Targeted protein degradation system and its applications

Abstract

The present disclosure provides endoplasmic reticulum-based chimeric protein constructs for targeted protein degradation and their applications in the treatment of cancer, viral infections, autoimmune diseases, neurodegenerative diseases, and the like. [Solution] The chimeric protein construct provided in the present disclosure includes an ERAD machinery protein binding domain and a targeting domain. The ERAD machinery protein binding domain of the chimeric protein construct can be a transmembrane domain of a viral endoplasmic reticulum resident protein or a functional variant thereof, and an endoplasmic reticulum resident domain or a functional variant thereof. The targeting domain of the chimeric protein construct can target any target protein of interest, and can be a natural ligand of the target protein, an antibody or an antigen-binding fragment thereof that specifically recognizes the target protein. The targeting domain of the chimeric protein construct can also be an antigen that is specifically recognized by an antibody.

Description

[Technical field]

[0001] The present disclosure relates to a targeted protein degradation system and its applications, particularly in disease treatment, including but not limited to cancer, viral infections, autoimmune diseases, and neurodegenerative diseases. [Background technology]

[0002] Targeted protein ablation is needed for at least the following reasons: 1) to determine the function of a protein in basic research; 2) to validate therapeutic targets; 3) to inhibit the action of pathogenic proteins (e.g., tumor proteins or proteins that cause neurodegeneration); 4) to promote the degradation of proteins that accumulate during the aging process; and 5) to reduce side effects of cell therapy (e.g., to inhibit the expression of immunogenic proteins). However, each of the conventional techniques for targeted protein ablation has its own technical problems. Gene editing techniques (e.g., CRISPR technology) can target and knock out genes encoding any target protein at the genetic level, but they have problems with off-target effects, low specificity in recognizing and cleaving the desired DNA site, and prone to side effects such as chromosomal instability.

[0003] Small interfering RNA (siRNA) can inhibit the synthesis of target proteins at the RNA level, but siRNA also has potential off-target effects. For example, the sense strand of siRNA can mediate expression silencing of cognate genes, causing sense strand-mediated off-target effects. Small mismatches in small nucleic acid sequences can also cause silencing of other genes, resulting in off-target effects and resulting in toxic and side effects. Protein targeted degradation chimera technology (PROTAC) can degrade target proteins at the protein level, and is usually added to cells as a biochemical reagent or administered to animals or humans to induce the degradation of target proteins in cells. Early PROTACs were based on polypeptide ligand molecules linked with E3 ligase, but PROTACs made with polypeptide ligand molecules have low cell permeability and instability, resulting in low degradation efficiency. PROTACs with small molecules as E3 ligase ligands have shown remarkable efficacy, but their membrane permeability and bioavailability are poor. Thus, there is an urgent need for novel targeted protein degradation systems to overcome at least one of the problems present in the various conventional techniques for targeted removal of target proteins. Summary of the Invention [Problem to be solved by the invention]

[0004] In one aspect, the present application provides a chimeric protein construct that degrades a target protein via the endoplasmic reticulum-associated degradation (ERAD) mechanism, comprising an ERAD mechanism protein binding domain and a targeting domain. In some embodiments, the present application provides a universal targeted protein degradation module that hijacks the ERAD mechanism to inhibit trafficking of the target protein in the ER and promotes translocation of the target protein to the cytoplasm, and then ubiquitinates and degrades the target protein via the ubiquitin proteasome-based UPS mechanism and / or endocytoses and degrades the target protein via the lysosome-based ALP mechanism. Such a design can effectively inhibit the expression and rapidly degrade any protein (including endogenous or exogenous proteins) to achieve therapeutic purposes. The present application redesigns viral elements to target various endogenous or exogenous target proteins and achieve retention and targeted degradation of the target protein in the endoplasmic reticulum. In another aspect, the present application provides a chimeric protein construct that synergistically degrades a target protein by the ERAD machinery and the ubiquitination machinery, and includes an ERAD machinery protein binding domain, a targeting domain and a protein degradation pathway member binding domain. Such a design can greatly improve the degradation efficiency of the target protein, and is suitable for many targets that cannot be degraded by PROTAC technology.

[0005] In the present application, the two chimeric protein constructs are also referred to as TPD (Targeted Protein Degradation) chimeric protein constructs. The present application further provides nucleic acids encoding the chimeric protein constructs, nucleic acid vectors, oncolytic viruses, and cells expressing the chimeric protein constructs, as well as their use in the treatment of diseases. Embodiment 1. A chimeric protein construct comprising an endoplasmic reticulum-associated degradation (ERAD) machinery protein-binding domain and a targeting domain. Embodiment 2. The chimeric protein construct described in embodiment 1, wherein the ERAD machinery protein binding domain comprises a transmembrane domain of a viral endoplasmic reticulum resident protein or a functional variant thereof and an endoplasmic reticulum resident domain or a functional variant thereof. Embodiment 3. A chimeric protein construct described in some embodiments 1 or 2, wherein the viral endoplasmic reticulum resident protein is adenovirus E3-19K. Embodiment 4. A chimeric protein construct described in embodiment 1 or 2, wherein the viral endoplasmic reticulum resident protein is not adenovirus E3-19K. Embodiment 5. The chimeric protein construct of some embodiment 4, wherein the viral endoplasmic reticulum resident protein is at least one selected from the group consisting of HCMV glycoproteins US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, and Cowpox Virus protein CPXV203.

[0006] Embodiment 6. A chimeric protein construct described in any one of embodiments 1 to 5, further comprising a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain, which may be linked to the ERAD machinery protein binding domain. Embodiment 7. The chimeric protein construct according to any one of embodiments 1 to 6, wherein the targeting domain comprises an antibody or a functional fragment thereof (e.g., Fd, Fv, Fab, Fab', F(ab')2, Fv(scFv), single chain antibody (scFv), nanobody, two chain antibody, three chain antibody and four chain antibody) that specifically targets the target protein. Embodiment 8. The chimeric protein construct of embodiment 7, wherein the target protein is a pathogenic protein, and the pathogenic protein may be a tumor-associated protein, a virus-associated protein, an immune function-associated protein (including an immunosuppressive protein and an immune activating protein), an autoantigen protein or a neurodegenerative disease-associated protein. 9. The autoantigen protein is selected from the group consisting of islet cell antigen (ICA), insulin (IAA), glutamic acid decarboxylase 65 (GAD65), insulinoma antigen-2 (IA-2), and other autoantigens associated with type I diabetes; citrullinated protein / peptide antibodies, heterogeneous nuclear ribonucleoprotein A2 / B1, aldolase, alpha-enolase, calreticulin, heat shock protein (HSP60), BiP, PGK1, stress-induced phosphoprotein 1, and FUSE-BP1 / 2, and other autoantigens associated with rheumatoid arthritis (RA); deoxyribonucleoproteins, SmD1, SmD3, Clq, lupus anticoagulant (LA), cardiolipin (CL), and beta 2 glycoprotein I (β2 GP autoantigens related to systemic lupus erythematosus (SLE), such as prothrombin (PT), and phosphatidylserine (PS); autoantigens related to systemic sclerosis (SSc) / scleroderma (SD), such as Scl-70, SSA, and Ro52; autoimmune liver disease-related antigens, such as mitochondrial antigens, Spl00, PML, gp210, and p62; autoantigens related to myasthenia gravis, such as acetylcholine receptors; central nervous system (CNS) autoantigens, such as the voltage-dependent potassium channel (VGKC) complex, voltage-dependent calcium channel receptors, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, gamma-aminobutyric acid-B (GABAB) receptors, and glycine receptors. Autoantigens related to nervous system autoimmune diseases (limbic encephalitis, encephalomyelitis, cerebellar ataxia); multiple sclerosis-related autoantigens such as myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG); polymyositis (PM) and dermatomyositis (DM)-related autoantigens such as Jo-1, Mi-2, PM-Scl, and Ro-52; gluten-sensitive enteropathy-related autoantigens such as endomysial antibody (EMA) and tissue transglutaminase (tTG); anti-NMDAR antibody encephalitis-related autoantigens such as N-methyl-D-aspartate receptor; and neuromyelitis optica (NMO)-related autoantigens such as aquaporin 4 (AQP4).9. The chimeric protein construct of embodiment 8, selected from the group consisting of reproduction-related autoantigens, such as ovarian antigens, sperm antigens, etc.;

[0007] Embodiment 10. The tumor-related protein is BCL-2, c-MYC, Ras, HER2, BCR / ABL, ABL1 / BCR, TGFB1, TLX1, P53, WNT1, WNT2, WT1, αv-β3, PKCa, ABL, BCL1, CD24, CDK4, EGFR / ERBB-1, HSTF1, INT1 / WNT1, INT2, MDM2, MET, MYB, MYC, MYCN, MYCL1, RAFI, NRAS, REL, AKT2, APC, BCL2-ALPHA, BCL2-BETA, BCL3, BCR, BRCA1, BRCA2, CBL, CCND1, CDKN1A, CDKN1C, CDKN2A, CDKN2B, CRK, CRK-II, CSF1R / FMS, DBL, DDOST, PMS-2, PRAD-1, RAF, RHOM-1, RHOM-2, SIS, TAL2, TANI, TIAM1, TSC2, TRK, TSC1, STK11, PTCH, MEN1, MEN2, P57 / KIP2, PTEN, HPC1, ATM, XPA / XPG, BCL6, DEK, AKAP13, CDH1, BLM, EWSR1 / FLI1, FES, FGF3, FER, FGR, FLI1 / ERGB2, FOS, FPS / FES, FRA1, FRA2, FYN, HCK, HEK, HER3 / ERBB-2, ERBB-3, HER4 / ERBB-4, HST2, INK4A, INK4B, JUN, JUNB, JUND, KIP2, KIT, KRAS2A, KRAS2B, LCK, LYN, MAS, MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P53, PDGFB, PIM1, PTC, RBI, RET, ROS1, SKI, SRC1, TALI, TGFBR2, THRA1, THRB, TIAM1, TRK, VAV, VHL, WAF1, WNT2, WT1, YES1, ALK / NPM1, AMI1, AXL, FMS, GIP, GLI, GSP, HOX11, HST, IL3, INT2, KS3, K-SAM, LBC, DCC, DPC4 / SMAD4, E-CAD, E2F1 / RBAP, ELK1, ELK3, EPH, EPHA1, E2F1, EPHA3, ERG, ETS1, ETS2, LMO-1, LMO-2, L-MYC, LYL1, LYT-10, MDM-2, MLH1, MLL, MLM, N-MYC, OST, PAX-5, PMS-1, FGF4, FGF6, FANCA,FLI1 / ERGB2, FOSL1, FOSL2, GLI, HRAS1, HRX / MLLT1, HRX / MLLT2, KRAS2, MADH4, MASI, MCF2, MLLT1 / MLL, MLLT2 / HRX, MTG8 / RUNX1, MYCLK1, MYH11 / CBFB, NFKB2, NOTCH1, NPM1 / ALK, NRG / REL, NTRK1, PBX1 / TCF3, PML / RARA, PRCA1, RUNX1, RUNX1 / CBFA2T1, SET, SHP2, TCF3 / PBX1, TNFα, clusterin, survivin, TOEβ, c-fos, c-SRC, estrogen receptor ER-α, androgen receptor and INT-1, and the tumor-associated protein is encoded by an oncogene selected from the group consisting of Bcl-2 family members (e.g., Bcl-2, Bcl-xL, and Bcl-w), VEGF / VEGFR, PDGFRβ, EGFR, EGFR mutants, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor ER-α, andro ... 10. The chimeric protein construct according to embodiment 9, which may be selected from the group consisting of: tyrosine kinases (c-ABL, BCR-ABL, BTK, FAK, PTK6, Wee1, TRK transmembrane receptors), serine / threonine kinase receptors (IRAK4, LRRK2, B-Raf, RIPK2, CDK4 / 6, CDK7, CDK8, CDK8 / 19, CDK9, TBK1), protein kinase II (CK2), epigenetic related proteins (BRD2, BRD3, BRD4, BRDT, TRIM24, BRD9, PBRM1, SMARCA2, SMARCA4, EP300, EZH2, WDR5), adrenomedullin (ADM), DPP3.

[0008] Embodiment 11. The pathogenic protein is selected from the group consisting of HBV surface antigen, HBV capsid glycoprotein, HBeAg, HBV DNA polymerase, HBV encoded X protein (HBx), HIV Gag protein, HIV Env protein, HIV gp120, HIV-1 reverse transcriptase, HIV gp120, HCV NS3-4A protease, HCV RNA polymerase, HCV envelope protein, EBV DNA polymerase, EBV EBNA1, coronavirus RNA synthetase, coronavirus spike protein, coronavirus envelope protein, coronavirus membrane protein, coronavirus nucleocapsid protein, RNA-dependent RNA polymerase (RdRp) such as novel coronavirus RNA-dependent RNA polymerase, Herpesviruses DNA RNA polymerase, Herpesvirus capsid glycoprotein, CMV The chimeric protein construct of some embodiments 8, wherein the chimeric protein construct is a viral associated protein selected from DNA polymerase, CMV capsid glycoprotein, RSV envelope protein, RSV capsid protein, RSV RNA polymerase, influenza virus RNA polymerase, influenza virus envelope protein, HPV DNA polymerase, and HPV capsid protein.

[0009] Embodiment 12. The chimeric protein construct of some embodiments 7, wherein the target protein is an immune function-related protein selected from antigen-presenting molecules (e.g., MHC class I molecules, MHC class II molecules, MICA / B molecules, etc.), antigen-recognition molecules (e.g., TCR, CD123, NKG2D, etc.), immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), and immune stimulatory / co-stimulatory molecules (e.g., CD3, CD80 / 86, CD28, etc.). Embodiment 13. The chimeric protein construct of some embodiment 7, wherein the target protein is a nervous system disease-related target protein selected from Tau, amyloid-β (Aβ), α-synuclein, mutant huntingtin (mHTT), α-synuclein, TAR RNA binding protein (TARDBP) and FUS RNA binding protein (FUS). Embodiment 14. The chimeric protein construct of embodiment 2, 4 or 5, wherein the targeting domain does not specifically bind to a TCR. Embodiment 15. The chimeric protein construct of some embodiment 14, wherein the targeting domain specifically binds to MHC I, MHC II, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and / or ULBP6, preferably wherein MHC I is HLA I, and preferably wherein MHC II is HLA II. Embodiment 16. The chimeric protein construct of embodiment 4, wherein the targeting domain specifically binds to a TCR.

[0010] Embodiment 17. A chimeric protein construct described in some embodiment 16, wherein the viral endoplasmic reticulum resident protein is at least one selected from the group consisting of HCMV glycoproteins US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, and Cowpox Virus protein CPXV203. Embodiment 18. A chimeric protein construct according to any one of embodiments 1 to 17, linked to at least one co-expression moiety. Embodiment 19. The chimeric protein construct of embodiment 18, wherein the at least one co-expressed portion is independently selected from an intact viral ER resident glycoprotein (e.g., HCMV US2, US3, US11, US10, adenovirus E3-Kl 9, HCMV US6, HSV ICP47), a chimeric antigen receptor (CAR), a functional T cell receptor (TCR), a chemokine receptor and an NK cell activating receptor. Embodiment 20. The chimeric protein construct of embodiment 19, wherein the chemokine receptor is selected from CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2 and CXCR4. Embodiment 21. The chimeric protein construct of embodiment 20, wherein the NK cell activating receptor comprises (a) an extracellular domain (ED) of an NK cell activating receptor or a functional variant thereof, (b) a transmembrane domain (TMD) of an NK cell activating receptor or a functional variant thereof, and (c) an intracellular domain (ICD) of an NK cell activating receptor or a functional variant thereof; and a hinge or linker may be included between the extracellular domain of an NK cell activating receptor or a functional variant thereof, the transmembrane domain of an NK cell activating receptor or a functional variant thereof and / or the intracellular domain of an NK cell activating receptor or a functional variant thereof. Embodiment 22. The chimeric protein construct of embodiment 21, wherein the NK cell activating receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxicity receptor, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC and NKp80.

[0011] Embodiment 23. A chimeric protein construct described in embodiment 21 or 22, wherein the NK cell activating receptor is complexed with a CNK signaling module. Embodiment 24. A chimeric protein construct described in any one of embodiments 18 to 23, wherein the at least one co-expression moiety is linked to the chimeric protein construct by a cleavable linker. Embodiment 25. A nucleic acid molecule encoding a chimeric protein construct according to any one of embodiments 1 to 24. Embodiment 26. The nucleic acid molecule according to any of embodiments 25, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (e.g., mRNA, circular RNA, ccRNA), threose nucleic acid (TNA), ethylene glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribose configuration, α-LNA (diastereoisomer of LNA) having an α-L-ribose configuration, 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-α-LNA having a 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof. Embodiment 27. A vector comprising a nucleic acid molecule described in embodiment 25 or 26, wherein the nucleic acid molecule is operably linked to at least one polynucleotide regulatory element so as to express a chimeric protein construct encoded by the nucleic acid molecule. Embodiment 28. The vector described in embodiment 27, wherein the vector is selected from a plasmid, a nanoplasmid, a cosmid, a viral vector (e.g., an oncolytic viral vector), a minicircle, an RNA vector, or a linear or circular DNA (e.g., transposon DNA) or an RNA molecule.

[0012] Embodiment 29. The vector of embodiment 28, wherein the vector is a viral vector selected from retroviruses, lentivirus vectors, adenoviruses, parvoviruses (e.g., adeno-associated viruses), adeno-associated virus (AAV) vectors, coronaviruses, negative-stranded RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., matchiviruses and Sendai viruses), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, wherein the double-stranded DNA viruses include adenoviruses, herpesviruses (e.g., herpes simplex viruses I and II, Epstein-Barr virus, cytomegalovirus), poxviruses (e.g., cowpox virus, fowlpox virus, and canarypox virus), Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, baculoviruses, hepatitis viruses, and virus-like particles (VLPs). Embodiment 30. The vector described in some embodiments 29, wherein the viral vector is a retroviral vector. Embodiment 31. The vector of embodiment 30, wherein the retroviral vector is selected from avian leukoproliferative sarcoma, mammalian C-type, B-type, D-type viruses, HTLV-BLV collection, lentiviruses, and foamy viruses. Embodiment 32. The vector described in some embodiments 29, wherein the viral vector is a lentiviral vector. Embodiment 33. The vector of embodiment 32, wherein the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or ovine demyelinating leukoencephalitis lentivirus. Embodiment 34. The vector according to any one of embodiments 27 to 33, further combined with another vector comprising another nucleic acid molecule encoding at least one coexpression moiety. Embodiment 35. An engineered cell expressing a chimeric protein construct described in any one of embodiments 1 to 25, or comprising a nucleic acid molecule described in embodiment 26, or comprising a vector described in any one of embodiments 27 to 33.

[0013] Embodiment 36. An engineered cell according to embodiment 35, expressing the chimeric protein construct and co-expression moiety according to any one of embodiments 1 to 18. Embodiment 37. The engineered cell of some embodiment 35 or 36, wherein the cell is an immune cell selected from a T cell, a natural killer (NK) cell, a B cell, a macrophage, a monocyte, a dendritic cell, a neutrophil or a γδ T cell. Embodiment 38. The engineered cell of embodiment 37, wherein the T cells are selected from the group of cells consisting of CD8+ T cells, CD4+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, gamma-delta T cells, cytokine-induced killer (CIK) T cells and tumor-infiltrating lymphocytes, and regulatory T cells. Embodiment 39. A method for producing an engineered cell described in any one of embodiments 35 to 38, comprising introducing into a starting cell a vector described in any one of embodiments 27 to 34 under conditions suitable for expressing the nucleic acid molecule described in embodiment 25 or 26. Embodiment 40. The method of some embodiment 39, wherein the starting cell is a stem cell or a cell differentiated from a stem cell.

[0014] Embodiment 41. The method of some embodiment 38, wherein the stem cells are hematopoietic progenitor cells (e.g., T cell progenitors, NK cell progenitors, macrophage progenitors), hematopoietic stem cells (HSCs), CD34+ cells, embryonic stem cells, mesenchymal stem cells or iPSC cells. Embodiment 42. The method of any of embodiments 37, wherein the cell is an immune cell. Embodiment 43. The method of some embodiment 40, wherein the immune cell is a T cell, a natural killer (NK) cell, a B cell, a macrophage, a monocyte or a dendritic cell. Embodiment 44. The method of some embodiments 41, wherein the cell is a T cell selected from the group consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, gamma-delta T cells, cytokine-induced killer (CIK) T cells and tumor-infiltrating lymphocytes, and regulatory T cells. Embodiment 45. A cell population generated ex vivo by a method according to any one of embodiments 39 to 44. Embodiment 46. A pharmaceutical composition or kit comprising (i) a chimeric protein construct according to any one of embodiments 1 to 24, or a nucleic acid molecule according to embodiment 25 or 26, or a vector according to any one of embodiments 27 to 34, or an engineered cell according to any one of embodiments 35 to 38, or a cell population according to embodiment 45, and (ii) a pharma- ceutically acceptable vehicle. Embodiment 47. A method for degrading a target protein, comprising delivering the vector described in any one of embodiments 27 to 34 to a cell expressing the target protein. Embodiment 48. The method of embodiment 47, wherein the cell is selected from a tumor cell, a virus-infected cell, a neuronal cell, a transplant cell, and an immune cell. Embodiment 49. The method of embodiment 47 or 48, wherein the delivery comprises delivery by a viral vector (e.g., an oncolytic virus). Embodiment 50. The method described in embodiment 49, wherein the vector expresses in the cell a chimeric protein construct described in any one of embodiments 1 to 24. Embodiment 51. The method described in embodiment 50, wherein the chimeric protein construct is capable of simultaneously binding to a target protein and an ERAD machinery protein, thereby degrading the target protein. Embodiment 52. A method of treating a condition or disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 46.

[0015] Embodiment 53. The disease includes various solid tumors, hematological tumors, viral infections, autoimmune diseases, neurological diseases, degenerative diseases (e.g., Alzheimer's disease), metabolic diseases (e.g., diabetes (e.g., type II diabetes), lipid metabolism-related diseases (e.g., tumor lipid metabolism, nonalcoholic fatty liver disease), atherosclerosis (AS), tumor glucose metabolism); Preferably, the solid tumor is selected from nervous system tumors, head and neck tumors, thoracic tumors, digestive system tumors, genitourinary system tumors, soft tissue and skin tumors, bone tumors, etc.; Preferably, the nervous system tumors include diffuse glioma, diffuse astrocytoma / anaplastic astrocytoma, glioblastoma, oligodendroglioma, oligoastrocytoma, childhood diffuse glioma, other astrocytoma, ependymoma, neuronal / mixed neuronal-glioma, medulloblastoma, other embryonal tumors, schwannoma, meningioma, solitary fibrous tumor, hemangiopericytoma, etc.; Preferably, the head and neck tumor includes nasal cavity and paranasal sinus malignant tumor, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumor, intracranial tumor, thyroid cancer, tongue cancer, etc.; Preferably, thoracic tumors include lung cancer, esophageal cancer, cardia cancer, breast cancer, mediastinal tumors, etc.; Preferably, the digestive system tumor includes gastric cancer, colon cancer, sigmoid colon / rectal cancer, liver cancer, pancreatic cancer / periampullary cancer, biliary tract cancer, small intestinal malignant tumor, etc.; Preferably, genitourinary system tumors include kidney cancer, prostate cancer, bladder cancer, testicular malignancies, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, etc.; Preferably, the soft tissue and skin tumors include malignant fibrous histocytoma, rhabdomyosarcoma, synovial sarcoma, cutaneous melanoma, etc.; Preferably, bone tumors include osteosarcoma, Ewing's sarcoma, and the like; Preferably, the colon cancer is a colon adenoma; Preferably, the breast cancer is a triple negative breast cancer cell; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the disease is a hematological tumor selected from leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), etc.; Preferably, the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, etc.; Preferably, the viral infection includes respiratory viral disease, gastrointestinal viral disease, liver viral disease, skin and mucous membrane viral disease, eye viral disease, central nervous system viral disease, lymphocytic viral disease, insect-borne viral disease, lentivirus infection disease, etc.; Preferably, the respiratory viral disease includes infections such as rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus and coronavirus; influenza; mumps; etc.; Preferably, the gastrointestinal viral disease includes polio; Cooksackie virus infection; ECHO virus infection; the viral gastroenteritis includes rotavirus gastroenteritis, Norwalk virus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis, calicivirus gastroenteritis, etc.; Preferably, the liver viral disease includes viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, EB virus hepatitis and cytomegalovirus hepatitis, etc.; Preferably, the skin and mucous membrane viral diseases include measles, rubella, infantile exanthema, chickenpox and shingles, smallpox, herpes simplex virus infection, rabies, and foot and mouth disease; Preferably, the ocular viral disease includes epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, etc.; Preferably, the central nervous system viral disease includes epidemic B encephalitis, western equine encephalitis, eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, hymenoptera encephalitis, and lymphocytic choroid plexus meningitis, etc.; Preferably, the lymphocytic viral disease includes infectious mononucleosis, cytomegalovirus infection, acquired immune deficiency syndrome, and the like; Preferably, the insect-borne viral disease includes viral hemorrhagic fevers such as epizootic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Omuscrub hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever; Dengue fever and Dengue hemorrhagic fever; West Nile fever; Colorado tick fever; Sandfly fever, etc.; Preferably, lentivirus infection diseases include subacute sclerosing panencephalitis, Kuhl's disease, progressive multifocal leukoencephalopathy, and subacute spongiform encephalopathy (corticostriatospinal degeneration), etc.; Preferably, the autoimmune disease includes organ-specific and systemic autoimmune diseases; Preferably, the organ-specific autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebrospinal sclerosis, acute idiopathic polyneuropathy, etc.; Preferably, the systemic autoimmune disease includes systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.; Preferably, the nerve disease is a nervous system disease or peripheral nerve disease such as trigeminal neuralgia, facial paralysis, facial muscle spasm, vestibular neuritis, glossopharyngeal neuralgia, mononeuropathy, brachial plexus neuralgia, multiple mononeuropathy, polyneuropathy, acute inflammatory demyelinating polyneuropathy, or chronic inflammatory demyelinating polyneuropathy; Spinal diseases such as spondylitis, compressive myelopathy, subacute complex degeneration of the spinal cord, syringomyelia, spinal vascular disease, and spinal arachnoiditis; Cerebrovascular diseases such as transient ischemic attack, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, and intracranial venous thrombosis; Central nervous system infections such as meningitis, encephalitis, etc. caused by infection with viruses, bacteria, fungi, or parasites, and lentiviral encephalitis caused by lentivirus infection; Demyelinating diseases of the central nervous system, such as multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, and leukodystrophies; Movement disorders such as Parkinson's disease, chorea, hepatolenticular degeneration, muscle tone disorders, idiopathic tremor, and tardive movement disorders; epilepsy; Headaches such as migraines, tension headaches, and cluster headaches; Degenerative diseases of the nervous system, such as motor neuron disease, Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, and multisystem atrophy; Genetic disorders of the nervous system, such as hereditary ataxia, hereditary spastic paraplegia, peroneal muscular atrophy, neurofibromatosis, tuberous sclerosis, and superficial cerebrovascular disease; Neurodevelopmental disorders such as congenital hydrocephalus, cerebral palsy, basilar depression, and subcerebellar tonsillar disease; Neuromuscular junction and muscle disorders such as myasthenia gravis, periodic paralysis, polymyositis, progressive muscular dystrophy, myotonic myopathy (tonic muscular dystrophy, myotonia congenita), metabolic myopathy (mitochondrial myopathy, lipid storage myopathy, glycogenosis); Autonomic nervous system disorders such as Raynaud's disease, erythematous limb pain, hemifacial atrophy, generalized autonomic dysfunction, spontaneous hyperhidrosis, and progressive lipodystrophy; Nervous system tumors such as brain gliomas, lymphomas, and spinal cord tumors; This includes paraneoplastic syndromes of the nervous system, such as paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, subacute necrotizing myelopathy, subacute motor neuron disease, and paraneoplastic sensory neuron disease, Preferably, the method according to embodiment 52, wherein the metabolic disease comprises diabetes, hyperlipidemia, gout, and the like. Embodiment 54. A method for stimulating an immune response in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 46. [Brief description of the drawings]

