial cells, positive for CD155 and negative for CEA) were used as target cells, respectively. Effector cells were plated at a 2:1 effector-target ratio, and the cytotoxicity of the different groups against target cells was measured 24 hours later. The results are shown in Figure 3. There was no significant difference in the killing of positive cells among the groups, but CD226-z with the primary stimulation signal CD3ζ also killed CEA-negative DLD1, while CEA CD226-CAR-T in the form of CD226 fusion protein and CEA CAR-T that did not express CD226 fusion protein only killed CEA-positive DLD1-CEA cells, and did not kill CEA-negative DLD1 compared with CT. This shows that the CD226 fusion protein described in this application does not affect the specificity of CAR when expressed in immune cells in combination with the CAR structure, and the CD226 fusion protein does not directly lead to the killing of tumor cells due to the recognition of CD155. After the addition of the primary stimulation signal CD3ζ, the CD226-z structure will recognize the CD155 molecules expressed by the target cells, thereby killing the target cells expressing CD155.

[0169] After 24 hours, the supernatant was collected and the secretion capacity of IFN-γ by immune cells after being stimulated by target cells was tested. The results are shown in Figure 4. The IFN-γ secretion of the CEA CD226-CAR-T group was much higher than that of the CEA CAR-T group, the CT group, and the CD226-z group. This shows that the combination of CD226 fusion protein and CAR can enhance the stimulation of T cells by the CAR structure and enhance the cytokine secretion capacity of CAR-T cells. This enhanced secretion capacity is very advantageous in solving the current defect of insufficient effectiveness of CAR-T in the treatment of solid tumors. However, CD226-z, which has added the primary stimulation signal CD3ζ, did not have this effect.

[0170] The above results show that compared with the control CEA CAR-T cells that do not express CD226 fusion protein, CEA CAR-T cells expressing CD226 fusion protein (CEA CD226-CAR-T cells) maintain the specificity of CEA CAR-T for CEA targets, improve the activation ability of CAR-T, and have higher factor secretion.

[0171] In vivo anti-tumor effect and persistence verification

[0172] For in vivo efficacy evaluation, 8-10 week-old NCG immunodeficient mice were injected intraperitoneally with 1e+6 DLD1-CEA-Luc-GFP cells per mouse to establish tumors. In vivo imaging was performed 7 days later, and mice were randomly divided into groups based on fluorescence levels. Each group received an intraperitoneal injection of 2.0e+6 copies of the CAR-T cell. In vivo imaging and orbital blood collection were performed every 7 days for copy number determination. The results are shown in Figures 5 and 6. Figure 5 demonstrates the effect of different CAR-T cells on tumors by fluorescence intensity in different tumors. The CEA CD226-CAR-T cell exhibited the best in vivo anti-tumor effect. Figure 6 examines CAR expression in tumor-bearing mice at different time points after infusion of different CAR-T cells. The persistence of CAR-T cells with different CAR structures in tumor-bearing tissues, as demonstrated by copy number, is further demonstrated. The CEA CD226-CAR-T cell persisted in the blood of tumor-bearing mice for at least 43 days, maintaining a high copy number. The results showed that CEA CAR-T cells expressing CD226 fusion protein (CEA CD226-CAR-T cells) prolonged mouse survival, inhibited tumor growth, slowed tumor recurrence cycle, and had better in vivo expansion than the control CEA CAR-T group, compared with the control CEA CAR-T group that did not express CD226 fusion.

[0173] Example 2: Functional verification of CAR-T cells targeting PSCA using CD226 fusion protein

[0174] Cell preparation

[0175] The CAR vector construction and CAR-T preparation methods are shown in Example 1. The constructed CAR structures include: three vectors containing the gene sequences of PSCA ScFv-8h-8TM-BBZ-P2A-CD226-28TM-28 (PSCA CD226-CAR), PSCA ScFv-8h-8TM-BBZ (PSCA CAR-T), and CD226-28TM-28z (CD226-z). The amino acid sequence of PSCA ScFv-8h-8TM-BBZ-P2A-CD226-28TM-28 (PSCA CD226-CAR) is shown in SEQ ID NO: 10, the amino acid sequence of PSCA ScFv-8h-8TM-BBZ (PSCA CAR-T) is shown in SEQ ID NO: 9, and the amino acid sequence of CD226 fusion protein (CD226-28TM-28) is shown in SEQ ID NO: 2. The amino acid sequence of PSCA scFv is shown in SEQ ID NO: 8, and the amino acid sequence of 8h is shown in SEQ ID NO: 10. NO:13, the amino acid sequence of 8TM is shown in SEQ ID NO:14, the amino acid sequence of BB is shown in SEQ ID NO:15, the amino acid sequence of Z is shown in SEQ ID NO:16, the amino acid sequence of P2A is shown in SEQ ID NO:24, the amino acid sequence of CD226 is shown in SEQ ID NO:1, the amino acid sequence of 28TM is shown in SEQ ID NO:17, and the amino acid sequence of 28 is shown in SEQ ID NO:18.

