NKG2D engineered cells and compositions thereof

Abstract

According to the engineering immune cell for immunotherapy and the preparation method thereof, the engineering immune cell is more durable in a host organism, and the transplantation survival rate is high.

Description

NKG2D engineered cells and compositions thereof Technical Field

[0001] The present application relates to a cell with anti-transplant rejection function, and also relates to a method for resisting transplant immune rejection, and in particular to a method for resisting NK cell immune rejection.

[0002] Cross-references

[0003] This application claims the benefit of Chinese patent application No. CN202211692424.X filed on December 27, 2022, Chinese patent application No. CN202310089423.4 filed on January 29, 2023, and Chinese patent application No. CN202310225670.2 filed on March 9, 2023, the contents of which are incorporated herein in their entirety.

[0004] Sequence listing file submitted at the same time

[0005] The entire contents of the following XML file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Format (CRF) (Name: FG00927PCT-SequenceListing.xml, Date: 20231227, Size: 157KB). Background Art

[0006] Due to the immunogenetic differences between the donor and the recipient, when performing an exogenous donor transplant, as an exogenous graft, the donor may also be recognized and attacked by the immune cells in the recipient's body, thereby inhibiting or eliminating the exogenous graft, resulting in a host-versus-graft reaction (HVGR). By knocking out the MHC molecules in the graft cells, the host T cell rejection of the graft can be effectively resisted, but it may cause rejection reactions by other immune cells in the host. For example, in allogeneic cell transplantation, when the MHC-I class molecules of allogeneic cells are missing, it will lead to NK cell rejection reactions in the host body, enhancing the clearance of allogeneic cells. Therefore, how to effectively prevent the immune rejection reaction of host NK cells is crucial to the development of allogeneic cell transplantation therapy.

[0007] Summary of the Invention

[0008] After extensive and in-depth research, the inventors unexpectedly discovered that expressing chimeric receptors targeting NKG2D ligands on immune cells can resist host NK cell attack, prolong the survival of these immune cells both inside and outside the host body, and enhance anti-tumor effects. Based on this, the present application was completed.

[0009] The first aspect of the present application provides a chimeric polypeptide comprising:

[0010] a) a binding domain capable of specifically binding to a target molecule;

[0011] b) a receptor regulatory domain comprising a cleavage site,

[0012] The receptor regulatory domain includes an extracellular region and a transmembrane region, wherein the extracellular region includes one or more target molecule binding inducible cleavage sites B, and the transmembrane region includes one or more target molecule binding inducible cleavage sites A; and

[0013] c) intracellular domain,

[0014] In which, the binding of the binding domain to the target molecule can induce the cleavage of the cleavage site A and / or B of the receptor regulatory domain, thereby releasing the intracellular domain; the extracellular region of the receptor regulatory domain does not originate from the Notch protein, and the non-target molecule binding-induced cleavage of the chimeric polypeptide is reduced by mutating the extracellular region of the receptor regulatory domain or changing the steric hindrance of the extracellular region.

[0015] In one example, by mutating the extracellular region of the receptor regulatory domain or changing the steric hindrance of the extracellular region, the non-target molecule binding-induced cleavage of the chimeric polypeptide is reduced, while the cleavage of the target molecule binding-induced cleavage site B is increased, thereby increasing the cleavage of the cleavage site A and releasing the intracellular domain.

[0016] In one example, by mutating the extracellular region of the receptor regulatory domain or changing the steric hindrance of the extracellular region, the non-target molecule binding-induced cleavage of the chimeric polypeptide is reduced, while the cleavage of the target molecule binding-induced cleavage site A is increased to release the intracellular domain.

[0017] In one example, the mutation is located at a non-target molecule binding-inducible cleavage site in the extracellular region.

[0018] In one example, the mutation includes a deletion, an insertion, and / or a point mutation.

[0019] In one example, the steric hindrance of the extracellular region is altered by adding EGF repeats to the extracellular region to reduce cleavage induced by non-target molecule binding.

[0020] In one example, the extracellular region contains an extracellular region derived from Jagged2, EphrinB2, APLP1, APLP2, APP, CD44, CSF1R, CXCL16, CX3CL1, Delta1, E-cadherin, EphB2, EphrinB1, Growth hormone receptor, HLA-A2, IFNaR2, IL1R2, L1, LRP, LRP2, LRP6, N-cadherin, Nectin1α, NRADD, p75-NTR, Pcdhα4, Pcdhγ-C3, PTPκ, PTP-LAR, SorCS1b, SorLA, Sortilin, ApoER2, PKHD1, ErbB4, IFNaR2, VEGF-R1, or VLDLR, or a combination thereof, or a fragment or combination thereof of the extracellular region of any of the foregoing proteins, or a variant or combination thereof of the extracellular region of any of the foregoing proteins, or a truncated structure or combination thereof of the extracellular region of any of the foregoing proteins.

[0021] In one example, the extracellular region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence homology to any one of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 10, or 65.

[0022] The second aspect of the present application provides a chimeric polypeptide, comprising:

[0023] a) a binding domain capable of specifically binding to a target molecule;

[0024] b) a receptor regulatory domain comprising one or more cleavage sites,

[0025] The receptor regulatory domain comprises an extracellular region and a transmembrane region, wherein the extracellular region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence homology to any one of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 10, or 65, and the transmembrane region comprises one or more target molecule binding inducible cleavage sites A; and

[0026] c) intracellular domain,

[0027] The binding of the binding domain to the target molecule can induce the cleavage of the receptor regulatory domain, thereby releasing the intracellular domain.

[0028] In one example, the transmembrane region further comprises a stop transfer sequence (STS).

[0029] In one example, the transmembrane region of the receptor regulatory domain comprises a γ-secretase cleavage site.

[0030] In one example, the transmembrane region of the receptor regulatory domain comprises a Notch transmembrane region.

[0031] In one embodiment, the STS comprises an amino acid sequence having at least 80% sequence homology to any one of SEQ ID NOs: 23, 24, 25, 26, and 27.

[0032] In one example, the transmembrane region of the receptor regulatory domain comprises an amino acid sequence having at least 80% sequence homology to any one of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22.

[0033] In one example, the binding domain comprises an antigen binding domain capable of binding to a target molecule on the surface of a target cell, or the binding domain comprises a ligand portion capable of binding to a receptor.

[0034] In one embodiment, the target molecule is selected from the group consisting of: differentiation marker clusters, cell surface receptors, adhesion proteins, integrins, mucins, lectins, tumor antigens, and tissue-specific antigens.

[0035] In one embodiment, the target molecule is a tumor antigen or a tissue-specific antigen.

[0036] In one embodiment, the binding domain is selected from the group consisting of: an antibody, a receptor, a ligand of a receptor, a cell adhesion molecule, a non-antibody molecular scaffold, or a combination thereof.

[0037] In one embodiment, the antibody is a single domain antibody, a single chain antibody, a double chain antibody, a three chain antibody, a mini antibody, a F(ab')2 fragment, a F(ab) v fragments, scFv, single domain antibodies (sdAbs), and functional fragments thereof, or combinations thereof.

[0038] In one embodiment, the binding domain specifically binds to a tumor antigen selected from the group consisting of: Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, EGFR, BCMA, CD7, NKG2D-Ligand, CD19, B7H3, ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2), IGLL1, IL 11Ra, IL13Ra2, CD 117, MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, cMet and Axl.

[0039] In one example, the binding domain specifically binds to a tissue-specific marker selected from the group consisting of: brain tissue marker MOG, liver tissue marker ASGR1, and prostate tissue marker PSA.

[0040] In one example, the binding domain comprises an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or homologous to any of the amino acid sequences shown in 43, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 or 83.

[0041] In one example, the intracellular domain comprises a transcription factor, a site-specific nuclease, a recombinase, an inhibitory immune receptor, an activating immune receptor, or a combination thereof.

[0042] In one example, the transcription factor is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16, or a combination thereof.

[0043] In one example, additional proteolytic cleavage sites, signal sequences, detectable tags, tumor-specific cleavage sites, disease-specific cleavage sites, and combinations thereof are also included.

[0044] In one example, the receptor regulatory domain comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence homology to any one of the amino acid sequences shown in the extracellular region of SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 65, and the transmembrane region of SEQ ID No: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, when sequentially connected.

[0045] In one example, the chimeric polypeptide comprises an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to any of the amino acid sequences of SEQ ID No: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 65, the transmembrane region of SEQ ID No: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, and the intracellular region of SEQ ID No: 28, when sequentially linked.

[0046] In one example, the chimeric polypeptide comprises an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to any one of the amino acid sequences set forth in SEQ ID NO:44, 45, 46, 47, 48 or 49.

[0047] In one embodiment, the target cell is a pathogen.

[0048] In one example, the target cell is a human cell.

[0049] In one example, the human cell is a tumor cell or a normal tissue cell.

[0050] In a third aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the chimeric polypeptide described in the first, second, and eighteenth aspects, the antibody described in the sixteenth aspect, or the immunoconjugate described in the seventeenth aspect.

[0051] In one example, the nucleic acid molecule is constructed in an expression cassette or expression vector.

[0052] In one example, the expression vector includes a viral vector or a transposon vector.

[0053] In one example, the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

[0054] The fourth aspect of the present application provides an engineered cell comprising the chimeric polypeptides described in the first, second, and eighteenth aspects, the antibody described in the sixteenth aspect, the immunoconjugate described in the seventeenth aspect, and / or the nucleic acid molecule described in the third aspect.

[0055] In one example, the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

[0056] In one example, the engineered cells are B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, or a combination thereof; preferably, the engineered cells are autologous or allogeneic cells.

[0057] In one example, the chimeric polypeptide further comprises an expression cassette encoding an exogenous gene operably linked to the intracellular domain of the chimeric polypeptide, wherein the intracellular domain of the chimeric polypeptide regulates the expression of the exogenous gene.

[0058] In one example, the exogenous gene is expressed under the control of a promoter regulated by GAL-4, tetR, ZFHD1, HNF1A or HAP1.

[0059] In one example, the exogenous gene expression product is selected from: non-coding RNA, cytokine, cytotoxin, chemokine, immunomodulator, pro-apoptotic factor, anti-apoptotic factor, hormone, differentiation factor, dedifferentiation factor, recombinant TCR, CAR, reporter gene or a combination thereof.

[0060] In one example, the binding domain of the chimeric polypeptide specifically binds to a first target molecule, the exogenous gene expression product is a CAR, and the CAR specifically binds to a second target molecule different from the first target molecule, wherein the first target molecule and the second target molecule can be respectively selected from: Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, EGFR, BCMA, CD7, NKG2D-Ligand, MOG, CD19, B7H3, , ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2(ERBB2), IGLL1, IL llRa, IL13Ra2, CD 117, MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, cMet and Axl.

[0061] In one example, the first target molecule and the second target molecule are any one selected from the following combinations: ASGR1 and GPC3, EGFRvⅢ and IL13Ra2, EGFRvⅢ and B7H3, Mesothelin and Claudin18.2, Claudin18.2 and Mesothelin, FAP and Claudin18.2, CLL1 and NKG2D, CD123 and NKG2D, CLL1 and NKG2D ligand, CD123 and NKG2D ligand, MOG and B7H3.

[0062] In one example, the chimeric polypeptide and CAR respectively include the sequences shown in SEQ ID NOs: 46 and 50; or the sequences shown in SEQ ID NOs: 47 and 50; or the sequences shown in SEQ ID NOs: 48 and 51; or the sequences shown in SEQ ID NOs: 48 and 52; or the sequences shown in SEQ ID NOs: 48 and 53; or the sequences shown in SEQ ID NOs: 48 and 54; or the sequences shown in SEQ ID NOs: 49 and 51; or the sequences shown in SEQ ID NOs: 49 and 52; or the sequences shown in SEQ ID NOs: 49 and 53; or the sequences shown in SEQ ID NOs: 49 and 54.

[0063] In one example, the cytokine is IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, CCL21, or a combination thereof.

[0064] In one example, the engineered cell comprises a nucleic acid sequence having at least 80% sequence homology to any one of SEQ ID NOs: 55, 56, 57, 58, or 59, or an amino acid sequence translated therefrom.

[0065] In one embodiment, the engineered cells further express an exogenous receptor targeting NK cells.

[0066] In one example, the exogenous receptor targets an NK cell marker selected from: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, 15KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCR), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specifically expressed antigens, such as CD38, CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, TIGIT, CS1, NKG2D ligand.

[0067] In one example, the exogenous receptor comprises a CAR or a recombinant TCR.

[0068] In one example, the exogenous receptor includes NKG2A-CAR, CD94-CAR, CS1-CAR, TIGIT-CAR, NKG2D-CAR that recognizes an NKG2D ligand, or a combination thereof.

[0069] The fifth aspect of the present application provides a composition comprising the engineered cells described in the fourth aspect, wherein the composition further comprises another engineered cell targeting an NK cell marker.

[0070] In one example, the other engineered cell targets an NK cell marker selected from: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, 15KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCR), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specifically expressed antigens, such as CD38, CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, TIGIT, CS1, NKG2D ligand.

[0071] In one example, the other cell expresses NKG2A-CAR, CD94-CAR, CS1-CAR, TIGIT-CAR, NKG2D-CAR that recognizes an NKG2D ligand, or a combination thereof.

[0072] In one example, it is characterized in that the engineered cell or the another engineered cell is independently selected from: immune cells, neurons, epithelial cells, endothelial cells or stem cells.

[0073] In one example, the immune cell is selected from: B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells or a combination thereof.

[0074] In one example, the T cells are selected from: allogeneic T cells or autologous T cells.

[0075] In one example, the engineered cell and / or another engineered cell is an engineered cell and / or another engineered cell comprising any one, two, three, or four of the following a)-d):

[0076] a) Low or no expression of endogenous HLA-I molecules;

[0077] b) low or no expression of endogenous TCR molecules;

[0078] c) low or no expression of endogenous HLA-II molecules; and / or

[0079] d) Endogenous NKG2A molecule is lowly expressed or absent.

[0080] In one example, the engineered cell and / or another engineered cell is an engineered cell and / or another engineered cell comprising any one, two, three, or four of the following a) to d):

[0081] a) Low or absent expression of endogenous HLA-I molecules, including knockout of genes encoding HLA-I proteins;

[0082] b) Low or no expression of endogenous TCR molecules, including knockout of genes encoding TCR proteins;

[0083] c) low or absent expression of endogenous HLA-II molecules, including knockout of genes encoding HLA-II proteins; and / or

[0084] d) Low or no expression of endogenous NKG2A molecules, including knockout of the gene encoding NKG2A protein.

[0085] In one example, the engineered cell and / or another engineered cell is an engineered cell and / or another engineered cell comprising any one of a) to e):

[0086] a) Knockout of endogenous B2M using CRISPR technology;

[0087] b) Knockout of endogenous B2M / TCR using CRISPR technology;

[0088] c) Knockout of endogenous B2M / TCR / CIITA using CRISPR technology;

[0089] d) Knockout of endogenous B2M / TCR / NKG2A using CRISPR technology;

[0090] e) Knockout of endogenous B2M / TCR / FAS using CRISPR technology; or

[0091] f) Use CRISPR technology to knock out endogenous B2M / TCR / CIITA / NKG2A.

[0092] The sixth aspect of the present application provides a pharmaceutical composition comprising: a pharmaceutically acceptable carrier; and also comprising the nucleic acid molecule described in the third aspect and / or the engineered cell described in the fourth aspect and / or the composition described in the fifth aspect and / or the biomaterial described in the nineteenth aspect.

[0093] A seventh aspect of the present application provides a method for regulating the activity of engineered cells, comprising:

[0094] a) providing the engineered cell according to the fourth aspect; and

[0095] b) contacting the engineered cell with a target molecule, wherein binding of the target molecule to the binding domain of the chimeric polypeptide on the engineered cell induces cleavage of the proteolytic cleavage site of the chimeric polypeptide and releases the transcription factor in the intracellular domain of the chimeric polypeptide, thereby regulating the activity of the engineered cell.

[0096] In one example, the engineered cell activity is selected from any one or a combination of the following: cell proliferation, cell apoptosis, non-apoptotic cell death, cell differentiation, cell dedifferentiation, cell migration, cell adhesion and / or cytolytic activity.

[0097] In one example, the transcription factor-regulated exogenous gene expression is selected from the group consisting of: chemokines, chemokine receptors, chimeric antigen receptors, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, hormones, metabolic enzymes, pathogen-derived proteins, proliferation inducers, receptors, RNA-guided nucleases, site-specific nucleases, T cell receptors, toxins, toxin-derived proteins, transcription regulators, transcription activators, transcription repressors, translation regulators, translation activators, translation repressors, activating immune receptors, antibodies, apoptosis inhibitors, apoptosis inducers, modified T cell receptors, immune activators, immunosuppressants, and inhibitory immune receptors.

[0098] In one example, the released transcription factor regulates differentiation of the engineered cell, which is an immune cell, a stem cell, a progenitor cell, or a precursor cell.

[0099] In one embodiment, it is a method of treating a tumor.

[0100] In an eighth aspect, the present application provides a method for activating engineered cells, the method comprising:

[0101] The engineered cell of the fourth aspect is contacted with a target molecule, wherein the binding domain comprising the chimeric polypeptide comprises an antibody that specifically binds to a first target molecule, and wherein the contact causes the chimeric polypeptide to release the transcription factor activator protein in the intracellular domain of the chimeric polypeptide to regulate the expression of CAR and / or recombinant TCR in the engineered cell, wherein the CAR and / or recombinant TCR specifically binds to a second target molecule different from the first target molecule and activates the engineered cell.

[0102] In one example, the first target molecule and the second target molecule are different tumor antigens; or the first target molecule is a tissue-specific molecule and the second target molecule is a tumor antigen.

[0103] The ninth aspect of the present application provides a method for inhibiting the activity of target cells in a subject, characterized in that it comprises: administering to the subject a therapeutically effective amount of the engineered cells described in the fourth aspect, wherein the engineered cells inhibit the activity of target cells in the subject.

[0104] In one example, the target cell is a tumor cell.

[0105] In one example, the target cell is an acute myeloma leukemia cell, an anaplastic lymphoma cell, an astrocytoma cell, a B cell cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a hepatocellular carcinoma cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T cell lymphoma cell, a renal cancer cell, a sarcoma cell, a gastric cancer cell, a liver cancer cell, a mesothelioma cell, or a sarcoma cell.

[0106] In one example, the target cell expresses low levels of the target molecule, and the binding domain of the chimeric polypeptide specifically binds to the target molecule.

[0107] In one example, the engineered cell further comprises a CAR, a recombinant TCR, an exogenous cytokine and / or a therapeutic monoclonal antibody that is triggered to undergo transcriptional activation by the chimeric polypeptide binding to a target molecule.

[0108] In one example, the target molecules include a first target molecule and a second target molecule, the chimeric polypeptide specifically binds to the first target molecule, and the CAR and / or recombinant TCR specifically binds to the second target molecule; the first target molecule and the second target molecule in the target cell express heterogeneous tumor antigens, or the first target molecule is a tissue-specific molecule and the second target molecule is a tumor antigen.

[0109] In one example, the positive rate of the first target molecule in the target cells is lower than the positive rate of the second target molecule in the target cells.

[0110] In one embodiment, it can further improve the anti-tumor specificity of the engineered cells.

[0111] In a tenth aspect, the present application provides a system, a kit, or a reagent kit for regulating cell activity, inhibiting target cells, or treating a health condition of a subject in need thereof, comprising: one or more of the following:

[0112] a) the chimeric polypeptides of aspects 1, 2, and 18;

[0113] b) the nucleic acid molecule according to the third aspect;

[0114] c) the cell according to the fourth aspect; and

[0115] d) the composition according to the fifth aspect;

[0116] e) the pharmaceutical composition according to the sixth aspect;

[0117] f) the antibody according to the sixteenth aspect;

[0118] g) the immunoconjugate according to the seventeenth aspect;

[0119] h) The biomaterial according to the nineteenth aspect.

[0120] In an eleventh aspect, the present application provides one or more of the following uses for treating tumors:

[0121] a) the chimeric polypeptides described in aspects 1 and 2;

[0122] b) the nucleic acid molecule according to the third aspect;

[0123] c) the cell according to the fourth aspect; and

[0124] d) the composition according to the fifth aspect;

[0125] e) the pharmaceutical composition according to the sixth aspect;

[0126] f) the antibody according to the sixteenth aspect;

[0127] g) the immunoconjugate according to the seventeenth aspect;

[0128] h) The biomaterial according to the nineteenth aspect.

[0129] In one embodiment, the tumor is a solid tumor, a hematological tumor, a soft tissue tumor, or a metastatic lesion.

[0130] In aspect 12, the present application provides the chimeric polypeptide described in aspects 1 and 2; the nucleic acid molecule described in aspect 3; the cells described in aspects 4 and 13; the composition described in aspect 5; and the pharmaceutical composition described in aspect 6, for use in the manufacture of a drug for treating a health condition.

[0131] In a thirteenth aspect, the present application provides an engineered cell that recognizes allogeneic immune cells, wherein the engineered cell expresses a molecule that recognizes an NKG2D ligand, and the expression of the molecule that recognizes an NKG2D ligand is regulatable.

[0132] In one embodiment, the molecule that recognizes an NKG2D ligand is a chimeric receptor.

[0133] In one example, the chimeric receptor comprises the full-length NKG2D polypeptide or a fragment thereof, or the chimeric receptor comprises an antibody or a fragment thereof that recognizes an NKG2D ligand; preferably, the NKG2D fragment comprises the extracellular region of NKG2D.

[0134] In one example, the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

[0135] In one example, the engineered cell expresses a second chimeric receptor that does not recognize the NKG2D ligand, and after the second chimeric receptor recognizes the target molecule, it induces the expression of the molecule that recognizes the NKG2D ligand.

[0136] In one example, the binding domain of the second chimeric receptor binds to the target molecule, thereby inducing cleavage of the second chimeric receptor and releasing transcription factors to regulate the expression of the molecule that recognizes the NKG2D ligand;

[0137] Preferably, the second chimeric receptor comprises the chimeric polypeptide described in the first aspect or the second aspect;

[0138] Preferably, the second chimeric receptor comprises a synNotch polypeptide;

[0139] Preferably, the second chimeric receptor comprises the sequence shown in SEQ ID NO: 44, 45, 46, 47, 48, 49, 60, 61, 62, 63, 64, 65, 66 or 67;

[0140] Preferably, the transcription factor is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16 or a combination thereof.

[0141] In one example, the target molecule includes a tumor antigen and / or a pathogen antigen.

[0142] In one example, the engineered cells are selected from the group consisting of autologous or allogeneic T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune cells; preferably, the engineered cells are autologous or allogeneic T cells.

[0143] In one example, the engineered cells further express a third chimeric receptor that recognizes a tumor antigen or a pathogen antigen.

[0144] In one example, the allogeneic immune cells include NK cells and / or T cells.

[0145] In one example, the engineered cells do not express HLA, or the HLA gene endogenously expressed in the cells is silenced; preferably, the HLA is an HLA-I class gene.

[0146] In one example, the HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M; preferably, the HLA-I gene is B2M.

[0147] In one example, the engineered cells are T cells with HLA-I gene and endogenous TCR gene silenced;

[0148] Preferably, the cells are B2M / TCR gene-silenced and B2M / TCR / FAS gene-silenced T cells; preferably, B2M and / or TRAC are knocked out using CRISPR / Cas9 technology, and B2M / TCR / FAS are knocked out using CRISPR / Cas9 technology.

[0149] In one example, the chimeric receptor, the third chimeric receptor comprises a chimeric antigen receptor (CAR) and / or a recombinant T cell receptor (recombinant TCR).

[0150] In one example, the chimeric receptor, the third chimeric receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain;

[0151] Preferably, the engineered cells transmit signals through the intracellular signaling domain to mediate killing of host immune cells, or mediate resistance to killing of host immune cells; preferably, the extracellular domain is combined with the transmembrane domain through a hinge.

[0152] In one embodiment, the chimeric receptor comprises:

[0153] (i) NKG2D polypeptide or fragment thereof, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28 and the intracellular domain of CD3ζ; and / or

[0154] (ii) NKG2D polypeptide or fragment thereof, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD137 and the intracellular domain of CD3ζ; and / or

[0155] (iii) NKG2D polypeptide or fragment thereof, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and the intracellular domain of CD3ζ; and / or

[0156] (iv) NKG2D polypeptide or a fragment thereof, CD28 or the transmembrane region of CD8 and the intracellular domain of CD3ζ.

[0157] In one example, the engineered cells can enhance the survival and proliferation of a second T cell and / or a second CAR-T cell carrying a targeted tumor antigen that is previously, simultaneously, or subsequently introduced into the subject; as well as the killing of tumor cells by the second T cell and / or the second CAR-T cell.

[0158] In a fourteenth aspect, the present application provides an engineered cell expressing a molecule that recognizes an NKG2D ligand, which is used for preparing a drug for eliminating allogeneic immune cells.

[0159] In one embodiment, the molecule that recognizes an NKG2D ligand is a chimeric receptor.

[0160] In one example, the chimeric receptor comprises a full-length NKG2D polypeptide or a fragment thereof, or the chimeric receptor comprises an antibody or a fragment thereof that recognizes an NKG2D ligand.

[0161] In one example, the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

[0162] In one embodiment, the expression of the molecule that recognizes the NKG2D ligand is regulatable.

[0163] In one example, the allogeneic immune cells include NK cells and / or T cells.

[0164] The fifteenth aspect of the present application provides a method for preventing or regulating transplant immune rejection, comprising administering the engineered cells described in the thirteenth aspect, or administering the engineered cells having the use described in the fourteenth aspect.

[0165] In one example, the method is used to kill allogeneic immune cells; preferably, the allogeneic immune cells include NK cells and / or T cells.

[0166] In a sixteenth aspect, the present application provides a fully human antibody that recognizes MOG, wherein the antibody is selected from:

[0167] (1) an antibody comprising a light chain variable region comprising LCDR1 as shown in SEQ ID NO: 84, and / or comprising LCDR2 as shown in SEQ ID NO: 85, and / or comprising LCDR3 as shown in any one of SEQ ID NOs: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98;

[0168] (2) an antibody comprising a heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO: 99 or 100, and / or a HCDR2 as set forth in SEQ ID NO: 101 or 102, and / or a HCDR3 as set forth in any one of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115;

[0169] (3) an antibody comprising (1) a light chain variable region of the antibody and (2) a heavy chain variable region of the antibody;

[0170] (4) An antibody, which is a variant of the antibody according to any one of (1) to (3), and has the same or similar activity as the antibody according to any one of (1) to (3).

[0171] In one embodiment, the antibody is selected from:

[0172] (1) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 86, 99, 101, and 103, respectively; or

[0173] (2) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 87, 99, 101, and 104, respectively; or

[0174] (3) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 88, 99, 101, and 105, respectively; or

[0175] (4) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 89, 99, 101, and 106, respectively; or

[0176] (5) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 90, 99, 101, and 107, respectively; or

[0177] (6) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 91, 100, 102, and 108, respectively; or

[0178] (7) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 92, 100, 102, and 109, respectively; or

[0179] (8) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 93, 100, 102, and 110, respectively; or

[0180] (9) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 94, 100, 102, and 111, respectively; or

[0181] (10) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 95, 100, 102, and 112, respectively; or

[0182] (11) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 96, 100, 102, and 113, respectively; or

[0183] (12) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 97, 100, 102, and 114, respectively; or

[0184] (13) an antibody comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 98, 100, 102, and 115, respectively;

[0185] (14) An antibody, which is a variant of the antibody according to any one of (1) to (13), and has the same or similar activity as the antibody according to any one of (1) to (13).

[0186] In one embodiment, the antibody is selected from:

[0187] (1) An antibody comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, or 128, or a nucleic acid sequence encoding the amino acid sequence, or a variant of any of the foregoing sequences, or an amino acid sequence having at least 80% identity with any of the foregoing sequences, or a nucleic acid sequence encoding the amino acid sequence;

[0188] (2) an antibody comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or 141, or a nucleic acid sequence encoding the amino acid sequence, or a variant of any of the foregoing sequences, or an amino acid sequence having at least 80% identity with any of the foregoing sequences, or a nucleic acid sequence encoding the amino acid sequence;

[0189] (3) An antibody comprising (1) the light chain variable region of the antibody and (2) the heavy chain variable region of the antibody.

