Polypeptide capable of being combined with immune checkpoint and application thereof

Abstract

The invention relates to a fusion protein which comprises a CD226 extracellular domain, a transmembrane domain and a co-stimulation domain, and the fusion protein can significantly enhance the tumor killing ability of cells expressing engineered receptors and prolong the in-vivo duration of the cells. The invention also provides a nucleic acid molecule containing the coding sequence of the fusion protein, an engineered cell for expressing the fusion protein, and applications of the fusion protein, the nucleic acid molecule and the engineered cell.

Description

Technical Field

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

[0002] CAR-T is a T lymphocyte that expresses a chimeric antigen receptor. It has made breakthrough progress in the treatment of hematological tumors. However, the complex structure of solid tumors, tumor heterogeneity and complex tumor microenvironment block the infiltration and function of CAR-T. At the same time, low oxygen and low pH affect the survival of CAR-T in the body. Studies have shown that in the treatment of solid tumors, improving the survival of CAR-T or enhancing the amplification of CAR-T in the body or in the tumor location can significantly improve the efficacy of CAR-T in the treatment of solid tumors.

[0003] CD226 is a transmembrane glycoprotein composed of three domains. The first domain outside the envelope of the CD226 molecule 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. The known ligands of CD226 are CD155 and CD112. When CD226 binds to its ligand, the CD226 molecule moves directionally to the lipid rafts on the cell membrane and recruits intracellular signaling molecules such as PTK and PKC, which are phosphorylated on four tyrosine residues to activate the cells. Summary of the invention

[0004] The present 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 an extracellular segment of CD226, a transmembrane structure and an intracellular signaling portion. On the other hand, the present application also constructs engineered immune cells, such as T cells, expressing CD226 fusion proteins and CAR structures. 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. CD226 fusion protein can enhance the killing ability of CAR-T cells, competitively bind to CD155 and CD112 with TIGIT, thereby alleviating immunosuppression in the tumor microenvironment.

[0005] Specifically, the present application provides:

[0006] 1. A fusion protein, which from N-terminus to C-terminus comprises or is:

[0007] 1) CD226 extracellular region, transmembrane domain and co-stimulatory domain; or

[0008] 2) Signal peptide, CD226 extracellular region, transmembrane domain and co-stimulatory domain.

[0009] In some embodiments, the signal peptide is a signal peptide derived from a secreted protein.

[0010] In some embodiments, the extracellular region and the transmembrane domain are connected by a hinge region. In some embodiments, the hinge region is a hinge region of IgG, IgD, CD8α or CD28, or a combination thereof.

[0011] 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 co-stimulatory 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 connector. In some embodiments, the connector 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 an amino acid sequence as shown in SEQ ID NO: 1 or 3, or a conservatively substituted variant of the amino acid sequence as shown in SEQ ID NO: 1 or 3, 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 as shown in SEQ ID NO: 1 or 3.

[0012] 2. A 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 shown in SEQ ID NO: 17, or a conservatively substituted variant of the amino acid sequence as shown in SEQ ID NO: 17, 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 as shown in SEQ ID NO: 17.

[0013] 3. The fusion protein according to item 1 or 2, wherein the co-stimulatory domain comprises a signal transduction domain selected from one or more of the following molecules or consists of a signal transduction domain of one or more signaling molecules selected from the following:

[0014] 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 more) sequence identity to the amino acid sequence as set forth in SEQ ID NO:18.

[0015] 4. A 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 an amino acid sequence as shown in SEQ ID NO: 4, 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 as shown in SEQ ID NO: 4.

[0016] 5. The fusion protein according to any one of items 1 to 4, which comprises or is from the N-terminus to the C-terminus:

[0017] 1) CD226 extracellular region, CD28 transmembrane domain and CD28 signal transduction domain; or

[0018] 2) CD226 signal peptide, CD226 extracellular region, CD28 transmembrane domain and CD28 signal transduction domain.

[0019] 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 as shown in SEQ ID NO: 1, a conservatively substituted variant of the amino acid sequence as 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 as shown in SEQ ID NO: 1.

[0020] 7. The fusion protein according to item 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 with the amino acid sequence as shown in SEQ ID NO: 2.

[0021] In some embodiments, the CD226 extracellular region is further directly or indirectly connected 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 connected to one or more polypeptides or proteins that bind to tumor antigens or immune checkpoint proteins.

[0022] 8. An engineered receptor comprising a fusion protein according to any one of items 1 to 6, and a primary signal transduction domain located at the C-terminus of the fusion protein.

[0023] 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.

[0024] 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 immune receptor.

[0025] 10. An engineered nucleic acid molecule comprising a fusion protein according to any one of items 1 to 7 or an engineered receptor according to 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.

[0026] 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 creatinine. 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 following: 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-carboxy-propyl)uridine ((ACP3)U), uridine-5-oxyacetic acid (O5U), uridine-5-oxyacetic acid methyl ester (MV), 5-methoxycarbonyl uridine (M5U), 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 following: wybutoxosine (osyw), ybutoxosine (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 following: 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) and 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 selected from 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 2'-O-methylation modification on the ribose of the nucleotide or 3'thiophosphate bond modification between nucleotides or both. In some embodiments, the modification is 2'-O-methylation modification on the first three nucleotide riboses at the 5' end, 2'-O-methylation modification on the last three nucleotide riboses at the 3' end, 3'thiophosphate modification between the first three nucleotides at the 5' end, and 3'thiophosphate modification between the last three nucleotides at the 3' end.

[0027] In some embodiments, the nucleic acid molecule further encodes one or more second engineered receptors, which 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.

[0028] 11. An engineered cell comprising the fusion protein according to any one of items 1 to 7 or the engineered receptor according to item 8 or 9 and / or the engineered nucleic acid molecule according to item 10.

[0029] 12. The engineered cell according to item 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,

[0030] 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; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit 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; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EG 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);Hexose moiety of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (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 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 myelocytic disease 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 receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1) and CD155;

[0031] 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.

[0032] In some embodiments, the one or more target molecules are selected from one or more of the following: CD70, PSCA, and CEA.

[0033] In some embodiments, the engineered receptor is selected from one or more of the following: CAR, TCR, and TAC.