[0176] The PSCA CAR nucleotide sequence is shown in SEQ ID NO: 31, the PSCA CD226 CAR nucleotide sequence is shown in SEQ ID NO: 32, the CD226 nucleotide sequence is shown in SEQ ID NO: 25, and the CD226 fusion protein nucleotide sequence is shown in SEQ ID NO: 26.

[0177] PBMCs were activated with anti-CD3 and anti-CD28 monoclonal antibodies for 24 hours and then infected with activated PBMCs at a multiplicity of infection (MOI) of 3-5. Total cells were counted on days 1, 2, 5, 6, 8, 10, and 12 after viral infection / transduction. The results are shown in Figure 7 , which shows the cell expansion fold.

[0178] The results showed that the PSCA-targeting CAR-T group expressing CD226 fusion protein had no difference from the control CAR-T group and the CT group, and all had excellent expansion and viability.

[0179] In vitro efficacy verification

[0180] HT-1376 (a human bladder cancer cell line, positive for PSCA and CD155) was used as the target cell line. Effector cells were plated at a 1:1 effector to target ratio, and the ability of different groups to kill target cells was tested 24 hours later. As shown in Figure 8, the CAR-T group expressing the CD226 fusion protein targeting the PSCA target (PSCA CD226-CAR-T) showed no significant difference in killing positive cells compared to the control CAR-T group.

[0181] After 24 hours, the supernatant was collected and the secretion of IFN-γ by immune cells after stimulation with target cells was tested. As shown in Figure 9, IFN-γ secretion in the PSCA CD226-CAR-T group was much higher than that in the PSCA CAR-T group, approximately five times that of the PSCA CAR-T group.

[0182] The above results show that PSCA CAR-T cells expressing CD226 fusion protein (PSCA CD226-CAR-T cells) have improved CAR-T activation ability and higher factor secretion compared with control PSCA CAR-T cells that do not express CD226 fusion protein.

[0183] In vivo anti-tumor effect and persistence verification

[0184] For in vivo efficacy evaluation, 8-10 week-old NCG immunodeficient mice were injected intraperitoneally with 3e+5 HT-1376-Luc-GFP cells per mouse to establish tumors. Live imaging was performed 14 days later, and mice were randomly divided into groups based on fluorescence values. Each group received an intraperitoneal injection of 5.0e+5 CAR-T cells per mouse. The total cell number in the control T group was the same as that in the experimental group. Live imaging analysis and orbital blood collection for copy number analysis were performed every 7 days. The results are shown in Figures 10 and 11. Figure 10 demonstrates the effect of different CAR-T cells on tumors by fluorescence intensity in different tumors. Higher fluorescence intensity indicates larger tumor volume and higher tumor cell viability. Figure 11 examines CAR expression in tumor-bearing mice at different time points after infusion of different CAR-T cells. The copy number analysis further demonstrates the persistence of CAR-T cells with different CAR structures in tumor-bearing tissues. The results in Figure 10 demonstrate that PSCA CD226 CAR-T cells co-expressing the CD226 fusion protein exhibit superior in vivo efficacy compared to CAR-T cells not expressing the CD226 fusion protein. Tumor recurred in the conventional PSCA CAR-T group on day 42 (the penultimate measurement point) after CAR-T infusion, and tumor cell fluorescence intensity significantly increased on day 49 (the last measurement point). However, the anti-tumor effect of the PSCA CD226-CAR-T cell group infused with CD226 fusion protein targeting the PSCA target was maintained on day 49, demonstrating superior anti-tumor efficacy. The results in Figure 11 demonstrate that the CAR copy number in the conventional PSCA CAR-T group began to be significantly lower than that in the PSCA CD226-CAR-T cell group infused with CD226 fusion protein targeting the PSCA target on day 21, and this difference significantly increased on day 28. Compared with control PSCA CAR-T cells that do not express CD226 fusion protein, PSCA CAR-T cells expressing CD226 fusion protein (PSCA CD226-CAR-T cells) inhibited tumor growth and slowed tumor recurrence cycles, and their in vivo expansion was superior to that of the control PSCA CAR-T group.