[0190] In one embodiment, the antibody is selected from:

[0191] (1) an antibody comprising a heavy chain variable region represented by SEQ ID NO: 129, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NOs: 84, 85, and 86, respectively; or an antibody comprising a light chain variable region represented by SEQ ID NO: 116, and HCDR1, HCDR2, and HCDR3 represented by SEQ ID NOs: 99, 101, and 103, respectively; or

[0192] (2) an antibody comprising a heavy chain variable region as set forth in SEQ ID NO: 130, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 84, 85, and 87, respectively; or an antibody comprising a light chain variable region as set forth in SEQ ID NO: 117, and HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 99, 101, and 104, respectively; or

[0193] (3) an antibody comprising a heavy chain variable region represented by SEQ ID NO: 131, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NOs: 84, 85, and 88, respectively; or an antibody comprising a light chain variable region represented by SEQ ID NO: 118, and HCDR1, HCDR2, and HCDR3 represented by SEQ ID NOs: 99, 101, and 105, respectively; or

[0194] (4) an antibody comprising a heavy chain variable region represented by SEQ ID NO: 132, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NOs: 84, 85, and 89, respectively; or an antibody comprising a light chain variable region represented by SEQ ID NO: 119, and HCDR1, HCDR2, and HCDR3 represented by SEQ ID NOs: 99, 101, and 106, respectively; or

[0195] (5) an antibody comprising a heavy chain variable region as shown in SEQ ID NO: 133, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 84, 85, and 90, respectively; or an antibody comprising a light chain variable region as shown in SEQ ID NO: 120, and HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 99, 101, and 107, respectively; or

[0196] (6) an antibody comprising a heavy chain variable region represented by SEQ ID NO: 134, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NOs: 84, 85, and 91, respectively; or an antibody comprising a light chain variable region represented by SEQ ID NO: 121, and HCDR1, HCDR2, and HCDR3 represented by SEQ ID NOs: 100, 102, and 108, respectively; or

[0197] (7) an antibody comprising a heavy chain variable region represented by SEQ ID NO: 135, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NOs: 84, 85, and 92, respectively; or an antibody comprising a light chain variable region represented by SEQ ID NO: 122, and HCDR1, HCDR2, and HCDR3 represented by SEQ ID NOs: 100, 102, and 109, respectively; or

[0198] (8) an antibody comprising a heavy chain variable region represented by SEQ ID NO: 136, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NOs: 84, 85, and 93, respectively; or an antibody comprising a light chain variable region represented by SEQ ID NO: 123, and HCDR1, HCDR2, and HCDR3 represented by SEQ ID NOs: 100, 102, and 110, respectively; or

[0199] (9) an antibody comprising a heavy chain variable region represented by SEQ ID NO: 137, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NOs: 84, 85, and 94, respectively; or an antibody comprising a light chain variable region represented by SEQ ID NO: 124, and HCDR1, HCDR2, and HCDR3 represented by SEQ ID NOs: 100, 102, and 111, respectively; or

[0200] (10) an antibody comprising a heavy chain variable region represented by SEQ ID NO: 138, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NOs: 84, 85, and 95, respectively; or an antibody comprising a light chain variable region represented by SEQ ID NO: 125, and HCDR1, HCDR2, and HCDR3 represented by SEQ ID NOs: 100, 102, and 112, respectively; or

[0201] (11) an antibody comprising a heavy chain variable region represented by SEQ ID NO: 139, and LCDR1, LCDR2, and LCDR3 represented by SEQ ID NOs: 84, 85, and 96, respectively; or an antibody comprising a light chain variable region represented by SEQ ID NO: 126, and HCDR1, HCDR2, and HCDR3 represented by SEQ ID NOs: 100, 102, and 113, respectively; or

[0202] (12) an antibody comprising a heavy chain variable region as shown in SEQ ID NO: 140, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 84, 85, and 97, respectively; or an antibody comprising a light chain variable region as shown in SEQ ID NO: 127, and HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 100, 102, and 114, respectively; or

[0203] (13) An antibody comprising a heavy chain variable region as shown in SEQ ID NO: 141, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 84, 85, and 98, respectively; or an antibody comprising a light chain variable region as shown in SEQ ID NO: 128, and HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 100, 102, and 115, respectively.

[0204] In one embodiment, the antibody is selected from:

[0205] (1) An antibody, wherein the light chain variable region and heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 116 and 129, respectively, or amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or nucleic acid sequences encoding the amino acid sequences;

[0206] (2) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 117 and 130, respectively, or amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences, or nucleic acid sequences encoding the aforementioned amino acid sequences;

[0207] (3) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences set forth in SEQ ID NOs: 118 and 131, respectively, or amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences, or nucleic acid sequences encoding the aforementioned amino acid sequences;

[0208] (4) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences set forth in SEQ ID NOs: 119 and 132, respectively, or amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences, or nucleic acid sequences encoding the aforementioned amino acid sequences;

[0209] (5) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 120 and 133, respectively, or amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences, or nucleic acid sequences encoding the aforementioned amino acid sequences;

[0210] (6) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences set forth in SEQ ID NOs: 121 and 134, respectively, or amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences, or nucleic acid sequences encoding the aforementioned amino acid sequences;

[0211] (7) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 122 and 135, respectively, or amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or nucleic acid sequences encoding the amino acid sequences;

[0212] (8) An antibody, wherein the light chain variable region and heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 123 and 136, respectively, or amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences, or nucleic acid sequences encoding the aforementioned amino acid sequences;

[0213] (9) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 124 and 137, respectively, or amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences, or nucleic acid sequences encoding the aforementioned amino acid sequences;

[0214] (10) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 125 and 138, respectively, or amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or nucleic acid sequences encoding the amino acid sequences;

[0215] (11) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 126 and 139, respectively, or amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or nucleic acid sequences encoding the amino acid sequences;

[0216] (12) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 127 and 140, respectively, or amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or nucleic acid sequences encoding the amino acid sequences;

[0217] (13) an antibody, wherein the light chain variable region and the heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NOs: 128 and 141, respectively, or amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or nucleic acid sequences encoding the amino acid sequences;

[0218] (14) An antibody, which is a variant of the antibody described in any one of (1) to (13), and has the same or similar activity as the antibody described in any one of (1) to (13).

[0219] In one example, the scFv of the antibody has an amino acid sequence as shown in SEQ ID NO: 43, 68, 77, 81, 82 or 83, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or a nucleic acid sequence encoding the amino acid sequence.

[0220] In one example, the antibody is a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, dAb fragment, multifunctional antibody, scFv-Fc antibody or IgG4 antibody.

[0221] In one example, the antibody binds to human or murine MOG; and / or, the antibody binds to cells expressing human or murine MOG.

[0222] The seventeenth aspect of the present application provides an immunoconjugate, which comprises: the antibody described in the sixteenth aspect, and a functional molecule connected thereto.

[0223] The eighteenth aspect of the present application provides a chimeric polypeptide,

[0224] a) the binding domain of the chimeric polypeptide comprises the antibody of aspect 16;

[0225] b) a receptor regulatory domain comprising one or more cleavage sites, the receptor regulatory domain comprising an extracellular region and a transmembrane region; and

[0226] c) intracellular domain,

[0227] The binding of the binding domain to MOG can induce the cleavage of the receptor regulatory domain, thereby releasing the intracellular domain.

[0228] A nineteenth aspect of the present application provides a biomaterial, which is any one of the following:

[0229] 1) A nucleic acid molecule encoding the chimeric polypeptides of aspects 1, 2, and 18, the molecule recognizing the NKG2D ligand of aspect 13, the antibody of aspect 16, or the immunoconjugate of aspect 17;

[0230] 2) comprising the vector or expression vector described in 1); or

[0231] 3) A virus comprising the nucleic acid molecule described in 1) or 2).

[0232] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0233] Incorporation by reference

[0234] All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a terminology herein and that of an incorporated reference, the terminology herein controls. BRIEF DESCRIPTION OF THE DRAWINGS

[0235] FIG1 shows that a chimeric polypeptide targeting GPC3, comprising the full-length EphrinB2 extracellular region, or a truncated version thereof, or a modified version thereof, regulates gene expression.

[0236] FIG2 shows that chimeric polypeptides targeting MOG, comprising the extracellular domain of full-length EphrinB2 or its truncated form, regulate gene expression.

[0237] Figure 3 shows that after co-incubation with MOG-positive cells, B7H3-CAR-T cells regulated by MOG-synE induced the expression of B7H3-CAR (Figure 3A), effectively killed glioma cells (Figure 3B), and released the cytokine IL2 (Figure 3C).

[0238] Figure 4A shows the expression level of CD123 in AML cells; Figure 4B shows the transcriptional activity triggered by the binding of a chimeric polypeptide targeting CD123.

[0239] FIG5 shows the expression levels of NKG2D ligands in tumor cells.

[0240] Figure 6 shows that NKG2D-CAR-T cells regulated by the chimeric polypeptide CD123-synE or CD123-synE-del3 kill NKG2D ligand-positive tumor cells.

[0241] Figure 7A detected the expression of NKG2D ligands on tumor cells; Figure 7B showed that constitutive NKG2D-CAR-T cells can effectively kill NKG2D ligand-positive tumor cells, while regulatory NKG2D-CAR-T cells have almost no killing effect.

[0242] Figure 8 shows that placing CD3Z-NKG2D-CAR under the regulation of CD123-synE or CD123-synE-del3 can improve the in vivo survival ability and anti-tumor effect of CAR-T cells.

[0243] FIG9 shows the expression of NKG2D-Ligand in resting NK cells from different donors detected by flow cytometry.

[0244] Figure 10 shows that after NK cells were co-incubated with tumor cells or NK cells were co-incubated with UCAR-T for 24 hours, the expression of NKG2D-Ligand of NK cells was upregulated.

[0245] FIG11 shows the killing effect of regulatory or constitutive NKG2D-UCAR T cells on NK cells.

[0246] Figure 12 shows that when NK cells are present, regulatory NKG2D-UCAR-T cells can also kill tumor cells, and the killing effect becomes stronger as the co-incubation time increases.

[0247] Figure 13 shows that when NK cells are present, regulatory NKG2D-UCAR-T cells significantly kill tumor cells. After co-incubation with tumor cells, regulatory NKG2D-UCAR-T cells have a strong killing effect on NK cells, and the longer the co-incubation time, the stronger the killing effect; they can also partially resist the killing effect of NK cells.

[0248] Figure 14 shows that when NK cells are present, regulatory NKG2D-UCAR-T cells can effectively inhibit the growth of in situ tumors in mice.

[0249] Figure 15 shows the binding of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, and A13 (Fab form) antibodies to 293T-hMOG cells and 293T-mMOG cells.

[0250] FIG16 shows SDS-PAGE images of eukaryotically expressed antibodies A2, A6, A8, A9, A10, and A13 (scFv-Fc format).

[0251] FIG17 shows the EC50 of antibodies A2, A6, A8, A9, A10, and A13 (scFv-Fc format) binding to MOG detected by ELISA.

[0252] FIG18 shows the EC50 of FACs detecting the binding of antibodies A2, A6, A8, A9, A10, and A13 (scFv-Fc format) to 293T-hMOG and 293T-mMOG. DETAILED DESCRIPTION

[0253] The following description and examples illustrate embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and therefore can vary. Those skilled in the art will recognize that there are many variations and modifications in the present disclosure, and these variations and modifications are within the scope thereof. Unless otherwise indicated, any embodiment may be combined with any other embodiment.

[0254] As used herein, unless otherwise indicated, some of the invention embodiments herein take into account numerical ranges. Various aspects of the application can be presented in range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the application. Therefore, it should be considered that the description of the range has been specifically disclosed as clearly written out all possible subranges and single numerical values ​​within the range. For example, it should be considered that the description of a range such as 1 to 6 has specifically disclosed subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and single digits within the range, for example, 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the scope, this applies. When there is a range, the range includes the range endpoints.

[0255] the term

[0256] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of this application, wherein suitable methods and materials are described herein. All publications, applications and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail. In addition, unless otherwise specified, the materials, methods and examples are illustrative only and are not intended to be limiting.

[0257] Unless otherwise indicated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art and are fully explained in the literature.

[0258] The term "NKG2D ligand (NKG2DL, NKG2D-L)" or "NKG2D-ligand" refers to a ligand that binds to NKG2D, including 8 polypeptides: MICA (Gene ID: 100507436), MICB (Gene ID: 4277), ULBP-1 (RAET1I, Gene ID: 80329), ULBP-2 (RAET1H, Gene ID: 80328), ULBP-3 (RAET1N, Gene ID: 79465), ULBP-4 (RAET1E, Gene ID: 135250), ULBP-5 (RAET1G, Gene ID: 353091) and ULBP-6 (RAET1L, Gene ID: 154064). The terms "anti-NKG2D ligand antibody", "NKG2D ligand binding protein", "NKG2D ligand antibody", "NKG2D ligand recognizing antibody" refer to a polypeptide that binds to an NKG2D ligand with sufficient affinity.

[0259] The term "NKG2D," also known as KLRK1 (Gene ID: 22914), is a C-type lectin family receptor expressed on the surface of NK cells. It is also expressed on NKT cells, activated CD8+ T cells, CD4+ T cells, and γδ+ T cells, and is an important activating receptor for NK cells. In one embodiment, the NKG2D polypeptide comprises the sequence set forth in SEQ ID NO: 42.

[0260] The term "EphrinB2" refers to erythropoietin-producing hepatoma interactor B2 (Gene ID: 1948), a cell surface ligand for an Eph receptor. Eph receptors are a class of tyrosine kinases that regulate cell migration, repulsion, and adhesion in neural, vascular, and epithelial development. In one embodiment, the EphrinB2 polypeptide comprises the sequence set forth in SEQ ID NO: 1. In another embodiment, the EphrinB2 polypeptide comprises the sequence set forth in SEQ ID NO: 3, 4, 5, or 6.

[0261] Jagged2, also known as JAG2, HJ2; SER2; LGMDR27, Gene ID: 3714, encodes a protein that is one of several ligands that activate Notch and related receptors. Two transcript variants encoding different isoforms of this gene have been identified.

[0262] The term "APLP2," also known as "APPH; APPL2; CDEBP; APLP-2," refers to amyloid precursor protein 2 (APLP2), a member of the APP (amyloid precursor protein) family that includes APLP1 (Gene ID: 333) and APLP2 (Gene ID: 334). This protein is ubiquitously expressed. It works synergistically with APP to mediate neuromuscular transmission, spatial learning, and synaptic plasticity. This protein has been implicated in the pathogenesis of Alzheimer's disease. Multiple alternatively spliced ​​transcript variants encoding different isoforms have been identified.

[0263] The term "antibody" is used in the broadest sense herein and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), domain antibodies, and antibody fragments thereof that can specifically bind to an antigen or antigenic determinant, as long as they exhibit the desired antigen-binding activity. The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, (i) Fab fragments consisting of the VL, VH, CL, and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of the VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region; (v) F(ab')2 fragments, bivalent fragments comprising two linked Fab fragments; (vi) single-chain Fv molecule antigen-binding sites; (vii) bispecific single-chain Fv dimers; (viii) "dibodies" or "tribodies," multivalent or multispecific fragments constructed by genetic fusion; and (ix) scFv genetically fused to the same or different antibodies.

[0264] The term "scFv" refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., connected by a synthetic linker, such as a short flexible polypeptide linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used herein, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL. The antigen binding function of an antibody can be performed by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen binding units." The term "antigen binding unit" also includes any molecular structure containing a polypeptide chain with a specific shape that is suitable for and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.

[0265] The term "variable region or variable domain" refers to the domain of an antibody heavy chain or light chain that participates in antibody antigen binding. The heavy chain variable domain (VH) and light chain variable domain (VL) of a natural antibody typically have similar structures, wherein each domain comprises four conserved FRs and three CDRs. A single VH or VL domain can confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to the antigen.

[0266] The term "hypervariable region" or "complementarity determining region" or "CDR" refers to each region of an antibody variable domain whose sequence is hypervariable, and / or forms structurally defined loops ("hypervariable loops"), and / or contains residues that contact the antigen ("antigen contacts"). Typically, an antibody comprises six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3).

[0267] The term "Fc region" or "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0268] "Framework (FR)" refers to variable domain residues other than the hypervariable region (CDR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. In VH (or VL), CDR and FR sequences typically appear in the following order:

[0269] FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0270] Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0271] The term "natural antibody" refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by a disulfide bond. From N-terminal to C-terminal, each heavy chain has a variable region (VH), which is also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), which is also referred to as a variable light chain domain or a light chain variable domain, followed by a light chain constant (CL) domain. The light chain of an antibody can be assigned to one of two types based on the amino acid sequence of its constant domain, referred to as κ (κ) and λ (λ).

[0272] The terms "whole antibody," "full-length antibody," and "intact antibody" are used interchangeably to refer to a full-length antibody having a structure substantially similar to a native antibody structure or having heavy chains containing an Fc region as defined herein or including an intact antigen-binding region.

[0273] The term "single domain antibody (sdAb)" refers to a type of antibody that lacks the antibody light chain and only has the heavy chain variable region. Due to its small molecular weight, it is also called a nanobody.

[0274] The term "single domain antibody" refers to an antibody comprising all or part of the heavy chain variable domain, or all or part of the light chain variable domain. The single domain antibody may be a human single domain antibody.

[0275] The terms "monoclonal antibody" and "single antibody" refer to an antibody obtained from a population of substantially homologous antibodies, that is, the antibody molecules comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or generated during the preparation of the monoclonal antibody preparation, which variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the term "monoclonal" indicates that the antibody is obtained from a population of substantially homologous antibodies and is not to be construed as requiring that the antibody be prepared by any particular method. For example, it can be prepared by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0276] The term "fully human antibody (or fully human antibody)" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or the amino acid sequence of an antibody derived from a non-human source using a human antibody library or other human antibody encoding sequence. The definition of a fully human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues. Fully human antibodies can be generated by phage display technology. Fully human antibodies can be produced by engineered strains and / or engineered cells.

[0277] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antigen binding unit. Antigens can include peptides, proteins, glycoproteins, polysaccharides and lipids, portions thereof and combinations thereof. Antigens can include tumor antigens or pathogen antigens. "Antigen" can also refer to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. The term "antigenic determinant," also known as "epitope" or "epitope" or "antigenic determinant," includes any determinant or region that can be bound by an antibody. An epitope is the region of an antigen to which an antibody targeting the antigen binds, including specific amino acids that are in direct contact with the antibody.

[0278] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear, cyclic, or branched, it may contain modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers, for example, those modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA-mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation to a labeling component. The term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including amino acids with optical isomers, as well as amino acid analogs and peptidomimetics.

[0279] The term "peptide-drug conjugate (PDC)" is mainly composed of three parts: a peptide, a linker, and a cytotoxic payload, which is used for targeted therapy. For example, payloads include doxorubicin, paclitaxel, MTX, and camptothecin.

[0280] The term "conservative modification" or "conservative sequence modification" refers to amino acid modifications that do not significantly affect or alter the desired activity or properties of a peptide containing the amino acid sequence, including amino acid substitutions, insertions, and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. For example, families of amino acid residues with similar side chains have been defined in the art, as shown in Table 1.

[0281] Table 1: Families of amino acid residues with similar side chains

[0282] In some embodiments, the present invention provides the antibody of the present invention.Therefore, the amino acid residues of other identical side chain families can be used to replace one or more amino acid residues in the CDR region of the present application antibody or in the framework region, and the function retained by the changed antibody (variant antibody) can be tested.Non-conservative substitutions need to replace the member of one of these groups with the member of another group.A kind of substitution variant comprises replacing one or more hypervariable region residues of parent antibody (for example, humanized or people's antibody).Generally, the variant of the gained being selected for further study will have the change (for example, improvement) of certain biological properties (for example, the affinity of increase, the immunogenicity of reduction) relative to parent antibody and / or will substantially keep certain biological properties of parent antibody.Exemplary substitution variant is affinity maturation antibody, for example, using affinity maturation technology (such as those described herein) based on phage display conventional preparation.In brief, one or more CDR residues are mutated and variant antibody is displayed on phage and screens specific biological activity (for example, binding affinity).

[0283] In some embodiments, the present invention relates to a CDR region or a VL region of the antibody. The CDR region is modified to modify the CDR region to improve the affinity of the antibody. The CDR region is modified to modify the codon-encoded residues that are mutated at a high frequency during the somatic maturation process, and / or to remove the antigen residues, and the resulting variant VH or VL is tested for binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variant with the desired affinity. Another method of introducing diversity includes a CDR-directed approach in which several CDR residues (e.g., 4-6 residues at the same time) are randomized.

[0284] Substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not significantly reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not significantly reduce binding affinity (e.g., conservative modifications described herein) may be made in the CDRs. Such changes may be outside of the residues that contact the antigen in the CDRs. In the variant VH and VL sequences provided above, each CDR may be unchanged, or contain no more than one, two or three amino acid substitutions.

[0285] The term "transplant rejection" refers to the condition in which, after a transplant of an allogeneic tissue, organ, or cell transplant, the host's immune system recognizes the foreign transplant as a "foreign component," initiating an immunological response that attacks, destroys, and eliminates the transplant. This application provides cells and methods for combating transplant rejection.

[0286] The term "graft" refers to a biological material or preparation derived from an individual other than a host, which is used to be implanted into a host. The graft may be from any animal source, such as a mammalian source, preferably from humans. The graft may be from a host, such as cells from a host that are cultured in vitro or transformed and then implanted into the host. The graft may be from another individual of the same species, such as cells from another person that are cultured in vitro or transformed and then implanted into the host. The graft may be from a xenogeneic individual, such as an organ from another species (such as a mouse, pig, or monkey) that is implanted into a human. Xenotransplantation includes, but is not limited to, vascularized xenotransplantation, partially vascularized xenotransplantation, non-vascularized xenotransplantation, xenodressing, xenobandage, and xenostructures.

[0287] The term "autologous" refers to an organism originating from the same organism. For example, a sample can be removed from a subject (e.g., a cell) at a later time, processed and returned to the subject (e.g., a patient). The autologous process is distinguished from the allogeneic process, in which the donor and the recipient are different subjects. The term "autotransplantation" includes any procedure involving the transplantation, implantation or infusion of cells, tissues or organs into a recipient, wherein the subject and the donor are identical individuals. The transplantation of cells, organs and / or tissues as described herein can be used for autologous transplantation into the mankind. Autologous transplantation includes but is not limited to vascularized autotransplantation, partially vascularized autotransplantation, non-vascularized autotransplantation, autologous dressings, autologous bandages and autologous structures.

[0288] The term "allogeneic transplantation" includes any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a subject, wherein the subject and the donor are different individuals of the same species. Transplantation of cells, organs, and / or tissues described herein can be used for allogeneic transplantation into humans. Allogeneic transplantation includes, but is not limited to, vascularized allografts, partially vascularized allografts, non-vascularized allografts, allografts, allobandages, and allogenic structures.

[0289] The term "cell" refers to a cell of human or non-human origin or of animal origin.

[0290] The term "host" refers to a subject into which a transplant is to be implanted. For example, it can be an individual, such as a human, into whom exogenous cells are implanted.

[0291] The term "subject" refers to any animal, such as a mammal or marsupial. The subject of the present application includes, but is not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.

[0292] The term "immune cell" refers to cells that participate in the immune response and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, etc. For example, the immune cells are T cells, NK cells, and NKT cells. For example, the T cells can be autologous T cells, xenogeneic T cells, or allogeneic T cells. For example, the NK cells can be autologous NK cells or allogeneic NK cells. The term "CIK cells", i.e. cytokine-induced killer cells (CIK), are a new type of immune active cells. CIK has strong proliferation ability, strong cytotoxicity, and certain immune characteristics. Because the cells express two membrane protein molecules, CD3 and CD56, they are also called NK cell-like T lymphocytes (NKT cells), which have the powerful anti-tumor activity of T lymphocytes and the non-MHC restricted tumor killing advantages of NK cells. For example, immune cells are obtained by sorting donor peripheral blood mononuclear cells (PBMC).

[0293] The term "artificially modified cells with immune effector cell function" refers to cells or cell lines that lack immune effector function that have been artificially modified or stimulated to acquire immune cell function. For example, 293T cells have been artificially modified to have immune effector cell function, and stem cells have been induced to differentiate into immune cells in vitro.

[0294] "T cells" described herein can be natural T cells obtained from PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and from infection sites, ascites, pleural effusion, spleen tissue, and tumor tissue. T cells can be a cell population with specific phenotypic characteristics obtained through sorting, etc., or a mixed cell population with different phenotypic characteristics. T cells can be cells comprising at least one T cell subset: memory stem cell-like memory T cells (stem cell-like memory T cells, Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and / or effector memory T cells (Tem). For example, T cells can be T cells of a certain specific subtype, such as or αβT cells, γδT cells. For example, any technique known to those skilled in the art, such as FicollTM separation and / or apheresis, can be used to obtain T cells from blood collected from an individual. For example, PBMC collected by apheresis are then screened to obtain T cells. T cells can be any type of T cells and can be at any developmental stage, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells, such as Th1 and Th2 cells, CD8+ T cells (such as cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc. T cells may be CD8+ T cells or CD4+ T cells. For example, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. For example, cells collected by apheresis can be washed to remove plasma molecules and the cells can be placed in a suitable buffer or culture medium for subsequent processing steps. For example, the T cells can be from a healthy donor, or from an individual diagnosed with cancer. The conventional method for preparing CAR-T in this area is to use PBMC cells collected by apheresis, and then continue to culture the obtained T cells after activation with magnetic beads of anti-CD3 and CD28 antibodies, and obtain CAR-T cells after lentiviral infection.

[0295] The “NK cells” described in this application include primary NK cells or NK cell lines. For example, NK cells are isolated from PBMCs of healthy donors by sorting CD56-positive cells; or NK cells are obtained by removing other types of cells from PBMCs by negative screening. Most of the NK cells isolated from PBMCs of healthy donors are resting NK cells, which do not undergo autonomous amplification. They are activated after being stimulated by target cells such as tumor cells or by IL2, IL15 and / or IL18; activated NK cells proliferate rapidly and have enhanced cell killing function. NKG2D ligand expression is almost undetectable in resting NK cells. After co-incubation with tumor cells, NKG2D ligand expression was detected on the NK cell membrane. After co-incubation with UCAR-T cells (B2M knockout, TCR / B2M knockout, TCR / B2M / FAS knockout), NKG2D ligand expression was detected on the NK cell membrane.

[0296] The terms "activation" and "activation" are used interchangeably and refer to the process by which cells transition from a resting state to an active state. This process may include responses to antigens, migration and / or phenotypic or genetic changes in functional activity state. For example, the term "activation" may refer to the process by which T cells are gradually activated. This activation process is regulated by the first stimulation signal and the co-stimulation signal. The activation of T cells is a dynamic process, and its duration and degree of activation are affected by external stimulation conditions. "T cell activation" or "T cell activation" refers to the state of T cells that are stimulated to induce detectable cell proliferation, cytokine production and / or detectable effector function. Using CD3 / CD28 magnetic beads, in vitro antigen stimulation or in vivo antigen stimulation will affect the degree and duration of T cell activation. For example, the engineered T cells are co-incubated with tumor cells containing specific target antigens or activated after viral infection.

[0297] The term "peripheral blood mononuclear cell (PBMC)" refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, etc. Density-based cell separation methods, for example, can be used to obtain PBMCs by centrifugation of peripheral blood or apheresis samples or leukapheresis samples with or without lysis of red blood cells using a Percoll-Ficoll gradient.

[0298] The term "engineering" or "engineering" refers to the application of the principles and methods of cell biology and molecular biology to change the genetic material in a cell or obtain a cell product at the overall cell level or at the organelle level through certain engineering means. For example, "engineering" refers to one or more changes in a nucleic acid (e.g., a nucleic acid in an organism's genome). "Engineering" can refer to the alteration, addition, and / or deletion of a gene. An engineered cell can also refer to a cell with an added, deleted, and / or altered gene.

[0299] " Low expression " described in this application is that the protein and / or RNA level of target gene expression in engineered cells is lower than the expression level before cell engineering treatment. For example, low expression of B2M, TCR, FAS, NKG2A or NKG2D ligand refers to that the expression of B2M, TCR, FAS, NKG2A or NKG2D ligand in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%. The expression or content of a specific protein in cells can be determined using specific antibodies by any suitable method known in the art, such as ELISA, immunohistochemistry, immunoblotting or flow cytometry.

[0300] The term "MHC" stands for the histocompatibility complex, which in human cells is referred to as the HLA antigen. Rejection is mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue. The term "B2M" stands for beta-2 microglobulin, also known as B2M, which is the light chain of the MHC class I molecule.

[0301] The term "recombinant T cell receptor (recombinant TCR)" includes chimeric receptors derived from one or more TCR subunits. For example, a recombinant TCR comprises an extracellular domain, a transmembrane domain, and a TCR intracellular domain of at least a portion of a TCR subunit, wherein the TCR subunit portion is effectively connected to an antigen binding domain. For example, the TCR subunits in the recombinant TCR are derived from CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRγ, and / or TCRδ subunits. For example, the recombinant TCR can be integrated into a TCR / CD3 complex expressed on a T cell. For example, a recombinant TCR comprises the constant regions and intracellular domains of TCRα and TCRβ subunits, and the subunit constant regions are effectively connected to an antigen binding domain. For example, a recombinant TCR comprises the constant regions and intracellular domains of TCRγ and TCRδ subunits, and the subunit constant regions are effectively connected to an antigen binding domain. For example, the recombinant TCR comprises a CD3ζ, CD3ε, CD3γ, or CD3δ subunit, and the extracellular domain of the subunit is operably linked to an antigen binding domain.

[0302] The term "gene editing" refers to a genetic engineering technology that uses site-specific nucleases to insert, knock out, modify or replace DNA at a specific location in the genome to change the DNA sequence. Gene knockout technology using nucleases includes CRISPR / Cas9 technology, ZFN technology, TALE technology and TALE-CRISPR / Cas9 technology, Base Editor technology, guide editing technology and / or homing endonuclease technology. The guide sequence (gRNA) is a polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence, and the gRNA can guide the CRISPR complex to sequence-specific binding to the target sequence. Whenever the sequence of gRNA is involved in this application, it can be a targeted DNA sequence, or it can be a complete Cas9 guide sequence formed by the ribonucleotides corresponding to the DNA and crRNA and TracrRNA. gRNA is used to guide, bind to or recognize Cas enzymes. For example, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. For example, CRISPR technology is used to construct endogenous TCR / B2M / FAS knockout or engineered cells with endogenous TCR / B2M knockout. The gRNA sequences targeting TCR, B2M, and FAS are shown in SEQ ID NOs: 142, 143, and 144, respectively.