[0034] 13. According to the engineered cell of item 12, the engineered receptor that binds other molecules is a CAR that binds CEA. In some embodiments, the CAR that binds CEA includes or is from N-terminus to C-terminus: a domain that binds CEA-hinge region-transmembrane region-signal transduction domain, or a domain that binds CEA-transmembrane region-signal transduction domain. In some embodiments, the CEA is human CEA. In some embodiments, the domain that binds CEA is an antibody or an antigen-binding fragment of the antibody. In some embodiments, the antigen-binding fragment is scFv. In some embodiments, the signal transduction domain includes or is a primary signal transduction domain. In some embodiments, the signal transduction domain includes 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 includes one or more costimulatory domains. In some embodiments, the CAR binding to CEA comprises or is CEA ScFv-8h-8TM-BBZ from N-terminus to C-terminus. Wherein, CEA scFv represents a scFv composed of a heavy chain variable region and a light chain variable region of an anti-CEA antibody, and "-" represents connection by a peptide bond or peptide chain. In some embodiments, the CEA scFv comprises or is an amino acid sequence as shown in SEQ ID NO: 5.

[0035] 14. An engineered cell according to item 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.

[0036] 15. The engineered cell according to item 12, wherein the engineered receptor that binds to other molecules 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 domain that binds to PSCA-hinge region-transmembrane region-signal transduction domain, or a domain that binds to PSCA-transmembrane region-signal transduction domain. In some embodiments, the PSCA is human PSCA. In some embodiments, the domain that binds to PSCA is an antibody or an antigen-binding fragment of the antibody. In some embodiments, the antigen-binding fragment is a 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 PSCA-binding CAR comprises or is: PSCAScFv-8h-8TM-BBZ from N-terminus to C-terminus. Wherein, PSCAscFv represents a scFv composed of a heavy chain variable region and a light chain variable region of an anti-PSCA antibody, and "-" represents connection by a peptide bond or peptide chain. In some embodiments, the PSCA scFv comprises or is the amino acid sequence shown in SEQ ID NO: 8.

[0037] 16. An engineered cell according to claim 15, wherein the PSCA-binding CAR comprises or is an amino acid sequence as shown 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.

[0038] 17. According to the engineered cell of item 12, 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 domain that binds to CD70-hinge region-transmembrane region-signal transduction domain, or a domain that binds to CD70-transmembrane region-signal transduction domain. In some embodiments, the CD70 is human CD70. In some embodiments, the domain that binds to CD70 is an antibody or an antigen-binding fragment of the antibody. In some embodiments, the antigen-binding fragment is 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: CD70ScFv-8h-8TM-BBZ from N-terminus to C-terminus. Wherein, CD70 scFv represents a scFv composed of a heavy chain variable region and a light chain variable region of an anti-CD70 antibody, and "-" represents connection by a peptide bond or peptide chain. In some embodiments, the CD70 scFv comprises or is an amino acid sequence as shown in SEQ ID NO: 35.

[0039] 18. An engineered cell according to item 17, wherein the CD70-binding CAR comprises or is an amino acid sequence as shown in SEQ ID NO: 12 or a conservative substitution variant of SEQ ID NO: 12, or an amino acid sequence of SEQ ID NO: 12 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.

[0040] The engineered cell according to any of the foregoing, wherein the costimulatory domain comprises a signal transduction 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, CD19, CD4 1d, 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.

[0041] 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.

[0042] 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.

[0043] An 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.

[0044] 19. The engineered cell according to any one of items 11-18, which is a T cell, a NK cell, a macrophage, a DC cell, a B cell, or a precursor cell thereof.

[0045] 20. Use 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 for preparing a drug for treating cancer. In some embodiments, the cancer is a human cancer.

[0046] 21. The method of 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. 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 renal clear cell adenocarcinoma.

[0047] In addition, the present application also provides a method for treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a fusion protein according to any one of items 1-7, an engineered receptor according to item 8 or 9, an engineered nucleic acid molecule according to item 10, or an engineered cell according to any one of items 11-19. In some embodiments of the method for treating cancer, the cancer is selected from one or more of the following:

[0048] In some embodiments, the subject or patient is a human patient.

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

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

[0051] 22. A method for prolonging the in vivo persistence of CAR-T cells, comprising expressing the fusion protein according to any one of items 1 to 7 or the engineered receptor according to item 8 or 9 on the membrane of the CAR-T cells.

[0052] 23. A method for improving the in vivo proliferation ability of CAR-T cells, comprising expressing the fusion protein according to any one of items 1 to 7 or the engineered receptor according to item 8 or 9 on the membrane of the CAR-T cells.

[0053] 24. A method for enhancing the in vivo killing ability of CAR-T against target cells, comprising expressing the fusion protein according to any one of items 1 to 7 or the engineered receptor according to item 8 or 9 on the CAR-T cell membrane.

[0054] In some embodiments, the aforementioned in vivo refers to a cancer patient or subject in vivo. 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:

[0055] 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.

[0056] In some embodiments, the CAR-T 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 is a CAR of 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 by a peptide bond or a peptide chain.

[0057] 2A self-cleaving polypeptide is a common multi-gene expression scheme that realizes multiple gene co-expression at a translation level. 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 vest rhinitis virus 2A (E2A). Its main working principle is that when the ribosome recognizes the 2A peptide end during translation, it will skip the glycyl-prolyl peptide bond synthesis, and slide, thereby directly generating 2 independent proteins. The purpose of the application using 2A peptides is to obtain engineered immune cells expressing CD226 fusion proteins, and finally the function of the engineered immune cells is verified by the test of the embodiment, and the unpredictable effect of CD226 fusion proteins on engineered immune cells is further verified. In addition to the above-mentioned 2A self-cleaving 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, and 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 transducing different genes into target cells separately, or by transducing the target genes simultaneously with the constructed vectors expressing different genes; whether the target cells are transduced using linkers or by separately constructing expression vectors of multiple genes, the purpose of obtaining engineered cells expressing CD226 can be achieved in the end, and the functions and unpredictable effects of engineered cells are mainly based on the protein molecules expressed and the final engineered cells themselves.

[0058] 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 embodiments herein are based on the verification of engineered T cells, the unpredictable effects of the engineered cells are mainly based on CD226 fusion protein, based on the combination of CD226 fusion protein and CAR, so 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

[0059] Figure 1A schematic diagram of the expression of engineered cells expressing CD226 fusion protein is shown.