[0016] [Figure 1]The chimeric protein construct of endoplasmic reticulum-based targeted protein degradation technology is shown. Here, FIG. 1A is a linear schematic and structural schematic of the basic ER-TPD chimeric protein construct TBD-TMD-ERD; FIG. 1B shows the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD. [Diagram 2] 2A shows the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L with E3 ubiquitin ligase ligand domain, where FIG. 2A is a linear schematic, FIG. 2B is a structural schematic, and FIG. 2C shows the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L. [Diagram 3] 3 shows the ER-TPD chimeric protein construct TBD-TMD-ERD-E2L with E2 ubiquitin-conjugating enzyme ligand domain, where FIG. 3A is a linear schematic, FIG. 3B is a structural schematic, and FIG. 3C shows the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L. [Figure 4] ER-TPD chimeric protein construct TBD-TMD-ERD-LL with lysosomal ligand domain, where FIG. 4A is a linear schematic, FIG. 4B is a structural schematic, and FIG. 4C shows the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-LL. [Diagram 5] ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL with combination of E3 ubiquitin ligase ligand and lysosomal ligand, where FIG. 5A is a linear schematic, FIG. 5B is a structural schematic, and FIG. 5C shows the mechanism of action of ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL. [Figure 6] ER-TPD chimeric nucleic acid constructs targeting TCR, TCR-ER-TPD, in which FIG. 6A shows chimeric nucleic acid construct TCR-TPD1; FIG. 6B shows chimeric nucleic acid construct TCR-TPD2. [Figure 7] The results of detecting the cell phenotype of TCR ER-TPD Jurkat cells at 48 h (A), 96 h (B), and day 6 (C) are shown. [Figure 8] The expression vectors designed using a single lentivirus expression vector with the EF1α promoter as the promoter are shown. Here, Figure 8A shows the CNK expression vector; Figure 8B shows the chimeric protein construct HLA-ER-TPD; Figure 8C shows the chimeric protein construct CNK-HLA-ER-TPD. [Figure 9] 1 shows the results of flow cytometry measurement of cell phenotype 48 hours after co-transfection of 293 cells with an ER-TPD chimeric nucleic acid construct targeting HLA-abc and a CNK expression vector. [Figure 10] 10A shows an ER-TPD chimeric nucleic acid construct targeting CD123, where FIG. 10A shows a CNK expression vector; FIG. 10B shows a chimeric protein construct HLA-TPD. [Figure 11] 1 shows the flow cytometry measurement of cell phenotype 48 hours after transfection of an scFv TPD chimeric nucleic acid construct targeting CD123 into acute myeloid leukemia cell line THP1 cells. [Figure 12] The application of ER-TPD-CD123 CAR-T cells targeting CD123 is shown. Here, Fig. 12A1 shows CD123 CAR, Fig. 12B1 shows the expression of CD123 in activated T cells, Fig. 12C1 shows that CAR-T cells targeting CD123 produce suicide effect in activation culture and tumor cell killing process; Fig. 12A2 shows CD123 CAR-T-T2A-CD123 ER-TPD chimeric protein construct, Fig. 12B2 shows that expressing this chimeric protein construct can degrade self-expressed CD123, and Fig. 12C2 shows the killing effect on tumor cells after expressing this chimeric protein construct. [Figure 13]The application of ER-TPD chimeric protein constructs targeting autoantibodies in the treatment of autoimmune diseases is shown. Here, Fig. 13A shows that insulin antibodies are expressed and released in plasma cells of type I diabetes patients, and these antibodies bind to insulin to remove insulin from peripheral blood, resulting in a significant decrease in the patient's insulin utilization rate; Fig. 13B shows that ER-TPD transposon DNA targeting insulin antibodies is delivered into plasma cells using liposomes targeting plasma cells, achieving integration and long-term expression of the IAA ER-TPD chimeric protein construct. [Figure 14] Exemplary chimeric fusion proteins comprising one or more transmembrane and cytoplasmic domains of TCRαβ or HLA molecule binding molecules, viral ER resident proteins are described. Here, FIG. 14A is a schematic diagram and composition of UT chimeric multidomains. The broad structure of one viral ER resident glycoprotein (VEP) consists of a luminal domain (LD), a transmembrane domain (TMD) and a cytoplasmic functional domain; the UT chimeric multidomain consists of a TCR or HLA binding domain, a hinge / spacer, a VEP transmembrane and cytoplasmic domain. FIG. 14B is a schematic diagram and composition of UT chimeric multidomains. The broad structures of different viral ER resident glycoproteins (VEP) consist of different intraluminal domains (LD), a transmembrane domain (TMD) and a cytoplasmic functional domain; the UT chimeric multidomain consists of a TCR or HLA binding domain, a hinge / spacer and a VEP transmembrane and cytoplasmic domain. FIG. 14C is a schematic diagram and composition of UT chimeric multidomains. The broad structure of different viral ER-resident glycoproteins (VEPs) consists of different luminal domains (LDs), transmembrane domains (TMDs) and cytoplasmic functional domains; UT chimeric multidomains consist of TCR or HLA binding domains, hinge / spacer, different VEP transmembrane domains and chimeric cytoplasmic domains are derived from different VEPs. [Figure 15]We show that forced expression of UT elements can effectively inhibit the expression of TCR in Jurkat cells. A lentiviral vector with truncated EGFR was constructed, which encodes anti-TCR scFv fused with different viral ER resident proteins, HCMV US2, US3, US11, adenovirus E19TM / CT domain, and encodes anti-TCR scFv fused with US2 and adenovirus E19TM / CT. The lentivirus was transduced into Jurkat cells, and non-transduced Jurkat cells served as control. 48 hours after transduction, the cells were subjected to flow cytometer to detect the expression of TCRαβ in transduced cells (EGFR+ cells). The results showed that: all EGFR(+) Jurkats showed obvious downregulation of TCRαβ expression; cells transduced in combination with UT elements (US2 / E19) can effectively inhibit the expression of TCRαβ. All tested UT chimeric elements (US2, US3, US11, E19) inhibited the expression of TCRαβ. The combination of UT elements (US2 and E19) significantly improved the inhibitory effect on the expression of TCRαβ on the surface of Jurkat cells. [Figure 16]Here, FIG. 16A shows that forced expression of UT elements can effectively inhibit the expression of TCR in human T cells. A lentiviral vector carrying an expression cassette containing a truncated EGFR encodes an anti-TCR scFv (anti-TCR-US2TM / CT-T2A-EGFRt) fused to the HCMV US3TM / CT domain. Human T cells were stimulated and transduced with this lentivirus, and untransduced T cells served as the control. Five days after transduction, the cells were subjected to a flow cytometer to detect the expression of TCRαβ in the transduced T cells (EGFR+ cells). The results showed that EGFR(+)T showed obvious downregulation of TCRαβ expression compared with untransduced T cells. This indicated that the US elements containing anti-TCR-US3TM / CT could effectively inhibit the expression of TCRαβ in human T cells. Figure 16B shows that forced expression of UT elements can effectively inhibit the expression of TCR in human T cells. A lentiviral vector carrying an expression cassette containing truncated EGFR encodes an anti-TCR scFv (anti-TCR-E19TM / CT-T2A-EGFRt) fused to the HCMV E19TM / CT domain. Human T cells were stimulated and transduced with this lentivirus, and untransduced T cells served as the control. Five days after transduction, the cells were subjected to a flow cytometer to detect the expression of TCRαβ in transduced T cells (EGFR+ cells). The results showed that EGFR(+)T showed obvious downregulation of TCRαβ expression compared with untransduced T cells. Increased viral transduction can further improve the inhibition on the expression of TCRαβ on the T cell surface. This indicated that the US element containing anti-TCR-E19TM / CT can effectively express TCRαβ in human T cells. [Figure 17]We show that CNK-T cells with UT elements downregulated the expression of TCR in CD8 / CD4 cells. The lentiviral vector contains an expression cassette (CNK-UT-E19) encoding a chimeric NK receptor and an anti-TCR scFv fused with E19TM / CT domains. The lentivirus was transduced into human T cells. Five days after transduction, the cells were subjected to flow cytometry to detect the expression of TCRαβ in transduced cells (NKG2D+ cells), T cells transduced with conventional chimeric NK receptors, and non-transduced T cells. The results showed that: The introduction of UT elements into the CNK expression cassette does not affect the expression of chimeric NK receptors. Also, it can significantly inhibit the expression of TCR in CD8+ and CD4+ CNK-T cells. [Figure 18] We show that CNK-T cells carrying UT elements showed strong cytotoxicity against tumor cells, similar to normal CNK-T cells. Non-transgenic T cells, CNK-T cells and CNK-UT(E19) cells were co-cultured with HepG2 cells at an E:T ratio of 1:5. After 24 hours of culture, cells were collected and analyzed by flow cytometer to detect the status of tumor lysis and T cell activation. The data showed that: CNK-UT cells showed strong cytotoxicity against HepG2 cells, which was equivalent to normal CNK-T cells. After 24 hours of co-culture, CD45(-) tumor cells were significantly decreased in the co-culture of CNK-T and CNK-UT. In addition, both CD25 and CD137 were up-regulated after co-culture of CD8 and CD4 CNK-UT cells with HepG2 cells. [Figure 19]Four structures of the CNK-UT multi-functional complex are shown, where Figure 19A is the basic CNK-UT multi-functional complex; Figure 19B is the CNK-UT multi-functional complex with the addition of a binding protein molecular domain that targets MHC I; Figure 19C is the CNK-UT multi-functional complex with the addition of a binding protein molecular domain that targets MHC I and a chimeric adaptor; and Figure 19D is the CNK-UT multi-functional complex with the addition of a binding protein molecular domain that targets MHC I and a receptor that targets and kills tumor cells, such as a CAR or TCR. [Figure 20] The structure of four CNK-UT elements expressing CNK-UT multifunctional complex is shown. Here, as shown in Figure 20A, a combination of CNK-UT elements, DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-AdE3 ERAD, is expressed by a single lentivirus EF1α promoter expression vector; as shown in Figure 20B, a combination of CNK-UT elements, DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-US2 ERAD, is expressed by a single lentivirus EF1α promoter expression vector; as shown in Figure 20C, a combination of CNK-UT elements, DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TAA scFv-DAP10 and anti-TCR-AdE3, is expressed by a single lentivirus expression vector, respectively, by EF1α and CMV promoters. The expression of multiple genes of ERAD-E2A-AdE3 was regulated to achieve the expression of multiple functional elements in one vector; as shown in Figure 20D, two different lentiviral expression vectors were used to regulate the expression of anti-TAA scFv-CD28 / 4-1BB-CD3ζ-T2A-CD3ζ-p2A-NKG2D and anti-TCR- AdE3 ERAD-T2A-AdE3, respectively, and co-transfected into T cells to achieve the expression of multiple CNK-UT functional elements in the same T cells. [Figure 21]1 shows the flow cytometry measurement results of the basic phenotype of CNK-UT (DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3) cells. [Figure 22] The results of the recognition and specific killing of CNK-UT cells against human colon adenoma cell line HT29 are shown, where Fig. 22A shows the expression status of different NK target proteins in human colon adenoma cell line HT29; Fig. 22B shows that CNK-UT cells have effective killing and activation functions against HT29. [Figure 23] CNK-UT cells recognize and specifically kill MDA-MB453. Here, Figure 23A shows the expression status of different NK target proteins in triple-negative breast cancer cell line MDA-MB453; Figure 23B shows that CNK-UT cells show effective killing and activation function against THP1. [Figure 24] Figure 25 shows that GPC3 CAR / CNK-UT cells have more effective tumor elimination ability in mice than GPC3 CAR-T cells. [Diagram 25] This shows that GPC3 CAR / CNK-UT cells have more effective tumor elimination ability than GPC3 CAR-T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary, and the scope of the present disclosure will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described herein.

[0018] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Also, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed. It will be apparent to one of skill in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein has distinct components and features that may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any method described can be carried out in the order of events described or in any other order that is logically possible.

[0019] I. Definition The following definitions are provided to assist the reader.Unless otherwise defined, all technical terms, symbols, and other scientific or medical terms or terminology used herein are intended to have the meanings that are commonly understood by those skilled in the art.In some cases, terms that have commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference to the definitions of the terms that are commonly understood in the art. It should be noted that in this disclosure, terms such as "comprises / comprised / comprising", "contains / containing", etc. have the meanings given to them in U.S. Patent Law, which are inclusive or open, and do not exclude the addition of unrecited elements or method steps. The terms "consisting essentially of / consists essentially of" have the meanings given to them in U.S. Patent Law, which may include additional components or steps that do not materially affect the basic and novel characteristics of the invention sought to be protected. The terms "consist of / consisting of" have the meanings given to them in U.S. Patent Law, which are closed. As used herein, the singular forms "a / an" and "said" include plural referents unless the context clearly dictates otherwise.

[0020] As used herein, the term "about" refers to a measurable value, such as an amount or duration, and includes variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or ±0.1% from the given value. "Antigen" refers to a molecule that can be specifically recognized and bound by an antibody. In some embodiments, an antigen as described herein can be a molecule that elicits an immune response. This immune response can be a humoral response, a cell-mediated response, or both. It should be understood by those skilled in the art that an antigen can be any large molecule, including nearly any protein or peptide. It is understood that antigens of the present disclosure include therapeutic antibodies that can initiate an immune response. "Antibody" refers to a polypeptide of the immunoglobulin (Ig) family that binds to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three regions: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one region (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR3), interspersed with more conservative regions called frame regions (FR).The CDR boundaries of the antibodies disclosed herein may be defined or identified according to the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., December 5, 186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J. Molecular Biology, 196, 901 (1987); Chothia, C. et al., Nature, December 21-28, 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481-514, (2015). VH and VL each consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0021] As used herein, the terms "functional fragment" and "antigen-binding fragment" of an antibody may be used interchangeably and refer to a fragment of an antibody that contains one or more CDRs, or any other antibody portion that binds to an antigen but does not contain the complete original antibody structure. Specific examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide bond stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen as the parent antibody binds. In some embodiments, an antigen-binding fragment can contain one or more CDRs from a particular parent antibody. The term "Fab" of an antibody refers to a monovalent antigen-binding fragment of an antibody, which consists of one light chain (variable and constant regions) linked by disulfide bonds to the variable region and first constant region of one heavy chain. Fab can be obtained by digesting an antibody with papain at the residues near the N-terminus of the disulfide bond between the heavy chains in the hinge region. "Fab'" refers to a Fab fragment containing a portion of the hinge region, which can be obtained by digesting an antibody with pepsin to residues near the C-terminus of the interheavy chain disulfide bond in the hinge region, and thus differs from Fab by a small number of residues in the hinge region, including one or more cysteines. "F(ab')2" refers to a dimer of Fab' containing two light chains and part of two heavy chains. The "Fv" of an antibody refers to the minimum antibody fragment that has a complete antigen-binding site. The Fv fragment consists of one light chain variable region bound to one heavy chain variable region. "dsFv" refers to a disulfide bond stabilized Fv fragment, in which the linkage between one light chain variable region and one heavy chain variable region is a disulfide bond.

[0022] A "single chain Fv antibody" or "scFv" refers to an engineered antibody in which the variable regions of a light chain and the variable regions of a heavy chain are linked together directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)). An "scFv dimer" refers to a single chain containing two heavy chain variable regions and two light chain variable regions with a linker. In some embodiments, an "scFv dimer" is a bivalent diabody or a bivalent scFv (BsFv) in which a VH-VL (linked by a peptide linker) dimerizes with another VH-VL to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes) by coordinating one VH with the other VL. In another embodiment, an "scFv dimer" is a bispecific diabody in which VH1-VL2 (linked by a peptide linker) and VL1-VH2 (also linked by a peptide linker) are linked together, coordinating VH1 with VL1 and VH2 with VL2, respectively, with each coordinating pair having a different antigen specificity. "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0023] "Camelized single domain antibody", "heavy chain antibody", "nanobody" or "HCAb" refers to an antibody that comprises two VH domains and no light chain (Riechmann L. and Muyldermans S., J Immunol Methods. Dec. 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun.;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally obtained from the Camelidae family (camels, dromedaries and llamas). Camelized antibodies do not contain light chains but have a true antigen-binding repertoire (Hamers-Casterman C. et al. Nature. June 3;363(6428):446-8 (1993); Nguyen VK. et al. Heavy-chain antibodies in Camelidae; a case of evolutionary innovation Immunogenetics. April;54(1):39-47 (2002); Nguyen VK. et al. Immunology. May;109(1):93-101 (2003)). The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated from an adaptive immune response (Koch-Nolte F. et al., FASEB J. November;21(13):3490-8. Epub 2007-06-15 (2007)). "Diabodies" comprise small antibody fragments with two antigen-binding sites, said fragments comprising a VH domain linked to a VL domain in a single polypeptide chain (VH-VL or VL-VH) (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. 15 Jul;90(14):6444-8 (1993); EP404097; WO93 / 11161). The linker is too short to pair the two domains in the same chain. The domains then pair with the complementary domains from another chain and generate two antigen-binding sites.The antigen-binding sites can target the same or different antigens (or epitopes).

[0024] "Domain antibody" refers to an antibody fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In some embodiments, two or more VH domains are covalently linked via a peptide linker to form a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens. In some embodiments, a "(dsFv)2" comprises three peptide chains, in which two VH portions are linked via a peptide linker and two VL portions are linked by disulfide bridges. In some embodiments, a "bispecific ds diabody" comprises a VH1-VL2 (linked by a peptide linker) linked to VL1-VH2 (also linked by a peptide linker) via a disulfide bridge between VH1 and VL1. In some embodiments, a "bispecific dsFv" or "dsFv-dsFv'" comprises three peptide chains, in which the heavy chains of the VH1-VH2 moieties are linked by a peptide linker (e.g., a long flexible linker) and paired with the VL1 and VL2 moieties, respectively, via a disulfide bridge. The heavy and light chains paired via a single disulfide bond have different antigen specificities. An "autologous cell" refers to any cell that is derived from the same individual as the recipient of the cell (ie, the recipient itself).

[0025] An "allogeneic cell" refers to any cell derived from another individual of the same species as the recipient of the cell (ie, not the recipient itself). As used herein, "construct" and "protein construct" can be used interchangeably and refer to a protein or protein complex. The term "complex" refers to an aggregate or assembly of more than one protein (e.g., two, three, four or more proteins) that are aggregated or assembled. The complex is preferably capable of performing a function that cannot be performed by the individual proteins therein. In a complex, the constituent proteins may or may not be in contact with each other. As used herein, the term "chimeric" when referring to a construct means that one part of the construct is derived from one species and another part of the construct is derived from another species.

[0026] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein is operably inserted to cause expression of said protein. A vector can be used to transform, transduce or transfect a host cell to express genetic elements contained therein. Specific examples of vectors include plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage; and animal viruses. Types of animal viruses as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses and papova viruses (e.g., SV40). A vector may contain multiple elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. A vector may also contain an origin of replication. A vector may further include materials that contribute to cell entry, such as, but not limited to, a viral particle, a liposome, or a protein coat layer. A vector may be an expression vector or a cloning vector. A vector provided in this disclosure (e.g., an expression vector) includes a nucleic acid sequence encoding a chimeric protein construct provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and / or at least one selectable marker.Specific examples of vectors include retroviruses (including lentivirus, herpes simplex virus), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage and M13 phage, and the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, and the like. , pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0027] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been or will be introduced. The term "functional variant" as used herein refers to a polypeptide / protein that has at least 70% sequence identity to a parent polypeptide / protein, but still retains the function of the parent polypeptide / protein. A variant differs from the parent polypeptide / protein in one or more amino acid residues. For example, a functional variant may have a substitution, addition, deletion, insertion or truncation of one or more amino acid residues of the parent polypeptide / protein.

[0028] In the present application, the percentage of "identity" or "sequence identity" of a polypeptide or polynucleotide is determined by comparing two sequences that are optimally aligned in a comparison window, and for optimal alignment of the two sequences, the portion of the comparison window of the polynucleotide or polypeptide sequence may contain additions or deletions (i.e., gaps) relative to the reference sequence (which does not contain additions or deletions). The percentage is calculated as follows: the number of positions in the two sequences where the same nucleic acid base or amino acid residue appears is determined, the number of matching positions is calculated, and the number of matching positions is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to calculate the percentage of sequence identity. Alignment for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available at the National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., Journal of Molecular Biology, 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics, Oxford, England, 23(21):2947-822 (1997)). (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the above tools or customize the parameters appropriate for the matching, such as by selecting an appropriate algorithm. In some embodiments, residue positions that are not identical may differ by conservative amino acid substitutions.A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). Generally speaking, a conservative amino acid substitution does not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Manners for making such adjustments are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference.

[0029] "Operably linked" refers to the functional relationship between two or more polynucleotide sequences. In the context of a polynucleotide encoding a fusion protein (e.g., a polypeptide chain of a CAR of the present disclosure), the term refers to the joining of two or more polynucleotide sequences such that the two polynucleotides are held in the same expression frame to translate a single amino acid sequence. In the context of transcriptional or translational regulation, the term refers to the functional relationship between a regulatory sequence and a coding sequence. For example, a promoter is positioned in the correct location and orientation of the coding sequence to regulate transcription. "Polynucleotide" or "nucleic acid" refers to a chain of nucleotides. As used herein, polynucleotide includes all polynucleotide sequences obtained by any means available in the art, including, but not limited to, recombinant and synthetic means. "Polypeptide" and "protein" may be used interchangeably and refer to a chain of amino acid residues covalently linked by peptide bonds. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0030] "T cell receptor" or "TCR" refers to a protein complex on the surface of a T cell that is responsible for recognizing antigen fragments, which are peptides that bind to MHC molecules. The term "chimeric natural killer receptor" (CNK receptor) refers to a composition comprising a chimeric NK activating receptor module and a chimeric NK signaling module. The term "CNKT cells" refers to T cells that contain a CNK construct. The term "chimeric natural killer receptor-Universal T cell" (CNK-UT) refers to a T cell expressing an NK activating receptor modular construct, a CNK signaling modular construct and a UT modular construct. The term "CAR / CNK-UT" refers to chimeric antigen receptor & chimeric natural killer receptor-Universal T cell (CNK-UT) T cells expressing a CAR construct, an NK activating receptor modular construct, a CNK signaling modular construct and a UT modular construct that target a specific tumor antigen.

[0031] As used herein, the term "costimulatory ligand" includes molecules on antigen-presenting cells (e.g., APCs, dendritic cells, B cells and other immune cells) that specifically bind cognate costimulatory molecules on T cells to provide signals that mediate T cell responses, such as, but not limited to, proliferation, activation, and differentiation, in addition to the primary signal provided, for example, by binding of the TCR / CD3 complex to a peptide-bearing MHC molecule. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind to B7-H3. Costimulatory ligands further include antibodies that specifically bind to costimulatory molecules present especially on T cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7LIGHT, NKG2C, B7-H3, and CD83. "Costimulatory molecule" or "costimulatory receptor" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand and mediates a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Costimulatory molecules include non-naturally occurring engineered proteins.

[0032] As used herein, a "costimulatory signal" refers to a signal that links to a primary signal, such as TCR / CD3, leading to T cell proliferation and / or up- or down-regulation of key molecules. The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as, for example, downregulation of TGF-B and / or reorganization of cytoskeletal structure. The term "stimulatory molecule" as used herein refers to a molecule that specifically binds, in a T cell, to a cognate stimulatory ligand present on an antigen-presenting cell. As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (herein referred to as a "stimulatory molecule") on a T cell and mediate a primary response of the T cell, such as, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include peptide-bearing MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies, among others.

[0033] As used herein, "activated" refers to a state of T cells that has been stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers specifically to T cells that are undergoing cell division. An "effective amount" or "therapeutically effective amount" refers to that amount of a cell, composition, formulation, or any material described herein to effectively achieve a desired biological result, including, but not limited to, the elimination of B cells expressing a particular B cell receptor (BCR) and the antibodies produced therefrom. As used herein, a "subject" is a mammal, e.g., a human or other animal, typically a human. In some embodiments, the subject, e.g., a patient, to whom the cell, cell population, or composition is administered is a mammal, typically a primate, e.g., a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, e.g., a rodent. As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to the complete or partial amelioration or alleviation of a disease or condition or disorder, or of symptoms, side effects or consequences or phenotypes associated therewith. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, alleviation of any direct or indirect pathological consequences of a disease, prevention of metastasis, slowing of disease progression, amelioration or alleviation of the disease state, and amelioration or improvement of prognosis. These terms do not imply complete cure of a disease or complete elimination of any symptoms or effects on all symptoms or consequences.

[0034] As used herein, "prevention" includes preventing the onset or recurrence of the disease in a subject susceptible to the disease but not diagnosed with the disease. In some embodiments, the cells and compositions provided are used to slow the progression of the disease or to slow the progression of the disease. As used herein, "inhibiting" a function or activity refers to reducing the function or activity when compared to the same conditions (excluding the condition or parameter of interest) or when compared to other conditions. For example, a cell that inhibits tumor growth reduces the rate of tumor growth compared to when the cell is not present. In the context of administration, an "effective amount" of a reagent (e.g., a chimeric antigen receptor, a polynucleotide, a vector, a pharmaceutical formulation, a cell, or a composition) refers to an amount that, at the dosages / dosages and time periods required, effectively achieves a desired result, e.g., a therapeutic or prophylactic result. A "therapeutically effective amount" of a reagent (e.g., a pharmaceutical formulation or cells) refers to an amount that effectively achieves a desired therapeutic result (e.g., treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effects of a treatment) at the dosage and time period required. A therapeutically effective amount can vary depending on factors such as, for example, the disease state, the age, sex, and weight of the subject, and the cell population being administered. In some embodiments, the methods provided include administering cells and / or compositions in an effective amount, e.g., a therapeutically effective amount. "Prophylactically effective amount" refers to an amount that effectively achieves the desired prophylactic result at the required dosage and time period. Usually, but not necessarily, a prophylactically effective amount is less than a therapeutically effective amount, since a prophylactic dose is usually used in subjects before or at an early stage of disease. In some respects, in the case of relatively low tumor burden, the prophylactically effective amount is higher than the therapeutically effective amount.