[0303] The term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells, which can be achieved by various means known in the art, such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0304] The terms "nucleic acid molecule encoding," "coding DNA sequence," and "coding DNA" refer to the sequence or order of deoxyribonucleotides along a deoxyribonucleic acid strand. For example, a nucleic acid sequence encodes an amino acid sequence. When referring to a nucleotide sequence, "sequence" can include DNA or RNA and can be single-stranded or double-stranded.

[0305] The term "homology" or "identity" refers to the subunit sequence identity between two polymer molecules (e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules). The term "substantial identity" or "substantial homology" refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence or nucleic acid sequence. For example, such a sequence is at least about 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the amino acid or nucleic acid sequence used for comparison. Sequence identity can be measured using sequence analysis software (e.g., BLAST, BESTFIT, GAP or PILEUP / PRETTYBOX programs).

[0306] The term "expression vector" refers to a vector containing a recombinant polynucleotide, which includes expression control sequences operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or an in vitro expression system. Expression vectors include plasmids and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0307] The term "vector" refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Examples include, but are not limited to, linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Examples include autonomously replicating plasmids or viruses. Also included are non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds and liposomes.

[0308] The term "isolated" refers to separation from cellular components or other components with which the polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof is normally associated in nature. As will be understood by those skilled in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof need not be "isolated" to distinguish it from its naturally occurring counterpart. Furthermore, a "concentrated," "isolated," or "diluted" polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof can be distinguished from its naturally occurring counterpart because the concentration or number of molecules per volume is greater ("concentrated") or less ("diluted") than that of its naturally occurring counterpart.

[0309] The term "exogenous" refers to a nucleic acid molecule, polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or is expressed at a level insufficient to achieve the function it would have when overexpressed.

[0310] The term "endogenous" refers to a nucleic acid molecule, polypeptide, etc. that originates from the organism itself.

[0311] The term "chimeric receptor" refers to a fusion molecule composed of extracellular, transmembrane, and intracellular domains, formed by connecting DNA fragments or protein-specific cDNAs from different sources using genetic recombination technology. Chimeric receptors include, but are not limited to, chimeric antigen receptors (CARs) and recombinant TCR receptors.

[0312] The term "chimeric antigen receptor" (CAR) includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and / or a costimulatory molecule. In one aspect, the stimulatory molecule is a ζ chain bound to a T cell receptor complex; in one aspect, the cytoplasmic signaling domain further includes a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (ie, CD137), CD27, and / or CD28. For example, the polypeptide groups are connected to each other.

[0313] The term "primary signal domain" or "primary signal domain" regulates the initial activation of the TCR complex in an irritating manner. On the one hand, the primary signal domain is caused by the combination of, for example, TCR / CD3 complexes and MHC molecules loaded with peptides, thereby mediating T cell responses (including but not limited to, proliferation, activation, differentiation, etc.). The primary signal domain that works in a stimulating manner may include an immunoreceptor tyrosine activation motif or an ITAM signaling motif. For example, the fragment of the primary signal domain comprising ITAM includes, but is not limited to, sequences derived from TCRε, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d. For example, in the present application CAR, the intracellular signaling domain includes an intracellular signaling sequence, such as the primary signal domain of CD3ζ.

[0314] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate the activity of the cell via a defined signaling pathway by generating a second messenger or by acting as an effector in response to such a messenger. The intracellular signaling domain can include the entire intracellular portion of a molecule, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0315] The term "costimulatory molecule" refers to a signal that is combined with a cell stimulation signal molecule, such as TCR / CD3, and the combination causes the increase or decrease of T cell proliferation and / or key molecules. It is an associated binding partner on a T cell that specifically binds to a co-stimulatory ligand and mediates the co-stimulatory response of T cells, including but not limited to cell proliferation. Co-stimulatory molecules are cell surface molecules or their ligands that are required for an effective immune response and are non-antigen receptors. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18) and 4-1BB (CD137).

[0316] The intracellular signaling domain (or structural region) can be selected from the intracellular co-stimulatory domains of any one or more of the following polypeptides: CD27, CD28, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, CD83.

[0317] The term "CD3ζ (also known as CD3Zeta)" includes the protein provided by GenBank Accession No. BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc. "CD3ζ" is used interchangeably with "CD3z" and "CD3Z" in this application.

[0318] The term "effective amount" or "therapeutically effective amount" refers to a dose sufficient to prevent or treat an individual disease (cancer). The effective dose for therapeutic or preventive use depends on the stage and severity of the disease being treated, the age, weight and general health of the subject, and the judgment of the prescribing physician. The size of the dose also depends on the active substance selected, the method of administration, the time and frequency of administration, the presence, nature and extent of adverse side effects that may accompany the administration of a specific active substance, and the desired physiological effect. According to the judgment of the prescribing physician or a person skilled in the art, one or more rounds, or multiple administrations of the engineered cells of the present application may be required. As an example and not a limitation of the present application, an exemplary dose of engineered cells may be at least one million cells (1×10 6 cells / dose).

[0319] The engineered cells provided by the present application (e.g., T, NK, NKT cells) can be used to treat, prevent or improve autoimmune diseases or inflammatory diseases, in particular inflammatory diseases associated with autoimmune diseases, such as arthritis (e.g., rheumatoid arthritis, chronic progressive arthritis and osteoarthritis) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarthritis (including ankylosing spondylitis), Reiter's syndrome, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis and enteropathic arthritis, enthesitis, hypersensitivity (including airway hypersensitivity and skin hypersensitivity) and allergies. The engineered T cells provided herein are useful for treating and preventing autoimmune hematological disorders (including, for example, hemolytic anemia, aplastic anemia, pure red blood cell anemia, and idiopathic thrombocytopenia), systemic lupus erythematosus (SLE), lupus nephritis, inflammatory muscle disease (dermatomyositis), periodontitis, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Stevens Johnson syndrome, spontaneous sprue, autoimmune inflammatory bowel disease (including, for example, ulcerative colitis, Crohn's disease, and irritable bowel syndrome), endocrine eye diseases, Graves' disease, sarcoidosis, multiple sclerosis, systemic sclerosis, fibrotic diseases, primary biliary cirrhosis, juvenile diabetes mellitus (type I diabetes), uveitis, keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, periprosthetic osteolysis, Glomerulonephritis (with and without nephrotic syndrome, including, for example, idiopathic nephrotic syndrome or minimal change disease), multiple myeloma, other types of tumors, inflammatory diseases of the skin and cornea, myositis, loosening of bone implants, metabolic disorders (such as obesity, atherosclerosis and other cardiovascular diseases, including dilated cardiomyopathy, myocarditis, type II diabetes and dyslipidemia) and autoimmune thyroid diseases (including Hashimoto's thyroiditis), primary vasculitis of small and medium vessels, large vessel vasculitis including giant cell arteritis, hidradenitis suppurativa, neuromyelitis optica, Sjogren's syndrome, Behçet's disease, atopic and contact dermatitis, bronchiolitis, inflammatory muscle diseases, autoimmune peripheral neuropathies, immune kidney, liver and thyroid diseases, inflammation and atherosclerosis, autoinflammatory febrile syndrome, immune hematological disorders and bullous diseases of the skin and mucous membranes.

[0320] The engineered cells provided herein (eg, T, NK, NKT cells) can be used to treat, prevent or improve asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema and other obstructive or inflammatory diseases of the airways.

[0321] Provided herein is an engineered cell (e.g., T, NK, NKT cell), which expresses a chimeric receptor that recognizes an NKG2D ligand, and optionally, the engineered cell further comprises a DAP10 polypeptide or a fragment thereof. The engineered cell may be a constitutively expressed chimeric receptor that recognizes an NKG2D ligand (e.g., comprising a constitutive promoter). The engineered cell may be an inducible expression chimeric receptor that recognizes an NKG2D ligand (e.g., comprising an inducible promoter). The engineered cell can resist killing by the host's immune cells (e.g., T, NK, NKT cells). In the presence of host immune cells (e.g., T, NK, NKT cells), the engineered cell has a long survival time and / or a high transplant survival rate compared to reference cells (e.g., cells that do not express NKG2D-CAR). The engineered cell may be an autologous cell or an allogeneic cell. The engineered cell can increase the persistence and / or transplant survival rate of another immune cell in the presence of a host's immune cells (e.g., T, NK, NKT cells). The engineered cell and / or another immune cell may be a cell from the same individual or an allogeneic cell.

[0322] Provided herein is an engineered cell (e.g., T, NK, NKT cell), which expresses: a chimeric polypeptide and a chimeric receptor that recognizes an NKG2D ligand, wherein the chimeric polypeptide can regulate the transcriptional activity of the chimeric receptor; optionally, the engineered cell further comprises a DAP10 polypeptide or a fragment thereof. The engineered cell can resist killing by the host's immune cells (e.g., T, NK, NKT cells). In the presence of host immune cells (e.g., T, NK, NKT cells), the engineered cell has a long survival time and / or a high transplant survival rate compared to reference cells (e.g., cells that do not express NKG2D-CAR). The engineered cell may be an autologous cell or an allogeneic cell. The engineered cell can increase the persistence and / or transplant survival rate of another immune cell in the presence of a host's immune cells (e.g., T, NK, NKT cells). The engineered cell and / or another immune cell may be a cell from the same individual or an allogeneic cell.

[0323] The engineered cells of the present application (e.g., T, NK, NKT cells) can be administered as the sole active ingredient or in combination with other drugs such as immunosuppressants or immunomodulators or other anti-inflammatory agents or other cytotoxic agents or anticancer agents (e.g., as adjuvants or in combination therewith), for example, to treat or prevent diseases related to immune disorders. For example, the engineered cells of the present application can be used in combination with the following drugs: DMARDs, such as gold salts, sulfasalazine, antimalarials, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, tacrolimus, sirolimus, minocycline, leflunomide, glucocorticoids; calcineurin inhibitors, such as cyclosporine A or FK 506; regulators of lymphocyte recirculation, such as FTY720 and FTY720 analogs; mTOR inhibitors, such as rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; ascomycins with immunosuppressive properties, such as ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprine; leflunomide; mizoribine; mycophenolate mofetil; 15-deoxyspergualin or its immunosuppressive homologs, analogs or derivatives; immunosuppressive monoclonal antibodies, for example, against leukocyte receptors, such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40.monoclonal antibodies to CD45, CD58, CD80, CD86, or their ligands; antibodies that bind to immune checkpoint inhibitors, for example, the checkpoint inhibitors comprise: (a) one or more antagonists of checkpoint molecules comprising PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rar a (retinoic acid receptor α), TLR3, VISTA, NKG2A / HLA-E or inhibitory KIR, (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab and derivatives or functional equivalents thereof, or (c) at least one of atezolizumab, nivolumab and pembrolizumab; TOLL-like receptor agonist poly(I:C); sorafenib, regorafenib; chemotherapeutic agents: cyclophosphamide, fludarabine, albumin-paclitaxel or a combination thereof, 5-fluorouracil or a prodrug or active metabolite thereof, oxaliplatin or a prodrug or active metabolite thereof, taxanes, or a combination thereof); olaparib; gemcitabine; other immunomodulatory compounds. The engineered cells can also be combined with whole-body irradiation or localized irradiation.

[0324] The engineered cells (e.g., T, NK, NKT cells) of the present application may also express one or more of the following polypeptides in any combination: 1) another chimeric receptor that recognizes tumor antigens and / or pathogen antigens, for example, another chimeric receptor is CAR and / or recombinant TCR; 2) cytokines, such as IL7 and / or CCL21, IL7+CCL19, sPD-1, IFNβ, IL4R extracellular domain+IL21R intracellular domain, IL-12, TGF-β receptor extracellular domain+IL-2R intracellular domain, RUNX3, RUNX3 and+IL15 (or IL18 or IL21), IL21, IL21+CCL19, IL15, IL18, CD11a fusion protein, HLA-E-B2M chimeric protein, CXCR4, or a combination thereof; 3) antibodies that bind to immune checkpoint inhibitors, for example, the checkpoint inhibitors comprise: (a) one or more antagonists of checkpoint molecules, which comprise PD-1, PDL-1, TIM -3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD16 0. CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab and their derivatives or functional equivalents; or (c) at least one of atezolizumab, nivolumab and pembrolizumab.

[0325] The method for obtaining suitable engineered cells may include selection based on specific markers. For example, the marker may include GFP, a resistance gene, a cell surface marker, or an endogenous tag. Any endogenous marker may be used to select cells. Applicable cell selection techniques include flow cytometry and / or magnetic columns. The screened engineered cells are then infused into the subject. The screened engineered cells can also be expanded to a large number. The screened engineered cells can be expanded before infusion. The effective amount of engineered cells used to treat a subject may vary depending on cell viability and the efficiency of genetic modification of the cells (e.g., the efficiency of transgenic integration into one or more cells, or the expression level of a protein encoded by the transgenic). Cell viability (e.g., proliferation capacity) and the efficiency of transgenic integration after genetic modification can be used to determine the effective amount of engineered cells administered to the subject. An increase in cell viability after genetic modification can reduce the dosage of the engineered cells. An increase in the efficiency of transgenic integration into one or more cells can reduce the dosage of the engineered cells. The effective amount of engineered cells for treatment can be determined by determining a function of cell viability over time. The therapeutically effective amount of engineering cells can be determined by determining that transgene is integrated into one or more cells relative to a time-related variable (e.g., cell culture time, electroporation time, cell stimulation time) The function of the change in efficiency. Effective cells in treatment can be cell colonies comprising about 30% to about 100% expression NKG2D-CAR or chimeric polypeptide on the cell surface. In one example, effective cells in treatment can express about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% to greater than about 99.9% NKG2D-CAR or chimeric polypeptide, as measured by flow cytometry.

[0326] The engineered cells provided by the present application (e.g., T, NK, NKT cells) can be used to treat, prevent or improve any tumor disease, including acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia (CLL), chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's disease, Lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (such as non-small cell lung cancer), lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia, B-precursor acute lymphoblastic leukemia (B-ALL), pre-B cell precursor acute lymphoblastic leukemia (BCP-ALL), B cell lymphoma, acute lymphoblastic leukemia (ALL), Burkitt's lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer. Preferably, the tumor is characterized by high expression of NKG2D-ligand. For example, a solid tumor with high expression of NKG2D-ligand. For example, a CD123-positive blood tumor. For example, a CLL1-positive blood tumor. For example, a B7H3-positive brain tissue tumor. For example, B7H3-positive gliomas.

[0327] The term "tumor antigen" refers to an antigen that emerges or is overexpressed during the development and progression of a hyperproliferative disease. Hyperproliferative conditions are called cancers or tumors. Tumor antigens include antigens from solid tumors and blood tumors.

[0328] The tumor antigens of the present application include, but are not limited to, thyroid stimulating hormone receptor (TSHR); CD171; CS-1 (CS1); C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); IL-13Rα; IL-11Rα; PSCA; PSMA; CEA; NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; Epstein-Barr virus (Epstein-Barr virus) CAM; protease serine 21 (PRSS21); VEGFR, VEGFR2; Lewis (Y) antigen; CD24; PDGFR-β; SSEA-4; MUC1; MUC6; EGFR; EGFR2; ERBB3; ERBB4; EGFRvIII; neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; EphA2; fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); OAcGD2; folate receptor; TEM1 / CD248; TEM7R; Claudin 6; Claudin18.2; Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6; BCMA; CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1; HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; oncofetal variant of tumor necrosis; GPRC5D; CXORF61; CD97; CD179a; ALK; polysialic acid; PLAC1; the hexose portion of globoH glycoceramide (GloboH); breast 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); WT1; ETV6-AML; SPA17; XAGE1; Tie2; MAD-CT-1; 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 (N-ACETYLGlucosaminyltransferase V) A17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; MYCN; RhoC; CYP1B1; CCCTC binding factor (zinc finger protein)-like (BORIS); SART3; PAX5; OYTES1; LCK; AKAP-4; SSX2; CD79a; CD79b; CD72; LAIR1; FCAR; LILRA2; CD300LF; CLEC12A; BST2; EMR2; lymphocyte antigen 75 (LY75); glypican-3 (GPC3); FCRL5; FcRH5, immunoglobulin lambda-like polypeptide 1 (IGLL1). Preferably, the tumor antigen is CD123, CLL1, CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, or Claudin18.2. FAP, Mesothelin, NKG2D-ligand, NKG2A, CD94. The term "pathogen antigen" is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; viral antigens are selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.

[0329] The terms "recognition", "binding" and "targeting" are used interchangeably and refer to selective binding to a target antigen. For example, recognizing a target cell refers to binding to a target antigen (e.g., a target molecule) on a target cell. In one example, an engineered cell recognizes a target cell, that is, the engineered cell targets a target cell. For example, an engineered cell inhibits or kills a target cell by binding to a target antigen on a target cell. For example, an engineered cell expressing a chimeric receptor that recognizes an NKG2D ligand achieves the purpose of inhibiting or killing a target cell (e.g., T, NK, NKT cell) by recognizing or targeting an NKG2D ligand on a target cell (e.g., T, NK, NKT cell). In one example, an NKG2D-CAR-T cell inhibits or kills a host NK cell (e.g., an allogeneic NK cell) by recognizing or targeting an NKG2D ligand on a host NK cell.

[0330] The present application provides isolated nucleic acids, vectors, expression vectors, and engineered cells (also referred to as host cells) encoding chimeric receptors that recognize NKG2D ligands, chimeric polypeptides that regulate transcriptional activity, chimeric receptors that recognize tumor antigens, antibodies that recognize MOG, or fragments thereof. The nucleic acids may be located in intact cells, in cell lysates, or in partially purified or substantially purified form. For example, the present application includes: GPC3-synE, GPC3-synE-del1, GPC3-synE-del2, GPC3-synE-del3, GPC3-synE-del23, GPC3-synE-EGFn (n=1, 2, 3, 4), MOG-synE, MOG-synE-del3, CD123-synE, CD123-synE-del3, CLDN18.2-synE, GPRC5D-synE, CLL1-synE, B7H3-CAR-MOG-s ynE, B7H3-CAR-MOG-synE-del3, ZNKG2D-CAR-CD123-synE, ZBB-NKG2D-CAR-CD123-synE, ZNKG2D-CAR-DAP10-CD123-synE, ZNKG2D-CAR-CD123-synE-del3, ZBB-NKG2D-CAR-CD123-synE-del3 or ZNKG2D-CAR-DAP10-CD123-synE-del3 nucleic acids and engineered cells comprising the nucleic acids.

[0331] The expression of a chimeric receptor that recognizes an NKG2D ligand and / or a chimeric polypeptide having transcriptional activity can be controlled by one or more promoters. The promoter can be ubiquitous, constitutive (an unregulated promoter that allows continuous transcription of the associated gene), tissue-specific, or inducible (a promoter with regulated transcriptional activity that, when stimulated by inducing conditions, can significantly and rapidly regulate transcription of the chimeric receptor gene).

[0332] The nucleic acids of the present application can be obtained using standard molecular biology techniques, such as cDNA encoding the light and heavy chains of the antibody or encoding the VH and VL segments by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), one or more nucleic acids encoding the antibody can be recovered from the library. Methods for introducing exogenous nucleic acids into host cells are generally known in the art and may vary depending on the host cell used.

[0333] A gene encoding a chimeric receptor that recognizes an NKG2D ligand and / or a gene encoding a chimeric polypeptide with transcriptional regulation activity can be introduced into a cell, optionally along with a gene encoding a DAP10 polypeptide or fragment thereof. For example, the genes are introduced into engineered cells (e.g., T, NK, or NKT cells). When introduced into a cell, the gene can be a complementary DNA (cDNA) segment, a copy of messenger RNA (mRNA), or the gene itself, residing within its original genomic DNA region (with or without introns). The gene encoding the chimeric receptor that recognizes an NKG2D ligand and the gene encoding the chimeric polypeptide with transcriptional regulation activity can be introduced into the cell simultaneously; optionally, a gene encoding a DAP10 polypeptide or fragment thereof can also be introduced. The gene encoding the chimeric receptor that recognizes an NKG2D ligand and the gene encoding the chimeric polypeptide with transcriptional regulation activity can be located on the same vector, optionally further comprising a gene encoding a DAP10 polypeptide or fragment thereof. In one example, the chimeric polypeptide can regulate the expression of both the chimeric receptor that recognizes an NKG2D ligand and the DAP10 polypeptide.

[0334] Compared with reference cells (e.g., cells that do not express NKG2D-CAR), engineered cells that constitutively express NKG2D-CAR or engineered cells that regulatedly express NKG2D-CAR have enhanced survival and expansion capabilities during in vitro and in vivo culture.

[0335] The DNA encoding the transgene can also be designed to include a reporter gene, so that the presence of the transgene or its expression product can be detected by activating the reporter gene. Any reporter gene can be used. By selecting cells in cell culture in which the reporter gene is activated, cells containing the transgene are detected.

[0336] The expression of the exogenous introduced gene can be verified by expression assay (e.g., qPCR) or by measuring RNA levels. Expression level can also indicate copy number. For example, if the expression level is very high, this indicates that the exogenous introduced gene may be integrated into the genome in a multi-copy form. Alternatively, the exogenous introduced gene is integrated in a high transcription region, for example, near a highly expressed promoter. Expression can also be verified by measuring protein levels such as by immunoblotting.

[0337] The chimeric receptor (eg, NKG2D-CAR) for recognizing NKG2D ligands provided herein refers to a fusion molecule formed by connecting cDNA or peptide segments corresponding to DNA fragments or proteins from different sources, including an extracellular domain, a transmembrane domain, and an intracellular domain; optionally, the extracellular domain is directly connected to the transmembrane domain or is connected by a hinge. For example, including but not limited to: chimeric antigen receptor (CAR), recombinant TCR receptor. NKG2D-CAR can be regulated by the chimeric polypeptide provided herein. Notch polypeptides or polypeptides derived therefrom well known to those skilled in the art can be used to regulate the chimeric receptor for recognizing NKG2D ligands of the present application.

[0338] The chimeric receptor that recognizes NKG2D ligands includes: 1) the extracellular domain can be selected from: the extracellular domain of NKG2D polypeptide or an antibody or fragment thereof that recognizes NKG2D ligand; 2) the transmembrane domain can be selected from: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, BAFFR, CEACAM1, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD137 (4-1BB), CD16, CD160, CD18 (CD11a , LFA-1), CD160 (BY55), CD162 (SELPLG), CD19, CD2, CD22, CD226 (DNAM1), CD229 (Ly9), CD244 (SLAMF4, 2B4), CD27, CD278 (ICOS ), CD28, CD29, CD33, CD37, CD4, CD40, CD45, CD49a, CD49D, CD49f, CD5, CD64, CD8, CD80, CD84, CD86, CD9, CD96(Tactile), CD134 , CD154, CRTAM, GITR, HLA-E, HLA-F, HLA-G, HVEM(LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGA L, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LTBR, ​​NKp80(KLRF1), NKp44, NKp30, NKp46, NKG2D, NKG2C, OCIL, OX40, PAG / Cbp, The transmembrane domain of PSGL1, SLAM (SLAMF1, CD150, IPO-3), SLAMF6 (NTB-A, Ly108), SLAMF7, BLAME (SLAMF8), TNFR2, VLA1, VLA-6, cadherin and / or collagen; 3) the intracellular domain can be selected from: TCRε, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, CD66d, NKG2D, CD28 or CD137 intracellular signaling domain or a combination thereof.

[0339] The chimeric receptor that recognizes NKG2D ligand can be selected from: 1) comprising the full length of NKG2D polypeptide or a fragment thereof; 2) comprising the full length of NKG2D polypeptide and an immunoreceptor tyrosine-based activation motif; 3) comprising the full length of NKG2D polypeptide and an ITAM signaling motif; 4) comprising the full length of NKG2D polypeptide and a sequence of a TCRε, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, CD66d, CD28 or CD137 intracellular signaling domain or a combination thereof; 5) comprising the full length of NKG2D polypeptide and a CD3ζ intracellular signaling domain; 6) comprising the full length of NKG2D polypeptide, a CD28 intracellular signaling domain, and a CD3ζ intracellular signaling domain. 7) including the full-length NKG2D polypeptide, the CD137 intracellular signaling domain and the CD3ζ intracellular signaling domain; 8) including the full-length NKG2D polypeptide, the CD28 intracellular signaling domain, the CD137 intracellular signaling domain and the CD3ζ intracellular signaling domain; 9) including the NKG2D polypeptide extracellular domain, the transmembrane domain, and the immunoreceptor tyrosine activation motif; 10) including the NKG2D polypeptide extracellular domain, the transmembrane domain, and the ITAM signaling motif; 11) including the NKG2D polypeptide extracellular domain, the transmembrane domain, and the TCRε, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD27 8, CD66d, CD28 or CD137 intracellular signaling domain or a combination thereof; 12) comprising an NKG2D polypeptide extracellular domain, a transmembrane domain, and a CD3ζ intracellular signaling domain; 13) comprising an NKG2D polypeptide extracellular domain, a transmembrane domain, a CD28 intracellular signaling domain and a CD3ζ intracellular signaling domain; 14) comprising an NKG2D polypeptide extracellular domain, a transmembrane domain, a CD137 intracellular signaling domain and a CD3ζ intracellular signaling domain; 15) comprising an NKG2D polypeptide extracellular domain, a transmembrane domain, a CD28 intracellular signaling domain, a CD137 intracellular signaling domain and a CD3ζ intracellular signaling domain; 16) comprising an NKG2D polypeptide extracellular domain, a CD28 or CD8 transmembrane domain, and an immunoreceptor tyrosine-based activation motif; 17) comprising an N 18) comprising a sequence comprising a NKG2D polypeptide extracellular domain, a CD28 or CD8 transmembrane domain, and an ITAM signaling motif; 19) comprising a NKG2D polypeptide extracellular domain, a CD28 or CD8 transmembrane domain, and a TCRε, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, CD66d, CD28 or CD137 intracellular signaling domain, or a combination thereof; 20) comprising a NKG2D polypeptide extracellular domain, a CD28 or CD8 transmembrane domain, a CD28 intracellular signaling domain, and a CD3ζ intracellular signaling domain;21) comprising the extracellular domain of an NKG2D polypeptide, a CD28 or CD8 transmembrane domain, a CD137 intracellular signaling domain, and a CD3ζ intracellular signaling domain; 22) comprising the extracellular domain of an NKG2D polypeptide, a CD28 or CD8 transmembrane domain, a CD28 intracellular signaling domain, a CD137 intracellular signaling domain, and a CD3ζ intracellular signaling domain.

[0340] In one example, the chimeric receptor recognizing the NKG2D ligand comprises a fragment having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the amino acid sequence shown in SEQ ID No: 51, 52, 53 and / or 54, and / or may optionally comprise substitutions of up to 1, 2, 3, 4, 5 or more amino acid residues by different amino acid residues.

[0341] In some examples, the chimeric receptor that recognizes an NKG2D ligand may have any combination of one or more of the following properties: 1) binding to a recombinant human NKG2D ligand (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, and / or ULBP-6); 2) binding to an endogenous NKG2D ligand (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, and / or ULBP-6) on the surface of a tumor cell; 3) binding to an endogenous NKG2D ligand (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and / or ULBP-6); 4) binds to endogenous NKG2D ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and / or ULBP-6) on the surface of human AML cells; 5) binds to endogenous NKG2D ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and / or ULBP-6) selected from THP1, KG-1, Molm13 cell surface; 6) Binds to endogenous NKG2D ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and / or ULBP-6) on the surface of human NK cells; 7) Binds to endogenous NKG2D ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and / or ULBP-6) on the surface of activated human NK cells; 8) Binds to endogenous NKG2D ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and / or ULBP-6) on the surface of human NK cells after co-incubation with tumor cells. -3, ULBP-4, ULBP-5 and / or ULBP-6); 9) binds to endogenous NKG2D ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and / or ULBP-6) on the surface of human NK cells after co-incubation with T cells; 10) binds to endogenous NKG2D ligands (MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and / or ULBP-6) on the surface of human NK cells after co-incubation with T cells (endogenous B2M knockout T cells).

[0342] The engineering cells that recognize NKG2D ligands may include one or more exogenous genes. One or more exogenous genes may express a chimeric receptor that binds to at least one NKG2D ligand (e.g., MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5 and / or ULBP-6). The engineering cells that recognize NKG2D ligands may also include one or more chimeric receptors, or they may include a single chimeric receptor and a secondary engineered receptor. The engineering cells that recognize NKG2D ligands may encode suicide gene transgenes. Suicide genes can induce the elimination of engineering cells that recognize NKG2D ligands. Suicide genes are any genes that induce apoptosis in the CAR immune response cells. Suicide genes can be encoded in viral vectors together with chimeric receptors that recognize NKG2D ligands.