[0185] Comparing the in vitro killing results of CEA CAR-T and CEA CD226 CAR-T on CEA-negative CD155-positive DLD1 cells in Figure 3, it can be seen that the CD226 fusion protein does not directly kill tumor cells; comparing the groups in Figure 3 and Figure 5, it can also be found that although the CAR structure CD226-z designed with CD226 as the extracellular recognition domain has a killing function in vitro, it has no tumor-killing effect on tumor-bearing mice, while the CEA CD226 CAR-T expressing CD226 fusion protein is not only effective in mice, but also has a significantly higher effectiveness than the single CEA CAR-T; it can be seen that the improvement of the effectiveness of CAR-T by CD226 fusion protein does not only depend on the extracellular region of CD226, and the CD226 fusion protein alone cannot produce effectiveness. Instead, it is an unexpected result caused by the combined use of the fusion protein and CAR structure described in the application, such as the increase in CAR-T activated cytokine secretion and the enhanced persistence of CAR-T in vivo.

[0186] The inventors are also validating the combination of CAR structures and fusion proteins targeting different targets, including CEA, PSCA, and CD70. These targets target tumors including, but not limited to, bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, testicular cancer, tongue cancer, and uterine cancer, among other malignancies. In tumor cells targeting these multiple targets, CAR-T cells combined with CD226 fusion protein and CAR demonstrated superior efficacy.

[0187] Example 3: Functional verification of CD226 fusion protein in CAR-T cells targeting CD70

[0188] The CAR vector construction and CAR-T preparation methods are shown in Example 1. Among them, the constructed CAR structure is: three vectors of CD70 ScFv-8h-8TM-BBZ-P2A-CD226-28TM-28 (CD70 CD226-CAR), CD70 ScFv-8h-8TM-BBZ (CD70 CAR-T), and CD226-28TM-28z (CD226-z) gene sequences, the amino acid sequence of CD70 ScFv-8h-8TM-BBZ-P2A-CD226-28TM-28 (CD70CD226-CAR) is shown in SEQ ID NO: 12, the amino acid sequence of CD70 ScFv-8h-8TM-BBZ (CD70 CAR-T) is shown in SEQ ID NO: 11, and the amino acid sequence of CD226 fusion protein (CD226-28TM-28) is shown in SEQ ID NO: 2; wherein, the amino acid sequence of CD70 scFv is shown in SEQ ID NO: NO:35, the amino acid sequence of 8h is shown in SEQ ID NO:13, the amino acid sequence of 8TM is shown in SEQ ID NO:14, the amino acid sequence of BB is shown in SEQ ID NO:15, the amino acid sequence of Z is shown in SEQ ID NO:16, the amino acid sequence of P2A is shown in SEQ ID NO:24, the amino acid sequence of CD226 is shown in SEQ ID NO:1, the amino acid sequence of 28TM is shown in SEQ ID NO:17, and the amino acid sequence of 28 is shown in SEQ ID NO:18.

[0189] Among them, the CD70 CAR nucleotide sequence is shown in SEQ ID NO: 33, the CD70 CD226 CAR nucleotide sequence is shown in SEQ ID NO: 34, the CD226 nucleotide sequence is shown in SEQ ID NO: 25, and the CD226 fusion protein nucleotide sequence is shown in SEQ ID NO: 26.

[0190] In vitro efficacy verification

[0191] 786-O (human renal clear cell adenocarcinoma cells, CD70 positive) were used as target cells. Effector cells were plated at a 1:2 effector-target ratio. After 24 hours, the supernatant was collected and tested for IFN-γ secretion after immune cell stimulation with target cells. The results, as shown in Figure 12, showed that IFN-γ secretion was higher in the CD70 CD226-CAR-T group than in the CD70 CAR-T group.

[0192] The above results show that CD70 CAR-T cells expressing CD226 fusion protein (CD70CD226-CAR-T cells) have improved CAR-T activation ability and higher factor secretion compared with control CD70 CAR-T cells that do not express CD226 fusion.

[0193] The amino acid sequences and corresponding nucleic acid sequences of each element in the proteins, polypeptides and CAR structures used in the examples of this application are shown in the following sequence table.

[0194] Sequence Listing

Claims

1. A fusion protein, which comprises or is from the N-terminus to the C-terminus: 1) CD226 extracellular region, transmembrane domain and co-stimulatory domain; or 2) Signal peptide, CD226 extracellular region, transmembrane domain and co-stimulatory domain.