[0060] Figure 2A and Figure 2B The proliferation rate and viability of CAR-T cells expressing CD226 fusion protein are shown. Figure 2A is the value-added multiple, Figure 2B For survival rate.

[0061] Figure 3 In vitro killing by CEA CAR-T expressing CD226 fusion protein is shown.

[0062] Figure 4 Shown is the in vitro cytokine secretion of CEA CAR-T expressing CD226 fusion protein.

[0063] Figure 5 The in vivo efficacy of CEA CAR-T expressing CD226 fusion protein against colorectal cancer tumor bearing models is shown.

[0064] Figure 6 The figure shows the copy number detection of CAR when CEA CAR-T expressing CD226 fusion protein was used to treat the colorectal cancer mouse tumor-bearing model.

[0065] Figure 7 The proliferation fold of PSCA CAR-T cells expressing CD226 fusion protein is shown.

[0066] Figure 8 In vitro killing by PSCA CAR-T expressing CD226 fusion protein is shown.

[0067] Fig. 9 Shown is the in vitro cytokine secretion of PSCA CAR-T expressing CD226 fusion protein.

[0068] Fig.10 The in vivo efficacy of PSCA CAR-T expressing CD226 fusion protein against bladder cancer tumor-bearing models is shown.

[0069] Fig.11 The figure shows the detection of CAR copy number when PSCA CAR-T expressing CD226 fusion protein was used to treat bladder cancer mouse tumor-bearing model.

[0070] Fig.12 Shown is the in vitro cytokine secretion of CD70 CAR-T expressing CD226 fusion protein. Specific implementation plan

[0071] The present application relates to a transmembrane protein with an extracellular segment of CD226 as an antigen binding domain, and the transmembrane protein is referred to as a fusion protein in the present application, which comprises an extracellular region of CD226, 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 to enhance the duration of the CAR-T cells in vivo. 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.

[0072] definition

[0073] It should be understood that the present disclosure is not limited to the aspects described herein, which may of course vary in nature. It should also be understood that the terminology used herein is only used to describe particular aspects and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0074] 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 techniques and patent disclosures cited herein are incorporated herein by reference in their entirety. Unless otherwise indicated, those skilled in the art will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art.

[0075] The term "CD226" is an immune activator receptor, also known as DNAM-1, which competes with CD226 (i.e., 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 (adhesion protein-2 or PVRL2). However, compared with the binding of CD226 to PVR, the binding affinity of CD225 to PVRL2 and PVRL3 is much weaker. In its natural state, CD226 is a glycoprotein that is mostly expressed on the surface of NK cells, platelets, monocytes, and some T cells, and is a member of the Ig superfamily. CD226 consists of three domains: the extracellular domain, the transmembrane domain, and the intracellular domain. The extracellular domain includes two immunoglobulin V-like domains and eight N-linked glycosylation sites. Its 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.

[0076] As used herein, "CD226 extracellular region" can be used to refer to any peptide segment in the CD226 extracellular region or any truncated body of the CD226 extracellular region, or the full length of the CD226 extracellular region, as long as it retains the ability to bind to CD155. In some embodiments, the CD226 extracellular region comprises an amino acid sequence as 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 to refers to the position of the corresponding amino acid by sequence alignment. After 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) so that the amino acid sequence of any CD226 molecule has the same residues as the amino acid sequence shown in SEQ ID NO: 1 or 3 at as many positions as possible, the amino acids shown in SEQ ID NO: 1 or 3 are numbered in sequence order starting from 1 at the first amino acid at the nitrogen terminus. In the amino acid sequence of any CD226 molecule, the first amino acid and the last amino acid at the same position as the amino acid sequence shown in SEQ ID NO: 1 or 3 and all the amino acids between the first amino acid and the last amino acid constitute the "amino acid sequence corresponding to SEQ ID NO: 1 or 3".

[0077] "CD155" is also known as "PVR", which 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 its 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 enhanced tumor progression.

[0078] "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.

[0079] As used herein, the percentage of "identity", such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to the degree of similarity between amino acid sequences or between nucleotide sequences determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, the proportion of the number of positions with the same base or amino acid residue to the total number of positions determined after two sequences have the same residue at as many positions as possible by introducing spaces, etc. The percentage of "identity" can be determined using software programs known in the art. Preferably, the comparison is performed using default parameters. A preferred comparison program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0080] As used herein, a "variant" has at least one amino acid difference relative to a reference amino acid sequence, for example, 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 include 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 foregoing 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% is optimal, and have the same or substantially similar properties or functions as the native or parent protein or enzyme.

[0081] As used herein, "amino acid" refers to any monomer 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 also includes non-natural amino acids or amino acid analogs derived or modified on the basis of any of the above natural amino acids. 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 a photosensitive crosslinker, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids containing 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), deoxyephedrine (Des), 2,2'-diaminopimelic acid (Dp m), 2,3-diaminopropanesulfonic acid (Dpr), ethylglycine (EtGly), N-ethylaspartic 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 one 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 amino acids selected from the group consisting of 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.

[0082] In the context of the application, the terms "DNA" and "RNA" refer to single-stranded or double-stranded DNA or RNA molecules. Unless otherwise specified, 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. In this article, the A, C, G, T and U nucleotides refer to nucleotides containing adenine, guanine, cytosine, thymine and uracil as their respective nitrogenous bases.

[0083] RNA molecules include coding RNA or non-coding RNA (ncRNA), such as Pre-mRNA, mature mRNA or long noncoding RNA (lncRNA).

[0084] 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:

[0085] Replace one or more deoxyribonucleotides in DNA with ribonucleotides;

[0086] Substituting one or more ribonucleotides in the RNA with deoxyribonucleotides; or

[0087] De novo synthesis using deoxyribonucleotides and ribonucleotides as raw materials by biological or chemical synthesis. It should be noted that the method of obtaining a hybrid molecule of DNA and RNA is not limited to the above method, and the hybrid molecule of DNA and RNA obtained by any method belongs to the category of "hybrid molecule of DNA and RNA" defined in this application.

[0088] As used herein, if 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 one or more thymines in the base sequence of one of the nucleic acid molecules are converted to uracil to obtain a nucleic acid molecule that is exactly the same as the base sequence of another nucleic acid molecule. 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 of arrangement of bases in a polynucleotide molecule. Those skilled in the art should know that, unless otherwise specified, the base sequence or polynucleotide sequence described in this application can be used to describe DNA sequences. "T" refers to thymine, but when the base sequence or polynucleotide sequence is used to describe RNA (such as 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 (such as mRNA or Poly (A) tail) sequence that is complementary or corresponding to the DNA, wherein each "T" of the DNA sequence is replaced by "U".