[0343] The engineered cells of the present application that constitutively express chimeric receptors that recognize NKG2D ligands (e.g., NKG2D-CAR) or that regulatedly express chimeric receptors that recognize NKG2D ligands (e.g., NKG2D-CAR) may also have any combination of one or more of the following characteristics: 1) low expression or no expression of endogenous NKG2D ligands; 2) low expression or no expression of endogenous B2M / TCR; 3) low expression or no expression of endogenous B2M / TCR / NKG2D ligands; 4) low expression or no expression of endogenous B2M / TCR / FAS; 5) low expression or no expression of endogenous B2M / TCR / FAS / NKG2D ligands; 6) knockout of endogenous immune checkpoints, for example, a polynucleotide fragment encoding NKG2D-CAR is inserted into an endogenous immune checkpoint gene so that the gene is interrupted, for example, the immune checkpoints are PD-1, PD-L1, and CTLA-4. For example, endogenous FAS, B2M, TCR, and / or NKG2D ligands are knocked out using gene editing technology. For example, endogenous FAS, B2M, TCR, and / or NKG2D ligands are knocked out using gene editing technology. For example, endogenous FAS, B2M, TCR, and / or NKG2D ligands are knocked out using CRISPR technology.

[0344] In one example, an engineered cell that recognizes an NKG2D ligand includes: a ZNKG2D-CAR, a ZBB-NKG2D-CAR, a ZNKG2D-CAR-DAP10, and / or an NKG2D-28Z-CAR polypeptide; optionally, the engineered cell further includes a gRNA fragment targeting B2M / TCR or B2M / TCR / FAS. An immune cell that recognizes an NKG2D ligand may include: a fragment having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity to the amino acid sequence shown in SEQ ID NO: 51, 52, 53, and / or 54, and / or may optionally include at most 1, 2, 3, 4, 5, or more amino acid residues substituted with different amino acid residues; optionally, the immune cell further includes a gRNA sequence (e.g., SEQ ID NO: 142, 143, and / or 144).

[0345] The engineered cells constitutively or regulatedly expressing NKG2D-CAR of the present application also express any combination of one or more of the following polypeptides: 1) another chimeric receptor that recognizes tumor antigens and / or pathogen antigens, for example, the other chimeric receptor is CAR and / or recombinant TCR; 2) cytokines; 3) antibodies that bind to immune checkpoint inhibitors.

[0346] The engineered cells constitutively or regulatedly expressing NKG2D-CAR of the present application are used in combination with one or more of the following anticancer agents: TOLL-like receptor agonist poly(I:C); sorafenib; regorafenib; chemotherapy agents: cyclophosphamide, fludarabine, albumin-paclitaxel, or a combination thereof; 5-fluorouracil or its prodrug or active metabolite, oxaliplatin or its prodrug or active metabolite, taxane drugs, or a combination thereof; olaparib; gemcitabine; mTOR inhibitor: rapamycin.

[0347] The engineered cells expressing regulated NKG2D-CAR of the present application also express one or more of the following polypeptides in any combination: 1) polypeptides for regulating NKG2D-CAR, for example, synNOTCH polypeptides and polypeptides derived therefrom; 2) polypeptides having binding-triggered transcriptional regulatory activity, for example, chimeric polypeptides provided herein; 3) chimeric polypeptides comprising a receptor regulatory domain composed of the EphrinB2 extracellular region, its modified form (for example, EphrinB2-(EGF-like domain) n, n=1-4), or its truncated form (for example, EphrinB2EC-del1, EphrinB2EC-del2, EphrinB2EC-del3 or EphrinB2EC-del23); 4) polypeptides that bind to tumor antigens to trigger transcriptional regulatory activity, for example, tumor antigens are ALPPL2, ALPI, Axl, B7H3, BCMA, CD4+ Tg, IL-6+ Tg, IL-7+ Tg, IL-8+ Tg, IL-9+ Tg, IL-10+ Tg, IL-11+ Tg, IL-12+ Tg, IL-13+ Tg, IL-14+ Tg, IL-15+ Tg, IL-16+ Tg, IL-17+ Tg, IL-18+ Tg, IL-19+ Tg, IL-20+ Tg, IL-21+ Tg, IL-22+ Tg, IL-23+ Tg, IL-24+ Tg, IL-25+ Tg, IL-26+ Tg, IL-27+ Tg, IL-28+ Tg, IL-29+ Tg, IL-30+ Tg, IL-31+ Tg, IL-32+ Tg, IL-33+ Tg, IL-34+ Tg, IL-35+ T 117. CD123, CD171, CD179a, CD19, CD213A2, CD20, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD7, CD71, CD94, CD97, CEA, Claudin18 .2, CLDN6, CLECL1, CLL1, cMet, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, FAP, FCRH5, FLT3, GD2, GD3, GM3, GPC3, GPRC5D, HER2(ERBB2), IGLL1, IL llRa, IL13Ra2, Mesothelin, MUC1, NCAM, NKG2D-Ligand, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2 or WT1; 5) polypeptides that bind to pathogen antigens to trigger transcriptional regulatory activity; 6) polypeptides that bind to tissue-specific antigens to trigger transcriptional regulatory activity, for example, brain tissue-specific marker MOG, liver tissue-specific marker ASGR1, prostate tissue marker PSA, etc. or a combination thereof; 7) polypeptides that bind to blood tumor antigens to trigger transcriptional regulatory activity, for example, CD123, CLL1, GPRC5D, FcRH5, CD38, BCMA, CD19, CD20, etc.; 8) polypeptides that bind to solid tumor antigens to trigger transcriptional regulatory activity, for example, B7H3, GPC3, Claudin 6. Claudin18.2, FAP, Mesothelin, NKG2D ligand, NKG2A, CD94; 9) Another chimeric receptor that recognizes tumor antigens and / or pathogen antigens, for example, the other chimeric receptor is CAR and / or recombinant TCR.

[0348] In one example, the regulated expression NKG2D-CAR engineered cells of the present application include: ZNKG2D-CAR, ZBB-NKG2D-CAR, ZNKG2D-CAR-DAP10 and / or NKG2D-28Z-CAR polypeptide fragments; further comprising a chimeric polypeptide that specifically binds to antigens such as Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvⅢ, BCMA, CD123, MOG, B7H3, NKG2A or FcRH5, and regulates NKG2D-CAR expression. Preferably, the engineered cells further include a gRNA fragment targeting B2M / TCR or B2M / TCR / FAS.

[0349] The regulated NKG2D-CAR-expressing engineered cells of the present application may include: ZNKG2D-CAR-CD123-synE (e.g., SEQ ID NO: 55), ZBB-NKG2D-CAR-CD123-synE (e.g., SEQ ID NO: 56), ZNKG2D-CAR-DAP10-CD123-synE (e.g., SEQ ID NO: 57), NKG2D-28Z-CAR-CD123-synE (e.g., including SEQ ID NOs: 48 and 54), ZNKG2D-CAR-CD123-synE-del3 (e.g., including SEQ ID NOs: 49 and 52), ZBB-NKG2D-CAR-CD123-synE-del3 (e.g., including SEQ ID NOs: 49 and 51), ZNKG2D-CAR-DAP10-CD123-synE-del3 (e.g., including SEQ ID NOs: 49 and 52), NO: 49 and 53), NKG2D-28Z-CAR-CD123-synE-del3 (e.g., including SEQ ID NO: 49 and 54) fragments. Preferably, the engineered cells further comprise a gRNA fragment targeting B2M / TCR or B2M / TCR / FAS. More preferably, the engineered cells further comprise a gRNA sequence (e.g., SEQ ID NO: 142, 143, and / or 144).

[0350] The regulated NKG2D-CAR-expressing engineered cells of the present application may include: a fragment of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the amino acid sequence shown in SEQ ID No: 51, 52, 53 and / or 54, and / or optionally including at most 1, 2, 3, 4, 5 or more amino acid residues replaced by different amino acid residues; and also including at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the amino acid sequence shown in SEQ ID No: 44, 45, 46, 47, 48 and / or 49, and / or optionally including at most 1, 2, 3, 4, 5 or more amino acid residues replaced by different amino acid residues. Preferably, the nucleic acid fragment encoding the chimeric polypeptide having transcriptional regulatory activity and the NKG2D-CAR regulated by the chimeric polypeptide are located in the same expression vector. Preferably, the engineered cell further comprises a gRNA fragment targeting B2M / TCR or B2M / TCR / FAS. More preferably, the engineered cell further comprises a gRNA sequence (e.g., SEQ ID NO: 142, 143 and / or 144).

[0351] The regulated expression NKG2D-CAR engineering cells of the present application may include: a fragment of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity of the polynucleotide sequence shown in SEQ ID No: 55, 56 and / or 57 or the amino acid sequence encoded therein, and / or may optionally include at most 1, 2, 3, 4, 5 or more amino acid residues replaced by different amino acid residues. Preferably, the engineering cell further includes a gRNA fragment targeting B2M / TCR or B2M / TCR / FAS. More preferably, the engineering cell further includes a gRNA sequence (e.g., SEQ ID NO: 142, 143 and / or 144). The present application comprises constitutively expressed NKG2D-CAR-T cells or regulatedly expressed NKG2D-CAR-T cells, and may also have any combination of one or more of the following characteristics: 1) low expression or no expression of endogenous NKG2D ligands; 2) low expression or no expression of endogenous B2M / TCR; 3) low expression or no expression of endogenous B2M / TCR / NKG2D ligands; 4) low expression or no expression of endogenous B2M / TCR / FAS; 5) low expression or no expression of endogenous B2M / TCR / FAS / NKG2D ligands; 6) knocking out endogenous immune checkpoints, for example, a polynucleotide fragment encoding NKG2D-CAR is inserted into an endogenous immune checkpoint gene so that the gene is interrupted, for example, the immune checkpoint is PD-1, PD-L1, CTLA-4. For example, endogenous FAS, B2M, TCR and / or NKG2D ligands are knocked out using gene editing technology. For example, endogenous FAS, B2M, TCR, and / or NKG2D ligands are knocked out using gene editing technology. For example, endogenous FAS, B2M, TCR, and / or NKG2D ligands are knocked out using CRISPR technology. For example, a polynucleotide fragment encoding an NKG2D-CAR is inserted into an endogenous TCR, B2M, or FAS gene such that the gene is interrupted.

[0352] The engineered cells of the present application comprising a constitutively expressed chimeric receptor (e.g., NKG2D-CAR) that recognizes an NKG2D ligand or an engineered cell that regulatedly expresses a chimeric receptor (e.g., NKG2D-CAR) that recognizes an NKG2D ligand have one or more of the following uses in any combination: 1) for preparing universal CAR-T cells; 2) for resisting autologous or allogeneic immune cell killing and having anti-tumor effects; 3) for resisting autologous or allogeneic NK cell killing; 3) for resisting host immune rejection and anti-solid tumor effects; 4) for enhancing the efficacy of another immune cell administered previously, simultaneously, or later. 5) Enhance the anti-tumor effect of another immune cell administered before, simultaneously, or after administration in vitro and in vivo; 6) In the absence of NK cells, regulatory NKG2D-CAR-T cells can significantly kill NKG2DL-positive tumor cells; 7) In the presence of NK cells, regulatory NKG2D-CAR-T cells can also significantly kill tumor cells; 8) After co-incubation with tumor cells, regulatory NKG2D-CAR-T cells have a strong cytotoxic effect on NK cells, and the longer the co-incubation time, the stronger the cytotoxic effect; and can partially resist the killing effect of NK cells. Regardless of the presence of NK cells, UCAR-T cells expressing ZNKG2D-CD123-synE, ZNKG2D-DAP10-CD123-synE, or ZBB-NKG2D-CD123-synE (e.g., with endogenous TCR / B2M or TCR / B2M / FAS knockout or low expression) can effectively inhibit the growth of leukemia xenografts.

[0353] The present application provides a combination of NKG2D-CAR-T cells and T cells expressing chimeric receptors that recognize tumor antigens (such as CAR, recombinant TCR), optionally, the endogenous TCR / B2M or TCR / B2M / FAS of the two cells used in combination are knocked out. The NKG2D-CAR-T cells provided in the present application can promote the survival and / or expansion of T cells expressing chimeric receptors that recognize tumor antigens (such as CAR, recombinant TCR) in the presence of autologous or allogeneic immune cells (T, NK, NKT cells).

[0354] The chimeric polypeptide provided herein is a receptor that regulates transcriptional activity in a target molecule-dependent manner. The chimeric polypeptide provided herein is a recombinant, non-naturally occurring receptor comprising a binding domain, a receptor regulatory domain, and an intracellular domain.

[0355] After the chimeric polypeptide binds to the target molecule, it triggers the hydrolysis of the chimeric polypeptide, releasing the intracellular domain. The chimeric polypeptide can bind to the target molecule (e.g., tumor antigen, tissue-specific marker) displayed on the surface of the target cell, triggering the hydrolysis of the chimeric polypeptide, releasing the intracellular domain. Exemplarily, after the chimeric polypeptide binds to the tumor antigen on the surface of the tumor cell, it regulates the transcription factor of the customized transcription program in the cell expressing the chimeric polypeptide. For example, after the chimeric polypeptide binds to the brain tissue marker, it regulates the transcription factor of the customized transcription program in the cell expressing the chimeric polypeptide.

[0356] The chimeric polypeptide of the present application comprises, from N-terminus to C-terminus: (a) a binding domain capable of specifically binding to a target molecule, (b) a receptor regulatory domain comprising one or more cleavage sites, wherein the extracellular region and the transmembrane region of the receptor regulatory domain are not simultaneously derived from Notch, and c) an intracellular domain; wherein the binding of the binding domain to the target molecule can induce the cleavage of the receptor regulatory domain, releasing the intracellular domain. The binding domain capable of specifically binding to the target molecule and the intracellular domain are heterologous to the Notch receptor polypeptide.

[0357] The receptor regulatory domain of the chimeric polypeptide includes an extracellular region and a transmembrane region (also known as a transmembrane domain). The receptor regulatory domain contains one or more ligand-inducible proteolytic cleavage sites, and the cleavage sites are selected from I-CLiPs (intramembranously cleaving proteases) enzyme cleavage sites or sheddase protease cleavage sites. I-CLiPs is a transmembrane cleavage protease that catalyzes the hydrolysis of specific sites on the transmembrane region of a transmembrane protein. The I-CliPs may contain a γ-secretase cleavage site. The γ-secretase cleavage site may contain a γ-secretase cleavage site of a Gly-Val dipeptide sequence. The sheddase protease may be selected from: BACE1, ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, MT1-MMP or a combination thereof.

[0358] For example, the receptor regulatory domain comprises one or more ligand-inducible proteolytic cleavage sites located in the transmembrane region, and the cleavage sites are selected from the I-CLiPs enzyme cleavage site.

[0359] The receptor regulatory domain comprises a transmembrane region that can be a single-pass transmembrane receptor transmembrane region, comprising at least one γ-secretase cleavage site. In one example, the transmembrane region includes, but is not limited to, the transmembrane region of CLSTN1, CLSTN2, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16, CX3CL1, DCC, DLL1, DSG2, DAG1, CDH1, EPCAM, EPHA4, EPHB2, EFNB1, EFNB2, ErbB4, GHR, HLA-A, and IFNAR2, wherein the transmembrane region comprises at least one γ-secretase cleavage site. In one example, transmembrane regions include, but are not limited to, IL1R1, IL1R2, IL6R, INSR, ERN1, ERN2, JAG2, KCNE1, KCNE2, KCNE3, KCNE4, KL, CHL1, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGR, ROBO1, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKHD1, NECTIN1, PCDHGC3, NRG1, LRP1B, CDH2, NRG2, PTPRK, SCN2B, Nradd, and PTPRM transmembrane regions. For example, the transmembrane region includes a Notch1 transmembrane region, a Notch2 transmembrane region, a Notch3 transmembrane region, or a Notch4 transmembrane region from a human or non-human animal (e.g., mouse, zebrafish, fruit fly, African clawed frog, or Gallus). For example, the transmembrane region includes an APLP1 transmembrane region or an APLP2 transmembrane region from a human or non-human animal. For example, the carboxyl terminus of the transmembrane region includes a stop transfer sequence (STS). The STS connects the intracellular domain of the chimeric polypeptide and prevents it from entering the endoplasmic reticulum lumen.

[0360] The extracellular region of the chimeric polypeptide provided herein includes the full-length extracellular region of Jagged2, EphrinB2, APLP1, APLP2, APP, CD44, CSF1R, CXCL16, CX3CL1, Delta1, E-cadherin, EphB2, EphrinB1, Growth hormone receptor, HLA-A2, IFNaR2, IL1R2, L1, LRP, LRP2, LRP6, N-cadherin, Nectin1α, NRADD, p75-NTR, Pcdhα4, Pcdhγ-C3, PTPκ, PTP-LAR, SorCS1b, SorLA, Sortilin, ApoER2, PKHD1, ErbB4, IFNaR2, VEGF-R1, or VLDLR, or a fragment of the extracellular region of any of the above proteins, or a truncated or truncated structure thereof, or a variant of the extracellular region of any of the above proteins.

[0361] Chimeric polypeptides synE, synE-del1, synE-del2, synE-del3, and synE-del23, comprising the extracellular region of the receptor regulatory domains of EphrinB2EC, EphrinB2EC-del1, EphrinB2EC-del2, EphrinB2EC-del3, or EphrinB2EC-del23, respectively, were capable of triggering cleavage of the chimeric polypeptides upon specific binding to target molecules, releasing the intracellular domain. Leakage induced by expression of synE-del3 or synE-del23 was significantly reduced.

[0362] This application constructs a modified EphrinB2 extracellular domain, EphrinB2-(EGF-like domain)n, with exemplary n=1 (SEQ ID No: 7), n=2 (SEQ ID No: 8), n=3 (SEQ ID No: 9), and n=4 (SEQ ID No: 10). The aforementioned EphrinB2 modifications, combined with the Notch1 transmembrane region (SEQ ID No: 15), form the corresponding receptor regulatory domains synE-EGF1, synE-EGF2, synE-EGF3, and synE-EGF4. Chimeric polypeptides comprising synE-EGF1, synE-EGF2, synE-EGF3, and synE-EGF4 can all trigger cleavage of the chimeric polypeptides and release the intracellular domains after specific binding to target molecules; compared to pre-modification, the background activation level of the chimeric polypeptides comprising the modifications is reduced in the absence of antigenic stimulation. EphrinB2 truncations include any of the aforementioned modifications. The truncated EphrinB2EC-del1, EphrinB2EC-del2, EphrinB2EC-del3 or EphrinB2EC-del23 comprises any of the modifications described above.

[0363] Fragments, variants, or truncated structures (also referred to as truncated forms or truncations) of the extracellular region of EphrinB2 may also be used interchangeably, and may have a sequence identity of at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to the sequence of the extracellular region of EphrinB2. The amino acid sequence of the truncated structure may be as shown in SEQ ID NO: 3, 4, 5, or 6.

[0364] In one example, the fragment, variant or truncated structure of the extracellular region of EphrinB2 is a structure in which the potential site of ADAM10 cleavage of the extracellular region of EphrinB2 is deleted or mutated.

[0365] In one example, the extracellular region comprises the full-length extracellular region of EphrinB2 from a human or non-human animal (e.g., a white-cheeked gibbon, a bonobo, a Sumatran orangutan, a chimpanzee, a gorilla, a rabbit, a Peruvian night monkey, a marmoset, a Cluys lemur, a small-eared macaque, a mouse, a rat, a cow, or an African clawed frog), or a truncated or mutant thereof. For example, the extracellular region comprises a fragment having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity to the amino acid sequence of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 10 or 65, and / or may optionally comprise substitutions of up to 1, 2, 3, 4, 5 or more amino acid residues with different amino acid residues.

[0366] The chimeric polypeptides provided herein comprise a receptor regulatory domain having an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or homologous to any one of the sequences set forth in SEQ ID No: 60, 61, 62, 63, 64, 66, or 67.

[0367] The chimeric polypeptide's short receptor regulatory domain facilitates viral packaging and expression, reducing the complexity of industrial production. The coding sequence for the chimeric polypeptide synE, its truncated forms, or its modified forms is shorter than the full-length coding sequence for synNotch. The resulting vector containing the complete chimeric polypeptide has high cell transduction efficiency, reducing the complexity of industrial production.

[0368] The chimeric polypeptide synE, or its mutants, truncations, or modifications, and the gene it binds to and triggers regulation are not placed in the same vector, creating a dual-vector system. A chimeric polypeptide synE, or its mutants, truncations, or modifications, and the gene it binds to and triggers regulation are placed in the same vector, creating a single-vector system. This single-vector system with synE, its mutants, truncations, or modifications exhibits higher infection efficiency than synNotch, reducing the complexity of industrial production.

[0369] The chimeric polypeptide synE, or its mutants, truncations, or modifications, induces significantly reduced levels of leaky expression in transcriptional regulation. The chimeric polypeptide synE, or its mutants, truncations, or modifications, combines stringent induction of expression with strong induction capacity. For example, the chimeric polypeptide synE, or its mutants, truncations, or modifications, can better distinguish between different expression levels of the same target molecule, making it suitable for identifying target molecules that are low in normal tissue but highly expressed in tumor tissue, and is less likely to cause off-target effects in normal tissue. For example, the chimeric polypeptide synE, or its mutants, truncations, or modifications, regulates gene expression more stringently and is less sensitive to low-expressing target molecules. When targeting target molecules that are highly expressed in tumors but low in normal tissue, synE, or its mutants, truncations, or modifications, will have greater selectivity for tumor tissue and improved safety.

[0370] The intracellular domain of the chimeric polypeptide of the present application comprises a protein fragment or a combination thereof selected from any one of the following proteins: transcription factors (including transcription activators, transcription repressors), transcription co-activators, transcription co-repressors, DNA-binding polypeptides, RNA-binding polypeptides, translation regulatory polypeptides, hormones, cytokines, toxins, antibodies, chromatin regulators, suicide proteins, organelle-specific polypeptides (such as nuclear pore regulators, mitochondrial regulators, endoplasmic reticulum regulators, etc.), pro-apoptotic polypeptides, anti-apoptotic polypeptides, other polypeptides that promote cell death by other mechanisms, pro-proliferation polypeptides, anti-proliferation polypeptides, immune co-stimulatory polypeptides, site-specific nucleases, recombinases, inhibitory immune receptors, activating immune receptors, variants of Cas9 and RNA-targeting nucleases, DNA recognition polypeptides, signaling polypeptides, receptor tyrosine kinases, non-receptor tyrosine kinases, and polypeptides that promote differentiation.

[0371] The intracellular domain of the chimeric polypeptide of the present application includes a transcriptional activator protein that promotes or inhibits transcription of a promoter-driven DNA sequence. For example, a transcription factor directly regulates cell differentiation. For example, a transcription factor indirectly regulates cell differentiation by regulating the expression of a second transcription factor. For example, the transcription factor is a transcriptional activator protein or a transcriptional repressor protein. For example, the transcription factor is a transcriptional repressor protein. For example, the transcription factor is a transcriptional activator protein. For example, the transcription factor also includes a nuclear localization signal. For example, the transcription factor is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP 1-VP16. For example, the transcription factor is Gal4. For example, the transcription factor is Gal4-VP64.

[0372] The antibodies induced by the intracellular domain of the chimeric polypeptide can be therapeutic antibodies for treating diseases (including immune diseases and tumors).

[0373] The intracellular domain of the chimeric polypeptide may comprise a protein fragment or combination thereof selected from any of the following proteins: a transcriptional activator, a transcriptional repressor, a site-specific nuclease, a recombinase, an inhibitory immune receptor, an activating immune receptor. For example, the transcriptional activator comprises GLA4, GLA4-VP64, or a fragment thereof. For example, the transcriptional activator comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology to the amino acid sequence shown in SEQ ID NO: 28. For example, the intracellular domain of the chimeric polypeptide is a Cas9 polypeptide. For example, the intracellular domain of the chimeric polypeptide is a recombinase. For example, the intracellular domain of the chimeric polypeptide is an inhibitory immune receptor. For example, the intracellular domain of the chimeric polypeptide is an activating immunoreceptor.

[0374] The chimeric polypeptide may comprise an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology to any one of the amino acid sequences set forth in SEQ ID No: 44, 45, 46, 47, 48 or 49.

[0375] The chimeric polypeptide may comprise an extracellular region as set forth in SEQ ID No: 1, 3, 4, 5, 6, 7, 8, 9, 10, or 65, a transmembrane region as set forth in SEQ ID No: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, and an intracellular region as set forth in SEQ ID No: 28, wherein any of the amino acid sequences shown in the sequence, when linked, has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology. The chimeric polypeptide provided herein includes a binding domain that specifically binds to a target molecule. The binding domain of the chimeric polypeptide may specifically bind to one or more target molecules. For example, the chimeric polypeptide includes a linker inserted between the binding domain and the receptor structure regulatory domain. For example, the binding domain includes an antibody, an antigen, a ligand, a receptor, a target (e.g., a tag FLAG), an Fc receptor, an extracellular matrix component, a cell adhesion molecule, a non-antibody molecule scaffold, or a combination thereof. For example, the binding domain of the chimeric polypeptide includes an antigen binding domain. For example, the antigen binding domain includes an antibody-based recognition scaffold. For example, the antigen binding domain includes an antibody. For example, the antibody included in the antigen binding domain specifically binds to a tumor antigen, a disease-associated antigen, or an extracellular matrix component. For example, the antibody included in the antigen binding domain specifically binds to a cell surface antigen, a soluble antigen, or an antigen fixed on an insoluble substrate. For example, the antigen binding domain includes a single-chain antibody Fv (scFv). For example, the antibody included in the antigen binding domain can specifically bind to multiple antigens. For example, the antigen binding domain includes a nanobody, a single domain antibody, a double-chain antibody, a three-chain antibody, or a minibody, or a combination thereof. For example, the antigen binding domain is a non-antibody based recognition scaffold, such as an avimer, DARPin, ad nectin, avimer, affibody, anticalin or affilin. For example, the antibody is a single domain antibody, a single chain antibody, a diabody, a three chain antibody, a mini antibody, a F(ab')2 fragment, a F(ab)v fragment, a scFv, a single domain antibody (sdAb) and a functional fragment thereof or a combination thereof.

[0376] The binding domain of the chimeric polypeptide can include an antigen, such as an endogenous antigen or an exogenous antigen. For example, the binding domain comprises a ligand for a receptor. For example, the binding domain comprises a receptor. For example, the binding domain comprises a cell adhesion molecule (e.g., all or a portion of the extracellular domain of a cell adhesion molecule). For example, the binding domain comprises a portion of a polymerizing domain.

[0377] The binding domain of the chimeric polypeptide specifically binds to an antigen as defined in the terms "tumor antigen" or "pathogen antigen" herein. For example, the binding domain comprises an antibody that specifically binds to CD38, GPRC5D, Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvIII, BCMA, CD123, MOG, B7H3, NKG2A, FcRH5, or a combination thereof. For example, the binding domain comprises an antibody VH or VL or scFV that specifically binds to Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvIII, BCMA, CD123, MOG, B7H3, NKG2A, FcRH5, or a combination thereof. For example, the binding domain comprises an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or homologous to a sequence of amino acids set forth in SEQ ID NO:43, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, or 83. For example, the binding domain comprises: SEQ ID NOs: 116 and 129, SEQ ID NOs: 117 and 130, SEQ ID NOs: 118 and 131, SEQ ID NOs: 119 and 132, SEQ ID NOs: 120 and 133, SEQ ID NOs: 121 and 134, SEQ ID NOs: 122 and 135, SEQ ID NOs: 123 and 136, SEQ ID NOs: 124 and 137, SEQ ID NOs: 125 and 138, SEQ ID NOs: 126 and 139, SEQ ID NOs: 127 and 140, and / or SEQ ID NOs: 128 and 129. or an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or homologous to any of the preceding proteins.

[0378] The present application provides chimeric polypeptides that specifically bind to Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvIII, BCMA, CD123, MOG, B7H3, NKG2A or FcRH5. For example, the chimeric polypeptide comprises: a binding domain as shown in SEQ ID NO: 43, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 or 83, an extracellular region as shown in SEQ ID No: 1, 3, 4, 5, 6, 7, 8, 9, 10 or 65, a transmembrane region as shown in SEQ ID No: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, and a transmembrane region as shown in SEQ ID The intracellular region shown in No: 28, any of the amino acid sequences shown after sequential connection has an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical or homologous to any of the amino acid sequences shown in No: 28.

[0379] Jurkat responder cells or human T cells containing the chimeric polypeptide GPC3-synE, GPC3-synE-del1, GPC3-synE-del2, GPC3-synE-del3, GPC3-synE-del23, or GPC3-synE-EGFn (n=1, 2, 3, 4) that recognizes the tumor antigen GPC3 have the following characteristics: gene expression can be induced after co-incubation with GPC3-positive tumor cells SK-Hep1-GPC3, HuH7, or PLC / PRF / 5; gene expression cannot be induced after co-incubation with GPC3-negative tumor cells SK-Hep1.

[0380] Jurkat responder cells or human T cells containing the chimeric polypeptide MOG-synE, MOG-synE-del1, MOG-synE-del2, MOG-synE-del3, or MOG-synE-del23 that recognizes the tumor antigen MOG have the following characteristics: gene expression can be induced after co-incubation with MOG-positive cells K562-mMOG, K562-hMOG (also known as K562-huMOG), 293T-mMOG, or 293T-Hmog (also known as 293T-huMOG); and gene expression cannot be induced after co-incubation with MOG-negative cells K562 or 293T.

[0381] Jurkat responder cells or human T cells containing the chimeric polypeptide CD123-synE, CD123-synE-del1, CD123-synE-del2, CD123-synE-del3 or CD123-synE-del23 that recognizes the tumor antigen CD123 have the following characteristics: gene expression can be induced after co-incubation with CD123-positive cells Molm13, MV-4-11 or THP-1; gene expression cannot be induced after co-incubation with CD123-negative cells.