[0035] II. Target Protein Degradation (TPD) The present invention provides a novel targeted protein degradation system that is used to target specific proteins (i.e., target proteins) to inhibit their expression (e.g., surface expression) and promote ubiquitination-mediated degradation via the endoplasmic reticulum-associated degradation (ERAD) machinery. Endoplasmic reticulum-associated protein degradation (ERAD) is a cellular pathway that targets misfolded or mutated proteins in the endoplasmic reticulum, ubiquitinates them, and then degrades them by the proteasome. The process of ERAD can include three steps: (1) recognition of misfolded or mutated proteins in the endoplasmic reticulum; (2) retrotranslocation of the recognized misfolded or mutated proteins from the endoplasmic reticulum to the cytoplasm, which contains the ubiquitin-proteasome system (UPS), which transports terminally misfolded proteins from the endoplasmic reticulum to the cytoplasm; and (3) ubiquitin-dependent degradation of the recognized misfolded or mutated proteins by the proteasome (see Annamaria et al., ER-associated degradation: Protein quality control and beyond. J. Cell Biol. Vol. 204 No. 6 869-879). Therefore, the present application provides a new chimeric strategy that combines a targeting domain (e.g., an antibody fragment (ScFv) targeting a specific protein) with an ERAD functional motif (e.g., a viral ER resident protein TMD and ED return signal domain), and demonstrates that this strategy can effectively inhibit the expression of a specific protein (e.g., the expression of a specific protein on the cell surface).

[0036] The ER-TPD technology platform provided in this application has the following advantages over conventional protein degradation / removal technologies (e.g., PROTAC, CRISPR, etc.): 1. The ER-TPD chimeric protein construct can be delivered to cells via vectors (e.g., liposomes, nanoparticles, lentiviruses, adenoviruses, oncolytic viruses, etc.), stably expressed, and exhibits its functions, and does not have the problems of reduced degradation efficiency due to low cell permeability and short half-life compared to PROTAC; 2. The ER-TPD technology is based on the ERAD mechanism of the endoplasmic reticulum, and directly intervenes in protein synthesis and effectively degrades proteins in the process of protein synthesis and assembly in the endoplasmic reticulum, so it has a more effective degradation efficiency than exogenous PROTAC technology; 3. Exogenous PROTAC cannot effectively degrade membrane proteins and secretory proteins, whereas ER-TPD directly intervenes in protein synthesis and effectively degrades proteins in the process of protein synthesis and assembly in the endoplasmic reticulum, so it has a more effective degradation efficiency than exogenous PROTAC technology; It can directly target, retain and degrade vacuolar membrane proteins and secretory proteins, so it can target many important disease-related target proteins that could not be targeted by conventional TPD technology, providing novel drug development rules for disease treatment;4. In the design method of ER-TPD, various single-chain antibody structures or artificial affinity ligand structures can be adopted as targeting domains, which greatly enhances the specific and accurate recognition and binding ability to target proteins; Meanwhile, conventional PROTAC technology adopts small molecule chemical structures, which makes it difficult to ensure the specificity of the targeting structure, so ER-TPD technology has the great advantage of targeting accuracy and specificity;5. ER-TPD technology can achieve multi-pathway mechanism of targeted protein degradation through downstream degradation guide ligand structure. Multi-pathway mechanisms include the UPS mechanism by ubiquitous proteasome and the ALP mechanism by autophagy lysosome. The downstream degradation guide ligand structure design can be flexibly linked to the intracellular degradation system by genetic engineering techniques to achieve effective targeted degradation.On the other hand, traditional PROTAC technology can only achieve degradation by targeting E3 ligase;6.ER-TPD technology degrades target proteins at the protein level, so compared with technologies that block the expression of target proteins at the gene level (e.g., CRISPR technology), there are no off-target effects or toxic and side effects such as chromosomal instability caused by the gene editing process;7.ER-TPD technology can be perfectly combined with gene and cell therapy technologies, greatly expanding the clinical indications of current gene and cell therapy and improving clinical efficacy;8.The development of targeted degradation drugs based on ER-TPD technology is highly achievable and has a short cycle. With nucleic acid and cell drugs based on ER-TPD technology, scale-up and industrialized production can be more easily achieved, which is an important foundational technology for the development of new drugs.

[0037] In one aspect, the disclosure provides an Endoplasmic Reticulum-based Targeted Protein Degradation (ER-TPD) chimeric protein construct, the chimeric protein construct comprising an ER-associated degradation (ERAD) machinery protein binding domain and a targeting domain. In some embodiments, the chimeric protein construct further comprises a protein degradation pathway member (e.g., a ubiquitin-proteasome system pathway member, an endosomal-lysosomal pathway member, an autophagy pathway member) binding domain. The protein degradation pathway member binding domain may be linked to a component of the chimeric protein construct by any suitable means, for example, linked to the ERAD machinery protein binding domain. In another aspect, the disclosure further provides a library of compositions, where each composition comprises a viral protein or a nucleic acid molecule encoding same, containing a functional motif that can hijack the ERAD machinery to retain it in the ER and accelerate proteasome-mediated degradation, and an affinity moiety that targets and degrades a specific protein, for use in therapeutically modifying cellular phenotype, improving cellular function, inducing apoptosis, or inhibiting viral replication.

[0038] 1. ERAD machinery protein binding domain As used herein, the term "ERAD machinery protein" refers to a protein that is involved in the ERAD machinery or pathway. The term "ERAD machinery protein binding domain", also referred to as "ERAD degradation domain", refers to a portion or domain that can bind to and / or utilize an ERAD machinery protein, e.g., the transmembrane domain (or a functional variant thereof) and the cytoplasmic domain (or a functional variant thereof) of a viral endoplasmic reticulum (ER) resident glycoprotein, the cytoplasmic domain also referred to as the "endoplasmic reticulum resident domain" or "ER resident domain". In some embodiments, the ERAD machinery protein binding domain comprises a transmembrane domain of a viral endoplasmic reticulum resident protein or a functional variant thereof and an endoplasmic reticulum resident domain or a functional variant thereof. In some embodiments, the viral ER resident glycoprotein can be any viral ER resident protein that is capable of hijacking the ERAD machinery and promoting ubiquitination and proteasome-mediated degradation of a target protein.

[0039] i. ERAD machinery protein binding domain derived from adenovirus E3-19K In some embodiments, the viral endoplasmic reticulum resident protein is adenovirus E3-19K. Adenovirus E3-19K (also called "E19") contains three functional modules: a luminal domain that interacts with MHC-I and MICA / B molecules, a transmembrane domain, and an endoplasmic reticulum resident domain, and the endoplasmic reticulum resident domain contains a dilysine motif in the cytoplasmic tail, which can return the Golgi apparatus to the endoplasmic reticulum, and is also called an "ER return signal motif". Research has revealed that the transmembrane domain and the ER return signal motif are required to ensure efficient ER localization, trafficking inhibition of histocompatibility complex class I (MHC-I) and MHC-I-related chains A and B (MICA / B molecules) and proteasomal degradation. Thus, adenovirus E3-19K can resident MHC class I molecules (e.g., MHC-I and MICA / B molecules) in the secretory pathway and disrupt antigen presentation. The chimeric protein construct provided in the present disclosure achieves the goal of degrading a target protein by utilizing the transmembrane domain and endoplasmic reticulum resident domain (i.e., ERAD degradation domain) of previral E3-19K to fuse with a target protein binding domain. In some preferred embodiments, the adenovirus E3-19K ERAD degradation domain comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 60, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity. The adenovirus E3-19K ERAD degradation domain has a di-lysine motif in the cytoplasmic tail and can return the Golgi apparatus to the endoplasmic reticulum. In some preferred embodiments, the amino acid sequence of the adenovirus E3-19K ERAD degradation domain is shown in SEQ ID NO: 60.

[0040] ii. ERAD machinery protein-binding domains from other viral glycoproteins In some embodiments, the viral endoplasmic reticulum resident protein is not adenovirus E3-19K. In some embodiments, the viral endoplasmic reticulum resident protein is at least one selected from the group consisting of HCMV glycoproteins US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, and Cowpox Virus protein CPXV203. In some embodiments, the viral endoplasmic reticulum resident protein comprises HCMV glycoproteins US2 and US11. In some embodiments, the viral endoplasmic reticulum resident proteins include E3-19K and at least one selected from the group consisting of HCMV glycoproteins US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, and Cowpox Virus protein CPXV203. The HIV-1 viral Vpu protein is an ER membrane protein that targets newly synthesized CD4 in the endoplasmic reticulum for degradation by the proteasome in the cytoplasm. The Vpu protein retains CD4 in the ER primarily through SCFβ-TrCP-dependent ubiquitination and transmembrane domain (TMD) interactions.

[0041] a) HCMV US2 and US11 The two HCMV proteins, US2 and US11, are ER-resident type I integral membrane glycoproteins that both select this ERAD pathway to promote the degradation of MHC class I heavy chains and inhibit MHC class I antigen presentation. Expression of either of these proteins results in rapid degradation of newly synthesized MHC class I heavy chains. US2 and US11 bind MHC class I heavy chains by their luminal domains and recruit host cell proteins that extract the polypeptides from the endoplasmic reticulum membrane by "pulling" the cytoplasmic tails of the heavy chains. After translocation to the cytoplasm, MHC class I molecules are ubiquitinated and degraded by the proteasome. In addition to class I molecules, US2 also causes the degradation of two proteins of the class II pathway, DR-α and DM-α, as well as HFE, a non-canonical major histocompatibility complex (MHC) class I protein involved in iron regulation. The luminal domain of US2 is responsible for binding MHC class I and class II molecules, while the transmembrane (TM) and cytoplasmic (CT) domains interact with cellular components of the ERAD machinery or pathway, contributing to promote the translocation and enzymatic degradation of both MHC class I and class II proteins (Chevalier MS et al., 2002, 2003). The cytoplasmic tail of US2 is sufficient to interact with signal peptide peptidase (SPP), an essential component of the US2-dependent MHC I dislocation complex (Loureiro J et al., 2006), which is required for US2-dependent MHC heavy chain translocation (see Joana et al., Signal peptide peptidase is required for dislocation from the endoplasmic reticulum. Nature volume 441, pages 894-897 (2006)). US2 also contributes to the ubiquitination and proteasomal degradation of US2 tail-anchored MHC I and II molecules by interacting with the endoplasmic reticulum-resident RING-type E3 ligase TRC8 through its TM domain (Stagg HR et al., 2009).

[0042] In contrast, US11-induced degradation of MHC-I molecules requires Derlin-1, but not SPP. The ER luminal domain of US11 interacts with the luminal domain of MHC-I heavy chain, whereas the TM domain of US11 binds Derlin-1. Thus, the primary function of US11 may be to deliver MHC-I molecules to Derlin-1 (Lilley BN et al., 2004; Cho S et al., 2013) and then induce their translocation to the cytosol for degradation by the proteasome. US11 also activates unfolded proteins. Through Derlin-1, US11 associates with the ERAD RING E3 ligase TMEM129 and recruits Ube2J2 to ubiquitinate MHC-I before US11-induced degradation. In some preferred embodiments, the ERAD degradation domain of HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 48, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV glycoprotein US2 is set forth in SEQ ID NO: 48.

[0043] In some preferred embodiments, the ERAD degradation domain of US2 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 76, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the ERAD degradation domain of US2 is set forth in SEQ ID NO: 76. In some preferred embodiments, the ERAD degradation domain of HCMV glycoprotein US11 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 54, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV glycoprotein US11 is set forth in SEQ ID NO: 54. In some preferred embodiments, the ERAD degradation domain of US11 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 82, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the ERAD degradation domain of US11 is set forth in SEQ ID NO: 82.

[0044] b) HCMV US3 Rather than promoting degradation of MHC proteins, the HCMV US3 glycoprotein physically binds peptide-bearing MHC class I heterodimers, retaining the class I complex in the ER and inhibiting the association of the invariant chain with class II DR-αβ dimers in the ER, leading to mislocalization of the class II complex and reduced peptide loading. Thus, US3 can interfere with the intracellular trafficking and maturation of MHC class I molecules at the early stage of HCMV infection. US3 is an endoplasmic reticulum-resident membrane protein. Domain swapping experiments revealed that the luminal domain of US3 allows US3 to retain itself in the endoplasmic reticulum, and that both the intraluminal and transmembrane domains are essential for the retention of class I MHC molecules in the endoplasmic reticulum. In some preferred embodiments, the ERAD degradation domain of HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 51, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV glycoprotein US3 is set forth in SEQ ID NO: 51. In some preferred embodiments, the ERAD degradation domain of US3 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 79, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the ERAD degradation domain of US3 is set forth in SEQ ID NO: 79.

[0045] c) HCMV US10 The HCMV US10 glycoprotein also interacts with components of MHC class I antigen presentation: US10 binds free class I heavy chains and slows their transport out of the ER, but US10 has no effect on US2 or US11. In some preferred embodiments, the ERAD degradation domain of HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 57, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV glycoprotein US10 is set forth in SEQ ID NO: 57.

[0046] d) HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a, BHV UL49.5 The HCMV L protein US6, unlike US2, US3, US10 and US11, affects antigen presentation through a completely different strategy. Contrary to interacting with free class I heavy chains or fully assembled class I complexes, US6 inhibits the translocation of cytoplasmic peptides through the TAP complex (TAP1 / 2). US6 binds to the ER luminal side of TAP1 and induces a conformational change that prevents ATP binding. Residues 89-108 in the ER-luminal domain of US6 contribute to and are necessary for the binding of US6 to TAP. Such inhibition of TAP activity not only affects the expression of canonical MHC class I alleles, but also the expression of the non-canonical alleles HLA-C and HLA-G in fetal cytotrophoblast cells. HSV ICP47, expressed early in the infection cycle as the HCMV US6 protein, may or may not be present in in vitro replication, can apply a similar strategy to block the assembly of class I molecules. ICP47 inhibits TAP-mediated peptide transport and binds tightly to the TAP1-TAP2 complex. One clue to the mechanism by which ICP47 inhibits TAP is that ICP47 exhibits high species selectivity. Both HSV1 and HSV2I CP47 inhibit gorilla, monkey, pig, dog, and cow TAP, and have little effect on mouse, rat, guinea pig, or rabbit TAP. ICP47 has approximately 100-fold higher affinity for human TAP than for mouse TAP. ICP47 inhibits peptide binding to TAP but does not affect ATP binding. ICP47 has an affinity for TAP that is 10 to 1,000 times higher than most peptides, and is used as a competitive inhibitor of peptide-TAP binding, and is thought to bind directly to the peptide-binding site.

[0047] In some preferred embodiments, the HHV-7 US6 TAP-binding domain comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 65, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and has an endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the HHV-7 US6 TAP-binding domain is set forth in SEQ ID NO: 65. In some preferred embodiments, the HSV ICP47 TAP-binding domain comprises an amino acid sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, to the amino acid sequence set forth in SEQ ID NO: 67, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the HSV ICP47 TAP-binding domain is set forth in SEQ ID NO: 67. In some preferred embodiments, the TAP-binding domain of CPXV012 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 69, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the TAP-binding domain of CPXV012 is set forth in SEQ ID NO: 69.

[0048] In some preferred embodiments, the full-length sequence of CPXV012 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:68, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of CPXV012 is set forth in SEQ ID NO:68. In some preferred embodiments, the EBV BNFL2a TAP-binding domain comprises an amino acid sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, to the amino acid sequence set forth in SEQ ID NO: 71, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the EBV BNFL2a TAP-binding domain is set forth in SEQ ID NO: 71. In some preferred embodiments, the full-length sequence of EBV BNFL2a comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:70, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of EBV BNFL2a is set forth in SEQ ID NO:70. In some preferred embodiments, the TAP-binding domain of BHV UL49.5 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 73, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the TAP-binding domain of BHV UL49.5 is set forth in SEQ ID NO: 73. In some preferred embodiments, the full-length sequence of BHV UL49.5 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:72, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of BHV UL49.5 is set forth in SEQ ID NO:72.

[0049] e) Other In some preferred embodiments, the ERAD degradation domain of HHV-7 US21 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 63, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and has an endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HHV-7 US21 is set forth in SEQ ID NO: 63. In some preferred embodiments, the full-length sequence of HHV-7 US21 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:61, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of HHV-7 US21 is set forth in SEQ ID NO:61. In some preferred embodiments, the HCMV UL16 ERAD degradation domain comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, to the amino acid sequence set forth in SEQ ID NO: 85, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the HCMV UL16 ERAD degradation domain is set forth in SEQ ID NO: 85.

[0050] In some preferred embodiments, the full-length sequence of HCMV UL16 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:83, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of HCMV UL16 is set forth in SEQ ID NO:83. In some preferred embodiments, the HCMV UL141 ERAD degradation domain comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 88, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the HCMV UL141 ERAD degradation domain is set forth in SEQ ID NO: 88. In some preferred embodiments, the full-length sequence of HCMV UL141 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:86, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of HCMV UL141 is set forth in SEQ ID NO:86. In some preferred embodiments, the Golgi resident domain of HCMV UL142 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 91, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the Golgi resident domain of HCMV UL142 is set forth in SEQ ID NO: 91. In some preferred embodiments, the HIV Nef comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 92, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the HIV Nef is set forth in SEQ ID NO: 92.

[0051] In some preferred embodiments, the full-length sequence of HCMV UL142 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:89, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of HCMV UL142 is set forth in SEQ ID NO:89. In some preferred embodiments, the HIV Vpu comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 93, and has endoplasmic reticulum resident function. In some embodiments, the HIV Vpu amino acid sequence is set forth in SEQ ID NO: 93. In some preferred embodiments, the HHV-8 KK3 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, to the amino acid sequence set forth in SEQ ID NO: 94, and has an endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the HHV-8 KK3 is set forth in SEQ ID NO: 94. In some preferred embodiments, the HHV-8 KK5 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, to the amino acid sequence set forth in SEQ ID NO: 95, and has an endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the HHV-8 KK5 is set forth in SEQ ID NO: 95. In some preferred embodiments, the MHV-68 MK3 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 96, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the MHV-68 MK3 is set forth in SEQ ID NO: 96. In some preferred embodiments, the HTLV-1 p12 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 97, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and has an endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the HTLV-1 p12 is set forth in SEQ ID NO: 97.

[0052] In some preferred embodiments, the KDEL receptor binding domain of cowpox virus protein CPXV203 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 100, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the KDEL receptor binding domain of cowpox virus protein CPXV203 is set forth in SEQ ID NO: 100. In some preferred embodiments, the KDEL receptor binding domain of cowpox virus protein CPXV203 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 103, and has endoplasmic reticulum resident function. In some embodiments, the amino acid sequence of the KDEL receptor binding domain of cowpox virus protein CPXV203 is set forth in SEQ ID NO: 103. In some preferred embodiments, the full length sequence of the cowpox virus protein CPXV203 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:98, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full length sequence of the cowpox virus protein CPXV203 is set forth in SEQ ID NO:98. In some preferred embodiments, the full length sequence of the cowpox virus protein CPXV203 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:101, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full length sequence of the cowpox virus protein CPXV203 is set forth in SEQ ID NO:101.

[0053] 2. Targeted Domains The targeting domain (target binding domain) of the chimeric protein constructs provided in this disclosure can be any structure that recognizes and binds to the target protein, such as an antibody, an antibody fragment, a functional motif derived from a naturally occurring protein (e.g., a natural ligand), a synthetic polypeptide or protein that has affinity for the target protein, or various variants, such as mutants, fusions, truncations, etc. of the above molecules. For example, the targeting domain can be the natural ligand of the target protein, an antibody or antigen-binding fragment thereof that specifically recognizes the target protein, or an antigen that is specifically recognized by the antibody. In some embodiments, the targeting domain comprises an antibody or functional fragment thereof that specifically targets a target protein (e.g., a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent diabody), Fv (scFv), multispecific antibody (e.g., bispecific antibody), camelized single domain antibody (e.g., VHH), nanobody, domain antibody, bivalent domain antibody, two-chain antibody, three-chain antibody, and four-chain antibody). The target protein domain can comprise any binding ligand capable of binding the target protein, including, but not limited to, a ligand, a portion that binds a ligand in a receptor, a portion that binds a host protein in a viral protein, and the like.

[0054] The target protein can be any protein whose level or activity is desired to be modulated. For example, the activity can be altered by the ERAD mechanism to block the protein in the ER and accelerate proteasome-mediated degradation. Such degradation can change the phenotype of a cell, enhance the function of the cell, cause cell apoptosis, and / or inhibit viral replication to achieve a therapeutic goal (e.g., improved therapeutic efficacy, reduced side effects, etc.). In some embodiments, the target protein can be a protein involved in cell cycle, apoptosis, signal transduction, cell differentiation, cell dedifferentiation, cell growth, production of a cytokine or bioregulator thereof, production of a cytokine or bioregulator, production of a regulatory or functional protein, pro-inflammatory signal transduction, or glucose regulatory pathway. In another embodiment, the target protein is a disease-associated protein (also referred to as a "pathogenic protein"), where "disease-associated protein" or "pathogenic protein" as used herein refers to a protein whose function or activity change leads to the onset of a disease or whose function is relatively important to the progression of a disease; exemplary pathogenic proteins include, but are not limited to, oncogenic proteins, viral proteins, and proteins that lead to autoimmune diseases (e.g., antibodies produced in plasma cells). In some embodiments, the target protein is selected from an immune function-related target protein, a nervous system disease-related target protein, an infectious disease-related target protein (e.g., a viral infection-related target protein), an autoantigen-related target protein, or a tumor-related target protein (or oncogenic protein), a metabolic disease-related target protein.

[0055] In some embodiments, the pathogenic protein is an oncogenic protein. The oncogenic protein may be encoded by an oncogenic gene, such as BCL2, c-MYC, Ras, HER2, BCR / ABL, ABL1 / BCR, TGFB1, TLX1, P53, WNT1, WNT2, WT1, αv-β3, PKCa, ABL, BCL1, CD24, CDK4, EGFR / ERBB-1, HSTF1, INT1 / WNT1, INT2, MDM2, MET, MYB, MYC, MYCN, MYCL1, RAFI, NRAS, REL, AKT2, APC, BCL2-ALPHA, BCL2-BETA, BCL3, BCR, BRCA1, BRCA2 , CBL, CCND1, CDKN1A, CDKN1C, CDKN2A, CDKN2B, CRK, CRK-II, CSF1R / FMS, DBL, DDOST, PMS-2, PRAD-1, RAF, RHOM-1, RHOM-2, SIS, TAL2, TANI, TI AM1, TSC2, TRK, TSC1, STK11, PTCH, MEN1, MEN2, P57 / KIP2, PTEN, HPC1, ATM, XPA / XPG, BCL6, DEK, AKAP13, CDH1, BLM, EWSR1 / FLI1, FES, FGF3, FE R, FGR, FLI1 / ERGB2, FOS, FPS / FES, FRA1, FRA2, FYN, HCK, HEK, HER3 / ERBB-2, ERBB-3, HER4 / ERBB-4, HST2, INK4A, INK4B, JUN, JUNB, JUND, KIP2 , KIT, KRAS2A, KRAS2B, LCK, LYN, MAS, MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P53, PDGFB, PIM1, PTC, RBI, RET, ROS1, SKI, SRC1, TALI, TGFBR2, THRA1, THRB, TIAM1, TRK, VAV, VHL, WAF1, WNT2, WT1, YES1, ALK / NPM1, AMI1, AXL, FMS, GIP, GLI, GSP, HOX11, HST, IL3, INT2, KS3, K-SAM , LBC, DCC, DPC4 / SMAD4, E-CAD, E2F1 / RBAP, ELK1, ELK3, EPH, EPHA1, E2F1, EPHA3, ERG, ETS1, ETS2, LMO-1, LMO-2, L-MYC, LYL1, LYT-10, MDM-2,MLH1, MLL, MLM, N-MYC, OST, PAX-5, PMS-1, FGF4, FGF6, FANCA, FLI1 / ERGB2, FOSL1, FOSL2, GLI, HRAS1 , HRX / MLLT1, HRX / MLLT2, KRAS2, MADH4, MASI, MCF2, MLLT1 / MLL, MLLT2 / HRX, MTG8 / RUNX1, MYCLK1, MY H11 / CBFB, NFKB2, NOTCH1, NPM1 / ALK, NRG / REL, NTRK1, PBX1 / TCF3, PML / RARA, PRCA1, RUNX1, RUNX1 / C BFA2T1, SET, SHP2, TCF3 / PBX1, TNFa, Clusterin, Survivin, TOEβ, c-fos, c-SRC and In some embodiments, the oncogenic protein is selected from the group consisting of Bcl-2 family members (e.g., Bcl-2, Bcl-xL, and Bcl-w), VEGF / VEGFR, PDGFRβ, EGFR, EGFR mutants, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor ER-α, androgen receptor (AR), tyrosine kinases (c-ABL, BCR-ABL, BTK, FAK, PTK6, Wee1, TRK transmembrane receptors), serine / threonine kinase receptors. (IRAK4, LRRK2, B-Raf, RIPK2, CDK4 / 6, CDK7, CDK8, CDK8 / 19, CDK9, TBK1), protein kinase II (CK2), epigenetic-related proteins (BRD2, BRD3, BRD4, BRDT, TRIM24, BRD9, PBRM1, SMARCA2, SMARCA4, EP300, EZH2, WDR5), adrenomedullin (ADM), DPP3.

[0056] In some embodiments, the target protein comprises an immune function-related protein. As used herein, the term "immune function-related protein" refers to a functional protein involved in the body's immune process, such as antigen-presenting molecules (e.g., MHC class I molecules, MHC class II molecules, HLA, etc.), antigen-recognition molecules (e.g., TCR, CD123, NKG2D, etc.), immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), immune stimulatory / co-stimulatory molecules (e.g., CD3, CD80 / 86, CD28, etc.), and other molecules involved in innate or adaptive immunity. In some embodiments, immune function associated target proteins include, for example, CD123, CD7, CD5, MHC class I molecules, MHC class II molecules, non-canonical MHC molecules (e.g., HLA-G, HLA-E), MICA / B, ULBP1-6, IL6, IL6 receptor, IL1 receptor, RANKL, TGF-β1, PD1, PD-L1, CTLA4, Tim3, LAG3, Siglec-15, TIGIT, CD47, IL4RA, CD94 / NKG2A, CXCR1 / 2, CXCL8, CCR2 / CCR5, CCR4, CXCR4, c-Rel, CCL2, CCL5, CCL20, CCL22, CSF-1, CCL2, CCL5 indolylamine-2,3-dioxygenase (IDO), or arginase 1 (ARG1). In some embodiments, the target protein comprises a nervous system disease-associated target protein. As used herein, the term "nervous system disease-associated" refers to the involvement of the protein, particularly a neurodegenerative disease protein, in a nervous system disease. In some embodiments, the nervous system disease-associated target protein includes, for example, Tau, amyloid-β (Aβ), α-synuclein, mutant huntingtin (mHTT), α-synuclein, TAR RNA binding protein (TARDBP) and FUS RNA binding protein (FUS).

[0057] In some embodiments, the target protein comprises a viral infection associated target protein. Examples of the viral infection-related target protein include HBV encoded X protein (HBx), HBV DNA polymerase, HBV capsid glycoprotein, HIV-1 reverse transcriptase, HIV gp120, HCV NS3-4A protease, HCV RNA polymerase, HCV envelope protein, EBV DNA polymerase, EBV EBNA1, coronavirus RNA synthetase, coronavirus spike protein, coronavirus envelope protein, coronavirus membrane protein, coronavirus nucleocapsid protein, and other RNA-dependent RNA polymerases (RNA-dependent RNA polymerases, RdRp) such as novel coronavirus RNA-dependent RNA polymerase, herpesviruses DNA, RNA polymerase, herpesvirus capsid glycoprotein, CMV DNA polymerase, CMV capsid glycoprotein, RSV envelope protein, RSV capsid protein, RSV RNA polymerase, influenza virus RNA polymerase, influenza virus envelope protein, HPV In another embodiment, the target protein is a viral protein, such as, for example, HBV surface antigen, HBeAg, HBV polymerase protein, HIV Gag protein, HIV Env protein, etc., whose function is believed to be important for viral replication, proliferation, function, and viral disease progression.