[0089] In this article, "coding" refers to i) genetic information contained in a DNA sequence that can be transcribed into an RNA molecule, and / or ii) genetic information contained in an 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 that can be translated into a protein in an mRNA precursor or mature mRNA, and can also refer to a complementary sequence or a fragment thereof of a deoxyribonucleotide (DNA) sequence that is used as a template to transcribe the mRNA precursor or mature mRNA. In addition, the "coding sequence" of the present application may further include polynucleotide sequences encoding 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.

[0090] Unless otherwise specified, "peptide", "polypeptide" and "protein" are used interchangeably in this application and can be used to refer to any natural active substance formed by covalently linking two or more amino acids through peptide bonds, which may or may not contain the secondary or tertiary structure of the protein molecule.

[0091] As used herein, the term "contacting" is used according to its simple common meaning, referring to the process of allowing at least two different substances to become close enough to react, interact or physically contact. It should be understood that the resulting reaction product can be directly produced by the reaction between the added reagents, or produced by the intermediate of one or more added reagents, which can be produced in the reaction mixture. The term "contacting" can include allowing two substances to react, interact or physically contact, wherein the two substances can be, for example, engineered receptors (or engineered nucleic acid molecules) and cells provided herein. In an embodiment, contacting includes, for example, allowing engineered nucleic acid molecules or engineered peptides described in the present application to enter cells.

[0092] As used herein, "CAR-T cell in vivo persistence" refers to the duration of CAR-T cells in the patient or subject's body, that is, the period from the in vivo administration of CAR-T cells to their final disappearance from the body. It can be calculated or measured by comparing the copy number of CAR. For example, after a specific period of time of use of CAR-T in vivo, an increase in its copy number can be used to indicate an extension of the in vivo persistence of CAR-T cells.

[0093] In addition, in the present application, 8h represents the CD8 hinge region, 8TM represents the CD8 transmembrane domain, BB represents the 4-1BB co-stimulatory domain (or called the 4-1BB signal transduction domain), and Z or z represents the CD3ζ signal transduction domain.

[0094] Unless otherwise defined 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.

[0095] Fusion Protein

[0096] The present application also provides a fusion protein comprising an extracellular region of CD226, wherein the fusion protein is a transmembrane protein, which comprises an extracellular region, a transmembrane domain and an intracellular region, and 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:

[0097] 1) CD226 extracellular region, transmembrane domain and co-stimulatory domain; or

[0098] 2) Signal peptide, CD226 extracellular region, transmembrane domain and co-stimulatory domain.

[0099] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus:

[0100] 1) CD226 extracellular region, transmembrane domain and co-stimulatory domain; or

[0101] 2) Secretory protein signal peptide, CD226 extracellular region, transmembrane domain and co-stimulatory domain;

[0102] Wherein the CD226 extracellular region and the transmembrane domain, the transmembrane domain and the co-stimulatory 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, 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 connector, and two proteins or polypeptides connected to each other using a connector are respectively connected to one end of the connector by a covalent or non-covalent bond. The "connector" can be a peptide connector (i.e., a peptide chain, such as a peptide chain composed of 1 to 50 amino acids or their derivatives) or a non-peptide connector, and the connector can be cleavable (i.e., it can be hydrolyzed by an enzyme in an organism, such as a mammal) 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.

[0103] In some embodiments, the CD226 extracellular region and the transmembrane domain contain or are a hinge region, which can be the hinge region of any molecule, or the portion between the recognition domain of any membrane receptor molecule and the transmembrane domain. As used herein, a "hinge region" is generally a flexible portion of a protein that can participate in the propagation of conformational changes, such as from an active site to other parts of the protein structure) including other subunits), such as the segment between the immunoglobulin heavy chain CH1 and CH2. 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.

[0104] As used herein, "costimulatory domain" is usually derived from the co-stimulatory receptor of immune cells, providing a second signal or secondary intracellular signal of activated 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 signal transduction domain of a ligand that specifically binds to CD83.

[0105] As used herein, "transmembrane domain" is the part of a transmembrane protein that connects the inside and outside of a 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 a cell membrane. Typically, a "transmembrane domain" can be involved in regulating and / or conducting transmembrane signals. An exemplary transmembrane domain may be selected from the transmembrane domains of any one or more of the following molecules, or may be composed of the transmembrane domains of any one or more of the following molecules: ICOS, CD226, 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.

[0106] In some embodiments, the extracellular region of the fusion protein further comprises one or more other 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 in any manner to form a protein complex. In some embodiments, the binding is that the CD226 extracellular region is connected to the other 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 other 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 other 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 other 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 is connected in series with the multiple proteins, polypeptides or protein functional domains. 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.

[0107] In some embodiments, the other one or more proteins, polypeptides or protein domains comprise a recognition polypeptide that specifically binds to another one or more proteins, and the other one or more proteins may 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; CD 22; CD30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13Rα); interleukin 11 receptor alpha (IL-11Rα); prostate-specific membrane antigen (PSMA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; Epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; carcinoembryonic variant of tumor necrosis area;G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; 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 virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma, breakpoint X 2 (SSX2); CD79a; CD79b; CD72; leukocyte phase Related immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5);Immunoglobulin lambda-like polypeptide 1 (IGLL1) and CD155. ;

[0108] 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:

[0109] 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.

[0110] In some embodiments, the other one or more proteins, polypeptides or protein functional domains contain a recognition polypeptide that specifically binds to another one or more proteins, and the other one or more proteins include any one or more immune checkpoint proteins selected from the above-mentioned, and any one or more tumor antigens mentioned above.

[0111] 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.

[0112] Engineered receptors

[0113] The 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.

[0114] In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a T cell antigen coupling (TAC).

[0115] As used herein, the term "CAR", i.e., a 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. Among them, the "intracellular signal transduction domain" comprises a primary signal transduction domain and / or a co-stimulatory domain. In some embodiments, the intracellular signal transduction domain comprises 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 signal transduction domain" generally comprises an immune-receptor tyrosine-based activation motif (ITAM), the basic composition of which is: YXXL / V. Wherein Y is tyrosine, L / V refers to leucine or valine, and X can be any amino acid. When the receptor binds to the corresponding ligand, the tyrosine in the ITMA connected thereto can be phosphorylated under the action of a class of protein tyrosine kinases PTKs connected to the cell membrane, thereby recruiting other free protein kinases or adapter proteins in the cell to transmit activation signals into the cell.