[0382] In one example, the chimeric polypeptide comprises a nucleotide sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity or homology to any one of the nucleotide sequences set forth in SEQ ID NO: 55, 56, 57, 58, or 59, or the amino acid sequence encoded thereto.

[0383] The target molecule that binds to the chimeric polypeptide of the present application is also called a ligand or a target antigen.

[0384] The target molecule bound by the chimeric polypeptide can be membrane-bound. The target molecule can be present on the cell surface. The target molecule can be immobilized on an insoluble substrate (e.g., polyethylene, polystyrene, polyvinyl pyrrolidone, polycarbonate, nitrocellulose, etc.). The target molecule can be soluble. The target molecule can be present in the extracellular environment (e.g., extracellular matrix). The target molecule can be present in an artificial matrix. The target molecule can be present in an acellular environment. The target molecule can be present in an insoluble support in various forms, such as a plate, a tissue culture dish, a column, etc. The target molecule can be present in the extracellular matrix (ECM) (e.g., the antigen is a component of the ECM). The target molecule can be present in an artificial matrix. The target molecule can be present in an acellular environment. Target molecules include polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, and lipopolysaccharides. For example, the target molecule is selected from the group consisting of: differentiation marker clusters, cell surface receptors, adhesion proteins, integrins, mucins, lectins, and tumor antigens.

[0385] The target molecule bound by the chimeric polypeptide can be a cluster of differentiation (CD) markers. For example, the CD markers are selected from the following group: CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placental-like 2 (ALPPL2), B cell maturation antigen (BCMA), blue fluorescent protein (BFP), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), signal regulatory protein alpha (SIRPα).

[0386] The target molecule bound by the chimeric polypeptide can be an antigen. For example, the target molecule includes a tumor antigen and / or a pathogen antigen. For example, the tumor antigen is selected from the group consisting of GPRC5D, Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, EGFR, , BCMA, B7H3, CD7, NKG2D-Ligand, CD19, B7H3, ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, FLT3, GD2, GD3, GM3, HER2 (ERBB2), IGLL1, IL 11Ra, IL13Ra2, CD 117, MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, FcRH5, cMet or Axl.

[0387] In one embodiment, the chimeric polypeptide and the molecule whose transcriptional expression is regulated by it recognize different tumor antigens.

[0388] In the treatment of tumors with antigenic heterogeneity, the proportion of cells positive for the first target molecule (exemplarily, EGFRvⅢ) (i.e., the positive rate) is low and the positive rate of the second target molecule (exemplarily, IL13Ra2) is high; such tumor cells will escape the treatment targeting the first target molecule, thereby reducing the efficacy; due to the widespread expression of the second target molecule, the treatment targeting the second target molecule may produce off-target toxicity. For example, a chimeric polypeptide that binds to the first target molecule is constructed to trigger transcriptional regulation to recognize the expression of an exogenous receptor for the second target molecule, thereby achieving specific and extensive killing of tumor cells that express the second target molecule and partially express the first target molecule. For example, the killing effect of the CAR targeting the second target molecule depends on the triggered transcriptional regulatory activity of the chimeric polypeptide that binds to the first target molecule, that is, the killing effect requires the presence of the first target molecule and the second target molecule in the environment. Using the chimeric polypeptide synE or its mutant or truncation or its modified body that binds to the first target molecule to trigger the expression of an exogenous receptor (e.g., CAR) targeting the second target molecule can significantly improve the anti-tumor effect of the CAR-T cells regulated by the chimeric polypeptide transcription against tumors with heterogeneous expression of the first target molecule compared to synNotch.

[0389] In the treatment of tumors with heterogeneous target molecules, the first target molecule (exemplary, Mesothelin) is highly expressed but also expressed in normal tissues, and the second target molecule (exemplary, Claudin18.2) is highly expressed but also expressed in other normal tissues; targeting the first target molecule or the second target molecule alone will lead to off-target toxicity. For example, using a chimeric polypeptide synE or its mutant or truncation or modified form, synJagged2EC, synE-APLP2(TM) that specifically binds to the first target molecule to trigger transcriptional regulation of the expression of an exogenous receptor targeting the second target molecule can significantly improve the anti-tumor effect of CAR-T cells that bind to the chimeric polypeptide and trigger transcriptional regulation against tumors with heterogeneous expression of the first and second target molecules, compared to synNotch. For example, using a chimeric polypeptide synE or its mutant or truncation or modified form, synJagged2EC, synE-APLP2(TM)) that specifically binds to a second target molecule to trigger and regulate the expression of an exogenous receptor targeting a first target molecule can significantly improve the anti-tumor effect of CAR-T cells triggered and regulated by the chimeric polypeptide against tumors with heterogeneous expression of the first and second target molecules compared to synNotch.

[0390] In one example, cells expressing CARs targeting a second target molecule will kill each other during culture, thereby affecting the cell activity and yield. For example, cells expressing a chimeric polypeptide targeting a first target molecule (e.g., a tumor antigen) are cultured in vitro. Due to the lack of contact with tumor cells expressing the tumor antigen, the chimeric polypeptide does not trigger transcriptional regulation of the chimeric receptor (e.g., CAR, recombinant TCR) targeting the second target molecule contained in the cell, thereby reducing the mutual killing phenomenon of the cells during in vitro and in vivo culture, thereby increasing the survival and / or expansion of the cells during in vitro culture. For example, compared with constitutively expressed NKG2D-CAR-T cells, immune cells comprising NKG2D-CARs expressing chimeric polypeptides that recognize CD123 have enhanced in vitro and in vivo survival and anti-tumor effects.

[0391] The immune cells of the regulated NKG2D-CAR expressed by the chimeric polypeptide can resist NK cell killing and increase the persistence and / or transplant survival rate of the immune cells expressing the regulated NKG2D-CAR in the presence of host immune cells. For example, it can be used to prepare universal CAR-T cells. For example, the combination of NKG2D-CAR-T cells expressed by the chimeric polypeptide and T cells expressing chimeric receptors (such as CAR) that recognize tumor antigens.

[0392] A chimeric polypeptide that specifically binds to a first target molecule is used to trigger transcriptional regulation of the expression of a CAR targeting a second target molecule. After the cell specifically binds to the first target molecule on the tumor cell, transcriptional regulation is triggered to express an exogenous receptor targeting the second target molecule in the cell, thereby killing the tumor cells expressing the second target molecule, and / or attacking the host immune cells expressing the second target molecule to increase the survival and proliferation of the cells, thereby further improving the anti-tumor activity. For example, the second target molecule includes an NK cell marker selected from: NKG2 receptor family, such as NKG2A, NKG2D, NKG2D ligand, NKG2C, NKG2D-ligand, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, 15KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCR), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specifically expressed antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, TIGIT, CS1. For example, a chimeric polypeptide triggering transcriptional regulation of the first target molecule specifically binding to leukemia cells (such as CLL1, CD123) recognizes the expression of an exogenous receptor for NKG2D ligand, thereby killing host NK cells, thereby resisting host immune rejection. For example, the second target molecule is an NKG2D ligand. In one example, the immune cells expressed by the CAR comprising a chimeric polypeptide regulating and recognizing a tumor antigen can kill host NK cells or resist the immune rejection of host NK cells. For example, the immune cells expressed by the CAR comprising a chimeric polypeptide regulating and recognizing NKG2D ligands for CD123 or CLL1 can kill host NK cells or resist the immune rejection of host NK cells, thereby improving the survival and amplification of the immune cells in vitro and in vivo, and enhancing anti-tumor effects. For example, the immune cells expressed by the CAR comprising a chimeric polypeptide regulating and recognizing a tumor antigen for a solid tumor can kill host NK cells or resist the immune rejection of host NK cells. For example, a chimeric polypeptide comprising a tumor antigen that recognizes a solid tumor regulates immune cells expressing a CAR that recognizes an NKG2D ligand, which can kill host NK cells or resist immune rejection by host NK cells, thereby improving the survival and proliferation of the immune cells in vivo and in vitro, and enhancing the anti-tumor effect. For example, the solid tumor cells express an NKG2D-ligand.

[0393] The chimeric polypeptides provided herein bind to trigger-regulated genes and are operably linked to transcriptional control elements that are activated or inhibited by the intracellular domain of the chimeric polypeptide. For example, the regulated gene is expressed by a promoter regulated by GAL-4, tetR, ZFHD1, HNF1A, or HAP1. For example, the regulated gene expression product is selected from the group consisting of: non-coding RNA, cytokines, cytotoxins, chemokines, immunomodulators, pro-apoptotic factors, anti-apoptotic factors, hormones, differentiation factors, dedifferentiation factors, recombinant TCR receptors, CARs, reporter genes, or combinations thereof.

[0394] Chimeric polypeptide binding trigger regulatory genes include but are not limited to: chemokines, chemokine receptors, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, hormones, metabolic enzymes, proliferation inducers, receptors, small molecule second messenger synthetases, CARs, recombinant TCR receptors, T cell receptors, second chimeric polypeptides, transcription activators, transcription repressors, transcription activators, transcription repressors, translation regulators, antibody molecules, translation activators, translation repressors, activating immune receptors, apoptosis inhibitors, apoptosis inducers, immune activators, immunosuppressants, and inhibitory immune receptors. For example, the nucleic acid sequence encoding the regulatory gene or a combination thereof is operably connected to a transcriptional control element, which is activated or inhibited by the intracellular domain of the chimeric polypeptide. For example, the transcriptional control element includes UAS (SEQ ID NO: 29). For example, the intracellular domain of the chimeric polypeptide includes GAL4-VP64 (SEQ ID NO: 28); the chimeric polypeptide binds to a trigger regulatory gene nucleic acid sequence and is operably connected to UAS. For example, the intracellular domain of the chimeric polypeptide includes GAL4-VP64; the chimeric polypeptide binds to a trigger regulatory gene nucleic acid sequence operably linked to a UAS-CMV promoter (SEQ ID NO: 30).

[0395] For example, after the chimeric polypeptide in the cell binds to the target molecule, it triggers the receptor to hydrolyze, causing the receptor to cleave and release the intracellular domain, thereby inducing the cell to express cytokines or chemokines including: interferons (α-interferon, β-interferon, γ-interferon), interleukins (IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15 , IL-16, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-24), tumor necrosis factor (TNF-α), transforming growth factor-β, TRAIL, MIP-1, MIP-1β, MCP-1, RANTES, IP10, CCL2, CCL3, CCL5, CCL17, CCL19, CCL21, CCR7, CXCL9, CXCL10, CXCL11, CXCL16, MCSF. For example, cytokines include IL-2, IL-7, IL-9, IL-12, IL-15, IL-18, CCL21, or a combination thereof.

[0396] Cells (e.g., T, NK and / or NKT cells) can be transduced with a viral vector encoding a chimeric polypeptide. The viral vector can be a lentiviral vector or a retroviral vector. The viral vector can be a pRRLSIN vector. The transduced cells can stably express the chimeric polypeptide. When the chimeric polypeptide is localized on the cell membrane, it can be hydrolyzed in the transmembrane region after recognizing the target molecule, and release the intracellular transcription regulatory domain GAL4-VP64, which is transferred into the cell nucleus and can activate transcription of the promoter containing the UAS sequence. By connecting the regulated gene to the downstream of the UAS promoter and transferring it into cells expressing the chimeric polypeptide, the target-dependent gene-specific expression of the target can be achieved, which is regulated by the chimeric polypeptide.

[0397] In one example, after the chimeric polypeptide in the cell binds to the first target molecule, it triggers the receptor to undergo hydrolysis, causing the receptor to cleave, releasing the intracellular domain, and inducing the cell to express a CAR or recombinant TCR that recognizes the second target molecule. The nucleic acid sequence of the CAR or recombinant TCR is operably linked to a transcriptional control element, which is activated or inhibited by the intracellular domain of the chimeric polypeptide. For example, the CAR or recombinant TCR specifically recognizes: tumor antigens, cancer cell-associated antigens, blood tumor antigens, solid tumor antigens, cell surface antigens, intracellular antigens, etc.

[0398] In one embodiment, the first target molecule and the second target molecule are different and are respectively selected from: 1) blood tumor antigens: CD19 (expressed on B cells), CD20 (expressed on B cells), CD22 (expressed on B cells), CD30 (expressed on B cells), CD33 (expressed on bone marrow cells), CD70 (expressed on B cells / T cells), CD123 (expressed on bone marrow cells), κ (expressed on B cells), Lewis Y (expressed in bone marrow cells), NKG2D ligand (expressed in bone marrow cells), ROR1 (expressed in B cells), SLAMF7 / CS1 (expressed in myeloma cells, natural killer cells, T cells and most B cells), CD138 (expressed in malignant plasma cells in multiple myeloma), CD56 (expressed in myeloma cells, nerve cells, natural killer cells, T cells and trabecular osteoblasts), CD38 (expressed in B cells / T cells) and CD160 (expressed in NK cells / T cells); 2) solid tumor antigens: B7H3 (expressed in malignant tumors and gliomas), CAIX (expressed in kidney), CD44v6 / v7 (expressed in cervix), CD171 (expressed in neuroblastoma), CEA (expressed in colon), EGFRvIII (expressed in gliomas), E GP2 (expressed in cancer), EGP40 (expressed in colon), EphA2 (expressed in glioma, lung), ErbB2 (HER2) (expressed in breast, lung, prostate, glioma), ErbB receptor family (expressed in breast, lung, prostate, glioma), ErbB3 / 4 (expressed in breast, ovary), HLA-A1 / MAGE1 (expressed in melanoma), HLA-A2 / NY-ESO-1 (expressed in malignant tumor, melanoma), FR-a (expressed in ovary), FAR (expressed in rhabdomyosarcoma), GD2 (expressed in neuroblastoma, malignant tumor, melanoma), GD3 (expressed in melanoma, lung cancer), HMW-MAA (expressed in melanoma), IL11Ra (expressed in osteosarcoma), IL13Ra2 (expressed in glioma), Lewis Y (expressed in breast / ovary / pancreas), mesothelin (expressed in mesothelioma, breast, pancreas), Muc1 (expressed in ovary, breast, prostate), NCAM (expressed in neuroblastoma, colorectal), NKG2D ligand (expressed in ovary, malignant tumors),tumors), PSCA (expressed in prostate and pancreas), PSMA (expressed in prostate), TAG72 (expressed in colon), VEGFR-2 (expressed in tumor vasculature), Axl (expressed in lung cancer), Met (expressed in lung cancer), α5β3 (expressed in tumor vasculature), α5β1 (expressed in tumor vasculature), TRAIL-R1 / TRAIL-R2 (expressed in solid tumors (colon, lung, pancreas) and hematological malignancies), RANKL (expressed in prostate cancer and bone metastasis), tenascin (expressed in glioma, Epithelial tumors (breast, prostate), EpCAM (expressed in epithelial tumors (breast, colon, lung)), CEA (expressed in epithelial tumors (breast, colon, lung)), gpA33 (expressed in colorectal cancer), mucin (expressed in epithelial tumors (breast, colon, lung, ovary)), TAG-72 (expressed in epithelial tumors (breast, colon, lung)), EphA3 (expressed in lung, kidney, melanoma, glioma, hematological malignancies), and IGF1R (expressed in lung, breast, head and neck, prostate, thyroid, glioma). Examples of surface and intracellular antigens include, for example, Her2 (ERBB2), MAGE-A1 (MAGEA1), MART-1 (MLANA), NY-ESO (CTAG1), WT1, MUC17, MOG, and MUC13. In one embodiment, the first and second target molecules are selected from the group consisting of BCMA, B7H6, CAIX, CD123, CD138, CD171, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CEA, CS1, EGFRvIII, EGP2, EGP40, Erb family members (ERBB1, ERBB2, ERBB3, ERBB4), FAP, fetal acetylcholine receptor (AChR), folate receptor alpha (FOLR1), , folate receptor beta (FOLR2), GD2, GD3, GPC3, IL-13Ra2 (IL13RA2), kappa light chain (IGK), Lewis-Y, mesothelin (MSLN), mucin-1 (MUC1), mucin-16 (MUC16), NKG2D ligand, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), and anaplastic lymphoma receptor tyrosine kinase (ALK), MOG.

[0399] In one example, the binding domain of the chimeric polypeptide specifically binds to a first target molecule, and the CAR specifically binds to a second target molecule different from the first target molecule, selected from the group consisting of: Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, EGFR, EGFRvIII, BCMA, CD7, NKG2D-Ligand, MOG, CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL 11Ra, IL13Ra2, CD117, MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPα, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, or Axl.

[0400] Regulatory B7H3-CAR-T cells: T cells containing a MOG-binding chimeric polypeptide and a B7H3-CAR. The MOG-binding chimeric polypeptides (MOG-synE, its mutants, truncations, or modified forms, synJagged2EC, synE-APLP2(TM)), synNTOCH, and syn-CD8) can trigger transcriptional regulation of B7H3-CAR expression. Regulatory B7H3-CAR-T cells exhibit one or more of the following properties: 1) They effectively kill B7H3-positive glioma cells (such as U87 and U251) after incubation with MOG-positive cells; 2) they release the cytokine IL-2 only in the presence of MOG; and 3) their cytotoxicity against B7H3-positive glioma cells is diminished or absent when co-incubated with cells that low or no MOG expression. MOG-synE-del3 demonstrates excellent regulatory capabilities for the initiation of anti-tumor effects by CAR-T cells. The binding domain of the chimeric polypeptide can be a ligand / receptor that binds MOG; or an antibody that binds MOG: scFv, Fab, single domain antibody, or fully human antibody or fragment thereof.

[0401] Regulatory NKG2D-CAR-T cells: T cells comprising a chimeric polypeptide that binds to CD123 and NKG2D-CAR (binding to NKG2D ligand); and the chimeric polypeptide that binds to CD123, CD123-synE or its mutant or truncation or its modified form, synJagged2EC, synE-APLP2(TM)), synNOTCH, syn-CD8, can trigger transcriptional regulation of NKG2D-CAR expression. Regulatory NKG2D-CAR-T cells possess one or more of the following characteristics: 1) After incubation with CD123-positive cells, they can effectively kill NKG2D ligand-positive tumor cells (e.g., MV-4-11, THP-1) or immune cells (e.g., NK cells, T cells, NKT cells), while having weak killing effects on cells with low NKG2D ligand expression (e.g., KG-1, Molm13); 2) When co-incubated with cells with low or no CD123 expression, the killing effect on NKG2D ligand-positive cells is weakened or absent (e.g., SK-Hep1, K562). The killing ability of NKG2D-CAR-T cells regulated by CD123-synE or CD123-synE-del3 is NKG2D ligand-dependent, while cells with high CD123 expression alone (e.g., Molm13) do not induce NKG2D ligand-independent killing. Placing NKG2D-CAR under the control of CD123-synE or CD123-synE-del3 can improve the in vivo survival and anti-tumor efficacy of regulated NKG2D-CAR-T cells. Regulated NKG2D-CAR-T cells can recognize target cells through CD123-synE or CD123-synE-del3. The killing effect of NKG2D-CAR-T cells still requires the activation of NKG2D ligands on the target cell surface, and the downstream signals of CD123-synE or CD123-synE-del3 do not cross-influence the downstream signals of NKG2D-CAR.

[0402] In one embodiment, a transcription factor triggered by a chimeric polypeptide in response to a first target molecule drives the expression of a CAR in response to a second target molecule, so that the CAR is active and activates T cells only in the presence of the first and second target molecules. For example, the first target molecule and the second target molecule are ASGR1 and GPC3, EGFRvⅢ and IL13Ra2, EGFRvⅢ and B7H3, Mesothelin and Claudin18.2, Claudin18.2 and Mesothelin, FAP and Claudin18.2, CLL1 and NKG2D ligand, CD123 and NKG2D ligand, MOG and B7H3. For example, the chimeric polypeptide and CAR respectively include: the sequence shown in SEQ ID NO: 46, 50; or the sequence shown in SEQ ID NO: 47, 50; or the sequence shown in SEQ ID NO: 48, 51; or the sequence shown in SEQ ID NO: 48, 52; or the sequence shown in SEQ ID NO: 48, 53; or the sequence shown in SEQ ID NO: 48, 54; or the sequence shown in SEQ ID NO: 49, 51; or the sequence shown in SEQ ID NO: 49, 52; or the sequence shown in SEQ ID NO: 49, 53; or the sequence shown in SEQ ID NO: 49, 54.

[0403] U87 and U251 cells express endogenous IL13Ra2 but not EGFRvIII. T cells containing a chimeric peptide that regulates CAR expression were tested for their ability to kill cells expressing at least two or more target molecules with varying positivity rates. For example, EGFRvIII-positive and EGFRvIII-negative cells were mixed in varying proportions to simulate natural gliomas that are IL13Ra2-positive and only partially EGFRvIII-positive. IL13Ra2-CAR-T cells, controlled by the chimeric peptide EGFRvIII-synE, were able to significantly kill EGFRvIII-heterogeneous tumors both in vitro and in vivo, and also had a killing effect on tumor cell populations with low EGFRvIII positivity rates.

[0404] The nucleic acid sequence encoding CAR is operably connected to a transcriptional control element, which is regulated by the intracellular domain of the chimeric polypeptide. For example, the nucleic acid sequence encoding CAR is operably connected to a UAS-CMV promoter, and the intracellular domain of the chimeric polypeptide includes GLA4-VP64. For example, the extracellular antigen binding region of CAR includes an antibody that recognizes B7H3, Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvⅢ, BCMA, CD7, CD123, NKG2D ligands, or a combination thereof. For example, the extracellular antigen binding region of CAR includes an antibody scFV that recognizes B7H3, Mesothelin, FAP, Claudin18.2, CLL1, CD19, GPC3, WT1, HER2, EGFR, EGFRvⅢ, BCMA, CD7, CD123, NKG2D ligands, or a combination thereof. For example, the extracellular antigen binding region of the CAR comprises an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or homologous to an amino acid sequence as set forth in SEQ ID NO: 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. For example, an IL13Ra2-CAR, B7H3-CAR, Claudin18.2-CAR, Mesothelin-CAR, or NKG2D-CAR whose expression is regulated by the chimeric polypeptide is operably linked to a transcriptional control element that is regulated by the intracellular domain of the chimeric polypeptide. For example, IL13Ra2-CAR, B7H3-CAR, Claudin18.2-CAR, Mesothelin-CAR or NKG2D-CAR whose expression is regulated by a chimeric polypeptide is inserted downstream of the UAS-CMV promoter, and the intracellular domain of the chimeric polypeptide includes Gal4-VP64. The “Z-CAR-T expressing X-chimeric polypeptide Y” or “X-chimeric polypeptide YZ-CAR-T” or “Z-CAR-X-chimeric polypeptide Y” described in the examples of the present application is used to describe T cells (also referred to as regulated Z-CAR-T cells) comprising a chimeric polypeptide Y targeting antigen X that regulates the expression of a CAR recognizing antigen Z. The Z-CAR is inserted downstream of the UAS-CMV promoter, and the intracellular domain of the chimeric polypeptide Y includes Gal4-VP64. In the above, the symbols X and Z are different tumor antigens, that is, the chimeric polypeptide Y can target antigen X, and the CAR can recognize antigen Z. When the chimeric polypeptide Y and CAR are used in combination in T cells, the expression of the CAR is regulated by the intracellular domain of the chimeric polypeptide Y.

[0405] For example, gastric cancer highly expresses both mesothelin and claudin18.2. To prevent claudin18.2-CAR-T cells from killing normal tissues expressing claudin18.2, the killing effect of claudin18.2-CAR-T cells is limited to the tumor area. For example, claudin18.2-CAR-T cells expressing mesothelin-synEphrinB2EC can significantly kill gastric cancer cells that are both mesothelin- and claudin18.2-positive.

[0406] The CAR extracellular antigen binding region and the transmembrane region can be directly connected or connected by a hinge. For example, the hinge includes a CD8 hinge, for example, a sequence with 95-100% identity to SEQ ID NO: 31. For example, the nucleic acid molecule encoding the CAR includes a polynucleotide encoding a signal peptide upstream. For example, the signal peptide includes a CD8 signal peptide, for example, a sequence with 95-100% identity to SEQ ID NO: 37.

[0407] The transmembrane domain of the CAR molecule of the present application may include a CD28 or CD8 transmembrane domain. For example, the CAR includes a sequence with 95-100% identity to SEQ ID NO: 32. For example, the CAR includes a sequence with 95-100% identity to SEQ ID NO: 33.

[0408] CAR includes intracellular signaling domain: primary signaling domain and / or costimulatory signaling domain. For example, the primary signaling domain includes CD3ζ intracellular domain, for example, with SEQ ID NO:36 having a sequence with 95-100% identity. For example, the costimulatory signaling domain includes CD28 and / or 4-1BB intracellular domain. For example, CAR includes a sequence with 95-100% identity to SEQ ID NO:34, 35, 38, 39 or 40.

[0409] The intracellular signaling domain of CAR may include a human CD3 ζ intracellular domain. The intracellular signaling domain of CAR may include a human CD3 ζ intracellular domain and a CD28 intracellular domain. The intracellular signaling domain of CAR may include a human CD3 ζ intracellular domain and a 4-1BB intracellular domain. The intracellular signaling domain of CAR may include a CD3 ζ intracellular domain, a CD28 intracellular domain and a 4-1BB intracellular domain.

[0410] Exemplarily, the CAR for transcriptional regulation of expression of the chimeric polypeptide of the present application includes the sequences shown in SEQ ID NO: 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, respectively, and the sequence sequentially connected to SEQ ID NO: 38, 39 or 40.

[0411] Chimeric polypeptide binding triggers regulatory genes including, but not limited to, trastuzumab (Herceptin, available from Chugai Pharmaceutical Co., Ltd.); bevacizumab (Avastin, available from Genentech, Inc.); infliximab (Remicade); rituximab (Rituxan, available from Biogen Idec Inc.); and adalimumab (Humira). For example, the above therapeutic antibodies are inserted into the UAS-CMV promoter regulated by Gal4-VP64.

[0412] The chimeric polypeptides and / or chimeric receptors that recognize NKG2D ligands of the present application may further include one or more other domains, including: a signal peptide, an epitope tag, an affinity domain, a nuclear localization signal (NLS), or a polypeptide that generates a detectable signal. For example, a nucleic acid molecule encoding the chimeric polypeptide may include a polynucleotide encoding a signal peptide upstream. For example, the signal peptide includes a CD8 signal peptide, e.g., a sequence having 95-100% identity to SEQ ID NO: 37.

[0413] The nucleotide sequence encoding the chimeric polypeptide of the present application is operably linked to a transcriptional control element (e.g., a promoter, enhancer, etc.). For example, the transcriptional control element is inducible. For example, the transcriptional control element is constitutive. For example, the promoter is functional in eukaryotic cells. For example, the promoter is a cell type-specific promoter. For example, the promoter is a tissue-specific promoter.

[0414] The expression vector of the chimeric polypeptide and / or chimeric receptor that recognizes NKG2D ligand of the present application can be a viral vector, for example, an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, a retroviral vector, etc. For example, a retroviral vector (gamma-retrovirus or lentivirus) is used to introduce nucleic acid molecules into cells. Non-viral vectors can also be used. Transduction can use any suitable viral vector or non-viral delivery system. In the specific embodiments of the present application, a pRRLSIN vector is constructed to express a chimeric polypeptide. In the specific embodiments of the present application, a pRRLSIN vector is constructed to express a chimeric polypeptide in combination with a triggering transcriptional regulatory gene, for example, a chimeric polypeptide in combination with a CAR that triggers transcriptional regulation. A chimeric polypeptide or CAR can be constructed with auxiliary molecules (e.g., cytokines) in a single polycistronic expression cassette, multiple expression cassettes of a single vector, or multiple vectors. Examples of elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Baculovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis Virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides).

[0415] Other viral vectors that can be used include, for example, adenoviral, lentiviral and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus.

[0416] Non-viral methods can also be used for genetic modification of engineered cells. For example, nucleic acid molecules can be introduced into immune cells by lipofection, asialomucoid-polylysine coupling, or microinjection under surgical conditions. Other non-viral gene transfer methods include in vitro transfection using transposons, liposomes, calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. It is also possible to first transfer nucleic acid molecules into cell types that can be cultured in vitro (e.g., autologous or allogeneic primary cells or their progeny), and then inject the cells (or their progeny) modified with the nucleic acid molecules into the target tissue of the subject or inject them systemically.

[0417] The present application provides engineered cells genetically modified with the nucleic acids of the present application: engineered cells genetically modified with nucleic acids encoding the chimeric polypeptides of the present application and / or chimeric receptors that recognize NKG2D ligands; or engineered cells genetically modified with nucleic acids comprising the chimeric polypeptides of the present application and chimeric polypeptides that bind to trigger transcriptional regulatory genes (e.g., chimeric receptors that recognize NKG2D ligands). The present application provides a method for regulating the activity of cells expressing the chimeric polypeptides of the present application. The method generally comprises contacting the engineered cells with a molecule of interest, inducing cleavage of the chimeric polypeptide, thereby releasing the intracellular domain, and the release of the intracellular domain regulates the activity of the cells.