2. The fusion protein according to claim 1, wherein the transmembrane domain comprises or consists of a transmembrane domain of one or more molecules selected from the following: ICOS, CD4, CD8α, CD28, CD3ζ and CD226.

3. The fusion protein according to claim 1 or 2, wherein the co-stimulatory domain comprises a signal transduction domain selected from any one or more of the following molecules or consists of a signal transduction domain of one or more signal molecules selected from the following: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80(KLRF1), CD 160. CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a ligand that specifically binds to CD83. The fusion protein according to any one of claims 1 to 3, wherein the signal peptide comprises or is a signal peptide of a CD226 or CD8α molecule.

5. The fusion protein according to any one of claims 1 to 4, which comprises or is from the N-terminus to the C-terminus: 1) CD226 extracellular region, CD28 transmembrane domain and CD28 signal transduction domain; or 2) CD226 signal peptide, CD226 extracellular region, CD28 transmembrane domain and CD28 signal transduction domain.

6. The fusion protein according to any one of claims 1 to 5, wherein the CD226 extracellular region comprises or is the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having a sequence identity of more than 85% with SEQ ID NO:

1. 7 . The fusion protein according to claim 6 , which comprises or is the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having a sequence identity of more than 85% with SEQ ID NO:

2.

8. An engineered receptor comprising the fusion protein of any one of claims 1 to 6, and a primary signal transduction domain located at the C-terminus of the fusion protein.

9. An engineered receptor according to claim 8, wherein the primary signaling domain comprises or consists of the signaling domains of one or more of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, CD79α, CD79β, CD66d, DAP10 and DAP12.

10. An engineered nucleic acid molecule comprising a nucleic acid encoding the fusion protein of any one of claims 1-7 or the engineered receptor of claim 8 or 9.

11. An engineered cell comprising the fusion protein of any one of claims 1 to 7 or the engineered receptor of claim 8 or 9 and / or the engineered nucleic acid molecule of claim 10.

12. The engineered cell according to claim 11, further comprising one or more engineered receptors that bind to one or more target molecules, wherein The one or more target molecules are selected from one or more of the following: CD70, PSCA and CEA; The engineered receptor is selected from one or more of the following: CAR, TCR and TAC.

13. An engineered cell according to claim 12, wherein the engineered receptor that binds other molecules is a CAR that binds CEA.

14. The engineered cell according to claim 13, wherein the CEA-binding CAR comprises or is an amino acid sequence as shown in SEQ ID NO: 6 or a conservatively substituted variant thereof, or an amino acid sequence having at least 85% sequence identity thereto.

15. The engineered cell according to claim 12, wherein the engineered receptor that binds other molecules is a CAR that binds PSCA.

16. The engineered cell according to claim 15, wherein the PSCA-binding CAR comprises or is the amino acid sequence shown in SEQ ID NO: 9 or a conservatively substituted variant thereof, or an amino acid sequence having at least 85% sequence identity thereto.

17. An engineered cell according to claim 12, wherein the engineered receptor that binds other molecules is a CAR that binds CD70.

18. The engineered cell according to claim 17, wherein the CD70-binding CAR comprises or is an amino acid sequence as shown in SEQ ID NO: 12 or a conservatively substituted variant thereof, or an amino acid sequence having at least 85% sequence identity thereto.

19. The engineered cell according to any one of claims 11-18, which is a T cell, a NK cell, a macrophage, a DC cell, a B cell, or a precursor cell thereof.

20. Use of the fusion protein of any one of claims 1 to 7, the engineered receptor of claim 8 or 9, the engineered nucleic acid molecule of claim 10, or the engineered cell of any one of claims 11 to 19 for preparing a drug for treating cancer.

21. The use according to claim 20, wherein the cancer is selected from one or more of the following: bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, and uterine cancer.

22. A method for extending the in vivo persistence of a CAR-T cell, comprising expressing the fusion protein of any one of claims 1 to 7 or the engineered receptor of claim 8 or 9 on the CAR-T cell membrane.

23. A method for improving the in vivo expansion ability of CAR-T cells, comprising expressing the fusion protein described in any one of claims 1 to 7 or the engineered receptor described in claim 8 or 9 on the CAR-T cell membrane.

24. A method for enhancing the in vivo killing ability of CAR-T on target cells, comprising expressing the fusion protein described in any one of claims 1 to 7 or the engineered receptor described in claim 8 or 9 on the CAR-T cell membrane.

Images