[0116] As used herein, an engineered "TCR" is an engineered T cell receptor comprising: (a) an antigen binding domain (as used in this application, 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 the same (e.g., all CD3ε, all TCRα or all TCRβ). In some embodiments, the first, second and third TCR subunits are different. In some embodiments, the engineered 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.

[0117] As used herein, "TAC" is 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, the TAC comprises: (a) an antigen binding domain (as used herein, the 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) that specifically recognizes an extracellular antigen binding domain of a TCR subunit (e.g., CD3ε); (d) optionally A second connector; (e) optionally an extracellular antigen binding domain or a portion thereof of a first TCR co-receptor (e.g., CD4, CD8); (f) a transmembrane domain comprising a transmembrane domain of a second TCR co-receptor (e.g., CD4, CD8); and (g) optionally an intracellular signal transduction domain comprising an intracellular signal transduction domain of a third TCR co-receptor (e.g., CD4, CD8); wherein the TCR subunit is selected from any one or more of the following groups: CD3ε, CD3δ, CD3γ, TCRα, TCRβ, TCRγ, and TCRδ; and wherein the first, second, and third TCR co-receptors are each independently selected from any one of the following groups: CD4, CD8, and CD28. In some embodiments, the first, second, and third TCR co-receptors are the same. In some embodiments, the first, second, and third TCR co-receptors are different. In some embodiments, the TAC further comprises a hinge domain (e.g., from CD8α) located between the C-terminus of the 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.

[0118] As used herein, the term "antigen binding domain" covers the concepts of "ligand binding domain" and "receptor binding domain", which are usually 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 subject to any restrictions. 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 a diabody.

[0119] Engineered Nucleic Acid Molecules

[0120] The present application also provides an engineered nucleic acid molecule encoding the aforementioned fusion protein or engineered receptor, the nucleic acid molecule comprising a target protein coding sequence. In some embodiments, after the engineered nucleic acid molecule is introduced into a suitable host cell, the engineered nucleic acid molecule 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 molecule". "Natural nucleic acid molecule" refers to a nucleic acid molecule that exists in a 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, an engineered nucleic acid molecule can be used to refer to any nucleic acid molecule that can be obtained by any or multiple bioengineering means, which can have a polynucleotide sequence that is exactly the same as a natural nucleic acid molecule, have a modification that is exactly the same as a natural nucleic acid molecule, and even form a structure that is exactly the same as a natural nucleic acid molecule, but the difference between the engineered nucleic acid molecule and its corresponding natural nucleic acid molecule, or the natural nucleic acid molecule with the same polynucleotide sequence at least includes: the engineered nucleic acid molecule is not directly purified or extracted from an animal or plant that exists naturally in nature in its natural form.

[0121] 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, the DNA molecule, in addition to comprising the target protein coding sequence, also comprises a gene manipulation or regulatory element for replication and / or expression in a prokaryotic and / or eukaryotic cell.

[0122] The necessary structural elements that make the engineered DNA molecule replicate or efficiently replicate in cells are known in the art, including, for example, an origin of replication (ORI). In some embodiments, the engineered DNA molecule further comprises a marker gene or its fragment and / or a reporter gene or its fragment and a unique restriction endonuclease site that allows insertion of DNA elements, preferably a restriction endonuclease site in the form of a multiple cloning site (MCS). The marker gene is conducive to identifying cells containing a plasmid comprising the marker gene, and can be selected from, for example, an antibiotic resistance gene. Each restriction endonuclease site in the MCS can be specifically recognized by different restriction endonucleases.

[0123] In some embodiments, the DNA molecule is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid composed of double-stranded DNA molecules. 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, and linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify in cells independently of the genomic genetic information stored in the nucleoid or nucleoid of prokaryotes, 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 such vectors: they are based on pUC, pTZ plasmids or any other plasmids (e.g., pMB1, pCoIE1) etc. that contain an ORI that supports high copies of plasmids.

[0124] In some embodiments, the engineered DNA molecule is a DNA molecule or a fragment thereof constituting a prokaryotic nucleoid or nucleoid, or a DNA molecule or a fragment thereof constituting a eukaryotic genome, that is, the target protein coding sequence or its complementary sequence can be replicated along with the prokaryotic genome.

[0125] In some embodiments, the engineered DNA molecule can be transcribed into mRNA. In some embodiments, the engineered DNA molecule also includes a coding sequence of an element that can be used to start or regulate the expression of the protein, polypeptide or its fragment after transcription, and the element includes but is not limited to 5'UTR, 3'UTR, poly (A) tail (or tailing signal), etc. In some embodiments, the engineered DNA molecule includes a coding sequence of at least one untranslated region (UTR). In some embodiments, the engineered DNA molecule includes at least the coding sequence of 5'UTR and the target protein coding sequence. In some embodiments, the engineered DNA molecule includes at least the coding sequence of 5'UTR, the target protein coding sequence, the coding sequence of 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) at both ends of the target protein coding sequence may be included, respectively, which are the first three nucleotides and the last three nucleotides that can be translated of the mRNA molecule, respectively. The 5'UTR usually contains 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. The RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes at the start of translation. 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, the 5'UTR includes an internal ribosome entry site or IRES. The 3'UTR may contain one or more regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of mRNA molecules, binding sites for regulatory RNA molecules (such as miRNA molecules), and / or signal sequences that participate in the intracellular transport of mRNA molecules.

[0126] On the basis of the aforementioned embodiments, 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 the stability of mRNA molecules, binding sites for amino acid sequences that enhance the translation of mRNA molecules, 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, an out-of-frame upstream translation initiation site, an out-of-frame upstream start codon, and / or a nucleotide sequence that produces a secondary structure that reduces or prevents translation. The presence of such nucleotide sequences in the 5'UTR can have a negative impact on translation.

[0127] The target protein coding sequence comprises codons that can be translated into an amino acid sequence. All the codons contained in the target protein coding sequence may be naturally occurring codons encoding amino acids, or may be partially or entirely composed of artificially synthesized codons. In some embodiments, some or all of the codons have been codon-optimized. In some embodiments, some or all of the codons encode non-natural amino acids.