[0418] In one example, the engineered cells are genetically modified to express the chimeric polypeptide of the present application, and are further genetically modified to express CAR. For example, the engineered cells are genetically modified with a nucleic acid comprising a nucleotide sequence encoding CAR, and the intracellular domain of the chimeric polypeptide is a transcriptional activator protein, and the nucleotide sequence encoding CAR is operably linked to a transcriptional control element activated by the intracellular domain of the chimeric polypeptide. Many CAR polypeptides have been described in the art, and any of them are applicable to the present application.

[0419] In one example, the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell or a stem cell. Stem cells include human pluripotent stem cells (including human induced pluripotent stem cells (iPSC) and human embryonic stem cells). For example, the engineered cell is an immune cell. For example, the engineered cell is a primary cell. For example, the engineered cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, other T cells or a combination thereof. The engineered cell can be a cell of autologous origin or a cell of allogeneic origin.

[0420] The present application provides a method for regulating the activity of an engineered cell expressing a chimeric polypeptide of the present application, comprising: contacting the engineered cell containing the chimeric polypeptide of the present application with a target molecule, wherein the target molecule, upon binding to the binding domain of the chimeric polypeptide, induces cleavage at a protease cleavage site in the receptor regulatory domain of the chimeric polypeptide, releasing the intracellular domain, wherein the release of the intracellular domain regulates the activity of the engineered cell. For example, the contacting is performed in vivo, ex vivo, or in vitro. For example, the target molecule is on the surface of the target cell, immobilized on an insoluble substrate, present in the extracellular matrix, present in an artificial matrix, or is soluble. For example, the release of the intracellular domain regulates the proliferation of the engineered cell. For example, the release of the intracellular domain regulates apoptosis of the engineered cell. For example, the release of the intracellular domain induces cell death through a mechanism other than apoptosis. For example, the release of the intracellular domain regulates gene expression in the engineered cell through transcriptional regulation, chromatin regulation, translation, trafficking, or post-translational processing. For example, the release of the intracellular domain regulates differentiation of the engineered cell. For example, the release of the intracellular domain regulates migration of the engineered cell. For example, the release of the intracellular domain regulates the expression and secretion of a molecule from the engineered cell. For example, the release of the intracellular domain regulates the adhesion of the engineered cell to the target cell or to the extracellular matrix. For example, the release of the intracellular domain induces the re-expression of the gene product in the engineered cell. For example, the release of the intracellular domain induces the cessation of expression of the gene product in the engineered cell. For example, the gene product is a transcriptional activator, a transcriptional repressor, a modified TCR, a chimeric antigen receptor, a translation regulator, a cytokine, a hormone, a chemokine or an antibody. For example, the released transcription factor regulates the differentiation of the engineered cell, and the engineered cell is an immune cell, a stem cell, a progenitor cell or a precursor cell. For example, the method is used to treat a tumor.

[0421] The present application provides a method for activating T cells, the method comprising: making T cells as described herein (wherein T cells are genetically modified with one or more nucleic acids comprising a nucleotide sequence encoding the following: i) the chimeric polypeptide to which the present application relates, and ii) CAR; wherein the domain of the chimeric polypeptide includes an antibody specific for a first target molecule, and wherein the contact causes the release of an intracellular domain (such as a transcriptional activator) of the chimeric polypeptide, expression of CAR polypeptides in T cells, wherein the CAR polypeptide activates T cells after binding to a second target molecule. For example, the first target molecule and the second target molecule are different tumor antigens; or the first target molecule is a molecule with tissue specificity and the second target molecule is a tumor antigen.

[0422] The present disclosure provides a method for regulating the activity of an engineered cell, the method comprising: contacting the engineered cell with an antigen immobilized on a surface, wherein the engineered cell expresses a chimeric polypeptide of the present disclosure, which can result in the release of an intracellular domain and regulate the activity of the engineered cell. For example, the intracellular domain is a transcription factor that regulates cell differentiation.

[0423] The present disclosure provides a method for locally regulating the activity of engineered cells, the method comprising: expressing a chimeric polypeptide comprising the present application in a cell; after the engineered cell contacts a target molecule, releasing the intracellular domain to regulate the activity of the engineered cell: expression of gene products of the cell, cell proliferation, apoptosis, non-apoptotic cell death, cell differentiation, cell dedifferentiation, cell migration, cell expression of secretory proteins, and cell adhesion.

[0424] The present application provides a method for treating a tumor in a subject having a tumor, the method comprising: i) genetically modifying T cells, NK cells and / or NKT cells obtained from an individual using a vector comprising a chimeric polypeptide encoding the present application, or a chimeric polypeptide and a CAR that binds to trigger transcriptional activity, wherein the chimeric polypeptide can specifically bind to an antigen expressed by tumor cells in the subject; ii) introducing the genetically modified T cells, NK cells and / or NKT cells into the subject, wherein the genetically modified T cells, NK cells and / or NKT cells recognize and kill tumor cells, thereby treating the tumor.

[0425] The present application provides a method for treating a tumor in a subject having a tumor, the method comprising: i) genetically modifying T cells, NK cells and / or NKT cells obtained from an individual using a vector comprising a chimeric polypeptide encoding the present application, or a chimeric polypeptide and a CAR that binds to trigger transcriptional activity, wherein the chimeric polypeptide can specifically bind to a certain tissue-specific marker in the subject; ii) introducing the genetically modified T cells, NK cells and / or NKT cells into the subject, wherein the genetically modified T cells, NK cells and / or NKT cells recognize and kill tumor cells, thereby treating the tumor.

[0426] The present application provides a method for inhibiting the activity of a target cell in a subject, the method comprising: administering to a subject a therapeutically effective amount of an engineered cell expressing a chimeric polypeptide of the present application, wherein the engineered cell inhibits the activity of the target cell in the subject and / or kills the target cell. For example, the target cell is a tumor cell. For example, the target cell includes, but is not limited to, acute myeloma leukemia cells, anaplastic lymphoma cells, astrocytoma cells, B cell cancer cells, breast cancer cells, colon cancer cells, ependymoma cells, esophageal cancer cells, glioblastoma cells, glioma cells, leiomyosarcoma cells, liposarcoma cells, hepatocellular carcinoma cells, lung cancer cells, mantle cell lymphoma cells, melanoma cells, neuroblastoma cells, non-small cell lung cancer cells, oligodendroglioma cells, ovarian cancer cells, pancreatic cancer cells, peripheral T cell lymphoma cells, renal cancer cells, sarcoma cells, gastric cancer cells, hepatocellular carcinoma cells, mesothelioma cells or sarcoma cells. For example, the target cell expresses a low level of the target molecule. For example, the engineered cells further comprise a CAR, a modified TCR, an exogenous cytokine, and / or a therapeutic monoclonal antibody that is transcriptionally activated upon binding of the chimeric polypeptide to a target molecule. For example, the chimeric polypeptide specifically binds to a first target molecule, and the CAR and / or modified TCR specifically binds to a second target molecule. For example, the first and second target molecules in the target cells are both tumor antigens, but there is heterogeneity in expression within the tumor. For example, the first and second target molecules in the target cells are both tumor antigens, and the first target molecule has a lower positive rate in tumor tissue than the second target molecule. For example, the first and second target molecules in the target cells are both tumor antigens, and the second target molecule has a lower positive rate in tumor tissue than the first target molecule. For example, the first target molecule is a tissue-specific molecule, and the second target molecule is a tumor antigen. For example, the first target molecule is not a tumor antigen but has tissue-specific expression, and the second target molecule is a tumor antigen. For example, the first target molecule has a lower positive rate than the second target molecule. For example, the method can improve the anti-tumor specificity of the engineered cells.

[0427] This application provides a method for preventing, alleviating, and / or treating tumors, comprising administering the cell composition and the pharmaceutical composition to a subject in need thereof. The cell composition and pharmaceutical composition provided herein have been described above, and the method for preventing, alleviating, and / or treating tumors provided herein includes all technical solutions thereof.

[0428] The present application provides a kit for inducing and / or enhancing an immune response and / or treating and / or preventing a tumor or pathogen infection in a subject. For example, the kit includes an effective amount of a chimeric polypeptide of the present application and / or a composition and pharmaceutical composition of a chimeric receptor that recognizes an NKG2D ligand. For example, the kit includes a sterile container; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container forms known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding drugs. For example, the kit includes a molecule encoding a chimeric receptor that recognizes an NKG2D ligand, a chimeric polypeptide, or a chimeric polypeptide that binds to a target molecule to trigger transcriptional activation (e.g., NKG2D-CAR), a modified TCR, an exogenous cytokine, and / or a therapeutic monoclonal antibody, which can be optionally included in one or more carriers.

[0429] In this application, the methods described in this application can also be interpreted as therapeutic uses, that is, the methods described in this application can all be considered as therapeutic uses of the compositions, nucleic acid molecules or engineered cells of this application or pharmaceutical uses for preparing corresponding therapeutic uses. For example, this application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for regulating the activity of engineered cells or the use of the above-mentioned engineered cells for preparing drugs for regulating the activity of engineered cells; this application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for activating engineered cells or the use of the above-mentioned engineered cells for preparing drugs for activating engineered cells; this application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for inhibiting the activity of target cells in a subject or for preparing drugs for inhibiting the activity of target cells in a subject; this application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for improving or treating the health status of subjects in need or for preparing drugs for improving or treating the health status of subjects in need. All the contents described in the above-mentioned application can also be applied to therapeutic uses or pharmaceutical uses.

[0430] The present application provides methods for inducing and / or increasing an immune response in a subject in need of a composition of the present application. The composition of the present application can be used to treat and / or prevent tumors in a subject. The composition of the present application can be used to prolong the survival of a subject suffering from a tumor. The composition of the present application can also be used to treat and / or prevent pathogen infection or other infectious diseases in human subjects such as those with immunosuppression. This method comprises administering an effective amount of the composition of the present application to achieve the desired effect, whether to alleviate an existing condition or to prevent recurrence. For treatment, the amount administered is an amount that effectively produces the desired effect. The effective amount can be provided by one or more administrations. The effective amount can be provided in large doses or by continuous infusion.

[0431] The present application provides a method for transducing a viral vector into a cell (e.g., an immune effector cell), the method being related to the activation and transduction of the cell to be transduced, the activation and transduction of the cell can be performed simultaneously, i.e., the input composition comprising the cell to be transduced, the cell stimulating agent to be transduced, and the viral vector particles carrying the recombinant nucleic acid are incubated together, or the activation can be performed first and then transduced, such as the input composition comprising the cell to be transduced, and the cell stimulating agent to be transduced are incubated together for activation, and then the viral vector particles carrying the recombinant nucleic acid are added and incubated, and the transduction activation and transduction total time of the recombinant nucleic acid are controlled to be completed within 72 hours, preferably within 48 hours, or within 36 hours, or within 24 hours. For example, the method provided is related to incubating and / or contacting a retroviral vector particle (e.g., a lentiviral vector) with a cell (e.g., an immune cell, e.g., a T cell) group, and before and / or simultaneously and / or after contacting or incubating the cell with the viral particle, using an ex vivo cell activation reagent (e.g., an anti-CD3 / anti-CD28 reagent) to activate and / or activate the T cell. Preferably, the cell is activated first and then viral transduction is performed. For example, when an input composition comprising cells to be transduced, a stimulatory agent for cells to be transduced, and viral vector particles carrying a recombinant nucleic acid are incubated together, the incubation time is no more than 72 hours for harvesting to obtain an output composition, wherein the output composition contains cells transduced with the recombinant nucleic acid; preferably, the incubation time is 1 hour to 72 hours; more preferably, the incubation time is 2 hours to 48 hours; more preferably, the incubation time is 2 hours to 36 hours; more preferably, the incubation time is 12 hours to 36 hours; more preferably, the incubation time is 12 hours to 24 hours; more preferably, the incubation time is 15 hours to 24 hours. For example, after purification treatment such as washing and centrifugation, the output composition can be prepared into a pharmaceutical preparation without further in vitro expansion and culture, that is, the drug prepared using the output composition does not require in vitro expansion before use in a subject (or patient). For example, a method for transducing cells with a viral vector comprises the following steps: step (1), incubating an input composition containing cells to be transduced and a stimulator for cells to be transduced for no more than 72 hours, step (2), adding viral vector particles containing a recombinant nucleic acid and incubating for no more than 24 hours, and step (3), harvesting to obtain an output composition, wherein the output composition contains cells transduced with the recombinant nucleic acid; preferably, the total incubation time of (1) and (2) does not exceed 72 hours; more preferably, the total incubation time of (1) and (2) does not exceed 60 hours, or does not exceed 48 hours, or does not exceed 32 hours, or does not exceed 28 hours, and more preferably, the total incubation time of (1) and (2) does not exceed 24 hours.For example, the incubation time of step (1) is 2-72 hours; preferably, the incubation time of step (1) is 2-71 hours; more preferably, the incubation time of step (1) is 2-48 hours; more preferably, the incubation time of step (1) is 2-32 hours; more preferably, the incubation time of step (1) is 2-28 hours; more preferably, the incubation time of step (1) is 3-24 hours; more preferably, the incubation time of step (1) is 5-24 hours; more preferably, the incubation time of step (1) is 7-24 hours; more preferably, the incubation time of step (1) is 7-23 hours; more preferably, the incubation time of step (1) is 10-23 hours; more preferably, the incubation time of step (1) is 15-23 hours; more preferably, the incubation time of step (1) is 15-22 hours. For example, the incubation time of step (2) is 30 minutes to 24 hours, preferably, the incubation time of step (2) is 30 minutes to 21 hours; preferably, the incubation time of step (2) is 30 minutes to 17 hours; preferably, the incubation time of step (2) is 30 minutes to 12 hours; preferably, the incubation time of step (2) is 30 minutes to 10 hours; preferably, the incubation time of step (2) is 30 minutes to 8 hours; preferably, the incubation time of step (2) is 1 hour to 8 hours; preferably, the incubation time of step (2) is 1 hour to 4 hours; more preferably, the incubation time of step (2) is 1 hour to 3 hours. For example, the recombinant nucleic acid can encode a receptor that recognizes a tumor antigen or a pathogen antigen, such as a recombinant T cell receptor. The resulting cells transduced with the recombinant nucleic acid can be used for adoptive immunotherapy. For example, the provided method can be used to prepare immune cells, such as T cells, for adoptive therapy, and the total activation transduction time of the method is controlled within 24 hours, or 36 hours, or 48 hours, or 72 hours. For example, the provided method shortens the engineering and / or preparation time of cells for adoptive cell therapy. For example, the input composition comprises a primary cell population, which has been obtained from a sample of a subject and / or enriched for a specific cell subset (such as a T cell). For example, a cell population (such as an input composition) can be a cell population that has been previously cryopreserved. For example, incubation and / or contact is started no more than or no more than about 1 hour, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 48 ​​hours or 72 hours after obtaining a sample containing primary cells (such as a single collection sample) from a subject. For example, the method produces an output composition wherein at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the total cells (or a particular target cell type, such as T cells) in the output composition are transduced with the viral vector and / or express the recombinant gene product encoded thereby.For example, at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the cells (e.g., T cells) in a cell population (e.g., an output composition) are transduced with retroviral vector particles according to the provided methods.

[0432] Activation or stimulation can be performed ex vivo or in vivo. For example, after incubating (e.g., transducing) cells with viral particles, the cells can be infused into the patient's body for in vivo activation and amplification. For example, the cell activator to be transduced can be one, two, or a combination of multiple. For example, the T cell activator can be a CD3 binding molecule (e.g., an antibody to CD3), a CD28 binding molecule (e.g., an antibody to CD28), recombinant IL-2, recombinant IL-15, recombinant IL-7, recombinant IL-21, or a mixture of at least two, such as an antibody to CD3 and an antibody to CD28, or an antibody to CD3, an antibody to CD28, or IL2. For example, the multiplicity of infection of the viral vector particles is not higher than 20; preferably, the multiplicity of infection is 0.5-20; more preferably, the multiplicity of infection is 1.5-20; more preferably, the multiplicity of infection is 3-20; more preferably, the multiplicity of infection is 3-12. For example, during or after incubation, the provided method may also include ex vivo culturing input compositions, output compositions and / or transduced cells, such as culturing under conditions of activated cells to induce their proliferation and / or activation. Activation is carried out in the presence of one or more activators. For example, the activator can be a CD3 binding molecule, a CD28 binding molecule, or a cytokine (such as recombinant IL-2, recombinant IL-15, recombinant IL-7 or recombinant IL-21). For example, the binding molecule is an antibody or an antigen binding fragment, such as an anti-CD3 antibody and / or an anti-CD28 antibody. For example, further culturing is carried out under conditions that achieve cell expansion, to produce cells of a therapeutically effective dose for administration to a subject by adoptive cell therapy. For example, the provided method avoids significantly changing the differentiation state of T cells and / or minimizing the change in the differentiation state of T cells in vitro during the introduction, transfer and / or transduction of T cells with nucleic acids encoding recombinant receptors (such as CARs). For example, memory T cells are produced according to the provided method, including stem cell memory T cells, central memory T cells, and effector memory T cells. For example, the proportion of cells transduced with recombinant nucleic acid (e.g., CAR T cells) in the cell output composition of the present application is lower than that in conventional processes. In a specific embodiment, the number of cells does not exceed 1*10 10 , 1*10 9 , 1*108 , 1*10 7 , 1*10 6 , 1*10 5 , or 1*10 4 . For example, the content of memory cell phenotype (such as memory T cells) in the output composition of cells transduced with recombinant nucleic acids in the present application is higher than that in conventional processes. For example, the content is at least 1.5 times, 2 times, 3 times, 4 times or 5 times higher. For example, memory T cells are cells with T central cell memory (TCM) phenotype, such as CD45RO+CCR7+CD62L+T cells and / or CD45RO+CCR7+CD27+CD28+CD62L+T cells. For example, one, more or all steps in the preparation of cells for clinical use (such as adoptive cell therapy) in the present application are carried out under sterile conditions. For example, one or more processes of enrichment, activation, transduction, or washing of cells are carried out in a closed system. For example, cells are treated in vitro for a shorter time, further shortening the time. For example, cells transduced with recombinant nucleic acids (such as CAR T cells) produced by the provided method exhibit longer persistence and / or reduced cell consumption when administered to a subject. For example, the provided methods produce cells transduced with recombinant nucleic acids (e.g., CAR T cells) that exhibit improved efficacy when administered to a subject. For example, the provided methods reduce the variability of cells during the preparation of cell therapy products. For example, eliminating the time required for ex vivo activation and transduction of cells improves the process of preparing cells transduced with recombinant nucleic acids for adoptive immunotherapy.

[0433] Provided herein is a method for incubating or contacting an input composition (including cells to be transduced) with retroviral vector particles (e.g., lentiviral vector particles). For example, the input composition is a composition of primary cells obtained from a subject, wherein, in some cases, a subpopulation or subset of cells has been selected and / or enriched. For example, when the cells to be transduced are T cells, the input composition can be a T cell population, an enriched T cell population, or PBMCs.

[0434] In one example, the method includes processing steps performed in the following order, wherein: first, primary cells are isolated (e.g., selected or separated) from a biological sample; the selected cells are activated, expanded or proliferated ex vivo in the presence of an activation reagent, and then viral vector particles are added and incubated together for transduction, and the total activation and transduction time does not exceed or does not exceed about 24 or 36 or 48 hours, wherein the transduction time is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

[0435] T cell activators are solid supports (e.g., beads, including magnetic beads and / or microbeads; polymer matrices, including polymer nanomatrix) coupled with anti-CD3 and / or anti-CD28 and / or anti-41-BB monoclonal antibodies. For example, the sample or composition of the cell to be separated is incubated with a small magnetizable or magnetically responsive material (such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., Dynabeads or MACS beads). For example, the magnetic particles or beads include magnetically responsive materials combined with specific binding members (such as antibodies or other binding partners). There are many well-known magnetically responsive materials used in magnetic separation methods. The incubation is typically carried out under such conditions that antibodies or binding partners or molecules specifically bound to such antibodies or binding partners attached to magnetic particles or microbeads are specifically bound to cell surface molecules (if present on cells in the sample). In some aspects, the sample is placed in a magnetic field, and those cells with magnetically responsive or magnetizable particles attached thereto are attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted by the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. For example, the magnetically responsive particles or microbeads remain attached to the cells, which are then incubated, cultured and / or engineered; in some aspects, the particles or microbeads remain attached to the cells for administration to a patient. For example, magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles or microbeads from cells are known and include, for example, the use of competing non-labeled antibodies and magnetizable particles or antibodies or microbeads conjugated to cleavable linkers. For example, the magnetizable particles are biodegradable.

[0436] Can be before cell transduction or transduction and activate and / or amplify simultaneously.For example, the method provided herein does not include further cultivation or hatching, for example, does not include ex vivo amplification step, or includes significantly shorter ex vivo amplification step.For example, make the whole process of cell engineering (for example selection and / or enrichment, hatching and activation transduction are combined and / or further culture or cultivate) be to carry out in the following time period after obtaining sample from experimenter: be no more than 9 days, be no more than 8 days, be no more than 7 days, be no more than 6 days, be no more than 5 days, be no more than 4 days, be no more than 3 days, be no more than 2 days or be no more than 1 day.It should be understood that the time of described cultivation or hatching can not include any time period that makes cell experience cryopreservation.

[0437] Alternatively, engineered cells (e.g., output compositions or formulated compositions) may be administered to the subject immediately or soon after transduction without significant ex vivo amplification. For example, engineered cells may be administered immediately after the transduction step. For example, engineered cells may be administered soon after the activation transduction step, for example, without significant ex vivo amplification or significantly shorter ex vivo amplification compared with conventional methods (which may require significant in vitro activation, amplification, and / or enrichment). For example, engineered cells may be administered within three days, two days, or one day of transduction. For example, engineered cells may be administered within 48 hours, 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, or shorter time of the activation transduction step. For example, engineered cells may be administered within 48 hours, 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, or shorter time compared with conventional methods. For example, the expansion and / or activation of cells can be performed in vivo after exposure to an antigen, e.g., the expansion of engineered cells can be performed in vivo after administration of the cells to a subject. For example, the extent, degree, or magnitude of in vivo expansion can be expanded, enhanced, or increased by various methods that can modulate (e.g., increase) the expansion, proliferation, survival, and / or efficacy of administered cells (e.g., cells expressing an exogenous gene).

[0438] The present application provides an antibody that specifically binds to MOG, which is a fully human antibody with low immunogenicity and few clinical side effects.

[0439] The term "MOG" (Myelin oligodendrocyte glycoprotein) is a myelin oligodendrocyte glycoprotein. MOG is specifically expressed in oligodendrocytes of the mammalian central nervous system and is a component of the central nervous system myelin sheath. As used herein, "MOG" includes any native MOG from any vertebrate source, including mammals such as primates (e.g., humans and monkeys) and rodents (e.g., mice and rats). "MOG" refers to any variant, derivative, or isoform of the MOG gene or the encoded protein. A human MOG polypeptide has an amino acid sequence or fragment thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% homologous or identical to the amino acid sequence encoded by the transcript expressed by the gene with NCBI GenBank Gene ID: 4340, and / or may optionally include at most one, at most two, or at most three conservative amino acid substitutions. Murine MOG polypeptides have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% homologous or identical to the amino acid sequence encoded by the transcript expressed by the gene with NCBI GenBank Gene ID: 17441, or a fragment thereof, and / or may optionally include up to one, up to two, or up to three conservative amino acid substitutions. The term encompasses "full-length," unprocessed MOG as well as any form of MOG derived from processing in cells. The term also encompasses naturally occurring variants of MOG, such as splice variants or allelic variants. The anti-MOG antibodies described herein can specifically bind to human MOG. Anti-MOG antibodies can also specifically bind to both human MOG and murine MOG. Exemplary, the amino acid sequence of full-length human MOG is set forth in SEQ ID NO: 41.

[0440] The antibodies of the present application can be isolated by screening combinatorial libraries for antibodies with the desired one or more activities.For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies with the desired binding properties.

[0441] In certain phage display methods, VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for phage binding to the antigen. Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, natural Libraries (e.g., from humans) thus provide a single source of antibodies to a variety of non-self antigens as well as self antigens without the need for any immunization. Finally, natural libraries can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve rearrangement in vitro.

[0442] In this article, antibodies or antibody fragments screened from a fully human antibody library are considered to be fully human antibodies or fully human antibody fragments.

[0443] Parent antibodies can refer to original antibodies screened from antibody libraries or hybridomas, antibodies used for antibody modification, or antibodies that require modification.

[0444] The term "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody, which alterations improve the affinity of the antibody for antigen.

[0445] The term "variant" refers to a polypeptide having substantially the same amino acid sequence or one or more activities encoded by substantially the same nucleotide sequence as the sequence of the antibody provided herein. The variant has the same or similar activities as the antibody provided in the Examples of this application.

[0446] The term "variant antibody" or "antibody variant" includes antibodies that differ by at least one amino acid modification compared to a parent antibody. The variant antibody sequences herein preferably have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity to the parent antibody sequence. Antibody variants may refer to the antibody itself or to a composition comprising the antibody variant. Amino acid sequence variants of an antibody may be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis.

[0447] The terms "anti-MOG antibody," "antibody that binds to MOG," "MOG antibody," and "antibody that recognizes MOG" refer to antibodies that bind MOG with sufficient affinity to be useful in the preparation of diagnostic and / or therapeutic agents targeting MOG. Anti-MOG antibodies may bind to unrelated, non-MOG proteins to an extent that is less than about 10% of the binding of the antibody to MOG, as measured by an enzyme-linked immunosorbent assay (ELISA). Anti-MOG antibodies bind to an epitope of MOG that may be conserved among MOG from different species.

[0448] Described herein are antigen-binding proteins, including Fab antibodies, having Fab-based antigen-binding regions. The human MOG extracellular domain was used, and Fabs were selected from a fully human, natural Fab phage library. These molecules exhibit exquisite specificity. For example, the antibodies only recognize 293T cells overexpressing MOG, but not wild-type 293T cells. Unless otherwise specified, MOG in this application refers to human or murine MOG.

[0449] The present application includes antibodies with Fab, scFv sequences, which are fused to one or more heavy chain constant regions to form antibodies with human immunoglobulin Fc regions to produce bivalent proteins, thereby increasing the overall affinity and stability of the antibody. In addition, the Fc portion allows other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to be directly conjugated to antibodies such as those used in antigen quantification studies, so that fixed antibodies are used for affinity measurements, for directed delivery of therapeutics, for testing Fc-mediated cytotoxicity and many other applications using immune effector cells.

[0450] The results presented herein highlight the specificity, sensitivity, and utility of the present antibodies in targeting MOG.

[0451] The antibodies or antibody fragments of the present application are based on the use of phage display to identify and select antigen-binding fragments (Fabs), the amino acid sequences of which confer specificity to the antibody or antibody fragment for MOG and form the basis of antigen-binding proteins. Thus, the Fabs can be used to design a range of different "antibodies or antibody fragments," including, for example, full-length antibodies, fragments thereof such as F(ab')2, fusion proteins, IgG4, multivalent antibodies (multifunctional antibodies), i.e., antibodies with more than one specificity for the same antigen or different antigens, e.g., bispecific T cell-binding antibodies (BiTEs), triabodies, etc.

[0452] The present application also provides full-length antibodies, whose heavy and light chains can be full-length (e.g., the antibody can include at least one, preferably two complete heavy chains, and at least one, preferably two complete light chains) or can include an antigen-binding portion (Fab, F(ab')2, Fv or scFv). The antibody heavy chain constant region can be selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE. The choice of antibody type will depend on the immune effector function that the designed antibody is intended to elicit. When constructing recombinant immunoglobulins, suitable amino acid sequences of the constant regions of various immunoglobulin isotypes and methods for producing a wide range of antibodies are known to those skilled in the art.

[0453] The present application provides an antibody that recognizes MOG, the antibody comprising a light chain variable region, the light chain variable region comprising the LCDR1 shown in SEQ ID NO: 84, and / or comprising the LCDR2 shown in SEQ ID NO: 85, and / or comprising the LCDR3 shown in any one of SEQ ID NOs: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98; and / or the antibody comprising a heavy chain variable region, the heavy chain variable region comprising the HCDR1 shown in SEQ ID NO: 99 or 100, and / or comprising the HCDR2 shown in SEQ ID NO: 101 or 102, and / or comprising the HCDR3 shown in any one of SEQ ID NOs: 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115. More preferably, the antibody that recognizes MOG comprises LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 84, 85, 86, 99, 101, and 103, respectively, or as shown in SEQ ID NOs: 84, 85, 87, 99, 101, and 104, respectively, or as shown in SEQ ID NOs: 84, 85, 88, 99, 101 and 105, respectively, or as shown in SEQ ID NOs: 84, 85, 89, 99, 101, and 106, respectively, or as shown in SEQ ID NOs: 84, 85, 90, 99, 101, and 107, respectively, or as shown in SEQ ID NOs: 84, 85, 91, 100, 102, and 108, respectively, or as shown in SEQ ID NOs: 84, 85, 92, 100, 102, and 109, respectively, or as shown in SEQ ID NOs: NO:84, 85, 93, 100, 102, 110, or as shown in SEQ ID NO:84, 85, 94, 100, 102, 111, respectively, or as shown in SEQ ID NO:84, 85, 95, 100, 102, 112, respectively, or as shown in SEQ ID NO:84, 85, 96, 100, 102, 113, respectively, or as shown in SEQ ID NO:84, 85, 97, 100, 102, 114, respectively, or as shown in SEQ ID NO:84, 85, 98, 100, 102, 115, respectively.