[0058] In some embodiments, the target protein comprises an autoantigen-associated target protein. Many autoimmune disease-related autoantigens are known in the art, including, for example, type I diabetes-related autoantigens such as islet cell antigen (ICA), insulin (IAA), glutamic acid decarboxylase 65 (GAD65), and insulinoma antigen-2 (IA-2); rheumatoid arthritis (RA)-related autoantigens such as citrullinated protein / peptide antibodies, heterogeneous nuclear ribonucleoprotein A2 / B1, aldolase, alpha-enolase, calreticulin, heat shock protein (HSP60), BiP, PGK1, stress-induced phosphoprotein 1, and FUSE-BP1 / 2; deoxyribonucleoproteins, SmD1, SmD3, Clq, lupus anticoagulant (LA), cardiolipin (CL), and β2 glycoprotein I (β2 GP). I), systemic lupus erythematosus (SLE)-related autoantigens such as prothrombin (PT) and phosphatidylserine (PS); systemic sclerosis (SSc) / scleroderma (SD)-related autoantigens such as Scl-70, SSA, and Ro52; autoimmune liver disease-related antigens such as mitochondrial antigen, Spl00, PML, gp210, and p62; myasthenia gravis-related autoantigens such as acetylcholine receptor; voltage-dependent potassium channel (VGKC) complex, voltage-dependent calcium channel receptor, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, and gamma-aminobutyric acid-B Autoantigens related to central nervous system autoimmune diseases (limbic encephalitis, encephalomyelitis, cerebellar ataxia) such as (GABAB) receptors and glycine receptors; autoantigens related to multiple sclerosis such as myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG); autoantigens related to polymyositis (PM) and dermatomyositis (DM) such as Jo-1, Mi-2, PM-Scl, and Ro-52; autoantigens related to gluten-sensitive enteropathy such as endomysial antibody (EMA) and tissue transglutaminase (tTG);Anti-NMDAR encephalitis-related autoantigens such as N-methyl-D-aspartate receptor; Neuromyelitis optica (NMO)-related autoantigens such as aquaporin 4 (AQP4); Reproduction-related autoantigens such as ovarian antigens and sperm antigens; In some embodiments, the pathogenic protein is a metabolic disease-associated target protein. Examples of the metabolic disease-related target protein include, but are not limited to, atherosclerosis (AS)-related target proteins such as CD36, low-density lipoprotein receptor (LDLR), ChemR23 (CMKLR1), and mitochondrial dehydrogenase ALDH4A1 (target protein); type II diabetes-related target proteins such as RalGAPα1 and dipeptidyl peptidase IV (DPP4); non-alcoholic fatty liver-related target proteins such as TMEM16A and VAMP3; tumor glucose metabolism-related target proteins such as, but not limited to, hexokinase (HK), glucose transporter 1 (GLUT1), glucose transporter 4 (GLUT4), phosphoglycerate dehydrogenase, and lactate dehydrogenase; and ATP citrate lyase (ATP citrate lyase). These include tumor lipid metabolism-related target proteins such as fatty acid synthase (FASN), cholesterol esterase (acetyl-CoA acetyltransferase 1, ACAT1), and fatty acid synthase (FASN).

[0059] In some embodiments, the targeting domain specifically targets a target protein other than TCR, HLA-I, MICA and MICB target proteins, and the viral endoplasmic reticulum resident protein is any of the viral endoplasmic reticulum resident proteins of the present disclosure. In some embodiments, the targeting domain specifically targets at least two target proteins selected from TCR, HLA-I, MICA, and MICB, and the viral endoplasmic reticulum resident protein is any of the viral endoplasmic reticulum resident proteins of the present disclosure. In some embodiments, the targeting domain specifically targets TCR, HLA-I, MICA or MICB and said viral endoplasmic reticulum resident protein is not adenovirus E3-K19. In some embodiments, the targeting domain of the chimeric protein construct is linked to the ERAD machinery protein binding domain by a hinge or linker. In some embodiments, the hinge comprises the amino acid sequence set forth in SEQ ID NO: 123 (IgG4 hinge) or SEQ ID NO: 133 ((Gly4Ser)2). In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:47, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US2 is set forth in SEQ ID NO:47. In some preferred embodiments, the HLA binding domain of US2 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:75, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the HLA binding domain of US2 is set forth in SEQ ID NO:75.

[0060] In some preferred embodiments, the MHC binding domain of HCMV glycoprotein US11 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:53, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the MHC binding domain of HCMV glycoprotein US11 is set forth in SEQ ID NO:53. In some preferred embodiments, the HLA-binding domain of US11 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:81, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the HLA-binding domain of US11 is set forth in SEQ ID NO:81. In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:50, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US3 is set forth in SEQ ID NO:50. In some preferred embodiments, the HLA binding domain of US3 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:78, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the HLA binding domain of US3 is set forth in SEQ ID NO:78. In some preferred embodiments, the HLA binding domain of HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:56, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the HLA binding domain of HCMV glycoprotein US10 is set forth in SEQ ID NO:56.

[0061] In some preferred embodiments, the MHC I and / or MHC II targeting binding protein molecule domain or functional variant thereof is derived from a viral glycoprotein that degrades MHC and / or MHC II molecules; preferably, the viral glycoprotein is selected from HCMV glycoprotein US2, US3, US11 or US10, adenovirus E3-19K or HHV-7 US21. In some preferred embodiments, the MHC I and / or MHC II targeting binding protein molecular domain or functional variant thereof further comprises a viral protein that directly inhibits or degrades the following NK target proteins: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6; preferably, the viral protein is selected from HCMV UL16, UL141, UL142 or adenovirus E3-19K. In some preferred embodiments, the MHC I and / or MHC II targeting binding protein molecule domain or functional variant thereof further comprises a viral protein that transports the MHC I molecule from the Golgi apparatus to lysosomes for degradation; preferably, the viral protein is selected from HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3 and HTLV-1 p12. In some preferred embodiments, the MHC I and / or MHC II targeting binding protein molecule domain or functional variant thereof further comprises a viral protein that mediates the return of the MHC-polypeptide molecule from the Golgi apparatus to the endoplasmic reticulum and promotes its degradation; preferably, the viral protein comprises an MHC binding structure and a KDEL receptor binding domain; preferably, the viral protein is the Cowpox Virus protein CPXV203. In some preferred embodiments, the HHV-7 US21 MHC binding domain comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:62, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the HHV-7 US21 MHC binding domain is set forth in SEQ ID NO:62.

[0062] In some preferred embodiments, the NK target protein binding domain of HCMV UL16 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:84, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the NK target protein binding domain of HCMV UL16 is set forth in SEQ ID NO:84. In some preferred embodiments, the NK target protein binding domain of HCMV UL141 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:87, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the NK target protein binding domain of HCMV UL141 is set forth in SEQ ID NO:87. In some preferred embodiments, the MICA, ULBP3-binding domain of HCMV UL142 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:90, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the MICA, ULBP3-binding domain of HCMV UL142 is set forth in SEQ ID NO:90. In some preferred embodiments, the MHC binding domain of the cowpox virus protein CPXV203 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:99, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the MHC binding domain of the cowpox virus protein CPXV203 is set forth in SEQ ID NO:99. In some preferred embodiments, the MHC binding domain of the cowpox virus protein CPXV203 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:102, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the MHC binding domain of the cowpox virus protein CPXV203 is set forth in SEQ ID NO:102. In some preferred embodiments, the adenovirus E3-19K MHC binding domain comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:59, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the adenovirus E3-19K MHC binding domain is set forth in SEQ ID NO:59.

[0063] 3. Protein Degradation Pathway Member Binding Domains The chimeric protein constructs provided herein may further comprise a proteolytic pathway member binding domain. As used herein, a "proteolytic pathway member binding domain" refers to any moiety that can directly or indirectly bind to a member in a proteolytic pathway. The protein degradation pathway can be any pathway that mediates the degradation of proteins in cells. Known protein degradation pathways include, but are not limited to, the ubiquitination-proteasome pathway, the endosome-lysosome pathway, and the autophagy degradation pathway. Ubiquitination-proteasome pathway members include, for example, E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases, and the proteasome. Examples of E1 ubiquitin activating enzymes include, for example, UBA1, UBA2, UBA3, UBA5, UBA6, UBA7, ATG7, NAE1, and SAE1. Examples of E2 ubiquitin conjugating enzymes include, for example, hCdc34, Ubc-Uev1A, UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2L6, UBE2M, UBE2N, UBE2O, UBE2Q1, UBE2Q2, UBE2R1 (CDC34), UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2W, UBE2Z, ATG3, BIRC6, and UFC1. Examples of E3 ubiquitin ligases include, for example, von Hippel-Lindau (VHL), Cereblon (CRBN), inhibitor of apoptosis protein (IAP), Kelch-like ECH-associated protein 1 (Keap1), RNF4, RNF114, MDM2, LUBAC, FBW7, Met30, HECT, SKP2, betaTRCP1, HUWEI, TRAF6, SMURF1, and E6AP. In some embodiments, examples of E3 ubiquitin ligases include, for example, E3A, mdm2, Anaphase-promoting complex (APC), UBR5 (EDD1), SOCS / BC-box / eloBC / CUL5 / RING, LNXp80, CBX4, CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECTD4, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, HERC6, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, VHL, WWP1, WWP2, Parkin, and MKRN1.

[0064] Examples of members of the endosomal-lysosomal pathway include AP-1, AP-2, AP-3, endosome, lysosome, HOPS, ESCRT, GASP, BLOC-1, ESCRT, Retromer, ESCRT, sorting nexin, Dapper2, SNX4, Pincher, Rap1-PDZ-GEF1, clathrin, C3G / CrkL / Shp2 / Gab2, and the like. Examples of members of the autophagy degradation pathway include chaperone-mediated autophagy (CMA), USP10, G3BP1, ULK1, ATG16L1, TRIM16, FBXO27VDAC, RHOT1, MFN1 / 2, BNIP3L, FUNDC1, BNIP3, AMBRA1, BCL2LI3, FKBP8, CHDH, DISC1, PHB2, cardiolipin, SEC62, RTN3, PEX5, PEX14, ABCD3, and NUFIP1. In some embodiments, the protein degradation pathway member binding domain is a VHL binding domain. In some embodiments, the VHL binding domain comprises the amino acid sequence of SEQ ID NO: 119 (DRHDSGLDSM) or SEQ ID NO: 120 (ALAPYIP). In some embodiments, the protein degradation pathway member binding domain is a Keap1 binding domain. In some embodiments, the Keap1 binding domain comprises the amino acid sequence of SEQ ID NO: 121 (LDPETGEYL). In some embodiments, the protein degradation pathway member binding domain is an E3 ubiquitin ligase binding domain. The E3 ubiquitin ligase binding domain comprises the amino acid sequence of SEQ ID NO: 119 (DRHDSGLDSM). In some embodiments, the protein degradation pathway member binding domain is a CMA binding domain. In some embodiments, the CMA binding domain comprises the amino acid sequence of SEQ ID NO: 122 (KFERQ). In some embodiments, the CMA binding domain comprises the amino acid sequence of SEQ ID NO: 123 (KFERQKILDQRFFE).

[0065] In some embodiments, the protein degradation pathway member binding domain is a proteasome binding domain. In some embodiments, the proteasome binding domain is selected from the proteasome binding domains of yeast Rad23 (e.g., S. cerevisiae Rad23), the ubiquitin-like (UbL) domain of human Rad23b (hHR23b), HPV E7, and ankyrin. In some embodiments, the proteasome binding domain comprises amino acids 1-77 of yeast Rad23. In some embodiments, the proteasome binding domain comprises amino acids 1-83 of human Rad23b. The inventors have found that chimeric protein constructs containing protein degradation pathway member binding domains (ligand for E3 Ligase (E3L), ligand for E2 Ubiquitin-conjugating enzyme (E2L) or ligand for lysosome (LL)) (e.g., as described above) significantly improve the degradation effect on the target protein compared to the basic TPD design (Targeted protein binding domain-Transmembrane domain-ER retention domain, TBD-TMD-ERD). Functional structures derived from viral ER resident proteins have different binding abilities to the ERAD retroporter complex, and the native structures also differ in their interactions with ER resident E3 ligases and compete with the normal degradation pathway of the ERAD machinery for misfolded proteins in the endoplasmic reticulum. Therefore, the natural TMD-ERD domains have differences and limitations in the ubiquitination and degradation of target proteins. To further improve the degradation efficiency of target proteins, the introduction of ubiquitin-proteasome system (UPS) ligand domains and / or autophagy-lysosome pathway (ALP) ligand domains can further promote the ubiquitination and proteasome degradation of the target proteins transported into the cytoplasm, or transport them to the lysosome to achieve effective hydrolysis of the target proteins. In some embodiments, the targeting domain or ERAD machinery protein binding domain of the chimeric protein construct is linked to the protein degradation pathway member binding domain by a hinge or linker. The hinge or linker can be one or more molecular sequence structures (GlyxSery)n, where n can be a number from 1 to 10, and x, y can be a number from 0 to 10, respectively, with the proviso that x and y are not 0 at the same time. In some embodiments, the hinge comprises the amino acid sequence shown in SEQ ID NO: 172 (IgG4 hinge) or SEQ ID NO: 173 ((Gly4Ser)2).

[0066] 4. TPD Chimeric Protein Constructs In some embodiments, the chimeric protein constructs provided herein comprise a target protein binding domain that targets the TCR, an adenovirus E3-19K, and one or more of the protein degradation pathway member binding domains described above. In some embodiments, the chimeric protein constructs provided herein comprise a target protein binding domain that targets the TCR, an adenovirus E3-19K, and an E3 ubiquitin ligase binding domain. In some embodiments, the E3 ubiquitin ligase binding domain comprises the amino acid sequence set forth in SEQ ID NO: 147 (DRHDSGLDSMGSGSGALAPYIP). In some embodiments, the chimeric protein construct provided herein comprises a target protein binding domain that targets a TCR, a transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum resident protein other than adenovirus E3-19K, and an endoplasmic reticulum resident domain or a functional variant thereof, wherein the viral endoplasmic reticulum resident protein can be at least one selected from HCMV glycoproteins US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, and cowpox virus protein CPXV203. In some embodiments, the chimeric protein constructs provided herein comprise a target protein binding domain that targets the target protein (e.g., TCR), a transmembrane domain of HCMV US2 and / or US11 or a functional variant thereof, and an endoplasmic reticulum resident domain or a functional variant thereof. In some embodiments, the chimeric protein construct provided herein comprises a target protein binding domain that targets the target protein (e.g., TCR), a transmembrane domain of HCMV US3 or a functional variant thereof, and an endoplasmic reticulum resident domain or a functional variant thereof. In some embodiments, the chimeric protein construct provided herein comprises a target protein binding domain that targets the target protein (e.g., TCR), a transmembrane domain of HCMV US10 or a functional variant thereof, and an endoplasmic reticulum resident domain or a functional variant thereof.

[0067] In some embodiments, the chimeric protein constructs provided herein comprise a target protein binding domain that targets the target protein (e.g., TCR), a transmembrane domain or functional variant thereof of HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a and / or BHV UL49.5, and an endoplasmic reticulum resident domain or functional variant thereof. In some embodiments, the chimeric protein construct provided herein comprises a target protein binding domain that targets the target protein (e.g., TCR), a transmembrane domain of HHV-7 US21 or a functional variant thereof, and an endoplasmic reticulum resident domain or a functional variant thereof. In some embodiments, the chimeric protein constructs provided herein comprise a target protein binding domain that targets the target protein (e.g., TCR), a transmembrane domain of HCMV UL16, UL141 and / or UL142 or a functional variant thereof, and an endoplasmic reticulum resident domain or a functional variant thereof. In some embodiments, the chimeric protein constructs provided herein comprise a target protein binding domain that targets the target protein (e.g., TCR), a transmembrane domain or functional variant thereof of HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3 and / or HTLV-1 p12, and an endoplasmic reticulum resident domain or functional variant thereof. In some embodiments, the chimeric protein construct provided herein comprises a target protein binding domain that targets the target protein (e.g., TCR), a transmembrane domain of Cowpox Virus protein CPXV203 or a functional variant thereof, and an endoplasmic reticulum resident domain or a functional variant thereof. In some embodiments, the chimeric protein constructs provided herein comprise a targeting protein binding domain that does not target TCR and an adenovirus E3-19K. In some embodiments, the chimeric protein constructs provided herein comprise a targeting protein binding domain that targets CD123 and an adenovirus E3-19K. In some embodiments, the chimeric protein construct further comprises one or more of the protein degradation pathway member binding domains described above.

[0068] 5. Co-expression part In some embodiments, the chimeric protein construct provided in the present disclosure and at least one co-expression moiety are co-expressed. Any suitable means for co-expression can be used. For example, the chimeric protein construct (or a part thereof) and the at least one co-expression moiety can be linked by a cleavable linker, and when the linker is cleaved, the chimeric protein construct and the co-expression moiety are co-expressed. In some embodiments, the chimeric protein constructs provided herein and at least one co-expression moiety are linked by, for example, a self-cleavable linkage. In some embodiments, the self-cleavable linker is a cleavable peptide, such as, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. In some preferred embodiments, the T2A comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:108, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of T2A is set forth in SEQ ID NO:108. In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is set forth in SEQ ID NO:109. In some preferred embodiments, the P2A comprises an amino acid sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably 98% or 99% or more identity, to the amino acid sequence set forth in SEQ ID NO:110; the amino acid sequence of P2A is set forth in SEQ ID NO:110. In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is set forth in SEQ ID NO:111. In some preferred embodiments, the E2A comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:112, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of E2A is set forth in SEQ ID NO:112.

[0069] In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is set forth in SEQ ID NO:113. In some preferred embodiments, the F2A comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:114, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of F2A is set forth in SEQ ID NO:114. In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is set forth in SEQ ID NO:115. The co-expression moiety provided herein may be any protein or polypeptide having a biological function. Depending on the desired biological function, an appropriate co-expression moiety may be selected. For example, a protein that degrades or reduces MHC class I or class II molecules may be selected as the co-expression moiety to reduce the immunogenicity or antigen presentation of cells. Also, for example, a binding domain that recognizes or binds to a target protein (e.g., a chemokine receptor, or a chimeric antigen receptor CAR) may be selected as the co-expression moiety to allow cells to recognize the target protein. Furthermore, for example, an immunostimulatory molecule may be selected as the co-expression moiety to enhance immune stimulatory activity. In some preferred embodiments, examples of the co-expressed moieties include intact viral ER resident glycoproteins (e.g., HCMV US2, US3, US11, US10, adenovirus E3-Kl 9, HCMV US6, HSV ICP47), chimeric antigen receptors (CARs), functional T cell receptors (TCRs), chemokine receptors (e.g., CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2, and CXCR4), NK cell activating receptors (e.g., NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxic receptors, and the like). receptor, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC and NKp80), CNK signaling modules, cytokines, CD7, immune stimulatory molecules (e.g., one or more selected from TNF-a, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and granulocyte-macrophage colony-stimulating factor).

[0070] i) Intact viral ER-resident glycoproteins In some embodiments, the co-expressed moieties are intact viral ER resident glycoproteins, including but not limited to HCMV US2, US3, US11, US10, adenovirus E3-Kl 9, HCMV US6 and HSV ICP47. In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:46, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US2 is set forth in SEQ ID NO:46. In some preferred embodiments, the full-length sequence of US2 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:74, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US2 is set forth in SEQ ID NO:74. In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:49, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US3 is set forth in SEQ ID NO:49. In some preferred embodiments, the full-length sequence of US3 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:77, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US3 is set forth in SEQ ID NO:77.

[0071] In some preferred embodiments, the full-length sequence of HCMV glycoprotein US11 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:52, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of HCMV glycoprotein US11 is set forth in SEQ ID NO:52. In some preferred embodiments, the full-length sequence of US11 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:80, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US11 is set forth in SEQ ID NO:80. In some preferred embodiments, the full-length sequence of HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:55, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of HCMV glycoprotein US10 is set forth in SEQ ID NO:55. In some preferred embodiments, the full-length sequence of the adenovirus E3-19K comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:58, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the adenovirus E3-19K is set forth in SEQ ID NO:58. In some preferred embodiments, the full-length sequence of HCMV US6 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:64, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of HCMV US6 is set forth in SEQ ID NO:64. In some preferred embodiments, the full-length sequence of HSV ICP47 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:66, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity, and preferably has activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of HSV ICP47 is set forth in SEQ ID NO:66.

[0072] ii) Chimeric Antigen Receptor (CAR) In some embodiments, the co-expression moiety is a chimeric antigen receptor (CAR). The CAR comprises a target protein binding domain (e.g., an extracellular recognition domain that targets tumors), a transmembrane domain, and an immunoreceptor activation signaling domain (ITAM) (also called an "intracellular signaling domain"). In some embodiments, the CAR may further comprise a co-stimulatory domain. In some embodiments, a hinge or linker is included between the extracellular recognition domain that targets tumors, the transmembrane domain, and / or the intracellular signaling domain. In some embodiments, the tumor-targeting extracellular recognition domain is selected from a tumor antigen-specific binding domain, a tumor microenvironment-targeting antigen-binding domain, and / or a chemokine receptor-targeting domain. In some preferred embodiments, the tumor-targeting extracellular recognition domain is selected from an antibody or functional fragment thereof, a TCR, or a combination thereof, that targets and recognizes a tumor-associated antigen; the antibody functional fragment is selected from an Fd, Fv, Fab, Fab', F(ab')2, Fv(scFv), a single-chain antibody (scFv) or a nanobody, a two-chain antibody, a three-chain antibody, and a four-chain antibody.

[0073] In some preferred embodiments, the transmembrane domain of the CAR described herein may be derived from any membrane-bound or transmembrane protein, including BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-1), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD4 9a, CD49d, CD49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF 1, SLAM), CD154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, CRT These include, but are not limited to, AM, GITR, HYEM (LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7R a, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, ​​OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, the α, β or ζ chains of the T cell receptor, TNFR2, VLA1 and VLA-6. In some preferred embodiments, the transmembrane domain of the CAR is selected from a transmembrane domain of an NK cell activating receptor, a DAP10 transmembrane domain, a DAP12 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, and combinations thereof.

[0074] In some preferred embodiments, the transmembrane domain of said CAR is selected from CD8 transmembrane domain, alpha and / or beta chain transmembrane domains of the T cell receptor, CD28 transmembrane domain, CD3 epsilon transmembrane domain, CD45 transmembrane domain, CD4 transmembrane domain, CD5 transmembrane domain, CD8 transmembrane domain, CD9 transmembrane domain, CD16 transmembrane domain, CD22 transmembrane domain, CD33 transmembrane domain, CD37 transmembrane domain, CD64 transmembrane domain, CD80 transmembrane domain, CD86 transmembrane domain, CD134 transmembrane domain, CD137 transmembrane domain, CD154 transmembrane domain, GITR transmembrane domain, and combinations thereof. In some preferred embodiments, the ITAM is derived from an intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from TCRζ, CD2, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, FcRγ, CD66d, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; preferably, the ITAM is fused to an NK cell signal adaptor or a functional variant thereof; preferably, the immune receptor is CD3ζ.

[0075] In some preferred embodiments, the immunoreceptor activating signaling domain (ITAM) is derived from an intracellular signaling domain, such as CD3zeta, common FcRgamma (FCER1G), FcγRIIa, FcRβ, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRICD66d, DAP10, and DAP12. In some preferred embodiments, the intracellular signaling domain of the CAR comprises the intracellular signaling domain and / or the costimulatory signaling domain of a NK cell activating receptor. In some preferred embodiments, the T cell costimulatory signaling domain is derived from an intracellular signaling domain of a costimulatory molecule; preferably, the costimulatory molecule is an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin protein, a lymphocyte activation signaling molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD16, CD27, CD28, CD30, CD40, CD38, CD35, CD79A, CD79B, CDS, ICAM-1, LFA-1, (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, or the like. , NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4 , IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NCR, DAP10, DAP12, TNFR2, TRANCE / RANKL, DNAM1( CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100 SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp, TRIM, ZAP70, and PTCH2.More preferably, the costimulatory signaling domain may be selected from the group consisting of NKG2D intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, NCR intracellular signaling domain, CD28 intracellular signaling domain, 4-1BB intracellular signaling domain, OX40 intracellular signaling domain, and ICOS intracellular signaling domain.

[0076] In some embodiments, the hinge and / or transmembrane domain of the CAR described herein provides for cell surface presentation of the extracellular domain of the CAR. The hinge of the CAR described herein may be derived from any membrane-bound or transmembrane protein, including BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-1), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD 49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLA M), CD154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, CRT Examples of suitable CARs include, but are not limited to, AM, GITR, HYEM (LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, ​​OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, the α, β or ζ chain of the T cell receptor, TNFR2, VLA1 and VLA-6. In some embodiments, the hinge of the CAR described herein comprises a CD8α hinge region, a human immunoglobulin (Ig) hinge region or a glycine-serine rich sequence. In some preferred embodiments, the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence (Gly(x)Ser(y))n, where n is an integer from 1 to 10, and x and y are independently integers from 0 to 10, with the proviso that x and y are not both 0; more preferably, the linker comprises the amino acid sequence set forth in SEQ ID NO:104 or the amino acid sequence set forth in SEQ ID NO:105.

[0077] In some preferred embodiments, the hinge is an IgG1 hinge or an IgG4 hinge. In some preferred embodiments, the IgG1 hinge comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity to the amino acid sequence set forth in SEQ ID NO: 106; the amino acid sequence of the IgG1 hinge is set forth in SEQ ID NO: 106. In some preferred embodiments, the IgG4 hinge comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity to the amino acid sequence set forth in SEQ ID NO: 107; the amino acid sequence of the IgG4 hinge is set forth in SEQ ID NO: 107. In some preferred embodiments, the NK activating receptor module, the CNK signaling module and / or the UT module include a cleavable peptide between them, such as, for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide or a GSG-P2A peptide.

[0078] In some preferred embodiments, the T2A comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:108, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of T2A is set forth in SEQ ID NO:108. In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is set forth in SEQ ID NO:109. In some preferred embodiments, the P2A comprises an amino acid sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably 98% or 99% or more identity, to the amino acid sequence set forth in SEQ ID NO:110; the amino acid sequence of P2A is set forth in SEQ ID NO:110. In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is set forth in SEQ ID NO:111. In some preferred embodiments, the E2A comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:112, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of E2A is set forth in SEQ ID NO:112. In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is set forth in SEQ ID NO:113. In some preferred embodiments, the F2A comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:114, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of F2A is set forth in SEQ ID NO:114. In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is set forth in SEQ ID NO:115.

[0079] iii) CNK receptor In some embodiments, the co-expression moiety is a CNK receptor. The CNK receptor comprises a chimeric NK activating receptor module and a chimeric NK signaling module (also referred to as a "CNK signaling module"). The chimeric NK activating receptor module may comprise at least an NK cell activating receptor or a functional variant thereof. NK cell activating receptor In some embodiments, the NK cell activating receptor comprises (a) an extracellular domain (ED) of an NK cell activating receptor or a functional variant thereof, (b) a transmembrane domain (TMD) of an NK cell activating receptor or a functional variant thereof, and (c) an intracellular domain (ICD) of an NK cell activating receptor or a functional variant thereof, and may comprise a hinge or linker between the extracellular domain of an NK cell activating receptor or a functional variant thereof, the transmembrane domain of an NK cell activating receptor or a functional variant thereof, and / or the intracellular domain of an NK cell activating receptor or a functional variant thereof. For details of the NK cell activating receptor and the CNK signaling module, see, for example, US 2020 / 0308248 A1, the entire contents of which are incorporated herein by reference. The NK cell activating receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, Natural Cytotoxicity Receptors (NCR), TRAIL, DNAM-1, signaling lymphocytic activation molecule (SLAM) family molecule 2B4 (also known as CD244), DNAX accessory molecule 1 (DNAM-1, also known as CD226), CD16a, 2B4, NTB-A, CRACC (CS1) and NKp80; wherein the natural cytotoxicity receptor includes NKp46 (also known as NCR1 or CD335), NKp44 (also known as NCR2 or CD336) and NKp30 (also known as NCR3 or CD337). In some embodiments, the natural cytotoxicity receptor is selected from NKp46, NKp44, and NKp30.