[0128] In some embodiments, the engineered DNA molecule further comprises a structural element necessary for initiating or regulating transcription of the RNA on the 5' end side of the target gene fragment, and the structural element is known in the art. In some embodiments, the structural element at least comprises a promoter. Promoters and their sequences are known in the art, including weak promoters, medium strength promoters, strong promoters, mini promoters or core promoters, etc. 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 and a subsequent transcription factor binding site. The recognition and binding site can interact with an amino acid sequence that mediates or regulates transcription. Compared with the recognition site, the binding site is closer to the aforementioned target gene fragment. 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 of the enhancing 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 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 other regulatory elements before it, such as an enhancer that can promote the transcription of the aforementioned target protein coding sequence in a DNA plasmid.

[0129] In some embodiments, the eukaryotic cell is a yeast cell. In some embodiments, the DNA molecule is a yeast display vector.

[0130] In addition, the present application also provides an engineered RNA molecule comprising an engineered receptor or fusion protein encoding the aforementioned. In some embodiments, the engineered RNA molecule is obtained by transcription of the aforementioned engineered DNA molecule. In some embodiments, the engineered RNA molecule has the same sequence as the RNA molecule obtained by transcription of the aforementioned engineered DNA molecule. In some embodiments, the engineered RNA is mRNA. As used herein, "mRNA" (messenger RNA) is any RNA that encodes at least one protein, polypeptide, or fragment thereof, naturally occurring, non-naturally occurring, or modified, and the mRNA has the ability to be translated to produce the encoded protein, polypeptide, or fragment thereof in vitro, in vivo, in situ, or in vitro. Therefore, the mRNA can be a mature mRNA or a pre-mature mRNA, and the elements or structures that must be included or selectively included are known in the art. In some embodiments, the mRNA comprises a plurality of necessary functional component coding sequences to express, regulate, or enhance the expression level of the protein, polypeptide, or fragment thereof. The functional components include, but are not limited to, 5' caps, 5' UTRs, 3' UTRs, etc. Both the 5'UTR and the 3'UTR are usually transcribed from genomic DNA and are elements present in the pre-mature mRNA.

[0131] The term "5' cap" is located at the 5' end of the mRNA and contains methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate to form a 5',5'-triphosphate connection with its adjacent nucleotide. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are called type O, type I and type II respectively. Type O refers to the ribose of the terminal nucleotide is not methylated, type I refers to the ribose of one terminal nucleotide is methylated, and type II refers to the ribose of both terminal nucleotides is methylated. 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 generate m7G(5')ppp(5')(2'-OMeA)pG. Type II cap structures can be generated from Type I cap structures followed 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 followed by 2'-O-methylation of the 5'-fourth to last nucleotide using 2'-O methyl-transferase.

[0132] In some embodiments, the mRNA further comprises a stabilizing element. Stabilizing elements may include, for example, a histone stem-loop. In some embodiments, the mRNA comprises a coding region, at least one histone stem-loop and optionally a poly(A) sequence or a polyadenylation signal. The poly(A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the mRNA comprises a combination of a poly(A) sequence or a polyadenylation signal and at least one histone stem-loop, although both have alternative mechanisms in nature, their synergistic effect can increase protein expression to a level that exceeds that observed for any single element. 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 generally derived from a histone gene and comprises two adjacent partial or complete reverse complementary sequences separated by a spacer (composed of a short sequence) and the intramolecular base pairing forms a loop. Unpaired loop regions are generally unable to base pair with any 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) may occur. In some embodiments, the at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length.

[0133] 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.

[0134] 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 before being administered (e.g., within a few minutes to an hour).

[0135] The lipid nanoparticles generally include 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 ... 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.

[0136] 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 has the same genetic information as the engineered DNA molecule or the engineered RNA molecule.

[0137] 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 more 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 more than 85% sequence identity to a polynucleotide sequence as set forth in any one or more of SEQ ID NOs: 25 to 34.

[0138] Engineered cells

[0139] The present application also provides an engineered cell, wherein the engineered cell expresses or contains the aforementioned fusion protein or the aforementioned engineered receptor on its cell membrane, or the engineered cell contains the aforementioned engineered nucleic acid molecule.

[0140] 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 precursor cells thereof. 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.

[0141] In some embodiments, the engineered cell is a CAR-T or CAR-NK cell targeting one or more different antigens, which comprises a fusion protein or engineered receptor as described above, and one or more CARs targeting other tumor antigens. In some embodiments, the engineered cell is a CAR-T or CAR-NK cell targeting one or more different antigens, which comprises a fusion protein or engineered receptor as described above, and one or more CARs targeting immune checkpoint proteins. In some embodiments, the engineered cell is a CAR-T or CAR-NK cell targeting one or more different antigens, which comprises 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.

[0142] In some embodiments, the engineered cell is a TCR-T cell targeting one or more different antigens, comprising 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, comprising 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, comprising 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.

[0143] In some embodiments, the engineered cell is a TAC-T cell targeting one or more different antigens, which comprises a fusion protein or engineered receptor as described above, and the TAC-T further comprises one or more TACs targeting other tumor antigens. In some embodiments, the engineered cell is a TAC-T cell targeting one or more different antigens, which comprises a fusion protein or engineered receptor as described above, and the TAC-T further comprises a TAC targeting one or more immune checkpoint proteins. In some embodiments, the engineered cell is a TAC-T cell targeting one or more different antigens, which comprises a fusion protein or engineered receptor as described above, and the TAC-T further comprises one or more TACs targeting other tumor antigens, and one or more TACs targeting immune checkpoint proteins.

[0144] 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.

[0145] In some embodiments, the engineered cells are selected from: T cells, NK cells, macrophages, DC cells, B cells, or precursor cells thereof.

[0146] In some embodiments, the tumor antigens described herein are selected from one or more of the following:

[0147] 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-1Gag, 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, carcinoembryonic variant of tumor necrosis area, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, (PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K , OR51E2, TARP, WT1, ETV6-AML, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG, NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, (PAX5, OYTES1, (LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and CD155.

[0148] 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.