[0454] The present application provides an antibody that recognizes MOG, comprising a light chain variable region that may comprise an amino acid sequence as shown in SEQ ID NO: 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 or 128, or a nucleic acid sequence encoding the amino acid sequence, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, or a variant thereof; and / or comprising a heavy chain variable region that comprises SEQ ID NO: The invention relates to an amino acid sequence as shown in NO:129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or 141, or a nucleic acid sequence encoding the amino acid sequence, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or a variant thereof.

[0455] Given that each of these heavy chain variable region and light chain variable region sequences can bind to MOG, the heavy chain and light chain variable region sequences can be "mixed and matched" to generate the anti-MOG binding molecules of the present application. The light chain variable region and heavy chain variable region of the antibody that recognizes MOG are as follows: SEQ ID NO: 116, 129; SEQ ID NO: 117, 130; SEQ ID NO: 118, 131; SEQ ID NO: 119, 132; SEQ ID NO: 120, 133; SEQ ID NO: 121, 134; SEQ ID NO: 122, 135; SEQ ID NO: 123, 136; SEQ ID NO: 124, 137; SEQ ID NO: 125, 138; SEQ ID NO: 126, 139; SEQ ID NO: 127, 140; or as follows: The amino acid sequence shown in NO:128, 141 or the nucleic acid sequence encoding the amino acid sequence, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the above sequence, or a variant of the above sequence.

[0456] The present application provides antibodies that recognize MOG, including scFvs, which may include the amino acid sequence shown in SEQ ID NO: 43, 68, 77, 81, 82 or 83, or a nucleic acid sequence encoding the amino acid sequence, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the above sequence, or a variant of the above sequence.

[0457] The present application provides an antibody that recognizes the same antigenic determinant as the aforementioned anti-MOG antibody.

[0458] The present application provides an antibody that specifically binds to MOG, which can be a whole antibody, scFv, single-domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, dAb fragment, multifunctional antibody, scFv-Fc antibody, or IgG4 antibody. The antibody binds to cells expressing MOG.

[0459] Antibody assay

[0460] The anti-MOG antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art, including, for example, ELISA, biacore, Western blot, and flow cytometric analysis. Suitable assays are described in detail in the Examples.

[0461] The term "affinity" refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the use of Biacore to determine antibody affinity as described herein. The "affinity" of an antibody for MOG herein is expressed as the antibody's KD. The KD of an antibody refers to the equilibrium dissociation constant for an antibody-antigen interaction. The larger the KD value for an antibody binding to its antigen, the weaker its binding affinity for that particular antigen.

[0462] The term "EC50", half maximal effect concentration, refers to the concentration that elicits 50% of the maximal effect.

[0463] Immunoconjugates

[0464] The present application also provides an immunoconjugate, which comprises the antibody described herein and a functional molecule linked thereto. The antibody and the functional molecule can form a conjugate by covalent bonding, coupling, attachment, cross-linking, etc.

[0465] The terms "connection" or "fusion" are used interchangeably herein. These terms refer to connecting two or more chemical elements or components together by any means including chemical conjugation or recombinant methods. "In-frame fusion" refers to connecting two or more ORFs to form a longer ORF that is continuous in a manner that maintains the correct reading frame of the original open reading frame (ORF). Therefore, the resulting recombinant fusion protein is a single protein containing two or more fragments that correspond to the polypeptides encoded by the original ORF (these fragments are usually not so connected in the natural state). Although the reading frame is therefore continuous throughout the fusion fragment, these fragments can be physically or spatially separated by, for example, in-frame connecting sequences (e.g., "flexons").

[0466] Another aspect of the present application provides a nucleic acid molecule encoding at least one antibody, functional variant thereof, or immunoconjugate of the present application. Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. This is typically done by cloning the sequence into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells using conventional methods.

[0467] The present application also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable protein expression. The host cells can be prokaryotes, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

[0468] A1-scFv, A2-scFv, A3-scFv, A4-scFv, A5-scFv, A6-scFv, A7-scFv, A8-scFv, A9-scFv, A10-scFv, A11-scFv, A12-scFv, and A13-scFv can be sequentially linked to the EphrinB2 extracellular region or the EphrinB2-del3 extracellular region, the Notch transmembrane region, and GAL4-VP64 to construct the chimeric polypeptide MOG-synE or MOG-synEdel3.

[0469] The present application provides one or more vectors (e.g., expression vectors) comprising the above-mentioned nucleic acids, and host cells comprising the above-mentioned nucleic acids. The host cells comprise (e.g., are transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody. The host cells can be eukaryotic, e.g., Chinese hamster ovary (CHO) cells or lymphocytes (e.g., YO, NSO, Sp20 cells).

[0470] The host cell expresses the MOG-targeting chimeric polypeptide described in the present application.

[0471] The host cell may include a T cell, a natural killer cell, a cytotoxic T lymphocyte, a natural killer T cell, a DN T cell, a regulatory T cell, a NK92 cell, and / or a stem cell-derived immune effector cell.

[0472] The present application provides a method for preparing an anti-MOG antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expressing the antibody as described above, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0473] In order to express proteins, the nucleic acid encoding the antibody of the present application can be integrated into an expression vector. A variety of expression vectors can be used for protein expression. An expression vector can include a self-replicating extrachromosomal vector, or a vector that is integrated into the host genome. Expression vectors used for the present application include, but are not limited to, those that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As known in the art, a variety of expression vectors are commercially available or otherwise obtainable. Can be used in the present application to express antibodies.

[0474] The host cells provided herein can be administered in combination with an agent that enhances their function, preferably, in combination with a chemotherapeutic drug; and / or the host cells can be administered in combination with an agent that improves one or more side effects associated therewith; and / or the host cells can be administered in combination with a host cell expressing a chimeric antigen receptor targeting a protein other than MOG.

[0475] It should be appreciated that certain features of the present application that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present application that are described in the context of a single embodiment for clarity may also be provided individually or in any suitable subcombination. All combinations of the embodiments of the present application are expressly included in the present application and disclosed herein, just as each and every combination is individually and clearly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly included in the present application and disclosed herein, just as each and every such subcombination is individually and clearly disclosed herein.

[0476] The biological materials used in the examples of this application are shown in Table 2.

[0477] Table 2

[0478] Example 1. Construction of chimeric polypeptide synE and its mutants, truncations, and modifications

[0479] This example constructs the composition of various exemplary chimeric polypeptides used in this application (Table 3).

[0480] Table 3

[0481] For example, the fragment sequences in Table 2 above are as follows: antiGPC3-scFv (SEQ ID No: 69), AntiCD123-scFv (SEQ ID No: 71), AntiMOG-scFv (SEQ ID No: 68), anti CLDN18.2-scFv (SEQ ID No: 75), AntiGPRC5D-scFv (SEQ ID No: 76), anti CLL1-scFv (SEQ ID No: 79), EphrinB2 extracellular region (SEQ ID No: 1, 2), EphrinB2-del1 (SEQ ID No: 3), EphrinB2-del2 (SEQ ID No: 4), EphrinB2-del3 (SEQ ID No: 5), EphrinB2-del23 (SEQ ID No: 6), EphrinB2-(EGF-like domain) 1 (SEQ ID No: 7), EphrinB2-(EGF-like domain) 2 (SEQ ID No: 8), EphrinB2-(EGF-like domain) domain)3 (SEQ ID No:9), EphrinB2-(EGF-like domain)4 (SEQ ID No:10), Notch1 (SEQ ID No:15), Notch1 (SEQ ID No:15), GAL4-VP64 (SEQ ID No:28).

[0482] The chimeric polypeptide expression vector was constructed using the pRRLSIN vector (Addgene).

[0483] After the chimeric polypeptide expression vectors were introduced into Jurkat cells, biotin-labeled antigen polypeptide (Kaixing Diagnostics, HD0832) + PE-labeled streptavidin (eBioscience, 12-4317-87) were used. Flow cytometry results showed that the chimeric polypeptides were stably expressed on the cell membrane.

[0484] BFP (genebank ID: QJR97815.1) was inserted downstream of the UAS-CMV promoter (SEQ ID NO: 30) controlled by Gal4-VP64 as shown in Table 2 to serve as a chimeric polypeptide-activated reporter gene. Green fluorescent protein (GFP) controlled by the pGK promoter (Addgene #79120, 7721-8220 bp) was inserted downstream of BFP to serve as a transduction-positive marker. The GFP protein sequence is shown in genebank ID: UDY80669.1. UAS-BFP-PGK-GFP was ligated according to the nucleic acid sequence and integrated into the pRRLSIN expression vector. Lentivirus was then prepared using standard molecular biology techniques and transduced into Jurkat cells to establish Jurkat responder cells.

[0485] Example 2. synE and its truncated forms regulate gene expression

[0486] SK-Hep1-GPC3 cells overexpressing human GPC3 were constructed using conventional molecular biological techniques.

[0487] The Jurkat responder cells containing GPC3-synE, GPC3-synE-del1, GPC3-synE-del2, GPC3-synE-del3, and GPC3-synE-del23 constructed in Example 1 were respectively mixed with 2×10 4 Hepatocellular carcinoma cells (SK-Hep1, SK-Hep1-GPC3, HuH7, and PLC / PRF / 5) were mixed in a 1:1 ratio and incubated at 37°C for 24 hours. BFP expression levels were then assessed by flow cytometry. Figure 1 shows that upon incubation with GPC3-positive target cells (SK-Hep1-GPC3, HuH7, and PLC / PRF / 5), the chimeric polypeptides GPC3-synE and its truncations all regulated gene expression. However, in the absence of target cells (control) or upon incubation with GPC3-negative target cells, synE and its truncations did not significantly induce BFP expression compared to background levels. Leakage induced by synE-del3 or synE-del23 was significantly reduced.

[0488] Example 3: MOG-synE regulates gene expression

[0489] Conventional molecular biology techniques were used to construct cells overexpressing human or mouse MOG: K562-mMOG, K562-huMOG, 293T-mMOG, or 293T-huMOG.

[0490] Jurkat responder cells containing the MOG-targeting chimeric polypeptides MOG-synE and MOG-synE-del3 were mixed with 2×10 4The target cells were mixed at a ratio of 1:1 and incubated at 37°C for 24 hours. The expression level of BFP was detected by flow cytometry. Figure 2 shows that after incubation with MOG-positive target cells (K562-mMOG, K562-huMOG, 293T-mMOG, and 293T-huMOG), MOG-synE or its truncated forms all regulated gene expression.

[0491] Example 4. Regulatory B7H3-CAR-T cells

[0492] B7H3-CAR (SEQ ID NO: 50) was inserted downstream of the UAS-CMV promoter (SEQ ID NO: 30) and then integrated into the chimeric polypeptide MOG-synE (SEQ ID NO: 46) expression vector (pRRLSIN) or the MOG-synE-del3 (SEQ ID NO: 47) expression vector (pRRLSIN) to construct the B7H3-CAR-MOG-synE (SEQ ID NO: 58) vector and the B7H3-CAR-MOG-synE-del3 (SEQ ID NO: 59) vector. The corresponding lentivirus of the above expression vectors was prepared using conventional molecular biology techniques.

[0493] Conventional methods for preparing CAR-T cells in the art were used: donor PBMC cells were collected, activated with magnetic beads containing anti-CD3 and CD28 antibodies (Life Technologies, 40203D), and then cultured to obtain T cells; after infecting T cells with a lentivirus containing the above-mentioned vector, T cells in which the chimeric polypeptide regulated the expression of B7H3-CAR were prepared, i.e., regulatory B7H3-CAR-T cells.

[0494] 2×10 4 The regulatory B7H3-CAR-T cells were mixed with target cells at a 1:1 ratio and incubated at 37°C for 24 hours. The B7H3-CAR was then labeled with the antigen huB7-H3 (prepared using conventional molecular biology techniques) and analyzed by flow cytometry. As shown in Figure 3A, regulatory B7H3-CAR-T cells induced B7H3-CAR expression after incubation with K562-huMOG; regulatory B7H3-CAR-T cells did not express B7H3-CAR after incubation with K562 or U251.

[0495] Example 5. In vitro killing of regulated B7H3-CAR-T cells

[0496] 10,000 U251 cells were added to a 96-well E-plate (Agilent, 300600900) and cultured for 24 hours. Then, 2,000 leukemia cells (K562, K562-huMOG) and 10,000 regulated B7H3-CAR-T cells were added, mixed, and incubated at 37°C for 72 hours. During the incubation process, the cell growth index was recorded in real time (Agilent, xCELLigence RTCA MP) to detect the killing effect.

[0497] 10,000 U87 cells, leukemia cells (K562, K562-huMOG), and regulated B7H3-CAR-T cells were mixed at a ratio of 3:3:1, 1:1:1, or 1:1:3 and incubated at 37°C. U87 cells served as target cells and T cells served as effector cells, with target-effector ratios of 3:1, 1:1, and 1:3, respectively. After incubation at 37°C for 24 hours, the killing was detected by the LDH assay (Roche, 11644793001).

[0498] 10,000 U87 or U251 cells: CAR-T cells: leukemia cells (K562, K562-huMOG) were mixed at a ratio of 1:1:0.2 and incubated at 37°C for 24 h. The supernatant was collected and cytokine levels were detected by Cytometric bead array (BD, 558264).

[0499] Figures 3B and 3C show that regulatory B7H3-CAR-T cells can effectively kill B7H3-positive glioma cells U87 and U251 in the presence of MOG; however, in the absence of MOG, the killing effect on B7H3-positive glioma cells is weakened or even absent; regulatory B7H3-CAR-T cells release the cytokine IL2 only in the presence of MOG.

[0500] Example 6.CD123-synE regulates gene expression

[0501] Jurkat responder cells containing the chimeric polypeptide CD123-synE (SEQ ID NO: 48) or CD123-synE-del3 (SEQ ID NO: 49) targeting CD123 were co-cultured with 2×10 4 AML cells were mixed and incubated at 37°C for 24 hours, and the expression level of BFP was detected by flow cytometry. Figure 4A, B shows that after incubation with CD123-positive target cells (Molm13, MV-4-11, THP-1), the chimeric polypeptides of CD123-synE or its truncated form all regulated gene expression.

[0502] Example 7. In vitro killing of regulatory NKG2D-CAR-T cells

[0503] ZBB-NKG2D-CAR (SEQ ID NO: 51), ZNKG2D-CAR-DAP10 (SEQ ID NO: 53), ZNKG2D-CAR (SEQ ID NO: 52), and NKG2D-28Z-CAR (SEQ ID NO: 54) were respectively inserted downstream of the UAS-CMV promoter (SEQ ID NO: 30), and then integrated into the chimeric polypeptide CD123-synE (SEQ ID NO: 48) expression vector or the CD123-synE-del3 (SEQ ID NO: 49) expression vector, and T cells were transduced to prepare NKG2D-CAR-T cells regulated by the chimeric polypeptide targeting CD123. Constitutive NKG2D-CAR-T cells were constructed using expression vectors (pRRLSIN) containing ZBB-NKG2D-CAR (SEQ ID NO: 51), ZNKG2D-CAR-DAP10 (SEQ ID NO: 53), ZNKG2D-CAR (SEQ ID NO: 52), and NKG2D-28Z-CAR (SEQ ID NO: 54), respectively.

[0504] The CAR-T cells constructed above were respectively mixed with 2×10 4 AML cells were mixed and co-cultured at 37°C for 18 hours, and the number of viable target cells was detected by flow cytometry (7-AAD negative (BD, 559925), CD3 negative (eBioscience, 12-0038-42)). Figure 6 shows that NKG2D-28Z-CAR-T cells, ZNKG2D-CAR, or ZNKG2D-CAR-DAP10-T cells regulated by the chimeric polypeptide significantly killed THP1 cells expressing NKG2D ligands, but had weak killing effects on KG-1 and Molm13 cells (Figure 5) with low expression of NKG2D ligands.

[0505] This indicates that the killing ability of NKG2D-CAR-T cells regulated by CD123-synE or CD123-synE-del3 is NKG2D ligand-dependent, while high expression of CD123 alone (such as Molm13) will not cause NKG2D ligand-independent killing.

[0506] Example 8. Specificity of in vitro killing by regulatory NKG2D-CAR-T cells

[0507] The regulatory NKG2D-CAR-T cells constructed in Example 7 were respectively mixed with 1×10 4SK-Hep1 or K562 cells expressing NKG2D ligands but not CD123 were mixed and co-cultured at 37°C for 18 hours. The supernatant was assayed for LDH (Roche, 11644793001). Figures 7A and 7B show that constitutive NKG2D-CAR-T cells can effectively kill target cells, while regulatory NKG2D-CAR-T cells have almost no killing effect. This result suggests that CD123-synE or CD123-synE-del3 further enhances the specificity of NKG2D-CAR-T cells targeting AML cells.

[0508] Example 9. In vivo survival and efficacy of NKG2D-CAR-T cells expressing CD123-synE chimeric polypeptides

[0509] Conventional molecular biological techniques were used to construct THP-1 cells expressing exogenous luciferase (THP1-Luci).

[0510] Immunodeficient (NPG) mice were inoculated with 1×10 7 THP-1-Luci cells / mouse, after confirming tumor formation by fluorescence imaging on day 15, the mice were grouped according to the diagram, with 5 mice in each group, and the corresponding number of NKG2D-CAR-T cells or UTD cells were intravenously infused, and tumor fluorescence imaging was performed weekly (IVIS LUMINA III SYSTEM). On the 9th day after injection of CAR-T cells, peripheral blood of the mice was collected and the number of CD4+ and CD8+ T cells surviving was detected by flow cytometry (CD3 antibody (Thermo, 46-0036-42), CD4 antibody (BD, 562424), CD8 antibody (BD, 555369)). Figure 8 shows that placing ZNKG2D-CAR under the control of CD123-synE or CD123-synE-del3 can improve the survival ability and anti-tumor effect of CAR-T cells in vivo.

[0511] Example 10. Construction of universal CAR (UCAR) T cells

[0512] In vitro synthesis included sgRNA sequences targeting TRAC, B2M, and FAS (SEQ ID NOs: 142, 143, and 144) (Kaixing Diagnostics). The endogenous TCR / B2M or TCR / B2M / FAS of the regulatory NKG2D-CAR-T cells constructed in Example 7 were knocked out using CRPSP / Cas9 technology (Cas9 protein, Kaika Biotechnology (Shanghai) Co., Ltd., CAS-EE109). NKG2D-UCAR-T-DKO or NKG2D-UCAR-T-TKO were obtained by magnetic bead sorting (Miltenyi Biotech, 130-048-801). Untransduced U-UTD cells with TCR / B2M knockout were used as controls. Constitutive NKG2D-UCAR-T cells were constructed in the same manner.

[0513] Example 11 Flow cytometry detection of NKG2D-Ligand (NKG2DL) expression in resting NK cells from different donors

[0514] Peripheral blood PBMCs from different donors were collected and NK cells were screened using CD56 magnetic beads (Miltenyi Biotec, 130-050-401) according to the manufacturer's instructions. 5×10 5 NK cells were plated with NKG2D-Fc protein (final concentration 5 μg / mL) (R&D, 1299-NK), with PBS used as a negative control, and incubated at room temperature for 15 minutes. Anti-human IgG Fc-PE fluorescent antibody (1:200 dilution) (eBioscience, 12-4998-82) was added to label cells bound to NKG2D-Fc protein, and NKG2DL expression was detected by flow cytometry. Figure 9 shows that NKG2DL is almost absent in resting NK cells from different donors.

[0515] Example 12 Flow cytometry detection of NKG2DL expression after 24 h of co-culture of NK cells and tumor cells, or NK cells and UCAR-T cells

[0516] The human primary NK cells (3×10 4 ) and tumor cells (HL60, KG-1, MV4-11, THP-1) at a ratio of 1:1, or 3×10 4Primary human NK cells were seeded with NKG2D-UCAR-T cells at a 1:1 ratio and incubated at 37°C for 24 hours. NKG2D-Fc protein (final concentration 5 μg / mL) (R&D, 1299-NK) was then added, with PBS used as a negative control, and incubated at room temperature for 15 minutes. Anti-human IgG Fc-PE fluorescent antibody (1:200 dilution) (eBioscience, 12-4998-82) was added to label cells bound to NKG2D-Fc protein, and NKG2DL expression was detected by flow cytometry. Figure 10 shows that NKG2DL expression on NK cells was upregulated after co-incubation with different tumor cells or with NK cells and NKG2D-UCAR-T cells.

[0517] Example 13. Cytotoxicity of NK cells by regulatory or constitutive NKG2D-UCAR-T (TRAC / B2M KO) cells

[0518] Take 2×10 4 Human primary NK cells were inoculated with UTD or NKG2D-UCART cells (regulatory or constitutive) at a 1:1 effector-target ratio. After incubation at 37°C for 16 hours, CAR-T cells were labeled with CD3-PE antibody (eBioscience, 12-0038-42) and NK cells were labeled with CD56-APC (eBioscience, 17-0567-42). 7-AAD fluorescent dye (BD, 559925) was used to distinguish between dead and live cells, and the number of NK cells was determined by flow cytometry. Figure 11 shows that both regulatory and constitutive NKG2D-UCAR-T cells were able to kill NK cells compared to the UTD group.

[0519] Example 14. Regulated NKG2D-UCAR-T (TRAC / B2M KO) cells kill NK cells

[0520] Take 2×10 4 Human primary NK cells: tumor cells (MV-4-11 or MOLM-13 cells expressing both CD123 and NKG2DL) and UCAR-T cells were mixed at a ratio of 1:1:1 and incubated at 37°C for 16, 36, or 72 hours. UCAR-T cells were labeled with HLA-ABC-PE (eBioscience, 12-9983-42) and NK cells with CD56-APC (eBioscience, 17-0567-42), respectively. 7-AAD fluorescent dye (BD, 559925) was used to distinguish live from dead cells. Flow cytometry was used to assess the cytotoxicity of UCAR-T cells against tumor cells, as well as the cytotoxicity of UCAR-T cells against NK cells and the cytotoxicity of NK cells against UCAR-T cells.

[0521] As shown in Figure 12A and B, in the absence of NK cells, regulated NKG2D-UCAR-T cells can significantly kill NKG2DL-positive tumor cells. When NK cells are present, regulated NKG2D-UCAR-T cells can also significantly kill tumor cells, and the killing effect becomes stronger with the extension of co-incubation time. After co-incubation with tumor cells, regulated NKG2D-UCAR-T cells have a strong killing effect on NK cells; and can partially resist the killing effect of NK cells.

[0522] Example 15. Regulated NKG2D-UCAR-T (TRAC / B2M / Fas KO) cells kill NK cells

[0523] Take 2×10 4 Individual primary NK cells: tumor cells (THP-1): regulatory NKG2D-UCAR-T cells were mixed at a ratio of 1:1:1 and incubated at 37°C for 24 or 72 hours. HLA-ABC-PE (eBioscience, 12-9983-42) was used to label UCAR-T cells, and CD56-APC (eBioscience, 17-0567-42) was used to label NK cells; 7-AAD fluorescent dye (BD, 559925) was used to distinguish between dead and live cells. Flow cytometry was used to detect the killing effect of UCAR-T cells on tumor cells, and the killing effect of UCAR-T cells on NK cells and NK cells on UCAR-T cells were also detected. As shown in Figure 13, in the absence of NK cells, regulatory NKG2D-UCAR-T cells can significantly kill NKG2DL-positive tumor cells. When NK cells are present, regulatory NKG2D-UCAR-T cells significantly kill tumor cells. After co-incubation with tumor cells, regulatory NKG2D-UCAR-T cells have a strong killing effect on NK cells, and the longer the co-incubation time, the stronger the killing effect; and can partially resist the killing effect of NK cells.

[0524] Example 16. In vivo anti-tumor efficacy of regulated NKG2D-UCAR-T (TRAC / B2M / Fas KO) cells

[0525] THP1-luci-GFP cells (8×10 6 / mouse) in NOG mice and divided them into 7 groups, 5 mice in each group. Fluorescence imaging (IVIS LUMINA III SYSTEM) 14 days after inoculation, the photon intensity reached an average of 1.1×10 4 p / s / cm 2 / sr, NK (2×10 6After the first NK injection, regulated NKG2D-UCAR-T (5×10 6 As shown in Figure 14, 27 days after UCAR-T infusion, regardless of the presence of NK cells, UCAR-T cells expressing ZNKG2D-CD123-synE, ZNKG2D-DAP10-CD123-synE, or ZBB-NKG2D-CD123-synE can effectively inhibit the growth of leukemia xenografts, among which UCAR-T cells expressing ZBB-NKG2D-CD123-synE have the best efficacy.

[0526] Example 17. Rapid preparation process of CAR-T cells targeting NKG2DL

[0527] T cells were activated with magnetic beads (Miltenyi Biotec, 170-076-156) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours and then transduced with a virus containing a regulatory or constitutive NKG2D-CAR (prepared using conventional molecular biology techniques). The transduction time was 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. The transduced T cells were collected and cryopreserved, or the T cell density was adjusted to approximately 1 × 10 6 cells / ml were inoculated into 24-well plates, cultured for more than 24 h, and then cryopreserved.

[0528] Example 18. Preparation of MOG recombinant protein

[0529] The extracellular segment of human MOG (positions 30-154 of the full-length human MOG as shown in SEQ ID NO: 41) was synthesized in vitro and fused with the Fc segment of the human IgG1 heavy chain constant region to form the fusion protein MOG_huFc. The MOG_huFc protein was expressed and purified by transfection into 293F cells using conventional protein purification techniques.

[0530] Example 19. Screening and identification of MOG antibodies

[0531] 1. Screening MOG-specific antibodies using a fully human phage display library

[0532] The phage display library used in this application is a fully human natural scFv phage library constructed by our company, with a library capacity of 1E+11. Fab fragments highly specific for MOG were obtained using screening methods known to those skilled in the art.

[0533] Briefly, immunotubes were coated with 10 μg / ml of the antigen MOG_huFc and human Fc fragments, respectively. To minimize the effects of the Fc fragment, the phage library was added to the immunotube coated with the human Fc fragment and allowed to bind for 1 hour. The supernatant was then added to the immunotube coated with MOG_huFc and allowed to bind for 1.5 hours. Nonspecific phage were then washed away, and bound phage were eluted and used to infect Escherichia coli TG1 cells in logarithmic growth phase. The eluted phage were expanded and purified using PEG / NaCl precipitation for the next round of screening. Screening was repeated for 3-4 cycles to enrich for Fab phage clones that specifically bound to MOG_huFc. Positive clones were identified by standard ELISA against MOG_huFc. The ELISA used the human Fc fragment as an irrelevant antigen to verify antibody specificity. A total of 376 clones were screened, of which 93 clones specifically bound to MOG_huFc but not to the human Fc region. ELISA retesting of these 93 clones showed high consistency with the initial results, with 90 of them specifically binding to MOG_huFc but not to the human Fc region. Forty-seven clones with high signal values ​​were sequenced, resulting in 13 clones designated A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, and A13.

[0534] Among them, the LCDR1 and LCDR2 sequences of 13 clones were identical, as shown in SEQ ID NO: 84 and 85, respectively; the HCDR1 and HCDR2 sequences of A1, A2, A3, A4, and A5 were identical, as shown in SEQ ID NO: 99 and 101, respectively; the HCDR1 and HCDR2 sequences of A6, A7, A8, A9, A10, A11, A12, and A13 were identical, as shown in SEQ ID NO: 100 and 102, respectively. The LCDR3 sequences of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, and A13 are shown in SEQ ID NOs: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98, respectively; the HCDR3 sequences are shown in SEQ ID NOs: 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, and 115, respectively; the VL sequences are shown in SEQ ID NOs: 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128, respectively; and the VH sequences are shown in SEQ ID NOs: 1 NO: 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141. The scFv sequences of A2, A6, A8, A9, A10, and A13 are shown in SEQ ID NO: 77, 81, 68, 82, 83, and 43, respectively.

[0535] 2. Using FACs to determine the specificity of antibody binding to target cells

[0536] 293T-hMOG and 293T-mMOG cells were counted and plated in U-bottom plates, with approximately 2×105 cells per well. The cells were incubated with primary antibodies (Fab formats A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, and A13, 5 μg / mL) and fluorescently labeled with secondary antibodies (Anti-Fc-FITC, 1:200, Jackson ImmunoResearch). Fluorescence intensity was then measured using a flow cytometer. Mean fluorescence intensity (MFI) was calculated using FlowJo analysis software. A blank control group (NA) was maintained without antibody addition.

[0537] The results are shown in Figure 15. Antibodies A1, A3, A9, A11, and A12 bound to 293T-hMOG cells; and antibodies A2, A4, A5, A6, A7, A8, A10, and A13 bound to 293T-hMOG cells and 293T-mMOG cells.

[0538] Example 20. Determination of the affinity of antibodies (Fab form) using surface plasmon resonance (SPR)

[0539] The affinity of different antibodies for MOG was determined using a biacore T200. The specific method was as follows: MOG_His was coated onto a CM5 chip via amino-coupling to a concentration of approximately 150 RU. A gradient of diluted antibody was then passed through the antigen-coated channel at a flow rate of 30 μl / min. The running buffer was HBS-N and the temperature was 25°C. The experimental data were analyzed using BIAevaluation 3.2, and the kinetic curves were fitted using a 1:1 Langmuir model. The results are shown in Table 4.