[0080] In some preferred embodiments, the NK cell activating receptor is an NK cell activating receptor from a mammal; preferably, the mammal is selected from a human, a primate, a mouse, a horse, a cow, a sheep, a goat, a cat, a pig, a dog, a llama, an alpaca, an elephant, a squirrel, or a guinea pig. In some preferred embodiments, the NK cell activating receptor is a recombinant NK cell activating receptor that comprises NK cell activating receptor domains of different origins. In some preferred embodiments, the NK cell activating receptor is an NK cell activating receptor of human origin; preferably, the NK cell activating receptor is a recombinant NK cell activating receptor comprising NK cell activating receptor domains of different human origin. In some preferred embodiments, the NK cell activating receptor is a mouse-derived NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant NK cell activating receptor comprising NK cell activating receptor domains from different mice. In some preferred embodiments, the NK cell activating receptor is a recombinant NK cell activating receptor comprising human and mouse derived NK cell activating receptor domains. In some preferred embodiments, the extracellular domain of the NK cell activating receptor is the extracellular domain of a human or mouse NK cell activating receptor. In some preferred embodiments, the transmembrane domain of the NK cell activating receptor is a transmembrane domain of a human or mouse NK cell activating receptor. In some preferred embodiments, the intracellular domain of the NK cell activating receptor is the intracellular domain of a human or mouse NK cell activating receptor. In some preferred embodiments, the NK cell activating receptor comprises the extracellular domain of a human NK cell activating receptor, the transmembrane domain of a mouse NK cell activating receptor, and the intracellular domain of a human NK cell activating receptor. In some preferred embodiments, the functional mutant of the NK cell activating receptor is selected from a mutant of the NK cell activating receptor, a wild-type fusion protein, or a fusion protein of the wild-type and mutant forms. In some preferred embodiments, the extracellular domain of human NKG2D comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:1, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the extracellular domain of human NKG2D is set forth in SEQ ID NO:1.

[0081] In some preferred embodiments, the full-length sequence of human NKG2D comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:2, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human NKG2D is set forth in SEQ ID NO:2. In some preferred embodiments, the extracellular domain of mouse NKG2D comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:3, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the extracellular domain of mouse NKG2D is set forth in SEQ ID NO:3. In some preferred embodiments, the full-length sequence of mouse NKG2D comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:4, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of mouse NKG2D is set forth in SEQ ID NO:4. In some preferred embodiments, the full-length sequence of human-mouse recombinant NKG2D comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:5, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human-mouse recombinant NKG2D is set forth in SEQ ID NO:5.

[0082] In some preferred embodiments, the full-length sequence of human NKG2C comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:6, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human NKG2C is set forth in SEQ ID NO:6. In some preferred embodiments, the full-length sequence of human NKG2E comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:7, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human NKG2E is set forth in SEQ ID NO:7. In some preferred embodiments, the full-length sequence of human NKG2F comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:8, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human NKG2F is set forth in SEQ ID NO:8. In some preferred embodiments, the full-length sequence of human CD94 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO:9; the amino acid sequence of the full-length sequence of human CD94 is set forth in SEQ ID NO:9. In some preferred embodiments, the full-length sequence of human KIR2DL4 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:10, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human KIR2DL4 is set forth in SEQ ID NO:10.

[0083] In some preferred embodiments, the full-length sequence of human KIR2DS1 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:11, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human KIR2DS1 is set forth in SEQ ID NO:11. In some preferred embodiments, the full-length sequence of human KIR2DS2 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:12, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human KIR2DS2 is set forth in SEQ ID NO:12. In some preferred embodiments, the full-length sequence of human KIR2DS4 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:13, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human KIR2DS4 is set forth in SEQ ID NO:13. In some preferred embodiments, the full-length sequence of human KIR3DS1 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:14, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human KIR3DS1 is set forth in SEQ ID NO:14.

[0084] In some preferred embodiments, the full-length sequence of human NKp46 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:15, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human NKp46 is set forth in SEQ ID NO:15. In some preferred embodiments, the full-length sequence of human NKp44 comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO:16; the amino acid sequence of the full-length sequence of human NKp44 is set forth in SEQ ID NO:16. In some preferred embodiments, the full-length sequence of human NKp30 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:17, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human NKp30 is set forth in SEQ ID NO:17. In some preferred embodiments, the full-length sequence of human DNAM1 comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO:18, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human DNAM1 is shown in SEQ ID NO:18. In some preferred embodiments, the full-length sequence of human TRAIL comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:19, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human TRAIL is set forth in SEQ ID NO:19.

[0085] In some preferred embodiments, the full-length sequence of human CD16a comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:20, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human CD16a is set forth in SEQ ID NO:20. In some preferred embodiments, the full-length sequence of human 2B4 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:21, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human 2B4 is set forth in SEQ ID NO:21. In some preferred embodiments, the full-length sequence of human NTB-A comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:22, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human NTB-A is set forth in SEQ ID NO:22. In some preferred embodiments, the full-length sequence of human CRACC comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:23, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human CRACC is set forth in SEQ ID NO:23. In some preferred embodiments, the full-length sequence of human NKp80 comprises an amino acid sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably 98% or 99% or more identity, to the amino acid sequence set forth in SEQ ID NO:24; the amino acid sequence of the full-length sequence of human NKp80 is set forth in SEQ ID NO:24.

[0086] CNK Signaling Module In some embodiments, the CNK signaling module comprises at least (i) an NK cell adaptor (e.g., DAP10 or DAP12) or a functional variant thereof. In some embodiments, the NK cell adaptor comprises (a) an extracellular domain (ED) of an NK cell adaptor or a functional variant thereof, (b) a transmembrane domain (TMD) of an NK cell adaptor or a functional variant thereof, and (c) an intracellular domain (ICD) of an NK cell adaptor or a functional variant thereof; and optionally a hinge or linker between the extracellular domain of the NK cell adaptor or a functional variant thereof, the transmembrane domain of the NK cell adaptor or a functional variant thereof, and / or the intracellular domain of the NK cell adaptor or a functional variant thereof. In some preferred embodiments, the NK cell signaling adaptor is an NK cell signaling adaptor from a mammal; preferably, the mammal is selected from a human, a primate, a mouse, a horse, a cow, a sheep, a goat, a cat, a pig, a dog, a llama, an alpaca, an elephant, a squirrel, or a guinea pig. In some preferred embodiments, the NK cell signaling adaptor is a recombinant NK cell signaling adaptor that comprises NK cell signaling adaptor domains of different origins. In some preferred embodiments, the NK cell signaling adaptor is an NK cell signaling adaptor of human origin; preferably, the NK cell signaling adaptor is a recombinant NK cell signaling adaptor comprising an NK cell signaling adaptor domain of a different human origin. In some preferred embodiments, the NK cell signaling adaptor is a mouse-derived NK cell signaling adaptor; preferably, the NK cell signaling adaptor is a recombinant NK cell signaling adaptor comprising an NK cell signaling adaptor domain derived from a different mouse.

[0087] In some preferred embodiments, the NK cell signaling adaptor is a recombinant NK cell signaling adaptor comprising human and mouse derived NK cell signaling adaptor domains. In some preferred embodiments, the extracellular domain of the NK cell signal adaptor is an extracellular domain of a human or mouse NK cell signal adaptor. In some preferred embodiments, the NK cell signaling adaptor transmembrane domain is a human or mouse NK cell signaling adaptor transmembrane domain. In some preferred embodiments, the intracellular domain of the NK cell signal adaptor is a human or mouse intracellular domain of the NK cell signal adaptor. The NK cell signal adaptor in the CNK signaling module is DAP10 or DAP12. In some preferred embodiments, the functional mutant of the CNK cell signal adaptor is selected from a mutant of DAP10 or DAP12, or a fusion protein of DAP10 and DAP12, or a fusion protein of wild-type DAP10 or DAP12 and a mutant DAP10 or DAP12. In some preferred embodiments, the CNK signaling module further comprises (ii) an immune receptor activation signaling domain (ITAM) and / or (iii) a T cell costimulatory signaling domain.

[0088] In some preferred embodiments, a hinge or linker is included between the NK cell signaling adaptor or functional variant thereof, the immunoreceptor activating signaling domain (ITAM) and / or the T cell costimulatory signaling domain; preferably, the NK cell signaling adaptor or functional variant thereof is fused to the immunoreceptor activating signaling domain (ITAM). In some preferred embodiments, the ITAM is derived from an intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from TCRζ, CD2, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, FcRγ, CD66d, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; preferably, the ITAM is fused to an NK cell signal adaptor or a functional variant thereof; preferably, the immune receptor is CD3ζ. In some preferred embodiments, the T cell costimulatory signaling domain is derived from the intracellular signaling domain of a costimulatory molecule. In some preferred embodiments, the full-length sequence of human DAP10 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:25, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human DAP10 is set forth in SEQ ID NO:25. In some preferred embodiments, the full-length sequence of human DAP10 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:26, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human DAP10 is set forth in SEQ ID NO:26.

[0089] In some preferred embodiments, the transmembrane domain of human DAP10 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:27, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the transmembrane domain of human DAP10 is set forth in SEQ ID NO:27. In some preferred embodiments, the full-length sequence of human DAP12 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:28, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the full-length sequence of human DAP12 is set forth in SEQ ID NO:28. In some preferred embodiments, the transmembrane domain of human DAP12 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:29, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the transmembrane domain of human DAP12 is set forth in SEQ ID NO:29. In some preferred embodiments, the human DAP10 and human DAP12 transmembrane domain fusion proteins comprise an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:30, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequences of the human DAP10 and human DAP12 transmembrane domains are set forth in SEQ ID NO:30. In some preferred embodiments, the human DAP10-DAP12 fusion protein sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:31, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP10-DAP12 fusion protein sequence is set forth in SEQ ID NO:31.

[0090] In some preferred embodiments, the human CD3zeta intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:32, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human CD3zeta intracellular signaling domain sequence is set forth in SEQ ID NO:32. In some preferred embodiments, the human DAP10-CD3zeta sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:33, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP10-CD3zeta sequence is set forth in SEQ ID NO:33. In some preferred embodiments, the human DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:34, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP12-CD3zeta sequence is set forth in SEQ ID NO:34. In some preferred embodiments, the human DAP10-DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:35, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP10-DAP12-CD3zeta sequence is set forth in SEQ ID NO:35. In some preferred embodiments, the human 41BB intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:36, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human 41BB intracellular signaling domain sequence is set forth in SEQ ID NO:36.

[0091] In some preferred embodiments, the human DAP10-41BB sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:37, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP10-41BB sequence is set forth in SEQ ID NO:37. In some preferred embodiments, the human DAP10-41BB-CD3zeta sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:38, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP10-41BB-CD3zeta sequence is set forth in SEQ ID NO:38. In some preferred embodiments, the human CD28 intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO:39; the amino acid sequence of the human CD28 intracellular signaling domain sequence is set forth in SEQ ID NO:39. In some preferred embodiments, the human DAP10-CD28 sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:40, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP10-CD28 sequence is set forth in SEQ ID NO:40. In some preferred embodiments, the human DAP10-CD28-CD3zeta sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:41, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP10-CD28-CD3zeta sequence is set forth in SEQ ID NO:41.

[0092] In some preferred embodiments, the human DAP12-41BB sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:42, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP12-41BB sequence is set forth in SEQ ID NO:42. In some preferred embodiments, the human DAP12-41BB-CD3zeta sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:43, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP12-41BB-CD3zeta sequence is set forth in SEQ ID NO:43. In some preferred embodiments, the human DAP12-CD28 sequence comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO:44; the amino acid sequence of the human DAP12-CD28 sequence is set forth in SEQ ID NO:44. In some preferred embodiments, the human DAP12-CD28-CD3zeta sequence comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:45, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity; the amino acid sequence of the human DAP12-CD28-CD3zeta sequence is set forth in SEQ ID NO:45. In some preferred embodiments, the CNK signaling module comprises an amino acid sequence selected from SEQ ID NOs:25-45. In some preferred embodiments, the chimeric protein construct, the NK cell activating receptor, and the CNK signaling module form a multifunctional complex, wherein the multifunctional complex comprises the amino acid sequence set forth in SEQ ID NO: 117. In some preferred embodiments, the multifunctional complex comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity, to the amino acid sequence set forth in SEQ ID NO: 117; the amino acid sequence of the multifunctional complex comprises the amino acid sequence set forth in SEQ ID NO: 117.

[0093] 6. Nucleic acid molecules In another aspect, the disclosure further provides a chimeric nucleic acid construct encoding any of the chimeric protein constructs described herein. The nucleic acid molecule may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), ethylene glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose configuration, α-LNA (diastereoisomer of LNA) with α-L-ribose configuration, 2′-amino-LNA with 2′-amino functionalization, and 2′-amino-α-LNA with 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), and / or chimeras and / or combinations thereof. Exemplary DNA includes, but is not limited to, plasmid DNA (pDNA), and the like. Exemplary RNA includes, but is not limited to, mRNA, circular RNA, ccRNA, and the like. In some preferred embodiments, the nucleic acid molecule is an mRNA. An mRNA is any RNA that encodes at least one protein and can produce the encoded protein in vitro, in vivo, in situ, or ex vivo. Unless otherwise specified, the nucleic acid sequences described herein may be exemplified by "T" in a representative DNA sequence, but it should be understood by those of skill in the art that when the sequence represents an RNA (e.g., an mRNA), "T" is replaced with "U". Thus, any DNA disclosed or identified herein by a particular sequence identification number also discloses the corresponding RNA (e.g., an mRNA) sequence complementary to that DNA, in which each "T" in the DNA sequence is replaced with "U". In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence having 80% or more identity to the nucleotide sequence set forth in SEQ ID NO:118, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably a nucleotide sequence having 98% or more identity.

[0094] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably 98% or 99% or more identity, to the nucleotide sequence set forth in SEQ ID NO: 160. In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably 98% or 99% or more identity, to the nucleotide sequence set forth in SEQ ID NO: 162. In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence having 80% or more identity to the nucleotide sequence set forth in SEQ ID NO:165, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably a nucleotide sequence having 98% or more identity. In some preferred embodiments, the nucleic acid molecule is an in vitro synthesized mRNA. In some preferred embodiments, the in vitro synthesized mRNA has modifications and may include one or more modifications selected from 5' UTR, 5' UTR, polyA tail, 5'-capping, and nucleotides having one or more modifications in the coding region. One or more modified nucleotides in the nucleotide sequence.

[0095] i) Modification of nucleic acid molecules (e.g., mRNA) Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including, but not limited to, untranslated regions (UTRs) at their 5'-ends (5' UTRs) and / or UTRs at their 3'-ends (3' UTRs), as well as other structural features such as 5'-cap structures and 3'-polyA tails. Both 5' UTRs and 3' UTRs are usually transcribed from genomic DNA and are part of the premature mRNA. The characteristic structures of mature mRNAs (e.g., 5'-cap structures and 3'-polyA tails) are usually added to the transcribed (premature) mRNA during mRNA processing. "5' UTR" refers to the region located immediately upstream (i.e., 5') of the start codon of an mRNA (i.e., the first codon of an mRNA transcript translated by a ribosome), which does not code for a polypeptide. In generating an RNA transcript, the 5' UTR may contain a promoter sequence. This promoter sequence is well known in the art. It should be understood that such promoter sequences are not present in the nucleic acid molecules of the present disclosure. A "3' UTR" is a region located immediately downstream (ie, 3') of an mRNA termination codon (ie, a codon in an mRNA transcript that directs translation termination) and that does not encode a polypeptide.

[0096] A "poly-A tail" is a downstream region of an mRNA, e.g., immediately downstream (i.e., 3') of the 3' UTR, that contains multiple consecutive adenosine monophosphates. A poly-A tail may contain 10 to 300 adenosine monophosphates. For example, a poly-A tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly-A tail contains 50 to 250 adenosine monophosphates. In relevant biological environments (e.g., within a cell or in vivo), the function of the polyA tail is to protect the mRNA from accelerated enzymatic degradation (e.g., accelerated enzymatic degradation in the cytoplasm) and to contribute to transcription termination and / or transport and translation of the mRNA from the cell nucleus. The 3'-polyA tail is an adenine nucleotide that is usually added to the 3'-end of the transcribed mRNA and can contain about 400 adenine nucleotides. The length of the 3'-polyA tail can affect the stability of the mRNA molecule itself. 5'-capping of polynucleotides may be completed simultaneously with the in vitro transcription reaction, using the following chemical RNA cap analogs according to manufacturer's protocols to generate a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLaboratory, Ipswich, MA). 5'-capping of modified RNAs may be completed post-transcriptionally using cowpox virus capping enzyme to generate the "cap 0" structure: m7G(5')ppp(5')G (New England BioLaboratory, Ipswich, MA). Cap 1 structure: m7G(5')ppp(5')G-2'-O-methyl can be generated using cowpox virus capping enzyme and a 2'-O methyltransferase. Cap 2 structure can be generated from Cap 1 structure, followed by 2'-O-methylation of the 5'-antepenultimate nucleotide with a 2'-O methyltransferase. Cap 3 structure can be generated from Cap 2 structure, followed by 2'-O-methylation of the 5'-preantepenultimate nucleotide with a 2'-O methyltransferase. The enzymes can be recombinant enzymes. An mRNA molecule containing at least one of the above stabilizing elements can significantly increase the expression level of the protein it encodes, and when two or more of the above stabilizing elements are used, the expression level of the protein exhibits a synergistic effect compared to mRNAs using a single stabilizing element.

[0097] In some embodiments, the mRNAs described in this disclosure have one or more AU-rich sequences deleted. AU-rich sequences, also called AURES, are destabilizing sequences found in the 3' UTR. In some embodiments, the mRNA described herein further comprises an open reading frame (ORF) encoding a signal peptide. The signal peptide may comprise the 15-60 amino acids at the N-terminus of a protein and is generally required for membrane transport across the secretory pathway, thus controlling the secretory pathway of many proteins into eukaryotes and prokaryotes. The signal peptide may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 amino acids in length. An "open reading frame" is a contiguous stretch of DNA that begins with a start codon (eg, methionine (ATG)) and ends with a stop codon (eg, TAA, TAG, or TGA) that encodes a polypeptide. Signal peptides from heterologous genes are known in the art and can be incorporated into the nucleic acids of the present disclosure after determining the desired properties. In some embodiments, the signal peptide can include any of MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQ ID NO: 148), MDWTWILFLVAAATRVHS (SEQ ID NO: 149), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 150), MLGSNSGQRVVFTILLVAPAYS (SEQ ID NO: 151), MKCLLYLAFLFIGVNCA (SEQ ID NO: 152), MWLVSLAIVTACAGA (SEQ ID NO: 153), and MFVFLVLLPLVSSQC (SEQ ID NO: 154). The nucleic acid molecules provided herein can be synthesized by solid-phase techniques, liquid-phase chemistry, enzymatic ligation and combinations thereof. The nucleic acid molecules after synthesis can be purified and quantified. See specifically WO2021222304, the entire contents of which are incorporated herein by reference.

[0098] 7. Vectors In another aspect, the present disclosure further provides a vector comprising a nucleic acid molecule provided herein, wherein the nucleic acid molecule is operably linked to at least one polynucleotide regulatory element (e.g., a promoter) so as to express a chimeric protein construct encoded by the nucleic acid molecule. In some embodiments, the vector is selected from a plasmid, a nanoplasmid, a cosmid, a viral vector, a minicircle, an RNA vector, or a linear or circular DNA (eg, transposon DNA) or RNA molecule. In some embodiments, the viral vector is selected from a viral vector selected from a retrovirus, a lentiviral vector, an adenovirus, a parvovirus (e.g., adeno-associated virus), an adeno-associated virus (AAV) vector, a coronavirus, a negative strand RNA virus such as an orthomyxovirus (e.g., influenza virus), a rhabdovirus (e.g., rabies and vesicular stomatitis virus), a paramyxovirus (e.g., matchivirus and Sendai virus), a positive strand RNA virus such as a picornavirus and an alphavirus, and a double-stranded DNA virus, including adenovirus, herpesvirus (e.g., herpes simplex virus I and II, Epstein-Barr virus, cytomegalovirus), poxvirus (e.g., cowpox virus, fowlpox, and canarypox), Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, baculovirus, hepatitis virus, and virus-like particles (VLPs).

[0099] In some embodiments, the viral vector is a retroviral vector. In some embodiments, the retrovirus is selected from avian leukoproliferative sarcoma viruses, mammalian C-type, B-type viruses, D-type viruses, the HTLV-BLV collection, lentiviruses, and foamy viruses. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an oncolytic viral vector. An oncolytic viral vector refers to an oncolytic virus-based viral vector into which an exogenous nucleic acid sequence may be inserted. In some embodiments, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or ovine demyelinating leukoencephalitis lentivirus. In some embodiments, the vector is a transposon-based expression vector. A transposon is a DNA sequence that can change its location in a genome. In a transposon system, the nucleic acid molecule encoding the chimeric protein construct provided herein is flanked by terminal repeat sequences that can be recognized by a transposase that mediates transposon movement. A transposase can be co-delivered as a protein and can be encoded in the same vector as the chimeric protein construct or in a separate vector. Non-limiting examples of transposon systems include Sleeping Beauty, Piggyback, Frog Prince, and Prince Charming. In some embodiments, the vectors provided in this disclosure may be combined with other vectors that include a nucleic acid molecule encoding at least one other co-expression moiety. In some preferred embodiments, the vector further comprises a promoter; preferably, the promoter is the EF1α promoter or the CMV promoter. In some preferred embodiments, the chimeric protein constructs provided in the present disclosure may be expressed under the control of the same promoter or different promoters in the same vector as the co-expressed moiety, or may be expressed in multiple vectors.

[0100] 8. Cell In another aspect, the present disclosure further provides engineered cells that express the chimeric protein constructs provided herein or contain the chimeric nucleic acid constructs or vectors provided herein. In some embodiments, the engineered cells co-express the chimeric protein constructs provided in the present disclosure and the co-expression moieties described above. The co-expression moieties and the target protein binding domains of the chimeric protein constructs may be combined to achieve a particular purpose. For example, when the target protein binding domain specifically recognizes a protein expressed in immune cells that may cause immune rejection, the co-expression moieties may be proteins that further suppress immune rejection (e.g., viral ER resident glycoproteins, etc.), proteins that target the tumor microenvironment (e.g., chemokine receptors, etc.), proteins that stimulate immune activity (e.g., cytokines, immune cell lineage proteins CD7, CD5, etc.), and the cells thus produced have reduced immunogenicity (or immune rejection) and / or enhanced immune activity when transplanted into the human body. This is very useful for cellular immunotherapy and other therapeutic cell transplants. Thus, the cells may be immune cells (e.g., T cells), stem cells, kidney cells, pancreatic islet cells, cardiomyocytes, etc. In some embodiments, the cell is an immune cell selected from a T cell, a natural killer (NK) cell, a B cell, a macrophage, a monocyte, a dendritic cell, a neutrophil, or a gamma delta T cell. The T cell may be selected from the group consisting of CD8+ T cells, CD4+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, gamma delta T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes, and regulatory T cells.

[0101] In some embodiments, the engineered cell is a T cell. When expressing the chimeric protein construct and co-expression moiety to obtain a T cell with reduced immunogenicity and / or enhanced immune activity, the T cell may further express a receptor that specifically targets a tumor-associated marker. The receptor may include, but is not limited to, a CAR, a TCR, a NK cell activating receptor module, etc. In some embodiments, the engineered cell is a T cell that expresses a chimeric protein construct provided herein and one or more selected from adenoviral E3-K19, a CAR, an NK cell activating receptor, and a CNK signaling module. In some embodiments, the engineered cell is a T cell that expresses a chimeric protein construct provided herein, together with an NK cell activating receptor and, optionally, a CNK signaling module. In some embodiments, the engineered cell is a T cell that expresses both the chimeric protein construct, adenoviral E3-K19 and a CAR provided in this disclosure. In another aspect, the disclosure further provides a method of producing an engineered cell (e.g., a T cell) described herein comprising introducing into a starting cell a vector described herein under conditions suitable for expressing a nucleic acid molecule described herein.

[0102] Many methods known in the art for producing CAR-T cells can also be applied to produce the engineered cells described herein. For example, Zhang et al., Engineering CAR-T cells, Biomarker Research (2017) 5:22, describes a method for producing CAR-T cells. The methods provided herein can include one or more steps selected from obtaining starting cells, culturing (including proliferation, and optionally activation) starting cells, and genetically modifying the cells. The starting cells can be stem cells, which can be hematopoietic progenitor cells (e.g., T cell progenitors, NK cell progenitors, macrophage progenitors), hematopoietic stem cells (HSCs), CD34+ cells, embryonic stem cells, mesenchymal stem cells, or iPSC cells. The starting cells can also be cells differentiated from stem cells, such as immune cells as described above. The starting cells may be obtained from any source, for example, immune cells (e.g., T cells) isolated from a subject (e.g., a human subject). In some embodiments, the immune cells are obtained from a subject of interest, for example, a subject suspected of having a particular disease or condition, a subject suspected of having a susceptibility to a particular disease or condition, a subject who may experience, has experienced, or has been treated for a particular disease or condition, and the subject may be a healthy volunteer or a healthy donor; the immune cells may be derived from a blood bank. The immune cells may be autologous or allogeneic with respect to the subject of interest. The immune cells may be collected from any location of the subject where they are present, including, but not limited to, blood, umbilical cord blood, spleen, thymus, lymph nodes, pleural effusion, splenic tissue, tumor, and bone marrow. The isolated immune cells may be used directly or may be stored for a period of time, such as by freezing.

[0103] Activation and / or proliferation of immune cells is one of the main steps of immune cell function. In some embodiments, immune cells are activated and expanded before or / and after they are genetically modified. In some embodiments, immune cells are activated and / or expanded in vitro, ex vivo or in vivo. Methods for activating and expanding immune cells have been described in the art and may be used in the methods described herein. For example, T cells may be activated and expanded by contacting a surface with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface. In particular, a T cell population can be stimulated by contacting with, for example, an anti-CD3 antibody or antigen-binding fragment thereof, an anti-CD2 antibody immobilized on the surface, or a protein kinase C activator (e.g., bryostatin) and a calcium ion carrier. To costimulate helper molecules on the surface of T cells, a ligand that binds to the helper molecule is used. For example, a T cell population may be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. Anti-CD3 and anti-CD28 antibodies can be used to stimulate proliferation of CD4+ or CD8+ T cells. In some embodiments, primary and costimulatory signals for T cells can be provided by different protocols.