[0149] In some embodiments, the engineered cell comprises the aforementioned fusion protein or engineered receptor, and a CAR, TCR or TAC that binds CEA, PSCA or CD70. In some embodiments, the engineered cell comprises the aforementioned fusion protein or engineered receptor and a CAR that binds CEA. In some embodiments, the structure of the CAR that binds CEA is as follows: CEA ScFv-8h-8TM-BBZ. In some embodiments, the engineered cell comprises the aforementioned fusion protein or engineered receptor and a CAR that binds PSCA. In some embodiments, the structure of the CAR that binds CEA is as follows: PSCAScFv-8h-8TM-BBZ. In some embodiments, the engineered cell comprises the aforementioned fusion protein or engineered receptor and a CAR that binds CD70. In some embodiments, the structure of the CAR that binds 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 chain and light chain of antibodies targeting CEA, PSCA and CD70, respectively.

[0150] use

[0151] The present 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:

[0152] 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.

[0153] In some embodiments, the cancer involves tumor cells that highly express CD155.

[0154] 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.

[0155] Example

[0156] Example 1: Application of CD226 fusion protein in functional verification of CAR-T cells targeting CEA Cell expansion and viability Force detection

[0157] Three vectors containing 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) gene sequences were constructed and viruses were prepared. Among them, CEA scFv is the scFv of anti-CEA antibody, 8h represents the CD8 hinge region, 8TM represents the CD8 transmembrane domain, BB represents the 4-1BB co-stimulatory 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 co-stimulatory 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 text.

[0158] The specific virus preparation method is as follows:

[0159] The present embodiment uses calcium phosphate method to package lentivirus, referring to the Molecular Cloning Experiment Guide (3rd edition, J. Sambrook et al.), specifically: 293T cells are cultured with DMEM medium containing 10% FBS (w / v) until they are in good condition, the above-mentioned target plasmids are respectively mixed with three lentivirus packaging plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G in appropriate proportions, CaCl2 and 2×HBS are added, mixed and allowed to stand at room temperature, and then added to the treated 293T cell culture medium, and the medium is changed to 10mL DMEM medium containing 10% FBS again after 4-6h, and the cell supernatant is collected after 48h or 72h. The virus supernatant is collected and purified, and the purified virus is divided into 1.5mL EP tubes and stored at -80℃ for standby use.

[0160] The concentrated lentivirus was titered using 293T and / or CHO cells. 1×105 293T and / or CHO cells were inoculated into a 24-well plate, and the concentrated virus was infected with 293T and / or CHO cells at a volume of 1, 2.5, and 10ul / well (DEAE promoter was required). Two days after infection, the infected 293T and / or CHO cells were collected for flow cytometry. Protein-L was used to detect total CAR expression and calculate the virus titer. The titer calculation method is: 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 2.

[0161] The lymphocytes were separated by gradient centrifugation. After centrifugation, the second white lymphocyte layer was taken and washed with 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 on the 4th, 6th and 8th day after virus infection / transduction. The results are shown in Figure 2: Figure 2A is the cell expansion multiple, Figure 2B is the cell viability.

[0162] The results showed that there was no significant difference between the CEA CD226-CAR-T group and the control CAR-T group (CEA CAR-T and CE226-z group) and the CT group, and all had excellent expansion and viability.

[0163] In vitro effectiveness validation

[0164] DLD1-Luc-GFP is a human colorectal adenocarcinoma epithelial cell, which expresses CD155 on the cell surface but is negative for CEA expression on the membrane. Based on DLD1 cells, we overexpressed CEA molecules on them to make them double-positive cells expressing CD155 and CEA. The above cells were modified with Luc-GFP, hereinafter referred to as DLD1 and DLD1-CEA. DLD1-CEA (human colorectal adenocarcinoma epithelial cells, CEA and CD155 positive) and DLD1 (human colorectal adenocarcinoma epithelial cells, CD155 positive, CEA negative) were used as target cells, respectively. The effector cells were plated at an effector-target ratio of 2:1, and the killing ability of different groups on target cells was tested after 24 hours. The results are shown in the following table. Figure 3 As shown, 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 the application does not affect the specificity of CAR when it is 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.

[0165] After 24 hours, the supernatant was collected to test the secretion capacity of IFN-γ by immune cells after being stimulated by target cells. Figure 4As shown in the figure, 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 ability of CAR-T cells. This enhanced secretion ability is very advantageous in solving the current defect of insufficient effectiveness of CAR-T in the treatment of solid tumors. However, CD226-z with the addition of the primary stimulation signal CD3ζ did not have this effect.

[0166] The above results show that compared with the control CEA CAR-T cells that do not express CD226 fusion, 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.

[0167] In vivo anti-tumor effect and persistence verification

[0168] In vivo efficacy evaluation, 8-10 week old NCG immunodeficient mice were injected with DLD1-CEA-Luc-GFP 1e+6 cells / mouse to form tumors. In vivo imaging was performed on day 7, and mice were randomly divided into groups according to the fluorescence value. Different groups were injected with 2.0e+6 copies / mouse. In vivo imaging analysis and orbital blood collection were performed every 7 days for copy number detection. The results are shown in Figure 2. Figure 5 and Figure 6 As shown, Figure 5 The effects of different CAR-T cells on tumors were demonstrated by the fluorescence intensity of different tumors, among which CEA CD226-CAR-T had the best in vivo anti-tumor effect; Figure 6 The expression of CAR in tumor-bearing mice at different time points after the infusion of different CAR-T cells was detected. The persistence of CAR-T cells with different CAR structures in tumor-bearing biological tissues was further reflected by the copy number. Among them, CEA CD226-CAR-T can persist in the blood of tumor-bearing mice for at least 43 days and still maintain a high copy number. The results showed that CEA CAR-T cells expressing CD226 fusion protein (CEA CD226-CAR-T cells) prolonged the survival of mice, inhibited tumor growth, slowed down the 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.

[0169] Example 2: Application of CD226 fusion protein in the functional verification of CAR-T cells targeting PSCA

[0170] Cell preparation

[0171] The CAR vector construction and CAR-T preparation methods refer to Example 1.

[0172] After PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, they were infected with activated PBMCs at a certain multiplicity of infection (3-5 MOI). The total number of cells was counted on the 1st, 2nd, 5th, 6th, 8th, 10th, and 12th day after virus infection / transduction. Figure 7 As shown: Figure 7 is the cell expansion multiple.

[0173] The results showed that there was no difference between the PSCA-targeting CAR-T group expressing CD226 fusion protein and the control CAR-T group and the CT group, and both had excellent expansion and viability.