[0540] Table 4 Antibody (Fab form) affinity

[0541] Example 21. Construction of anti-MOG scFv_hFc fusion antibody and its transient expression and purification in eukaryotic cells

[0542] Primers were designed for the VH and VL segments of A2, A6, A8, A9, A10, and A13, and the corresponding scFv sequences were ligated into appropriate eukaryotic expression vectors. 293F cells in the logarithmic growth phase were transiently transfected using 293fectin™ Transfection reagent (Invitrogen, 12347-019) or polyethyleneimine (PEI) (Sigma-Aldrich, 408727). Culture supernatants were collected 5-7 days after transfection and affinity purified using Protein A. The resulting antibodies were quantitatively and qualitatively analyzed by SDS-PAGE (Figure 16).

[0543] Example 22. Determination of EC50 of Antibody (scFv_hFc Form) Binding to MOG by ELISA

[0544] The EC50 of binding of antibodies A2, A6, A8, A9, A10, and A13 to human MOG antigen was determined by standard ELISA. Immunoplates were coated with 2 μg / ml recombinant hMOG_his at 4°C overnight. Blocking was performed with 2% MPBS at room temperature for 2 hours, followed by three washes with PBS. A serial dilution of the test antibodies (A2, A6, A8, A9, A10, A13, and 12 5-fold serial dilutions starting at 100 nM) was added and incubated at room temperature for 1 hour. The plates were then washed three times with PBST (PBS containing 0.05% Tween-20) and three times with PBS. HRP-conjugated anti-huFc tag antibody (1:4000, Sigma) was then added and incubated at room temperature for 1 hour. The plates were then washed three times with PBST and three times with PBS. The reaction was terminated by the addition of TMBS substrate, color development was performed for 10 minutes, and the results were read on a microplate reader (Figure 17). EC50 values ​​are shown in Table 5.

[0545] Table 5 EC50 values ​​of antibodies (scFv_hFc format) binding to MOG

[0546] Example 23. Determination of EC50 of Antibody Binding (scFv_hFC Form) to 293T-hMOG Cells and 293T-mMOG Cells Using FACs

[0547] Take 2x10 5 292T-hMOG or 293T-mMOG cells were plated into 96-well plates and tested antibodies (A2, A6, A8, A9, A10, and A13, starting at 600 nM and diluted 11 times in a 3-fold series) were added. The cells were incubated at 4°C for 45 minutes. After washing twice with PBS (1% FBS), the cells were incubated with a FITC-conjugated anti-human IgG secondary antibody. After two washes, the cells were detected using a BD FACSLyric instrument. The results are shown in Figure 18, and the specific EC50 values ​​are shown in Table 6.

[0548] Table 6 EC50 of antibody (scFv form) binding to cells

[0549] Example 24: MOG-synE and MOG-synE-del3 regulate gene expression

[0550] Jurkat responder cells expressing MOG-binding chimeric polypeptides MOG-synE or MOG-synE-del3 were constructed using MOG antibodies A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, and A13, respectively, as described in the previous examples. These cells were mixed with target cells at a 1:1 ratio. After incubation for 24 hours, BFP expression was measured by flow cytometry. Target cells included 293T-hMOG, K562-hMOG, 293T-mMOG, and K562-mMOG. Results showed that after incubation with target cells, Jurkat responder cells expressing the MOG-binding chimeric polypeptides activated BFP expression in the presence of MOG protein.

[0551] The embodiments described in this application include any embodiment as a single embodiment or in combination with any other embodiment or part thereof. In addition, it should be understood that after reading the above teachings of this application, those skilled in the art may make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached hereto.

[0552] Sequence information

Claims

1. An engineered cell targeting allogeneic immune cells, wherein: The engineered cells express molecules that recognize NKG2D ligands, and the expression of the molecules that recognize NKG2D ligands is regulatable.

2. The engineered cell according to claim 1, wherein The molecules that recognize NKG2D ligands include: bispecific molecules, polypeptide-coupled drugs, and a first chimeric receptor.

3. The engineered cell according to claim 1 or 2, wherein: The molecule that recognizes the NKG2D ligand includes the full-length NKG2D polypeptide or a fragment thereof, or an antibody that recognizes the NKG2D ligand or a fragment thereof; preferably, the NKG2D polypeptide includes the sequence shown in SEQ ID NO:

42.

4. The engineered cell according to claim 2 or 3, wherein The first chimeric receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain; the extracellular domain comprises the extracellular domain of an NKG2D polypeptide, or an antibody or a fragment thereof that recognizes an NKG2D ligand; preferably, the extracellular domain is connected to the transmembrane domain via a hinge.

5. The engineered cell according to any one of claims 2 to 4, wherein: The intracellular domain of the first chimeric receptor includes an immunoreceptor tyrosine-based activation motif or an ITAM signaling motif.

6. The engineered cell according to any one of claims 2 to 5, wherein: The intracellular domain of the first chimeric receptor is selected from the group consisting of: TCRε, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, CD66d, CD28, CD137 intracellular signaling domains or sequences of combinations thereof.

7. The engineered cell according to any one of claims 2 to 6, wherein: The transmembrane domain of the first chimeric receptor is selected from the group consisting of: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, BAFFR, CEACAM1, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD137 (4-1BB), CD16, CD160, CD18 (CD11a, LFA-1), CD1 60(BY55), CD162(SELPLG), CD19, CD2, CD22, CD226(DNAM1), CD229(Ly9), CD244(SLAMF4, 2B4), CD27, C D278(ICOS), CD28, CD29, CD33, CD37, CD4, CD40, CD45, CD49a, CD49D, CD49f, CD5, CD64, CD8, CD80, CD84, CD86, CD9, CD96(Tactile), CD134, CD154, CRTAM, GITR, HLA-E, HLA-F, HLA-G, HVEM(LIGHTR), IA4, IL2R β, IL2Rγ, IL7Rα, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2 , LTBR, ​​NKp80 (KLRF1), NKp44, NKp30, NKp46, NKG2D, NKG2C, OCIL, OX40, PAG / Cbp, PSGL1, SLAM (SLAMF1, CD150, IPO-3), SLAMF6 (NTB-A, Ly108), SLAMF7, BLAME (SLAMF8), TNFR2, VLA1, VLA-6, cadherin and / or transmembrane domains of collagen.

8. The engineered cell according to any one of claims 2 to 7, wherein: The extracellular domain of the first chimeric receptor includes the extracellular region of the NKG2D polypeptide or an antibody or a fragment thereof that recognizes the NKG2D ligand, the transmembrane domain includes the NKG2D transmembrane domain, the CD8 transmembrane domain or the CD28 transmembrane domain, and the intracellular domain includes the NKG2D polypeptide intracellular domain, the CD28 intracellular domain, the CD137 intracellular domain, the CD3ζ intracellular domain or a combination thereof; optionally, the engineered cell also expresses the DAP10 polypeptide.

9. The engineered cell according to any one of claims 2 to 8, wherein: The first chimeric receptor includes the full length of NKG2D polypeptide; preferably, the first chimeric receptor intracellular domain also includes CD28, CD137, CD3ζ intracellular domain or a combination thereof; more preferably, the first chimeric receptor includes the sequence shown in SEQ ID NO: 51, 52, 53 and / or 54.

10. The engineered cell according to any one of claims 1 to 9, wherein: The engineered cells are immune cells, neurons, epithelial cells, endothelial cells or stem cells.

11. The engineered cell according to any one of claims 1 to 10, wherein: The engineered cells are autologous or allogeneic T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune cells; preferably, the engineered cells are autologous or allogeneic T cells.

12. The engineered cell according to any one of claims 1 to 11, wherein The engineered cell also expresses a second chimeric polypeptide that does not recognize the NKG2D ligand, and after the second chimeric polypeptide recognizes the first target molecule, it induces the expression of the molecule that recognizes the NKG2D ligand; Preferably, after the binding domain of the second chimeric polypeptide binds to the first target molecule, it induces the cleavage of the second chimeric polypeptide, releasing a transcription factor, and the transcription factor regulates the expression of the molecule that recognizes the NKG2D ligand.

13. The engineered cell according to claim 12, wherein The second chimeric polypeptide comprises a synNotch polypeptide; or the second chimeric polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 44, 45, 46, 47, 48, 49, 60, 61, 62, 63, 64, 65, 66 or 67.

14. The engineered cell according to claim 12 or 13, wherein The first target molecule recognized by the second chimeric polypeptide includes a tumor antigen, a tissue-specific marker, and / or a pathogen antigen.

15. The engineered cell according to any one of claims 1 to 14, wherein The engineered cells also express a third chimeric receptor that recognizes a tumor antigen and / or a pathogen antigen.

16. The engineered cell according to claim 15, wherein The third chimeric receptor comprises a chimeric antigen receptor (CAR) and / or a recombinant T cell receptor (recombinant TCR).

17. The engineered cell according to any one of claims 14 to 16, wherein: The tissue-specific antigens include: brain tissue-specific marker MOG, liver tissue-specific marker ASGR1, prostate tissue marker PSA or a combination thereof.

18. The engineered cell according to any one of claims 14 to 17, wherein: Tumor antigens are selected from: ALPPL2, ALPI, Axl, B7H3, BCMA, CD 117, CD123, CD171, CD179a, CD19, CD213A2, CD20, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD7, CD71, CD94, CD97, CEA, Claudin18.2, CLDN6, CLECL1, CLL1, cMet, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, FAP, FCRH5, FLT3, GD2, GD3, GM3, GPC3, GPRC5D, HER2 (ERBB2), IGLL1, IL llRa, IL13Ra2, Mesothelin, MUC1, NCAM, NKG2D-Ligand, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2 or WT1.

19. The engineered cell according to any one of claims 1 to 18, wherein: The engineered cells target allogeneic NK, NKT and / or T cells, or the engineered cells kill allogeneic NK, NKT and / or T cells or resist immune rejection of allogeneic NK, NKT and / or T cells.

20. The engineered cell according to any one of claims 1 to 19, wherein: The first chimeric receptor and the second chimeric polypeptide are located in the same expression vector; preferably, the vector comprises the nucleotide sequence shown in SEQ ID NO: 55, 56, 57.

21. The engineered cell according to any one of claims 1 to 20, wherein: The endogenous B2M, TCR, MHC-II, FAS, NKG2A and / or NKG2D ligand molecules of the engineered cells are low-expressed or not expressed, or a combination thereof; Preferably, the endogenous B2M / TCR / FAS of the engineered cells is lowly expressed or not expressed; Preferably, the sgRNA sequences targeting TRAC, B2M, and FAS are shown in SEQ ID NOs: 142, 143, and 144, respectively.

22. The engineered cell according to any one of claims 1 to 21, wherein: Compared with reference cells, the engineered cells have improved survival and proliferation capabilities during in vivo and in vitro culture.

23. The engineered cell according to any one of claims 1 to 22, wherein: The engineered cells are administered in combination with a second immune cell expressing a fourth chimeric receptor that recognizes a tumor antigen and / or a pathogen antigen.

24. The engineered cell of claim 23, wherein The engineered cells enhance the survival and proliferation of the second immune cells administered previously, simultaneously or later; or enhance the killing of tumor cells by the second immune cells.

25. The engineered cell according to any one of claims 1 to 24, wherein: The NKG2D ligands include: MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6 or a combination thereof.

26. An engineered cell targeting NK, NKT and / or T cells, wherein: The engineered cells express molecules that recognize NKG2D ligands.

27. A cell composition comprising the engineered cell of any one of claims 1-26, and a second immune cell expressing a fourth chimeric receptor that recognizes a tumor antigen and / or a pathogen antigen, preferably, the antigen recognized by the fourth chimeric receptor is different from the antigen recognized by the second chimeric polypeptide.

28. The composition of claim 27, wherein The tumor antigen recognized by the fourth chimeric antigen receptor is selected from: ALPPL2, ALPI, Axl, B7H3, BCMA, CD 117, CD123, CD171, CD179a, CD19, CD213A2, CD20, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD7, CD71, CD94, CD97, CEA, Claudin18.2, CLDN6, CLECL1, CLL1, cMet, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, FAP, FCRH5, FLT3, GD2, GD3, GM3, GPC3, GPRC5D, HER2 (ERBB2), IGLL1, IL llRa, IL13Ra2, Mesothelin, MUC1, NCAM, NKG2D-Ligand, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2 or WT1.

29. The composition of claim 27 or 28, wherein The fourth chimeric receptor comprises a chimeric antigen receptor (CAR) and / or a recombinant T cell receptor (recombinant TCR).

30. A composition according to any one of claims 27 to 29, wherein The endogenous B2M, TCR, MHC-II, FAS, NKG2A and / or NKG2D ligand molecules of the engineered cells and the second immune cells are low-expressed or not expressed, or any combination thereof; Preferably, the endogenous B2M / TCR / FAS of the engineered cells is lowly expressed or not expressed; Preferably, the sgRNA sequences targeting TRAC, B2M, and FAS are shown in SEQ ID NOs: 142, 143, and 144, respectively.

31. A composition according to any one of claims 27 to 30, wherein The engineered cells and / or second immune cells are autologous or allogeneic T, NK, and / or NKT cells.

32. A polynucleotide, wherein The polynucleotide encodes the bispecific molecule, the first chimeric receptor, the second chimeric polypeptide, the third chimeric receptor, the fourth chimeric receptor or a combination thereof included in the engineered cell of any one of claims 1-26 or the composition of any one of claims 27-31.

33. A vector, wherein The vector comprises the polynucleotide according to claim 32.

34. The engineered cell according to any one of claims 1-26, the composition according to any one of claims 27-31, the polynucleotide according to claim 32, and / or the vector according to claim 33, for use in clearing autologous or allogeneic NK, NKT and / or T cells.

35. Use of the engineered cells according to any one of claims 1-26, the composition according to any one of claims 27-31, the polynucleotide according to claim 32, and / or the vector according to claim 33 for preparing a drug for eliminating autologous or allogeneic NK, NKT and / or T cells.

36. The use according to claim 34 or 35, wherein The NK cells include resting NK cells and / or activated NK cells.

37. Use of the engineered cell according to any one of claims 1 to 26, the composition according to any one of claims 27 to 31, the polynucleotide according to claim 32, and / or the vector according to claim 33 for preparing an anti-tumor drug.

38. The use according to claim 37, wherein The tumors include: blood tumors and / or solid tumors; Preferably, the blood tumor is selected from: leukemia, lymphoma and myeloma or a combination thereof; the solid tumor is selected from: liver cancer, gastric cancer, esophageal cancer, gastroesophageal junction tumor, pancreatic cancer, bile duct cancer, gallbladder cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, glioma, melanoma or a combination thereof.

39. A method for preventing or regulating transplant rejection, wherein: Administering the engineered cells of any one of claims 1-26, the composition of any one of claims 27-31, the polynucleotide of claim 32, and / or the vector of claim 33; preferably, the method is used to kill autologous or allogeneic NK, NKT and / or T cells.

40. A method for increasing the survival time and / or expansion capacity of engineered cells targeting tumors and / or pathogens in a subject in the presence of host immune cells, wherein: The engineered cells of any one of claims 1-26, the composition of any one of claims 27-31, the polynucleotide of claim 32, and / or the vector of claim 33 are administered to a subject; preferably, the subject is a human; preferably, wherein the engineered cells are autologous or allogeneic T cells, NK cells, NKT cells; preferably, the host immune cells are NK, NKT and / or T cells.

41. A chimeric polypeptide comprising: a) a binding domain capable of specifically binding to a first target molecule; b) a receptor regulatory domain comprising a cleavage site, The receptor regulatory domain includes an extracellular region and a transmembrane region, wherein the extracellular region includes an EphrinB2 extracellular region or a fragment thereof, and the transmembrane region contains one or more target molecule binding inducible cleavage sites; and c) intracellular domain, The binding of the binding domain to the first target molecule can induce the cleavage of the receptor regulatory domain, thereby releasing the intracellular domain.

42. The chimeric polypeptide of claim 41, wherein The transmembrane region also includes a stop transfer sequence (STS).

43. The chimeric polypeptide of claim 41 or 42, wherein The transmembrane region of the receptor regulatory domain includes the Notch transmembrane region.

44. The chimeric polypeptide of any one of claims 41 to 43, wherein: The receptor regulatory domain is selected from: an extracellular domain comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 65, and a transmembrane region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology to SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology to SEQ ID NO: 60, 61, 62, 63, 64, 66, 67.

45. The chimeric polypeptide of any one of claims 41-44, wherein The intracellular domain includes a transcription factor, a site-specific nuclease, a recombinase, an inhibitory immune receptor, an activating immune receptor, or a combination thereof.

46. ​​The chimeric polypeptide of claim 45, wherein The transcription factor is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16 or a combination thereof.

47. The chimeric polypeptide of any one of claims 41-46, wherein The intracellular domain of the chimeric polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology to SEQ ID NO:

28.

48. The chimeric polypeptide of any one of claims 41-47, wherein The first target molecule is a tumor antigen or a tissue-specific marker.

49. The chimeric polypeptide of any one of claims 41-48, wherein The first target molecule is a tumor antigen selected from the group consisting of ALPPL2, ALPI, Axl, B7H3, BCMA, CD 117, CD123, CD171, CD179a, CD19, CD213A2, CD20, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD7, CD71, CD94, CD97, CEA, Claudin18.2, CLDN6, CLECL1, CLL1, cMet, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FCRH5, FLT3, GD2, GD3, GM3, GPC3, GPRC5D, HER2 (ERBB2), IGLL1, IL llRa, IL13Ra2, Mesothelin, MUC1, NCAM, NKG2D-Ligand, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2 or WT1.

50. The chimeric polypeptide of any one of claims 41-49, wherein: The first target molecule is a tissue-specific marker, selected from: a brain tissue-specific marker MOG, a liver tissue-specific marker ASGR1, a prostate tissue marker PSA, or a combination thereof.

51. The chimeric polypeptide of any one of claims 41-50, wherein: The binding domain of the chimeric polypeptide comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or homologous to any of the amino acid sequences set forth in SEQ ID NO:63, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, or 83.

52. The chimeric polypeptide of any one of claims 41-51, wherein The chimeric polypeptide comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or homologous to any of the amino acid sequences shown in SEQ ID NO:55, 56, 57, 58 or 59.

53. A polynucleotide, wherein The polynucleotide encodes the chimeric polypeptide according to any one of claims 41-52.

54. The polynucleotide of claim 53, wherein The polynucleotide further comprises an expression cassette encoding an exogenous gene operably linked to the intracellular domain of the chimeric polypeptide; preferably, the chimeric polypeptide and the expression cassette of the exogenous gene are located in one vector.

55. An engineered cell comprising the chimeric polypeptide of any one of claims 41-52, and / or the polynucleotide of claim 53 or 54.

56. An engineered cell, wherein: The engineered cell comprises the chimeric polypeptide of any one of claims 41-52, and optionally, further comprises an expression cassette encoding an exogenous gene operably linked to the intracellular domain of the chimeric polypeptide, wherein the intracellular domain of the chimeric polypeptide regulates the expression of the exogenous gene.

57. The engineered cell of claim 56, wherein The binding domain of the chimeric polypeptide specifically binds to a first target molecule, the expression product of the exogenous gene is CAR, and the CAR specifically binds to a second target molecule different from the first target molecule; preferably, the second target molecule is a tumor antigen or an NK cell marker.

58. The engineered cell of claim 57, wherein The second target molecule is selected from: ALPPL2, ALPI, Axl, B7H3, BCMA, CD 117, CD123, CD171, CD179a, CD19, CD213A2, CD20, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD7, CD71, CD94, CD97, CEA, Claudin18.2, CLDN6, CLECL1, CLL1, cMet, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FCRH5, FLT3, GD2, GD3, GM3, GPC3, GPRC5D, HER2 (ERBB2), IGLL1, IL llRa, IL13Ra2, Mesothelin, MUC1, NCAM, NKG2A, NKG2D, NKG2D ligand, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TIGIT, TSHR, VEGFR2 or WT1.

59. The engineered cell according to any one of claims 56 to 58, wherein The first target molecule and the second target molecule are respectively selected from the following combinations: ASGR1 and GPC3, EGFRvⅢ and IL13Ra2, EGFRvⅢ and B7H3, Mesothelin and Claudin18.2, Claudin18.2 and Mesothelin, FAP and Claudin18.2, CLL1 and NKG2D, CD123 and NKG2D, MOG and B7H3, CLL1 and NKG2D ligand, CD123 and NKG2D ligand.

60. The engineered cell according to any one of claims 56 to 59, wherein The chimeric polypeptide comprises the sequence shown in SEQ ID NO: 46 and / or 47, and the CAR comprises the sequence shown in 50; the chimeric polypeptide comprises the sequence shown in SEQ ID NO: 44, 45, 46, 47, 48 and / or 49, the promoter encoding the CAR comprises the nucleotide sequence shown in SEQ ID NO: 30, and the CAR comprises the sequence shown in SEQ ID NO: 51, 52, 53 and / or 54.

61. The engineered cell according to any one of claims 56 to 60, wherein: The engineered cells are immune cells, preferably, the engineered cells are autologous or allogeneic T, NK and / or NKT cells.

62. The engineered cell according to any one of claims 56 to 61, wherein The endogenous B2M, TCR, MHC-II, FAS, NKG2A and / or NKG2D ligand molecules of the engineered cells are low-expressed or not expressed, or a combination thereof; Preferably, the endogenous B2M / TCR / FAS of the engineered cells is lowly expressed or not expressed; Preferably, the sgRNA sequences targeting TRAC, B2M, and FAS are shown in SEQ ID NOs: 142, 143, and 144, respectively.

63. A pharmaceutical composition comprising the engineered cell of any one of claims 1-26 and 56-62, the composition of any one of claims 27-31, the polynucleotide of claim 32, and / or the vector of claim 33, and a pharmaceutically acceptable excipient.

64. A method for preparing the engineered cell according to any one of claims 1-26, 56-62, comprising the following steps: (i) adding an input composition comprising cells to be transduced and a stimulator for cells to be transduced, and incubating; (ii) adding viral vector particles comprising the polynucleotides of claims 32, 53 and / or 54, and incubating; the total incubation time of steps (i) and (ii) does not exceed 24 hours, to obtain the engineered cells.

65. The method of claim 64, wherein: The incubation time of step (i) is not less than 3 hours; the incubation time of step (i) is not less than 1 hour.

66. The method of claim 64 or 65, wherein: The cells to be transduced are T cells; preferably, the cells to be transduced are enriched or separated CD3+T cells, enriched or separated CD4+T cells, or enriched or separated CD8+T cells, or a combination thereof.

67. The method of any one of claims 64 to 66, wherein: The vector is a lentiviral vector, and / or the cell stimulator to be transduced comprises an anti-CD3 antibody and / or an anti-CD28 antibody.

68. The method of any one of claims 64 to 67, wherein: The engineered cells do not need to be expanded in vitro or the in vitro expansion does not exceed 24 hours before being administered to a subject in need.

69. A gRNA construct, comprising a gRNA, wherein the gRNA comprises the sequence shown in SEQ ID NO:

144.

70. The construct of claim 69, wherein The gRNA introduced into the cell together with the gRNA, either before or after, includes the sequences shown in SEQ ID NOs: 142 and 143.

71. A cell comprising a gRNA construct as described in claim 69 or 70. 72.An antibody that recognizes MOG, wherein The antibody is selected from: (1) an antibody comprising a light chain variable region, wherein the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 84, and / or comprises LCDR2 as shown in SEQ ID NO: 85, and / or comprises LCDR3 as shown in any one of SEQ ID NO: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98; (2) an antibody comprising a heavy chain variable region, wherein the heavy chain variable region comprises the HCDR1 shown in SEQ ID NO: 99 or 100, and / or comprises the HCDR2 shown in SEQ ID NO: 101 or 102, and / or comprises the HCDR3 shown in any one of SEQ ID NO: 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115; (3) an antibody comprising (1) a light chain variable region of the antibody and (2) a heavy chain variable region of the antibody; (4) An antibody, which is a variant of the antibody according to any one of (1) to (3), and has the same or similar activity as the antibody according to any one of (1) to (3).

73. The antibody of claim 72, wherein The LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 contained in the antibody are selected from: (1) the amino acid sequence shown in SEQ ID NO: 84, 85, 86, 99, 101, 103; or (2) the amino acid sequence shown in SEQ ID NO: 84, 85, 87, 99, 101, 104; or (3) the amino acid sequence shown in SEQ ID NO: 84, 85, 88, 99, 101, 105; or (4) the amino acid sequence shown in SEQ ID NO: 84, 85, 89, 99, 101, 106; or (5) the amino acid sequence shown in SEQ ID NO: 84, 85, 90, 99, 101, or 107; or (6) the amino acid sequence shown in SEQ ID NO: 84, 85, 91, 100, 102, 108; or (7) the amino acid sequence shown in SEQ ID NO: 84, 85, 92, 100, 102, 109; or (8) the amino acid sequence shown in SEQ ID NO: 84, 85, 93, 100, 102, or 110; or (9) the amino acid sequence shown in SEQ ID NO: 84, 85, 94, 100, 102, 111; or (10) the amino acid sequence shown in SEQ ID NO: 84, 85, 95, 100, 102, or 112; or (11) the amino acid sequence shown in SEQ ID NO: 84, 85, 96, 100, 102, or 113; or (12) the amino acid sequence shown in SEQ ID NO: 84, 85, 97, 100, 102, or 114; or (13) The amino acid sequences shown in SEQ ID NOs: 84, 85, 98, 100, 102, and 115.

74. The antibody of claim 72 or 73, wherein The antibody is selected from: (1) an antibody comprising a light chain variable region, wherein the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 or 128, or a nucleic acid sequence encoding the amino acid sequence, or a variant of any of the above sequences, or an amino acid sequence having at least 80% identity with any of the above sequences, or a nucleic acid sequence encoding the amino acid sequence; (2) an antibody comprising a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or 141, or a nucleic acid sequence encoding the amino acid sequence, or a variant of any of the foregoing sequences, or an amino acid sequence having at least 80% identity with any of the foregoing sequences, or a nucleic acid sequence encoding the amino acid sequence; (3) An antibody comprising (1) the light chain variable region of the antibody and (2) the heavy chain variable region of the antibody.

75. The antibody of any one of claims 72-74, wherein The light chain variable region and heavy chain variable region of the antibody are respectively selected from: SEQ ID NO: 116, 129; SEQ ID NO: 117, 130; SEQ ID NO: 118, 131; SEQ ID NO: 119, 132; SEQ ID NO: 120, 133; SEQ ID NO: 121, 134; SEQ ID NO: 122, 135; SEQ ID NO: 123, 136; SEQ ID NO: 124, 137; SEQ ID NO: 125, 138; SEQ ID NO: 126, 139; SEQ ID NO: 127, 140; or SEQ ID NO: 128, 141, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the above sequence, or a nucleic acid sequence encoding the amino acid sequence.

76. The antibody of any one of claims 72-75, wherein The scFv of the antibody has an amino acid sequence shown in SEQ ID NO: 43, 68, 77, 81, 82 or 83, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the above sequence, or a nucleic acid sequence encoding the amino acid sequence.

77. The antibody of any one of claims 72-76, wherein The antibody is a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, dAb fragment, multifunctional antibody, scFv-Fc antibody or IgG4 antibody.

78. The antibody of any one of claims 72-77, wherein The antibody binds to human or mouse MOG; and / or, the antibody binds to cells expressing human or mouse MOG.

79. The antibody of any one of claims 72-78, wherein The antibodies are fully human antibodies.

80. An immunoconjugate, wherein The immunoconjugate comprises: the antibody according to any one of claims 72-79, and a functional molecule linked thereto.

81. A chimeric polypeptide, wherein a) the binding domain of the chimeric polypeptide comprises the antibody of any one of claims 72-79; b) a receptor regulatory domain comprising one or more cleavage sites, the receptor regulatory domain comprising an extracellular region and a transmembrane region; and c) intracellular domain, The binding of the binding domain to MOG can induce the cleavage of the receptor regulatory domain, thereby releasing the intracellular domain.

82. A biological material, which is any one of the following: 1) A nucleic acid encoding the antibody of any one of claims 72 to 79, the immunoconjugate of claim 80, or the chimeric polypeptide of claim 81; 2) comprising the expression vector described in 1); or 3) comprising the virus described in 1) or 2).

83. An engineered cell comprising the chimeric polypeptide of claim 81.

84. A pharmaceutical composition comprising: The antibody of any one of claims 72 to 79, the immunoconjugate of claim 80, the chimeric polypeptide of claim 81, the biomaterial of claim 82, the engineered cell of claim 83, or a combination thereof.

85. A method for regulating the activity of the engineered cell of claim 83, wherein: The method comprises contacting the engineered cell with a MOG polypeptide, wherein the MOG binds to the binding domain of the chimeric polypeptide on the engineered cell, thereby inducing the cleavage of the proteolytic cleavage site of the chimeric polypeptide and releasing the transcription factor in the intracellular domain of the chimeric polypeptide, thereby regulating the activity of the engineered cell.

86. The method of claim 85, wherein: It is a method for treating a disease; preferably, it is a method for treating a tumor; preferably, it is a method for treating a central nervous system tumor; preferably, it is a method for treating a glioma.

87. A kit, wherein It includes one or more of the following: a) the antibody according to any one of claims 72 to 79; b) the immunoconjugate of claim 80; c) the chimeric polypeptide of claim 81; d) the biological material according to claim 82; e) The engineered cell of claim 83; f) The pharmaceutical composition of claim 84.

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