[0104] Genetically modified cells can be achieved by transducing a nucleic acid molecule encoding a chimeric protein construct provided herein into a substantially homogeneous starting cell. In some embodiments, a retroviral vector (e.g., a lentiviral vector) is used to introduce a nucleic acid molecule provided herein into a starting cell. For example, a nucleic acid molecule provided herein can be cloned into a lentiviral vector and expressed from its endogenous promoter, a lentiviral long terminal repeat sequence, or a promoter specific to the target cell type of interest. Common delivery methods for delivering viral vectors include, but are not limited to, electroporation, microinjection, gene gun, and magnetic transfection. Genetically modified cells can also be achieved by delivering nucleic acid molecules in LNPs. See specifically the contents of the above subsection "6. Nucleic acid molecules". Other non-viral methods for delivering nucleic acids include in vitro transfection using calcium phosphate, DEAE glucan, electroporation and protoplast fusion. Transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases or TALE nucleases, CRISPR) can be used to achieve genetic modification to starting cells to obtain the engineered cells described in this disclosure.

[0105] In some embodiments, the engineered cells provided herein are generated by transfecting a starting cell with a nucleic acid molecule encoding a chimeric protein construct provided herein prior to administration. In some embodiments, the engineered cells provided herein may be generated by transfecting an immune cell with a nucleic acid molecule encoding a chimeric protein construct provided herein, for example, via a viral vector. The engineered cells provided herein exhibit reduced expression of immunogenic molecules (e.g., TCR, HLA, etc.) and / or reduced expression of immunosuppressive molecules (e.g., PD-1, etc.) and / or increased expression of tumor targeting receptors (e.g., CAR, engineered TCR, CNK receptor, etc.) on the cell surface. In another aspect, the disclosure further provides a cell population generated ex vivo by the above method. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cell population expresses detectable levels of an expression marker peptide provided herein (e.g., an EGFR truncation (EGFRt) cleavably linked to a chimeric protein construct described herein). The expression level of the EGFR truncation can represent the expression level of the chimeric protein construct described herein in the cell population.

[0106] 9. Pharmaceutical Compositions and Kits In another aspect, the present disclosure further provides a pharmaceutical composition or kit comprising (i) a chimeric protein construct, a nucleic acid molecule, a vector, or a cell population as described herein, and (ii) a pharma- ceutically acceptable vehicle. The term "pharmaceutically acceptable vehicle" refers to any and all solvents, dispersion media, coating layers, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that may facilitate the storage and administration of the chimeric protein construct, nucleic acid molecule, expression vector, and / or cell population provided herein. The pharma- ceutically acceptable vehicle may contain any suitable component, such as, but not limited to, an aqueous saline solution, a liposome, a polymeric excipient, a colloid, or a vector particle. In some embodiments, the pharma- ceutically acceptable medium is an aqueous salt solution capable of dissolving or dispersing the chimeric protein constructs, nucleic acid molecules, expression vectors and / or cell populations provided herein.Specific examples of aqueous salt solutions include, but are not limited to, buffered salt solutions, sucrose solutions, saline, acetate buffers, phosphate buffers, citrate buffers, bicarbonate buffers, salt solutions and polysorbate solutions.

[0107] i) Nucleic acid pharmaceutical composition In some embodiments, the pharma- ceutically acceptable vehicle is a liposome. Liposomes are unilamellar or multilamellar vesicles with a membrane formed of a lipophilic material and an aqueous interior. The nucleic acid molecules and / or vectors provided in the present disclosure may be encapsulated in the aqueous interior of the liposome. Exemplary liposomes include, but are not limited to, 3[N-(N',N'-dimethylaminoethane)aminoformyl]cholesterol (DC-Chlo)-based liposomes, N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA)-based liposomes, and 1,2-dioleoyloxy-3-trimethylethane (DOTAP)-based liposomes. Methods for making liposomes and encapsulating nucleic acid molecules and / or vectors in liposomes are known in the art (see, for example, DD Lasic et al, Liposomes in gene delivery, published by CRC Press, 1997). In some embodiments, the pharma- ceutically acceptable vehicle is a polymeric excipient, including, but not limited to, a microsphere, a microcapsule, a polymeric micelle, and a dendritic polymer. The nucleic acid molecules and / or vectors provided herein can be encapsulated, attached, or coated into polymeric moieties by methods known in the art (see, e.g., W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; U.S. Patent 6025337; Advanced Drug Delivery Reviews, 57(15): 2177-2202 (2005)).

[0108] In some embodiments, the pharma- ceutically acceptable vehicle is a colloid or carrier particle, such as gold colloids, gold nanoparticles, silica nanoparticles, and multi-segment nanorods. The nucleic acid molecules and / or vectors provided herein can be coated, attached, or bound to the carrier by any suitable means known in the art (see, e.g., M. Sullivan et al., Gene Therapy, 10: 1882-1890 (2003), C. McIntosh et al., J. Am. Chem. Soc., 123 (31): 7626-7629 (2001), D. Luo et al., Nature Biotechnology, 18: 893 - 895 (2000), and A. Salem et al., Nature Materials, 2: 668 - 671 (2003)). In some embodiments, the pharmaceutical composition further comprises additives, including but not limited to stabilizers, preservatives, and transfection facilitators that contribute to cellular uptake of the pharmaceutical. Suitable stabilizers include but are not limited to sodium glutamate, glycine, EDTA, and albumin. Suitable preservatives include but are not limited to 2-phenoxyethanol, sodium benzoate, potassium sorbate, methyl hydroxybenzoate, phenols, thimerosal, and antibiotics. Suitable transfection facilitators include but are not limited to calcium ions.

[0109] The pharmaceutical compositions provided herein can be administered by any suitable route known in the art, including, but not limited to, parenteral, oral, enteral, buccal, nasal, topical, rectal, vaginal, intramuscular, intranasal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary and subcutaneous routes of administration. The pharmaceutical compositions provided herein can be administered to a subject in the form of a formulation or formulation suitable for each administration route. Formulations suitable for administering the pharmaceutical composition include, but are not limited to, solutions, dispersions, emulsions, powders, suspensions, aerosols, sprays, nasal drops, liposomal formulations, patches, implants and suppositories. The formulations may be conveniently present in unit dosage form and may be prepared by any method known in the pharmaceutical art. The method of preparing these formulations or pharmaceutical compositions includes providing the nucleic acid molecules described in this disclosure with one or more pharma- ceutically acceptable vehicles and, optionally, one or more excipients. Methods for making such formulations are described, for example, in Remington's Pharmaceutical Sciences (Remington:The Science and Practice of Pharmacy, 19th ed., A. R. Gennaro (ed), Mack Publishing Co., N.J., 1995; R. Stribling et al., Proc. Natl. Acad. Sci. USA, 89:11277-11281 (1992); A. Barnes et al., Current Opinion in Molecular Therapeutics 2000 2:87-93(2000); T. W. Kim et al., The Journal of Gene Medicine, 7(6): 749-758(2005); and S. F. Jia et al., Clinical Cancer Research, 9:3462 (2003); A. Shahiwala et al., Recent patents on drug delivery and formulation, 1:1-9 (2007), the entireties of which are incorporated herein by reference.

[0110] In some embodiments, the nucleic acid molecule (e.g., mRNA) can be delivered by physical, biological, or chemical methods (see, e.g., S. Guan, J. Rosenecker, Gene Ther. 2017, 24, 133.). Physical methods include, but are not limited to, delivery by gene gun (e.g., Au-particle gene gun), electroporation, sonoporation, etc. (see, e.g., Kutzler et al., (2008) DNA vaccines: Ready for prime time? Nat Rev Genet 9: 776-788; Geall et al., Nonviral delivery of self -amplifying RNA vaccines. Proc Natl Acad Sci US A. Sep. 4, 2012;109(36 ):14604-9.). Biological methods include, but are not limited to, delivery by viral vectors (eg, retroviral vectors, adenoviral vectors, adeno-associated viral vectors). Chemical methods include, but are not limited to, natural proteins / glycans, polymers, and lipids. Exemplary natural proteins / glycans include protamine and chitosan (see, for example, AE Skold et al., Cancer Immunol. Immunother. 2015, 64, 1461; US ​​Kumar et al. ACS Nano 2021, 11, 17582). Exemplary polymers include polyethyleneimine (PEI) (e.g., linear PEI, branched PEI, and dendritic PEI), poly(β-amino ester) (PBAE) (see, for example, K. Singha et al., Nucleic Acid Ther. 2011, 21, 133; AA Eltoukhy et al., Biomaterials 2012, 33, 3594).

[0111] Exemplary lipids include cationic lipids such as 1,2-diocta-decenyl-3-trimethylammoniumpropane (DOTMA) and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) (see, e.g., X. Hou et al., Nat. Rev. Mater. 2021, 10, 1078.); liposomes formed with DOTMA, DOTAP and DOPE (dioleoyl-sn-glycero-3-phosphoethanolamine, a colipid) that can self-assemble with mRNA to form colloidally stabilized nanoparticles (see, e.g., LM Kranz et al., Nature 2016, 534, 396.). An exemplary lipid may be an ionizable lipid. Ionizable lipids (pKa 6.5-6.9) are alternative lipid materials that are neutral at physiological pH but positively charged in an acidic environment due to protonation of free amines (see, for example, SC Semple et al., Nat. Biotechnol. 2010, 28, 172). After intracellular translocation, the nanoparticles formed with ionizable lipids are encapsulated in endosomes. Then, as the pH value in endosomes and lysosomes continues to decrease, the ionizable lipids gain protons and become ionized, promoting the fusion of lipid nanoparticles (LNPs) with the endosomal membrane, and finally releasing the mRNA loaded on the lipid nanoparticles into the cytoplasm (see, for example, L. Miao et al., Mol. Cancer 2021, 20, 41.).

[0112] Ionizable lipids can form lipid nanoparticle formulations with cholesterol, auxiliary lipids and polyethylene glycolated lipids (i.e., PEGylated lipids). Cholesterol is a naturally rigid and hydrophobic lipid that can maintain the structure and stability of lipid nanoparticles. Cholesterol can also promote the fusion of mRNA-loaded lipid nanoparticles (i.e., mRNA nanoparticles) with endosomal membranes. Auxiliary lipids (e.g., zwitterionic lipids DOPE, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) are widely used to promote cell membrane permeation and endosomal membrane escape (see, e.g., N. Chaudhary et al., Nat. Rev. Drug Discovery 2021, 20, 817.). PEGylated lipids consist of PEG and anchor lipids. The hydrophilic PEG is mainly distributed on the surface of the mRNA complex, while the hydrophobic region is embedded inside the lipid bilayer. The introduction of PEGylated lipids can not only extend the half-life of lipid nanoparticles, but also adjust the particle size by changing the molecular weight of the PEG chain. Typically, the molecular weight and lipid tail length ranges from 350 to 3000 Da and 10 to 18 carbons, respectively (see, for example, N. Chaudhary et al., Nat. Rev. Drug Discovery 2021, 20, 817.).

[0113] Over the past few decades, researchers have developed a number of new synthetic methods, including DLin-MC3-DMA, SM-102, TT3, C12-200, 306O i10and ALC-0315, have developed a large library of ionizable lipids for mRNA delivery (see, e.g., M. Yanez Arteta et al., Proc. Natl. Acad. Sci. USA 2018, 115, E3351; R. Verbeke et al., Controlled Release 2021, 333, 511; B. Li et al., Nano Lett. 2015, 15, 8099; KA Hajj et al., Nano Lett. 2020, 20, 5167; KA Hajj et al., Small 2019, 15, 1805097; AB Vogel et al., Nature 2021, 592, 283). Some of these have achieved notable success in clinical applications. A typical example is DLin-MC3-DMA, a key component of Onpattro for siRNA delivery approved by the US Food and Drug Administration (FDA) (see, for example, A. Akinc et al., Nat. Nanotechnol. 2019, 14, 1084). DLin-MC3-DMA is also widely used for mRNA delivery, including protein and peptide replacement, gene editing and antiviral infection (see, for example, RS Riley et al., Sci. Adv. 2021, 7, eaba1028.). Two “star molecules”, SM-102 and ALC-0315, were approved by the FDA as key components of the BNT162b and mRNA-1273 vaccines, respectively, to prevent COVID-19 (see, for example, X. Hou et al., Nat. Rev. Mater. 2021, 10, 1078.).

[0114] An ideal lipid-based mRNA vector should meet the following criteria: 1) naked mRNA can form a stable complex that protects the mRNA from degradation; 2) four key modules (ionizable lipid, cholesterol, auxiliary lipid and PEGylated lipid) are added to stabilize the mRNA complex; 3) the lipid nanoparticle components are protonated to cause membrane instability and facilitate endosomal escape of the mRNA complex; and 4) all lipid materials are biodegradable and do not cause any damage to the patient. The following points should be considered when optimizing lipid delivery platforms: 1) Degradation ability of ionizable lipids: The lipid backbone structure can be improved by introducing alkyne and ester groups into the lipid tail to facilitate lipid removal and reduce toxicity; 2) Immunogenicity of lipid nanoparticles: Heterocyclic lipids in lipid nanoparticles improve the efficiency of mRNA vaccines by activating the stimulator of interferon genes (STING) pathway in dendritic cells (DCs) (see, for example, L. Miao et al., Nat. Biotechnol. 2019, 37, 1174.); 3) Stability of lipid nanoparticles: Several promising strategies, such as pKa optimization, excipient introduction, and mRNA modification, are expected to improve the stability of mRNA vaccines.

[0115] Lipid nanoparticles can be produced using ingredients, compositions and methods known in the art, e.g., PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016 / 000129, PCT / US2016 / 014280, PCT / US2016 / 014 280, PCT / US2017 / 038426, PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 052117, PCT / US2012 / 069610, PCT / US2017 / 027492, CT / US2016 / 059575, and PCT / US2016 / 069491, the contents of which are incorporated herein by reference in their entireties.

[0116] ii) Oncolytic viruses and pharmaceutical compositions thereof In some embodiments, the present application provides an oncolytic virus capable of expressing the chimeric protein constructs described herein. As used herein, "oncolytic virus" refers to any virus that can infect, replicate within, and lyse tumor cells. In some embodiments, the oncolytic virus can further spread to other tumor cells in successive replication cycles. Oncolytic viruses may be derived from a number of viruses, including, but not limited to, cowpox virus, adenovirus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), Lassa virus (LASV), Newcastle disease virus (NDV), and variants of these viruses. In some embodiments, the present application provides an oncolytic virus comprising a nucleic acid molecule provided herein, wherein the nucleic acid molecule is operably linked to at least one polynucleotide regulatory element (e.g., a promoter) so as to express a chimeric protein construct encoded by the nucleic acid molecule. In some embodiments, the oncolytic viruses described herein can infect virtually any cell type. In some embodiments, the oncolytic viruses can be replication-selective. Replication-selective oncolytic viruses replicate more in tumor cells than in non-tumor cells.

[0117] It should be understood that oncolytic viruses can be replication-selective when viral replication is under the control of gene expression regulatory factors, such as an enhancer / promoter region derived from the 5' side of the albumin gene (see, e.g., Miyatake et al., 1997, J. Virol. 71:5124-5132). In some embodiments, the oncolytic virus can have a tumor cell-specific promoter or tumor cell-specific transcriptional regulatory sequences in its genome. "Tumor cell specific" refers to promoters or transcriptional term sequences that are normally present at higher levels in the target tumor cells than in normal cells. In this way, the oncolytic virus can be endowed with an enhanced level of tumor cell specificity. For example, the HSV main transcription unit can be placed under the transcriptional control of the tumor growth factor-β (TGF-β) promoter by operably linking the HSV gene to the TGF-β promoter. Certain tumor cells are known to overexpress TGF-β more than non-tumor cells of the same type. Thus, an oncolytic virus whose replication is under the transcriptional control of the TGF-β promoter is replication-selective because it can replicate better in certain tumor cells than in non-tumor cells of the same type. Similar replication-selective oncolytic viruses can be made using any gene expression regulator known to cause selective overexpression in affected cells. Replication-selective oncolytic viruses can be, for example, HSV-1 mutants in which the gene encoding ICP34.5 has been mutated or deleted. Oncolytic viruses can further include other modifications in their genome. For example, oncolytic viruses can include additional DNA inserted into the UL44 gene. This insertion can result in functional inactivation of the UL44 gene and a resulting truncated phenotype. Alternatively, it may be inserted into an already inactivated gene or may replace a deleted gene.

[0118] In some embodiments, the oncolytic virus is modified to place a nucleic acid sequence encoding a chimeric protein construct described herein under the control of a tumor cell specific promoter. In some embodiments, the oncolytic virus is modified to place a gene encoding at least one protein required for viral replication under the control of a tumor cell specific promoter. In some embodiments, the oncolytic viral vector can express a chimeric protein construct described herein. The targeting domain of the chimeric protein construct can recognize and bind to a tumor protein. The tumor protein can be a protein highly expressed in tumor cells (e.g., c-Myc, Bcl-2, Bcl-xL, Bcl-w, KRAS), a protein expressed in tumor cells that promotes tumor cell growth (e.g., estrogen receptor, androgen receptor, Her2, VEGF, VEGFR, PDGFRβ, EGFR, EGFR mutant), or a protein that promotes tumor cell evasion from normal immune responses (e.g., PD-L1, TGF-β1).

[0119] In some embodiments, the targeting domain of the chimeric protein construct can bind to Bcl-2. Any targeting domain that can target and bind to bcl-2 can be used. For example, but not limited to, a nanobody sequence against the highly conservative and homologous BH1, BH2 motif of Bcl-2 anti-apoptotic family member protein is developed as a targeting domain, and an ER-TPD element that targets Bcl-2 family protein is constructed and inserted behind a strong promoter of an oncolytic virus by cloning. After specifically infecting tumor cells, the oncolytic virus can grow in large quantities and express the ER-TPD element of the Bcl-2 family protein, thereby achieving the degradation of Bcl-2 anti-apoptotic member protein in cells and directly causing apoptosis of tumor cells. In some embodiments, the targeting domain of the chimeric protein construct can bind to VEGFR. Any targeting domain that can target and bind to VEGFR can be used. For example, but not limited to, the recombinant scFv using the heavy and light chains of VEGFR antibodies (anti-VEGF-A antibody Bevacizumab and anti-VEGFR2 antibody Ramucizumab) is connected in series with the ER-TPD element to construct the ER-TPD element that targets VEGFR, and then inserted behind the strong promoter of oncolytic virus by cloning, and after specifically infecting tumor cells, the oncolytic virus grows in large quantities and expresses the VEGFR ER-TPD element, thereby directly inhibiting the expression of VEGF in tumor cells, and the tumor cells are growth-suppressed due to lack of sufficient nutrients, and apoptosis is induced.

[0120] In some embodiments, the targeting domain of the chimeric protein construct can bind to TGF-β1 and PD-L1. Any targeting domain capable of targeting and binding to bcl-2 can be used. For example, but not limited to, a recombinant scFv using the heavy and light chains of TGF-β1 and PD-L1 antibodies can be connected in series with an ER-TPD element to construct an ER-TPD element targeting TGF-β1 and PD-L1, which can be inserted behind a strong promoter of an oncolytic virus by cloning and then specifically infected into tumor cells, whereby the oncolytic virus can proliferate in large quantities and express the TGF-β1 and PD-L1 ER-TPD elements, thereby directly inhibiting and degrading the expression of TGF-β1 and PD-L1 in tumor cells, destroying the immunosuppressive microenvironment of the tumor, and achieving the therapeutic effect of the tumor. Without being limited by theory, it is believed that the chimeric protein constructs of the present application have unique advantages when expressed in tumor cells by oncolytic viral vectors. The chimeric protein constructs of the present application can promote apoptosis or cell death of tumor cells by targeting and degrading proteins in tumor cells that aid in tumor cell growth or immune evasion or resist apoptosis. In addition, oncolytic viral vectors can specifically replicate in tumor cells and promote tumor cell lysis through viral replication and degradation. By incorporating two different tumor killing means into oncolytic viral vectors, a synergistic killing effect can be achieved in one pharmaceutical form.

[0121] In another aspect, the present disclosure further provides a pharmaceutical composition comprising an oncolytic virus provided herein and a pharma- ceutically acceptable vehicle, which in some embodiments includes any and all delivery media, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorbents, and the like, suitable for administration to a mammalian, particularly a human, subject. In some embodiments, the oncolytic virus or pharmaceutical composition thereof described herein is prepared for intravenous or intratumoral administration. In some embodiments, the oncolytic virus or pharmaceutical composition thereof may be in a human or animal compatible solvent or diluent. The solvent or diluent is preferably isotonic, hypotonic or weakly hypotonic and has a relatively low ionic strength. Typical examples include sterile water, physiological saline (e.g., sodium chloride), Ringer's solution, glucose, trehalose or lactose solution, Hank's solution and other physiologically balanced salt solutions. In one embodiment, the oncolytic virus or pharmaceutical composition thereof described herein may contain a buffer. Suitable buffers include, but are not limited to, phosphate buffer (e.g., PBS), bicarbonate buffer, and / or Tris buffer capable of maintaining a physiological pH or a slightly alkaline pH (e.g., about pH 7 to about pH 9). In one embodiment, the oncolytic virus composition of the present invention can be formulated to improve its stability, particularly under manufacturing conditions and long term (i.e., at least 6 months, preferably at least 2 years) frozen (e.g., -70°C, -20°C), refrigerated (e.g., 4°C), or room temperature storage conditions. Various virus formulations known in the art are in frozen liquid form or lyophilized form (e.g., WO98 / 02522 and WO2008 / 114021, etc.). Solid (e.g., dry powder or lyophilized) compositions can be obtained by processes including vacuum drying and lyophilization. By way of illustration, buffered formulations with added NaCl and sugars are particularly suitable for virus storage.

[0122] iii) Cellular medicine composition In another aspect, the present disclosure further provides a cellular medicine composition comprising the engineered cell or cell population provided herein and a pharma- ceutically acceptable vehicle.Exemplary vehicles include buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or glucan, mannitol, proteins, peptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.In one aspect, the cellular medicine composition of the present invention is prepared for intravenous administration.

[0123] III. Application of TPD In some aspects, the present disclosure further provides for the use of a chimeric protein construct, nucleic acid molecule, or vector described herein in the generation of therapeutic cells. In some aspects, the present disclosure further provides for the use of a chimeric protein construct, nucleic acid molecule, or vector described herein in the treatment of disease. In some aspects, the present disclosure further provides for the use of a cell expressing a chimeric protein construct, nucleic acid molecule, or vector described herein in the treatment of disease.

[0124] 1. Cell transplantation In certain aspects, the present disclosure further provides the use of the chimeric protein constructs, nucleic acid molecules, or vectors described herein in the generation of transplanted cells (e.g., allogeneic cell transplantation). In some embodiments, the transplanted cells are immune cells (e.g., T cells), stem cells (and cells differentiated therefrom), kidney cells, pancreatic islet cells, cardiomyocytes, etc. In some embodiments, the transplanted cells can be autologous or allogeneic cells. In some aspects, the present application further provides a transplanted cell expressing a chimeric protein construct, nucleic acid molecule, or vector as described above, wherein the chimeric protein construct expresses a chimeric protein construct described herein, the chimeric protein construct comprising an ERAD machinery protein binding domain and a targeting domain, the targeting domain comprising a domain that specifically targets or binds to a target protein, the target protein being a transplant rejection-associated protein. In some embodiments, the chimeric protein construct further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain described herein. Any of the protein degradation pathway member binding domains described herein can be used.

[0125] In some embodiments, the transplant rejection-associated proteins include antigen-presenting molecules (e.g., MHC class I molecules, MHC class II molecules, MICA / B molecules, etc.), antigen recognition molecules (e.g., TCR, CD123, NKG2D, etc.), immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), and the like. In some embodiments, the transplant rejection-associated protein comprises an immunogenic protein (eg, HLA (HLA alpha / beta), TCR (alphabetaTCR), NKG2D (natural killer cell family 2 member D) ligand, etc.). The present disclosure further provides transplanted cells and methods of treatment thereof that can overcome problems in conventional cell transplantation and cell therapy, such as sources of individualized transplanted and therapeutic cells, impossibility of standardizing and scaling up production, and reduced therapeutic efficiency.

[0126] The immune system is highly plastic and has an almost limitless ability to detect invading viruses, bacteria, exogenous cells, and disease cells. This remarkable ability in immunosurveillance is achieved primarily through humoral and cellular immunity, which includes two key molecular structures: immunoglobulins and the T cell receptor (TCR). The TCR is the defining structure of T cells and is a transmembrane heterodimer consisting of α and β chains or δ and γ chains linked by disulfide bonds. In these chains, the complementarity determining regions (CDRs) determine which antigens the TCR will bind. In the TCR, the TCRα and TCRβ subunits (or TCRγ and TCRδ in γδ T cells) are responsible for recognizing major histocompatibility complex (MHC) / antigen ligands. The human MHC class I gene region contains alleles of HLA-A, B, C sites and codes for typical class I antigens (molecules) such as HLA-A, B and C antigens, called HLA-A, HLA-B and HLA-C. These antigen molecules are present on the surface of all body cells and bind to intracellular protein epitope peptides for recognition by the immune system. When cells produce mutant proteins or foreign bacteria or viruses invade, the cells present these mutant proteins or foreign protein antigen epitopes, and immune cells recognize them and perform immune attack and killing to eliminate diseased cells, pathogens and viruses that have invaded the cells. α / β T lymphocytes recognize peptide-MHC ligands through a multimeric protein assembly called the αβ T cell antigen receptor (TCR) CD3 complex. This structure consists of a variable αβTCR dimer that binds antigen and three invariant dimers (CD3γε, δε and ζζ) that are involved in TCR.CD3 surface trafficking, stabilization and signal transduction. The αβ T cell receptor (αβTCR) is expressed on most (approximately 95%) T cells and plays a key role in T cell activation by recognizing major histocompatibility complex (MHC)-anchored antigens. Therefore, TCR-mediated T cell activation is a key step in the mechanism of graft-versus-host disease (GVHD) pathogenesis in allogeneic hematopoietic cell transplantation (allo-HCT) and allogeneic CAR-T cell therapy processes.

[0127] The human leukocyte antigen (HLA) system or complex is a group of related proteins encoded by the human major histocompatibility complex (MHC) gene complex. These cell surface proteins are involved in regulating the immune system. In a therapeutic transplantation setting, an "HLA mismatch" occurs when the donor HLA on the allogeneic graft is different from that of the recipient. HLA mismatch can lead to activation of alloreactive T cells, causing acute cellular rejection (ACR) within 6 months of transplantation. Mismatched donor HLA antigens are also targets for the development of novel donor-specific HLA antibodies (dnDSA), which exert an enhancing effect in acute and chronic transplanted cell (e.g., T cell) rejection. Therefore, to generate universal transplant cells (e.g., T cells) for safe allogeneic transfusion and therapeutic purposes, genetic disruption of TCR is recommended to effectively block graft-versus-host disease (GVHD). There is also a need to inhibit HLA expression on allogeneic cells (eg, T cells) to reduce the rejection response of the recipient immune system to allogeneic T cells TCRαβ and / or HLA class I. In some embodiments, the transplanted cells expressing the above-described chimeric protein constructs, nucleic acid molecules or vectors have reduced immunogenicity.

[0128] In some embodiments, the transplanted cells having reduced immunogenicity (or high compatibility) described herein are T cells, and the TPD chimeric protein construct they express comprises an scFv that specifically recognizes TCR and / or HLA and the transmembrane domain or functional variant thereof of HCMV glycoproteins US2 and / or US11, and an endoplasmic reticulum resident domain or functional variant thereof. The ERAD machinery protein binding domain comprises an amino acid sequence set forth in SEQ ID NO: 76 or SEQ ID NO: 82, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identity thereto. In some embodiments, the transplanted cells with reduced immunogenicity are T cells, which express an ER-TPD chimeric protein construct comprising an scFv that specifically recognizes a TCR and a transmembrane domain or a functional variant of adenovirus E3-K19, and an endoplasmic reticulum resident domain or a functional variant thereof. In some embodiments, the transplanted cells with reduced immunogenicity are T cells, which express an ER-TPD chimeric protein construct comprising a target protein binding domain and an ERAD machinery protein binding domain, wherein the target protein binding domain comprises six CDRs comprised in the amino acid sequence set forth in SEQ ID NO: 116, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identity thereto. The ERAD machinery protein binding domain comprises the amino acid sequence set forth in SEQ ID NO: 60, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identity thereto.