[0174] In vitro effectiveness validation

[0175] HT-1376 (human bladder cancer cell line, PSCA and CD155 positive) was used as the target cell. The effector cells were plated at a 1:1 effector-target ratio, and the killing ability of different groups on target cells was tested after 24 hours. The results are shown in Figure 8 As shown, there was no significant difference in killing positive cells between the PSCA-targeted CAR-T group expressing CD226 fusion protein (PSCA CD226-CAR-T) and the control CAR-T.

[0176] After 24 hours, the supernatant was collected to test the secretion capacity of IFN-γ by immune cells after being stimulated by target cells. Fig. 9 As shown, the IFN-γ secretion in the PSCA CD226-CAR-T group was much higher than that in the PSCA CAR-T group, which was about 5 times that of the factor secretion in the PSCA CAR-T group.

[0177] 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.

[0178] In vivo anti-tumor effect and persistence verification

[0179] In vivo efficacy evaluation, 8-10 week old NCG immunodeficient mice were injected with HT-1376-Luc-GFP 3e+5 cells / mouse to form tumors. In vivo imaging was performed 14 days later, and mice were randomly divided into groups according to the fluorescence value. Different groups were injected with 5.0e+5 CAR-T / mouse, and the total number of cells in the Control T group was the same as that in the experimental group. In vivo imaging analysis and orbital blood collection were performed every 7 days for copy number detection. The results are shown in Fig.10 and Fig.11 As shown, Fig.10 The fluorescence intensity of different tumors is used to show the effects of different CAR-T cells on tumors. The higher the fluorescence intensity, the larger the tumor volume and the higher the tumor cell activity. Fig.11 The expression of CAR in tumor-bearing mice was detected at different time points after the reinfusion of different CAR-T cells. The persistence of CAR-T cells with different CAR structures in tumor-bearing biological tissues was further reflected by the copy number. Fig.10 The results showed that PSCA CD226 CAR-T cells co-expressing CD226 fusion protein had better in vivo efficacy than PSCACAR-T cells that did not express CD226 fusion protein. The conventional structure of PSCA CAR-T had tumor recurrence on the 42nd day (the second to last measurement point) after CAR-T transfusion, and the fluorescence intensity of tumor cells increased significantly on the 49th day (the last measurement point). However, the anti-tumor effect of the PSCA CD226-CAR-T cell group targeting PSCA expressing CD226 fusion protein was still maintained on the 49th day, showing better anti-tumor cells. Fig.11 The results showed that the CAR copy number of the PSCA CAR-T group with conventional structure began to be significantly lower than that of the PSCA CD226-CAR-T cell group with PSCA target expressed CD226 fusion protein on day 21, and this gap increased significantly on day 28. Compared with the control PSCA CAR-T that does not express CD226 fusion, PSCA CAR-T cells expressing CD226 fusion protein (PSCA CD226-CAR-T cells) inhibited tumor growth and slowed down the tumor recurrence cycle, and their in vivo expansion was better than that of the control PSCA CAR-T group.

[0180] contrast Figure 3 The results of in vitro killing of CEA CAR-T and CEA CD226 CAR-T on CEA-negative CD155-positive DLD1 cells showed that CD226 fusion protein did not directly kill tumor cells. Figure 3 and Figure 5In each group, it can also be found that the CAR structure CD226-z designed with CD226 as the extracellular recognition domain has a killing function in vitro, but has no killing effect on tumor-bearing mice, while the CEA CD226 CAR-T expressing the CD226 fusion protein is not only effective in mice, but also significantly more effective 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 single CD226 fusion protein cannot produce effectiveness, but is an unexpected result of the combined use of the fusion protein and CAR structure described in the application, such as the increase in CAR-T activated cytokine secretion, the enhancement of CAR-T persistence in vivo, and other comprehensive effects.

[0181] The inventors are also verifying the combination of CAR structures and fusion proteins for different targets of CEA, PSCA, and CD70. The tumors involved in the above targets include but are 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 and other malignant tumors. In the tumor cells of the above-mentioned multiple targets, CAR-T cells combined with CD226 fusion protein and CAR all show better effects.

[0182] Example 3: Application of CD226 fusion protein in functional verification of CAR-T cells targeting CD70

[0183] In vitro effectiveness validation

[0184] 786-O (human renal clear cell adenocarcinoma cells, CD70 positive) was used as the target cell. The effector cells were plated at a ratio of 1:2. After 24 hours, the supernatant was collected and tested for the secretion capacity of IFN-γ after the immune cells were stimulated by the target cells. The results are shown in Fig.12 As shown, the IFN-γ secretion in the CD70CD226-CAR-T group was higher than that in the CD70CAR-T group.

[0185] The above results show that CD70 CAR-T cells expressing CD226 fusion protein (CD70 CD226-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.

[0186] The amino acid sequences and corresponding nucleic acid sequences of each element in the proteins, polypeptides and CAR structures used in the examples of the present application are shown in the following sequence table.

[0187] Sequence Listing

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

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 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.

4. An engineered receptor comprising a fusion protein according to any one of claims 1 to 3, and a primary signal transduction domain located at the C-terminus of the fusion protein.

5. An engineered nucleic acid molecule comprising a nucleic acid encoding a fusion protein according to any one of claims 1 to 3 or an engineered receptor according to claim 4.

6. An engineered cell comprising the fusion protein according to any one of claims 1 to 3 or the engineered receptor according to claim 4 and / or the engineered nucleic acid molecule according to claim 5.

7. Use of the fusion protein according to any one of claims 1 to 3, the engineered receptor according to claim 4, the engineered nucleic acid molecule according to claim 5, and the engineered cell according to claim 6 for preparing a drug for treating cancer.

8. A method for extending the in vivo persistence of CAR-T cells, comprising expressing the fusion protein according to any one of claims 1 to 3 or the engineered receptor according to claim 4 on the CAR-T cell membrane.

9. A method for improving the in vivo expansion ability of CAR-T cells, comprising expressing the fusion protein according to any one of claims 1 to 3 or the engineered receptor according to claim 4 on the CAR-T cell membrane.

10. A method for enhancing the in vivo killing ability of CAR-T on target cells, comprising expressing the fusion protein according to any one of claims 1 to 3 or the engineered receptor according to claim 4 on the CAR-T cell membrane.

Images