[0129] In some embodiments, the ER-TPD chimeric protein construct expressed by the T cells with reduced immunogenicity further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome) binding domain that may be linked to the ERAD machinery protein binding domain. In some embodiments, the ER-TPD chimeric protein construct expressed by the T cells with reduced immunogenicity further comprises an E3 ubiquitin ligase binding domain having the amino acid sequence set forth in SEQ ID NO: 147. In some embodiments, the transplanted cells (eg, T cells) with reduced immunogenicity further express a co-expression moiety as described above. For example, to reduce the immunogenicity or antigen presentation of cells, proteins capable of degrading or reducing MHC class I or class II molecules in transplanted cells (e.g., T cells) with reduced immunogenicity may be expressed as co-expression moieties (e.g., intact viral ER resident glycoproteins such as, but not limited to, HCMV US2, US3, US11, US10, adenovirus E3-K19, HCMV US6, and HSV ICP47). Effective downregulation of TCR significantly inhibits TCR-mediated immune attack and reduces GVHD during allogeneic transfusion of T cells. Glycoproteins containing native viral ER resident can further inhibit MHC molecules, preventing peptide presentation to receptor CD8+ T cells and inhibiting immune recognition by allogeneic T cells. Therefore, the above-mentioned highly compatible T cells are also called "autologous universal T cells (UT)" or "universal T cells (UT)", which can achieve the therapeutic purpose of allogeneic transfusion and can improve the compatibility and long-term persistence of allogeneic T cells after transfusion.

[0130] In another aspect, the disclosure provides a method of reducing a subject's immune response to a cell transplant, the method comprising administering to the subject a therapeutically effective amount of the transplanted cells having the reduced immunogenicity, thereby reducing the subject's immune response to the transplant or the modified cells. In some embodiments, the transplanted cells intracellularly express a TPD-chimeric protein construct as described herein, the TPD-chimeric protein construct comprising a domain targeting an immunogenic protein (e.g., HLA (HLA alpha / beta), TCR (alphabetaTCR), NKG2D (natural killer cell family 2 member D) ligand, etc.), an ERAD machinery protein binding domain as described above, and optionally a protein degradation pathway member binding domain.

[0131] 2. Cell therapy Cell therapy is a method of modifying cells (e.g., immune cells) by genetic engineering to present tumor-associated antigens or express receptors that specifically recognize diseased cells, and then exponentially growing them in vitro. The cells are then injected into the patient's body to activate the immune system in the body to attack tumor cells or directly recognize and kill diseased cells. In 2012, CAR-T cells targeting CD19 achieved targeted removal of tumor cells in B-cell leukemia patients for the first time in the history of human medicine, becoming another treatment technique that can completely treat leukemia following bone marrow stem cell transplantation treatment techniques, and starting a new era of precision medicine cell therapy. This technology is expected to be applied to the treatment of various blood tumors and solid tumors. However, the clinical treatment effect of conventional CAR-T on solid tumors is poor, for the following reasons. (1) The killing function of CAR-T depends largely on the recognition of tumor-associated antigens (TAA) by the CAR structure, but due to the heterogeneity of solid tumors, the expression of target proteins on the surface of tumor cells varies greatly; conventional single-targeting CAR-T cannot completely recognize and kill all tumor cells, which leads to tumor immune evasion and recurrence / metastasis; (2) The direct immunosuppressive effect of solid tumors, the expression of PD-L1, B3H7, etc., directly inhibits the activation of T cells; (3) There are many immunosuppressive cells such as TAM, Treg, MDSC, etc. in the immunosuppressive microenvironment of solid tumors, and the design of conventional CAR-T makes it difficult to break through the constraints imposed by the immunosuppressive cells inside the tumor; (4) Conventional CAR-T is an individualized cell therapy, and the source of T cells is the patient himself. When a patient receives a large number of radiation therapy, chemotherapy, etc., the function of the immune system is impaired, and a sufficient amount of T cells cannot be isolated from the patient's peripheral blood, and even if they are isolated and modified, the proliferation and killing functions of T cells are weak, making it difficult to exert a therapeutic effect. The transplanted cells (e.g., T cells) provided herein can overcome problems faced by conventional cell therapy, such as the difference in tumor targeting, the inhibitory environment of tumor immunity, and cell sources for personalized therapy.

[0132] For cell therapy purposes, the transplanted cells (e.g., T cells, and even universal T cells with deficient TCRαβ and MHC molecule surface expression) may further be engineered to have reduced immunogenicity, e.g., to express the co-expression moieties described above to achieve therapeutic purposes. For example, a binding domain or molecule that recognizes and binds to a disease-associated target protein, such as a chimeric antigen receptor (CAR), an engineered TCR, a CNK receptor provided herein, or an antiviral protein binding domain, may be expressed in the transplanted cells (e.g., T cells) having the above-mentioned reduced immunogenicity so that the transplanted cells (e.g., T cells) can specifically act on a disease-associated target protein or cells expressing the disease-associated target protein. In some embodiments, the transplanted cells (e.g., T cells) having reduced immunogenicity may express a molecule (e.g., a chemokine receptor or a cytokine receptor) that can mediate migration of the transplanted disease to the disease microenvironment. Also, for example, the transplanted cells (e.g., T cells) having the reduced immunogenicity may be caused to express an immunostimulatory molecule so that the transplanted cells (e.g., T cells) have additional immunostimulatory activity.

[0133] In another aspect, the present application further provides that by introducing NK elements, particularly optimized recombinant NK elements, into the transplanted cells (e.g., UT cells) provided by the present application, T cells can effectively and broadly recognize all virus-infected cells and tumor cells in the same way as NK cells, and optionally, by further optimizing the transduction elements, CNK-T cells can effectively activate and kill tumor cells. NK targets include family member proteins such as MICA, MICB and ULBP1-6, which are widely expressed in various tumor cells and cover each stage of tumor progression, so that CNK technology can effectively solve the off-target effect of a single CAR-T and eliminate the possibility of tumor immune evasion. In addition, the chimeric adaptor introduced into the design of CNK can effectively transmit and amplify NK signals, overcome the constraints of immune checkpoints such as PD1 signals, effectively activate T cells, and achieve tumor cell killing. Chimeric adaptors can amplify CNK-T cell signals, resist immune suppression in tumor environments, achieve T cell activation, and kill tumor cells; CNK-T cells can also eliminate immune suppressive cells such as MDSCs by recognizing targets through NK. After infecting cells, viruses express specific functional proteins that directly promote the assembly and transport of MHCI or the targeted degradation of MHCI molecules, thereby inhibiting the presentation of viral antigen epitopes and causing immune evasion.

[0134] In one aspect, the present disclosure provides UT cells that further express a CNK receptor and optionally a CAR. Such UT cells are also referred to as "CNKT-UT." In the present application, it has been verified that CNK-UT has broad-spectrum tumor recognition and killing capabilities. The present application further designs a CAR / CNK-UT product tailored to a specific target, and the CAR / CNK-UT product has stronger killing and activation functions on tumor cells compared to conventional CAR-T; animal experiments have verified that the CAR / CNK-UT product also has more effective tumor elimination capabilities. The UT technology achieves heterologous universal modification, and since T cells are provided from healthy donors, it achieves standardization and scale-up production of CNK-UT products, which can be made in advance, and ensures the killing and activation functions of T cells on tumor cells. In some preferred embodiments, the CNKT-UT comprises a nucleic acid molecule encoding an amino acid sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, to the amino acid sequence set forth in SEQ ID NO: 117. In some preferred embodiments, the CNKT-UT comprises a nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO: 117. In another aspect, the present disclosure further provides a use of CNKT-UT in the manufacture of a medicament for treating a disease.

[0135] In some aspects, the disclosure further provides an immune cell comprising or expressing a chimeric protein construct described herein, or encoding a nucleic acid molecule or vector thereof, wherein the chimeric protein construct comprises an ERAD machinery protein binding domain and a targeting domain, the targeting domain comprising a domain that specifically targets or binds to a target protein, and the target protein is expressed in both the immune cell and a target cell. In some embodiments, the immune cell further comprises or expresses a binding domain or molecule (e.g., a CAR) that targets the target protein, a nucleic acid molecule or vector encoding the same. In some embodiments, examples of such target proteins include CD123, CD5, CD7, CD38, or CD4.

[0136] Without being limited by theory, a target protein is expressed in both the target cell (e.g., cancer cell or virus-infected cell) and the therapeutic immune cell. Usually, such a target is difficult to treat by cell therapy. Therefore, when the therapeutic immune cell expresses a CAR targeting such a target, the CAR also recognizes the same target expressed by the immune cell itself, so the immune cell undergoes autophagy, and cannot proliferate normally, or has a limited killing effect on the target cell. The chimeric protein construct provided in the present application can degrade the target protein expressed by the immune cell itself, thereby avoiding the autophagy effect and maintaining a specific killing effect on the target cell. Another advantage of using the chimeric protein construct of the present application is that the chimeric protein construct targeting this target and the binding domain (e.g., CAR, TCR, etc.) targeting this target can be simultaneously expressed by coexpression, which is more convenient and has a better effect than conventional methods such as gene knockout. In some embodiments, the chimeric protein construct further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain described herein. Any of the protein degradation pathway member binding domains described herein can be used.

[0137] Methods of cell administration for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, methods of adoptive T cell therapy are disclosed, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85. See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments, the cells or populations or subtypes of cells are analyzed in a range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the above values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, or a range defined by any two of the above values). 0 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the above values), and in some cases, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value therebetween.

[0138] In some embodiments, the total cell dosage and / or the individual subpopulation dosage of cells is 10 4 individual cells or approximately 10 4 ~10 cells / kilogram (kg) body weight 9 individual cells or approximately 109 In the range of 10 cells / kilogram (kg) of body weight, e.g., 10 5 ~10 6 In some embodiments, the range is at least about 1×10 cells / kg body weight, e.g., at least about 1×10 5 Individual cells / kg, 1.5×10 5 Individual cells / kg, 2×10 5 cells / kg or 1×10 6 cells / kg body weight or 1×10 5 Individual cells / kg, 1.5×10 5 Individual cells / kg, 2×10 5 cells / kg or 1×10 6 cells / kg body weight or approximately 1 x 10 5 Individual cells / kg, 1.5×10 5 Individual cells / kg, 2×10 5 cells / kg or 1×10 6 For example, in some embodiments, cells are cultured at 10 4 T cells or approximately 10 4 ~10 T cells / kilogram (kg) body weight 9 T cells or approximately 10 9 The T cells are administered in the range of 10 T cells / kilogram (kg) of body weight or within a certain margin of error therebetween, for example, 10 5 ~10 6 In some embodiments, the T cells are administered in the range of 1×10 T cells / kg body weight, e.g., at least or at least about 1×10 5 T cells / kg, 1.5×10 5 T cells / kg, 2 x 10 5 T cells / kg or 1×10 6 T cells / kg body weight or 1×10 5 T cells / kg, 1.5×10 5 T cells / kg, 2 x 10 5 T cells / kg or 1×10 6 T cells / kg body weight or approximately 1 x 10 5 T cells / kg, 1.5×10 5 T cells / kg, 2 x 10 5 T cells / kg or 1×10 6 The dose will be 1 T cell / kg body weight.

[0139] The cells may be administered by any suitable means, for example, by bolus, intravenous or subcutaneous injection, intraocular, periocular, subretinal, intravitreal, transseptal, subscleral, intrachoroidal, anterior cavity, subperineal, subconjunctival, sub-Tenon, retrobulbar, peribulbar, or posterior juxtascleral injection. In some embodiments, they are administered extra-gastrointestinal, intrapulmonary, intranasal, and, when localized treatment is desired, intralesional. Extra-gastrointestinal infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous. In some embodiments, a fixed dose of cells is administered by a single bolus. In some embodiments, the cells are administered by several boluses, for example, within 3 days, or by continuous infusion. In some embodiments, a method of repeated administration is provided in which a first dose of cells is administered followed by one or more second consecutive doses. When administered to a subject in an adoptive therapy setting, the timing and size of multiple administrations of cells is typically designed to increase the efficacy and / or activity and / or function of T cells expressing an antigen (e.g., T cells expressing a CAR). In some embodiments, repeated administration reduces downregulation or inhibitory activity that can occur when inhibitory immune molecules such as PD-1 and / or PD-L1 are upregulated in T cells expressing an antigen such as a CAR. Such methods include administering a first dose, typically followed by one or more consecutive doses, with a specific time window between each dose.

[0140] In the context of adoptive cell therapy, administration of a given "dose" includes administration of a given amount or number of cells as a single composition and / or a single continuous administration (e.g., as a single injection or continuous infusion), and also includes administration of a given amount or number of doses of cells provided by multiple individual compositions or infusions over a given period of time (3 days or less). Thus, in some cases, a first or continuous dose is a single or continuous administration of a particular number of cells administered or beginning at one time. However, in some cases, a first or continuous dose is administered over a period of 3 days or less, e.g., 3 or 2 days, with multiple injections or infusions, one each day, or multiple infusions per day.

[0141] 3. Targeted Degradation of Pathogenic Proteins In another aspect, the disclosure further provides a method of degrading a target protein in vivo or in vitro, the method comprising delivering a nucleic acid molecule and / or vector as described herein to a cell or subject expressing the target protein, the nucleic acid molecule and / or the vector causing the cell or subject to express and produce a chimeric protein construct as described herein. The target protein binding domain of the chimeric protein construct can bind to a target protein, and the ERAD machinery protein binding domain of the chimeric protein construct can induce spatial proximity of the ubiquitin-proteasome system (UPS) via the ERAD pathway, thereby allowing the target protein to be degraded by the proteasome in the UPS. When the chimeric protein construct further comprises a protein degradation pathway member binding domain, the method induces an additional degradation system in the vicinity of the target protein, significantly improving the degradation effect. In another aspect, the present disclosure further provides a method of treating and / or preventing the recurrence of a condition or disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the above-described pharmaceutical composition (e.g., comprising a nucleic acid molecule described herein, such as an mRNA, viral vector, oncolytic virus, chimeric protein construct, etc.).

[0142] In some embodiments, the methods include introducing a nucleic acid molecule (e.g., mRNA) or a vector (e.g., a viral vector) encoding a chimeric protein construct described herein into an individual in need thereof, such that the nucleic acid molecule or vector expresses the chimeric protein construct in cells of the individual. Any of the conventional routes of administration, such as non-intestinal, topical or mucosal routes, may be used in the present invention. Non-intestinal routes are injection or infusion administration, including systemic and local routes. In general, types of non-intestinal injections are intravenous (into a vein), intra-arterial (into an artery), intradermal (into the dermis layer), subcutaneous (below the epidermis), intramuscular (into a muscle) and intratumoral (into a tumor or a site very close to the tumor). A typical infusion is by an intravenous route. Mucosal administration includes, but is not limited to, oral / esophageal, intranasal, tracheal, intrapulmonary, intravaginal or intrarectal routes. Topical administration may also be by transdermal means (e.g., patches, etc.). Administration may be by conventional syringes and needles or any compound or device known in the art to facilitate or improve delivery of an active agent to a subject. Depending on the disease to be treated and the pathogenic protein to be degraded in the individual, an appropriate chimeric protein construct, a nucleic acid molecule thereof, and a vector thereof may be selected. In some preferred embodiments, the disease includes various solid tumors, hematological tumors, infectious diseases (e.g., viral infectious diseases), autoimmune diseases, neurodegenerative diseases, and metabolic diseases.

[0143] In some embodiments, the disease is a tumor or cancer, and the targeting domain of the chimeric protein construct specifically targets a tumor-associated target or an immune function-associated target, wherein the tumor-associated target or the immune function-associated target is as described above. In some preferred embodiments, the solid tumor is selected from nervous system tumors, head and neck tumors, thoracic tumors, gastrointestinal tumors, genitourinary system tumors, soft tissue and skin tumors, bone tumors, and the like. In some preferred embodiments, nervous system tumors include diffuse glioma, diffuse astrocytoma / anaplastic astrocytoma, glioblastoma, oligodendroglioma, oligoastrocytoma, childhood diffuse glioma, other astrocytoma, ependymoma, neuronal / mixed neuronal-glioma, medulloblastoma, other embryonal tumors, schwannoma, meningioma, solitary fibrous tumor, and hemangiopericytoma. In some preferred embodiments, head and neck tumors include nasal and paranasal malignancies, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, and the like. In some preferred embodiments, thoracic tumors include lung cancer, esophageal cancer, cardia cancer, breast cancer, mediastinal tumors, and the like. In some preferred embodiments, gastrointestinal tumors include gastric cancer, colon cancer, sigmoid / rectal cancer, hepatic cancer, pancreatic / periampullary cancer, biliary tract cancer, small intestine malignant tumors, and the like. In some preferred embodiments, genitourinary system tumors include renal cancer, prostate cancer, bladder cancer, testicular malignancies, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, and the like. In some preferred embodiments, soft tissue and skin tumors include malignant fibrous histocytoma, rhabdomyosarcoma, synovial sarcoma, cutaneous melanoma, and the like.

[0144] In some preferred embodiments, bone tumors include osteosarcoma, Ewing's sarcoma, and the like. In some preferred embodiments, the colon cancer is a colon adenoma. In some preferred embodiments, the breast cancer is a triple negative breast cancer cell. In some preferred embodiments, the liver cancer is hepatocellular carcinoma. In some preferred embodiments, the disease is a hematological malignancy selected from leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), and the like. In some preferred embodiments, the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, and the like. In another aspect, the present disclosure further provides a method of treating a tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an oncolytic virus or oncolytic virus pharmaceutical composition described herein. The oncolytic virus or composition of the present invention can be administered to a subject once or multiple times. When administered multiple times, it can be administered by the same or different routes and can be administered to the same or different sites. It may be administered repeatedly after a rest period in successive cycles. The interval between each administration can range from a few hours to a year (e.g., 24h, 48h, 72h, weekly, every two weeks, monthly or yearly). The interval can also be irregular (e.g., after tumor progression). The amount of a single administration can vary within the above ranges. In some embodiments, the route of administration of the oncolytic virus can include an intravenous route and an intratumoral route.

[0145] In the present invention, the appropriate dosage (e.g., 10 7 ~5x10 9 The oncolytic virus can be administered in one or more doses at approximately 10 pfu. The interval between each viral administration can vary from about 1 day to about 8 weeks. In another preferred treatment protocol, 10 8 or 10 9 The oncolytic cowpox virus of Pfu is administered intravenously or intratumorally for 2 to 5 times (e.g., 3 times), and the physician can select based on the actual situation. In some embodiments, the method includes administering an oncolytic viral vector comprising the above-mentioned nucleic acid molecule to the subject with cancer. The pharmaceutical composition or the oncolytic viral vector comprising the above-mentioned nucleic acid molecule can target and degrade pathogenic proteins, such as oncogenic proteins, viral proteins, immune-related proteins, etc. In some embodiments, the disease is an infectious disease and the targeting domain of the chimeric protein construct specifically targets an infectious disease-associated target, the infectious disease-associated target being as described above. In some preferred embodiments, the viral infection includes respiratory viral diseases, gastrointestinal viral diseases, liver viral diseases, skin and mucous membrane viral diseases, eye viral diseases, central nervous system viral diseases, lymphocytic viral diseases, insect-borne viral diseases, lentiviral infections, and the like. In some preferred embodiments, respiratory viral diseases include infections such as rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus and coronavirus; influenza; mumps; and the like.

[0146] In some preferred embodiments, the gastrointestinal viral diseases include polio; Cooksackie virus infection; ECHO virus infection; and the viral gastroenteritis includes rotavirus gastroenteritis, Norwalk virus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis, and calicivirus gastroenteritis, and the like. In some preferred embodiments, the liver viral diseases include viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, EB virus hepatitis, and cytomegalovirus hepatitis. In some preferred embodiments, the cutaneous and mucosal viral diseases include measles, rubella, exanthema in infancy, chickenpox and shingles, smallpox, herpes simplex virus infection, rabies, and foot and mouth disease. In some preferred embodiments, the ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, and the like. In some preferred embodiments, the central nervous system viral diseases include epizootic B encephalitis, western equine encephalitis, eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, hymenopteran encephalitis, and lymphocytic choroid plexus meningitis. In some preferred embodiments, lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, acquired immune deficiency syndrome, and the like.

[0147] In some preferred embodiments, the insect-borne viral diseases include viral hemorrhagic fevers such as epizootic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Ossus brevis hemorrhagic fever, Marburg disease, and Ebola hemorrhagic fever; dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick fever; sandfly fever, and the like. Preferably, lentiviral infection diseases include subacute sclerosing panencephalitis, Kuhl's disease, progressive multifocal leukoencephalopathy, and subacute spongiform encephalopathy (corticostriatospinal degeneration), and the like. In some embodiments, the disease is an autoimmune disease and the targeting domain of the chimeric protein construct specifically targets an autoantigen-associated target, the autoantigen-associated target being as described above. In some preferred embodiments, the autoimmune disease includes organ-specific and systemic autoimmune diseases. Preferably, organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis associated with pernicious anemia, pulmonary hemorrhagic nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebrospinal sclerosis, acute idiopathic polyneuropathy, and the like. In some preferred embodiments, the systemic autoimmune disease includes systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, and the like. In some embodiments, the disease is a neurological or degenerative disease, and the targeting domain of the chimeric protein construct specifically targets a neurological disease-associated target, the neurological disease-associated target being as described above.

[0148] In some embodiments, the neurological disorder is a nervous system disorder or peripheral nerve disorder, such as trigeminal neuralgia, facial paralysis, facial muscle spasm, vestibular neuritis, glossopharyngeal neuralgia, mononeuropathy, brachial plexus neuralgia, multiple mononeuropathy, polyneuropathy, acute inflammatory demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy; Spinal diseases such as spondylitis, compressive myelopathy, subacute complex degeneration of the spinal cord, syringomyelia, spinal vascular disease, and spinal arachnoiditis; Cerebrovascular diseases such as transient ischemic attack, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, and intracranial venous thrombosis; Central nervous system infections such as meningitis, encephalitis, etc. caused by infection with viruses, bacteria, fungi, or parasites, and lentiviral encephalitis caused by lentivirus infection; Demyelinating diseases of the central nervous system, such as multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, and leukodystrophies; Movement disorders such as Parkinson's disease, chorea, hepatolenticular degeneration, muscle tone disorders, idiopathic tremor, and tardive movement disorders; epilepsy; Headaches such as migraines, tension headaches, and cluster headaches; Degenerative diseases of the nervous system, such as motor neuron disease, Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, and multisystem atrophy; Genetic disorders of the nervous system, such as hereditary ataxia, hereditary spastic paraplegia, peroneal muscular atrophy, neurofibromatosis, tuberous sclerosis, and superficial cerebrovascular disease; Neurodevelopmental disorders such as congenital hydrocephalus, cerebral palsy, basilar depression, and subcerebellar tonsillar disease; Neuromuscular junction and muscle disorders such as myasthenia gravis, periodic paralysis, polymyositis, progressive muscular dystrophy, myotonic myopathy (tonic muscular dystrophy, myotonia congenita), metabolic myopathy (mitochondrial myopathy, lipid storage myopathy, glycogenosis); Autonomic nervous system disorders such as Raynaud's disease, erythematous limb pain, hemifacial atrophy, generalized autonomic dysfunction, spontaneous hyperhidrosis, and progressive lipodystrophy; Nervous system tumors such as brain gliomas, lymphomas, and spinal cord tumors; This includes nervous system paraneoplastic syndromes such as paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, subacute necrotizing myelopathy, subacute motor neuron disease, and paraneoplastic sensory neuron disease. In some preferred embodiments, neurodegenerative diseases can cause a gradual loss of neuronal structure and function, such as neuronal death and glial cell balance, leading to cognitive impairment, such as dementia. Exemplary neurodegenerative diseases include Alzheimer's disease, Parkinson's disease (PD), Huntington's disease, early onset AD or PD, and amyotrophic lateral sclerosis (ALS). In some embodiments, the disease is a metabolic disease, e.g., diabetes, hyperlipidemia, gout, etc. The metabolic disease-related targets are as described above. EXAMPLES

[0149] Example 1: Design of ER-TPD chimeric protein constructs In this example, five chimeric protein constructs were designed using Endoplasmic Reticulum-based Targeted Protein Degradation (ER-TPD). The first structure is a basic ER-TPD chimeric protein construct, and the other four structures are ER-TPD chimeric protein constructs with stronger functions, which are formed by adding E3 ubiquitin ligase ligands, E2 ubiquitin-binding enzyme ligands, or lysosomal ligands, or combinations thereof, to the first structure. The ER-TPD chimeric protein constructs contain integrated multifunctional domains or functional variants of recombinant protein molecules that bind to and target and degrade the target protein.

[0150] 1.1 Basic ER-TPD chimeric protein construct In some embodiments, a basic ER-TPD chimeric protein construct TBD-TMD-ERD is used, the linear and structural schematic of which is shown in FIG. 1A. It comprises three domains: (1) a targeting domain (targeted protein binding domain, TBD) or a functional variant thereof, which may be a target affinity ligand protein molecule or a functional variant thereof (which may be an antibody fragment, a polypeptide, a natural receptor, a ligand, or an artificial recombinant protein or a binding domain thereof having affinity to a target protein); (2) a transmembrane domain (transmembrane domain, TMD) or a functional variant thereof of a viral endoplasmic reticulum (ER) resident glycoprotein; and (3) a cytoplasmic domain (ER retention domain, ERD) or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein. The targeting domain or a functional variant thereof, the transmembrane domain (transmembrane domain) or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein and the cytoplasmic domain (ER retention domain, ERD) or a functional variant thereof may be connected by a hinge or a linker.

[0151] As shown in Figure 1B, the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD is as follows: [1] Specific binding and retention: After the chimeric protein construct is expressed, TBD-TMD-ERD resides in the endoplasmic reticulum and binds to the target protein through the targeting domain to retain the target protein in the endoplasmic reticulum and prevent the transport of the synthesized target protein to the Golgi apparatus. [2] Retrotransport: After the chimeric protein construct binds to the target protein, the complex formed by the chimeric protein construct and the target protein is transported from the endoplasmic reticulum to the cytoplasm by the retrotransporter in the ERAD mechanism. A typical retrotransporter contains Derlin, E3 ligase Hrd1 and its chaperonin Sel1L, and can recruit ATPase p97 in the cytoplasm. Derlin has six transmembrane domains, both of which are located in the cytoplasm at their N- and C-termini, forming transport channels, and the retrotransport process requires the involvement of ATPase p97 (Byun H et al. ERAD and how viruses exploit it. Front Microbiol. 2014 Jul 3; 5:330). [3] Ubiquitination and degradation by the proteasome: The retrotransporter acts with endoplasmic reticulum-specific E3 ubiquitination ligase to promote the ubiquitination of target proteins, and the ubiquitinated target proteins are transported into the cytoplasm by the retrotransporter and recognized and degraded by the proteasome.

[0152] 1.2 ER-TPD chimeric protein construct with E3 ubiquitin ligase ligand domain In some embodiments, the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L with E3 ubiquitin ligase ligand domain is used. Its linear and structural schematics are shown in Figures 2A and 2B, respectively. It comprises four domains: (1) a targeting domain or a functional variant thereof, which can be a target affinity ligand protein molecule or a functional variant thereof (can be an antibody fragment, a polypeptide, a natural receptor, a ligand, or an artificial recombinant protein domain having affinity for a target protein); (2) a transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein; (3) a cytoplasmic domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein; and (4) a ligand for E3 ligase (E3L) or a functional variant thereof, which can be a polypeptide or a recombinant protein and binds to multiple E3 ubiquitin ligases outside the endoplasmic reticulum. The targeting domain or a functional variant thereof, the transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein, the cytoplasmic domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein, and the E3 ubiquitin ligase ligand domain or a functional variant thereof may be linked by a hinge or a linker. Since the number of E3 ubiquitin ligases inherent to the endoplasmic reticulum is limited, and the ERAD pathway of misfolded proteins in the endoplasmic reticulum competes with E3 ubiquitin ligases, effective degradation of the target protein cannot be achieved unless the pathway of ubiquitination and proteasome degradation outside the endoplasmic reticulum is increased. Thus, the present inventors have found that effective ubiquitination and degradation of the target protein can be achieved by exogenously increasing the E3 ubiquitin ligase ligand.

[0153] As shown in Figure 2C, the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L is as follows: [1] Specific binding and retention: After the chimeric protein construct is expressed, TBD-TMD-ERD-E3L resides in the endoplasmic reticulum and binds to the target protein through the targeting domain to retain the target protein in the endoplasmic reticulum and prevent the transport of the synthesized target protein to the Golgi apparatus. [2] Retrotransport: After the chimeric protein construct binds to the target protein, the complex formed by the chimeric protein construct and the target protein is transported from the endoplasmic reticulum to the cytoplasm by the retrotransporter in the ERAD mechanism. A typical retrotransporter includes Derlin, E3 ligase Hrd1 and its chaperonin Sel1L, and can recruit cytoplasmic ATPase p97. Derlin has six transmembrane domains, both its N-terminus and C-terminus are located in the cytoplasm, forming a transport channel, and the retrotransport process requires the involvement of ATPase p97. [3] Ubiquitination and degradation by the proteasome: The E3 ligase ligand in the chimeric protein construct binds to the E3 ligase complex, and the target protein is ubiquitinated by the E2 ubiquitin conjugating enzyme in the complex and transported to the proteasome for degradation. Here, the ligand that targets the E3 ligase complex may be an artificially screened or constructed protein sequence, or may be a naturally occurring protein sequence such as the IκBα-derived phosphopolypeptide (DRHDSGLDSM, SEQ ID NO: 119) or a recombinant sequence thereof.The IκBα-derived phosphopolypeptide recruits SCFβ-TRCP in the E3 ubiquitin ligase complex and mediates the ubiquitination and degradation pathway of the target protein (Sakamoto KM et al. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc Natl Acad Sci US A. 2001;98:8554-9); it may further contain an E3 ligase recognition peptide fragment (ALAPYIP, SEQ ID NO: 120) derived from HIF-1α, which is linked to the E3 ligase VHL to achieve the ubiquitination and degradation pathway (Ivan M et al. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science. 2001;292:464-8).

[0154] 1.3 ER-TPD chimeric protein constructs with E2 ubiquitin-conjugating enzyme ligand domains In some embodiments, the ER-TPD chimeric protein construct TBD-TMD-ERD-E2L with E2 ubiquitin-conjugating enzyme ligand domain is used. Its linear and structural schematics are shown in Figures 3A and 3B, respectively. It comprises four domains: (1) a targeting domain or a functional variant thereof, which may be a target affinity ligand protein molecule or a functional variant thereof (which may be an antibody fragment, a polypeptide, a natural receptor, a ligand, or an artificial recombinant protein domain having affinity for a target protein); (2) a transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein; (3) a cytoplasmic domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein; and (4) an E2 ubiquitin-conjugating enzyme ligand domain or a functional variant thereof, which may be a polypeptide or a recombinant protein. The targeting domain or a functional variant thereof, the transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein, the cytoplasmic domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein, and the E2 ubiquitin-conjugating enzyme ligand domain or a functional variant thereof may be connected by a hinge or a linker.

[0155] As shown in Figure 3C, the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E2L is as follows: [1] Specific binding and retention: After the chimeric protein construct is expressed, TBD-TMD-ERD-E2L resides in the endoplasmic reticulum and binds to the target protein through the targeting domain to retain the target protein in the endoplasmic reticulum and prevent the synthesized target protein from being transported to the Golgi apparatus. [2] Retrotransport: After the chimeric protein construct binds to the target protein, the complex formed by the chimeric protein construct and the target protein is transported from the endoplasmic reticulum to the cytoplasm by the retrotransporter in the ERAD mechanism. A typical retrotransporter includes Derlin, E3 ligase Hrd1 and its chaperonin Sel1L, and can recruit cytoplasmic ATPase p97. Derlin has six transmembrane domains, both its N-terminus and C-terminus are located in the cytoplasm, forming a transport channel, and the retrotransport process requires the involvement of ATPase p97. [3] Ubiquitination and degradation by proteasome: The chimeric protein construct directly binds to E2 ubiquitin-conjugating enzyme by the E2 ubiquitin-conjugating enzyme ligand domain, and the target protein is ubiquitinated by the E2 ubiquitin-conjugating enzyme and transported to the proteasome for degradation. The E2 ubiquitin-conjugating enzyme ligand domain can achieve ubiquitination and degradation by proteasome of the target protein by binding to the UBC structure of E2 ubiquitin-conjugating enzyme by the RING zinc finger domain from IAP family protein. Here, the ligand targeting E2 ubiquitin-conjugating enzyme can be a RING zinc finger domain from IAP family protein or a recombinant protein sequence with a RING zinc finger domain. IAP (inhibitor of apoptosis proteins) is a family of apoptosis protein inhibitor molecules.IAPs, as E3 ligases of pro-apoptotic proteins (e.g., caspases, SMAC, ARTS), can inhibit cell apoptosis by achieving ubiquitination and degradation of pro-apoptotic proteins; they can also regulate NOD signaling and immune responses by ubiquitination of RIPK2 (receptor-interacting serine / threonine-protein kinase 2). Five members of the IAP family (XIAP, cIAP1, cIAP2, Livin, and ILP2) all have RING zinc finger domains and can bind to ubiquitination conjugating enzymes E2 and Apollon proteins (Mikihiko Naito et al. SNIPERs-Hijacking IAP activity to induce protein degradation.Drug Discov Today Technol. 2019 Apr;31:35-42). Therefore, by linking the chimeric protein construct to the ubiquitin conjugating enzyme E2 via the RING zinc finger domain derived from an IAP family member, more efficient ubiquitination and proteasome-mediated degradation of the target protein can be achieved. The following sequences can be used for designing the E2 ligand:

[0156] RING domain from XIAP: CKICMDRNIAIVFVPCGHLVTCKQCAEAVDKCPMCY (SEQ ID NO: 155); RING domain from cIAP1: CKVCMDKEVSVVFIPCGHLVVCQECAPSLRKCPICR (SEQ ID NO: 156); RING domain from cIAP2: CKVCMDKEVSVVFIPCGHLVVCQECAPSLRKCPICR (SEQ ID NO: 157); RING domain from Livin: CKVCLDRAVSIVFVPCGHLVCAECAPGLQLCPICR (SEQ ID NO: 158); RING domain from ILP-2: CKICMDRHIAVVFIPCGHLVTCKQCAEAVDRCPMCS (sequence number 159).

[0157] 1.4 ER-TPD chimeric protein constructs with lysosomal ligand domains In some embodiments, the ER-TPD chimeric protein construct TBD-TMD-ERD-LL with lysosomal ligand domain is used. Its linear and structural schematics are shown in Figures 4A and 4B, respectively. It comprises four domains: (1) a targeting domain or a functional variant thereof, which may be a target affinity ligand protein molecule or a functional variant thereof (which may be an antibody fragment, a polypeptide, a natural receptor, a ligand, or an artificial recombinant protein domain having affinity for a target protein); (2) a transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein; (3) a cytoplasmic domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein; and (4) a lysosomal ligand domain or a functional variant thereof, which may be a polypeptide or a recombinant protein. The targeting domain or a functional variant thereof, the transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein, the cytoplasmic domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein, and the lysosomal ligand domain or a functional variant thereof may be connected by a hinge or a linker.

[0158] As shown in Figure 4C, the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-LL is as follows: [1] Specific binding and retention: TBD-TMD-ERD-LL is expressed and resides in the endoplasmic reticulum membrane, and targets the target protein ligand and binds to the target protein to retain it in the endoplasmic reticulum, preventing the transport of the synthesized target protein to the Golgi apparatus. [2] Retrotransport: After the chimeric protein construct binds to the target protein, the complex formed by the chimeric protein construct and the target protein is transported from the endoplasmic reticulum to the cytoplasm by the retrotransporter in the ERAD machinery. [3] Lysosomal endocytosis and hydrolysis: The chimeric protein construct directly binds to lysosomal chaperones via the lysosomal ligand domain and is transported to lysosomes via the chaperones, where the target protein is degraded by various hydrolases. The lysosomal ligand domain can be a KFERQ-like motif or a recombinant protein sequence with a KFERQ-like motif, which can be specifically recognized and bound to the chaperone Hsc70, and the target protein ER-TPD complex is transported to the lysosome by the chaperone mediated autophagy (CMA) mechanism and degraded by various hydrolases.

[0159] Chaperone mediated autophagy (CMA) is a process of selective protein autophagy and lysosomal degradation. The chaperone Hsc70 recognizes and targets KFERQ-like motifs on target proteins and also recognizes the receptor protein LAMP2A on the lysosomal membrane, thereby transporting the target proteins to the lysosome for degradation. Here, the KFERQ-like motif is KFERQ derived from RNaseA (Backer J et al. Regulation of catabolism of microinjected ribonuclease A requires the amino-terminal 20 amino acids. Proc Natl Acad Sci. 1983; 80:2166-2170); QKILD (SEQ ID NO: 124) and QRDKV (SEQ ID NO: 125) derived from Hsc70 (Cuervo AM et al. Unique properties of lamp2a compared to other lamp2 isoforms. J Cell Sci. 2000; 113(Pt 24):4441-4450); QRFFE (SEQ ID NO: 126) derived from hemoglobin (Slot LA et al. Intracellular protein degradation in serum-deprived human fibroblasts. Journal of Biochemistry.1986; 237:491-498; QKKEL (SEQ ID NO:127), QFREL (SEQ ID NO:128) and IKLDQ (SEQ ID NO:129) from aldolase B; EFLKQ (SEQ ID NO:130) from Annexin I; QKVFD (SEQ ID NO:131) from Annexin II; QELLR (SEQ ID NO:132) from Annexin IV; QEFIK (SEQ ID NO: 133) from VI; RKVEQ (SEQ ID NO: 134) from aspartate aminotransferase; NLLKE from c-fos; NRVVD (SEQ ID NO: 135) from GAPDH; NKKFE (SEQ ID NO: 136) from glutathione transferase; VKELQ (SEQ ID NO: 137) from IκB; VDKLN (SEQ ID NO: 138) and RIKEN (SEQ ID NO: 139) from α-2-microglobulin; DVVRQ (SEQ ID NO: 140), QRIVE (SEQ ID NO: 141) and QLLRE (SEQ ID NO: 142) from Pax-2; IEKLQ (SEQ ID NO: 143) from 26S proteasome (C8); QEKVF (SEQ ID NO: 144) from 19S proteasome (PA28); QDLKF (SEQ ID NO: 145) from pyruvate kinase; VKKDQ (SEQ ID NO: 146) from α-synuclein (Massey AC. et al. Chaperone-Mediated Autophagy in Aging and Disease. Current Topics in Developmental Biology,2006; 73:205-235).

[0160] 1.5 ER-TPD chimeric protein construct with E3 ubiquitination ligase and lysosomal ligand domains In some embodiments, the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL is used, which has a combination of E3 ubiquitination ligase ligand and lysosomal ligand. Its linear and structural schematics are shown in Figure 5A and 5B, respectively. It comprises five domains: (1) a targeting domain or a functional variant thereof, which can be a target affinity ligand protein molecule or a functional variant thereof (can be an antibody fragment, a polypeptide, a natural receptor, a ligand, or an artificial recombinant protein domain having affinity for a target protein); (2) a transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein; (3) a cytoplasmic domain or a functional variant thereof of a viral endoplasmic reticulum resident glycoprotein; (4) an E3 ubiquitin ligase ligand or a functional variant thereof (ligand for E3 ligase, E3L) and (5) a lysosomal ligand or a functional variant thereof (Ligand for lysosome). The targeting domain or its functional variant, the transmembrane domain or its functional variant of a viral endoplasmic reticulum-resident glycoprotein, the cytoplasmic domain or its functional variant of a viral endoplasmic reticulum-resident glycoprotein, the E3 ubiquitin ligase ligand or its functional variant, and the lysosomal ligand domain or its functional variant may be linked together by a hinge or a linker.

[0161] As shown in Figure 5C, the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL is as follows: [1] Specific binding and retention: After the chimeric protein construct is expressed, TBD-TMD-ERD-E3L-LL resides in the endoplasmic reticulum and binds to the target protein through the targeting domain to retain the target protein in the endoplasmic reticulum and prevent the synthesized target protein from being transported to the Golgi apparatus. [2] Retrotransport: After the chimeric protein construct binds to the target protein, the complex formed by the chimeric protein construct and the target protein is transported from the endoplasmic reticulum to the cytoplasm by the retrotransporter in the ERAD machinery. [3] Lysosomal endocytosis and enzymatic degradation and ubiquitination / degradation by proteasome: The chimeric protein construct is transported out of the endoplasmic reticulum, directly binds to lysosomal chaperones via the lysosomal ligand domain, and is transported to lysosomes via the chaperones, where the target protein is degraded by various hydrolases; part of the chimeric protein is transported out of the endoplasmic reticulum; the E3 ligase ligand in the chimeric protein construct can bind to the E3 ligase complex, and the target protein is ubiquitinated by the E2 ubiquitin conjugating enzyme in the complex and transported to the proteasome for degradation.

[0162] Example 2: Design of ER-TPD chimeric nucleic acid constructs targeting TCR In this example, two ER-TPD chimeric nucleic acid constructs targeting TCR, TCR-ER-TPD, were designed. In some embodiments, as shown in FIG. 6, a single lentiviral expression vector and the EF1α promoter were used to design an ER-TPD chimeric protein construct, TCR-ER-TPD, targeting two TCRs as follows: (A) TCR-TPD1:anti-TCR-E3 / 19KTMD-ERD-P2A-EGFRt (Figure 6A); (B) TCR-TPD2:anti-TCR-E3 / 19KTMD-ERD-G4S-E3L-P2A-EGFRt (Figure 6B).

[0163] Example 3: Generation of expression plasmids for TCR-targeting ER-TPD chimeric protein constructs The coding nucleotide sequence of the ER-TPD chimeric protein construct TCR-ER-TPD (TCR-TPD1 and TCR-TPD2) targeting TCR designed in Example 2 was synthesized by total gene synthesis. Here, the nucleotide sequence of TCR-TPD1 is shown in SEQ ID NO: 160, and the protein it encodes is shown in SEQ ID NO: 161; the nucleotide sequence of TCR-TPD2 is shown in SEQ ID NO: 162, and the protein it encodes is shown in SEQ ID NO: 163. The nucleotide sequence of TCR-TPD1 and the nucleotide sequence of TCR-TPD2 were inserted into the lentiviral vector pCDH-CMV-MCS-EF1α-Puro (purchased from Youbio) by molecular cloning, respectively, to construct the TCR-ER-TPD lentiviral target vector, and the accuracy was confirmed by sequencing. Then, the plasmids were transformed into DH5α competent cells (Thermo Fisher), and single colonies were picked up, and after large-scale cultivation, the plasmids were extracted using the PureLink plasmid extraction kit. TM Plasmids were produced and purified using a HiPure Plasmid Maxiprep Kit (Thermo Fisher) to obtain the TCR-ER-TPD lentiviral plasmids, i.e., the TCR-TPD1 lentiviral plasmid and the TCR-TPD2 lentiviral plasmid, respectively.

[0164] Example 4: Viral generation of TCR-targeting ER-TPD chimeric protein constructs The TCR-ER-TPD lentiviral plasmid obtained in Example 3 and the packaging plasmids psPAX2 and pMD2.G (purchased from Addgene, product numbers: 12259 & 12260) in a ratio of 1.64 pmol: 1.3 pmol: 0.72 pmol were co-transfected into 293T cells (ATCC product, product number CRL3216TM) in a ratio of DNA: μg PEI = 1: 3 using polyethyleneimine transfection reagent (408727, Sigma). For specific methods of preparing packaging plasmids, see PureLinkTM Please refer to the instructions for the HiPure Plasmid Maxiprep Kit (K210006, Thermo). For specific transfection procedures, please refer to the Sigma transfection instructions. 16 hours after transfection, the medium was replaced with complete medium (purchased from Life Technologies, product number: 11995-065), and after culturing for 24, 48, and 72 hours, the lentivirus supernatants were collected, combined, centrifuged at -80°C and 3000 rpm for 10-15 minutes, filtered through a 0.45 μm filter, and finally, ultracentrifuged at 4°C and 25000 rpm for 2-3 hours to concentrate the virus, and the concentrated solution of TCR-ER-TPD lentivirus (TCR-TPD1 lentivirus or TCR-TPD2 lentivirus) was collected, and the collected TCR-ER-TPD lentivirus concentrated solution was stored at -80°C.

[0165] Example 5: Generation of TCR ER-TPD Jurkat cells Thawed Jurkat cells were cultured in RMPI1640 medium supplemented with 5% FBS in an incubator at 37°C and 5% CO2. The medium was replaced every 2 to 3 days, and the cell density was maintained at 1 × 10 6 The concentration of each cell was maintained at 0.5×10 single cells / ml. After the cell state recovered, the lentivirus carrying the TCR-ER-TPD lentivirus plasmid prepared in Example 4 was transfected into the recovered cells. The specific procedure is as follows. 6Jurkat cells were diluted to 1 single cell / ml and spread on a 24-well plate, and the TCR-TPD1 lentivirus and TCR-TPD2 lentivirus concentrates prepared in Example 4 were taken, and the virus solution was added to the cells so that the MOI was 1. Polybrene 5μg / ml was added, and the cells were placed in a flat-angle centrifuge, and then centrifuged at low speed (500g to 1000g / min) for 30 to 60 minutes. After that, the cells were placed in an incubator and cultured at 37°C, and 48 hours after transfection, Jurkat cells expressing TCR-ER-TPD (TCR ER-TPD Jurkat cells) were obtained. Then, the cell phenotype was detected by flow cytometry. After continuing the culture for 96h and 6 days, the following phenotype detection and cell function experiments were performed.

[0166] Example 6: Phenotype detection of ER-TPD transfected cells Flow cytometry was performed on the TCR ER-TPD Jurkat cells prepared in Example 5. Figure 7 shows the results of cell phenotype detection of TCR ER-TPD Jurkat cells at 48 h (A), 96 h (B), and day 6 (C). In cell culture and flow cytometry, 5 × 10 5Untransfected Jurkat cells and Jurkat cells transfected with TCR-ER-TPD lentivirus vector were cultured and stained with antibodies CD8-Pacific Blue, CD4-APC, EGFR-PE, and TCR-αβ-APC-Cy7 (all purchased from Biolegend), and cell phenotypes were identified using a flow cytometer (BD FACSCanto II). The results showed that: (A) after 48 h of culture, untransfected Jurkat cells expressed TCR-αβ but not EGFR; in Jurkat cells transfected with TCR-TPD1 lentivirus, EGFR was highly expressed, TCR-αβ began to be degraded, and its expression level on the cell membrane was reduced; in Jurkat cells transfected with TCR-TPD2 lentivirus, EGFR was highly expressed, and the degree of reduction in the expression level of TCR-αβ on the cell membrane was significantly higher than that of TCR-TPD1 lentivirus; (B) (A) After 96 h of culture, Jurkat cells transfected with the two TPD viruses both showed reduced surface TCR, and the degree of TCR reduction after transfection with the TCR-TPD2 lentivirus was significantly greater than that after transfection with the TCR-TPD1 lentivirus; (B) After 6 days of culture, Jurkat cells transfected with the two TPD viruses both showed reduced surface TCR, and the degree of TCR reduction after transfection with the TCR-TPD2 lentivirus was significantly greater than that after transfection with the TCR-TPD1 lentivirus.

[0167] Example 7: Design of HLA-targeting ER-TPD chimeric nucleic acid constructs In this example, an ER-TPD chimeric nucleic acid construct targeting HLA was designed. In some embodiments, as shown in FIG. 8, a single lentiviral expression vector and the EF1α promoter were used to design three expression vectors: (A) CNK:DAP10-CD3ζ-T2A-NKG2D (Figure 8A); (B) HLA-TPD:E3 / 19K-E3L (Figure 8B); (C) CNK-HLA-TPD:DAP10-CD3ζ-T2A-NKG2D-p2A-E3 / 19K-E3L (Figure 8C). Wherein HLA-TPD is the HLA targeting ER-TPD chimeric protein construct HLA-ER-TPD; CNK-HLA-TPD is the HLA targeting ER-TPD chimeric protein construct CNK-HLA-ER-TPD.

[0168] Example 8: Generation of expression plasmids for HLA-targeting ER-TPD chimeric protein constructs The coding nucleotide sequence of the HLA-targeting ER-TPD chimeric protein construct designed in Example 7 (the E3 / 19K-E3L nucleotide sequence is shown in SEQ ID NO: 164 and the protein it encodes is shown in SEQ ID NO: 165; the DAP10-CD3ζ-T2A-NKG2D-p2A-E3 / 19K-E3L nucleotide sequence is shown in SEQ ID NO: 166 and the protein it encodes is shown in SEQ ID NO: 167) was synthesized by total gene synthesis. The CNK nucleotide sequence designed in Example 7 (the nucleotide sequence is shown in SEQ ID NO: 168 and the protein it encodes is shown in SEQ ID NO: 169) was synthesized by total gene synthesis. The coding nucleotide sequence of the ER-TPD chimeric protein construct targeting HLA and the CNK nucleotide sequence were inserted into the lentiviral vector pCDH-CMV-MCS-EF1α-Puro (purchased from Youbio) by molecular cloning to construct the HLA-TPD lentiviral target vector, the CNK-HLA-TPD lentiviral target vector, and the CNK lentiviral target vector were constructed by confirming the accuracy by sequencing. The plasmids were then transformed into DH5α competent cells (Thermo Fisher), and a single colony was picked. After large-scale cultivation, the plasmids were...

Claims

1. A chimeric protein construct comprising an endoplasmic reticulum-associated degradation (ERAD) machinery protein-binding domain and a targeting domain.

2. The chimeric protein construct of claim 1, wherein the ERAD mechanism protein binding domain comprises a transmembrane domain of a viral endoplasmic reticulum resident protein or a functional variant thereof and an endoplasmic reticulum resident domain or a functional variant thereof.

3. The chimeric protein construct of claim 1, wherein the viral endoplasmic reticulum resident protein is adenovirus E3-19K.

4. The chimeric protein construct of claim 1, wherein the viral endoplasmic reticulum resident protein is not adenovirus E3-19K.

5. The chimeric protein construct of claim 4, wherein the viral endoplasmic reticulum resident protein is at least one selected from the group consisting of HCMV glycoproteins US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, and Cowpox Virus protein CPXV203.

6. The chimeric protein construct of claim 1, further comprising a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain, which may be linked to the ERAD machinery protein binding domain.

7. The chimeric protein construct of claim 1, wherein the targeting domain comprises an antibody or a functional fragment thereof (e.g., Fd, Fv, Fab, Fab', F(ab')2, Fv(scFv), single chain antibody (scFv), nanobody, two chain antibody, three chain antibody and four chain antibody) that specifically targets the target protein.

8. The chimeric protein construct of claim 7, wherein the target protein is a pathogenic protein, which may be a tumor-associated protein, a virus-associated protein, an immune function-associated protein (including an immunosuppressive protein and an immune activating protein), an autoantigen protein or a neurodegenerative disease-associated protein.

9. The chimeric protein construct of claim 7, wherein the target protein is an immune function-related protein selected from antigen-presenting molecules (e.g., MHC class I molecules, MHC class II molecules, MICA / B molecules, etc.), antigen-recognition molecules (e.g., TCR, CD123, NKG2D, etc.), immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), and immune stimulatory / co-stimulatory molecules (e.g., CD3, CD80 / 86, CD28, etc.).

10. The chimeric protein construct of claim 2, wherein the targeting domain does not specifically bind to a TCR.

11. 11. The chimeric protein construct of claim 10, wherein the targeting domain specifically binds to MHC I, MHC II, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and / or ULBP6, preferably wherein MHC I is HLA I and preferably wherein MHC II is HLA II.

12. The chimeric protein construct of claim 4, wherein the targeting domain specifically binds to a TCR.

13. The chimeric protein construct of claim 12, wherein the viral endoplasmic reticulum resident protein is at least one selected from the group consisting of HCMV glycoproteins US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, and Cowpox Virus protein CPXV203.

14. The chimeric protein construct of claim 1 linked to at least one co-expression moiety.

15. The chimeric protein construct of claim 14, wherein the at least one co-expression moiety is independently selected from an intact viral ER resident glycoprotein (e.g., HCMV US2, US3, US11, US10, adenovirus E3-Kl 9, HCMV US6, HSV ICP47), a chimeric antigen receptor (CAR), a functional T cell receptor (TCR), a chemokine receptor, and a NK cell activating receptor.

16. The chimeric protein construct of claim 15, wherein the chemokine receptor is selected from CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2 and CXCR4.

17. The chimeric protein construct of claim 16, wherein the NK cell activating receptor comprises (a) an extracellular domain (ED) of an NK cell activating receptor or a functional variant thereof, (b) a transmembrane domain (TMD) of an NK cell activating receptor or a functional variant thereof, and (c) an intracellular domain (ICD) of an NK cell activating receptor or a functional variant thereof; and a hinge or linker may be included between the extracellular domain of an NK cell activating receptor or a functional variant thereof, the transmembrane domain of an NK cell activating receptor or a functional variant thereof, and / or the intracellular domain of an NK cell activating receptor or a functional variant thereof.

18. 18. The chimeric protein construct of claim 17, wherein the NK cell activating receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxicity receptor, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC and NKp80.

19. The chimeric protein construct of claim 17, wherein the NK cell activating receptor is complexed with a CNK signaling module.

20. The chimeric protein construct of claim 14, wherein the at least one co-expression moiety is linked to the chimeric protein construct by a cleavable linker.

21. A nucleic acid molecule encoding the chimeric protein construct of claim 1.

22. A vector comprising the nucleic acid molecule described in claim 21, wherein the nucleic acid molecule is operably linked to at least one polynucleotide regulatory element so as to express a chimeric protein construct encoded by the nucleic acid molecule.

23. An engineered cell that expresses the chimeric protein construct of claim 1, or that contains the nucleic acid molecule of claim 21, or that contains the vector of claim 22.

24. 24. The engineered cell of claim 23, wherein the cell is an immune cell selected from a T cell, a natural killer (NK) cell, a B cell, a macrophage, a monocyte, a dendritic cell, a neutrophil, or a γδ T cell.

25. 23. A pharmaceutical composition or kit comprising (i) the chimeric protein construct of claim 1 or the nucleic acid molecule of claim 21 or the vector of claim 22 or the engineered cell of claim 23, and (ii) a pharma- ceutically acceptable vehicle.

Images